Adequate delivery of thyroid hormones to the brain is crucial for normal neurological development. MCT8 and OATP1C1, two solute carrier (SLC) transporters, mediate the passage of thyroid hormones across the blood-brain barrier and into the central nervous system. Mutations in MCT8 result in Allan-Herndon-Dudley syndrome (AHDS), an X-linked birth defect characterized by neurodevelopmental impairments and peripheral hyperthyroidism, whereas OATP1C1 deficiency is linked to brain hypometabolism and progressive neurodegeneration. Here, we report cryoelectron microscopy (cryo-EM) structures of MCT8 and OATP1C1 bound with the active thyroid hormone triiodothyronine (T3) and the prohormone thyroxine (T4) at 2.9 and 2.3 Å resolutions, respectively. Combined with functional studies, we elucidate their distinct thyroid hormone recognition and transport mechanisms and explain disease mutations. Although extracellular allosteric sites are not a common feature of SLC transporters, we identify one in OATP1C1. Collectively, these findings illuminate key aspects of thyroid hormone transport, a fundamental process in development and disease.

To meet the brain's moment-to-moment energy demand, neural activation rapidly increases local blood flow. This process, known as neurovascular coupling, involves rapid, coordinated vasodilation of the brain's arterial network. Here, we demonstrate that endothelial gap junction coupling enables long-range propagation of vasodilation signals through the vasculature during neurovascular coupling. The molecular composition of these gap junctions is zonated along the arterio-venous axis, with arteries being the most strongly coupled segment. Using optogenetics and visual stimuli in awake mice, we found that acute, arterial endothelial cell type-specific deletion of Cx37 and Cx40 abolishes arterial gap junction coupling and results in impaired vasodilation. Specifically, we demonstrated that arterial endothelial gap junction coupling determines both the speed and the spatial extent of vasodilation propagation elicited by neural activity. These findings indicate that endothelial gap junctions serve as a signaling highway for neurovascular coupling, enabling flexible and efficient distribution of limited energetic resources.

Fluorescent genetically encoded voltage indicators report transmembrane potentials of targeted cell types. However, voltage-imaging instrumentation has lacked the sensitivity to track spontaneous or evoked high-frequency voltage oscillations in neural populations. Here, we describe two complementary TEMPO (transmembrane electrical measurements performed optically) voltage-sensing technologies that capture neural oscillations up to ∼100 Hz. Fiber-optic TEMPO achieves ∼10-fold greater sensitivity than prior photometric voltage sensing, allows hour-long recordings, and monitors two neuron classes per fiber-optic probe in freely moving mice. With it, we uncovered cross-frequency-coupled theta- and gamma-range oscillations and characterized excitatory-inhibitory neural dynamics during hippocampal ripples and visual cortical processing. The TEMPO mesoscope images voltage activity in two cell classes across an ∼8-mm-wide field of view in head-fixed animals. In awake mice, it revealed sensory-evoked excitatory-inhibitory neural interactions and traveling gamma and 3-7 Hz waves in visual cortex and bidirectional propagation directions for both hippocampal theta and beta waves. These technologies have widespread applications probing diverse oscillations and neuron-type interactions in healthy and diseased brains.

Cortical neurons are specified during embryonic development but often acquire their mature properties at relatively late stages of postnatal development. This delay in terminal differentiation is particularly prominent for fast-spiking parvalbumin-expressing (PV+) interneurons, which play critical roles in regulating the function of the cerebral cortex. We found that the maturation of PV+ interneurons is triggered by neuronal activity and mediated by the transcriptional cofactor peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α). Developmental loss of PGC-1α prevents PV+ interneurons from acquiring unique structural, electrophysiological, synaptic, and metabolic features and disrupts their diversification into distinct subtypes. PGC-1α functions as a master regulator of the differentiation of PV+ interneurons by directly controlling gene expression through a transcriptional complex that includes ERRγ and Mef2c transcription factors. Our results uncover a molecular switch that translates neural activity into a specific transcriptional program, promoting the maturation of PV+ interneurons at the appropriate developmental stage.

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.

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.

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.

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.

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.

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.

The vasculature and mesenchyme exhibit distinct organ-specific characteristics adapted to local physiological needs, shaped by microenvironmental and cell-cell interactions from early development. To recapitulate this entire process, we co-differentiated mesoderm and endoderm within the same spheroid to vascularize lung and intestinal organoids from induced pluripotent stem cells (iPSCs). Bone morphogenetic protein (BMP) signaling fine-tuned the endoderm-to-mesoderm ratio, a critical step in generating appropriate proportions of endothelial and epithelial progenitors with tissue specificity. Single-cell RNA sequencing (scRNA-seq) revealed organ-specific gene signatures of endothelium and mesenchyme and identified key ligands driving endothelial specification. The endothelium exhibited tissue-specific barrier function, enhanced organoid maturation, cellular diversity, and alveolar formation on the engineered lung scaffold. Upon transplantation into mice, the organoid vasculature integrated with the host circulation while preserving organ specificity, further promoting organoid maturation. Leveraging these vascularized organoids, we uncovered abnormal endothelial-epithelial crosstalk in patients with forkhead box F1 (FOXF1) mutations. Multilineage organoids provide an advanced platform to study intricate cell-to-cell communications in human organogenesis and disease.

Vascular dementia (VaD), the second-leading cause of dementia, is primarily a white matter ischemic disease with no direct therapies. Cell-cell interactions within lesion sites dictate disease progression or repair. To elucidate key intercellular pathways, we employ a VaD mouse model with focal ischemia replicating many elements of the complex pathophysiology of human VaD combined with transcriptomic and functional analyses. By integrating cell-type-specific mouse VaD transcriptomes and human VaD single-nucleus RNA sequencing (snRNA-seq) data plus a custom ligand-receptor database (4,053 human and 2,032 mouse pairs), conserved dysregulated intercellular pathways in both species are identified. We demonstrate that two intercellular signaling systems, Serpine2-Lrp1 and CD39-A3AR, are disrupted in VaD. Reduced Serpine2 expression enhances oligodendrocyte progenitor cell (OPC) differentiation, promoting repair, while an A3AR-specific agonist-currently in clinical trials for psoriasis-restores tissue integrity and behavioral function in the VaD model. This study reveals intercellular signaling targets and provides a foundation for developing innovative therapies for VaD.

Recognition of exogenous RNA by Toll-like receptors (TLRs) is central to pathogen defense. Using two distinct binding pockets, TLR7 and TLR8 recognize RNA degradation products generated by endolysosomal nucleases. RNA modifications present in endogenous RNA prevent TLR activation; notably, pseudouridine-containing RNA lacks immunostimulatory activity. Indeed, this property has been critical to the successful implementation of mRNA technology for medical purposes. However, the molecular mechanism for this immune evasion has remained elusive. Here, we report that RNase T2 and PLD exonucleases do not adequately process pseudouridine-containing RNA to generate TLR-agonistic ligands. As a second safety mechanism, TLR8 neglects pseudouridine as a ligand for its first binding pocket and TLR7 neglects pseudouridine-containing RNA as a ligand for its second pocket. Interestingly, the medically used N1-methylpseudouridine also evades RNase T2, PLD3, and PLD4 processing but is able to directly activate TLR8. Taken together, our findings provide a molecular basis for self-avoidance by RNA-sensing TLRs.