Rebuilding human naïve pluripotency from primed stem cells is essential for generating pre-implantation epiblast-like cells, a key source for regenerative medicine. Here we present a defined, feeder-free protocol that efficiently converts primed human pluripotent stem cells (hPSCs) to a naïve-like state within 12 days. Integrated ATAC-seq, RNA-seq and miRNA-seq analyses reveal a rapid chromatin rewiring marked by increased accessibility at OCT/KLF motifs, closure at ZIC/RFX/NFκB sites, upregulation of miR-372/373 and miR-182/183, and downregulation of miR-302 and miR-363/106 clusters. Functional assays demonstrate that the inflammatory transcription factor NFκB/RELA blocks naïve induction by activating peri-implantation barrier genes, including IKBKE and VEGFR1. Conversely, enforced miR-372/373 expression accelerates conversion by directly targeting RELA. These data delineate an NFκB/RELA axis that orchestrates the epigenetic switch to human naïve pluripotency, offering a tractable fra

Neural circuit development in the human cortex is considerably prolonged in comparison to non-human primates, a trait that contributes to the remarkable cognitive capacity of modern humans. Here, we explore the regulatory role of non-coding RNAs, which dramatically expanded during brain evolution, in synapse development of human induced pluripotent stem-cell derived neurons. We found that inhibition of a human-specific microRNA, miR-1229-3p, alters the trajectory of human neuronal maturation and enhances excitatory synaptic transmission. Transcriptome analysis following miR-1229 knockdown revealed a downregulation of mitochondrial DNA (mtDNA) encoded genes. We further show that miR-1229 regulates mitochondrial morphology, mtDNA abundance as well as mitophagy, and that stimulation of mitochondrial metabolism rescues decreased calcium buffering in miR-1229-3p depleted neurons. Accordingly, miR-1229 directly targets an entire network of genes involved in mitochondrial function and ER

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DNA methylation is a critical epigenetic mark across numerous species, and identifying differentially methylated regions (DMRs) is essential for understanding genome regulation. Most existing DMR detection methods require predefined sample conditions, limiting the discovery of new epigenetic patterns, especially when group identities are unknown or uncertain, as is common in clinical settings. Additionally, only a very few approaches enable comparisons across multiple conditions. To address this significant gap, we present metilene3, a method for rapid, multi-condition DMR detection that operates in both supervised and unsupervised modes, using user-provided labels or autonomously clustering unlabeled samples. By segmenting the genome based on multiple pairwise methylation difference signals, metilene3 enables sample classification and DMR-anchored inference of epigenetic relationships. Using simulated and diverse human datasets, we show that metilene3 accurately detects DMRs, robustly

High-sensitivity glycomic analysis is essential for advancing both basic and translational biomedical research, yet remains methodologically challenging for limited-quantity samples, primarily due to complex workflows and reliance on specialized instrumentation. Here, we introduce solution-enhanced glycan reduction and permethylation (seGRAP), a streamlined, accessible, and high-sensitive method that enables glycan profiling from sub-nanogram protein inputs and picoliter-scale human plasma using widely available mass spectrometry (MS) platforms. seGRAP-MS demonstrates consistently robust and reproducible glycomic performance and enables comprehensive N-glycome characterization of human oocytes at single-cell resolution. Our single-oocyte analysis uncovers a highly conserved N-glycome across both individuals and developmental stages, revealing a previously uncharacterized layer of molecular stability in human reproduction. By significantly lowering technical and logistical barriers, seG

Perturbations in lysosome integrity are tightly linked to neurological disorders and ageing, but the underlying pathogenic mechanisms are incompletely understood. Using an unbiased proteomic approach, we here identified the bridge-like lipid transport protein VPS13C/PARK23 as a key component of a global early response pathway to lysosome damage. VPS13C readily binds lysosomes under mechanical or osmotic tension in anticipation of membrane lesions. The latter trigger a conformational change in the protein’s C-terminus, involving its ATG2C domain acting as sensor of damage-induced lipid packing defects. We show that ER-lysosome contacts formed by VPS13C provide critical binding platforms for OSBP/ORPs to enable efficient ER wrapping of damaged lysosomes. A chemical approach to assess directional ER-to-lysosome lipid transport revealed that VPS13C is essential for large-scale lipid delivery to acutely damaged lysosomes to facilitate their repair. Our findings offer new mechanistic insight

Biobank-scale imaging provides an unprecedented opportunity to characterise how thousands of organ phenotypes vary in populations. However, deriving specific phenotypes from imaging data requires time-consuming expert annotation, limiting scalability. In this study, we develop a 3D diffusion autoencoder to derive latent phenotypes from temporally resolved cardiac MRI data of 71,017 UK Biobank participants. These phenotypes are reproducible, heritable (h2 = [4—18%]), and significantly associated with cardiometabolic traits. To establish the genetic basis of such traits, we perform a genome-wide association study, identifying 89 significant common variants (P < 2.3 × 10−9) across 42 loci, including seven novel loci. Extensive multi-trait colocalisation analyses (PP.H4 > 0.8) link variants across phenotypic scales, from intermediate cardiac traits to cardiac disease endpoints. In conclusion, this study showcases the use of diffusion autoencoding methods as p

Human (h)IgG2 monoclonal antibodies (mAbs) are potent agonists due, in part, to their ability to undergo disulfide shuffling within their hinge regions. Herein, we describe a rapid, sensitive, collision-induced unfolding (CIU) assay that possesses a predictive relationship between gas-phase protein unfolding and agonism in hIgG2 variants. Furthermore, our results highlight the significance of hinge engineering in tuning mAb structure-function relationships for the development of future biotherapeutics. The agonistic activity of IgG2 antibodies depends, in part, on the flexibility of their hinge region, which is slow to measure in full-length antibodies. Here, the authors develop a rapid mass spectrometry assay that gauges this flexibility and predicts the agonism of engineered antibody variants.

Understanding genome regulation is limited by the complexity of molecular interactions in living cells. Cell-free systems provide a simplified platform for studying gene expression, but low mRNA levels have prevented RNA-seq. To address this, we develop an active learning workflow combining Bayesian optimization with automated high-throughput experimentation to systematically explore over 1.6 million buffer compositions, experimentally testing 653. We identify a “mRNA-optimized” buffer (20-fold increase in mRNA yield) and a “trade-off” buffer (13-fold increase while maintaining protein production). Using direct RNA-seq, we profile the T7 phage transcriptome in cell-free systems and compare it with a purified T7-RNAP transcription system and phage-infected bacteria. This comparative analysis reveals distinct regulatory layers: the T7-RNAP system captures promoter-strength hierarchies but lacks RNA degradation, whereas cell-free systems provide an accurate estimation of in vivo expressio

RNAs serve versatile functional roles by virtue of their structures and dynamics. RNA computational models are typically tailored to either perform structural modeling or solve a specific class of folding problems. Here, we present PlanarFold, a coarse-grained RNA model that integrates molecular dynamics simulation in two-dimensional space with dynamic programming to explore the diverse dynamic behaviours of RNAs, achieving a speedup of more than four orders of magnitude compared with all-atom molecular dynamics models. We demonstrate that, at the secondary structure level, PlanarFold quantitatively reproduces experimental results across diverse scenarios, including the native secondary structures, thermodynamics and kinetics, mechanical properties, and co-transcriptional and de novo folding pathways. The conformational dynamics revealed by PlanarFold can provide mechanistic insight into how RNAs perform or lose functions, and offer potential targets for mutagenesis and therapeutics de

Addicted individuals prefer drug rewards over natural rewards. Dysregulated dopamine (DA) signaling in the nucleus accumbens is thought to trigger this phenomenon, but there is little supporting evidence. Using a genetically encoded sensor, we monitored DA dynamics in mice in an operant associative task with natural (fat solution) or artificial (cocaine or optogenetic DA self-stimulation) rewards. During learning, DA transients emerged at predictive cues for both reward types, vanished at natural reward delivery, but persisted at artificial reward delivery. In choice sessions, animals’ preferences varied from exclusive natural reward to exclusive artificial reward. Individual preferences were predicted by cue-evoked DA response magnitudes. Revaluation of reward contingencies shifted reward-type preference and DA signaling accordingly, but not in mice showing compulsive drug seeking, indicating impaired value updating in these mice. These results support a model in which DA drives adapt

Progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is accompanied by profound remodeling of the bone marrow microenvironment (BME), yet the contribution of its non-immune compartment remains unclear. Using single-cell RNA sequencing in genetically engineered mouse models that recapitulate disease evolution, we transcriptionally profile endothelial cells (EC) and mesenchymal stem cells (MSC). EC adopt a stress-associated program at MGUS that precedes angiogenesis in MM, while MSC undergo early and sustained loss of differentiation capacity. We identify a coordinated interferon (IFN)-driven program across EC and MSC that defines MM in the BIcγ1 model but is absent in the more aggressive MIcγ1 model. Treatment with bortezomib, lenalidomide, and dexamethasone suppresses this IFN signature, promotes endothelial adaptation, and restores osteogenic potential in MSC. Validation in patient samples reveals enrichment of this IFN-signature across d

Long-term survival in breast cancer is often limited by metastatic recurrence arising from disseminated cancer cells that persist in a dormant state. The mechanisms that enable these dormant cells to survive and subsequently reawaken remain incompletely understood. Here an unbiased genome-scale genetic screen identified Med4 as a cancer cell-intrinsic gatekeeper in metastatic reactivation. Correspondingly, MED4 haploinsufficiency was found to be prevalent in metastatic breast cancer and associated with poorer clinical outcomes. Syngeneic mouse metastasis models revealed that MED4 enforces metastatic dormancy. Mechanistically, and unexpectedly given the canonical role of the Mediator complex in transcriptional activation, MED4 suppresses enhancer priming (H3K4me1) and activation (H3K27ac). Loss of a single Med4 allele disrupts enhancer poise, leading to extracellular matrix remodelling and integrin-mediated mechanotransduction programmes that ultimately drive metastatic outgrowth. Toget

Epitranscriptomic regulation of cellular RNAs is a major mechanism of gene expression control in the brain. N6-Methyladenosine (m6A) is installed on thousands of mRNAs and non-coding RNAs, where it functions as a context-dependent regulator of RNA–protein interactions to control the amplitude and kinetics of gene expression. In the nervous system, m6A is critical for neurodevelopment, synaptic plasticity and adaptive responses to physiological stimuli, and its dysregulation has been linked to various brain disorders. In this Review, we present a comprehensive synthesis of how m6A is deposited, interpreted and dynamically regulated, and integrate recent advances to present a unified framework for its function in neural cells. We discuss how m6A coordinates RNA stability, translation, localization and chromatin-associated processes across developmental and adult contexts and how disruption of these pathways contributes to neurological disease. Finally, we explore challenges and future di

In meiosis, homologous recombination (HR) facilitates the halving of genomic content in diploid parents to produce haploid gametes, while also reshuffling genetic material. However, studies in yeast and mammals have suggested that HR during meiosis also makes the process mutagenic, and it has been proposed that the single-strand (ss) DNA formed by HR of programmed double-strand breaks (DSBs) contributes to the observed mutagenesis. To determine the full mutagenic potential of ssDNA formed during meiosis, we expressed human APOBEC3A (A3A), a deaminase that specifically attacks and therefore allows detection of ssDNA, in meiotic yeast cells. We demonstrate that meiotic cells accumulate long tracts of ssDNA manifested by A3A-mutation clusters, of up to 134 mutations spanning more than 25 kb. We show that the formation of mutation clusters during meiosis require Spo11-induced DSBs, and that break-induced replication and hyper-resection of DSBs are the primary mechanisms underlying the form

Mitochondrial diseases frequently affect the brain leading to severe and disabling neurological symptoms. The heteroplasmic m.3243 A > G mutation in MT-TL1, encoding mt-tRNALeu, is responsible for ~80% of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), which is one of the most characteristic mitochondrial syndromes, leading to disability and early death. There are no animal models harbouring this mutation to provide precise mechanistic insights informing therapeutic interventions. Here, we generate a human iPSC-derived cerebral organoid slice model that recapitulates cortical architecture and mitochondrial pathology. Using biological assays and single-cell RNA sequencing, we uncover heteroplasmy-dependent transcriptional shifts and changes in key cellular processes in cortical neurons. Organoids with high heteroplasmy show a predominant impairment of deep-layer neurons triggered by mitochondrial stress, leading to axonal degeneration and apoptosis

Mass spectrometry-based chemical proteomics enables unbiased assessment of ligand potency and selectivity across the proteome. However, current approaches remain limited by the low throughput of single-compound screening and reliance on pre-synthesized libraries. Here we devise a mechanism-driven “library-versus-proteome” platform that couples dynamic combinatorial libraries with activity-based protein profiling, enabling real-time selection and optimization of ligands in complex biological systems. This approach increases screening throughput by 10- to 20-fold, streamlines library generation, and adopts a “screen first, synthesize later” paradigm. Applying this platform, we discover covalent inhibitors of serine hydrolases including PPME1, ABHD11 and PNPLA6, and reveal uncharacterized roles of PNPLA6 in lipid metabolism and cancer cell proliferation. We further extend the strategy to cysteine-targeting ligands by designing tailored warheads, enabling proteome-wide EC50 profiling of ov

The unfolded protein response (UPR) is a critical adaptive program triggered upon cellular stresses that profoundly reshapes the transcriptome and translatome. In the very first minutes of cellular stress, translation blockage, RNA decay and RNA granules formation prompt the synthesis of proteins essential to the stress response. Due to the dynamic nature of these processes, investigating translation upon stress has proven to be challenging; therefore, our understanding of these mechanisms and translatome rewiring upon stress remains limited. Here, we exploit O-Propargyl-puromycin (OPP) labelling of de novo peptides followed by LC-MS/MS to identify de novo proteins translated upon endoplasmic reticulum (ER) stress. Combined with transcriptomic analyses, our approach reveals that ER stress profoundly impacts the synthesis of core splicing factor proteins leading to a significant reshaping of the splicing landscape. We identify a signature of seven splicing events consistently occurring

Wnt proteins are lipid-modified morphogens fundamental in development and disease. During Wnt biogenesis, the G-protein-coupled receptor (GPCR)-like transporter Wntless (WLS) escorts lipidated Wnts from the endoplasmic reticulum to the plasma membrane, then transfers them to extracellular carriers, forming active and soluble morphogen-carrier complexes. To dissect the mechanisms involved, we solve cryo-EM structures of Wnt-bound WLS and unliganded WLS, and perform structure-guided functional experiments. Wnts engage WLS via three conserved hairpins, which are all required for Wnt trafficking to the cell surface and carrier-mediated secretion. Wnt release from cells is driven by dramatic conformational changes in the WLS transmembrane domain, reminiscent of GPCR activation, together with WLS extracellular rearrangements. Unexpectedly, we find that Wnt5a bound to WLS forms dimers, with implications for Wnt signaling. These findings define the mechanism of WLS conformational cycling that

Serine/threonine phosphoprotein phosphatases (PPPs) are conserved metalloenzymes and key regulators of intracellular signaling. Here, we systematically map the components and residues required for PP2A-like phosphatase (PP2A/PP4/PP6) function using genome-wide CRISPR knockout and focused base editing screens and provide this as a comprehensive resource. We uncover the reductase CYB5R4 as an evolutionarily conserved activator of PP4 and PP6, but not PP2A. We demonstrate that PP4 and PP6 are redox sensitive and require CYB5R4 to reduce the active site metal ions for activation. Mechanistically, this involves the selective binding of PP4 and PP6 through binding elements in the N-terminal tail of CYB5R4 and CYB5R4-bound heme serving as an electron donor for the active site. We further show that this activation is critical for regulating the response to DNA damage. Our work hints that active site metal ion redox regulation mediates crosstalk between the oxidative state of the cell and speci

A detailed, spatially resolved quantitative map of the human proteome is essential for a deeper understanding of human biology and disease1–4. Here we present a comprehensive human proteomic landscape, generated by profiling more than 13,000 proteins across 2,856 samples using data-independent acquisition mass spectrometry. The dataset spans 58 major tissue types, 251 specific tissue subtypes and 25 distinct carcinomas. This resource enables the depiction of spatially resolved proteome trajectories across tissue types and physiological states, including fetal, tumour, adjacent non-tumour and healthy adult tissue, thereby providing insight into both developmental processes and oncogenic progression. Furthermore, quantitative proteomics comparisons across diverse tissue types and states facilitate the indication of organ-specific toxicity, the identification of repurposable anticancer drug candidates and the prioritization of therapeutic targets for cancers. This study establishes a quan