Using a mouse tumor model with inducible cancer-cell-intrinsic cyclic GMP-AMP (cGAMP) synthase (cGAS) expression, we show that cancer-cell-derived cGAMP is essential and sufficient to trigger a sustained type I interferon response within the tumor microenvironment. This leads to improved CD8 T cell-dependent tumor restriction. However, cGAMP limits the proliferation, survival, and function of stimulator of IFN genes (STING)-expressing, but not of STING-deficient, CD8 T cells. In vivo, STING deficiency in CD8 T cells enhances tumor restriction. Consequently, cancer-cell-derived cGAMP both drives and limits the anti-tumor potential of CD8 T cells. Mechanistically, T cell-intrinsic STING is associated with pro-apoptotic and antiproliferative gene signatures. Our findings suggest that STING signaling acts as a checkpoint in CD8 T cells that balances tumor immunity.

Blood cancers are generally more common in males, and the prevalence of most mutations that drive clonal hematopoiesis and myeloid malignancies is higher in males. In contrast, hematopoietic DNMT3A mutations are more common in females. Among ∼450,000 participants in the UK Biobank, the prevalence of DNMT3A mutations and copy-number abnormalities is higher in females than males. In a murine model, Dnmt3a-mutant hematopoietic stem cells (HSCs) from unperturbed female mice had increased stemness gene expression compared to male and wild-type (WT) mice. Estrogen regulates HSCs, and we found that Dnmt3a mutations maintain stemness in the setting of estrogen-induced proliferative stress. Dnmt3a-mutant myeloid cells outcompeted WT cells under chronic estrogen treatment, an effect that was dependent on cell-intrinsic estrogen receptor alpha activity. Our studies indicate that estrogen might contribute to the female predominance of DNMT3A-mutant clonal hematopoiesis.

Sex chromosomes are expected to coevolve with their respective sex, potentially disfavoring their co-occurrence as cosexuality evolves. This effect is expected to be stronger where sex chromosomes are restricted to one sex, such as in plants expressing sex in their haploid stage. We assess this hypothesis in liverworts with U/V sex chromosomes, ancestral dioicy, and several independent transitions to monoicy (cosexuality). We report the chromosome-level genome assembly of Marchantia quadrata, which recently evolved monoicy, and perform comparative genomic analyses with its dioicous relative M. polymorpha. We find that monoicy evolved via retention of the V chromosome as a small ninth chromosome, complete loss of the U chromosome, and translocation of key U-linked genes to autosomes, among which the major sex-determining gene (Feminizer) acquired environmental/developmental regulation. Our findings parallel recent observations on Ricciocarpos natans, which evolved monoicy independently, suggesting genetic constraints that may make transitions to monoicy predictable in liverworts.

The heart and lung co-orchestrate their development during organogenesis. The mesoderm surrounding both the developing heart and anterior foregut endoderm provides instructive cues guiding cardiopulmonary development. Additionally, it serves as a source of cardiopulmonary progenitor cells (CPPs) expressing Wnt2 that give rise to both cardiac and lung mesodermal cell lineages. Despite the mesoderm's critical importance to both heart and lung development, mechanisms guiding CPP specification are unclear. To address this, we lineage traced Wnt2 CPPs at E8.5 and performed single-cell RNA sequencing on collected progeny across the developmental lifespan. Using computational analyses, we created a CPP-derived cell atlas that revealed a previously underappreciated spectrum of CPP-derived cell lineages, including all lung mesodermal lineages, ventricular cardiomyocytes, and epicardial and pericardial cells. By integrating spatial mapping with computational cell trajectory analysis and transcriptional profiling, we have provided a potential molecular and cellular roadmap for cardiopulmonary development.

Non-mammalian vertebrates maintain a proliferative cell population at the far periphery of their retina called the ciliary marginal zone (CMZ), which contributes to retinal regeneration upon injury. Humans do not maintain a proliferative CMZ into adulthood; however, it is unknown how long in development this region continues to generate neurons. Here, we identify a population of cells in the far-peripheral retina of the human that continues to proliferate after the rest of the retina is quiescent. Single-cell RNA sequencing and 5-ethynyl-2'-deoxyuridine tracing at late developmental time points reveal that this region has the capacity to produce both early- and late-born cell types late in development and a longer cell cycle than more centrally located retinal progenitor cells (RPCs). Moreover, while most RPCs exit the cell cycle with the addition of a transforming growth factor β inhibitor, early RPCs within the CMZ do not. These findings define the late stages of neurogenesis in human retinal development.

Autophagic lysosome reformation (ALR) is crucial for lysosomal homeostasis and therefore for different autophagic processes. Despite recent advances, the signaling mechanisms regulating ALR are incompletely understood. We show that RAF1, a member of the RAS/RAF/MEK/ERK pathway initiated by growth factors, has an essential, kinase-dependent role in lysosomal biology. RAF1 ablation impairs autophagy, and a proxisome screen identifies several proteins involved in autophagic and lysosomal pathways in the RAF1 molecular space. Two of these, SPG11 and the lipid phosphatase MTMR4, are RAF1 substrates. RAF1 ablation causes the appearance of enlarged autolysosomes and alters the phosphoinositide composition of autolysosomes. RAF1 and MTMR4 colocalize on autolysosomes, and overexpression of a MTMR4 mutant mimicking phosphorylation of the RAF1-dependent site rescues the lysosomal phenotypes induced by RAF1 ablation. Our data identify an RAF1 function in lysosomal homeostasis and a substrate through which the kinase regulates phospholipid metabolism at the lysosome, ALR, and autophagy.

Ticks ingest over 100 times their body weight in blood. As the primary tissue for blood storage and digestion, the tick midgut's regulation in response to this substantial blood volume remains unclear. Here, we show that blood intake triggers stem cell proliferation and mitochondrial fission in the midgut of Haemaphysalis longicornis. While inhibiting stem cell proliferation does not impact feeding behavior, disruption of mitochondrial fission impairs tick feeding capacity. Mitochondrial fission mediated by dynamin 2 (DNM2) regulates ATP generation, which in turn influences the expression of the tropomyosin-anchoring subunit troponin T (TNT). Knockdown of TNT disrupts muscle fiber assembly, hindering midgut enlargement and contraction, thereby preventing blood ingestion. These findings underscore the indispensable role of musculature in facilitating midgut expansion during feeding in ticks.

Topoisomerases typically function in the nucleus to relieve topological stress in DNA. Here, we show that a dual-activity topoisomerase, Top3b, and its partner, TDRD3, largely localize in the cytoplasm and interact biochemically and genetically with PIWI-interacting RNA (piRNA) processing enzymes to promote piRNA biogenesis, post-transcriptional gene silencing (PTGS) of transposons, and Drosophila germ cell development. Top3b requires its topoisomerase activity to promote PTGS of a transposon reporter and preferentially silences long and highly expressed transposons, suggesting that RNAs with these features may produce more topological stress for topoisomerases to solve. The double mutants between Top3b and piRNA processing enzymes exhibit stronger disruption of the signatures and levels of germline piRNAs, more de-silenced transposons, and larger defects in germ cells than either single mutant. Our data suggest that Top3b can act in an RNA-based process-piRNA biogenesis and PTGS of transposons-and this function is required for Top3b to promote normal germ cell function.

The eukaryotic ribosome is highly modified by protein methylation, yet many of the responsible methyltransferases remain unknown. Here, we identify SET and MYND domain-containing protein 5 (SMYD5) as a ribosomal protein methyltransferase that catalyzes trimethylation of RPL40/eL40 at lysine 22. Through a systematic mass spectrometry-based approach, we identify 12 primary sites of protein methylation in ribosomes from K562 cells, including at RPL40 K22. Through in vitro methylation of synthetic RPL40 using fractionated lysate, we then identify SMYD5 as a candidate RPL40 K22 methyltransferase. We show that recombinant SMYD5 has robust activity toward RPL40 K22 in vitro and that active site mutations ablate this activity. Knockouts of SMYD5 in K562 cells show a complete loss of RPL40 K22 methylation and decreased polysome levels. We show that SMYD5 does not methylate histones in vitro, and by systematic analysis of its recognition motif, we find that SMYD5 requires a KXY motif for methylation, explaining its lack of activity toward histones.

The endoplasmic reticulum (ER) is structurally and functionally diverse, yet how its functions are organized within morphological subdomains is incompletely understood. Utilizing TurboID-based proximity labeling and CRISPR knockin technologies, we map the proteomic landscape of the human ER network. Sub-organelle proteomics reveals enrichments of proteins into ER tubules, sheets, and the nuclear envelope. We uncover an ER-enriched actin-binding protein, calmin/CLMN, and define it as an ER-actin tether that localizes to focal adhesions adjacent to ER tubules. Mechanistically, we find that CLMN depletion perturbs adhesion disassembly, actin dynamics, and cell movement. CLMN-depleted cells display decreased polarization of ER-plasma membrane contacts and calcium signaling factor STIM1 and altered calcium signaling near ER-actin interfaces, suggesting that CLMN influences calcium signaling to facilitate F-actin/adhesion dynamics. Collectively, we map the sub-organelle proteome landscape of the ER, identify CLMN as an ER-actin tether, and describe a non-canonical mechanism by which ER tubules engage actin to regulate cell migration.

The Coronavirus Immunotherapeutic Consortium (CoVIC) conducted side-by-side comparisons of over 400 anti-SARS-CoV-2 spike therapeutic antibody candidates contributed by large and small companies as well as academic groups on multiple continents. Nine reference labs analyzed antibody features, including in vivo protection in a mouse model of infection, spike protein affinity, high-resolution epitope binning, ACE-2 binding blockage, structures, and neutralization of pseudovirus and authentic virus infection, to build a publicly accessible dataset in the database CoVIC-DB. High-throughput, high-resolution binning of CoVIC antibodies defines a broad and predictive landscape of antibody epitopes on the SARS-CoV-2 spike protein and identifies features associated with durable potency against multiple SARS-CoV-2 variants of concern and high in vivo efficacy. Results of the CoVIC studies provide a guide for selecting effective and durable antibody therapeutics and for immunogen design as well as providing a framework for rapid response to future viral disease outbreaks.

The hypothalamo-neurohypophyseal system is a neuroendocrine conduit through which the neurohormones oxytocin and arginine vasopressin are released from the brain into the general circulation, influencing functions like salt balance and reproduction. However, the precise mechanism for rapid neurohormone transport to the periphery remains unclear. We show, using live imaging in zebrafish, that both hyperosmotic physiological challenge and optogenetic stimulation of oxytocin neurons elicit a local increase in neurohypophyseal blood flow velocities and a change in capillary diameter. This response is dictated by the geometry of the hypophyseal vascular microcircuit. Genetic ablation of oxytocin neurons and inhibition of oxytocin receptor signaling attenuate the changes in capillary blood flow and diameter. Both the osmotic challenge and oxytocin neuronal activation elicit a local rise in neurohypophyseal capillary permeability in an oxytocin-signaling-dependent manner. We propose that oxytocin-dependent neurovascular coupling facilitates its efficient uptake into the blood circulation, suggesting a self-perpetuating stimulus-secretion-uptake mechanism for peripheral hormone transfer.

The lymphoid cycle serves as a sentinel of the immune response, yet the cell subtypes and immune properties within lymph fluid remain unclear. This study describes a comprehensive characterization of immune cells in rat lymph fluid using single-cell RNA sequencing, identifying a unique subset of CD4 T cells (CD4_Icos) that suppresses inflammation in early sepsis. Trajectory analysis reveals that CD4+Icos+ T cells can differentiate into regulatory T cells (Tregs). Transferring CD4+Icos+ T cells alleviates CLP-induced organ injury, while CD4 Icos-knockout (KO) mice show reduced Treg numbers, increased inflammation, and higher mortality. Further experiments identify Npas2 as an Icos-specific transcription factor regulating Icos expression and promoting the differentiation of CD4+Icos+ T cells. Clinical data show a negative correlation between ICOS expression in CD4 T cells and clinical outcomes in septic patients. These findings highlight the protective role of CD4 T cells in modulating immune responses and mitigating sepsis progression.

Intrinsic enteric neurons (iENs) form a crucial neuronal network within the myenteric and submucosal plexus of the gastrointestinal tract, primarily responsible for regulating gut peristalsis. The mechanisms by which iENs sense and integrate dietary and microbial signals to regulate intestinal homeostasis and inflammation remain unclear. Here, we showed that environmental sensor aryl hydrocarbon receptor (AHR) was expressed in different iEN subsets in the ileum and colon and that AHR ligands differentially modulated iEN activity in these regions. Genetic perturbation of Ahr in neurons increased iEN activation in the ileum but, conversely, decreased it in the colon in response to different intestinal pathogens. Furthermore, neuronal AHR deficiency enhanced the clearance of bacterial pathogens, which was associated with increased proliferation and abundance of group 3 innate lymphoid cells in the ileum. Together, our findings demonstrate the region-specific functions of AHR in neurons in response to infections.

Trained immunity refers to memory-like responses of innate immune cells when they re-encounter pathogenic stimuli. Bacillus Calmette-Guérin (BCG) vaccination implies enhanced antiviral immunity, whereas the underlying mechanisms remain unclear. Herein, we have uncovered elevated expression of low-density lipoprotein receptor (LDLR) on BCG-trained macrophages with robust type I interferon (IFNI) production and antiviral effects both in vivo and in vitro. Consequently, cholesterol is accumulated in BCG-trained macrophages, leading to the augmentation of NFE2L1 expression and the formation of NFE2L1/IRAK1/TRIM25 complex where TRIM25 mediates IRAK1 K63 polyubiquitination to exaggerate IFNI responses in an RIG-I-dependent manner. We have also observed LDLR macrophages displaying heightened IFNI responses in BCG-treated human macrophages. To antagonize LDLR degradation by PCSK9 inhibitors increases IFNI responses in the macrophages and accelerated viral clearance. Our study thus couples LDLR upregulation to antiviral activity in BCG-trained macrophages, making commercial PCSK9 inhibitors potential antiviral indications in clinic.

Painful physical symptoms in major depressive disorder (MDD) patients lead to poor outcomes during MDD treatment. Here, we report that decreased Na/K-ATPase β1 subunit (NKAβ1) expression in anterior cingulate cortex glutamatergic (ACC) neurons promotes ion dyshomeostasis, leading to hyperactivity of ACC-insular cortex circuits in chronic stress mice. This ultimately primes allodynia. Mechanistically, we reveal that chronic stress strengthens LAMP2A-driven chaperone-mediated autophagy (CMA) and subsequently promotes the degradation of NKAβ1. We further identify NKAβ1 as a CMA substrate. Accordingly, genetically LAMP2A loss in ACC neurons reverses chronic-stress-induced neuronal hyperexcitability, subsequently ameliorating allodynia. Additionally, we develop a trans-activating transcription (TAT)-LAMP2A peptide that significantly alleviates depression-induced allodynia. Taken together, our results reveal a mechanistic connection between CMA and neuronal excitability. TAT-LAMP2A peptide intervention, by disturbing CMA-dependent NKAβ1 elimination, could be a potential pharmacological treatment for depression-induced allodynia and further facilitate the efficacy of antidepressant treatment.

Spatial sequences encoded by cells in the hippocampal-entorhinal region have been observed to spontaneously alternate across the animal's midline during navigation in the open field, but it is unknown how this occurs. We show that sinusoidal sampling patterns emerge rapidly and robustly in a simple model of the hippocampal place-cell sequences based on spike frequency adaptation that makes no assumptions about sequence direction. We corroborate our findings using hippocampal data from rats performing a spatial memory task in the open field.

Some pathogen-derived effectors reprogram mRNA splicing in host plants to regulate plant immune responses. Whether effectors from Exserohilum turcicum, which causes northern corn leaf blight (NLB), interfere with RNA splicing remains unknown. We identify that the secreted protein EtEC81 (Exserohilum turcicum effector 81) modulates the alternative splicing (AS) of maize (Zea mays) pre-mRNAs and negatively regulates the pathogenicity of E. turcicum. EtEC81 physically interacts with MAIZE EtEC81-INTERACTING PROTEIN 1 (ZmEIP1), which associates with maize spliceosome components, modulates AS in host cells, and positively regulates defense responses against E. turcicum. Transcriptome analysis identifies 119 common events with altered AS in maize plants transiently overexpressing ZmEIP1 or EtEC81, suggesting that these factors cause the misregulation of cellular activities and thus induce immune responses. Together, our results suggest that the EtEC81 effector targets ZmEIP1 to reprogram pre-mRNA splicing in maize. These findings provide a mechanistic basis and potential target gene for preventing NLB.

Circular RNAs (circRNAs) increase in the brain with age across various animal systems. To elucidate the reasons behind this phenomenon, we profile circRNAs from fly heads at six time points throughout their lifespan. Our results reveal a linear increase in circRNA levels with age, independent of changes in mRNA levels, overall transcription, intron retention, or host gene splicing, demonstrating that the age-related accumulation is due to high stability rather than increased biogenesis. This remarkable stability suggests that circRNAs can serve as markers of environmental experience. Indeed, flies exposed to a 10-day regimen at 29°C exhibit higher levels of specific circRNAs even 6 weeks after returning to standard conditions, indicating that circRNAs can reveal past environmental stimuli. Moreover, half-life measurements show circRNA stability exceeding 20 days, with some displaying virtually no degradation. These findings underscore the remarkable stability of circRNAs in vivo and their potential as markers for stress and life experiences.