The 2018-2020 Ebola virus disease (EVD) epidemic facilitated the emergence of viral mutations, enhancing the potential for host adaptation during sustained human transmission. Here, we identified the Ebola virus (EBOV) glycoprotein V75A (GP-V75A) substitution as a dominant variant during the epidemic. This substitution, located within the receptor-binding domain, emerged early in the outbreak and rapidly reached high prevalence. GP-V75A demonstrated enhanced infectivity in multiple cell lines and murine models. Mechanistically, GP-V75A increased viral GP binding affinity to the host receptor Niemann-Pick C1 (NPC1) and reduced the dependency on endosomal cysteine proteases for entry. Notably, GP-V75A also significantly reduced the efficacy of NPC1-targeting compounds and neutralizing antibodies. Epidemiological analysis indicated that the rise in GP-V75A prevalence coincided with the increase in case number during the outbreak. These findings provide crucial insights into the evolutionary adaptation of EBOV during large-scale outbreaks and underscore the importance of real-time genomic surveillance for improving epidemic preparedness.

In response to perturbed transcription elongation, the MYC oncoprotein multimerizes and undergoes a phase transition. Here, we demonstrate that MYC globally relocalizes from its canonical positions on DNA to nascent RNA upon accumulation of intronic RNA. Upon binding to RNA, MYC forms multimers that concentrate the nuclear exosome, an RNA exonuclease, and its targeting complexes around double-stranded RNA and R-loops. MYC harbors four RNA-binding regions (RBRI-IV). RBRIII promotes MYC multimerization and is necessary for recruiting the exosome to R-loops. RBRIII is dispensable for transcriptional activation and pancreatic tumor cell proliferation in culture, but it is indispensable for sustaining tumor growth in vivo. Via RBRIII, MYC suppresses the accumulation of R-loop-derived RNA-DNA hybrids and prevents them from activating the innate immune kinase TBK1 via the TLR3 pattern recognition receptor. Our data demonstrate that the phase transition of MYC is an RNA-driven stress response that suppresses the accumulation of immunogenic RNA-DNA hybrids.

Mutations in lysosomal genes cause neurodegeneration and neuronopathic lysosomal storage disorders (LSDs). Despite their essential role in brain homeostasis, the cell-type-specific composition and function of lysosomes remain poorly understood. Here, we report a quantitative protein atlas of lysosomes from mouse neurons, astrocytes, oligodendrocytes, and microglia. We identify dozens of proteins not previously annotated as lysosomal and reveal the diversity of lysosomal composition across brain cell types. Notably, we identified SLC45A1, a gene whose mutations cause a monogenic neurological disease, as a neuron-specific lysosomal protein. Loss of SLC45A1 causes lysosomal dysfunction in vitro and in vivo. SLC45A1 functions as a lysosomal sugar transporter and impacts the stability of the V1 subunits of the vacuolar ATPase (V-ATPase). Consistently, SLC45A1 loss reduces lysosomal V1 subunits, elevates lysosomal pH, and disrupts iron homeostasis, causing mitochondrial dysfunction. Altogether, our work redefines SLC45A1-associated disease as an LSD and establishes a comprehensive map to study lysosome biology at cell-type resolution.

The ability to design and engineer mammalian genomes across arbitrary length scales would transform biology and medicine. Such capabilities would enable the systematic dissection of mechanisms governing gene regulation and the influence of complex haplotypes on human traits and disease. They would also facilitate the engineering of disease models that more faithfully recapitulate human physiology and of next-generation cell therapies harboring sophisticated genetic circuits. Over the past decade, advances in genome editing have made small, targeted modifications at single sites routine. However, achieving multiple coordinated alterations across long sequence windows (>10 kb) or installing large synthetic DNA segments in mammalian cells remains a major challenge. Recent advances in mammalian genome writing-the bottom-up design, assembly, and targeted integration of large custom DNA sequences, independent of any natural template-offer a potential solution. Here, we review key technological developments, highlight emerging applications, and discuss current bottlenecks and strategies for overcoming them.

Essential for eukaryotes, multiple copies of the exocyst complex tether each secretory vesicle to the plasma membrane (PM) in constitutive exocytosis. The exocyst higher-order structure (ExHOS) that coordinates the action of these multiple exocysts remains unexplored. We integrated particle tracking, super-resolution microscopy, and cryo-electron tomography to time-resolve the continuum conformational landscape of the ExHOS and to functionally annotate its different conformations. We found that 7 exocysts form a flexible ring-shaped ExHOS that tethers vesicles at <45 nm from the PM. The ExHOS rapidly expands while pulling the vesicle toward the PM in a stepwise mechanism comprising three metastable states at 27, 18, and 5 nm from the PM. After fusion, Sec18 mediates the disassembly of the stationary ExHOS, a function that controls the rate of exocytosis. By resolving biophysical principles in situ, we reconstructed the spatiotemporal dynamics of the multimeric architecture controlling vesicle tethering in exocytosis.

The rapid accessibility of calvarial immune cells to the brain, in principle, may offer transformative opportunities for overcoming drug delivery barriers in central nervous system (CNS) disorders. Here, we hijacked calvarial immune cells using drug-loaded nanoparticles (NPs) and leveraged their unique migration mechanism through skull-meninges microchannels to bypass the blood-brain barrier (BBB) for CNS drug delivery. We constructed NP-loaded immune cells in situ via intracalvariosseous (ICO) injection, validated their prompt migration in response to CNS perturbation, and targeted therapeutic delivery to CNS lesions. Compared with conventional delivery approaches, this strategy achieved promising therapeutic efficacy in improving both short- and long-term outcomes in preclinical stroke models. Our prospective clinical trial further supports the translational feasibility of ICO immune access in treating malignant stroke. These findings establish skull-based delivery as a promising, clinically translatable route for CNS drug delivery and highlight immune-assisted transport as a potentially transformative strategy for improving therapeutic outcomes in neurological disorders.

The efficacy of B cell depletion therapies, and their association with Epstein-Barr virus (EBV), implicate B cells in the pathogenesis of multiple sclerosis (MS). In mice, we observed that viral infections induce infiltration of B cells into the brain, independent of phenotype and specificity, and that myelin-reactive B cells then capture antigens directly from parenchyma. Trafficking of these antigen-loaded B cells to draining lymph nodes was not observed, and without T cell help, antigen-capturing B cells die rapidly. CD40L signaling or EBV latent membrane protein 1 (LMP1) can override this checkpoint, leading to B cell-receptor- and/or antibody-dependent inflammatory demyelination. Myelin-reactive B cells were identified in the healthy human B cell repertoire, and expression of LMP1 was observed in the brains of a subset of MS patients. These observations can explain the dependency of disease incidence on prior EBV infection, and the increased risk associated with brain infections, and suggest possible treatment strategies.

Epstein-Barr virus (EBV) is involved in causing and probably also in perpetuating multiple sclerosis (MS). Among several mechanisms of how EBV may contribute are transcriptome alterations, including changes of antigen processing and preferential presentation of both viral and self-antigens. Here, we report that EBV reprograms the transcriptome and immunopeptidome presented on the MS-associated human leukocyte antigen (HLA)-DR15 molecules of infected B cells. Identical myelin basic protein (MBP) peptides were found to be presented on both EBV-infected B cells and MS brain tissue but not primary B cells and thymic tissue. Peripheral memory and cerebrospinal fluid (CSF)-derived CD4+ T cells of HLA-DR15+ MS patients responded to MBP peptides, MBP(78-90) and/or MBP(83-90), and T cell clones raised with these peptides recognized all MBP peptides ending at amino acid MBP90 in MS brain tissue. Our study provides a new mechanistic link for how the environmental and genetic risk factors, EBV infection and HLA-DR15 haplotype, may contribute jointly to MS.

Epstein-Barr virus (EBV) infection constitutes a prerequisite for multiple sclerosis (MS) development, and cross-reactivity between EBV nuclear antigen 1 (EBNA1) and anoctamin-2 (ANO2) antibodies was previously demonstrated in persons with MS (pwMS). Here, we show that ANO2-specific CD4+ T cells are more frequent in pwMS. Immunization of SJL/J mice with ANO2 or EBNA1 led to cross-reactive CD4+ T cell and antibody responses. ANO2 pre-immunization led to exacerbated experimental autoimmune encephalomyelitis (EAE), an effect mediated by CD4+ T cells, as confirmed by adoptive transfer experiments. T cell clones with cross-reactivity to EBNA1 and ANO2 could be isolated from natalizumab-treated pwMS, and sequencing of EBNA1- and ANO2-specific T cell receptors (TCRs) revealed a significant repertoire overlap. We thus report the first mechanistic evidence that EBNA1 CD4+ T cells can target the MS autoantigen ANO2, thereby establishing a link between EBV infection and neuroinflammation.

The guanosine triphosphate (GTP)-bound state of the heterodimeric Rag GTPases functions as a molecular switch regulating mechanistic target of rapamycin complex 1 (mTORC1) activation at the lysosome downstream of amino acid fluctuations. Under low amino acid conditions, GTPase-activating protein (GAP) activity toward Rags 1 (GATOR1) promotes RagA GTP hydrolysis, preventing mTORC1 activation. KICSTOR recruits and regulates GATOR1 at the lysosome by undefined mechanisms. Here, we resolve the KICSTOR-GATOR1 structure, revealing a striking ∼60-nm crescent-shaped assembly. GATOR1 anchors to KICSTOR via an extensive interface, and mutations that disrupt this interaction impair mTORC1 regulation. The S-adenosylmethionine sensor SAMTOR binds KICSTOR in a manner incompatible with metabolite binding, providing structural insight into methionine sensing via SAMTOR-KICSTOR association. We discover that KICSTOR and GATOR1 form a dimeric supercomplex. This assembly restricts GATOR1 to an orientation that favors the low-affinity active GAP mode of Rag GTPase engagement while sterically restricting access to the high-affinity inhibitory mode, consistent with a model of an active lysosomal GATOR1 docking complex.

Extrachromosomal DNA (ecDNA) amplifications are key drivers of human cancers. Here, we show that ecDNAs are major platforms for generating and amplifying oncogene fusion transcripts across diverse cancer types. By integrating analysis of whole-genome and transcriptome sequences from tumor samples and cancer cell lines of a wide variety of tissue types, we reveal that ecDNAs have the highest rate of oncogene fusion events of any copy-number alteration. Focusing on the most common ecDNA fusion hotspot, we find that fusion of the 5' end of the long noncoding RNA gene, PVT1-with exon 1 joined to diverse 3' partners-confers increased RNA stability, potentially via an SRSF1-dependent mechanism, and enhances MYC-dependent transcription and cancer cell survival. These results demonstrate that ecDNA fosters genome instability and frequent oncogene fusion formation in cancer.

The evolutionary history of angiosperms (flowering plants) is characterized by a highly accelerated rate of diversification. Although a number of hypotheses have been proposed to explain the ecological success and rapid rise of angiosperms, the molecular mechanisms underlying their speciation are only partially understood. In this study, we analyzed the developmental transcriptomes of seven angiosperm species spanning 160 million years of evolution. We demonstrate that angiosperm protein-coding gene expression patterns diverged rapidly. Particularly high rates of expression changes were found for genes that are involved in the response to endogenous and environmental stimuli, promoting broad ecological tolerances, adaptive evolution, and speciation. This study highlights how ecological and evolutionary dynamics are highly interrelated, shows that the rates of expression divergence differ from those previously described for mammals, and provides a comprehensive resource to perform cross-kingdom comparative studies of transcriptome evolution.

Homeostasis and repair in the nervous system are thought to rely on distinct molecular programs. Here, we uncover an unexpected role for the pentose phosphate pathway (PPP) in peripheral sensory axons, where it supports both homeostatic mechanosensation and axonal regeneration after injury. We show that the PPP is enriched and active in sciatic nerve axoplasms, where it maintains redox balance via NADPH production, enabling physiological mechanical sensation. However, following sciatic nerve injury, the PPP is required for regeneration by fueling ribonucleotide synthesis through ribose-5-phosphate. In contrast, this pathway remains inactive after spinal cord injury (SCI), contributing to regenerative failure. Reactivation of the PPP, through neuronal transketolase overexpression or oral ribose supplementation, promotes metabolic reprogramming, restores sensory and motor axonal growth, and improves neurological recovery after SCI. These findings propose the PPP as a metabolic checkpoint in sensory neuron physiology and regeneration, highlighting its therapeutic potential for central nervous system repair.

Herpesviruses are widespread double-stranded DNA viruses that establish lifelong latency and cause various diseases. Although DNA-polymerase-targeting antivirals are effective, increasing drug resistance underscores the need for alternatives. Helicase-primase inhibitors (HPIs) are promising antivirals, but their mechanisms of action are poorly defined. Furthermore, how the helicase-primase (H/P) complex and DNA polymerase coordinate genome replication is not well understood for herpesviruses. Here, we report cryo-electron microscopy (cryo-EM) structures of the herpes simplex virus 1 H/P complex bound to HPIs, showing that these lock the H/P complex in an inactive state. Single-molecule assays reveal that HPIs cause H/P complexes to pause in unwinding activity on DNA. The structure of an HPI-bound replication fork complex, comprising the H/P complex (UL5, UL52, and UL8) and the polymerase holoenzyme (UL30 and UL42), reveals a previously uncharacterized interface bridging these complexes. These findings provide a structural framework for understanding herpesvirus replisome assembly and advancing inhibitor development.

A typical neuron signals to downstream cells when it is depolarized and fires sodium spikes. Some neurons, however, also fire calcium spikes when hyperpolarized. The function of such bidirectional signaling remains unclear in most circuits. Here, we show how a neuron class that participates in vector computation in the fly central complex employs hyperpolarization-elicited calcium spikes to invert two-dimensional mathematical vectors. By switching from firing sodium to calcium spikes, these neurons implement a ∼180° realignment between the vector encoded in the neuronal population and the fly's internal compass signal, thus inverting the vector. We show that calcium spikes rely on the T-type calcium channel Ca-α1T and argue via analytical and experimental approaches that these spikes enable vector computations in portions of angular space that would otherwise be inaccessible. These results reveal a seamless interaction between molecular, cellular, and circuit properties for implementing vector mathematics in the brain.

Mechanisms of adaptation of regulatory T cells (Tregs) to harsh tumor metabolic microenvironments for suppression of anti-tumor immunity remain largely unclear. Here, using spatial metabolomics and transcriptomics, we show that human hepatocellular carcinoma harbored metabolically heterogeneous subregions characterized by high glutaminolysis and ammonia contents, where Tregs were frequently present but CD8+ and CD4+ effector T cells die. We found Tregs used the urea cycle to detoxify ammonia by upregulating argininosuccinate lyase (ASL); meanwhile, ammonia was also converted to spermine by the FOXP3 transcription factor regulated spermine synthase (SMS). A direct interaction between spermine and PPARγ was verified by X-ray crystallography, leading to comprehensively modulating the transcription of multiple mitochondrial complex proteins to enhance oxidative phosphorylation and immunosuppression of Tregs. Clinically, anti-PD-1-treated dying tumor cells used transdeamination to release ammonia, which reinforced Treg function, leading to immunotherapeutic resistance. Targeting ammonia production to suppress Tregs presents a potential strategy for anti-tumor immunotherapy.

Prescription stimulants (e.g., methylphenidate) are thought to improve attention, but evidence from prior fMRI studies is conflicted. We utilized resting-state fMRI data from the Adolescent Brain Cognitive Development Study (n = 11,875; 8-11 years old) and validated the functional connectivity findings in a precision imaging drug trial with highly sampled (n = 5, 165-210 min each) healthy adults (methylphenidate 40 mg). Stimulant-related connectivity differences in sensorimotor regions matched fMRI patterns of daytime arousal, sleeping longer at night, and norepinephrine transporter expression. Taking stimulants reversed the effects of sleep deprivation on connectivity and school grades. Connectivity was also changed in salience and parietal memory networks, which are important for dopamine-mediated, reward-motivated learning, but not the brain's attention systems (e.g., dorsal attention network). The combined noradrenergic and dopaminergic effects of stimulants may drive brain organization towards a more wakeful and rewarded configuration, improving task effort and persistence without effects on attention networks.

A marked evolution in Alzheimer's disease (AD) therapy research is ongoing. In this perspective, we highlight emerging outcomes of tau-targeting approaches with disease-modifying potential evidenced by PET-based slowing of tau accumulation and early signs of cognitive benefit. We outline how decades of iterative amyloid β (Aβ)-trial refinement leading to the recent successes of approved anti-Aβ therapies have set the stage for accelerated optimization of next-generation trials. We summarize key learnings from first-generation tau immunotherapies and how these paved the way for early achievements in tau trials, while many challenges remain. Finally, we discuss the back-translation of clinical outcomes into fundamental insights on human tau pathobiology, and we outline challenges and future directions for AD therapy development including combination therapy and targets beyond Aβ/tau. Together, this provides a framework for next-generation AD and tau-therapy development toward increasingly efficient disease-halting interventions.

Implantation of a human embryo into the endometrium is a crucial event in gestation, as it marks the initiation of a pregnancy and is prone to high failure rates. We have limited understanding of these stages because of the inaccessibility of implanting embryos and the lack of suitable model systems. Here, we establish an in vitro model that recapitulates the luminal, glandular, and stromal compartments of the superficial layer of receptive human endometrium. Human embryos and blastoids implant into the endometrial model, achieving post-implantation hallmarks including advanced trophoblast structures that underlie early events in placental development. Single-cell RNA sequencing of the embryo-endometrial interface at day 14 uncovers predicted molecular interactions between conceptus and endometrium. Disrupting signaling interactions between extravillous trophoblast and endometrial stromal cells caused defects in trophoblast outgrowth, demonstrating the importance of crosstalk processes to sustain embryogenesis. This platform opens the opportunity to investigate early stages of human embryo implantation.