Biology subjects

Zhu, Y.

Publications and source records attributed to Zhu, Y..

At least 235 records · Page 13Linked to original sources

bioRxiv2025

Development of a Novel Benzodiazepine to Delineate Peripheral GABA-A Signaling Mechanisms in Visceral Pain Syndromes

Background and AimsVisceral pain is a cardinal symptom of many disorders affecting the gut. Modulators of gamma-aminobutyric acid (GABA) such as benzodiazepines may attenuate colonic pain but the specific contribution of peripheral GABAA receptors remains unclear as these agents have prominent central effects. MethodsUsing medicinal chemistry optimization of the benzodiazepine scaffold, we developed a novel and potent benzodiazepine-based positive allosteric modulator (PAM) of GABAA receptors, Li633, with no significant central nervous system (CNS) penetration. ResultsThe locomotor activity of rats placed in an open field was unchanged with Li633 at doses up to 30 mg/kg, confirming its lack of a CNS effect. LI-633 produced robust potentiation of GABA-induced inward current, with EC50 values ranging from 8 nM (5{beta}2{gamma}2) to 128 nM (3{beta}2{gamma}2). In vitro electrophysiological studies confirmed its ability to reduce excitability of human dorsal root ganglion (DRG) neurons. LI-633 potentiated muscimol-induced GABAergic currents in rat DRG neurons in a dose-dependent manner, with an EC50 of 70.4 nM. In vivo, LI-633 significantly attenuated visceral hypersensitivity and pain behavior in a rat model of irritable bowel syndrome (IBS) and functional dyspepsia (FD). In the IBS model, administration of the drug also resulted in decreased excitability of colon-specific DRG neurons and significantly reduced the colonic afferent response to balloon distention as measured by recordings of neural activity in dorsal ganglia rootlets. ConclusionsThese findings highlight the potential of targeting peripheral GABAA receptors for pain management in IBS and other disorders associated with visceral hypersensitivity.

neuroscience↗

bioRxiv2025

Senescent mast cells contribute to the progression of benign prostatic hyperplasia via SCF/c-KIT mediated endothelial-mesenchymal transition

Benign prostatic hyperplasia (BPH) is a common age-associated urological condition characterized by stromal expansion, but its cellular origins and regulatory mechanisms remain unclear. In this study, we identified endothelial-to-mesenchymal transition (EndMT) as a contributor to stromal cell accumulation in BPH. Using single-cell transcriptomic analysis, endothelial lineage tracing in mice, and validation in human samples, we showed that senescence-associated mast cells increased the expression of vascular endothelial growth factor A (VEGFA) and transforming growth factor-beta 1 (TGF-{beta}1) through the stem cell factor (SCF)/c-KIT-MAPK-JUND signaling pathway, thereby inducing EndMT in endothelial progenitor cells. Stromal fibroblasts express SCF, promoting mast cell activation and establishing a feedback loop that supports continued stromal proliferation. Intriguingly, inhibition of mast cell activation reduces EndMT and attenuates prostate enlargement in vivo. Thus, these findings revealed a senescence-linked immune-stromal interaction in the aging prostate and identify potential targets for therapeutic intervention in BPH.

cell biology↗

bioRxiv2025

GENERanno: A Genomic Foundation Model for Metagenomic Annotation

The rapid growth of genomic and metagenomic data has underscored the pressing need for advanced computational tools capable of deciphering complex biological sequences. In this study, we introduce Generanno, a compact yet powerful genomic foundation model (GFM) specifically optimized for metagenomic annotation. Trained on an extensive dataset comprising 715 billion base pairs (bp) of prokaryotic DNA, Generanno employs a transformer encoder architecture with 500 million parameters, enabling bidirectional attention over sequences up to 8192 bp at single-nucleotide resolution. This design addresses key limitations of existing methods, including the inability of traditional Hidden Markov Models (HMMs) to handle fragmented DNA sequences from multi-species microbial communities, as well as the suboptimal tokenization schemes of existing GFMs that compromise fine-grained analysis. At its core, Generanno excels in identifying coding regions from fragmented and mixed DNA sequences--a hallmark of metagenomic analysis. It achieves superior accuracy compared to traditional HMM-based methods (e.g., GLIMMER3, GeneMarkS2, Prodigal) and recent LLM-based approaches (e.g., GeneLM), while demonstrating robust generalization ability on archaeal genomes. Leveraging its advanced contextual understanding capability, Generanno further enables two essential functions: pseudogene prediction and taxonomic classification--both performed based solely on raw sequence data, without reliance on reference databases or comparative genomics. These functionalities collectively streamline the metagenomic analysis pipeline, significantly reducing preprocessing requirements and enabling end-to-end interpretation of sequencing data. Beyond its primary role in metagenomic annotation, Generanno also serves as a powerful GFM. To evaluate its broader utility, we curated the Prokaryotic Gener Tasks--a comprehensive benchmark suite specifically tailored for prokaryotic genomic analysis. It includes gene fitness prediction, antibiotic resistance identification, gene classification, and taxonomic classification, reflecting diverse aspects of functional genomics. On this benchmark, Generanno consistently outperforms existing GFMs such as DNABERT-2, NT-v2, and GenomeOcean, demonstrating strong generalization capabilities across a wide range of genomic tasks. Overall, Generanno provides a unified framework that integrates multiple critical functions for metagenomic annotation and beyond. By eliminating dependencies on external resources and offering rich contextual understanding of genomic sequences, this work delivers a foundational tool for advancing functional genomics in complex microbial communities. Implementation details and supplementary resources are available at https://github.com/GenerTeam/GENERanno.

genomics↗

bioRxiv2025

Intratumoral cDC1-T Cell Clusters Serve as Sites of Local Costimulation to Enhance CTL-Mediated Tumor Rejection

T cells are essential for anti-tumor immunity, but their ability to eliminate tumors depends on coordinated interactions with type 1 conventional dendritic cells (cDC1s). While cDC1s are known for cross-presenting tumor-derived antigens in lymph nodes to prime CD8+ T cells, their role within the tumor itself remains less well understood. Here, we use the Skin Tumor Array by Micro-Poration (STAMP) model to investigate how cDC1-T cell interactions shape immune responses and influence tumor fate. Our data reveal that it is the spatial distribution of both cDC1s and T cells that determines whether a tumor can be rejected. We defined three primary immunotypes based on the spatial distribution of T cells and cDC1s: T cell-inflamed/dendritic cell-inflamed (TC-In/DC-In) tumors, where T cells and cDC1s co-infiltrate the tumor; T cell-inflamed/dendritic cell-excluded (TC-In/DC-Ex) tumors, where T cells infiltrate but cDC1s remain at the periphery; and T cell-excluded/dendritic cell-excluded (TC-Ex/DC-Ex) tumors, which lack both cDC1 and T cell infiltration. Notably, TC-In/DC-In tumors are more likely to undergo rejection, whereas TC-In/DC-Ex tumors persist despite T cell infiltration. Within TC-In/DC-In tumors, cDC1s engage in direct interactions with T cells, upregulate co-stimulatory molecules, and sustain effector T cell responses, while cDC1s in TC-In/DC-Ex tumors express higher migration-associated genes, suggesting a propensity to exit the tumor. We further show that chemokine modulation, particularly through CXCL9, CCL5, and XCL1, can reshape immune infiltration patterns to promote intra-tumoral cDC1-T cell clustering and improve tumor rejection. These findings underscore the unexpectedly important role of cDC1 positioning and function in sustaining effective anti-tumor immunity and highlight spatially organized cDC1-T cell clusters as critical hubs for local T cell activation.

immunology↗

bioRxiv2025

The potential of 4-Methylumbelliferone to be repurposed for treating liver fibrosis

4-Methylumbelliferone (4-MU) is the active component of hymecromone, a choleretic and antispasmodic drug with an excellent safety profile. In rodent studies, high doses of 4-MU are also used to inhibit the production of hyaluronan (HA), a biomarker of liver fibrosis. Further, 4-MU shows excellent efficacy in inhibiting liver fibrosis of different etiologies in animal studies, eliciting interest in its repurposing for this condition. However, 4-MUs mechanism of action, and whether it inhibits liver fibrosis by impeding HA synthesis, remains unclear. Using several transgenic mouse models with HA overproduction or degradation in different types of liver cells, we found that both directions of perturbation reduced liver fibrosis levels. In addition, degrading HA via hyaluronidase PH20 overexpression impaired liver function manifested by increased serum aminotransferase (ALT) activity levels. These findings challenge both the role of HA modulation in 4-MUs action and the strategy of targeting HA to treat liver fibrosis. Additional mouse models also excluded the possibility that 4-MU modulates intestinal farnesoid X receptor (FXR) to inhibit liver fibrosis. Ablation of gut microbiota partially abolishes 4-MUs anti-liver fibrosis effect. However, the anti-liver fibrosis effect of 4-MU was lost in the lower-dose group, and the high doses effect in reducing ALT disappeared over time despite its liver fibrosis-reducing effect. Based on these findings, we argue that the lack of efficacy of 4-MU at a translatable dose and lack of a clear mechanism that allows further improvement of 4-MUs efficacy make 4-MU impractical for development as an anti-liver fibrosis treatment. HighlightsO_LI4-MU inhibits MASH fibrosis. C_LIO_LIBoth HA overproduction and digestion in the liver reduce liver fibrosis. C_LIO_LIIntestinal epithelial cell FXR is not required for 4-MU to reduce liver fibrosis. C_LIO_LIThe gut microbiota is partially responsible for 4-MUs antifibrotic effect in the liver. C_LIO_LI4-MU lacks the potential to be further developed into anti-liver fibrosis treatment. C_LI

molecular biology↗

bioRxiv2025

High-Sensitivity Top-Down Proteomics Reveals Enhanced Maturation of Micropatterned Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used for disease modeling, drug discovery, and precision medicine, yet their utility is often limited by their immature phenotype. One promising maturation strategy involves using micropatterned substrates that mimic native cardiomyocytes organizational growth and stiffness. However, the molecular maturity of this model has not yet been assessed, and there is currently no method to extract proteins from micropatterned hiPSC-CMs for top-down proteomic analysis. Herein, we present a high-sensitivity, surfactant-free protein extraction protocol for the top-down proteomic analysis of hiPSC-CMs. Through this method, we assessed the maturation of micropatterned hiPSC-CMs compared to traditional unstructured monoculture and co-culture monolayers at the proteoform level. We found that micropatterned hiPSC-CMs display molecular signatures of cardiomyocyte maturations including increased expression of ventricular myosin light chain isoforms, reduced expression of the fetal troponin T isoform, and decreased phosphorylation of alpha-tropomyosin. This surfactant-free, high-sensitivity approach enables robust top-down proteomics from limited, heterogeneous cell populations, and identifies the micropattern hiPSC-CM as a more adult-like CM model, broadening the utility of structured culture systems for cardiac disease modeling and translational research.

molecular biology↗

bioRxiv2025

An embryonic artery-forming niche reactivates in pulmonary arterial hypertension

Developmental programs that orchestrate cell fate and tissue architecture during organogenesis can cause disease when reactivated in adults. Here we identify a population of endothelial cells (ECs) defined by the pioneer factor early B cell factor 1 (EBF1) that controls both pulmonary artery (PA) morphogenesis and pulmonary arterial hypertension (PAH). During embryonic development, the PA emerges from an endothelial niche harbored within the vascular plexus, where Aplnr+ endothelial progenitors give rise to both arterial ECs and EBF1+ ECs. Rather than directly incorporating into the PA intima, EBF1+ ECs control the branching and maturation of the PA tree through paracrine vasculotrophic signals that direct plexus expansion, arterialization, and mural cell recruitment. Although essential for development, most EBF1+ ECs disappear upon completion of PA morphogenesis. In adult PAH, vascular injury reactivates this developmental program: normally quiescent general capillary ECs re-enter the cell cycle and differentiate into arterial ECs and EBF1+ ECs, reconstituting this artery-forming niche in a maladaptive reprise. Unlike their transient embryonic counterparts, EBF1+ ECs persist within neointimal lesions and express vasculotrophic signals associated with pathological PA remodeling. Capillary-restricted Ebf1 induction combined with endothelial injury recapitulates this program, driving neo-arterialization, neointimal formation, and severe PAH. Conversely, endothelial-targeted AAV-mediated Ebf1 knockdown achieves complete protection in a preclinical model of disease, demonstrating that endothelial EBF1 is necessary for PAH pathogenesis. These findings demonstrate that reactivation and persistence of a transient embryonic artery-forming niche in adulthood can promote pathological vascular remodeling and PAH.

developmental biology↗

bioRxiv2025

ArchLips: A comprehensive in silico database for high-throughput identification of archaeal lipids

Archaeal membrane lipids are markedly distinct from those in bacteria and eukaryotes, serving as unique biomarkers for unraveling their ecological and biogeochemical roles. Recent advancements in lipidomics have facilitated detailed cellular-level characterizations of lipid compounds. However, the lack of a comprehensive and dedicated database has severely limited large-scale, high-throughput investigations of archaeal lipids. We present ArchLips, a comprehensive database containing 218,868 in silico molecular structures and tandem mass spectra of 199,248 corresponding archaeal lipids. ArchLips enables the rapid and accurate identification of characterized archaeal lipid compounds from both pure cultures and environmental samples, serving as a transformative tool for enhancing our understanding of archaeal diversity and their ecological and evolutionary significance within global ecosystems.

biochemistry↗

bioRxiv2025

Bridging Hearts and Muscles: Genetic Insights into Cardiac Structure and Sarcopenia

BackgroundCurrently, the link between cardiovascular diseases and sarcopenia is increasingly garnering attention from researchers. However, studies exploring the association between cardiac structure and sarcopenia are sparse. This study aims to investigate the potential genetic links between cardiac structural features and sarcopenia. MethodsDuring the discovery phase, we employed Linkage Disequilibrium Score Regression (LDSC) and Mendelian Randomization (MR) to assess the genetic correlation and causality between traits related to cardiac structure and sarcopenia, with further validation of the results using validation set data. In the study, we established a scoring system to identify high-confidence trait pairs. For these pairs, we conducted sensitivity analyses to assess their heterogeneity and pleiotropy. Additionally, we undertook follow-up studies of these trait pairs, including using mediation analysis to evaluate the potential mediating effects of lipids, and enrichment analysis to explore possible shared biological pathways linking these characteristics. ResultsThe genetic correlation analyses during the discovery and validation phases identified 5 pairs (in forward analysis) and 16 pairs (in reverse analysis) of high-confidence trait pairs. As a key sarcopenia-related feature, appendicular lean mass (ALM) exhibited positive causal relationships with several cardiac structural features, including the volumes of the left and right atria. Mediation analysis suggested that certain lipids, such as phosphatidylcholine, might mediate these causal relationships. Notably, gene and pathway enrichment analyses revealed that genes associated with significant SNPs in high-confidence trait pairs were enriched in multiple key biological processes and pathways, such as tube morphogenesis, cardiac right ventricle morphogenesis, and muscle system processes in the forward analysis, as well as skeletal system development, heart development, and hemopoiesis in the reverse analysis. Cell-type enrichment analysis pointed to endothelial cells, smooth muscle cells, fibroblasts, and mesenchymal stem cells. ConclusionsThis study provides evidence for genetic relationships between cardiac structures and sarcopenia-related traits. These findings may enhance our understanding of the biological mechanisms underlying age-related diseases and provide scientific basis for developing future preventive and therapeutic strategies.

genetics↗

bioRxiv2025

Gene Editing and Aging: A Bibliometric Analysis of Global Trends and Frontier Themes (2015-2024)

ObjectiveThe accumulation of DNA damage and mutations is a key contributor to aging. Recent studies have shown that disrupting the Beclin 1-BCL2 autophagy regulatory complex through gene editing can extend lifespan in mice. The precise application of gene editing technologies offers a promising strategy for aging. This study conducted a bibliometric analysis to map the knowledge landscape of gene editing and aging. MethodsWe retrieved publications related to genome editing and aging from the Web of Science Core Collection, covering the period from 2015 to 2024. The data were analyzed using VOSviewer and R package Bibliometrix. These tools enabled us to identify the most productive researchers, journals, institutions, countries and visualized current trends, emerging research hotspots. ResultsA total of 982 publications on genome editing and aging were identified. The United States (n=285) and China (n=214) form a dual-core structure leading global output. Harvard University (n=116) emerged as the most prolific institution. Scientific Reports was the top-publishing journal, with 23 articles in 2024. ZHANG Y (n=12, citations=102, H-index=6) was identified as the most productive author. KIM Es 2017 publication in Nature Communications (TC=494, TC/year=54.9, NTC=9.33) has had a significant and ongoing impact. The analysis indicates that future directions will include CRISPR optimization and AI-assisted genomic analysis. ConclusionThis study presents the first comprehensive bibliometric analysis and visualization of the knowledge structure in gene editing and aging research up to 2024. It offers researchers a detailed overview of current developments, trends, and emerging frontiers in this rapidly evolving domain.

scientific communication and education↗

bioRxiv2025

The China Brain Multi-omics Atlas Project (CBMAP)

The China Brain Multi-omics Atlas Project (CBMAP) aims to generate a comprehensive molecular reference map of over 1,000 human brains (Phase I), spanning a broad age range and multiple regions in China, to address the underrepresentation of East Asian populations in brain research. By integrating genome, epigenome, transcriptome, proteome (including multiple post-translational modifications), and metabolome data, CBMAP is set to provide a rich and invaluable resource for investigating the molecular underpinnings of aging-related brain phenotypes and neuropsychiatric disorders. Leveraging high-throughput omics data and advanced technologies, such as spatial transcriptomics, proteomics, and single-nucleus 3D chromatin structure analysis, this atlas will serve as a crucial resource for the brain science community, illuminating disease mechanisms and enhancing the utility of data from genome-wide association studies (GWAS). CBMAP is also poised to accelerate drug discovery and precision medicine for brain disorders.

neuroscience↗

bioRxiv2025

Systemic trafficking of mRNA lipid nanoparticle vaccine following intramuscular injection generates potent tissue-specific T cell response

The mRNA lipid nanoparticles (LNPs) represent a new generation of vaccine carriers designed to elicit potent immune responses against infectious diseases and cancer. Despite the clinical success and rapid advancements in mRNA LNP technologies, the trafficking patterns of LNPs after intramuscular (i.m.) administration and the subsequent tissue-specific immunological effects have not been systematically characterized. Here, we report that trafficking of mRNA LNPs to different organs following i.m. injection is crucial for the induction of tissue-specific immunity beyond systemic immune response, particularly in tissue-resident CD8+ T cell generation, which is important for localized defense. By fine-tuning the composition of mRNA LNPs, trafficking patterns to systemic organs can be modulated, which can alter the resulting tissue-specific immune response. Formulations with a greater ability to enter the bloodstream can preferentially localize and transfect cells in specific organs like the liver, elicit stronger tissue-specific CD8+ T cell immune responses, and achieve enhanced efficacy in a liver tumor model. These findings highlight the potential to tailor mRNA LNP compositions to modulate trafficking following i.m. injection, thereby providing novel strategies for designing tissue-specific vaccines. Such strategies are particularly valuable for organ-specific diseases like cancer and infectious diseases, where tissue targeting and long-lasting immunity are essential for therapeutic success.

bioengineering↗

bioRxiv2025

Targeting protein glycosylation and cholesterol metabolism in chemoresistant pancreatic cancer

Chemotherapy remains the cornerstone of pancreatic ductal adenocarcinoma (PDAC) treatment. However, most cases of PDAC finally progress to advanced chemoresistant disease. This highlights an urgent need to develop new pharmacological strategies to overcome chemotherapy resistance using clinical grade inhibitors. Here, we established a biobank comprising 260 organoid lines derived from 322 pancreatic cancer patients. These organoids underwent extensive multi-omics profiling and drug sensitivity testing for both chemotherapy and targeted therapy. Increased protein glycosylation and cholesterol metabolism signaling pathways were especially enriched in chemoresistant organoid lines. Importantly, chemoresistant PDAC organoids were effectively targeted with statins, inhibitors of cholesterol synthesis. Statins treatment attenuated protein glycosylation, cholesterol levels, and the epithelial-to-mesenchymal transition (EMT) signature in PDAC organoids. We further conducted a phase 2 clinical trial (NCT06241352) combining atorvastatin with chemotherapy in patients with advanced chemoresistant pancreatic cancer. Among 29 patients, 21 (72.4%) demonstrated a response, with tumor markers decreasing by more than 20%, suggesting durable responses and potential clinical benefits in this challenging patient population. ClinicalTrials.gov identifier: NCT06241352.

cancer biology↗

bioRxiv2025

Novel Strategy against AML by Rescuing Loss of Phase Separation of Pathogenic NPM1c

Nucleophosmin (NPM1) is a nucleolar protein that participates in many biological processes through phase separation. In acute myeloid leukemia (AML), nearly one-third of cases harbor NPM1c mutations. Previous studies mainly focused on the contribution of the additional NES in NPM1c mislocalization and AML development; however, the mechanisms underlying the relationship between aberrant localization and phase separation of NPM1c remain poorly understood. In this study, we demonstrate that loss of phase separation (LoPS) with RNA is associated with NPM1c mislocalization, which uncovers a new strategy for therapeutic targeting of NPM1c. Leveraging high-throughput screening, we identify a potent small molecule capable of rescuing NPM1c LoPS in vitro and effectively restoring the nucleolar localization of NPM1c within cells. Remarkably, this compound demonstrates anti-leukemia activity in both AML cell lines and patient-derived blasts. Overall, these findings indicate a promising avenue for developing new AML therapies against NPM1c by reinstating its phase separation capabilities. One-Sentence SummaryRescuing the lost phase separation capacity of NPM1c unveils a promising AML therapy.

cancer biology↗

bioRxiv2025

Spatial Metagene Discovery and Associated Molecular Pattern Characterization in Spatial Transcriptomics and Multi-Omics using SEPAR

Spatially resolved transcriptomics (SRT) has transformed biomedical research by enabling gene expression profiling at near- or sub-cellular resolution while preserving spatial context. However, interpreting SRT data to understand cellular and gene organization remains challenging. Current methods focus on identifying global spatial domains across all genes, but often miss localized structures driven by specific gene subsets. Here, we introduce SEPAR, an unsupervised computational framework designed to analyze SRT data using a limited number of spatial metagenes and their expression patterns. SEPAR integrates gene activity and spatial neighborhood relationships to summarize gene expression per spot/- cell, decomposing data into contributions from spatial metagenes. It supports downstream analyses, including metagene expression pattern-specific gene identification, spatially variable gene (SVG) detection, spatial domain delineation, and gene expression refinement. Applied to diverse datasets, SEPAR demonstrates high efficacy. Gene sets linked to spatial metagene patterns are enriched with meaningful cell types and gene ontologies. SVGs are detected with higher accuracy, and gene refinement enhances biological signals, improving correlation analysis of functionally related genes. In spatial multi-omics data, SEPAR excels at revealing strong correlations between co-localized molecules in spatial CITE-seq data and identifies coordinated gene-peak relationships in MISAR-seq data, offering insights into spatial molecular interactions.

bioinformatics↗

bioRxiv2025

Context-dependent Interactors Regulate TDP-43 Dysfunction in ALS/FTLD

TDP-43 mislocalization, aggregation, and loss of splicing function are neuropathological hallmarks in over 97% of Amyotrophic Lateral Sclerosis (ALS), 45% of Frontotemporal Lobar Degeneration (FTLD), and 60% of Alzheimers Disease, which has been reclassified as LATE-NC. However, the mechanisms underlying TDP-43 dysfunction remain elusive. Here, we utilize APEX2-driven proximity labeling and mass spectrometry to characterize the context-dependent TDP-43 interactome in conditions of cytoplasmic mislocalization, impaired RNA-binding contributing to aggregation, and oxidative stress. We describe context-dependent interactors, including disrupted interactions with splicing-related proteins and altered biomolecular condensate (BMC) associations. By integrating ALS and FTLD snRNA-seq data, we uncover disease-relevant molecular alterations and validate our dataset through a functional screen that identifies key TDP- 43 regulators. We demonstrate that disrupting nuclear speckle integrity, particularly through the downregulation of the splicing factor SRRM2, promotes TDP-43 mislocalization and loss of function. Additionally, we identify NUFIP2 as an interactor associated with mislocalization that sequesters TDP-43 into cytoplasmic aggregates and co-localizes with TDP-43 pathology in patient tissue. We also highlight HNRNPC as a potent TDP-43 splicing regulator, where precise modulation of TDP-43 or HNRNPC can rescue cryptic exon splicing. These findings provide mechanistic insights and potential therapeutic targets for TDP-43 dysfunction.

molecular biology↗

bioRxiv2025

Perturbing RNA localization for functional study in neurons

Spatial RNA organization plays a pivotal role in diverse cellular processes and diseases, but the functional implications of spatial RNA localization remain underexplored. We present CRISPR-mediated transcriptome organization (CRISPR-TO) that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of RNA localization in live cells. CRISPR-TO enables targeted localization of RNAs to diverse subcellular compartments, including p-bodies, stress granules, telomeres, and nuclear stress bodies, across cell types. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips and co-transport with ribosomes, with {beta}-actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. Furthermore, CRISPR-TO-enabled parallel screening in primary neurons identifies Stmn2 mRNA localization as a driver of neurite outgrowth. By enabling large-scale perturbation of the spatial transcriptome, CRISPR-TO bridges a critical gap left by current sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.

bioengineering↗

bioRxiv2025

Nanosecond pulsed electric field precisely manipulates cytoplasmic Ca2+ oscillations during oocyte activation

Oocyte activation deficiency (OAD) is a primary cause of fertilization failure following intracytoplasmic sperm injection (ICSI), a problem that can potentially be overcome through artificial oocyte activation (AOA). However, concerns persist regarding the safety and efficacy of AOA techniques in clinical practice. In this study, nanosecond pulsed electric field (nsPEF) is proposed as a safe and controllable method for oocyte activation in vitro by precisely manipulating Ca2+ signaling in the ooplasm. Mouse oocytes collected from oviducts were exposed to nsPEF stimulation at varying intensities to induce distinct Ca2+ signaling patterns. Subsequently, these oocytes underwent parthenogenetic activation and were cultured to assess developmental potential up to the blastocyst stage. The sperm-initiated physiological Ca2+ oscillations are successfully mimicked by one series of Ca2+ signals induced by nsPEF at low or medium intensities, and improve activation efficiency and developmental potential compared to calcium ionophore A23187. Low-intensity nsPEF pulses mediated repetitive extracellular Ca2+ influx in a non-invasive, electro-permeable manner. Medium-intensity nsPEF stimulation triggered periodic Ca2+ release from the endoplasmic reticulum (ER) via the PIP2-IP3-IP3R pathway, producing physiological-like Ca2+ oscillations. The non-invasive nsPEF method ensures safe oocyte activation by preserving cellular integrity and minimizing stress responses. The efficacy of nsPEF in precisely manipulating Ca2+ signaling patterns was further validated in human unfertilized oocytes. Our findings present a novel AOA approach with enhanced safety and efficacy, especially for patients experiencing repeated fertilization failures. It is anticipated that nsPEF is emerging as a promising technology for addressing male infertility issues, and may offer a potential alternative fertility treatment, bypassing sperm for initiation of oocyte activation.

developmental biology↗