Biology subjects

Liang, R.

Publications and source records attributed to Liang, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Five decades of human H1N1 evolution reveal conserved glycan receptor specificity

H1N1 influenza viruses have circulated continuously among human populations for five decades. During evolution, the hemagglutinin has accumulated amino acid substitutions in and around the receptor-binding site (RBS), mainly due to antigenic pressure, potentially altering receptor recognition and binding profiles. Here, we investigated the glycan-binding properties of H1N1 strains collected from 1977 through 2024 using ELISA, flow cytometry, and tissue histochemistry to characterize changes in receptor-binding specificity over time. Across the strains examined, we observe a recurring preference for binding 2,6-sialylated di-LacNAc N-glycans. Binding to 2,3-sialylated glycans was generally undetectable, although A/Hongkong/117/1977 and A/New Caledonia/20/1999 bind chicken tracheal tissues. Interestingly, post-pandemic H1N1 viruses fluctuate in their relative binding avidities. The responsible amino acids, in turn, have different effects in pre-pandemic HA proteins. We also report a classical egg-adapted H1N1 mutant that shows null binding across platforms. These findings highlight the contribution of RBS mutations during initial adaptation and evolution of human H1N1 influenza viruses to glycan-binding properties, which, in contrast to H3N2 viruses, never fail to bind glycans that correlate with avian erythrocyte hemagglutination.

microbiology↗

bioRxiv2026

Overexpression of miR-424(322)/-503 induces severe dilated cardiomyopathy by regulating the fatty acid oxidation gene expression program

Fatty acid oxidation (FAO) is a major energy source in the adult heart, and disruption of cardiac metabolism is closely associated with heart failure. Here, we investigated the effects of cardiac-specific overexpression of the H19X-encoded miR-424(322)/-503 cluster using an inducible transgenic mouse model. Sustained miR-424(322)/-503 overexpression caused rapid metabolic and functional deterioration, with early impairment of fatty acid oxidation. Short-term induction reduced FAO activity and downregulated genes involved in lipid metabolism, while glycolytic enzyme activity remained largely unchanged. Continued miR-424(322)/-503 expression subsequently led to severe dilated cardiomyopathy characterized by ventricular dilation, wall thinning, fibrosis, reduced contractility, and high mortality. Importantly, disease progression was dependent on the level and duration of miR-424(322)/-503 expression, as intermittent or lower-dose induction delayed cardiac dysfunction and prolonged survival. Withdrawal of miR-424(322)/-503 expression after the onset of dysfunction promoted substantial functional recovery. Together, these findings identify miR-424(322)/-503 as a potent regulator of cardiac metabolic reprogramming that disrupts fatty acid metabolism and drives progressive heart failure.

molecular biology↗

bioRxiv2026

Loss of hemagglutination ability by H3N2 influenza A virus, subclade K.

Seasonal human H3N2 influenza viruses, subclade K (J.2.4.1), have been the predominant influenza A viruses in the Northern hemisphere influenza season of 2025/2026. Since 2024, the vaccine virus A/Darwin/6/21 has emerged in different antigenic variants. Antigenic changes are frequently caused by amino acid substitutions near the hemagglutinin (HA) receptor-binding pocket, which can also affect receptor binding properties, such as hemagglutination. Hemagglutination is crucial for assessing antigenicity using the hemagglutination inhibition (HAI) assay, and a loss of binding to turkey erythrocytes could significantly hamper this process. In this study, we explored how substitutions in or around the HA receptor-binding site affect binding to glycans at the molecular level. We employed ELISA, glycan array, flow cytometry, hemagglutination assays, and tissue staining. Substitutions at positions 140, 192, and 223 establish clade J viruses that emerged in 2024. Computational analysis of HA in complex with an elongated glycan reveals that mutation F192 forms a CH-Pi interaction to stabilize the binding. Based on this background, substitutions in antigenic sites A and B within subclade K viruses exhibit a binding preference for elongated glycans, which are not displayed on turkey erythrocytes. Conversely, our previously established glyco-remodeled erythrocytes are efficiently bound by these subclade K H3N2 viruses and could support influenza surveillance and vaccine development.

microbiology↗

bioRxiv2026

Architecture of a portal complex embedded in the poxvirus core

Gene transcription within the viral core is a unique feature of poxvirus replication. Following entry into the cytoplasm, the poxvirus core dissociates from the lateral bodies and undergoes expansion, functioning as a compartment for early transcription. However, the mechanisms governing molecular exchange between the viral core and the host cytoplasm remain poorly understood. Here, we determine the structures of the portal complex and its pore on the poxvirus core at 7.1 [A] and 4.9 [A] resolution, respectively, using cryo-electron tomography and sub-tomogram averaging. The pore is assembled from three viral proteins, E8, E6, and L3, for which we constructed an atomic model. Structural and channel analyses reveal that the pore satisfies the geometric and electrostatic requirements for the transport of RNA and smaller negatively charged molecules, while excluding double-stranded DNA and cytosolic DNA sensors. Together, our findings establish a structural framework for understanding the assembly and function of the poxvirus portal complex and identify potential targets for antiviral intervention.

biophysics↗

bioRxiv2026

Daikenchuto modulates gut microbial metabolism to mitigate irinotecan-induced enterotoxicity

Gut microbiota metabolic remodeling is a pivotal determinant in irinotecan-induced enterotoxicity and epithelial damage, although the underlying mechanisms remain unclear. Herein, we discovered that Daikenchuto (DKT), a traditional Chinese prescription for intestinal disorders, alleviated irinotecan-induced enterotoxicity without compromising its anti-tumor efficacy by improving weight loss, diarrhea, intestinal inflammation, and barrier damage, and these effects were partially dependent on gut microbiota. DKT significantly restored microbial tryptophan metabolism in irinotecan-treated rats, which was characterized by the enrichment of Limosilactobacillus reuteri, and elevated levels of indole-3-ethanol (IE) and indole-3-propionic acid (IPA). Multi-omics analysis further revealed a positive correlation between L. reuteri and IE and IPA. Consistent with this, DKT promoted L. reuteri proliferation, leading to the conversion of tryptophan to IE and IPA, which improved epithelial barrier damage in the irinotecan-treated Caco-2 cells. In addition, DKT suppressed the growth of Loop 1 {beta}-glucuronidase ({beta}-GUS)-producing bacteria, such as Escherichia coli. Furthermore, the main constituents of DKT selectively inhibited Loop 1 {beta}-GUS activity independent of the gut microbiota, which reduced the intra-luminal level of 7-ethyl-10-hydroxycamptothecin, the toxic metabolite of irinotecan. Taken together, this study reveals a dual gut microbiota-driven mechanism by which DKT mitigates irinotecan-induced enterotoxicity, which provides a promising strategy for managing chemotherapy-related enterotoxicity.

pharmacology and toxicology↗

bioRxiv2026

Multiscale Free-Energy Methods for Protonation-Coupled Light-Responsive Binding of Ionizable Photoswitchable eDHFR Inhibitors

Photoswitchable ligands enable photocontrol of biomolecular activity by binding to targets in an isomer-dependent, light-responsive manner. Recent developments in ionizable photoswitchable ligands greatly expand their applications but introduce a major design challenge: light-responsive binding can depend on isomeric form, chemical substitution, and binding-induced shifts in protonation equilibria. These effects are tightly coupled, subtle in magnitude, and difficult to predict. Consequently, few computational methods have been developed and systematically benchmarked for quantitatively predicting them. Here, we establish a multiscale free-energy method and benchmark it against experimental data for a series of recently developed photoswitchable inhibitors of Escherichia coli dihydrofolate reductase (eDHFR), a crucial target in photopharmacology. Constant pH replica-exchange molecular dynamics and quantum mechanics/molecular mechanics umbrella sampling quantitatively characterize the ligands protonation-state change upon binding to the eDHFR active site. Thermodynamic integration simulations using alternative alchemical pathways, thermodynamic cycles, and protonation-state assignments were evaluated for predicting light-responsive affinity differentials and substituent effects. Direct cis-to-trans transformations with explicit treatment of environment-dependent protonation states best reproduce experimental trends. Compound-to-compound pathways are less reliable because force-field inaccuracies introduce large pK errors that are difficult to correct when protonation/deprotonation processes implicitly enter the thermodynamic cycle. TI simulations that ignore binding-induced protonation-state changes fail to consistently reproduce experimental trends. Protein-ligand and ligand-water interaction analyses further reveal the energetic and structural origins of isomer-dependent binding. This study establishes a systematic free-energy method for designing ionizable photoswitches in photopharmacology.

pharmacology and toxicology↗

bioRxiv2026

A Dynamic Oligomerization Network Coordinates Hemagglutinin-Mediated Membrane Fusion on Influenza Virions

Influenza A virus (IAV) entry is mediated by the trimeric glycoprotein hemagglutinin (HA), which undergoes low-pH-triggered conformational rearrangements to drive membrane fusion. Although biochemical and biophysical studies have long suggested that HA trimers function cooperatively, the structural basis for this cooperativity on intact virions has remained unclear. Here, using cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), we determine the in situ structure of prefusion HA on native virions at 3.58 [A] resolution, enabling atomic modeling of membrane-anchored HA. We show that neighbouring HA trimers engage in lateral interactions mediated by HA1 subunits, assembling into flexible dimeric, pentameric, and hexameric organizations that form locally ordered lattices on the viral surface. A 6.0 [A] reconstruction of the HA-dimer reveals the molecular interfaces underlying these assemblies. Disruption of the dominant HA1-HA1 interaction markedly impairs viral entry and slows membrane fusion kinetics. Together, these findings define a virion-level mechanism for coordinated HA activation and establish a structural framework for understanding cooperative membrane fusion by class-I viral fusion proteins.

biophysics↗

bioRxiv2026

Relaxase Asymmetry Drives Initiation of Bacterial Conjugation

Bacterial conjugation is the major route of horizontal gene transfer and a key driver of the dissemination of antibiotic resistance, exacerbating the global health crisis. Central to this process is TraI, a bifunctional relaxase-helicase encoded by the iconic F-plasmid family. TraI initiates single-stranded DNA (ssDNA) transfer by covalently attaching to plasmid DNA through its trans-esterase (TE) activity and subsequently unwinds the plasmid via its helicase activity. Although genetic and biochemical studies have postulated that two TraI molecules are required for efficient conjugative transfer, how these molecules coordinate their activities in space and time - and how strand nicking is coupled to helicase loading - has remained unknown. Here, we capture the elusive, functionally asymmetric TraI homodimer and report its cryo-EM structure, revealing that the interaction between the two TraI molecules is mediated predominantly by ssDNA. We further show that TE-driven duplex melting at the origin of transfer (oriT) by one TraI molecule enables helicase loading by a second TraI molecule on the transfer strand of the unnicked plasmid DNA. Although the TE domain is intrinsically competent for strand nicking, its activity is inhibited by the host factor IHF, and this inhibition is relieved upon helicase loading. Together, these findings define a regulatory loop that coordinates helicase loading with strand nicking, providing mechanistic insight into the earliest stages of conjugative DNA processing.

microbiology↗

bioRxiv2026

Emergence and antigenic characterisation of influenza A(H3N2) viruses with hemagglutinin substitutions N158K and K189R during the 2024/25 influenza season

BackgroundSeasonal human influenza viruses can escape from antibody-mediated neutralization when amino acid changes occur in the hemagglutinin protein. Routine surveillance identified circulation of an A(H3N2) virus variant in the Netherlands with amino acid substitutions at hemagglutinin positions 158 and 189. These amino acid positions were previously responsible for antigenic change of influenza A(H3N2) viruses and potentially lead to escape of this variant from vaccine-mediated immunity. AimTo characterize the emergence and antigenic properties of N158K and K189R double substitution virus variants. MethodsWe analyzed the geographical and temporal dynamics of the double-substitution variant using a phylogeographic approach and used hemagglutination inhibition assays and antigenic cartography methods to map its antigenic properties. ResultsA(H3N2) viruses carrying K189R were first detected in Guatemala in June 2024, before subsequently gaining the N158K substitution, which was intially detected in Colombia in November 2024, followed by detection in the Netherlands in December 2024. However, detections within Europe remained almost entirely confined to the Netherlands. The proportion of viruses carrying the N158K and K189R substitutions increased to 16% - 24% per collection week of sequenced Dutch viruses during the peak of the epidemic of the 2024-2025 respiratory season. Antigenic characterization of viruses with N158K and K189R substitutions indicated that these are antigenically distinct from the A(H3N2) components of 2025-2026 Northern Hemisphere vaccines, showing 8-192-fold reduction in hemagglutination inhibition titers with antisera against the vaccine strain compared to antisera against the homologous virus. ConclusionsInfluenza A(H3N2) viruses with N158K and K189R escaped recognition by antibodies raised against the 2024-2025 and 2025/2026 Northern Hemipshere vaccine strains in hemagglutination inhibition assays. These variants circulated widely in the Netherlands during the 2024-2025 influenza season, raising concerns about reduced vaccine-mediated protection if such variants would spread more broadly during 2025-2026 Northern Hemipshere season.

microbiology↗

bioRxiv2026

eScreen: a deep learning framework for functionally decoding the regulatory genome at single-nucleotide resolution

The human genome is densely populated with cis-regulatory elements (CREs), yet deciphering their functional regulatory syntax and combinational logic remains a fundamental challenge. Here, we integrate 379 genome-scale CRISPR screen experiments, encompassing 21 million perturbations across 23 cell types, to construct a compendium of 41,239 high-confidence functional CREs from 530,527 candidates. Leveraging this resource, we develop eScreen, a deep learning model built on the StripedHyena2 architecture to functionally decode the regulatory genome at single-nucleotide resolution. eScreen achieves three primary functions: (1) predicts genome-wide cell-type-specific CRE functional activity with high accuracy, outperforming existing models; (2) provides mechanistic interpretation of regulatory syntax at single-nucleotide resolution; (3) dissects the functional organization of enhancer clusters through in silico perturbation analysis. We perform multiple independent CRISPR knockout, CRISPR interference (CRISPRi), and base editing screens to validate these functions of eScreen both at scale and on individual cases. Furthermore, we provide an interactive web server (https://escreen.huanglabxmu.com/) for the community to access the integrated CRISPR screen resources and eScreen functions. Collectively, our work establishes a highly precise and convenient tool to decode the causal effects of the regulatory genome.

genomics↗

bioRxiv2026

A spatial-temporal atlas of human islet pathophysiology identifies a size-dependent trajectory from compensation to decompensation

Type 2 diabetes (T2D) is characterized by progressive islet dysfunction, yet the transition from functional adaptation to failure remains poorly defined within the native tissue architecture. Using multiplex imaging mass cytometry, we systematically analyzed human pancreatic islets across a spectrum of non-diabetic (ND), prediabetic (PreD), and T2D donors, revealing that pathological remodeling is profoundly size-dependent. This remodeling is manifested as a coordinated evolution of subpopulation abundance, endocrine cell proportions, structural integrity, and protein expression profiles, revealing that islets of different sizes undergo divergent fates during disease progression. We identified a size-dependent vulnerability spectrum where medium-and large-sized islets (>100 m in diameter) serve as the primary histopathological correlates of glycemic failure (HbA1c), exhibiting early density loss and structural disintegration. In contrast, small islets (30-100 {micro}m in diameter) exhibited compensatory hormone upregulation during PreD. Notably, diverging from the death of the small paradigm in Type 1 Diabetes, our data reveal a significant expansion of extra-islet endocrine clusters (EECs) that initiates during the compensatory stage, effectively preceding the onset of overt hyperglycemia. Finally, t-SNE clustering reconstructed a continuous trajectory of islet remodeling, capturing the phenotypic evolution of islets from normoglycemia through compensation to clinical decompensation. This study provides a high-resolution atlas of islet pathophysiology, offering new insights into T2D progression. HighlightsO_LIIslet remodeling during T2D progression is critically dependent on islet size. C_LIO_LIMedium and large islets density negatively correlates with HbA1c. C_LIO_LISmall islets exhibit resilience to metabolic stress during T2D progression. C_LIO_LIEECs expansion suggests a process of -cell-biased neogenesis. C_LIO_LIt-SNE maps the islet trajectory from compensation to decompensation. C_LI

pathology↗

bioRxiv2025

Mechanistic Understanding of Protein-MOF Integration through Surfactant-Driven Interfacial Design

Integration of proteins into metal-organic frameworks (Protein@MOF) represents an effective method for protein stabilization, with rising demand across material and biomedical sciences. However, the molecular mechanism of protein-MOF interactions remains unsettled due to challenges in developing a general platform to systematically investigate such interactions, hindering improvements in their chemical and physical properties. Here, we develop a surfactant-guided strategy to modulate the assembly of protein@MOF through interfacial design. We discovered that the interfacial environment between proteins and MOFs is the primary factor determining encapsulation efficiency, structural retention, and functional performance. Lipid-based non-ionic surfactants such as glycerol monooleate (GMO) increase the proteins solvent-accessible surface area (SASA), suggesting partial remodeling of the protein surface and hydration shell. GMO at the interface of protein@MOF results in a 20% improvement in protein encapsulation and a 30% increase in MOF growth rate. All-atom molecular dynamics simulations reveal domain-specific interactions between GMO and flexible surface residues on protein in a concentration-dependent manner, involving both electrostatic and hydrophobic contacts. This work offers new molecular insights into how surfactant-driven interfacial design fine-tunes the stability of protein@MOF, laying the foundation for robust alternatives to lipid nanodiscs for membrane protein stabilization, and protein-based platforms for drug-delivery, biocatalysis, and biosensing.

biophysics↗

bioRxiv2025

Benchmarking Perturbation Tools for the Noncoding Genome

Deciphering the functionality of the noncoding genome which includes important cis-regulatory elements (CREs) and transcribed noncoding RNA genes remains technically challenging. Here, using massively parallel genetic screening, we systematically benchmark the performance of five representative loss-of-function perturbation tools, including single guide RNA (gRNA) mediated SpCas9 cleavage or CRISPR interference, and paired gRNA (pgRNA) involved dual-SpCas9, Big Papi (paired SpCas9 and SaCas9) or dual-enAsCas12a fragment deletion methods, in decoding the roles of the noncoding genome. For targeting CREs such as enhancer, dual-SpCas9 outperforms other methods with superior efficiency of destroying functional genomic regions. For perturbing noncoding RNA genes, in addition to dual-SpCas9, other RNA-targeting methods such as RNA interference are recommended to discriminate transcript-dependent or -independent roles. A deep learning model DeepDC with associated web server is built to facilitate optimal dual-SpCas9 pgRNA design for efficiently deleting a genomic fragment. Together, our work provides practical guidance on selecting appropriate loss-of-function tools to resolve the functional complexity of the noncoding genome.

genetics↗

bioRxiv2025

De Novo Design of Anti-Aging Peptides

Aging, a universal biological process in complex organisms, is increasingly recognized to be driven by progressive loss of epigenetic information, as proposed in the Information Theory of Aging (ITOA). However, research on anti-aging peptides remains scarce, with most existing efforts confined to derivatives of natural proteins, while systematic design attempts are virtually absent. This limitation not only restricts discovery within the evolutionary sequence space but also hampers the identification of candidates with novel mechanisms and improved efficacy. Here, we present ElixirSeeker2, the first computational framework for de novo design of anti-aging peptides. By integrating modeling of known anti-aging peptides, activity scoring from the IC50 database, and penalty constraints from toxic peptides, ElixirSeeker2 enables large-scale virtual screening and identification of novel peptide candidates. Several lead peptides demonstrated significant effects in delaying cellular senescence, restoring cellular functions in vitro and in enhancing locomotor activity of aged Caenorhabditis elegans. This study not only validates the feasibility of de novo design in anti-aging interventions but also establishes a strategy for the development of next-generation biologics.

bioinformatics↗

bioRxiv2025

Chemoenzymatically Synthesized O-Acetylated GD3 Gangliosides to Examine Viral Receptor Specificities in a Cellular Context

Gangliosides are a class of sialic acid-containing glycosphingolipids involved in a wide range of biological processes. The terminal sialic acid of gangliosides can be O-acetylated at C7 and/or C9 hydroxyl, contributing to ganglioside structural complexity and function. It has been difficult to obtain panels of structurally well-defined O-acetylated gangliosides for binding and functional studies. We describe here a chemoenzymatic strategy that can provide, for the first time, 7-O-, 9-O-, and 7,9-di-O-acetylated GD3 gangliosides. It is based on the chemical assembly of a common tetrasaccharide precursor as -glycosyl fluoride that is coupled to sphingosine by a glycosynthase, followed by O-acetyl editing by coronaviral hemagglutinin-esterases. The resulting synthetic glycosphingolipids have been employed for cell surface remodeling of erythrocytes. Analysis by liquid chromatography and ion mobility mass spectrometry (LC-IM-MS) demonstrated successful integration of the glycosphingolipids into the plasma membrane with preservation of acetyl ester patterns. Using human coronavirus HKU1 spike-functionalized virus-like particles, we demonstrate that the resulting glycan-remodeled erythrocytes can be utilized in hemagglutination studies as a label free method to investigate viral protein binding to individual glycoforms in a cellular environment. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/670503v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@37de01org.highwire.dtl.DTLVardef@1d043faorg.highwire.dtl.DTLVardef@1ee57borg.highwire.dtl.DTLVardef@6bf474_HPS_FORMAT_FIGEXP M_FIG Table of Contents 7-O-, 9-O-, and 7,9-di-O-acetylated GD3 gangliosides can be prepared by chemical assembly of a tetrasaccharide as -glycosyl fluoride that is coupled to sphingosine using a glycosynthase, followed by O-acetyl editing by coronaviral hemagglutinin-esterases. Cell surface remodeling of erythrocytes using the GD3 derivatives provide a tool to examine viral receptor specificities. C_FIG

biochemistry↗

bioRxiv2025

FlyTomo: A streamlined software for on-the-fly cryo-ET data processing and diagnosis

Cryo-electron tomography (Cryo-ET) combined with subtomogram averaging (STA) enables the structural elucidation of macromolecular assemblies in their native environments. However, the widespread adoption of cryo-ET has been limited by its labor-intensive, expertise-dependent data processing workflow. Here we present FlyTomo, a software that streamlines data processing from frame alignment to STA with high-throughput for authentic cryo-ET scenarios. During data acquisition, FlyTomo performs real-time diagnosis, enabling prompt feedback on sample quality, microscope performance and structural features. After acquisition, it aggregates diagnostic metrics into an interactive overview, guiding users through data review and refinement. FlyTomo also curates raw and processed data into structured directories to simplify data management and archiving. We validated FlyTomo across a diverse set of authentic cryo-ET samples, including purified enveloped viruses and cryo-lamellae, on multiple microscopes and cameras, achieving structures at resolutions ranging from 3.4 to 5.7 [A]. Collectively, by integrating accuracy, scalability and usability, FlyTomo reduces the technical barrier for in situ structural biology using cryo-ET.

biophysics↗

bioRxiv2025

LSH-mediated resolution of R-loops mitigates transcription-replication conflicts to preserve genomic stability in prostate cancer cells

Prostate cancer cells exhibit heightened transcriptional activity to sustain their aggressive phenotype, yet this creates DNA topological stress, leading to RNA: DNA hybrids (R-loops) and DNA damage that compromise genomic stability. LSH, an SNF2-family chromatin remodeler, is frequently dysregulated in cancers and linked to tumor progression, but its mechanistic role in genome maintenance remains unclear. Here, we demonstrate that LSH resolves R-loops in an ATP-dependent manner, mitigating transcription- replication (TR) conflicts in prostate cancer cells. LSH depletion caused R- loop accumulation due to reduced FANCD2 recruitment, exacerbating DNA damage and impairing Rad51 filament formation. Strikingly, LSH deficiency also increased R-loops at promoters of MYC and E2F target genes, stalling RNA polymerase II elongation and disrupting transcriptional programs critical for proliferation. Our findings reveal LSH is a key guardian of genome integrity, resolving R-loops to prevent TR conflicts and maintaining DNA repair fidelity. By linking LSH to R-loop resolution and transcriptional regulation, this study reveals its dual role in prostate cancer pathogenesis-- supporting malignant proliferation while safeguarding genomic stability. These insights nominate LSH as a potential therapeutic target to disrupt oncogenic transcription and induce synthetic lethality in prostate cancer.

cancer biology↗

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↗