Biology subjects

Zhuang, Z.

Publications and source records attributed to Zhuang, Z..

At least 19 recordsLinked to original sources

bioRxiv2026

Establishment of Rabbit Formative-like Pluripotent Cells by Bidirectional Modulation of WNT Signaling Pathway

The generation of embryonic stem cells (ESCs) with full developmental capacity and robust chimera-forming potential remains a major bottleneck for most non-rodent mammalian species including rabbits, an important animal both for biomedicine and agriculture. Formative pluripotency is a unique intermediate state bridging the naive and primed pluripotent states, which is defined by distinct molecular signatures, intrinsic competence for primordial germ cell (PGC) specification, and unique developmental adaptability to early embryonic microenvironments. Here, we established a novel culture medium termed KFiC, which could bidirectionally and coordinately modulate the WNT/{beta}-catenin signaling pathway via combined treatment of WNT activator CHIR99021 and inhibitor IWR-1. The KFiC culture system enables efficient derivation and stable long-term self-renewal of rbESCs with consistent formative-like pluripotent characteristics. KFiC-cultured rbESCs (KFiC-rbESCs) form compact, undifferentiated colonies, maintain intact genomic stability, and recapitulate the transcriptional landscape of pre-implantation epiblast cells, accompanied by sustained high expression of the core formative pluripotency marker OTX2. Functionally, KFiC-rbESCs possess efficient PGC differentiation competence and extensively contribute to embryonic development after microinjection into rabbit morulae, displaying their superior developmental plasticity. Notably, the KFiC system can stabilize an intermediate formative-like pluripotent state in mouse, human, and porcine pluripotent stem cells, and KFiC-adapted mouse ESCs retain potent chimera-forming ability. Collectively, this study establishes a reliable culture platform for generating and maintaining formative-like rbESCs. This cross-species compatible system provides a promising foundation for further optimizing naive-level pluripotency in rbESCs and facilitates the generation of genetically engineered rabbit disease models.

developmental biology↗

bioRxiv2026

A multi-tissue epigenomic atlas links the sheep non-coding genome to domestication and complex traits

Non-coding regulatory variation drives complex traits, domestication, and evolutionary adaptation, yet the sheep genome lacks high-resolution functional annotation. Here we present SheepEpimap, a multi-tissue regulatory atlas harmonizing 516 CUT&Tag histone modifications, ATAC-seq, and RNA-seq datasets across 43 adult tissues in sheep. We annotated 2.93 million cis-regulatory elements, yielding 557,441 enhancer-gene pairs and 145,407 variants with allele-specific effects. By training a sequence-to-function deep-learning model, we decoded the base-pair syntax of chromatin accessibility, annotated transcription factor motif instances genome-wide, and constructed 12,210 tissue-specific gene regulatory networks (GRNs). Integrating this resource with multi-tissue expression quantitative trait loci, selection sweeps, and genome-wide association studies prioritized non-coding variants driving domestication and complex traits. Finally, cross-species analysis revealed that sequence-conserved, tissue-matched enhancers were significantly enriched in the heritability of complex traits and diseases in humans. In summary, SheepEpimap (https://genome.ucsc.edu/s/mengzhu/SheepEpimap) provides an open-access foundational ecosystem for sheep functional genomics, precision breeding, and comparative biology.

genetics↗

bioRxiv2026

MG2Act: A Mechanism-Inspired Sequential Attention Framework for Molecular Glue Degradation Prediction

Molecular glue degraders act by inducing productive proximity between an E3 ligase and a substrate protein. For most characterized degradative glues, a small molecule first engages the E3, conditions its substrate-recognition surface, and only then enables recruitment of a compatible neo-substrate. This directionality is rarely encoded explicitly in computational models, which typically fuse molecule, E3 and target representations simultaneously. We present MG2Act, a structure-independent framework that translates this two-step logic into sequential cross-attention, using CRBN-mediated degradation as the most data-rich representative system. Starting from a curated continuous-valued benchmark of 1,207 pairs across 47 targets, a refined subset of 1,159 pairs was selected to train MG2Act after excluding rare targets. On identical processed data, MG2Act consistently outperforms machine learning baselines, robustly generalizes under strict redundancy-filtering, and responds coherently to mechanism-based perturbations. Prospective screening and zero-shot target-conditioned prioritization identified nanomolar degraders of IKZF1, CK1 and CDK4, including the non-classical IMiD-core CDK4 degrader SWC-202.

bioinformatics↗

bioRxiv2026

Spaceland: Histology-Guided Reconstruction of High-Resolution Whole-Organ 3D Molecular Atlases from Sparse Spatial Transcriptomics

Reconstructing whole organs in three-dimensional molecular detail is a key step toward building virtual organs for modeling tissue organization, disease progression and drug perturbation responses. However, high-resolution whole-organ spatial transcriptomic profiling remains impractical, forcing a trade-off between reconstruction fidelity and sampling density. Here, we introduce Spaceland, a morphology-guided framework that reconstructs continuous, high-resolution 3D molecular landscapes from sparsely sampled spatial transcriptomic sections and serial H&E histology. Spaceland formulates this task as learning continuous gene-expression fields within a morphology-informed histological space. It constructs a dense 3D morphological scaffold by optical-flow interpolation of foundation model-derived H&E representations and decodes sparse spot-level transcriptomic measurements onto an 8 m histology-aligned grid. Across mouse olfactory bulb, mouse hemibrain and spatiotemporal planarian regeneration, Spaceland generalized across platforms, tissue scales and biological contexts. In mouse benchmarks, Spaceland bridged 400 m molecular gaps, resolved sub-spot organization, outperformed ST-based interpolation and 2D H&E-based prediction methods, and remained robust with 160-320 m H&E intervals. In planarian regeneration, it enabled time-resolved whole-organism analysis from only four Visium sections per stage, revealing dynamic neoblast-neural spatial remodeling. Together, Spaceland shifts 3D molecular atlas construction from exhaustive experimental sampling toward data-driven virtual tissue and organ modeling, providing a scalable route to whole-organ molecular reconstruction.

bioinformatics↗

bioRxiv2026

Evolutionary Origins of Molecular Programs Underlying Brain Circuitry

The vertebrate pallium harbors independently evolved structures that, nevertheless, support strikingly similar sensory and cognitive circuit architectures. The mechanisms and evolutionary timing driving the emergence of these parallel pallial circuits remain unelucidated. Here, we integrated spatial transcriptomic and single-nucleus RNA-seq datasets from eight representative vertebrate species spanning approximately 500 million years of evolution to reconstruct the evolutionary assembly of the primary sensory-allocortical (Pr-Al) molecular axis, a conserved cortical hierarchical axis defined in our prior work. We uncovered that Pr- and Al-like neuronal identities are deeply conserved across sarcopterygians, encompassing all tetrapods and lobe-finned fishes. Intriguingly, this ancestral neuronal homology is uncoupled from spatially partitioned patterning: only tetrapods further compartmentalized these Pr- and Al- neurons into distinct pallium regions. Functional enrichment of Pr- and Al- gene programs uncovered a conserved tetrapod genetic core suite including MAPK signaling and axon guidance pathways. This core toolkit underwent sequential functional refinement from amphibians through mammals. Notably, mammals and birds convergently evolved association-cortical molecular profiles enriched for synaptic regulatory genes to support advanced cognitive functions. Collectively, this work delineates a stepwise vertebrate pallium evolutionary paradigm that explains how conserved molecular modules shape spatially organized brain circuitry across deep evolutionary time.

evolutionary biology↗

bioRxiv2026

Small-molecule modulators of HIPK4 activity and proteostasis

Homeodomain-interacting protein kinase 4 (HIPK4) is a dual-specificity kinase that is predominantly expressed in differentiating spermatids, required for sperm development, and a promising target for nonhormonal male contraception. Genetic and functional studies have established an essential role for HIPK4 in spermiogenesis, where it acts at least in part through regulation of the F-actin-scaffolded acroplaxome during spermatid head shaping. The direct molecular targets of HIPK4 and their downstream effectors remain poorly defined, and small-molecule probes would be versatile tools for further investigating HIPK4 functions. Synthetic HIPK4 ligands could also be valuable leads for the development of nonhormonal male contraceptives. Here, we report the discovery of a cyanoquinoline-based series of HIPK4 inhibitors with nanomolar potency. Our lead compounds are selective for HIPK4, both within the HIPK family and across the broader kinome, establishing this scaffold as a useful starting point for probe and lead development. Unexpectedly, we found that a subset of these cyanoquinolines also perturbs HIPK4 proteostasis in a cell type-specific manner. In spermatids, these compounds induce the formation of detergent-insoluble HIPK4 aggregates and promote interactions between this kinase and the autophagy receptor Tax1-binding protein 1 (TAX1BP1). Together, our findings establish cyanoquinoline ligands as a new chemotype for probing HIPK4 biology and advancing male contraceptive discovery.

biochemistry↗

bioRxiv2026

Macrocyclization of Broad-Spectrum Kinase Inhibitor Bosutinib leads to Potent and Selective Quinoline-based HIPK4 Inhibitor AZ137

Homeodomain-interacting protein kinase 4 (HIPK4) remains an understudied member of the dark kinome. While genetic knockout studies suggest roles for HIPK4 in spermiogenesis and cutaneous squamous cell carcinoma, whether these cellular functions can be recapitulated by pharmacological inhibition remains to be determined. However, such investigations have been hampered by a lack of high-quality chemical tools. To address this, we employed a rational design strategy utilizing macrocyclization of a bosutinib-based scaffold. Systematic optimization led to the discovery of AZ137 (28e), a potent and selective HIPK4 inhibitor (IC50 = 11 nM; cellular EC50 = 76 nM). AZ137 exhibits exceptional selectivity across three comprehensive orthogonal panels, high solubility, and no detectable cytotoxicity. Its cellular activity was confirmed in cell-based assays of HIPK4-dependent F-actin remodeling. Together with a negative control compound, this probe set provides a foundational framework for the validating HIPK4 as a therapeutic target and a high-quality resource to elucidate its roles in normal physiology and disease.

biochemistry↗

bioRxiv2026

From nucleotides to semantics: genomic representation learning via joint-embedding predictive architecture

Decoding the regulatory syntax encoded in genomic sequences is a central objective in computational biology. Most existing genomic foundation models treat DNA as a language and adopt pretraining objectives from natural language processing. DNA sequences, however, lack explicit semantic boundaries and contain substantial evolutionary noise. Nucleotide-level reconstruction in a low-dimensional input space can therefore increase computational overhead and may yield representations with limited discriminative capacity. Downstream tasks often depend on expensive finetuning, which restricts practical use in many biology laboratories. Here we present GenoJEPA, a genomic representation learning framework based on joint-embedding predictive architecture. GenoJEPA combines continuous patching with semantic alignment, shifting the optimization from local base reconstruction to semantic alignment in latent space. Across 55 downstream tasks, GenoJEPA shows strong representational capacity and robust generalization while reducing parameter count and computational cost. The resulting semantic vectors from frozen GenoJEPA support lightweight GPU-free classifiers to achieve competitive accuracy. These results suggest a practical route towards efficient training and broad application of larger-scale genomic foundation models.

bioinformatics↗

bioRxiv2026

Towards a Cytometry Foundation Model: Interpretable Sample-level Predictive Modelling via Pretrained Transformers

Foundation models have transformed scientific data modelling across domains, yet flow cytometry has lacked one. Despite the abundance of high-dimensional cellular data, automated analysis remains bottlenecked by marker variability: prior studies are typically confined to fixed marker panels and homogeneous data, limiting scalability and generalisation due to architectural constraints. We present the Generalised Pretrained Cytometry Transformer (GPCT), an interpretable framework designed to learn from heterogeneous marker panels for sample-level predictive modelling. Through a novel cytometry-specific pretraining regime, GPCT learns transferable cellular representations that achieve high classification accuracy across diverse datasets. Notably, pretraining significantly boosts performance on data-scarce downstream tasks, marking a pivotal step towards a cytometry foundation model. Furthermore, GPCT maintains interpretability and identifies the specific cell subsets most influential to its predictions. This enables direct biological validation of learned patterns and provides a data-driven basis for refining traditional gating strategies.

bioinformatics↗

bioRxiv2025

Complex Genomic Structural Variation Underlies Climate Adaptation among Eucalyptus species

Climate change threatens global natural forest ecosystems, however, our limited understanding of adaptive genomic variation in wild forest trees restricts our ability to address this threat. Eucalyptus species likely harbour many climate adaptive alleles considering their long-standing evolutionary success across the Australian continent, yet their complex genomic variation and adaptive implications remain undercharacterised. Here, we present a comprehensive pangenome analysis of Eucalyptus viminalis using haplotype-resolved genomes and population-level long-read sequencing spanning the species natural distribution. We discovered complex and simple structural variants (SVs) that doubled the haploid genome length (530 Mbp haploid to 1.1 Gbp pan-genome). Landscape genomic analyses with SVs identified CHILL1, a 400-kb cold-adaptation locus underlined by complex structural variation. Validation across 434 accessions from three Eucalyptus species confirmed the large-effects of CHILL1, which outperforms species classification at predicting cold adaptation. These findings demonstrate that wild tree populations harbour critical adaptive alleles that can expedite climate-informed restoration.

genomics↗

bioRxiv2025

Immunotherapy with Pro-regenerative Macrophages from Embryonic Stem Cells Ameliorate Osteoarthritis via TNFAIP3-Mediated Chondroprotection

Immune cell-based therapies for osteoarthritis (OA) remains underexplored. Macrophages coordinate tissue repair, yet their functional heterogeneity and therapeutic applicability, particularly for designed stem cell-derived populations with tailored reparative properties are not fully delineated. Inspired by embryonic skeletal development as a paradigm of scarless regeneration, we identified a pro-regenerative macrophage population through a single-cell RNA atlas of developing skeletal tissues. We subsequently established a mechanically dynamic induction approach to efficiently generate pro-regenerative macrophages from human embryonic stem cells (hESCs), mimicking this developmental phenotype. In vitro, these macrophages mitigate cartilage degradation in OA explants and suppress pro-inflammatory, catabolic signaling in chondrocytes. Injection into murine OA joints robustly attenuates disease progression, reduces extracellular matrix breakdown, and resolves synovial inflammation, representing the first demonstration of immune cell-based therapy for OA. Mechanistically, these macrophages orchestrate chondroprotection not through broad immunosuppression, but by activating a TNFAIP3-dependent signaling hub that restrains inflammatory and catabolic responses in chondrocytes. This TNFAIP3 activation precisely reversed the OA-associated gene signature, suppressing catabolic mediators while enhancing reparative factors, thereby restoring the anabolic-catabolic balance. Our findings uncover a developmental blueprint for engineering pro-regenerative macrophages from stem cells and establish a pioneering targeted immune cell therapy that engages an intrinsic chondroprotective program, offering a transformative and translationally relevant strategy for OA treatment.

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

A multi-tissue atlas of genetic regulatory effects in sheep

Characterizing the impact of genomic variants on genome function and ultimately complex traits in livestock is essential for the development of sustainable precision agriculture and comparative genomics. Here, as part of the Farm animal Genotype-Tissue Expression (FarmGTEx) project, we present the pilot phase of the SheepGTEx resource through analyzing 6,761 RNA-sequencing samples of 51 primary tissues in a multi-breed population of sheep. We identify millions of regulatory variants associated with seven types of molecular phenotypes, and fine-map 322,468 primary and 114,017 non-primary effects, revealing a high degree of regulatory allelic heterogeneity. We systematically characterize the pleiotropic effects of these variants on molecular phenotypes, assess their context-specific regulatory patterns across tissues, breeds, sexes, and developmental stages, as well as explore their evolutionary constraints across mammals. Finally, we demonstrate the substantial potential of the SheepGTEx resource (https://sheepgtex.farmgtex.org), by providing examples of regulatory mechanisms underpinning 34 complex traits, population divergence between European and Asian breeds, and adaptive evolution in sheep over the past ten millennia.

genomics↗

bioRxiv2025

Development of Degraders and 2-pyridinecarboxyaldehyde (2-PCA) as a recruitment Ligand for FBXO22

Targeted protein degradation (TPD) is a promising therapeutic strategy that requires the discovery of small molecules that induce proximity between E3 ubiquitin ligases and proteins of interest. FBXO22 is an E3 ligase that is overexpressed in many cancers and implicated in tumorigenesis. While FBXO22 was previously identified as capable of recognizing ligands bearing a primary amine degron, further investigation and development of recruitment ligands is required to enable its broader utility for TPD. Here, we describe the discovery of chemical probes that can either selectively degrade FBXO22 or recruit this ligase for TPD applications. First, we describe AHPC(Me)-C6-NH2 as a potent and selective FBXO22 degrader (DC50 = 77 nM, Dmax = 99%) that is suitable for interrogating the effects of FBXO22 loss of function. Further, we discovered that the simple hexane-1,6-diamine acts as a minimal FBXO22 self-degrader, whereas shorter C4 (putrescine) to C5 (cadaverine) analogs, found in mammalian cells, do not induce degradation. Finally, we found that 2-pyridinecarboxaldehyde (2-PCA) functions as a novel electrophilic degron capable of forming a reversible thioketal with cysteine 326 for recruiting FBXO22. Conjugating 2-PCA to various ligands successfully induced FBXO22-dependent degradation of BRD4 and CDK12. Collectively, these chemical probes will facilitate the study of FBXO22 biology and broaden its applicability in TPD.

biochemistry↗

bioRxiv2025

Spatial Transcriptomics Reveal Developmental Dynamics of the Human Cerebral Cortex and Striatum

The human fetal brain undergoes morphological changes that contribute to the development of regional functionalities. However, the features of structural development, the underlying molecular and cellular signatures in the fetal brain remain unclear. With spatial transcriptomics and snRNA-seq, we identified 25 forebrain regions and characterized the dynamic changes in the cortex and striatum during the late first and early second trimesters. In particular, we discovered that temporal lobe enriched NPY-expressing L2/3 EX neuron potentially interacted with L4 EX neurons during cortical expansion and arealization. Additionally, the gyrus and sulcus were developmental asynchronous, in which HOPX and SPARC genes were potentially involved. Further investigation on the striatum showed specific genes and cell types that enriched in patch and matrix compartments, and SST-positive interneurons potentially involved in the development of these structures. Together, our results give insights into the understanding of early fetal brain development.

bioinformatics↗

bioRxiv2025

Superior batch alignment and hyper-dimensional cytometry representations allow ultra-sensitive classification of disease phenotypes

Analysis of cytometry data predominantly relies on clustering and dimensionality reduction approaches for computational tractability. This is particularly relevant for modern spectral flow cytometers, which can simultaneously measure an increasingly large number of antibody marker channels. While dimensionality reduction provides for more efficient data processing, this comes at the expense of data loss that may miss subtle patterns among rare cell types that may be critical for disease detection. Maintaining analysis at full dimensions presents opportunities to preserve resolution and provides complete downstream data interpretability. However, a significant obstacle to analysis of cytometry data without dimensionality reduction is the significant batch effects observed in this data between experiment days, operators and equipment types. Here we show a new strategy to denoise batch variation from both flow- and mass-cytometry datasets using an autoencoder neural network architecture. We generated a benchmark flow cytometry dataset in mice to compare this approach to current toolsets and find our approach shows superior preservation of biological signals, whilst also performing batch correction equal to current best methodology. Our batch alignment approach works to such an extent that it becomes practically possible to project batch aligned data into multidimensional space to generate a novel representation of cellular phenotype for downstream model building. This hyperdimensional approach maintains original data resolution without requiring dimensionality reduction, and thus any resultant cell populations that differentiate phenotypes remain fully interpretable. We show with two large clinical datasets that our batch-alignment approach coupled with the multi-dimensional representation successfully detects meaningful patterns in cases where the original analysis methods struggled. This new framework removes some of the inherent technical limitations encountered in the integration of large, multi-batch cytometry datasets and provides a framework for machine learning model building from this modality. An implementation of this framework and an associated web application accompanies this manuscript at http://voxelcoder.cloud. Significance StatementThis work addresses a critical bottleneck in cytometry analysis by introducing a neural network-based approach that effectively removes technical batch variation while preserving biological signals. The novel hyperdimensional representation maintains full data resolution without dimensionality reduction, enabling more sensitive detection of disease-associated cellular signatures than current methods. Importantly, this framework enables reliable batch normalization of fresh samples processed at different times and locations, overcoming the practical constraints of clinical sample collection where simultaneous processing is often impossible. The enhanced sensitivity for identifying pathogenic cellular patterns has immediate implications for biomarker discovery and personalized medicine applications.

systems biology↗

bioRxiv2025

Structural innovations and neurogenic continuity define avian brain development and evolution

Understanding how neurogenic diversity and brain architecture emerge is crucial for deciphering the mechanisms of brain plasticity and cognitive evolution. Here, we present a comprehensive single-cell and spatial transcriptomic atlas of the budgerigar brain, a highly cognitive avian species. We uncover a striking dorsoventral symmetry in excitatory neuron distribution, precisely organized along the lamina mesopallialis intermedia (LMI), a key boundary structure. Cross-species comparisons further reveal the origins of this unique avian pallial organization, offering new insight into avian brain evolution. Our study also highlights distinct developmental trajectories and asynchronous maturation patterns between telencephalic and optic tectum excitatory neurons, underscoring their contribution to innate circuit assembly. Furthermore, we identify adult neural stem cells (NSCs) with evolutionarily conserved transcriptomic signatures across avian species, emphasizing their role in brain plasticity and adaptation. These findings refine fundamental models of avian brain development and elucidate conserved principles of cognitive plasticity.

evolutionary biology↗

bioRxiv2025

Design and Biological Activity of a Novel Brain Penetrant Urea Compound Against Glioblastoma

Glioblastoma (GBM) remains the most lethal primary brain tumor, largely due to therapy-resistant glioma stem cells (GSCs) and the ability of non-stem cells to dedifferentiate under therapeutic pressure. We developed MXC-017, a novel urea-based compound that crosses the blood-brain-barrier, directly targets GSCs, and prevents radiation-induced GSC formation. Using click chemistry pull-down and mass spectrometry, we identified vimentin as the target of MXC-017, further validated by in silico docking. Global transcriptomic profiling (bulk RNA-seq) and single-cell RNA-seq analyses revealed MXC-017s efficacy with minimal off-target effects, supported by metabolic and kinome assays. Normal cell toxicity was negligible in fibroblasts, microglia, astrocytes, and murine neural progenitors. Maximum tolerated dose was identified and we observed significantly extended median survival in 17 PDOX GBM models when treated with MXC-017 plus radiation, benchmarked against standard-of-care temozolomide. These findings underscore the therapeutic potential of vimentin-targeting agents to overcome radiation resistance and improve outcomes for GBM patients. Statement of SignificanceGlioblastomas distinctive nature and the blood-brain barrier hamper therapies targeting therapy-resistant GSCs. We developed a novel urea-based agent that crosses the barrier, targets GSCs, and prevents radiation-induced GSC formation. With minimal off-target effects, reduced toxicity, and superior survival in PDOX models, it offers potential to improve outcome in GBM.

neuroscience↗