Mammalian organs continuously produce and consume circulating metabolites for organismal health and survival. However, the landscape of this fundamental process and its perturbation by diet and disease is unknown. Using arteriovenous metabolomics, tissue transcriptomics, and hormone arrays in multiple pathophysiological conditions in pigs, we generated an atlas of 10 cross-organ metabolite production and consumption during fasting/feeding, Western diet, and cardiovascular disease progression induced by low-density lipoprotein receptor (LDLR) deficiency. We discovered numerous instances of feeding-dependent and -independent metabolite production and consumption by organs and proposed mechanisms by which these are disrupted by Western diet via altered metabolite concentration gradients and hormones. Both Western diet and LDLR deficiency trigger the release of bile acids (BAs) by extra-hepatic organs, likely contributing to abnormally elevated circulating BA levels and consequent vascular inflammation and atherosclerosis development. These resources reveal intricate inter-organ metabolic crosstalk across pathophysiological conditions, offering biochemical insights into diet effects and cardiometabolic diseases.

A universal strategy to precisely control protein activation in living animals is crucial for gain-of-function study of proteins under in vivo settings. We herein report CAGE-Proxvivo, a computer-aided proximal decaging strategy for on-demand protein activation as well as protein-protein interaction modulations in living mice. Through machine-learning-assisted evolution of desired aminoacyl-tRNA synthetases (aaRSs), we successfully incorporated chemically caged amino acids into rationally designed "decaging sites" to transiently block target proteins' function, which can be restored in situ via a small-molecule-triggered bioorthogonal cleavage reaction. This method demonstrates broad applicability ranging from activating proteins of interest to cell-type-specific modulation of distinct phenotypes in living systems. Beyond the active-pocket decaging, CAGE-Proxvivo also enables precise control of protein-protein interactions, as exemplified by a "gated" anti-CD3 antibody that permits chemically regulated T cell recruitment and activation at tumor sites. Overall, CAGE-Proxvivo offers a universal platform for time-resolved biological studies and on-demand therapeutic interventions under living conditions.

Insulin resistance is a hallmark of type 2 diabetes, which is a highly heterogeneous disease with diverse pathology. Understanding the molecular signatures of insulin resistance and its association with individual phenotypic traits is crucial for advancing precision medicine in type 2 diabetes. Utilizing cutting-edge proteomics technology, we mapped the proteome and phosphoproteome of skeletal muscle from >120 men and women with normal glucose tolerance or type 2 diabetes, with varying degrees of insulin sensitivity. Leveraging deep in vivo phenotyping, we reveal that fasting proteome and phosphoproteome signatures strongly predict insulin sensitivity. Furthermore, the insulin-stimulated phosphoproteome revealed both dysregulated and preserved signaling nodes-even in individuals with severe insulin resistance. While substantial sex-specific differences in the proteome and phosphoproteome were identified, molecular signatures of insulin resistance remained largely similar between men and women. These findings emphasize the necessity of incorporating disease heterogeneity into type 2 diabetes care strategies.

The membrane-less nuclear stress bodies (nSBs), with satellite III (SatIII) RNAs as the hallmark, are present in primates upon sensing stresses. We report that SatⅢ DNAs, SatⅢ RNAs, and 30 nSB proteins assemble into well-organized structures shortly after stresses. The activated SatⅢ heterochromatin loci rapidly expand, resulting in reduced spatial distance and enhanced expression of adjacent genes, including the transcription suppressor NFIL3, which is known to dampen proinflammatory cytokine production. Rearranging NFIL3 loci within the nSB territory enhances NFIL3 chromatin accessibility and makes NFIL3 promoters more accessible to transcription factors heat shock transcription factor 1 (HSF1) and bromodomain containing 4 (BRD4), which are also recruited to nSBs upon stresses. Human peripheral blood mononuclear cell (PBMC)-derived macrophages under heat shock plus pathogen-associated molecular pattern treatments exhibit increased SatⅢ and NFIL3 expression, the latter of which suppresses key inflammatory cytokines. Importantly, NFIL3 expression positively correlates with SatⅢ activation in septic patients, a process positively correlated to patient survival, highlighting a role of nSBs in restraining inflammatory responses.

Cytosolic aggregation of the nuclear protein TAR DNA-binding protein 43 (TDP-43) is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43-enriched phase within stress granules, which subsequently transition into pathological aggregates. Intra-condensate demixing of TDP-43 is observed in iPS-motor neurons, a disease mouse model, and patient samples. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We suggest that up-concentration inside condensates followed by intra-condensate demixing could be a general pathway for protein aggregation.

Metagenomic analysis has recently unveiled the widespread presence of pelagic fungi in the global ocean, yet their quantitative contribution to carbon stocks remains elusive, hindering their incorporation into biogeochemical models. Here, we revealed the biomass of pelagic fungi in the open-ocean water column by combining ergosterol extraction, Calcofluor-White staining, catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), and microfluidic mass sensor techniques. We compared fungal biomass with the biomass of other more studied microbial groups in the ocean such as archaea and bacteria. Globally, fungi contributed 0.32 Gt C (CI: 0.19-0.46), refining previous uncertainty estimates from two orders of magnitude to less than one. While fungal biomass was lower than that of bacteria, it exceeded that of the archaea (archaea:fungi:bacteria biomass ratio of 1:9:44). Collectively, our findings reveal the important contribution of fungi to open-ocean biomass and, consequently, the marine carbon cycle, emphasizing the need for their inclusion in biogeochemical models.

Humans cannot perceive infrared light due to the physical thermodynamic properties of photon-detecting opsins. However, the capability to detect invisible multispectral infrared light with the naked eye is highly desirable. Here, we report wearable near-infrared (NIR) upconversion contact lenses (UCLs) with suitable optical properties, hydrophilicity, flexibility, and biocompatibility. Mice with UCLs could recognize NIR temporal and spatial information and make behavioral decisions. Furthermore, human participants wearing UCLs could discriminate NIR information, including temporal coding and spatial images. Notably, we have developed trichromatic UCLs (tUCLs), allowing humans to distinguish multiple spectra of NIR light, which can function as three primary colors, thereby achieving human NIR spatiotemporal color vision. Our research opens up the potential of wearable polymeric materials for non-invasive NIR vision, assisting humans in perceiving and transmitting temporal, spatial, and color dimensions of NIR light.

In a human cell, DNA is packed with histones, RNA, and chromatin-associated proteins, forming a cohesive gel. At any given moment, only a subset of the proteome has physical access to the DNA and organizes its structure, transcription, replication, repair, and other essential molecular functions. We have developed a "zero-distance" photo-crosslinking approach to quantify proteins in direct contact with DNA in living cells. Collecting DNA interactomes from human breast cancer cells, we present an atlas of over one thousand proteins with physical access to DNA and hundreds of peptide-nucleotide crosslinks pinpointing protein-DNA interfaces with single-amino-acid resolution. Quantitative comparisons of DNA interactomes from differentially treated cells recapitulate the recruitment of key transcription factors as well as DNA repair proteins and uncover fast-acting restrictors of chromatin accessibility on a timescale of minutes. This opens a direct way to explore genomic regulation in a hypothesis-free manner, applicable to many organisms and systems.

Epigenetic pathways could provide a mechanistic explanation for the inheritance of acquired characteristics, as proposed by Lamarck in 1802, but epigenetic alterations that endow adaptive hereditary traits have rarely been observed. Here, in cultivated Asian rice (Oryzasativa L.), we identified an epiallele conferring acquired and heritable cold tolerance, an adaptive trait enabling northward spread from its tropical origins. We subjected cold-sensitive rice to multigenerational cold stress and identified a line with acquired stable inheritance of cold tolerance. DNA-hypomethylation variation in the acquiredcoldtolerance 1 (ACT1) promoter region rendered its expression insensitive to cold. This change is, in large part, responsible for the acquired cold tolerance, as confirmed by DNA-methylation editing. Natural variation in ACT1 DNA hypomethylation is associated with cold tolerance and rice geographic distribution. Hypomethylation at ACT1 triggers adaptive cold tolerance, presenting a route to epigenetic-variation-driven inheritance of acquired characteristics.

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell-type taxonomies from mouse and human to define marker genes and putative enhancers and create a large toolkit of transgenic lines and enhancer adeno-associated viruses (AAVs) for selective targeting of cortical cell populations. We report creation and evaluation of fifteen transgenic driver lines, two reporter lines, and >1,000 different enhancer AAV vectors covering most subclasses of cortical cells. The tools reported here have been made publicly available, and along with the scaled process of tool creation, evaluation, and modification, they will enable diverse experimental strategies toward understanding mammalian cortex and brain function.

Identifying additional immune checkpoints hindering antitumor T cell responses is key to the development of next-generation cancer immunotherapies. Here, we report the induction of serotonin transporter (SERT), a regulator of serotonin levels and physiological functions in the brain and peripheral tissues, in tumor-infiltrating CD8 T cells. Inhibition of SERT using selective serotonin reuptake inhibitors (SSRIs), the most widely prescribed antidepressants, significantly suppressed tumor growth and enhanced T cell antitumor immunity in various mouse syngeneic and human xenograft tumor models. Importantly, SSRI treatment exhibited significant therapeutic synergy with programmed cell death protein 1 (PD-1) blockade, and clinical data correlation studies negatively associated intratumoral SERT expression with patient survival in a range of cancers. Mechanistically, SERT functions as a negative-feedback regulator inhibiting CD8 T cell reactivities by depleting intratumoral T cell-autocrine serotonin. These findings highlight the significance of the intratumoral serotonin axis and identify SERT as an immune checkpoint, positioning SSRIs as promising candidates for cancer immunotherapy.

Female sociosexual behaviors, essential for survival and reproduction, are modulated by ovarian hormones and triggered in the context of appropriate social cues. Here, we identify primary estrous-sensitive Cacna1h-expressing medial prefrontal cortex (mPFCCacna1h+) neurons that integrate hormonal states with recognition of potential mates to orchestrate these complex cognitive behaviors. Bidirectional manipulation of mPFCCacna1h+ neurons shifts opposite-sex-directed social behaviors between estrus and diestrus females via anterior hypothalamic outputs. In males, these neurons serve opposite functions compared with estrus females. Miniscope imaging reveals mixed representation of self-estrous states and social target sex in distinct mPFCCacna1h+ subpopulations, with biased encoding of opposite-sex cues in estrus females and males. Mechanistically, ovarian-hormone-induced Cacna1h upregulation enhances T-type rebound excitation after oxytocin inhibition, driving estrus-specific activity changes and the sexually dimorphic function of mPFCCacna1h+ neurons. These findings uncover a prefrontal circuit that integrates internal hormonal states and target-sex information to exert sexually bivalent top-down control over adaptive social behaviors.

Phosphatidylinositol 3-kinase (PI3K) signaling is both the effector pathway of insulin and among the most frequently activated pathways in human cancer. In murine cancer models, the efficacy of PI3K inhibitors is dramatically enhanced by a ketogenic diet, with a proposed mechanism involving dietary suppression of insulin. Here, we confirm profound diet-PI3K anticancer synergy but show that it is, surprisingly, unrelated to diet macronutrient composition. Instead, the diet-PI3K interaction involves microbiome metabolism of ingested phytochemicals. Specifically, murine ketogenic diet lacks the complex spectrum of phytochemicals found in standard chow, including the soy phytochemicals soyasaponins. We find that soyasaponins are converted by the microbiome into inducers of hepatic cytochrome P450 enzymes, and thereby lower PI3K inhibitor blood levels and anticancer activity. A high-carbohydrate, low-phytochemical diet synergizes with PI3K inhibition to treat cancer in mice, as do antibiotics that curtail the gut microbiome. Thus, diet impacts anticancer drug activity through phytochemical-microbiome-liver interactions.

G-protein-biased agonists have been shown to enhance opioid analgesia by circumventing β-arrestin-2 (βarr2) signaling. We previously reported that SBI-553, a neurotensin receptor 1 (NTSR1)-positive allosteric modulator biased toward βarr2 signaling, attenuates psychostimulant effects in mice. Here, we demonstrate that its analog, SBI-810, exhibits potent antinociceptive properties in rodent models of postoperative pain, inflammatory pain, and neuropathic pain via systemic and local administration. SBI-810's analgesic effects require NTSR1 and βarr2 but not NTSR2 or βarr1. Mechanistically, SBI-810 suppresses excitatory synaptic transmission, inhibits NMDA receptor and extracellular-regulated signal kinase (ERK) signaling in spinal cord nociceptive neurons, reduces Nav1.7 surface expression and action potential firing in primary sensory neurons, and dampens C-fiber responses. Behaviorally, it reduces opioid-induced conditioned place preference, alleviates constipation, and mitigates chronic opioid withdrawal symptoms. These findings highlight NTSR1-biased allosteric modulators as a promising, non-addictive therapeutic strategy for acute and chronic pain management, acting through both peripheral and central mechanisms.

Neuronal growth and regeneration are regulated by local translation of mRNAs in axons. We examined RNA polyadenylation changes upon sensory neuron injury and found upregulation of a subset of polyadenylated B2-SINE repeat elements, hereby termed GI-SINEs (growth-inducing B2-SINEs). GI-SINEs are induced from ATF3 and other AP-1 promoter-associated extragenic loci in injured sensory neurons but are not upregulated in lesioned retinal ganglion neurons. Exogenous GI-SINE expression elicited axonal growth in injured sensory, retinal, and corticospinal tract neurons. GI-SINEs interact with ribosomal proteins and nucleolin, an axon-growth-regulating RNA-binding protein, to regulate translation in neuronal cytoplasm. Finally, antisense oligos against GI-SINEs perturb sensory neuron outgrowth and nucleolin-ribosome interactions. Thus, a specific subfamily of transposable elements is integral to a physiological circuit linking AP-1 transcription with localized RNA translation.

Auxin is crucial in orchestrating diverse aspects of plant growth and development and modulating responses to environmental signals. The asymmetric spatiotemporal distribution of auxin generates local gradient patterns, which are regulated by both cellular auxin influx and efflux. The AUXIN1/LIKE-AUX1 (AUX1/LAX) family transporters have been identified as major auxin influx carriers. Here, we characterize the auxin uptake mediated by AUX1 from Arabidopsis thaliana. Using cryoelectron microscopy (cryo-EM), we determine its structure in three states: the auxin-unbound, the auxin-bound, and the competitive inhibitor, 3-chloro-4-hydroxyphenylacetic acid (CHPAA)-bound state. All structures adopt an inward-facing conformation. In the auxin-bound structure, indole-3-acetic acid (IAA) is coordinated to AUX1 primarily through hydrogen bonds with its carboxyl group. The functional roles of key residues in IAA binding are validated by in vitro and in planta analyses. CHPAA binds to the same site as IAA. These findings advance our understanding of auxin transport in plants.

Much remains to be learned about the clonal fate of mammalian epiblast cells. Here, we develop high-diversity Cre recombinase-driven LoxCode barcoding for in vivo clonal lineage tracing for bulk tissue and single-cell readout. Embryonic day (E) 5.5 pre-gastrulation embryos were barcoded in utero, and epiblast clones were assessed for their contribution to a wide range of tissues in E12.5 embryos. Some epiblast clones contributed broadly across germ layers, while many were biased toward either blood, ectoderm, mesenchyme, or limbs, across tissue compartments and body axes. Using a stochastic agent-based model of embryogenesis and LoxCode barcoding, we inferred and experimentally validated cell fate biases across tissues in line with shared and segregating differentiation trajectories. Single-cell readout revealed numerous instances of asymmetry in epiblast contribution, including left-versus-right and kidney-versus-gonad fate. LoxCode barcoding enables clonal fate analysis for the study of development and broader questions of clonality in murine biology.

The amnion, an extra-embryonic tissue in mammalian embryos, is thought to provide crucial signaling, structural, and nutritional support during pregnancy. Despite its pivotal importance, studying human amnion formation and function has been hampered by the lack of accurate in vitro models. Here, we present an embryonic stem cell-derived 3D model of the post-gastrulation amnion, post-gastrulation amnioids (PGAs), that faithfully recapitulates extra-embryonic development up to 4 weeks post-fertilization, closely mimicking the functional traits of the human amniotic sac. PGAs self-organize, forming the amnion and the yolk sac, and are surrounded by the extra-embryonic mesoderm. Using PGAs, we show that GATA3 is required and sufficient for amniogenesis and that an autoregulatory feedback loop governs amnion formation, whereby extra-embryonic signals promote amnion specification. The reproducibility and scalability of the PGA system, with its precise cellular, structural, and functional integrity, opens avenues for investigating embryo-amnion interactions beyond gastrulation and offers an ideal platform for large-scale pharmacological and clinical studies.

Several organs undergo changes during pregnancy to accommodate fetal growth, including the small intestine. However, the signals that trigger this intestinal remodeling are not well understood. In a recent study, Ameku et al. describe an anticipatory intestinal growth program that is partially irreversible and supported by SGLT3a-dependent increase of Fgfbp1-positive progenitor cells.

Human DNA is unavoidably present in metagenomic analyses of human microbiomes. While current protocols remove human DNA before submission to public repositories, mitochondrial DNA (mtDNA) has been overlooked and frequently persists. We discuss the privacy risks and research opportunities associated with mtDNA, urging consideration by the scientific, ethics, and legal communities.