National Science Review

The axon initial segment (AIS) undergoes structural plasticity and refines neuronal excitability, yet the underlying mechanisms remain unclear. We here developed an in vivo CRISPR/Cas9 knockout platform using an all-in-one triple-guide RNA vector introduced via electroporation and employed this approach to seek molecules that regulate the developmental shortening of AIS in the chicken nucleus magnocellularis. We have targeted fourteen molecules associated with microtubules and found that knockouts of glycogen synthase kinase 3{beta} (GSK3{beta}) and Tau disabled the AIS shortening. Conversely, overexpression of constitutively active form of GSK3{beta} facilitated the AIS shortening in vivo. This extensive shortening was replicated in slice cultures, which was occluded by stabilization of microtubules. These results suggested that microtubule remodeling by GSK3{beta} activity contributed to the AIS shortening. This study thus provides a genetic approach suitable for genetic screening that allows identifying regulators of the AIS plasticity in the chicken brain.

Deep-sea corals are vital in maintaining coral ecosystem biodiversity, yet their genetic characteristics remain largely unexplored. Here, we present 11 deep-sea coral genome assemblies, including four Hexacorallia and seven Octocorallia species, significantly contributing new genomic information across two orders. Our analysis reveals the historical dynamics of coral speciation and the influence of environmental factors on the evolution of coral reef ecosystems.Total of 126 horizontal gene transfer (HGT) events were detected, among which genes from the ancestor of symbiodiniaceae indicate that the ancestors of deep-sea corals may have inhabited shallow-sea environments. Notably, several of these HGTs are involved in phosphorus (PhnX/PhnW) and cholesterol (DHCR7) metabolisms within corals, indicating that HGTs may serve as an adaptive survival strategy for the coral holobionts. Deep-sea corals also rely on symbiotic bacteria to synthesize 10 essential amino acids (such as valine and tyrosine), retaining only partial amino acid synthesis capacity. In addition, we investigated the evolution of key biological rhythm genes and temperature adaptation in corals. The loss of key rhythm genes (e.g., clock and cry) in deep-sea corals and copy number difference of genes related to heat stress (e.g., Cbl-b and Rchy) revealed genetic difference between deep-sea and shallow-sea corals. Our new genome assemblies enhance the understanding of deep-sea coral evolution, biodiversity, and adaptation, providing a genetic foundation for coral conservation.

The bodies of pterosaurs, the first flying vertebrates, are covered with integumentary filaments (pycnofibres) thought to be homologous to feathers in dinosaurs, but their coloration remains unknown. Here, we report a layered internal arrangement of melanosomes containing a photonic nanostructure within the monofilaments in a previously undescribed specimen of tapejarid pterosaur Sinopterus dongi from the Early Cretaceous Jehol Biota. Optical simulations showed that this structure reflects green to magenta iridescent coloration, confirming the presence of melanosome-based iridescent coloration previously thought to be unique to birds. This finding deepens our understanding of structure/color gamut relationships in amniotes, while supporting further shared characteristics associated with derived genetic and regulatory shifts in archosaurs.

Current treatment of IDH-wildtype glioblastoma (GBM) relies on the first-line chemotherapy-temozolomide. Although MGMT methylation is routinely conducted to predict chemosensitivity, its efficacy is often compromised. Thus, there is an urgent need to discover more accurate prognostic biomarkers. Cholesteryl ester (CE) has been recently recognized as a key feature of GBM, however, its role in GBM prognosis remains poorly understood. We first employed label-free stimulated Raman scattering (SRS) imaging to quantitatively analyze CE level in intact tumor tissues obtained from IDH-wildtype GBM patients. Our result revealed significantly prolonged 2-year overall survival (OS) in patients with CE level [&ge;] 40% compared to those with CE level < 40%. CE outperformed MGMT methylation for 2-year OS prognosis (AUC: 0.836 vs. 0.763). Importantly, CE also achieved superior prognostic performance over MGMT methylation on an independent cohort, with higher sensitivity (0.856 vs. 0.667), specificity (0.833 vs. 0.583), NPV (1.00 vs. 0.667), PPV (0.833 vs. 0.583). Given synergistic effects between CE and MGMT methylation, we developed a prognostic model combining these two biomarkers. Specially, machine learning (XGBoost) model exhibited optimal performance in the training cohort (AUC: 0.920), and maintained its superior performance on the independent cohort (sensitivity: 0.946, specificity: 0.873, NPV: 1.00; PPV: 0.917). Mechanistically, integrative analysis of TCGA database linked poor prognosis to the coordinated upregulation of genes involved in cholesterol efflux, hydrolysis, transport, and inhibition of de novo synthesis, unraveling a possible underlying mechanism between poor prognosis and cholesterol metabolism. This work identified CE as a prognostic biomarker for IDH-wildtype GBM.

Connexin36 (Cx36) is broadly expressed in neurons and serves as the principal protein that forms interneuronal gap junctions (GJs), also known as electrical synapses. Recent high-resolution structures of human Cx36 GJ have revealed crucial electrostatic interactions (ESIs) of charged residues between two docked Cx36 hemichannels at the second extracellular (E2) loops. Despite their structural importance, the mechanistic roles of these ESIs remain poorly understood. To investigate their significance, we systematically designed and tested a series of missense variants targeting key E2 interface residues, aiming to disrupt or modulate the electrostatic landscape at the docking interface. Based on the ESI pairs defined from the crystal structure, our combined computational calculations and dual patch-clamp experiments in engineered HEK293 cell pairs suggest that at least three ESI residual pairs per E2-E2 interface are required to support functional GJ formation. Furthermore, we found that these unique ESIs of Cx36 could play a role in its docking specificity to itself, as they rarely form heterotypic GJs with other brain connexins. Overall, these findings provide essential molecular and functional insights into the mechanisms governing Cx36 GJ formation and partner specificity, paving the way for future therapeutic approaches targeting connexin dysfunction in human diseases.

Head and neck squamous cell carcinoma (HNSCC) ranks as the seventh most common cancer, with increasing incidence and mortality rates and limited therapeutic progress. The heterohexameric prefoldin complex, a highly conserved co-chaperone assembly composed of six PFDN subunits, exhibits expression levels strongly correlated with cancer progression. Among these subunits, the PFDN5 gene presents a paradoxical role in cancer biology, demonstrating both tumor-promoting and tumor-suppressive activities. Notably, the PFDN5 gene generates two distinct protein isoforms through alternative splicing, yet their individual contributions to cancer remain unexplored. In this study, we reveal that an elevated short-to-long PFDN5 alternative splice variants ratio is significantly associated with improved overall survival in HNSCC patients. Using proximity-dependent biotin identification (BioID), we mapped shared and isoform-specific protein-protein interaction networks for PFDN5. Our analysis uncovered novel proximal interactors, implicating PFDN5 isoforms in unexpected functions, including spindle organization, transcriptional complexes, and NF-{kappa}B signaling. These results provide a foundation for exploring PFDN5 isoforms as potential therapeutic targets in HNSCC.

The Caenorhabditis elegans AWC olfactory neuron pair specifies asymmetric subtypes, AWCOFF and AWCON, through stochastic and coordinated cell signaling events. UNC-104/kinesin-3 (KIF1A) and UNC-116/kinesin-1 motor proteins act in the AWCON cell to regulate the synaptic localization of the TIR-1/SARM1-assembled calcium signaling complex in the AWCOFF cell to promote AWCOFF. However, the molecular mechanism in the AWCON cell that acts non-cell autonomously to control synaptic TIR-1 calcium signaling to promote AWCOFF remains unclear. Here, we show that JIP-1, a conserved c-Jun N-terminal kinase (JNK)-interacting protein 1, mediates the synaptic localization of TIR-1 in the AWC axon to specify the AWCOFF subtype. A jip-1 loss-of-function mutant, identified from an unbiased forward genetic screen, has reduced localization of TIR-1 at synapses in the AWC axon and accumulation of TIR-1 in the AWC cell body. jip-1 mutants significantly enhance the 2AWCON phenotype of a hypomorphic tir-1 mutant. JIP-1, like UNC-104 and UNC-116, mainly acts non-cell autonomously in AWCON to specify the AWCOFF subtype. Our findings provide mechanistic insights into how cell-specific Ca2+ signaling proteins, such as TIR-1, target synaptic regions via intercellular signaling to promote neuronal diversification.

PUF60 is a splicing factor related to the polypyrimidine-tract binding protein U2AF2. PUF60 is deleted in developmental disorders such as Verheij syndrome and amplified in approximately 8% of cancers. Thus, both increases and decreases in PUF60 expression can have profound physiological effects. However, little is known about how changes in PUF60 expression impact global splicing patterns. Here, we created a model system of CRISPRa/i in mouse stem cells (mESCs) to transcriptionally upregulate or downregulate Puf60. Our results uncovered extensive transcriptional, post-transcriptional, and post-translational regulation of Puf60 protein expression. We observed that Puf60 protein levels in normal mESCs drop dramatically at a critical cell density, leading to cell death. Puf60 is very essential in stem cells, and its repression causes cell death and impacts specific splicing events, including its own splicing autoregulation, providing valuable insights into the functional consequences of PUF60 dysregulation. Analysis of phosphoprotein data revealed phosphorylation of threonine at the N-terminus of PUF60. Our results showed that mutating threonine to glutamate downregulates the protein and alters its localization. Thus, our study reveals a novel regulatory mechanism of Puf60 phosphorylation that mediates its function and may be related to its frequent overexpression in cancer cells.

Consciousness recovery from general anesthesia represents a fundamental brain state transition. The absence of objective, continuous metrics to monitor the shift from unconsciousness to full awareness has left its dynamic, multidimensional nature unresolved, fracturing consciousness theoretical framework and posing clinical risks due to unreliable assessment of emergence. To address this, we developed a multiscale framework integrating AI-driven behavioral analysis, continuous neuro-physiology recording, and whole-brain functional imaging. This decodes recovery into three hierarchical stages, reflex restitution, level restoration, and content re-establishment, each with unique multimodal signatures. We identified heart rate stabilization as a potential noninvasive biomarker for the restoration of consciousness level. Crucially, the centromedian nucleus of thalamus acts as a dynamic hub, actively orchestrating staged whole-brain reconfiguration via stage-dependent network routing. These findings resolve the temporal orchestration of consciousness recovery, ground disparate theories within a unified mechanistic sequence, and open transformative paths for real-time monitoring and neuromodulation in anesthesiology. HighlightsA multimodal framework decodes consciousness recovery into three hierarchical stages Recovery stages span from reflex restitution, to level restoration, and finally to content re-establishment Heart rate stabilization serves as a noninvasive biomarker for consciousness level restoration The CM paces this staged reconfiguration as a dynamic network hub In BriefZhang et al. develop a multiscale framework that delineates consciousness recovery from anesthesia as a three-stage process, identifies heart rate stabilization as a biomarker for the restoration of consciousness level, and reveals the CM as a dynamic hub orchestrating this progression. These findings clarify the temporal orchestration of consciousness recovery, pave the way for real-time monitoring and neuromodulation in anesthesiology and other fields.

The transcription factor FOXP2 is the most well-known language-related gene in humans, yet its role in primate vocalization remains poorly understood. Here we report that knockdown of FOXP2 in the striatum markedly disrupts vocalization stability in the marmoset monkey, a valuable non-human primate model for studying vocal behavior. FOXP2 exhibited high expression in the marmoset striatum, especially during early development. Using the CRISPR-Cas12 system, we achieved specific in vivo editing of the FOXP2 gene and effective knockdown of FOXP2 protein expression in the marmoset striatum. Two neonatal marmosets received bilateral striatal injections of the gene-editing and control virus, respectively, and were raised together in the same family. In three such marmoset pairs, analysis of vocalizations recorded during 6-15 weeks post-injection revealed that striatal FOXP2 knockdown significantly altered vocal features and increased intra-individual variability in phee syllables--the most common marmoset vocalization, often produced repetitively as multi-syllable phee calls. Notably, in FOXP2-edited marmosets, acoustic alterations were minimal in the first syllable of phee calls but became progressively more pronounced in subsequent syllables, which exhibited a marked upward shift in the frequency spectrum over time with progressively steeper slopes. These temporal dynamics in vocal features reflect a reduction in the stability of continuous vocal production. In line with the known striatal functions in motor control, our findings provide the first evidence of FOXP2 in controlling vocalization in non-human primates, thereby opening new avenues for investigating the neural mechanisms underlying FOXP2 function.

Cerebellar neural circuit dynamics rely on a rich repertoire of synaptic and excitability mechanisms, which are thought to determine network computation in physiological and pathological conditions. In this work, we develop and validate a biologically-grounded spiking neural network of the cerebellar cortex, embedding key mechanisms of cellular excitability and synaptic transmission, and assess their impact on signal processing. Neuronal input-output functions, short-term synaptic plasticity, receptor-specific kinetics, and NMDA channel voltage-dependent gating were calibrated against detailed multicompartmental models through automatic tuning procedures. Incorporating these realistic biological properties allowed the network model to simulate key features observed in recordings from acute cerebellar slices. The neuronal discharge and local field potentials elicited by mossy fiber stimulation faithfully reproduced the natural patterns with millisecond precision. Then, selective receptor switch-off revealed the contribution of NMDA, GABA, and AMPA receptors to the frequency-dependent input-output function of the granular layer and Purkinje cells, linking previous empirical findings to specific synaptic mechanisms. This model combines high computational performance with biological realism and offers a computationally efficient framework to investigate neurophysiological phenomena and the neural correlates of behavior in large-scale long-lasting simulations, such as those needed to address the neural underpinnings of learning and of cerebellar pathologies.

The developmental program governing meibomian gland (MG) morphogenesis and proliferation remains poorly understood, largely due to the lack of physiologically relevant model systems. Here, we established a novel high-fidelity, three-dimensional organoids model derived from mouse meibomian gland (mMGO) epithelium. Transcriptomic and phenotypic analyses demonstrated that mMGOs faithfully recapitulate postnatal gland development in vivo, including dynamic transcription program, branching morphogenesis, lineage differentiation, and functional lipid accumulation. Leveraging this model, we identified the Hippo-YAP pathway as a pivotal regulator of MG epithelial proliferation and homeostasis for the first time. YAP inhibition severely impaired organoids growth, while pharmacological inhibition of Hippo pathway with XMU-MP-1 enhanced proliferation and progenitor clonogenicity. Crucially, in inflammation-induced atrophic organoids, XMU-MP-1 treatment rescued YAP nuclear localization and stimulated regrowth and functional restoration. Our study provided new mechanistic insights and a robust organoids platform for MG development research, and nominated targeted Hippo pathway inhibition as a promising strategy to reverse glandular atrophy in meibomian gland dysfunction.

Accurate prediction of drug synergy is paramount for developing effective combination therapies and advancing personalized medicine. Although methods based on graph neural networks (GNNs) have become a prevalent approach, they often treat molecules as flat graphs of connected atoms, thus overlooking their inherent hierarchical structure (i.e., atoms forming functional groups) and the critical topological information that governs molecular interactions. To address this limitation, we introduce TopoFuseNet, a novel hierarchical graph representation learning framework that integrates multi-scale topological features. The core innovations of TopoFuseNet include: 1) The first-ever application of "Group Centrality" from network science to cheminformatics, enabling the identification and quantification of functional groups crucial to drug activity; 2) A systematic, multi- path strategy to seamlessly integrate node-level (atom) and group-level (functional group) topological features into a Graph Attention Network (GAT) via feature augmentation, attention biasing, and hierarchical pooling; 3) A Differential Transformer module to deeply fuse multi-modal features learned from sequences, fingerprints, and our proposed hierarchical graph representations. Extensive experiments on two large-scale benchmark datasets, DrugComb and DrugCombDB, demonstrate that TopoFuseNet significantly outperforms state-of-the-art methods across multiple key metrics, including AUC, AUPRC, and F1-score, while exhibiting exceptional generalization robustness under various stringent cold-start scenarios. In-depth ablation studies further confirm the effectiveness and necessity of each proposed innovative module. Furthermore, multi-scale interpretability analysis and zero-shot cross-domain transfer experiments reveal that the model successfully captures molecular interaction rules with clear pharmacological significance, demonstrating immense practical potential for discovering novel combination therapies through large-scale virtual screening. Our work not only delivers a superior model for drug synergy prediction, but more importantly, it establishes a novel and scalable paradigm for effectively integrating hierarchical molecular structures and topological information into GNNs.

Personalized T cell therapy empowered by chimeric antigen receptor (CAR) that recognizes specific tumor antigen has cured numerous blood cancer patients since its initial approval in 2017. However, its access to a broader population has been limited by the unavailability of an off-the-shelf product derived from an allogeneic donor that can evade immune rejection, which is mediated by polymorphic class I and class II human leukocyte antigens (HLAs). Since class II HLAs are only expressed in specialized antigen-presenting cells but not T cells, it might suffice to evade T cells by deleting the common class I HLA light chain Beta-2 Microglobulin (B2M) (1). However, B2M-deficient cells can trigger a "missing-self" response to activate natural killer (NK) cells (2), a second function that was evolved to compensate loss of T cell response. Inserting a less polymorphic class I HLA gene encoding a known NK inhibitory ligand, namely HLA-E or HLA-G (3), into the B2M locus so that the endogenous B2M expression is disrupted could theoretically allow evasion of both T and NK cells. Despite being a seemingly better candidate in that HLA-G is uniquely expressed in immune-privileged sites such as the placenta with a believed function in protecting the fetus from immune rejection by the pregnant mother, whereas ubiquitously-expressing HLA-E is known to bind both inhibitory and activating NK receptors (4, 5), only HLA-E engineering has been attempted yet without convincing success in vivo (6, 7). Here, we generate an off-the-shelf CAR-T product with B2M replaced by a gene fusion encoding an HLA-G single-chain trimer under minimally impacted B2M epigenetic landscape, and observe its immune evasion property and a tumor-inhibitory function that is equivalent to its autologous control using a humanized mouse model for the first time with T and NK cells reconstituted from a donor with a distant HLA haplotype. HLA-G engineering may thus reprogram T cells into an immune-privileged state that can be utilized for all cell-based therapies.

Artificial intelligence has reached a pivotal threshold. Multimodal large models can approach human-level speech comprehension by rapidly transforming sound into meaning. However, whether this process relies on human-like mechanisms remains unknown. Here, we compared the human brain with twelve speech language models (SLMs) using a phonology-semantics confusion paradigm. Stereo-electroencephalography revealed two mechanisms of phonology-to-semantics (P2S) transfer in the human brain: a local sequential transformation within specific neuronal populations, and a global cross-regional hierarchy of P2S representations. Only brain-model alignment in the local sequential manner predicted model performance. Correspondingly, targeted lesioning of local sequential P2S-transfer model units markedly impaired comprehension performance, while activation steering of these units improved performance. In addition, such local sequential P2S-transfer model units were identified across languages. Together, this study establishes local sequential P2S transformation as a fundamental computational principle shared across biological and artificial intelligence, offering a mechanistic bridge for future brain-inspired speech systems.

Clot resistance to pharmacological thrombolysis remains a critical challenge in ischemic stroke (IS) management. Thrombus heterogeneity, particularly the presence of thrombolysis-resistant domains composed of dense fibrin and non-fibrin components, including neutrophil extracellular traps (NETs), significantly limits the efficacy of recombinant tissue-type plasminogen activator (r-tPA) and its variant, Tenecteplase (TNK). Consequently, novel therapeutic strategies are urgently required. Emerging evidence suggests that co-administration of deoxyribonuclease I (DNase I) with r-tPA can degrade DNA fibers and enhance clot lysis. In this study, we optimized a previously developed theranostic agent--iron oxide microparticles coated with polydopamine--by dual-grafting both r-tPA and DNase to target resistant thrombi. Using functional ultrasound imaging (fUS) during the acute phase of IS, we demonstrated accelerated reperfusion with this dual-functionalized platform in a r-tPA resistant IS model. Furthermore, MRI analysis confirmed a significant reduction in lesion volume at 24 hours, correlating with improved functional recovery five days post-ischemia.

Virtual screening has become an indispensable tool in modern structure-based drug discovery, enabling the identification of candidate molecules by computationally evaluating their potential to bind target proteins. The accuracy of such screenings critically depends on the quality of the target structures employed. Recent advances in protein structure prediction, particularly AlphaFold2, have revolutionized this field with unprecedented accuracy. However, AlphaFold2 models often exhibit limitations in local structural details, especially within binding pockets, which limit their utility for small molecule docking. In contrast, molecular dynamics simulations with accurate atomistic force fields can refine protein structures, but lack the ability to leverage the structural information provided by deep learning approaches. Here, we introduce bAIes, an integrative method that bridges this gap by combining physics-based force fields with data-driven predictions through Bayesian inference. Crucially, bAIes demonstrates a superior ability to discriminate between binders and non-binders in virtual screening campaigns, outperforming both AlphaFold2 and molecular dynamics-refined models. By enhancing the usability of AlphaFold2 models without requiring extensive experimental or computational resources, bAIes offers a convenient solution to a longstanding challenge in structure-based drug design, potentially accelerating the early phases of drug discovery.

Improving the reconstituted translation system is a key requirement for bottom-up synthetic biology. Here, we developed a two-step in vitro evolutionary method that can be used for improving translational proteins. In this method, two distinct conditions were sequentially applied while maintaining genotype-phenotype linkage in water-in-oil droplets. Using this method, we performed in vitro evolution of four translation factors, IleRS, PheRS, EF-G, and EF-Tu, and identified mutations that modestly enhanced translation activity in in vitro expression assays. One of the EF-G mutations (P610S) increased activity per protein approximately 2-fold for the recombinant protein purified from E. coli. This selection method is useful for improving translational proteins for bottom-up synthetic biology.

Major depressive disorder (MDD) seriously affects human physical and mental health and causes global socioeconomic burdens. Stress-induced depressive etiology is linked to dendritic atrophy in the hippocampus, however, the underlying mechanism was poorly understood. Here, we identify that glucuronyl C5-epimerase (Glce), a heparan sulfae-modifying enzyme, as a critical regulator of hippocampal dendritic integrity and stress resilience. Genetic ablation of Glce in the hippocampal excitatory neurons is sufficient to induce dendritic atrophy and depressive-like behaviors, whereas its restoration rescues these deficits. Notably, Glce levels are reduced in the plasma of depressed individuals. We further find that Glce functions independently of its enzymatic activity, instead binding directly to and activating PI3K (p85/p110), thereby triggering an Akt/CREB/BDNF signaling cascade. Together, our findings uncover a non-canonical role for Glce and establish that Glce-PI3K-BDNF axis is essential for maintaining hippocampal structure and behavioral resilience, thereby highlighting a critical role of Glce in the pathophysiology of depression.

Bifidobacteriaceae and Lactobacillaceae are key probiotic families and widely used in food production, yet a comprehensive understanding of strain functions and their gut microbial interactions based on complete genomes remain understudied. Here we constructed a complete-genome dataset of 3,300 strains from these two families, including 1,151 newly isolated from China. Compared with draft assemblies, complete genomes substantially recovered a gene functional landscape encompassing stress tolerance, surface exopolysaccharide synthesis, nutrient utilization, and mobile genetic elements. Major species from both families exhibited a prevalence >60% in the Chinese population, far higher than that in US/Dutch cohorts. Notably, as a core probiotic species with remarkable genomic plasticity and gut-adaptive potential, Lactiplantibacillus plantarum stood out in our dataset for its enriched functional profile and was particularly abundant in the Chinese population. Moreover, compared with non-Chinese genomes, our isolates of key species displayed less metabolic complementarity and stronger competition with potentially pathogenic keystone species in the gut, thereby linking strain origin to enhanced probiotic potential and ecological fitness to benefit human gut health.