Developmental gene expression is regulated by the dynamic interplay of histone H3 lysine 4 (H3K4) and histone H3 lysine 27 (H3K27) methylation, yet the physiological roles of these epigenetic modifications remain incompletely understood. Here, we show that mice depleted for all forms of H3K4 methylation, using a dominant histone H3-lysine-4-to-methionine (H3K4M) mutation, succumb to a severe loss of all major blood cell types. H3K4M-expressing hematopoietic stem cells (HSCs) and committed progenitors are present at normal numbers, indicating that H3K4 methylation is dispensable for HSC maintenance and commitment but essential for progenitor cell maturation. Mechanistically, we reveal that H3K4 methylation opposes the deposition of repressive H3K27 methylation at differentiation-associated genes enriched for a bivalent (i.e., H3K4/H3K27-methylated) chromatin state in HSCs and progenitors. Indeed, by concomitantly suppressing H3K27 methylation in H3K4-methylation-depleted mice, we rescue the acute lethality, hematopoietic failure, and gene dysregulation. Our results provide functional evidence for the interaction between two crucial chromatin marks in mammalian tissue homeostasis.

Inflammation, aberrant proteostasis, and energy depletion are hallmarks of neurodegenerative diseases such as multiple sclerosis (MS). However, the interplay between inflammation, proteasomal dysfunction in neurons, and its consequences for neuronal integrity remains unclear. Using transcriptional, proteomic, and functional analyses of proteasomal subunits in inflamed neurons, we found that interferon-γ-mediated induction of the immunoproteasome subunit, proteasome 20S beta 8 (PSMB8) impairs the proteasomal balance, resulting in reduced proteasome activity. This reduction causes the accumulation of phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key metabolic regulator, leading to enhanced neuronal glycolysis, reduced pentose phosphate pathway activity, oxidative injury, and ferroptosis. Neuron-specific genetic and systemic pharmacological targeting of PSMB8 or PFKFB3 protected neurons in vitro and in a mouse model of MS. Our findings provide a unifying explanation for proteasomal dysfunction in MS and possibly other neurodegenerative diseases, linking inflammation to metabolic disruption, and presenting an opportunity for targeted neuroprotective therapies.

Plant nucleotide-binding, leucine-rich repeat (NLR) immune receptors recognize pathogen effectors and activate defense. NLR genes can be non-functional in distantly related plants (restricted taxonomic functionality, RTF). Here, we enable Solanaceae NLR gene function in rice, soybean, and Arabidopsis by co-delivering sensor NLR genes with their cognate NLR required for cell death (NRC)-type helper NLRs. In soybean protoplasts and in Arabidopsis plants, Solanum americanum Rpi-amr1, Rpi-amr3, and pepper Bs2 sensor NLRs confer cognate effector responsiveness if co-expressed with NRC helper NLRs. Rice carrying pepper Bs2 and NRCs recognizes the conserved effector, AvrBs2, and resists an important pathogen, Xanthomonas oryzae pv. oryzicola, for which no resistance gene is available in rice. Rice lines carrying sensor and helper NLR genes otherwise resemble wild type, with unaltered basal resistance or field fitness. Thus, interfamily co-transfer of sensor and helper NLRs can broaden the utility of sensor NLRs, extending the tools available to control diseases of rice, soybean, Brassica, and other crops.

Single-cell RNA sequencing has revolutionized our understanding of cellular diversity but remains constrained by scalability, high costs, and the destruction of cells during analysis. To overcome these challenges, we developed STAMP (single-cell transcriptomics analysis and multimodal profiling), a highly scalable approach for the profiling of single cells. By leveraging transcriptomics and proteomics imaging platforms, STAMP eliminates sequencing costs, enabling cost-efficient single-cell genomics of millions of cells. Immobilizing (stamping) cells in suspension onto imaging slides, STAMP supports multimodal (RNA, protein, and H&E) profiling, while retaining cellular structure and morphology. We demonstrate STAMP's versatility by profiling peripheral blood mononuclear cells, cell lines, and stem cells. We highlight the capability of STAMP to identify ultra-rare cell populations, simulate clinical applications, and show its utility for large-scale perturbation studies. In total, we present data for 10,962,092 high-quality cells/nuclei and 6,030,429,954 transcripts. STAMP makes high-resolution cellular profiling more accessible, scalable, and affordable.

Recent outbreaks of multidrug-resistant fungi infecting human skin emphasize the importance of understanding fungal pathophysiology and spread. In efforts to address health concerns with various Indigenous Peninsular Malaysians (Orang Asli [OA]), tinea imbricata-a Trichophyton concentricum fungal skin infection-emerged as a particular concern. We investigated the etiology and transmission of tinea imbricata by culturing, testing antifungal sensitivities, and sequencing T. concentricum isolates in remote OA villages. Among regionally conserved isolates, we identified the emergence of terbinafine-resistant T. concentricum microbiologically and genomically. Investigating the skin microbiomes of 82 Indigenous OA, we found unique microbiota and lower relative abundances of bacterial commensals (Cutibacterium acnes, Staphylococcus epidermidis) among OA versus Malaysian and US urban populations, emphasizing how understudied populations provide unprecedented knowledge on host-microbiome co-evolution. These findings provide valuable insights into clinical, microbiological, and genomic features of chronic fungal skin infections, offering the potential to inform strategies to address drug resistance and effective therapy.

Microbial communities coat nearly every surface in the environment and have co-existed with animals throughout evolution. Whether animals exploit omnipresent microbial cues to navigate their surroundings is not well understood. Octopuses use "taste-by-touch" chemotactile receptors (CRs) to explore the seafloor, but how they distinguish meaningful surfaces from the rocks and crevices they encounter is unknown. Here, we report that secreted signals from microbiomes of ecologically relevant surfaces activate CRs to guide octopus behavior. Distinct molecules isolated from individual bacterial strains located on prey or eggs bind single CRs in subtly different structural conformations to elicit specific mechanisms of receptor activation, ion permeation and signal transduction, and maternal care and predation behavior. Thus, microbiomes on ecological surfaces act at the level of primary sensory receptors to inform behavior. Our study demonstrates that uncovering interkingdom interactions is essential to understanding how animal sensory systems evolved in a microbe-rich world.

Metazoan life requires the coordinated activities of thousands of genes in spatially organized cell types. Understanding the basis of tissue function requires approaches to dissect the genetic control of diverse cellular and tissue phenotypes in vivo. Here, we present Perturb-Multimodal (Perturb-Multi), a paired imaging and sequencing method to construct large-scale, multimodal genotype-phenotype maps in tissues with pooled genetic perturbations. Using imaging, we identify perturbations in individual cells while simultaneously measuring their gene expression profiles and subcellular morphology. Using single-cell sequencing, we measure full transcriptomic responses to the same perturbations. We apply Perturb-Multi to study hundreds of genetic perturbations in the mouse liver. Our data suggest the genetic regulators and mechanisms underlying the dynamic control of hepatocyte zonation, the unfolded protein response, and steatosis. Perturb-Multi accelerates discoveries of the genetic basis of complex cell and tissue physiology and provides critical training data for emerging machine learning models of cellular function.

Striatal dopamine plays fundamental roles in fine-tuning learned decisions. However, when learning from naive to expert, individuals often exhibit diverse learning trajectories, defying understanding of its underlying dopaminergic mechanisms. Here, we longitudinally measure and manipulate dorsal striatal dopamine signals in mice learning a decision task from naive to expert. Mice learning trajectories transitioned through sequences of strategies, showing substantial individual diversity. Remarkably, the transitions were systematic; each mouse's early strategy determined its strategy weeks later. Dopamine signals reflected strategies each animal transitioned through, encoding a subset of stimulus-choice associations. Optogenetic manipulations selectively updated these associations, leading to learning effects distinct from that of reward. A deep neural network using heterogeneous teaching signals, each updating a subset of network association weights, captured our results. Analyzing the model's fixed points explained learning diversity and systematicity. Altogether, this work provides insights into the biological and mathematical principles underlying individual long-term learning trajectories.

Cryptococcus neoformans is the most common cause of fungal meningitis and the top-ranking WHO fungal priority pathogen. Only distantly related to model fungi, C. neoformans is also a powerful experimental system for exploring conserved eukaryotic mechanisms lost from specialist model yeast lineages. To decipher its biology globally, we constructed 4,328 gene deletions and measured-with exceptional precision-the fitness of each mutant under 141 diverse growth-limiting in vitro conditions and during murine infection. We defined functional modules by clustering genes based on their phenotypic signatures. In-depth studies leveraged these data in two ways. First, we defined and investigated new components of key signaling pathways, which revealed metazoan-like cellular machinery not present in model yeasts. Second, we identified environmental adaptation mechanisms repurposed to promote mammalian virulence by C. neoformans, which lacks a known animal reservoir. Our work provides an unprecedented resource for deciphering a deadly human pathogen.

Social insects offer powerful models to investigate mechanisms of elaborate individual behaviors comprising a cooperative community. Workers of the leafcutter ant genus Atta are extreme examples of behavioral segregation among phenotypically distinct worker types. We utilize this worker system to test the molecular underpinnings of behavioral programming and the extent of plasticity to reprogramming. We identify specific neuropeptides mediating worker division of labor in A. cephalotes, finding two neuropeptides associated with characteristic behaviors of leaf cutting and of brood care. Genetic knockdown or injection of these neuropeptides led to a stark gain or loss of each behavior and to transcriptomic shifts toward gene pathways expressed in the natural castes. We also reveal global similarities between worker transcriptomes of the eusocial mammal, the naked mole-rat H. glaber, with orthologous A. cephalotes workers. This work underscores the essential function of neuropeptides in establishing complex social behavior and a remarkable plasticity among individual behavioral types.

Glioblastoma (GBM) is the most lethal of primary brain tumors. Here, we report our studies of MT-125, a small-molecule inhibitor of non-muscle myosin II. MT-125 has high brain penetrance and an excellent safety profile, blocks GBM invasion and cytokinesis, and prolongs survival in murine GBM models. By impairing mitochondrial fission, MT-125 increases redox stress and consequent DNA damage, and it synergizes with radiotherapy. MT-125 also induces oncogene addiction to PDGFR signaling through a mechanism that is driven by redox stress, and it synergizes with FDA-approved PDGFR and mTOR inhibitors in vitro. Consistent with this, we find that combining MT-125 with sunitinib, a PDGFR inhibitor, or paxalisib, a combined phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor, significantly improves survival in orthotopic GBM models over either drug alone. Our results demonstrate that MT-125 is a first-in-class therapeutic that has strong clinical potential for the treatment of GBM.

Mitochondrial abundance and genome are crucial for cellular function, with disruptions often associated with disease. However, methods to modulate these parameters for direct functional dissection remain limited. Here, we eliminate mitochondria from pluripotent stem cells (PSCs) by enforced mitophagy and show that PSCs survived for several days in culture without mitochondria. We then leverage enforced mitophagy to generate interspecies PSC fusions that harbor either human or non-human hominid (NHH) mitochondrial DNA (mtDNA). Comparative analyses indicate that human and NHH mtDNA are largely interchangeable in supporting pluripotency in these PSC fusions. However, species divergence between nuclear and mtDNA leads to subtle species-specific transcriptional and metabolic variations. By developing a transgenic enforced mitophagy approach, we further show that reducing mitochondrial abundance leads to delayed development in pre-implantation mouse embryos. Our study opens avenues for investigating the roles of mitochondria in development, disease, and interspecies biology.

N-Acyl lipids are important mediators of several biological processes including immune function and stress response. To enhance the detection of N-acyl lipids with untargeted mass spectrometry-based metabolomics, we created a reference spectral library retrieving N-acyl lipid patterns from 2,700 public datasets, identifying 851 N-acyl lipids that were detected 356,542 times. 777 are not documented in lipid structural databases, with 18% of these derived from short-chain fatty acids and found in the digestive tract and other organs. Their levels varied with diet and microbial colonization and in people living with diabetes. We used the library to link microbial N-acyl lipids, including histamine and polyamine conjugates, to HIV status and cognitive impairment. This resource will enhance the annotation of these compounds in future studies to further the understanding of their roles in health and disease and to highlight the value of large-scale untargeted metabolomics data for metabolite discovery.

The nomadic Sarmatians dominated the Pontic Steppe from the 3rd century BCE and the Great Hungarian Plain from 50 CE until the Huns' 4th-century expansion. In this study, we present a large-scale genetic analysis of 156 genomes from 1st- to 5th-century Hungary and the Carpathian foothills. Our findings reveal minor East Asian ancestry in the Carpathian Basin (CB) Sarmatians, distinguishing them from other regional populations. Using F4 statistics, qpAdm, and identity-by-descent (IBD) analysis, we show that CB Sarmatians descended from Steppe Sarmatians originating in the Ural and Kazakhstan regions, with Romanian Sarmatians serving as a possible genetic bridge between the two groups. We also identify two previously unknown migration waves during the Sarmatian era and a notable continuity of the Sarmatian population into the Hunnic period despite a smaller influx of Asian-origin individuals. These results shed new light on Sarmatian migrations and the genetic history of a key population neighboring the Roman Empire.

Understanding the mechanisms underlying mammalian regeneration may enable development of novel regenerative therapies. We present a mechanism wherein Desert hedgehog (Dhh), secreted from epithelial neuroendocrine cells, elicits a regenerative/protective response from mesenchymal cells. In mammalian airway, this mesenchymal response strikingly amplifies the initial signal from rare neuroendocrine cells to activate the entire tissue for survival and regeneration upon injury from SO2 gas inhalation or following influenza or SARS-CoV-2 infection. Similar epithelial-mesenchymal feedback (EMF) signaling directed by Dhh from neuroendocrine β cells likewise protects mouse pancreatic islets from streptozotocin (STZ) injury. A role for EMF signaling in human pancreatic islets is suggested by higher incidence of diabetes in patients treated with Hedgehog pathway inhibitors. Remarkably, EMF augmentation by small-molecule Hedgehog pathway agonism protects against STZ injury of pancreatic β cells and shields against airway injury from SO2 and influenza infection, with potential protective/therapeutic utility in chemical or infectious airway injury and in diabetes.

Early-life susceptibility to respiratory viral infections remains a major public health concern, yet the underlying mechanisms are poorly understood. We demonstrate that antibiotic-induced dysbiosis impairs influenza-specific CD8+ T cell immunity in infant mice and humans through the disruption of nuclear factor interleukin 3 (NFIL3)-dependent T cell programming. Mechanistically, we show that dysbiosis reduces intestinal and circulating inosine levels, disrupting NFIL3's epigenetic regulation of T cell factor 1 (TCF1) expression. This leads to intrinsic defects in CD8+ T cell proliferation and differentiation, diminished effector responses, and impaired formation of tissue-resident memory cells. Bifidobacterium colonization restores intestinal and pulmonary inosine levels, establishing a specific pathway of gut-lung metabolic communication. Notably, inosine supplementation rescues NFIL3-dependent regulation of TCF1, enhancing CD8+ T cell responses and protection against influenza infection in dysbiotic infants. Our findings reveal how early-life microbial communities shape antiviral immunity and identify inosine as a therapeutic target for enhancing respiratory defenses in infants.

Fecal microbiota transplant (FMT) is an increasingly used intervention, but its suitability to restore regional gut microbiota, particularly in the small bowel (SB), must be questioned because of its predominant anaerobic composition. In human subjects receiving FMT by upper endoscopy, duodenal engraftment of anaerobes was observed after 4 weeks. We hypothesized that peroral FMTs create host-microbe mismatches that impact SB homeostasis. To test this, antibiotic-treated specific-pathogen-free (SPF) mice were given jejunal, cecal, or fecal microbiota transplants (JMTs, CMTs, or FMTs, respectively) and studied 1 or 3 months later. JMT and FMT altered regional microbiota membership and function, energy balance, and intestinal and hepatic transcriptomes; JMT favored host metabolic pathways and FMT favored immune pathways. MTs drove regional intestinal identity (Gata4, Gata6, and Satb2) and downstream differentiation markers. RNA sequencing (RNA-seq) of metabolite-exposed human enteroids and duodenal biopsies post-FMT confirmed transcriptional changes in mice. Thus, regional microbial mismatches after FMTs can lead to unintended consequences and require rethinking of microbiome-based interventions.

The cGAS-cGAMP-STING pathway is crucial for antiviral immunity. While cytosolic cGAS detects viral DNA, most DNA viruses shield their genome and invade the nucleus, where chromatin restricts cGAS activation. How viruses may activate nuclear cGAS is not well understood. Here, we show that several herpesvirus proteins trigger nuclear cGAS activation by perturbing centromeres, where cGAS is enriched. The herpes simplex virus type 1 (HSV-1) ubiquitin ligase infected cell protein 0 (ICP0), which degrades centromeric proteins, promotes centromeric DNA amplification through the translesion DNA synthesis (TLS) pathway in quiescent monocyte-derived cells, thereby activating nuclear cGAS. During infection, HSV-1 evades this detection by also expressing UL36USP, a suppressor of TLS. Similarly to ICP0, the cytomegalovirus IE1 protein causes centromeric DNA amplification and cGAS activation. We define this mechanism as viral-induced centromeric DNA amplification and recognition (VICAR), uncovering a non-mitotic, immune-activating role of centromeres.

How does circuit wiring constrain neural computation? Recent work has leveraged connectomic datasets to predict the functions of cells and circuits in the brains of multiple species. However, many of these hypotheses have not been compared with physiological measurements, obscuring the limits of connectome-based functional predictions. To explore these limits, we characterized the visual responses of 43 cell types in the fruit fly and quantitatively compared them with connectomic predictions. We show that these predictions are accurate for some response properties, such as orientation tuning, but are surprisingly poor for other properties, such as receptive field size. Importantly, strong synaptic inputs are more functionally homogeneous than expected by chance and exert a disproportionately large influence on postsynaptic responses. Finally, we quantitatively define the subset of connections that best describe the functional differences between cell types. Our results establish a powerful set of constraints for improving the accuracy of connectomic predictions.