Biology subjects

Ouyang, Z.

Publications and source records attributed to Ouyang, Z..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural Basis of Ligand-Specific Orthosteric-Allosteric Coupling and Sensory Tuning in the Human Bitter Taste Receptor TAS2R14

The bitter taste receptor TAS2R14 recognizes hundreds of structurally diverse ligands, yet the mechanisms governing its broad promiscuity, graded agonist efficacy, and multi-site regulation remain unclear. Here, we report cryo-EM structures of TAS2R14-Gi1 complexes bound to 3,5-diiodosalicylic acid (DA), flufenamic acid (FA) and aristolochic acid (AA) at resolutions of 2.44 [A], 2.39 [A], and 2.69 [A], with a clear efficacy hierarchy of AA > FA > DA. Transmembrane helix 6 (TM6) acts as a core regulatory hub, with agonist binding triggering 12 [A] lever-like TM6 rearrangements to reshape receptor interfaces and modulate G-protein coupling strength. We also identify cholesterol hemisuccinate (CHS) as a new TAS2R14 orthosteric agonist, selectively potentiating DA-mediated signaling potency by [~]5-fold via allosteric-orthosteric coupling without affecting FA or AA. Our findings show TM6 dynamics unify TAS2R14s key functional features, advancing insights into bitter receptor activation and sterol regulation.

molecular biology↗

bioRxiv2026

Development of a BM7G((TKO/hCD46/hCD55/hTHBD/hEPCR) Donor Pig with Endogenous Promoter-Driven Transgenes for Xenotransplantation

Xenotransplantation offers a potential solution to the organ shortage crisis. Multi-gene modification of pigs, such as knockout of three carbohydrate antigen-related genes and expression of immunoprotective proteins, can significantly improve xenograft survival. However, existing strategies face challenges: transposon-based transgenesis may lead to unstable expression, while exogenous promoters used in site-specific integration are susceptible to epigenetic silencing, hindering long-term stable expression. Therefore, developing a donor pig model capable of sustained multi-gene expression is critical. To address this, CRISPR-Cas9 was used to knockout three major glycan antigen genes to eliminate hyperacute rejection. Subsequently, four human protective genes were site-specifically integrated into the porcine Rosa26 safe-harbor locus, with expression driven by the endogenous Rosa26 promoter and the THBD core promoter for long-term stable and tissue-specific expression, and the selection marker was removed using Cre/loxP. Results showed complete absence of the three glycan antigens in BM7G pigs, while the four protective proteins were stably expressed in vascular endothelial cells and major organs. Among them, hCD55 and hCD46 were widely expressed, while hTHBD and hEPCR showed vascular-specific expression. In-vitro assays confirmed that BM7G porcine endothelial cells significantly reduced human antibody binding, effectively inhibited complement-dependent cytotoxicity, and decreased thrombin-antithrombin complex formation. In conclusion, by combining xenoantigen knockout with endogenous promoter-driven expression of multiple human protective genes, a seven-gene modified pig model with low immunogenicity and synergistic protective function was successfully constructed, providing an important donor resource for xenotransplantation preclinical research.

immunology↗

bioRxiv2025

Derivation, Characterizations, and Applications of Rabbit Haploid Embryonic Stem Cells

Haploid embryonic stem cells (haESCs), which contain a single set of chromosomes, provide powerful systems for investigating gene function and creating gene-edited animal models. haESCs have been derived from several mammalian species including human, mouse and rats, yet not from rabbits. Here, we report the derivation of parthenogenetic haESCs of rabbit (rbPhESCs) using an optimized culture medium. These cells maintain a stable haploid karyotype during long-term culture and own pluripotency features comparable to diploid embryonic stem cells derived from rabbit fertilized embryos. By integrating gene trapping cassette into ROSA26 locus, rbPhESCs offer an ideal platform for high-throughput functional genomic screening, as shown in haESCs from other mammals. Consistent with their parthenogenetic origin, rbPhESCs durably preserve maternal-specific imprinting patterns even after extended culture in serum-containing conditions, providing a valuable platform for generating disease models deficient in maternally expressed imprinted genes. Therefore, our findings expand the repertoire of mammalian haESCs and establish rbPhESCs as a valuable platform for genetic studies and biotechnological applications in rabbits.

cell biology↗

bioRxiv2025

SpaceSequest: A unified pipeline for spatial transcriptomics data analysis

BackgroundSpatial transcriptomics has emerged as one of the most powerful tools for gaining biological insights, enabling researchers to uncover intricate relationships between gene expression patterns and tissue architecture. Recent advances in the field have resulted in a variety of new platforms, including Visium, Visium HD, and Xenium from 10x Genomics, as well as GeoMx and CosMx from NanoString Technologies, which has now been acquired by the Bruker Corporation. However, the existence of diverse spatial transcriptomics platforms and various data formats poses challenges in standardizing data analysis. Thus, there remains a critical gap in the availability of a comprehensive pipeline capable of conducting end-to-end analysis that is necessary to extract biological insights from multiple spatial transcriptomics platforms. ResultsHere, we present SpaceSequest, a tailored pipeline that utilizes cutting-edge computational methodologies to conduct a thorough analysis, enabling the extraction of crucial biological insights from five major spatial transcriptomics technologies. SpaceSequest performs (1) standardized quality control and general data processing, (2) key analyses customized for each spatial platform, (3) automated cell type annotation and deconvolution, and (4) high-quality figure and analysis result generation. In addition, SpaceSequest allows for smooth integration with cellxgene VIP and Quickomics for user-friendly data access and interactive visualization. ConclusionsSpaceSequest is a unified and comprehensive pipeline designed for the analysis, visualization, and publication of spatial transcriptomics data from various platforms. The source code is available at https://github.com/interactivereport/SpaceSequest. To facilitate seamless installation and usage, we have also created a detailed Bookdown tutorial that can be accessed through https://interactivereport.github.io/SpaceSequest/tutorial/docs/index.html.

bioinformatics↗

bioRxiv2025

Hypoimmunogenic human motor neurons induced from iPSCs in vivo substantially ameliorate ALS disease in large animal models

Stem cell-based therapy holds great potential for substituting degenerated motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). Missing protocols for advanced differentiation of transplanted cells into MNs, immune rejection, and the lack of suitable ALS models for preclinical trials have slowed the development of effective therapies. Here, we employed multiplex genetic-editing to generate a novel human pluripotent stem cell line containing doxycycline (Dox)-inducible MNs-specific transcription factors and comprehensively modified immunomodulatory genes. We transplanted these cells into the spinal cord of ALS large animal models (SOD1G93A pigs and TIA1P362L rabbits), which faithfully recapitulate pathologies and symptoms observed in ALS patients. The transplanted cells could efficiently differentiate into functional MNs upon Dox treatment in vivo, distribute throughout the spinal cord and motor cortex via extensive migration, survive long-term without the need for immunosuppression. Notably, these MNs integrated into host neural circuits, as evidenced by their long projection of peripheral axons to target muscle and reformation of neuromuscular junctions. As result, pathologies and motor deficits were substantially ameliorated in both animal models. One Sentence SummaryHypoimmunogenic human motor neurons induced from iPSCs in vivo reform neuromuscular junctions and ameliorate ALS disease in pig and rabbit models.

neuroscience↗

bioRxiv2025

CompBioAgent: an LLM-powered agent for single-cell RNA-seq data exploration

AO_SCPLOWBSTRACTC_SCPLOWAdvancements in high-throughput biological technologies, particularly single-cell RNA sequencing (scRNA-seq), have generated vast amounts of complex data that offer valuable insights into gene expression, cellular behavior, and disease mechanisms. However, the analysis of such data often requires specialized computational skills and expertise, which can pose a barrier to many researchers. To address this challenge, we developed CompBioAgent, a user-friendly web application designed to democratize access to bioinformatics resources, powered by Large Language Models (LLMs). By integrating with the CellDepot, CompBioAgent allows users to easily query and explore gene expression data related to various diseases, cell types, and experimental conditions. Moreover, the tool employs Cellxgene Visualization In Plugin (Cellxgene VIP) platform to generate a range of intuitive visualizations, including violin plots, UMAP embeddings, heatmaps, etc. With its natural language interface, CompBioAgent automatically converts scientific queries into structured data requests and returns the plot of interest, enabling seamless exploration of biological data without the need for programming expertise. Such integration enhances the ability of researchers to visualize complex biological insights and identify key patterns, making high-level data analysis more accessible and efficient. A demo website and a list of examples are available at https://celldepot.bxgenomics.com/compbioagent, and the source code is released at https://github.com/interactivereport/compbioagent.

bioinformatics↗

bioRxiv2025

Neuromodulation in neural organoids with shell MEAs

Neural organoids (NOs) have emerged as important tissue engineering models for brain sciences and biocomputing. Establishing reliable relationships between stimulation and recording traces of electrical activity is essential to monitor the functionality of NOs, especially as it relates to realizing biocomputing paradigms such as reinforcement learning or stimulus discrimination. While researchers have demonstrated neuromodulation in NOs, they have primarily used 2D microelectrode arrays (MEAs) with limited access to the entire 3D contour of the NOs. Here, we report neuromodulation using tiny mimics of macroscale EEG caps or shell MEAs. Specifically, we observe that stimulating current within a specific range (20 to 30 {micro}A) induced a statistically significant increase in neuron firing rate when comparing the activity five seconds before and after stimulation. We observed neuromodulatory behavior using both three- and 16-electrode shells and could generate 3D spatiotemporal maps of neuromodulatory activity around the surface of the NO. Our studies demonstrate a methodology for investigating 3D spatiotemporal neuromodulation in organoids of broad relevance to biomedical engineering and biocomputing. One-Sentence SummaryNeuromodulation, an essential intelligence feature, was observed using 3D stimulation and recording from neural organoids.

bioengineering↗

bioRxiv2025

Spatial transcriptomics reveals heterogeneous cell-cell interactions among brain regions in a cuprizone model consistent with multiple sclerosis lesions

The cuprizone (CPZ) model is widely used for modeling demyelination in multiple sclerosis (MS) and for testing potential remyelination therapies. We integrated single-cell and spatial transcriptomics (ST) to fine map the spatial cellular and molecular responses during de and remyelination. ST revealed global demyelination and neuroinflammation in the brain beyond the corpus callosum, with region-specific differences. We identified oligodendroglia and microglia as two major cell types with significant transcriptomic changes in the model. Ligand receptor pairing analyses predicted growth factor and phagocytic pathway enrichment during demyelination, which is consistent with changes in MS lesions. During remyelination, while mature oligodendrocytes nearly reversed their phenotype back to the control state, microglia remained associated with the demyelination phenotype. Finally, astrocytes in the CPZ model had the greatest preservation of disease-associated modules to MS lesions, while the MOL, OPC, and microglia showed moderate to low preservation, which overall suggested that the CPZ model had moderate translatability to chronically active MS lesions.

neuroscience↗

bioRxiv2024

Non-continuous neuromodulation in awake, unrestrained felines increases bladder capacity

ObjectiveNeuromodulation is a standard therapy for bladder symptoms such as overactive bladder. Previous studies have demonstrated that non-continuous stimulation (NCS) can increase bladder capacity and that bladder pressure can be estimated from dorsal root ganglia (DRG) neural activity in anesthetized animal models. Our goal is to determine if NCS elicits similar bladder capacity effects as continuous stimulation (CS) and if bladder pressure can be estimated from DRG signals in an awake, unrestrained animal model. ApproachWe performed aseptic, chronic implant surgeries with seven adult, male felines. Three animals were used to establish procedures, three for experimental testing, and one did not yield data. Bipolar stimulating electrodes were placed on the pudendal nerve and sacral nerve on the same side. Microelectrode arrays were inserted in two ipsilateral sacral DRG. Two single-lumen catheters were implanted in the bladder dome for recording bladder pressure and infusing saline. Fixed-sequence, repeated bladder fills were performed in four awake felines to evaluate the bladder capacity during no-stimulation (NS), NCS, and CS at either the pudendal or sacral nerve. NCS was performed based on increases in bladder pressure estimated from DRG recordings or when 50% of the average NS bladder capacity was reached. Main resultsWe observed similar bladder capacity increases for NCS (122 {+/-} 31% of NS control) as for CS (121 {+/-} 33%) in the four animals. NCS paradigms reduced stimulation time by 46% on average. Median correlation coefficients of 0.46 and 0.64 (maximum 0.93) between the predicted and measured bladder pressure were obtained for awake trials with DRG bladder units in two animals. SignificanceThis study demonstrated the feasibility of using NCS to increase bladder capacity in awake, unrestrained felines and for decoding bladder pressure from DRG recordings. Further studies are needed to optimize NCS timing for clinical translation.

bioengineering↗

bioRxiv2024

Stacked Human Arterial Endothelial Cells Generate Atherosclerotic Fatty Streaks and Release Proinflammatory Cytokines

Fatty streaks are the first sign of atherosclerosis. They consist of lipid-containing foam cells, which were believed to be derived from the monocytes in the blood through the leaky endothelium, and from the vascular smooth muscle cells migrating into the intima from the media. Here, we showed that fatty streaks can also be formed by the stacked human arterial endothelial cells (HAECs) cultured in vitro. Via SEM we revealed a novel cell phenotype (coralthelial) that forms a streak/coral-like structure. We observed accumulation of lipids in the coralthelial cell and increased Golgi and coat protein II markers in its nucleus. Additionally, proinflammatory cytokines were upregulated in these cells, likely due to Golgi nuclear translocation and subsequently increased expression of the ribosomal protein RPL23 in the nucleus. We demonstrated, for the first time, that the atherosclerotic fatty streak-like structure can be generated from the stacked HAECs, which also create a proinflammatory microenvironment. Significance StatementOur work presents a novel perspective on atherosclerotic fatty streak formation, a critical early event in atherosclerosis development. We demonstrate, for the first time, that human arterial endothelial cells (HAECs) can transform into a novel phenotype, termed "coralthelial cells". These cells contribute to fatty streak formation, and produce proinflammatory cytokines through the relocation of RPL23 with the Golgi apparatus to the nucleus. This finding provides a new cellular mechanism by which endothelial cells initiate atherosclerosis, independent of traditional monocyte-derived foam cells. By enriching established paradigms, our research not only opens new avenues for therapeutic intervention targeting endothelial cells but also enhances understanding of vascular diseases and provides potential diagnostic and prognostic markers for atherosclerosis.

cell biology↗

bioRxiv2023

Chromosome-scale genome assembly of Tinospora sagittata (Oliv.) Gagnep. enhances identifying genes involved in the biosynthesis of jatrorrhizine

Tinospora sagittata (Oliv.) Gagnep. is an important medicinal tetraploid plant in the Menispermaceae family. Its tuber, namely "Radix Tinosporae" used in Traditional Chinese Medicine, is rich in medicinal terpenoids and benzylisoquinoline alkaloids (BIAs), To enhance understanding the biosynthesis of medicinal compounds, we, herein, report the assembly of a high quality chromosome-scale genome with both PacBio HiFi and Illumina sequencing technologies. The size of assembled genome was 2.33 Gb consisting of 4070 scaffolds (N50=42.06Mb), of which 92.05% were assigned to 26 pseudochromosomes in A and B sub-genomes. A phylogenetic analysis with the T. sagittata and other 16 plant genomes estimated the evolutionary placement of T. sagittata and its divergence time in Ranunculales. Further genome evolution analysis characterized one round tandem duplication about 1.5 million years ago (MYA) and one whole-genome duplication (WGD) about 86.9 MYA. WGD contributed to the duplication of clade-specific cytochrome P450 gene family in Ranunculales. Moreover, sequencing mining obtained genome-wide genes involved in the biosynthesis of alkaloids and terpenoids. TsA02G014550, one candidate, was functionally characterized to catalyze the formation of (S)-canadine in the jatrorrhizine biosynthetic pathway. Taken together, the assembled genome of T. sagittata provides useful sequences to understand the biosynthesis of jatrorrhizine and other BIAs in plants.

genomics↗

bioRxiv2023

Acetylated dietary protein contributes to the acetylome in cells via KARS-mediated intra-translational deposition of acetyl-lysine in nascent protein

N6-acetyl-L-lysine residue is abundant in dietary protein but less is known about its potential influences on the diet-consumers. We herein report that KARS mediates intra- translational deposition of diet-derived N6-acetyl-L-lysine in nascent proteins to contribute the acetylome in cells. Acetylated dietary protein is a direct source of N6-acetyl-L-lysine that can widely and substantially contribute the acetylome in multiple organs of mice. By analyzing the co-crystal structure of Lysyl-tRNA synthetase (KARS) in complex with N6- acetyl-L-lysyl-AMP and pyrophosphate, together with in vitro biochemical assays, we learned that KARS can utilize N6-acetyl-L-lysine to produce N6-acetyl-L-lysyl-AMP and transfers the N6-acetyl-L-lysyl-moiety to lysine cognate tRNA to generate N6-acetyl-L- lysyl-tRNA, which introduces N6-acetyl-L-lysine into growing nascent polypeptide and intra-translationally results in protein acetylation. This undocumented protein modification mechanism is inherently different from post-translational modification (PTM) and termed as intra-translational modification (ITM). ITM can functionally mimic PTM mechanisms to deposit acetylation in histones to decondense chromatin. It can also modify PTM- inaccessible regions that are buried inside and functionally important to proteins. ITM is expected to extend the repertoire of acetylome and improve our understandings in protein modification modes in cells.

biochemistry↗

bioRxiv2022

Citrus genomic resources unravel putative genetic determinants of Huanglongbing, a pathogen-triggered immune disease

Citrus is one of the most important tree crops worldwide and citrus production is threatened by Huanglongbing (HLB), a devasting citrus disease caused by Candidatus Liberibacter. HLB is a pathogen-triggered immune disease, in which the pathogen initiates systemic and chronic immune responses including the excessive production of reactive oxidative species, which subsequently lead to cell death of phloem tissues and HLB disease symptoms. Here, we identified putative genetic determinants of HLB pathogenicity by integrating citrus genomic resources to characterize the pan-genome of accessions that differ in their response to HLB. Genome-wide association mapping and analysis of allele-specific expression between susceptible, tolerant, and resistant accessions further refined candidates underlying the response to HLB. To enable these analyses we first developed a phased diploid assembly of Citrus sinensis Newhall genome and produced resequencing data for 91 citrus accessions that differ in their response to HLB. These data were combined with previous resequencing data from 356 sequenced accessions for genome-wide association mapping of the HLB response. Genes with HLB pathogenicity were associated with the host immune response, ROS production, and antioxidants. Overall, this study has provided a significant recourse of citrus genomic data and we have identified candidate genes to be further explored to understand the genetic determinants of HLB pathogenicity and to generate HLB resistant/tolerant citrus varieties.

genomics↗

bioRxiv2022

CIRKO: A chemical-induced reversible gene knockout system for studying gene function in situ

Conditional loss and restoration of function are becoming important approaches for investigating gene function. Given that reversible conditional gene knockouts in cells required complicated manipulation, conditional inactivation and reactivation of a gene in primary somatic cells with limited proliferative capacity and in animal models remain difficult to achieve. Here, we first developed a reportable and reversible conditional intronic cassette (ReCOIN), wherein inactivation and reactivation of the gene are mediated via sequential expression of Cre and Flp recombinases, respectively. The expression pattern of the target gene can be monitored by direct visualization. To simply and tightly control temporal expression of the recombinases, on the basis of ReCOIN, we further presented a dual chemical-induced reversible gene knockout system (CIRKO) by insertion of reverse tetracycline transcriptional activator (rtTA) and tetracycline response element (TRE)-controlled Cre and FlpoERT2 recombinases cassettes into Rosa26 and Hipp11 loci of cells, respectively, in which transcription termination of the target gene can be induced at a specific stage in the presence of doxycycline, while gene restoration is achieved in the presence of doxycycline and tamoxifen simultaneously. This system provides a simple, rapid, and flexible gene switch for studying gene function in situ both in vitro and in vivo. Impact statementA novel chemical-induced reversible gene knockout system provides a simple, rapid, and flexible gene switch to facilitate the study of gene function in primary somatic cells in vitro, embryos in vitro or in vivo, and animals in vivo.

genetics↗

bioRxiv2022

Generating Functional Cells Through Enhanced Interspecies Chimerism with Human Pluripotent Stem Cells

Obtaining functional human cells through interspecies chimerism with human pluripotent stem cells (hPSCs) remains unsuccessful due to its extreamly low efficiency. Here, we show that hPSCs failed to differentiate and contribute teratoma in presence of mouse PSCs (mPSCs). While, MYCN, a pro-growth factor dramatically promotes hPSC contribution in teratoma co-formation by hPSCs/mPSCs. MYCN combined with BCL2 (M/B) greatly enhanced conventional hPSCs to integrate into preimplantation embryos of different species, such as mice, rabbits and pigs and substrantially contributed to mouse post-implantation chimera in embryonic and extra-embyonic tissues. Strikingly, M/B-hPSCs injected into preimplantation Flk-1+/- mouse embryos show further enhanced chimerism that allows obtaining live human CD34+ blood progenitor cells from chimeras through cell sorting. The chimera derived human CD34+ cells further gave rise to various subtype blood cells in typical colony forming unit(CFU) assay. Thus, we proof the concept to obtain functional human cells through enchanced interspecies chimerism with hPSCs.

cell biology↗

bioRxiv2021

Systemic influences of mammary cancer on monocytes in mice

There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and blood, particularly the myeloid compartment. Here we show using mouse models of breast cancer caused by the mammary epithelial expression of the Polyoma middle T antigen that there is an increase in the number of circulating and splenic monocytes. In the circulation, cancer does not affect ratios of classical to non-classical populations monocytes nor their halflives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. In the bone marrow cancer does not change monocytic progenitor number is unaffected but the proliferation rate of monocytes is higher thus providing an explanation for expansion in the circulating number. Deep RNA sequencing of these monocytic populations reveals cancer causes changes in the classical monocyte compartment with changes evident in bone marrow monocytes but more in the blood suggesting influences in both compartments. Down regulation of interferon type 1 signalling and antigen presentation were the most prominent. Consistent with this analysis down regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, transcription factors required for type 1 interferon signalling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, mouse models of cancer may be insufficient to study the systemic influences of human cancer.

cancer biology↗

bioRxiv2021

CellDepot: A unified repository for scRNA-seq data and visual exploration

CellDepot serves as an integrated web application to assist users in exploring single-cell RNA-seq (scRNA-seq) datasets and comparing the datasets among various studies through a user-friendly interface with advanced visualization and analytical tools. To begin with, it provides an efficient data management system that users can upload single cell datasets and query the database by multiple attributes such as species and cell types. In addition, the advanced query function incorporated in MySQL database system and its conditional filtering, allows users to quickly query and compare the expression of gene(s) across the datasets of interest. Moreover, by embedding the cellxgene VIP tool, CellDepot enables fast exploration of individual dataset in the manner of interactivity and scalability to gain more refined insights such as cell composition, gene expression profiles, and differentially expressed genes among cell types. In summary, the web portal allows large scale single cell data sharing, analysis and visualization for supporting decision-making, and encouraging scientists to contribute to the single-cell community in a tractable and collaborative way. Finally, CellDepot is released as open-source software to motivate crowd contribution, broad adoption, and local deployment for private data.

bioinformatics↗

bioRxiv2021

CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

bioinformatics↗