Biology subjects

Ouyang, Q.

Publications and source records attributed to Ouyang, Q..

At least 19 recordsLinked to original sources

bioRxiv2026

ctDNA Release Heterogeneity in Luminal Breast Cancer Revealed by Single-Cell Multi-Omics Spatial Analysis

Backgroud Breast cancer is a leading cause of cancer-related mortality, and HR+/HER2-; is the predominant subtype. Molecular pathology, including PIK3CA and ESR1 mutation, informs prognosis and treatment selection. Circulating tumor DNA (ctDNA) offers a non-invasive approach for cancer monitoring, but its clinical utility is limited by tissue plasma discordance, reflecting heterogeneous ctDNA release. The cellular and spatial determinants of this release remain poorly understood, particularly in HR+/HER2-; subtype, where heterogeneity across subclones complicates liquid biopsy interpretation. Results To identify the cellular and spatial determinants of ctDNA release, we developed scMASTER, integrating single-cell transcriptomes and mutations, spatial transcriptomics and matched tumor and plasma DNA sequencing. In ten patients with non-metastatic HR+/HER2- breast cancer, eight recurrent malignant epithelial states showed distinct ctDNA release potential. Antigen-presentation, stress, mitosis and mesenchymal-like states showed high release. PIK3CA and ESR1 mutations were linked to distinct functional states and altered ctDNA detectability. Three spatial architectures, EMT-enriched regions, intraductal luminal progenitors and avascular hypoxic zones, showed high release. Tumor-microenvironment interactions were bidirectional: anti-tumoral immunity correlated with reduced release, whereas immunomodulatory components correlated with increased release. A stratification model based on these features identified high-risk, immunosuppressed patients with poor prognosis, validated across external cohorts. Conclusions Our study establishes a single-cell spatial framework for decoding ctDNA release heterogeneity, linking tumor states, somatic mutations, spatial architecture, and immune interactions to ctDNA release. These findings provide mechanistic insights into tissue-plasma discordance, offer a biological rationale for interpreting ctDNA dynamics, refine patient risk stratification and liquid biopsy-based monitoring in HR+/HER2-; breast cancer.

cancer biology↗

bioRxiv2026

SparseSeg: Target-Conditioned Discovery Segmentation of Cryo-Volume Electron Microscopy Under Sparse Annotation

Cryo-volume electron microscopy (cryo-vEM) enables near-native visualization of cellular ultrastructure, but its broad use is limited by low image contrast and the high cost of dense voxel-level annotation. Existing automated segmentation methods often generalize poorly across cell types, organelles, and imaging conditions. Here, we introduce SparseSeg, a target-conditioned, sparsity-driven segmentation framework that treats organelle segmentation as a discovery process rather than a closed-set classification task. SparseSeg uses a small number of context-specific exemplars to iteratively propagate reliable supervision through the volume. It combines sparse patch-based sampling, a multi-kernel U-Net, and geometry-consistent refinement to expand accurate segmentation while suppressing context-dependent false positives. Across serial cryo-FIB-SEM and conventional vEM datasets, SparseSeg achieves robust segmentation under extreme sparse annotation, including settings with less than 1% labeled slices. This framework reduces annotation burden while preserving morphological fidelity for quantitative cryo-vEM analysis.

bioengineering↗

bioRxiv2026

Trem2hi macrophages bridge inflammation resolution and fibrosis initiation after ischemia-reperfusion injury in the kidney

Maladaptive repair of acute kidney injury (AKI) may lead to the development of chronic kidney disease (CKD) characterized by renal fibrosis. Macrophages play roles in AKI-to-CKD progression; however, the interplay between inflammation and fibrosis after AKI remains controversial and the precise role of the distinct macrophage subsets remains elusive. In the present study we identified a unique population of Trem2hi macrophages derived from the bone marrow as a mediator bridging inflammation resolution and fibrosis establishment after kidney injury. Trem2 deficient mice exhibited mitigated renal fibrosis after ischemia-reperfusion injury (IRI) while the renal injury and inflammation persisted. Mechanistically, Trem2 promoted renal inflammation resolution by facilitating macrophage efferocytosis to remove apoptotic tubule cells and reshaping the macrophage cytokine production profile. Loss of Trem2 expression led to excessive cholesterol accumulation in macrophages via Lxr-Abca1/Abcg1 axis and thus sustained pro-inflammatory cytokines production. Moreover, Trem2hi macrophages orchestrated the pro-fibrotic tubular epithelial cells and the activation of myofibroblasts through SPP1 to promote the establishment of renal fibrotic niche. Based on our findings, Trem2hi macrophages may serve as a potential therapeutic target for AKI-to-CKD in combination with anti-inflammatory remedies.

immunology↗

bioRxiv2026

Sex-specific Genetic Regulatory Effects in Chickens

Sexual dimorphism is a defining vertebrate feature, yet its sex-specific molecular architecture remains poorly understood. Here we established a sex-balanced, uniformly reared chicken cohort to map this landscape, integrating individual whole-genome sequencing with 7,969 bulk and 779,380 single nucleus transcriptomes across 32 tissues from 280 birds. We identified 495,098 independent expression quantitative trait loci for 20,194 genes, including 10,937 loci modulated by cell-type composition. Notably, 340 genes were regulated by 449 loci in a sex-dependent manner, significantly enrichment in endocrine tissues like adipose and the adrenal gland. Furthermore, we fine-mapped 1,219 structural variants, demonstrating their unique roles to tissue- and sex-specific expression beyond SNPs. Ultimately, we showed the utility of these regulatory effects in elucidating the molecular basis of metabolism and complex traits in both chickens and humans. This comprehensive atlas of regulatory effects provides profound insights into the genomic and molecular basis of sexual dimorphism in vertebrates.

genomics↗

bioRxiv2025

TUPAD: A Tn5-Mediated, Pre-amplification/Adapter-Dimer-Free Platform for Ultra-Uniform, One-Step PGT Profiling with High Stability and Broad Compatibility

High-throughput sequencing stands as a cornerstone of clinical molecular diagnostics, particularly in preimplantation genetic testing (PGT)--a key application where the selection of chromosomally normal embryos is critical to enhancing in vitro fertilization (IVF) success rates. A fundamental challenge inherent to PGT, however, lies in the ultra-low gDNA input (typically <100 pg) from embryo biopsies. To mitigate this limitation, traditional workflows rely on whole genome amplification (WGA) technologies, such as MDA, PicoPlex, and MALBAC. Yet these WGA-based protocols inherently entail pre-amplification of gDNA prior to standard library construction---a step that introduces significant coverage bias and compromised uniformity. These are intrinsic flaws irreparable by downstream library preparation or bioinformatics analysis, ultimately leading to clinical uncertainty in the interpretation of copy number variations (CNVs). In this study, we developed TUPAD (Tn5-Mediated, Uniform Pre-amplification-free and Adapter-Dimer-Free Library preparation), a transformative technology that bypasses the limitations of traditional WGA. By engineering a novel adapter-dimer-free transposon system, TUPAD enables high-efficiency library construction from as little as 1 picogram (pg) of gDNA. This breakthrough allows for a pre-amplification-free workflow, effectively eliminating the primary source of amplification bias. Compared to existing WGA standards, these advantages are fully retained in embryo biopsy and positive sample testing, TUPAD consistently generates highly uniform library data, significantly enhancing the detection limit and accuracy of CNVs and other genomic variants. Beyond these performance advantages, TUPAD also cuts wet-lab processing time by 50%. Practical validation confirms TUPAD results are stable, reliable, with proven practical utility in PGT testing.

molecular biology↗

bioRxiv2025

Modeling liquid-liquid phase separation and its impact on proteasomal substrate degradation

The proteasome, central to ubiquitin-proteasome mediated degradation, undergoes liquid-liquid phase separation (LLPS) during substrate processing. However, the mechanisms governing proteasomal LLPS and its functional interplay with degradation dynamics remain elusive. Here, we developed a reaction-diffusion model with thermodynamic constraints to quantitatively characterize proteasome-associated LLPS. The model successfully captures experimental LLPS dynamics, revealing a concentration-dependent phase transition boundary influenced by proteasome activity and molecular interactions. Significantly, LLPS was found to have bidirectional (enhancing or suppressing), context-dependent effects on degradation efficiency. This work elucidates regulatory mechanisms and functional roles of LLPS in proteasome-mediated degradation, offering a predictive quantitative model and testable hypotheses for future investigation.

biophysics↗

bioRxiv2025

Dynamic mechanisms of time-of-day-dependent adaptive immunity and vaccination responses

The timing of vaccine administration during the day significantly affects immunogenicity and efficacy, yet the mechanism governing the time-of-day dependent adaptive immunity and vaccine response remains elusive. Using mathematical modeling, we elucidate that the bistability arising from the self-enhancing homing process of antigen-presenting dendritic cells plays a key role in the time-of-day-dependent adaptive immune response. Modeling analyses of circadian-controlled immune responses to three vaccine types demonstrate that vaccine-specific differences in time-of-day-dependent immunity originate from distinctions in the circadian-regulated activation of antigen-presenting dendritic cells. This divergence is amplified by bistability in the dendritic cell homing process, resulting in long-term distinctions in adaptive immunity across vaccine types. The model results reveal a dynamic mechanism by which adaptive immune responses maintain circadian variations over extended time periods, suggesting that the timing of vaccine administration within the day is a promising strategy for effective disease prevention. Author SummaryInfection with the same pathogen in the morning or afternoon, or receiving the same vaccine at different times of the day, often leads to significantly different consequences in terms of infection severity or vaccine efficacy. This phenomenon is known as the time-of-day-dependent immune response. Although it is relatively common, the underlying dynamic mechanisms remain mostly unclear. In this paper, we employ mathematical modeling to investigate in detail the dynamics of adaptive immune responses following vaccination. We reveal that the kinetic origin of the time-of-day-dependent immune response lies in the positive feedback process during the homing of antigen-presenting cells, particularly dendritic cells. The bistability induced by self-activation, under the regulation of the circadian clock, plays a decisive role in generating this diurnal time dependency. The discovery of this mechanism suggests that time-of-day-specific vaccination is an effective strategy to optimize vaccine efficacy.

systems biology↗

bioRxiv2025

A transcriptional biosensor reveals mechanisms of α-ketoglutarate signaling to chromatin

Alpha-ketoglutarate (KG) is required for chromatin demethylation but mechanisms controlling KG abundance in the nucleus are poorly defined. Therefore, we designed a biosensor system to monitor this metabolite pool in human cells using an KG-responsive cyanobacterial transcription factor, NtcA. We then coupled this system with a genetic screen to identify genes that regulate KG in the nucleus, defining an inter-organelle pathway in which sequential mitochondrial activities of the GPT2 transaminase and SLC25A11 transporter supply nuclear KG. Using a mouse model of GPT2 deficiency, a human inborn error of metabolism, we found that this pathway controls chromatin methylation in the developing brain. Our work provides a tool to assess KG signaling to chromatin and a framework for leveraging forward genetics to study nuclear metabolite pools.

synthetic biology↗

bioRxiv2025

Subunit shuffling dynamics in KaiC's central hub reveal the synchronization mechanism of the cyanobacterial circadian clock

Protein complexes are critical for cellular functions, and subunit exchange within these complexes is increasingly recognized as a key regulatory mechanism. In the cyanobacterial circadian clock, subunits shuffling of the core clock protein KaiC is thought to synchronize the clock, though the underlying mechanism remains unclear. We developed a chromatography-based method to monitor the shuffling dynamics of hexamerization domain of KaiC (KaiC-CI) and found that ATPase activity is essential for this process. By analyzing experiment data with quantitative models, we found that KaiC-CI hexamer stochastically disassembles into two oligomers for shuffling after hydrolysis. Further, by assuming a hidden conformation for post-hydrolysis hexamers, we established an ATPase activity-dependent model that quantitatively describes the shuffling dynamics of KaiC-CI hexamers, linking the shuffling rate to ATP hydrolysis and nucleotide exchange rates. Using this model, we estimated the shuffling dynamics of full-length KaiC with indirect experimental data. Our findings suggest that KaiCs phosphorylation states regulate nucleotide exchange rates in the CI domain, thereby modulating ATPase activity and influencing subunit shuffling. This study provides a mechanistic framework for understanding the role of ATPase activity in subunit exchange and its implications for circadian clock regulation.

biophysics↗

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

Endosome maturation is orchestrated by inside-out proton signaling through a Na+/H+ exchanger and pH-dependent Rab GTPase cycling

Endosome maturation requires progressive lumen acidification. To what extent is lumen acidification sensed by cytosolic-side molecules that drive endosome maturation? We show here that "inside-out" proton signaling through the endosomal Na+/H+ Exchanger 6 (NHE6) activates the late endosome master regulator Rab7. The mechanism involves potent inactivation of the Rab7 GTPase-activating protein (GAP) TBC1D5 with decreasing pH. NHE6 interacts with TBC1D5 in a complex with Rab7. Neurons from NHE6-null mice or mice engineered with a selective defect in NHE6 proton efflux exhibit blocked endosome maturation and decreased active Rab7, consistent with an overactive Rab7 GAP. Finally, epistatic knock-down of TBC1D5, thereby reducing Rab7 GAP activity, in NHE6-null neurons rescues Rab7 GTPase cycling and endosome maturation. Importantly, NHE6 is mutated in Christianson Syndrome underscoring the significance of these mechanisms to neurodegeneration. We conclude that lumen acidification regulates pH-dependent Rab GTPase cycling to coordinate late endosome maturation by a process involving proton signaling.

cell biology↗

bioRxiv2024

Protocol for Simulating the Effect of THz Electromagnetic Field on Ion Channels

Terahertz (THz) electromagnetic fields are increasingly recognized for their crucial roles in various aspects of medical research and treatment. Recent computational studies have demon-strated that THz waves can modulate ion channel function by interacting with either the channel proteins or the bound ions through distinct mechanisms. Here we outline a universal simulation protocol to identify the THz frequencies that may affect ion channels, which consists of frequency spectrum analysis and ion conductance analysis. Following this protocol, we studied the effect of THz field on a CaV channel and found a broad frequency band in 1 to 20 THz range. We believe that this protocol, along with the identified characteristic frequencies, will provide a theoretical foundation for future terahertz experimental studies.

biophysics↗

bioRxiv2024

The Effect of THz Electromagnetic Field on the Conductance of Potassium and Sodium Channels

Ion channels are essential to various physiological processes and their defects are associated with many diseases. Previous research has revealed that Terahertz electromagnetic field can alter the channel conductance by affecting the motion of chemical groups of ion channels, and hence regulate the electric signals of neurons. In this study, we conducted molecular dynamics simulations to systematically investigate the effects of terahertz electromagnetic fields on the conductance of voltage-gated potassium and sodium channels, particularly focusing on the bound ions in the selectivity filters that have not been studied previously. Our results identified multiple new characteristic frequencies and showed that 1.4, 2.2, or 2.9 THz field increases the conductance of Kv1.2, and 2.5 or 48.6 THz field increases the conductance of Nav1.5. The conductance-enhancing effects are specific to the frequencies and directions of the electric field, which are determined by the intrinsic oscillation motions of the permeating ions in the selectivity filter or certain chemical groups of the ion channels. The amplitude of the THz field positively correlates with the change in ion conductance. Therefore, this study demonstrates that THz fields can specifically regulate ion channel conductances, which may carry great potential in biomedical applications.

biophysics↗

bioRxiv2024

Molecular regulation of Bna1205ams1 required for male fertility and development of a recessive genic male-sterility system in Brassica napus

Through the comprehensive use of two pollination control systems of cytoplasmic male sterility (CMS) and genic male sterility (GMS), the rapeseed yield was increased by more than 20%. However, more hybrid production systems and detailed mechanisms underlying male sterility are required. Here, we reported a novel two-line hybrid production system of 1205A for GMS and also investigated the underlying mechanism for male sterility. Five co-segregated kompetitive allele specific PCR (KASP) markers were developed and validated, which could be used for transferring the male sterility trait into external B. napus breeding lines and developing further two-line hybrid production systems of GMS. Inheritance studies detected one gene locus of Bna1205ams1 for regulating the male sterility of 1205A. As a potential candidate gene of Bna1205ams1, BnaC03g27700D was fine mapped and narrowed down to a 181.47 kb region on chrC03 and validated by functional analysis. The mutation of BnaC03g27700D in 1205A resulted in large metabolic fluctuations, most of which were involved in aborted tapetal PCD, which could lead to reduced pollen fertility with abnormal pollen exine. The developed new GMS line of 1205AB provided us with the opportunity to identify a new male sterility gene of BnaC03g27700D in B. napus. The study of BnaC03g27700D aims to renew the annotation of the gene and provide new resources for basic research on the genetic control of male sterility.

molecular biology↗

bioRxiv2024

Disease resistance of Brassica juncea to Sclerotinia sclerotiorum is established through the induction of indole glucosinolate biosynthesis

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is the main disease threat to oilseeds in Brassiceae, causing significant yield losses and reduction in oil content and quality. The studies on S. sclerotiorum require a great focus and extensive research on B. juncea compared to those on B. napus and B. oleracea. Transcriptome analysis revealed a large number of defense-related genes and response processes in B. napus and B. oleracea. However, similarities and differences in the defense responses to S. sclerotiorum on B. juncea are rarely reported. In the present study, we reported a B. juncea breeding line of H83 with high S. sclerotiorum resistance, which was used for transcriptome analysis compared to L36 with low resistance. A novel regulatory network was proposed to defend against S. sclerotiorum invasion in B. juncea. Upon infection of S. sclerotiorum, a series of auxin and MAPK signaling pathways were initiated within 12 h, and then defenses were activated to restrict the development and spread of S. sclerotiorum by inducing the massive synthesis of indole glucosinolates after 24 h. Twelve hub genes involved in the network were identified by the weighted gene co-expression network (WGCNA), which are involved in plant-pathogen interaction, signaling pathway genes, indole glucosinolate biosynthesis and cell wall formation. The hub genes were further validated by qRT-PCR. The research revealed a new resistant line of H83 against S. sclerotiorum and a different regulatory network in B. juncea, which would be beneficial for the future effective breeding of Sclerotinia-resistant varieties.

plant biology↗

bioRxiv2023

Parallel Molecular Data Storage by Printing Epigenetic Bits on DNA

DNA storage has shown potential to transcend current silicon-based data storage technologies in storage density, lifetime and energy consumption. However, writing large-scale data directly into DNA sequences by de novo synthesis remains uneconomical in time and cost. Inspired by the natural genomic modifications, in this work, we report an alternative, parallel strategy that enables the writing of arbitrary data on DNA using premade nucleic acids. With epigenetic modifications as information bits, our system employed DNA self-assembly guided enzymatic methylation to perform movable-type printing on universal DNA templates. By programming with a finite set of 700 DNA movable types and 5 templates, we achieved the synthesis-free writing of [~]270000 bits on an automated platform with 350 bits written per reaction. The data encoded in complex epigenetic patterns were retrieved high-throughput by nanopore sequencing, and algorithms were developed to finely resolve 240 modification patterns per sequencing reaction. Our framework presents a new modality of DNA-based data storage that is parallel, programmable, stable and scalable. Such a modality opens up avenues towards practical data storage and dual-mode data functions in biomolecular systems.

biochemistry↗

bioRxiv2023

GGA1 interacts with the endosomal Na+/H+ Exchanger NHE6 governing localization to the endosome compartment

Mutations in the endosomal Na+/H+ exchanger (NHE6) cause Christianson syndrome (CS), an X-linked neurological disorder. Previous studies have shown that NHE6 functions in regulation of endosome acidification and maturation in neurons. Using yeast two-hybrid screening with the NHE6 carboxyl-terminus as bait, we identify Golgi-associated, Gamma adaptin ear containing, ARF binding protein 1 (GGA1) as an interacting partner for NHE6. We corroborated the NHE6-GGA1 interaction using co-immunoprecipitation (co-IP): using over-expressed constructs in mammalian cells; and co-IP of endogenously-expressed GGA1 and NHE6 from neuroblastoma cells, as well as from mouse brain. We demonstrate that GGA1 interacts with organellar NHEs (NHE6, NHE7 and NHE9) but not with cell-surface localized NHEs (NHE1 and NHE5). By constructing hybrid NHE1/NHE6 exchangers, we demonstrate that the cytoplasmic tail of NHE6 is necessary and sufficient for interactions with GGA1. We demonstrate the co-localization of NHE6 and GGA1 in cultured, primary hippocampal neurons, using super-resolution microscopy. We test the hypothesis that the interaction of NHE6 and GGA1 functions in the localization of NHE6 to the endosome compartment. Using subcellular fractionation experiments, we show that NHE6 is mis-localized in GGA1 knockout cells wherein we find less NHE6 in endosomes but more NHE6 transport to lysosomes, and more Golgi retention of NHE6 with increased exocytosis to the surface plasma membrane. Consistent with NHE6 mis-localization, and Golgi retention, we find the intra-luminal pH in Golgi to be alkalinized. Our study demonstrates a new interaction between NHE6 and GGA1 which functions in the localization of this intra-cellular NHE to the endosome compartment.

cell biology↗

bioRxiv2023

Molecular mechanisms for activation of the 26S proteasome

Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated protein can increase, we studied ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5s AN1 Zn finger interacts with the Rpt5 ATPase and its C-terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Surprisingly, these C-terminal interactions are sufficient to activate proteolysis. With ZFAND5 bound, entry into the proteasomes protein translocation channel is wider, and ZFAND5 dissociation causes opening of the 20S gate for substrate entry. Using single-molecular microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

molecular biology↗