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.

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.

Red blood cell (RBC) lysis can cause morbidity and mortality. However, the molecular mechanisms underlying RBC lysis are not fully characterized, limiting therapeutic options. In this issue of Cell, Chen et al. identify a crucial role for the NLRP3-ASC-caspase-8 complex in driving programmed lytic cell death in RBCs.

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.

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.

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.