Neuronal axons have traditionally been considered to be the primary mediators of functional connectivity among brain regions. However, the role of astrocyte-mediated communication has been largely underappreciated. Astrocytes communicate with one another through gap junctions, but the extent and specificity of this communication remain poorly understood. Astrocyte gap junctions are necessary for memory formation1,2, synaptic plasticity3-5, coordination of neuronal signalling6, and closing the visual and motor critical periods7,8. These findings indicate that this form of communication is essential for proper central nervous system development and function. Despite the importance of astrocyte gap junctional networks, studying them has been challenging. Current methods such as slice electrophysiology disrupt network connectivity and introduce artefacts due to tissue damage. Here, we developed a vector-based approach that labels molecules as they are fluxed by astrocyte gap junctions in awake, behaving animals to overcome these limitations. We then used whole-brain tissue clearing9,10 to image these intact, three-dimensional astrocyte networks. We show that multiple astrocyte networks traverse the mouse brain. These networks selectively connect specific regions, rather than diffusing indiscriminately, and vary in size and organization. We observe local networks that are confined to single brain regions and long-range networks that robustly interconnect multiple regions across hemispheres, often exhibiting patterns distinct from known neuronal networks. We also demonstrate that astrocyte networks undergo structural reorganization in the adult brain after sensory deprivation. These findings reveal a mode of communication between distant brain regions that is mediated by plastic networks of gap junction-coupled astrocytes.

Alternative lengthening of telomeres (ALT) is a specialized telomere extension mechanism associated with 5-10% of all cancers1. Although ALT has been linked to epigenetic dysregulation and genome instability, specific genomic and epigenetic rearrangements generated after ALT activation have not been identified. Here we report the insertion of centromeric α-satellite repeats and CENP-B boxes at telomeric locations specifically in ALT cancer cell lines and primary ALT paediatric neuroblastomas, indicating a pathological link for this alteration. Analysis using directed methylation with long-read sequencing (DiMeLo-seq) revealed discrete footprints of CENP-A chromatin assembled at telomeric locations on subsets of chromosomes. By modelling ALT activation, we show that epigenetic dysregulation due to ATRX loss and DNA hypomethylation facilitates the acquisition of these centromeric chromatin signatures. Functionally, interfering with HJURP-mediated CENP-A deposition compromises telomere integrity and ALT, leading to aberrant telomeric mitotic DNA synthesis (MiDAS). We propose that, while originally generated by illegitimate recombination, these centromeric signatures became integral by maintaining telomeric chromatin integrity in the unique context of ALT cancer cells.

The benign-to-malignant transition is a defining step in cancer progression. To investigate when and how malignancy initiation occurs and tissue reorganization proceeds, we combine single-cell and spatial transcriptomic profiling in mouse models of pancreatic ductal adenocarcinoma (PDAC) that capture spontaneous p53 loss. Among Kras-mutant cells, we find that oncogenic and tumor-suppressive programs, including those controlled by p53, CDKN2A, and SMAD4, are co-activated in a discrete progenitor-like population, engaging senescence-like responses. Using a framework we developed for spatial analysis, we show that a niche centered on these cells undergoes stepwise remodeling during tumor progression, mirroring invasive PDAC. Transient KRAS inhibition depletes progenitor-like cells and dismantles their niche, delaying malignancy initiation. Conversely, p53 suppression enables progenitor cell expansion, epithelial-mesenchymal reprogramming, and immune-privileged niche formation. These findings position the progenitor-like state at the convergence of cancer-driving mutations, plasticity, and tissue remodeling, revealing a critical window for intercepting malignancy.

Pediatric low-grade gliomas (pLGGs) are the most common type of brain tumors in children, characterized by their typically slow growth and oncogene-induced senescence. Preclinical models provide the opportunity to investigate the effects of various treatments in a controlled setting before they are tested in human patients; however, reliable models for pLGGs are limited. Here we developed two organoid models for pLGGs, which, after engraftment into mice, exhibited low-grade features. Furthermore, the genome-wide DNA methylation and RNA profiles of the organoids demonstrated closer similarity to low-grade glioma entities compared to high-grade counterparts. Additionally, pLGG organoid-derived cells align with oligodendrocyte-like, astrocyte-like and MAPK signature clusters seen in patient tumors, indicating that the organoids generate a heterogeneous population of cancer cells, where cellular diversity may influence disease progression and treatment response.

Sequence-based deep learning models have become the state of the art for analyzing the genomic regulatory code. Particularly for enhancers, these models excel at deciphering sequence grammar that underlies their activity. To enable end-to-end enhancer modeling and design, we developed a software package called CREsted (cis-regulatory element sequence training, explanation and design). It combines preprocessing and analysis of single-cell assay for transposase-accessible chromatin using sequencing data, modeling chromatin accessibility from sequence, sequence design and downstream analysis to decipher enhancer grammar. We demonstrate CREsted’s functionality on a mouse cortex and a human peripheral blood mononuclear cell dataset. Additionally, we use CREsted to compare mesenchymal-like cancer cell states between tumor types, and we investigate different fine-tuning strategies of genomic foundation models within CREsted. Finally, we train a model on a zebrafish development atlas and use this to design and in vivo validate cell-type-specific enhancers. For varying datasets, we demonstrate that CREsted facilitates efficient training and analyses, enabling scrutinization of the enhancer logic and design of synthetic enhancers across tissues and species.

Fluorescence microscopy is constrained by optical limits, fluorophore chemistry and finite photon budgets, imposing trade-offs between imaging speed, resolution and phototoxicity. Here we introduce Micro S plit , a deep learning-based computational multiplexing method that enables multiple cellular structures to be imaged simultaneously in a single fluorescent channel and then computationally unmixed. We show that M i c r o S p l i t separates up to four superimposed noisy structures into distinct, denoised image channels, enabling faster and more photon-efficient imaging. Built on Variational Splitting Encoder-Decoder networks, M i c r o S p l i t models a posterior distribution over solutions, allowing uncertainty-aware predictions and the estimation of spatially resolved prediction errors from posterior variability. We demonstrate robust performance across diverse datasets, noise levels and imaging conditions, and show that M i c r o S p l i t improves downstream analysis while reducing photon exposure. All methods, data and trained models are released as open resources, enabling immediate adoption of computational multiplexing in biological imaging.

Mitochondrial matrix Ca2+ concentration ([Ca2+]m) is theorized to be an essential regulator of mitochondrial metabolism by positively regulating key mitochondrial dehydrogenases. However, ablation or functional inhibition of the mitochondrial calcium uniporter channel (mtCU) fails to significantly perturb basal metabolism and is largely phenotypically silent in the absence of stress. Here we demonstrate that MICU proteins, the reported gatekeepers of mtCU, function in coordination to impart calcium-dependent regulation to FADH2-dependent mitochondrial dehydrogenases through metabolon formation independently of the mtCU and [Ca2+]m. Our results demonstrate that MICU proteins differentially localize to mitochondrial microdomains and form heterodimers and interactomes in response to intermembrane space Ca2+ binding their respective EF-hand domains. Using an equimolar expression platform coupled with unbiased proteomics, we reveal unique interactomes for MICU1/MICU2 versus MICU1/MICU3 heterodimers and demonstrate that MICU proteins control coupling of mitochondrial glycerol-3-phosphate dehydrogenase and succinate dehydrogenase/complex II and impart calcium-dependent changes in activity. We propose that MICU-mediated mitochondrial metabolons are a fundamental system facilitating matching of mitochondrial energy production with cellular demand and is the primary physiological calcium signaling mechanism regulating homeostatic energetics, not mtCU-dependent changes in [Ca2+]m.

The nucleus pulposus (NP), the core shock-absorbing component of the intervertebral disc (IVD), plays a vital role in the pathogenesis of intervertebral disc degeneration (IVDD). Here, we uncover that degenerative NP tissue under mechanical stress is characterized by upregulated PIEZO1 and accumulation of cell-free DNA (cfDNA). Specific inhibition or knockout of PIEZO1 suppresses the cfDNA-induced NP degeneration. Mechanistically, PIEZO1-mediated calcium overload triggers mitochondrial DNA (mtDNA) leakage, initiating a cascade that culminates in MAFB-dependent activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome pathway. Targeting the PIEZO1-cfDNA-MAFB-NLRP3 axis, we developed a hydrogel nano composite system (HA-F127@MCC950 SiNPs) containing amino-functionalized SiO2 nanoparticles (SiNPs) loaded with pyroptosis antagonist MCC950, embedded within hyaluronic acid (HA) and pluronic F127 (F127). The engineered hydrogel possesses dual effects in cfDNA scavenging and NLRP3 suppression in NP cells (NPCs). Moreover, this approach markedly attenuated NP degeneration progression in rat models of cfDNA-induced and temporary compression model (TCM)-induced IVDD. Collectively, this nucleic acid clearance strategy provides new insights for IVDD treatment.

Modulating how macrophages sense mechanical cues offers a novel strategy to control fibrosis around implanted biomaterials. We term this approach ‘mechano-immunotherapy’, which involves the desensitization of immune mechanosensory pathways to control the host response. Here, we use RN-1734 (RN), a model small molecule to demonstrate the proof-of-concept that pharmacologically disrupting macrophage mechanosensation can mitigate fibrosis. In vitro, RN reduced calcium influx and pro-inflammatory cytokine secretion in J774. a2 macrophages. These effects were strictly context-dependent with efficacy observed only in macrophages on high-stiffness (10% w/v) GelMA hydrogels, with no significant impact on those in softer (5% w/v) hydrogels. In vivo, RN selectively attenuated fibrotic capsule formation around implanted electrospun scaffolds but not smooth hydrogels. Notably, despite hydrogels releasing ∼6-fold more drug than scaffolds, fibrosis was reduced only in the scaffold group, suggesting that therapeutic efficacy is driven by the inhibition of high mechanosensory input rather than the loaded drug concentration alone. Spatial transcriptomics revealed that macrophages acted as the primary mechanosensors at the tissue-implant interface. Unsupervised global principal component analysis revealed that RN acted predominantly on early day 3 macrophages. The strongest effect was observed in surface-adhered mechanosensitive macrophages, where RN treatment enhanced their M2-like phenotype and promoted their dispersal from clustered aggregates into broader distribution within the scaffold. This redistribution was accompanied by a marked reduction in the recruitment of interstitial macrophages from the surrounding tissue, which were enriched for matrix-forming gene signatures. Together, these findings suggest that pharmacological desensitization of immune mechanosensors may represent a promising, context-specific approach to improve biomaterial integration.

In migrating cells, the GTPase Rac organizes a protrusive front, whereas Rho organizes a contractile back. How these GTPases are positioned at opposite poles remains unclear. We leverage optogenetics, mechanical perturbations, and mathematical modelling to reveal a surprising mechanochemical long-range mutual activation between front and back polarity programmes that complements their well-known local mutual inhibition. Rac-based protrusions elevate membrane tension, stimulating an mTORC2-dependent activation of Rho at the opposite side of the cell. Conversely, Rho-mediated contractility induces cortical-flow-based regulation of phosphoinositide signalling that triggers Rac activation distally. We develop a minimal mechanochemical model to explain how long-range facilitation, together with local inhibition, enables robust Rho and Rac partitioning. Our findings demonstrate how the actin cortex and plasma membrane interact as an integrated mechanochemical system for long-range Rac-Rho patterning. This circuit is required for efficient polarity and migration in primary human T cells and is conserved in epithelial cells, highlighting the generality of this mechanism.

ABSTRACT Near‐infrared (NIR) fluorescent heptamethine cyanine (HMC) dyes with intrinsic tumor‐targeting ability are promising probes for solid tumor detection. However, HMC dyes are typically amphiphilic molecules, leading to non‐specific binding to blood proteins, primarily albumin, following systemic administration. While albumin binding can extend blood circulation time and enhance tumor accumulation, it can also lead to significant healthy tissue uptake, resulting in low tumor‐to‐background signal ratios (TBR) and potential toxicity. Here, we report a molecular shielding strategy to prevent non‐specific protein binding of tumor‐targeting HMC dyes. We develop a rationally designed library of molecularly shielded NIR fluorescent probes based on a clinically approved HMC dye, indocyanine green (ICG). To shield non‐specific protein interactions of ICG, its sidechain is modified with varying numbers of charged amino acid residues, which transformed ICG into a low protein binding, renal‐clearable pan‐cancer targeting probe. These shielded ICG molecules showed strong tumor accumulation and optimal tissue clearance in a few days, enabling high‐contrast tumor imaging with TBR values reaching 8 and clear tumor margins in orthotopic rodent tumor models. These new tumor‐targeting NIR probes represent a clinically translatable platform for cancer detection and therapy with a modular molecular structure composed of non‐toxic, FDA‐approved components.

Abnormal mechanical stress is closely linked to intervertebral disc degeneration (IVDD). Iron homeostasis disorder occurs in various degenerative diseases, including IVDD. PIEZO1 serves as a mechanosensitive cation channel, involving in multiple physiological and pathological processes; however, its potential association with iron homeostasis and IVDD remain to be elucidated. Here, it is discovered that PIEZO1 accumulates in cartilage endplate (CEP) during IVDD, accompanied by intensive ferritinophagy. Specific activation of PIEZO1 or NCOA4 (nuclear receptor coactivator 4) aggravates CEP degeneration. Conversely, chondrocyte-specific knockout of Piezo1 mitigates CEP degeneration by restoring the labile iron pool and stabilizing the mitochondrial genome. Mechanistically, PIEZO1-mediated nuclear translocation of YAP1 (Yes1 associated transcriptional regulator) enhances NCOA4-dependent ferritinophagy by promoting extracellular Ca2+ influx under oxidative stress. Moreover, ferritinophagy induces in the accumulation and release of Z-form mitochondrial DNA (Z-mtDNA), resulting in the activation of ZBP1 (Z-DNA binding protein 1), ultimately leading to NFKB-dependent inflammatory cascade. Therapeutically, blocking PIEZO1-mediated calcium influx or suppressing YAP1 activation alleviates ferritinophagy. Additionally, Ncoa4 silencing attenuates Z-mtDNA-ZBP1-NFKB axis-driven IVDD. Collectively, our findings suggest that mechanical overload induces ferritinophagy-dependent CEP degeneration via PIEZO1 activation and subsequent upregulation of the Z-mtDNA-ZBP1-NFKB axis, which might furnish a therapeutic target for IVDD.Abbreviation: AAV: adeno-associated virus; ALP: alkaline phosphatase; ARS: alizarin red S; BV:TV: bone volume:total volume; CAMK2/CaMKII: calcium/calmodulin dependent protein kinase II; CEP: cartilage endplate; CEPCs: cartilage endplate chondrocytes; ChIP: chromatin immunoprecipitation; CKO: conditional knockout; CsA: cyclosporin A; Co-IP: co-immunoprecipitation; DHI: disc height index; ECM: extracellular matrix; EtBr: ethidium bromide; HIF: hypoxia inducible factor; IVDD: intervertebral disc degeneration; KD: knockdown; LAT: large tumor suppressor kinase; LSI: lumbar spine instability; MDA: malondialdehyde; Mito-ROS: mitochondrial reactive oxygen species; MRI: magnetic resonance imaging; mtDNA: mitochondrial DNA; NCOA4: nuclear receptor coactivator 4; PCBP: poly(rC) binding protein; ROS: reactive oxygen species; RT-qPCR: real-time quantitative reverse transcription; SOFG: safranin O and fast green; WWTR1/TAZ: WW domain containing transcription regulator 1; TEAD1: TEA domain transcription factor 1; TEM: transmission electron microscopy; YAP1: Yes1 associated transcriptional regulator; ZBP1: Z-DNA binding protein 1; Z-DNA: Z-form DNA; Z-mtDNA: Z-form mitochondrial DNA.

The fine balance between cellular homeostasis and stress response is crucial for cell survival under conditions of genotoxic stress. Here, we identify a regulatory role for the translation repressor Sbp1 in modulating autophagy during hydroxyurea (HU)-induced replication stress. We observe that Sbp1 localizes to reversible, mRNA-containing cytoplasmic granules specifically upon HU treatment in an RGG motif-dependent manner. Loss of Sbp1 leads to selective translational upregulation of key autophagy genes ATG1, ATG2, and ATG9. Consistent with these translational changes, sbp1∆ cells exhibit increased selective macroautophagy/autophagy and enhanced bulk autophagy, whereas Sbp1 overexpression suppresses both processes. Interestingly, overexpression of Sbp1 shifts DNA repair toward non-homologous end joining (NHEJ) repair, linking altered autophagy to genome maintenance. Together, these findings identify Sbp1 as a negative regulator of autophagy during replication stress and suggest a regulatory axis linking granule-mediated mRNA sequestration, translational control of autophagy factors, and the cellular response to genotoxic stress.Abbreviations: CHX: cycloheximide; CPT: camptothecin; DDR: DNA damage response; GTA: genotoxin-associated targeted autophagy; HR: homologous recombination; HU: hydroxyurea; MMS: methyl methanesulfonate; mRNPs: mRNA-protein complexes; NHEJ: non-homologous end joining; P-bodies: processing bodies; RBPs: RNA binding proteins.

Stress granules (SGs) are biomolecular condensates that form transiently in the cytosol of mammalian cells in response to stress. Dysregulation of their assembly or disassembly is implicated in human age-related diseases. While phase separation is the key process underlying SG assembly, understanding of their function, composition and regulation in response to physiological stimuli is limited. This knowledge gap reflects the challenge of gaining comprehensive and quantitative insights into the dynamic regulation of the complex composition of SGs at the single-cell level. Here we present an emulsion-based microfluidics method to overcome this limitation. “Cytosolic extracts-in-oil droplets” (CEODs) recreate a confined active cytosolic milieu that undergoes phase separation and SG formation in response to stress under physiological conditions. This approach led to the discovery of seven previously unrecognised SG components involved in signalling pathways. CEODs provide a versatile and cost-effective screening platform for future mechanistic and therapeutic studies.

Neural circuits are shaped by the diverse axonal branching patterns of neurons across different cell types. To map these patterns, here we introduce POINTseq (projections of interest by sequencing), a barcoded connectomics method for rapid, cell-type-specific mapping of thousands of single-cell projections per animal. POINTseq leverages viral pseudotyping and cell-type-specific infection to integrate MAPseq-style high-throughput barcoded projection mapping with the established viral-genetic neural circuit analysis toolbox. We validated POINTseq by mapping genetically and projection-defined cell populations in the mouse motor cortex. We then used POINTseq to reconstruct the brain-wide projections of 5,902 individual dopaminergic neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). These neurons fall into >25 connectomic cell types, vastly exceeding the known diversity of dopaminergic cells, and form stereotyped projection motifs that may mediate parallel dopamine signaling. These data constitute the anatomical substrate on which the diverse functions of dopamine in the brain are built.

Nanobodies (Nbs) are considered promising antibody fragments for overcoming limitations in precision oncology due to their high specificity and deep tissue penetration. However, their therapeutic potential remains limited by their rapid renal clearance. In this study, anti-HER2 Nb-loaded liposomes with dual functionalization combining polyethylene glycol 2000 (PEG) and cyclic cell-penetrating peptides are developed to ameliorate their pharmacokinetic behavior while retaining binding specificity. Liposomal formulations with high encapsulation efficiencies are produced with dual centrifugation. Biophysical characterization reveals that PEGylation effectively mitigates cCPP-induced membrane destabilization, ensuring structural integrity. In vitro assays confirm that, despite the steric shielding by PEG, the encapsulated Nbs retain their functionality and specific binding to HER2-overexpressing cells. In vivo studies in zebrafish larvae demonstrate excellent biocompatibility and lack of immunogenicity. Crucially, liposomal encapsulation significantly modulates the pharmacokinetic profile of Nbs in rats, reducing renal accumulation compared to free Nbs. This study presents a robust liposomal platform that successfully balances the stealth properties of PEG with the functional benefits of cCPPs. Consequently, this platform offers an effective strategy to enhance the therapeutic window of low-molecular-weight biologics.

Until recently, it was widely assumed that oxytocin signaling occurred exclusively through the activation of neuronal oxytocin receptors, with neurons being the primary targets of released oxytocin. However, this view was challenged by the discovery of functional oxytocin receptors in central amygdala astrocytes, which are essential for the proper function of local neuronal microcircuits. Since then, astrocytic oxytocin receptors have been implicated in various aspects of rodent physiology and behavior, yet it remains unclear whether this mechanism is region-specific or widespread across the brain. Here, we provide extensive anatomical data on oxytocin receptor expression in mice and rats, functionally validated through calcium imaging. Based on this mapping and using genetic, calcium imaging and behavioral approaches, we further demonstrate a critical role for oxytocin receptor-expressing astrocytes in the nucleus accumbens in social behavior. In summary, our findings demonstrate that oxytocin receptors are widely expressed in astrocytes across different brain regions. In the nucleus accumbens, these receptors modulate social behavior-an observation with significant implications for the current model of oxytocinergic modulation in the brain.

Acute myeloid leukemia (AML) is an aggressive leukemia with high rates of chemoresistance and relapse. Patients with AML undergoing induction chemotherapy often have delayed erythropoietic recovery and febrile neutropenia. Infection is a leading cause of mortality in this population. There is an unmet need to improve disease-specific outcomes in patients with AML undergoing cytotoxic chemotherapy. AML, at diagnosis, is characterized by increased levels of circulating iron due to erythroid block and cell death, which is further aggravated upon intensive chemotherapy. We hypothesized that iron, particularly toxic non-transferrin-bound iron (NTBI), can be redistributed away from AML cells and bacteria into nonmalignant transferrin receptor (CD71)-expressing cells by administering exogenous iron-free apotransferrin (apoTF). Using mouse models of AML, we show that mice treated with human apoTF had decreased NTBI and increased bone marrow erythropoiesis and B cell responses. ApoTF treatment resulted in normalization of bone marrow blood vessels and reduction of lipid peroxidation in endothelial cells. Crucially, apoTF combined with chemotherapy resulted in a reduction of AML cells and in improved survival, which was dependent on adaptive immunity. We established a murine model of Escherichia coli sepsis in leukemic mice receiving chemotherapy. We show that apoTF administration increased the survival of E. coli-infected mice. Mechanistically, apoTF treatment decreased the levels of circulating C-C motif chemokine ligand 2 (CCL2) and interleukin-6 through reduced expression of CCL2 in lipopolysaccharide-polarized macrophages. Our results demonstrate an overall benefit of iron redistribution induced by transferrin in combination with cytotoxic chemotherapy in AML.

DNA sliding clamps are central coordinators of genome replication and maintenance, yet the full binding network (“interactome”) of the bacterial β-clamp remains incompletely defined. Here, we report a novel interaction between Escherichia coli β-clamp and the helicase-nuclease RecBCD complex. Using bacterial two-hybrid assays and co-immunoprecipitation, supported by fluorescence microscopy, we show that RecB associates with β-clamp. Nuclear magnetic resonance spectroscopy maps the interaction to the canonical ligand pocket of β-clamp and identifies a clamp-binding motif in RecB (residues 1018-1023, QVEMEF), whose mutation abolishes binding. Functional assays indicate that this interaction occurs upon conformational switching of RecBCD at a Chi site, and disruption of the motif reduces survival after DNA damage. We also find indications of a second binding site in the helicase domain of RecB. These findings expand the β-clamp interactome and suggest a previously unappreciated role for β-clamp in DNA double-strand break repair, with potential implications for antibacterial strategies.

DNA-damaging antibiotics like ciprofloxacin (CIP) induce extensive double-strand breaks in Escherichia coli, triggering both the SOS response and rapid DNA supercompaction. To uncover genes involved in the latter process beyond the previously identified key orchestrators encoded by recN and recA, we developed a novel machine learning-assisted high-throughput screening workflow and applied it to nearly 4000 E. coli strains, including the Keio collection’s single-gene deletion strains and additional in-house strains. Conservative validation identified 15 hit strains with impaired DNA supercompaction. While defects in recombinational repair genes were associated with the most severe impairments, our investigation also revealed genes not previously associated with DNA compaction or repair that had milder and more heterogeneous effects on supercompaction, including yaiW, which encodes a membrane-associated protein. Notably, several non-DNA-repair gene deletions affected RecN colocalization with the nucleoid, recN expression, SOS response activity, or survival after CIP exposure, supporting indirect or modulatory roles. Altogether, this work confirms RecN and RecA as primary drivers of DNA supercompaction and demonstrates that high-content imaging combined with machine learning-assisted analysis provides a scalable approach to explore bacterial DNA organization phenotypes and DNA damage responses.