Twenty-five years ago, two Cell papers reported the key missing functional piece in three molecular puzzles. The genetic swapping of immunoglobulin constant regions, the mutational fine-tuning of antibody specificity, and a baffling human immunodeficiency were traced to the action of one enzyme: activation-induced cytidine deaminase (AID).

Sick animals exhibit behavioral changes that extend beyond physiological symptoms, such as appetite loss and hypoactivity, and include a decline in social interactions. While social isolation during sickness has been recognized to have the evolutionary benefit of staving off disease spread, the molecular and neural mechanisms underlying this response remain unclear. Cytokines-immune-derived signaling molecules-have emerged as neuromodulators impacting brain function during inflammation. Through behavioral screening, we identify a unique role for the cytokine interleukin-1β (IL-1β) in promoting social withdrawal during sickness. IL-1β directly modulates the activity of IL-1R1-expressing neurons in the dorsal raphe nucleus (DRN) (IL-1R1DRN). Activation of these neurons is sufficient to elicit social withdrawal, while their inhibition or genetic deletion of IL-1R1 rescues self-imposed social isolation during systemic inflammation. Our findings reveal a neural mechanism that actively promotes social disengagement in sick animals, highlighting the role of IL-1R1DRN neurons in driving these behavioral adaptations.

The influence of the nervous system on the intestine is carried out by a combination of enteric, sensory, and autonomic innervation. However, disambiguating the functions of these physiologically distinct intestine-innervating neuronal populations has been a challenge. Here, we develop a collection of mouse genetic tools that enable precise manipulation and examination of intestine-innervating neurons, particularly those in the enteric nervous system, which represent the most frequent of the intestine-innervation neural populations. We report that an array of transcriptionally distinct enteric neuron subpopulations has distinct morphological specializations and influences on intestinal function, including controlling fecal output, fecal hydration, and food intake. We also report that subpopulations within the enteric nervous system require extrinsic innervation to exert control over intestinal transit or food intake. Collectively, these genetic approaches enable interrogation of the enteric nervous system and further study of its interactions with broader neural networks in the body.

Creating interspecies chimeras with human pluripotent stem cells (hPSCs) offers a promising strategy for modeling human development and generating donor organs; however, poor human cell integration remains a major barrier. Most existing efforts to improve human chimerism focus on genetically modifying donor hPSCs, while altering the host embryo remains largely unexplored. Using an interspecies PSC competition model, we discovered that RNA innate immunity in "winner" mouse cells drives the competitive elimination of hPSCs. Disrupting RNA-sensing pathways reduced the competitiveness and viability of mouse PSCs, and mouse embryos lacking Mavs-a key gene in RNA innate immunity-led to markedly improved human cell survival and chimerism. We also found that contact-dependent horizontal RNA transfer likely underlies this immune activation. Overall, our study uncovers a previously unrecognized role for RNA innate immunity in cell competition and demonstrates that targeting host immune pathways represents a powerful approach to improve human chimerism in animals.

BAX is a pro-apoptotic BCL-2 protein that resides in the cytosol as a monomer until triggered by cellular stress to form an oligomer that permeabilizes mitochondria and induces apoptosis. The paradigm for apoptotic blockade involves heterodimeric interactions between pro- and anti-apoptotic monomers. Here, we find that full-length BCL-w forms a distinctive, symmetric dimer (BCL-wD) that dissociates oligomeric BAX (BAXO), inhibits mitochondrial translocation, promotes retrotranslocation, blocks membrane-porating activity, and influences apoptosis induction of cells. Structure-function analyses revealed discrete conformational changes upon BCL-w dimerization and reciprocal structural impacts upon BCL-wD and BAXO interaction. Small-angle X-ray scattering (SAXS) analysis demonstrated that BAXO disrupts membranes by inducing negative Gaussian curvature, which is reversed by positive Gaussian curvature exerted by BCL-wD. Systematic truncation and mutagenesis dissected the core features of BCL-wD activity-dimerization, BAXO engagement, and membrane interaction. Our studies reveal a downstream layer of apoptotic control mediated by protein and membrane interactions of higher-order BCL-2 family multimers.

Expressed in the entorhinal cortex (EC), the cholecystokinin (CCK) B receptor (CCKBR) plays an important role in memory and learning. Here, we identify that CCKBR-Gs and -Gq signaling, rather than CCKBR-Gi signaling, are beneficial for Alzheimer's disease (AD) treatment. Clinically, patients with more severe AD associated with lower CCKBR-Gq activity. The cryo-electron microscopy (cryo-EM) structures of CCKBR in complex with the endogenous agonist sulfated CCK8 (CCK8s) and 3 different G protein subtypes revealed that distinct receptor conformations contribute to selective G protein bias. Leveraging these structural insights, we rationally develop synthetic CCKBR agonists, including a Gi-biased agonist (z-44) and a Gq-biased agonist (3r1). Notably, 3r1 demonstrates therapeutic potential by ameliorating cognitive decline in 5×FAD mice, reducing the number of amyloid-β plaques, and promoting long-term potentiation (LTP) via upregulation of the α-secretase (ADAM10) and the calcium signaling molecule PLCB4. Our findings suggest that synthetic biased agonists targeting CCKBR-Gq signaling have therapeutic potential for AD.

Genome-wide assessment of genetic variation is becoming routine in genetics, yet functional interpretation of non-coding single nucleotide variants in both common and rare diseases remains a major challenge. Here, we used chromatin immunoprecipitation coupled to self-transcribing active regulatory region sequencing (ChIP-STARR-seq) to functionally annotate non-coding regulatory elements (NCREs) in cellular models of human brain development. This provides gene regulatory insights into neural stem cells and evidence of NCRE priming already in embryonic stem cells for later neural activity. Based on this functional genomics atlas, we developed BRAIN-MAGNET (brain-focused artificial intelligence method to analyze genomes for non-coding regulatory element mutation targets), a functionally validated convolutional neural network that predicts NCRE activity from DNA sequence composition and identifies nucleotides required for NCRE function. BRAIN-MAGNET allows fine-mapping of genome-wide association study (GWAS) loci for common neurological traits and prioritizing candidate disease-causing rare non-coding variants in genetically unexplained individuals with neurogenetic disorders. Together, this NCRE atlas and BRAIN-MAGNET represent a powerful resource for the interpretation of non-coding genetic variation, possibly aiding the identification of previously unrecognized enhanceropathies.

Tumor-associated macrophages (TAMs) expressing the myeloid checkpoint TREM2 are key immunosuppressive cells in the tumor microenvironment (TME), driving tumor progression and contributing to poor prognosis in cancer patients. Due to their pivotal role, TAMs have emerged as a promising target for immunotherapies. However, current TAM-targeting monotherapies show limited efficacy, highlighting the need for strategies engaging multiple immune modalities. Here, we developed myeloid-targeted immunocytokines and natural killer (NK)/T cell enhancers (MiTEs) harnessing myeloid and lymphoid synergy for immunotherapy. MiTEs are trans-acting immunocytokines with tumor-specific activation, allowing dual targeting of TAMs and lymphocytes by TREM2 antagonism and cytotoxic effector cell activation through interleukin (IL)-2. To avoid off-target toxicities, MiTEs contain an IL-2 masking moiety, which is cleaved by a TAM-specific protease. MiTEs demonstrate high efficacy in preclinical tumor models through extensive immune reprogramming spanning TAM, T, and NK cell compartments. MiTEs show transformative potential for treating solid cancers by inducing potent multi-axis anti-tumor immunity while minimizing toxicities.

The ubiquitous metabolite heme has diverse enzymatic and signaling functions in most mammalian cells. Through integrated analyses of mouse models, human cell lines, and primary patient samples, we identify de novo heme biosynthesis as a selective dependency in acute myeloid leukemia (AML). The dependency is underpinned by a propensity of AML cells, and especially leukemic stem cells (LSCs), to downregulate heme biosynthesis enzymes (HBEs), which promotes their self-renewal. Inhibition of HBEs causes the collapse of mitochondrial Complex IV and dysregulates the copper-chaperone system, inducing cuproptosis, a form of programmed cell death brought about by the oligomerization of lipoylated proteins by copper. Moreover, we identify pathways that are synthetic lethal with heme biosynthesis, including glycolysis, which can be leveraged for combination strategies. Altogether, our work uncovers a heme rheostat that is connected to gene expression and drug sensitivity in AML and implicates HBE inhibition as a trigger of cuproptosis.

Although immunoglobulin A (IgA)+ long-lived plasma cells (LLPCs) generated following mucosal viral infection provide durable protection against reinfection, little is known about their generation. Here, we show that rotavirus (RV) infection induces gut-resident LLPCs that produce highly mutated, protective IgA. Unlike RV-specific immunoglobulin G (IgG)+ LLPCs, IgA+ LLPCs were generated independently of major histocompatibility complex class II (MHC class II) expression by dendritic cells-rather, MHC class II on B cells was both necessary and sufficient. B cell-MHC class II was also sufficient to induce T-bet+ follicular helper T (TFH1) cells, which were crucial for RV-specific IgA+ LLPC accumulation in the gut via interferon γ (IFNγ)- and CXCR3-dependent mechanisms. Similar to RV infection, TFH1 cells were required for an influenza-specific IgA response in the airway. However, unlike RV infection, B cell-MHC class II was not sufficient to induce influenza-specific IgA+ LLPCs, suggesting the operation of mucosal-site-specific priming mechanisms. Collectively, our data reveal that unconventionally primed TFH1 cells support IgA responses to mucosal viral infections.

Human gut bacteria are routinely exposed to stresses, and community-level responses are difficult to predict. To interrogate these effects, we screened stool-derived in vitro communities with 707 clinically relevant drugs. Across ∼5,000 community-drug conditions, compositional and metabolomic responses were shaped by nutrient competition, with certain species expanding due to the suppression of competitors. Most compositional changes arose from strain extinction and were reversed by reintroducing extinct species, although certain drugs promoted alternative states long after treatment. Despite strong selection pressures, resistance emergence was infrequent. Responses to drugs were qualitatively conserved across communities, while nutrient competition quantitatively tuned species abundances, consistent with consumer-resource model predictions. Together, nutrient competition provides a predictive framework to anticipate and potentially mitigate drug side effects on the gut microbiota.

Ancient DNA has revolutionized the study of extinct and extant organisms that lived up to 2 million years ago, enabling the reconstruction of genomes from multiple extinct species, as well as the ecosystems where they once thrived. However, current DNA sequencing techniques alone cannot directly provide insights into tissue identity, gene expression dynamics, or transcriptional regulation, as these are encoded in the RNA fraction. Here, we report transcriptional profiles from 10 Late Pleistocene woolly mammoths. One of these, dated to be ∼39,000 years old, yielded sufficient detail to recover tissue-specific regulatory mechanisms and biological functions essential for skeletal muscle metabolism, representing the oldest ancient RNA sequences recorded to date. We showcase the potential to study ancient RNA molecules beyond preconceived limitations, providing an analytical framework for validating and decoding preserved transcriptomes through time. With our findings, we anticipate the emergence of integrative paleo-studies combining genomics, proteomics, and transcriptomics.

The ability of a virus to infect a cell is partly determined by host factors required for the viral life cycle. However, not every cell of a given type is equally susceptible to infection. Profiling susceptible subsets of cells could reveal additional host factors, but viral infection obscures and remodels the state of these cells prior to infection. We used single-cell clone tracing to retrospectively identify cells that were highly susceptible to infection with SARS-CoV-2. Depletion of certain factors identified by our approach revealed roles in viral entry, whereas others exerted control over the infectable cell state itself. Patient lung samples revealed heterogeneous expression of these factors in vivo, with heightened expression in particular inflammatory pathologies. We further found a distinct cell state that was susceptible to the influenza A virus. Thus, intrinsic variability in state is a major determinant of whether individual cells can be infected by a virus.

Recent studies at molecular and genomic scales have enriched our understanding of life's most fundamental building block: the cell. However, bridging the gap between single-cell phenotypes and the emergent functions of tissues and organs remains a formidable challenge. Here, we suggest that the conceptual span from cells to tissues and organs is so large as to warrant intermediate stepping stones. Drawing inspiration from "network motifs"-discrete units of cell-level function that emerge from the interactions of a handful of genes or enzymes-we argue that similarly identifiable units of tissue-level function, which we term "mesoscale modules," emerge from coordinated "interactions" among relatively small numbers of cells and their extracellular milieu. We outline several such modules and propose that a concerted effort to study them will deepen our foundational understanding of tissue and organ functions. By developing these mesoscale insights, we anticipate a more tractable and mechanistic approach to complex human conditions rooted in tissue- and organ-scale dysregulation, including developmental defects, cancer, cardiovascular disease, immune-related disorders, infectious disease, and aging.

Age-dependent exhaustion of endogenous stem cell pools-and the resulting decline in tissue regeneration and homeostatic maintenance-is a hallmark of organismal aging and age-related pathology. In a study published in Cell, Liu and collaborators engineered human ESC-derived mesenchymal progenitor cells to give the ability to resist senescence, environmental stress, and malignant transformation.

Intrinsically disordered regions (IDRs) of proteins are defined by molecular grammars. This refers to IDR-specific non-random amino acid compositions and non-random patterning of distinct pairs of amino acid types. Here, we introduce grammars inferred using NARDINI+ (GIN) as a resource that uncovers IDR-specific and IDRome-spanning grammars. Using GIN-enabled analyses, we find that specific IDR features and GIN clusters are associated with distinct biological processes, intra-cellular localization preferences, specialized molecular functions, and functionalization as assessed by cellular fitness correlations. IDRs with exceptional grammars, defined as sequences with high-scoring non-random features, are harbored in proteins and complexes that enable spatial and temporal sorting of biochemical activities within the nucleus. Overall, GIN can be used to extract sequence-function relationships of individual IDRs or clusters of IDRs, to redesign extant IDRs or design de novo IDRs, to perform evolutionary analyses through the lens of molecular grammars and GIN clusters, and to make sense of IDR-specific disease-associated mutations.

Presynaptic 5-HT1AR autoreceptors predominantly signal through Gi3 protein, mediating feedback inhibition that hampers the therapeutic efficacy of conventional antidepressants. By contrast, postsynaptic heteroreceptors mainly couple to Go, which promotes antidepressant responses. However, selectively activating heteroreceptors while bypassing the negative feedback induced by autoreceptors remains a significant challenge. Here, we characterized the Gi/o subtype signaling profiles of 5-HT1AR and determined its structures in complex with six agonists and three distinct Gi/o family proteins: GoA, Gi3, and Gz. Combined with functional analysis, we elucidated the mechanisms underlying diverse agonist recognition modes and Gi/o subtype signaling selectivity of 5-HT1AR. Furthermore, we designed a pathway-selective agonist, TMU4142, which exhibits high GoA activity while minimizing Gi3 activation. Remarkably, TMU4142 demonstrated rapid antidepressant-like effects in a mouse model of depression. Collectively, these findings suggest that distinguishing heteroreceptors from autoreceptors based on their distinct downstream Gi/o signaling pathways could be a promising strategy to develop fast-acting antidepressants.

Fluctuations in host cell growth pose a critical challenge for maintaining reliable function in synthetic gene circuits. Growth-mediated dilution causes a global reduction in circuit component concentrations, which can significantly destabilize circuit behavior. However, effective strategies to counteract this problem remain lacking. Here, we present a phase-separation-based strategy to directly mitigate dilution effects. By fusing transcription factors (TFs) to intrinsically disordered regions (IDRs), we drive the formation of transcriptional condensates that concentrate TFs at their target promoters. These condensates buffer against prolonged rapid dilution of TF concentration and preserve bistable memory in self-activation circuits across variable growth conditions. We further show that this approach improves production efficiency in a cinnamic acid biosynthesis pathway. Together, our results establish liquid-liquid phase separation as an emerging design principle for constructing resilient synthetic circuits that maintain robust performance under dynamic growth conditions.