Despite promising evidence in diagnostics and therapeutics, microbiome research is not yet implemented into clinical medicine. Several initiatives, including the standardization of microbiome research, the refinement of microbiome clinical trial design, and the development of communication between microbiome researchers and clinicians, are crucial to move microbiome science toward clinical practice.

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.

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.

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.

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.

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.

Immunotherapies have revolutionized cancer care for many tumor types, but their potential long-term cognitive impacts are incompletely understood. Here, we demonstrated in mouse models that chimeric antigen receptor (CAR) T cell therapy for both central nervous system (CNS) and non-CNS cancers impaired cognitive function and induced a persistent CNS immune response characterized by white matter microglial reactivity, microglial chemokine expression, and elevated cerebrospinal fluid (CSF) cytokines and chemokines. Consequently, oligodendroglial homeostasis and hippocampal neurogenesis were disrupted. Single-nucleus sequencing studies of human frontal lobe from patients with or without previous CAR T cell therapy for brainstem tumors confirmed reactive states of microglia and oligodendrocytes following treatment. In mice, transient microglial depletion or CCR3 chemokine receptor blockade rescued oligodendroglial deficits and cognitive performance in a behavioral test of attention and short-term memory function following CAR T cell therapy. Taken together, these findings illustrate targetable neural-immune mechanisms underlying immunotherapy-related cognitive impairment.

During cellular differentiation, enhancers transform overlapping gradients of transcription factors (TFs) to highly specific gene expression patterns. However, the vast complexity of regulatory DNA impedes the identification of the underlying cis-regulatory rules. Here, we characterized 64,400 fully synthetic DNA sequences to bottom-up dissect design principles of cell-state-specific enhancers in the context of the differentiation of blood stem cells to seven myeloid lineages. Focusing on binding sites for 38 TFs and their pairwise interactions, we found that identical sites displayed both repressive and activating function as a consequence of cell state, site combinatorics, or simply predicted occupancy of a TF on an enhancer. Surprisingly, combinations of activating sites frequently neutralized one another or gained repressive function. These negative synergies convert quantitative imbalances in TF expression into binary activity patterns. We exploit this principle to automatically create enhancers with specificity to user-defined combinations of hematopoietic progenitor cell states from scratch.

The emergence of SARS-CoV in 2002 and SARS-CoV-2 in 2019 led to increased sampling of sarbecoviruses circulating in horseshoe bats. Employing phylogenetic inference while accounting for recombination of bat sarbecoviruses, we find that the closest-inferred bat virus ancestors of SARS-CoV and SARS-CoV-2 existed less than a decade prior to their emergence in humans. Phylogeographic analyses show bat sarbecoviruses traveled at rates approximating their horseshoe bat hosts and circulated in Asia for millennia. We find that the direct ancestors of SARS-CoV and SARS-CoV-2 are unlikely to have reached their respective sites of emergence via dispersal in the bat reservoir alone, supporting interactions with intermediate hosts through wildlife trade playing a role in zoonotic spillover. These results can guide future sampling efforts and demonstrate that viral genomic regions extremely closely related to SARS-CoV and SARS-CoV-2 were circulating in horseshoe bats, confirming their importance as the reservoir species for SARS viruses.

In humans, the detection and ultimately the perception of sweetness begin in the oral cavity, where taste receptor cells (TRCs) dedicated to sweet-sensing interact with sugars, artificial sweeteners, and other sweet-tasting chemicals. Human sweet TRCs express on their cell surface a sweet receptor that initiates the cascade of signaling events responsible for our strong attraction to sweet stimuli. Here, we describe the cryo-electron microscopy (cryo-EM) structure of the human sweet receptor bound to two of the most widely used artificial sweeteners-sucralose and aspartame. Our results reveal the structural basis for sweet detection, provide insights into how a single receptor mediates all our responses to such a wide range of sweet-tasting compounds, and open up unique possibilities for designing a generation of taste modulators informed by the structure of the human receptor.

The saola is one of the most elusive large mammals, standing at the brink of extinction. We constructed a reference genome and resequenced 26 saola individuals, confirming the saola as a basal member of the Bovini. Despite its small geographic range, we found that the saola is partitioned into two populations with high genetic differentiation (FST = 0.49). We estimate that these populations diverged and started declining 5,000-20,000 years ago, possibly due to climate changes and exacerbated by increasing human activities. The saola has long tracts without genomic diversity; however, most of these tracts are not shared by the two populations. Saolas carry a high genetic load, yet their gradual decline resulted in the purging of the most deleterious genetic variation. Finally, we find that combining the two populations, e.g., in an eventual captive breeding program, would mitigate the genetic load and increase the odds of species survival.

The chemokine receptor variant CCR5delta32 is linked to HIV-1 resistance and other conditions. Its evolutionary history and allele frequency (10%-16%) in European populations have been extensively debated. We provide a detailed perspective of the evolutionary history of the deletion through time and space. We discovered that the CCR5delta32 allele arose on a pre-existing haplotype consisting of 84 variants. Using this information, we developed a haplotype-aware probabilistic model to screen 934 low-coverage ancient genomes and traced the origin of the CCR5delta32 deletion to at least 6,700 years before the present (BP) in the Western Eurasian Steppe region. Furthermore, we present strong evidence for positive selection acting upon the CCR5delta32 haplotype between 8,000 and 2,000 years BP in Western Eurasia and show that the presence of the haplotype in Latin America can be explained by post-Columbian genetic exchanges. Finally, we point to complex CCR5delta32 genotype-haplotype-phenotype relationships, which demand consideration when targeting the CCR5 receptor for therapeutic strategies.

Chemically modified nucleotides in mRNA are critical regulators of gene expression, primarily through interactions with reader proteins that bind to these modifications. Here, we present a mechanism by which the epitranscriptomic mark N6-methyladenosine (m6A) is read by tRNAs during translation. Codons that are modified with m6A are decoded inefficiently by the ribosome, rendering them "non-optimal" and inducing ribosome collisions on cellular transcripts. This couples mRNA translation to decay. 5-Methoxycarbonylmethyl-2-thiouridine (mcm5s2U) in the tRNA anticodon loop counteracts this effect. This unanticipated link between the mRNA and tRNA epitranscriptomes enables the coordinated decay of mRNA regulons, including those encoding oncogenic signaling pathways. In cancer, dysregulation of the m6A and mcm5s2U biogenesis pathways-marked by a shift toward more mcm5s2U-is associated with more aggressive tumors and poor prognosis. Overall, this pan-epitranscriptomic interaction represents a novel mechanism of post-transcriptional gene regulation with implications for human health.

The human gut harbors thousands of microbial species, each exhibiting significant inter-individual genetic variability. Although many studies have associated microbial relative abundances with human-health-related phenotypes, the substantial intraspecies genetic variability of gut microbes has not yet been comprehensively considered, limiting the potential of linking such genetic traits with host conditions. Here, we analyzed 32,152 metagenomes from 94 microbiome studies across the globe to investigate the human microbiome intraspecies genetic diversity. We reconstructed 583 species-specific phylogenies and linked them to geographic information and species' horizontal transmissibility. We identified 484 microbial-strain-level associations with 241 host phenotypes, encompassing human anthropometric factors, biochemical measurements, diseases, and lifestyle. We observed a higher prevalence of a Ruminococcus gnavus clade in nonagenarians correlated with distinct plasma bile acid profiles and a melanoma and prostate-cancer-associated Collinsella clade. Our large-scale intraspecies genetic analysis highlights the relevance of strain diversity as it relates to human health.

Vaccines generate long-lived plasma cells and memory B cells (Bmems) that may re-enter secondary germinal centers (GCs) to further mutate their B cell receptor upon boosting and re-exposure to antigen. We show in mouse models that lymph nodes draining the site of primary vaccination harbor a subset of Bmems that reside in the subcapsular niche, generate larger recall responses, and are more likely to re-enter GCs compared with circulating Bmems in non-draining lymph nodes. This location-dependent recall of Bmems into the GC in the draining lymph node was dependent on CD169+ subcapsular sinus macrophages (SSMs) in the subcapsular niche. In human participants, boosting of the BNT162b2 vaccine in the same arm generated more rapid secretion of broadly neutralizing antibodies, GC participation, and clonal expansion of SARS-CoV-2-specific B cells than boosting of the opposite arm. These data reveal an unappreciated role for primed draining lymph node SSMs in Bmem cell fate determination.