Systemic gene therapy using adeno-associated virus (AAV) vectors is approved for the treatment of several genetic disorders, but challenges and toxicities associated with high vector doses remain. We report an alternate receptor for AAV (AAVR2, carboxypeptidase D [CPD]), which is distinct from the multi-serotype AAV receptor (AAVR). AAVR2 enables the transduction of clade E AAVs, including AAV8, and determines an exclusive AAVR-independent transduction pathway for AAV11 and AAV12. We characterized direct binding between the AAV8 capsid and AAVR2 by cryo-electron microscopy (cryo-EM) and identified contact residues. We observed that AAV8 directly binds to the carboxypeptidase-like domain 1 of AAVR2 via its variable region VIII and demonstrated that AAV capsids that lack AAVR2 binding can be bioengineered to engage with AAVR2. Finally, we overexpressed a minimal functional AAVR2 to enhance AAV transduction in vivo. Our study provides insights into AAV biology and clinically deployable solutions to reduce dose-related toxicities associated with AAV vectors.

The integrated stress response (ISR) is a conserved stress response that maintains homeostasis in eukaryotic cells. Modulating the ISR holds therapeutic potential for diseases including viral infection, cancer, and neurodegeneration, but few known compounds can do so without toxicity. Here, we present an optogenetic platform for the discovery of compounds that selectively modulate the ISR. Optogenetic clustering of PKR induces ISR-mediated cell death, enabling the high-throughput screening of 370,830 compounds. We identify compounds that potentiate cell death without cytotoxicity across diverse cell types and stressors. Mechanistic studies reveal that these compounds upregulate activating transcription factor 4 (ATF4), sensitizing cells to stress and apoptosis, and identify GCN2 as a molecular target. Additionally, these compounds exhibit antiviral activity, and one compound reduced viral titers in a mouse model of herpesvirus infection. Structure-activity and toxicology studies highlight opportunities to optimize therapeutic efficacy. This work demonstrates an optogenetic approach to drug discovery and introduces ISR potentiators with therapeutic potential.

Multicellular coordination enhances biological complexity, yet the widely used yeast Saccharomyces cerevisiae possesses limited multicellular capabilities. Here, we expand the possibilities for engineering multicellular behaviors in yeast by developing modular toolkits for two key mechanisms in multicellularity, contact-dependent signaling and specific cell-cell adhesion. MARS (mating-peptide anchored response system) enables contact-dependent signaling via surface-displayed peptides and G protein-coupled receptors, mimicking juxtacrine communication, while Saccharomyces SATURN (adhesion toolkit for multicellular patterning) uses adhesion-protein pairs for the creation of programmable cell aggregation patterns. Combining these allows the construction of multicellular logic circuits, equivalent to developmental programs that lead to cell differentiation based on local population. We further created JUPITER (juxtacrine sensor for protein-protein interaction), a genetic sensor based on MARS and SATURN, for assaying protein-protein interactions and selecting high-affinity nanobody binders. Collectively, these toolkits present versatile building blocks for constructing complex, user-defined multicellular yeast systems and expand the scope of its biotechnological applications.

Personalized neoantigen-targeting vaccines have demonstrated great promise; however, improved immunogenicity is still needed. Since antigen availability and effective T cell priming are critical for maximal immunogenicity, we tested a synthetic long peptide vaccine formulated with Montanide, poly-ICLC, and locally administered ipilimumab in addition to systemic nivolumab in 10 patients with melanoma. These personalized vaccines generated de novo ex vivo T cell responses against the majority of immunizing neoepitopes in all 9 fully vaccinated patients and ex vivo CD8+ T cell responses in 6 of 9. Vaccination induced hundreds of circulating and intratumoral T cell receptor (TCR) clonotypes that were distinct from those arising after PD-1 inhibition. By linking the vaccine neoantigen specificity of T cell clonotypes with single-cell phenotypes in tumors, we demonstrate remodeling of the intratumoral T cell repertoire following vaccination. These observations show that multi-pronged immune adjuvanticity can boost T cell responses to neoantigen-targeting vaccines.

Genetic targeting methods for monitoring and manipulating neuronal activity are not widely used for studying the primate brain, largely owing to the lack of a cell-type-specific targeting method. Using single-cell RNA and ATAC sequencing of macaque brains combined with in vivo screening, we identified a large set of enhancers capable of driving targeted gene expression in specific cell types. AAV vectors driven by these enhancers successfully targeted layer-specific glutamatergic neurons, GABAergic interneuron subtypes, astrocytes, and oligodendrocytes with high specificity. Cross-species comparison revealed that some macaque enhancers are conserved and functional across species, but enhancers with layer-specific targeting in macaques did not label neurons in mice, highlighting evolutionary differences in cortical CREs. Targeting precision was further improved using a FLPo-dependent intersectional approach with two enhancers. These enhancer-AAVs were validated by monitoring and manipulating activity in macaque visual cortex, providing valuable tools to dissect primate neural circuit functions.

This study investigated preventative and therapeutic agents against human T cell lymphotropic virus type-1 subtype-C (HTLV-1c) infection. We established and characterized a humanized mouse model of HTLV-1c infection and identified that HTLV-1c disease appears slightly more aggressive than the prevalent HTLV-1 subtype-A (HTLV-1a), which may underpin increased risk for infection-associated pulmonary complications in HTLV-1c. Combination antiretroviral therapy with tenofovir and dolutegravir at clinically relevant doses significantly reduced HTLV-1c transmission and disease progression in vivo. Single-cell RNA sequencing (scRNA-seq) and intracellular flow cytometry identified that HTLV-1c infection leads to dysregulated intrinsic apoptosis in infected cells in vivo. Pharmacological inhibition using BH3 mimetic compounds against MCL-1, but not BCL-2, BCL-XL, or BCL-w, killed HTLV-1c-infected cells in vitro and in vivo and significantly delayed disease progression when combined with tenofovir and dolutegravir in mice. Our data suggest that combination antiretroviral therapy with MCL-1 antagonism may represent an effective, clinically relevant, and potentially curative strategy against HTLV-1c.

In contrast to the rapid advancements in mesoscale connectomic mapping of the mammalian brain, similar mapping of the peripheral nervous system has remained challenging due to the body size and complexity. Here, we present a high-speed blockface volumetric imaging system with an optimized workflow of whole-body clearing, capable of imaging the entire adult mouse at micrometer resolution within 40 h. Three-dimensional reconstruction of individual spinal fibers in Thy1-EGFP mice reveals distinct morphological features of sensory and motor projections along the ventral and dorsal rami. Immunostaining facilitates body-wide mapping of sympathetic nerves and their branches, highlighting their perivascular patterns in limb muscles, bones, and most visceral organs. Viral tracing elucidates the fine architecture of vagus nerves and individual vagal fibers, revealing unexpected projection routes to various organs. Our approach offers an effective means to achieve a holistic understanding of cellular-level interactions among different systems that underlie body physiology and disease.

Cortical expansion endows advanced cognitive functions in primates, and whole-brain single-neuron projection analysis helps to elucidate underlying neural circuit mechanisms. Here, we reconstructed 2,231 single-neuron projectomes for the macaque prefrontal cortex (PFC) and identified 32 projectome-based subtypes of intra-telencephalic, pyramidal-tract, and cortico-thalamic neurons. Each subtype exhibited distinct topography in their soma distribution within the PFC, a characteristic pattern of axon targeting, and subregion-specific patchy terminal arborization in the targeted area, with putative functions annotated. Furthermore, we identified a subdomain connectivity network and extensive local axons within the PFC. Compared with those in mice, macaque PFC projectomes exhibited a similar topographic gradient of terminal arborization at the targeted regions but much higher target specificity, fewer collaterals, and smaller brain size-normalized arbors. Thus, whole-brain single-axon macaque projectomes revealed highly refined axon targeting and arborization, providing key insights into the structural basis for complex brain functions in primates.

Rapid advancements in artificial intelligence (AI), particularly large language models (LLMs) and multimodal AI, are transforming medicine through enhancements in diagnostics, patient interaction, and medical forecasting. LLMs enable conversational interfaces, simplify medical reports, and assist clinicians with decision making. Multimodal AI integrates diverse data like images and genetic data for superior performance in pathology and medical screening. AI-driven tools promise proactive, personalized healthcare through continuous monitoring and multiscale forecasting. However, challenges like bias, privacy, regulatory hurdles, and integration into healthcare systems must be addressed for widespread clinical adoption.

In a matter of years, single-cell omics has matured from a pioneering technique employed by just a handful of specialized laboratories to become a ubiquitous feature of biological research and a key driver of scientific discovery. The widespread adoption and development of single-cell omic assays has sparked mounting enthusiasm that these technologies are poised to also enhance the precision of diagnosis, the monitoring of disease progression, and the personalization of therapeutic strategies. Despite initial forays into clinical settings, however, single-cell technologies are not yet routinely used to inform medical or surgical decision-making. Here, we identify and categorize key experimental, computational, and conceptual barriers that currently hinder the clinical deployment of single-cell omics. We focus on the potential for single-cell transcriptomics to guide clinical decision-making through the development of combinatorial biomarkers that simultaneously quantify multiple cell-type-specific pathophysiological processes. We articulate a framework to identify patient subpopulations that stand to benefit from such biomarkers, and we outline the experimental and computational requirements to derive reproducible and actionable clinical readouts from single-cell omics.

In this issue of Cell, Yang and colleagues demonstrate that autocrine activation of serotonin receptors on tumor-infiltrating CD8+ T cells enhances antitumor immunity. Modulating serotonin signaling may provide a new approach to therapy for cancer. Serotonin-targeting drugs such as SSRIs and others, developed to fight depression, may thus be repurposed for cancer immunotherapy.

In this issue of Cell, Harris et al. reveal that high-molecular-weight soluble tau-rather than amyloid-beta-impairs burst firing in hippocampal neurons, providing a mechanistic link to cognitive decline in Alzheimer's disease. This disruption, linked to CaV2.3 downregulation, highlights soluble tau as a key driver of neuronal dysfunction and a promising therapeutic target.

Advances in brain mapping technologies have made substantial progress in identifying diverse cell types and their connectivity. Focusing on papers recently contributed by the Mesoscopic Brain Mapping Consortium to Cell, Neuron, and Developmental Cell, this commentary discusses insights into brain organization, development, evolution, and diseases, as well as future research directions.

Exercise improves immune checkpoint inhibitor (ICI) efficacy in cancers such as melanoma; however, the mechanisms through which exercise mediates this antitumor effect remain obscure. Here, we identify that the gut microbiota plays a critical role in how exercise improves ICI efficacy in preclinical melanoma. Our study demonstrates that exercise stimulates microbial one-carbon metabolism, increasing levels of the metabolite formate, which subsequently enhances cytotoxic CD8 T cell (Tc1)-mediated ICI efficacy. We further establish that microbiota-derived formate is both sufficient and required to enhance Tc1 cell fate in vitro and promote tumor antigen-specific Tc1 immunity in vivo. Mechanistically, we identify the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) as a crucial mediator of formate-driven Tc1 function enhancement in vitro and a key player in the exercise-mediated antitumor effect in vivo. Finally, we uncover human microbiota-derived formate as a potential biomarker of enhanced Tc1-mediated antitumor immunity, supporting its functional role in melanoma suppression.

The regulatory sequences of vertebrate genomes remain incompletely understood. To address this, we developed an ultra-throughput, ultra-sensitive single-nucleus assay for transposase-accessible chromatin using sequencing (UUATAC-seq) protocol that enables the construction of chromatin accessibility landscapes for one species in a 1-day experiment. Using UUATAC-seq, we mapped candidate cis-regulatory elements (cCREs) across five representative vertebrate species. Our analysis revealed that genome size differences across species influence the number but not the size of cCREs. We introduced Nvwa cis-regulatory element (NvwaCE), a mega-task deep-learning model designed to interpret cis-regulatory grammar and predict cCRE landscapes directly from genomic sequences with high precision. NvwaCE demonstrated that regulatory grammar is more conserved than nucleotide sequences and that this grammar organizes cCREs into distinct functional modules. Moreover, NvwaCE accurately predicted the effects of synthetic mutations on lineage-specific cCRE function, aligning with causal quantitative trait loci (QTLs) and genome editing results. Together, our study provides a valuable resource for decoding the vertebrate regulatory language.

The measles virus (MeV), a highly contagious non-segmented negative-sense RNA virus in the Paramyxoviridae family, causes millions of infections annually, with no approved antivirals available. The viral polymerase complex, comprising the large (L) protein and the tetrameric phosphoprotein (P), is a key antiviral target. We determined the cryo-electron microscopy structures of the MeV polymerase complex alone and bound to two non-nucleoside inhibitors, ERDRP-0519 and AS-136A. Inhibitor binding induces a conformational change in the catalytic loop, allosterically locking the polymerase in an inactive "GDN-out" state. These findings led to the proposal that ERDRP-0519 would also be effective against Nipah virus (NiV), a highly pathogenic virus with no available antivirals. This proposal was confirmed by structure determination of the NiV polymerase complex and by inhibition of transcription.

Protein engineering enables artificial protein evolution through iterative sequence changes, but current methods often suffer from low success rates and limited cost effectiveness. Here, we present AI-informed constraints for protein engineering (AiCE), an approach that facilitates efficient protein evolution using generic protein inverse folding models, reducing dependence on human heuristics and task-specific models. By sampling sequences from inverse folding models and integrating structural and evolutionary constraints, AiCE identifies high-fitness single and multi-mutations. We applied AiCE to eight protein engineering tasks, including deaminases, a nuclear localization sequence, nucleases, and a reverse transcriptase, spanning proteins from tens to thousands of residues, with success rates of 11%-88%. We also developed base editors for precision medicine and agriculture, including enABE8e (5-bp window), enSdd6-CBE (1.3-fold improved fidelity), and enDdd1-DdCBE (up to 14.3-fold enhanced mitochondrial activity). These results demonstrate that AiCE is a versatile, user-friendly mutation-design method that outperforms conventional approaches in efficiency, scalability, and generalizability.

Synonymous mutations, once known as "silent" mutations, are increasingly attracting the interest of biologists. Although they may affect transcriptional or post-transcriptional processes, their impact on biological traits remains under-investigated, particularly at the organismal level. Here, we identified two closely linked, epistatically interacting genes: YTH1, an RNA N6-methyladenosine (m6A) reader, and ACS2, an aminocyclopropane-1-carboxylic acid (ACC) synthase, which contribute to cucumber fruit length domestication. The causative mutation in ACS2 is a synonymous substitution at 1287C>T. In wild cucumber, ACS21287C results in m6A modification on nearby adenosine residues and the formation of loose RNA structural conformations. YTH1 recognizes the m6A modification, alters the folding equilibrium toward the weakest RNA structural conformation, and increases the ACS2 protein level, resulting in shorter fruit. In cultivated cucumber, ACS21287T disrupts m6A methylation and forms compact RNA structural conformations, leading to attenuated protein production and fruit elongation. This study provides genetic evidence of synonymous variation shaping a biological trait through epitranscriptomic regulations.

Non-muscle myosin II (NMII), a molecular motor that regulates critical processes such as cytokinesis and neuronal plasticity, has substantial therapeutic potential. However, translating this potential to in vivo use has been hampered by a lack of selective tools. The most prototypical non-selective inhibitor inactivates both NMII and cardiac muscle myosin II (CMII), a key regulator of heart function. Using rational drug design, we developed a series of NMII inhibitors that markedly improve tolerability by selectively targeting NMII over CMII, including MT-228 and clinical candidate MT-110. MT-228 and MT-110 have excellent properties, including high brain penetration and efficacy in preclinical models of methamphetamine use disorder (MUD), which has no current FDA-approved therapies. The structure of MT-228 bound to myosin II provides insight into its selectivity for NMII over CMII. The broad therapeutic windows of these NMII inhibitors provide valuable tools for the scientific community and a promising clinical candidate for the treatment of MUD.