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Poliovirus (PV) genome encodes a large single polyprotein that is processed by viral proteases to form an active replication complex through either cis or trans interactions between the viral proteins (i.e., interactions between viral proteins encoded on the same polyprotein molecule or between those encoded on different polyprotein molecules, respectively). In the processing of polyprotein, the cleavage of viral 3AB into 3A and 3B is unique, as it requires host factors (PI4KB/OSBP) and viral protease (3Cpro/3 CDpro) in cultured cells (i.e., in vivo). Here, we show viral/host requirements for the cleavage of 3AB in vivo. In a polyprotein encoding 2BC3ABCD of PV, cleavage of 3AB requires the activity of PI4KB as well as the entire 3Dpol region; even a partial deletion of the 3Dpol region severely affects the cleavage in the polyprotein. The activity of OSBP and the binding activity of 3 CDpro to negatively charged molecules are not required for the cleavage in the polyprotein. PV mutants with premature termination codons or in-frame deletions in the 3Dpol-coding region are generally quasi-infectious in trans-rescued replication with 3 CDpro, causing extensive in-frame genome duplication or deletion. Surprisingly, some PV mutants lacking the C-terminal peptides of 3Dpol showed stable replication without any reversion in the presence of 3 CDpro provided in trans; 3Dpol provided in trans could rescue the defect in 3AB cleavage via amino acid residues involved in 3Dpol-3AB and 3Dpol-3Dpol interactions, indicating a remarkable overlap with those required for the uridylylation of 3B. This work reveals novel roles of the 3Dpol region, offering insights into the polyprotein processing and recombination.

Anti-IgLON5 disease is an autoimmune disease, in which autoantibodies (AABs) against the neuronal cell surface protein IgLON5 lead to profound brain dysfunction and Tau pathology. How α-IgLON5 AABs cause neuronal Tau protein pathology and neurodegeneration remains unclear. We find that patient-derived α-IgLON5 AABs cluster IgLON5 proteins with other cell surface proteins, leading to neuronal hyperactivity that triggers pathological Tau missorting and phosphorylation, typically observed early in Tau-related neurodegenerative diseases. In wild-type mice, α-IgLON5 AABs induce hippocampal Tau phosphorylation and neuroinflammatory responses. Our findings establish a causal link between the α-IgLON5 AABs and Tau pathology in anti-IgLON5 disease patients and highlight the role of neuronal hyperactivity as a disease-overarching driver of Tau pathology and provide a potential target for therapeutic intervention.

Meiosis is a fundamental process responsible for sexual reproduction and generating genetic diversity in the progeny. Its successful completion requires fine-tuning of expression programs of many genes: promoting expression of genes involved in meiotic processes and suppressing genes whose expression may interfere with meiosis. Molecular mechanisms involved in meiotic transcriptome regulation and controlling meiosis progression vary between plants, animals, and fungi and remain elusive. We found that the Plural abnormalities of meiosis1 (Pam1) gene in maize controls meiosis progression by tethering transcriptome processing to the meiosis-specific chromosome axis. Pam1 encodes an RNA binding protein that becomes associated with chromosomes during early meiotic prophase I, binds transcripts of a large number of meiosis-related genes, and affects their splicing by interacting with the CCR4-NOT RNA processing protein complex. Disrupting Pam1 function results in a wide array of severe meiosis defects affecting chromosome condensation and dynamics, nuclear envelope and cytoskeleton organization, as well as the overall meiosis progression. Pam1 controls only a subset of meiotic genes and processes, indicating that several programs directing transcriptome architecture collectively regulate meiosis. RNA-binding proteins have been found to control meiosis progression in fungi and animals, and it is now shown to be also the case in plants. Interestingly, these proteins all exhibit distinct modes of action and evolutionary origins, presenting a remarkable case of convergent evolution. Uncovering mechanisms controlling meiosis progression should enable engineering meiosis to benefit crop improvement efforts.

Glioblastoma (GBM) contains mesenchymal cancer stem cells that drive tumor aggressiveness and recurrence and exhibit aberrant glycosylation during proneural-to-mesenchymal transition. A comprehensive computational analysis of human GBM transcriptomic datasets revealed an up-regulation of 13 genes involved in glycan mannosylation compared to normal brain, and histopathological staining of a tissue array representing 35 GBM cases revealed elevated mannose levels that correlated with increased expression of the mesenchymal marker CD44. Hydroxyl proton transfer-weighted magnetic resonance imaging (HPTw MRI) detected elevated mannose levels in aggressive human mesenchymal GBM in vitro and in vivo but not in GBM with a less aggressive nonmesenchymal phenotype. To establish causation over correlation, inhibiting expression of the mannose-binding lectins LMAN1/2 that regulate intracellular processing of mannosylated proteins decreased the glioma cell HPTw MRI signal. Our findings indicate that HPTw MRI correlates with high mannose and possibly other saccharide levels in mesenchymal GBM cells, serving as a surrogate imaging biomarker for predicting tumor aggressiveness and recurrence.

Immune checkpoint inhibitors show promise in the treatment of melanoma brain metastases but are limited by CD73/adenosine axis-mediated immune evasion. Directly targeting CD73 with antibodies faces challenges due to poor blood-brain barrier permeability and metabolic regulators within the tumor microenvironment (IL-17-driven HIF-1α/VEGF-A). To overcome these barriers, we developed a nose-to-brain delivery platform using glycerol as a mucosal penetration enhancer to codeliver anti-IL-17 and anti-CD73 antibodies. Glycerol reversibly opened nasal epithelial tight junction proteins, enhancing the brain delivery of anti-IL-17 and anti-CD73 antibodies by 19.4- and 17.1-fold, respectively, while minimizing systemic exposure. Critically, anti-IL-17 attenuated CD73/adenosine axis-mediated immune evasion, significantly boosting anti-CD73 targeting efficacy. Ultimately, this combination promoted CD8+ T cell activation and residency, pro-inflammatory macrophage polarization, and reduced Treg cell infiltration, thereby eliciting a strong antitumor effect. Our results establish an efficient nose-to-brain delivery platform for macromolecules and propose a therapeutic strategy for tumors using anti-IL-17 to overcome TME-imposed limitations in CD73-targeted immunotherapy.

Isoprenylcysteine carboxyl methyltransferase (ICMT) catalyzes C-terminal methylation of prenylated CAAX proteins, a final processing step promoting membrane association and signaling. Although ICMT has been pursued to disrupt RAS membrane targeting, its role in BRAFV600E-driven cancers and critical substrates remains unclear. Here, genetic and pharmacologic (UCM-1336) ICMT inhibition suppressed proliferation and invasion in BRAFV600E-mutant melanoma cells and reduced tumor growth in xenografts and mice. ICMT knockdown inhibited proliferation of BRAF-inhibitor-resistant melanoma cells. We identify INPP5E as an ICMT-dependent substrate: ICMT inhibition reduced INPP5E methylation, displaced it from membranes, and increased PI(4,5)P2. Forced INPP5E membrane targeting partially rescued growth defects caused by ICMT inhibition. These findings implicate an ICMT-INPP5E-axis that supports BRAFV600E-driven tumor growth.

Erosive hand osteoarthritis (EHOA) is a chronic joint disease characterized by severe inflammation and degeneration of cartilage and bone tissue. As this disease is multifactorial in nature, the molecular mechanisms that influence its pathogenesis are unclear, leading to a lack of disease-modifying therapies. However, by screening 40 families with a dominant inheritance pattern for EHOA, we identified two independent germline heterozygous mutations that associated with EHOA onset: PANX1 [c.G455A:p.R152H] and PANX3 [c.G71A:p.R24H]. Pannexin 1 (PANX1) and Pannexin 3 (PANX3) are mechanosensitive channel-forming glycoproteins that pass various metabolites and ions such as adenosine triphosphate (ATP) and calcium to regulate numerous physiological and cellular processes including tissue development, cell differentiation, and homeostasis. In this study, we report that electrophysiological recordings, ATP release, and basal dye uptake assays revealed increased channel activity of the PANX1 R152H variant, which led to increased cytotoxicity following long-term expression. In contrast, R24H mutant PANX3 channels exhibited a loss-of-function in mechanically stimulated dye uptake assays. Under stable, moderate expression conditions, this reduction in channel activity was associated with decreased cell growth, whereas overexpression led to increased cell death. In vivo, R24H expression in zebrafish embryos increased apoptosis and upregulation of p21 and osteogenic genes. Together these findings demonstrate that two mutations with opposing alterations in PANX channel activity, hyperactivity in R152H PANX1 and loss-of-function in R24H PANX3, can converge on degenerative cellular outcomes. Collectively, we report the first germline PANX3 mutation associated with disease and provide the first evidence linking PANX1 and PANX3 mutations to human erosive osteoarthritis.

The rise of Klebsiella pneumoniae (Kp) as an MDR pathogen has become a pressing global health challenge, contributing to UTIs and Pneumonia. A major factor underlying its persistence and antibiotic resistance is its inherent ability to form protective biofilm matrices. This study explores a natural approach to combat Kp biofilms by leveraging the antibacterial potential of plant-derived essential oils. Among bioactive compounds, 2-Phenyl ethyl methyl ether, a constituent of kewda oil, appears as a viable option. Our investigation revealed that 2-PEME exhibits significant antibacterial activity against Kp with a MIC of 50 mM, and the clinical isolate was further confirmed by morphological and molecular characterization. Notably, at a sub-MIC concentration of 25 mM, 2-PEME disrupted biofilm formation, reducing biofilm biomass by 61.17% ± 3.75% and 59.30% ± 6.24% in Kp and MTCC-109, respectively. These findings were further corroborated through microscopic analysis, providing strong evidence of 2-PEME's biofilm-inhibiting potential. To elucidate the mechanism of action, molecular docking studies were performed, which predicted strong binding affinities between 2-PEME and key biofilm-associated and drug-resistant proteins in Kp. Cell viability assays conducted on HEK-293, 3T3L1, and L929 cell lines demonstrated no cytotoxic effects, confirming their viability as a safe alternative. Computational pharmacokinetic analysis further supported its drug-like properties.

Collectin-11 (CL-11) is a complement-activating pattern recognition molecule with structural and functional similarities to mannose-binding lectin (MBL), produced in different tissues, including lung epithelium. Given its tissue localization and role in innate immunity, we investigated its potential to recognize and neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to activate complement. We produced recombinant CL-11, MBL, as well as wild-type, variant, and glycan-mutated SARS-CoV-2 Spike (S) proteins. We evaluated CL-11 binding to the S protein, complement activation, and inhibition of S protein-receptor binding using ELISA, as well as neutralization of SARS-CoV-2 in cell-based neutralization assays. CL-11 bound different SARS-CoV-2 S protein variants with similar binding preferences as MBL, targeting multiple glycan sites. Upon S protein binding, CL-11 mediated activation of both the lectin and alternative complement pathways, and inhibited S protein-receptor binding. Notably, CL-11 neutralized SARS-CoV-2, inhibiting infection of permissive cells. CL-11 binds different SARS-CoV-2 variants and neutralizes SARS-CoV-2 in an antibody-independent manner, suggesting a crucial role in early-stage infection control.

Ulcerative colitis (UC) is a chronic inflammatory bowel illness with few treatment options, which means that new ways to treat it are needed. This study examined the protective effects and mechanisms of Paragonimus proliferus metacercaria-derived antigens (PmAg) in a dextran sulfate sodium (DSS)-induced mouse ulcerative colitis model. We discovered that intraperitoneal delivery of PmAg substantially mitigated colitis severity, as demonstrated by decreased weight loss, lower disease activity index scores, maintained colon length, and enhanced histopathological findings. Mechanistically, PmAg inhibited pro-inflammatory cytokines (IL-1β, TNF-α), increased the anti-inflammatory cytokine IL-10, and bolstered antioxidant defenses (SOD, GSH). It also restored the integrity of the intestinal barrier by boosting the number of goblet cells and the expression of tight junction proteins (Occludin, Claudin-1), while stopping the activation of the nuclear factor-kappa B (NF-κB) signaling pathway. Moreover, 16S rRNA sequencing demonstrated that PmAg reinstated gut microbiota α-diversity, diminished pathogenic genera (e.g., Escherichia-Shigella), and enhanced beneficial taxa (e.g., Lachnospiraceae_NK4A136_group and Alistipes). Integrated fecal metabolomics research revealed that PmAg altered metabolic profiles, specifically as significantly enriched the primary bile acid biosynthesis pathway, alpha-Linolenic acid metabolism pathway and Ubiquinone and other terpenoid-quinone biosynthesis pathways. In conclusion, our results suggested that PmAg could mitigates experimental colitis in mice by anti-inflammatory, improving gut microbiota and modulating fecal metabolomics.

Alaska pollock protein (APP) promotes skeletal muscle hypertrophy, particularly in fast-twitch fibers. Using a time-controlled feeding model, plasma collected 90 min after APP intake markedly enhanced C2C12 myotube hypertrophy and contractility. At this time point, plasma glucagon-like peptide-1 levels were significantly elevated, suggesting the involvement of circulating factors associated with APP intake. Proteomic and lipidomic analyses of skeletal muscle identified 2302 proteins and 538 lipids, and revealed pronounced remodeling of lipid metabolism, including a 127% increase in docosahexaenoic acid (DHA)-containing phospholipids in hypertrophied fast-twitch fibers, partially resembling endurance training adaptations. APP also upregulated adipose triglyceride lipase and hormone-sensitive lipase in the skeletal muscle, indicating enhanced lipid catabolism. Although APP contained only trace amounts of DHA (~0.008% of the diet), this level was substantially lower than the dietary DHA level previously reported to alter tissue lipid composition. Therefore, APP-derived DHA was unlikely to be the primary driver of the observed phenotype. Together, these findings indicate that APP functions primarily as a high-quality protein source that promotes muscle anabolic responses and is associated with remodeling of lipid metabolic pathways, thereby potentially contributing to a circulating environment favorable for skeletal muscle hypertrophy and metabolic regulation.

The provisioning of royal jelly for developing larvae by nurse bees is fundamental to social interaction in honey bee colonies. While royal jelly production is regulated by collective larval demand, it remains unclear how colony-level needs are translated into individual worker behavior. Here, we show that the larval pheromone E-β-ocimene (EBO), a volatile compound also used by pollinators as a floral food cue, elicits an intrinsic craving for protein in nurse bees that drives increased pollen consumption. Through in vitro and in vivo experiments, we demonstrate that this response is mediated by the leucokinin (Lk) and leucokinin receptor (Lkr) system, acting through the PKA-CREB-IRS signaling pathway to modulate the expression of the insulin receptor substrate gene (Irs). Elevated pollen intake then promotes the enlargement of the hypopharyngeal glands and enhanced production of major royal jelly proteins. Our findings uncover a molecular mechanism linking larval signaling to worker nutrition, highlighting how social bonds between honey bee larvae and nurses are rooted in ancestral pathways of protein hunger that predate eusociality.

Hyperosmolarity caused by drought, high salinity, or cold stress inhibits plant growth and crop productivity. A conserved protein-kinase cascade of cytosolic B-RAFs and SnRK2s is rapidly activated upon osmotic stresses to initiate downstream adaptive responses, which represents one of the fastest known responses to osmotic stress in plants. How the kinase cascade is activated by osmotic stress is unknown. Here, we show that Arabidopsis B4 subgroup RAFs have intrinsically disordered regions and directly sense both ionic and nonionic hyperosmolarity by reversible condensation. B4-RAFs recruit and cocondense with subclass-I SnRK2s to phosphorylate and turn on SnRK2s, evading the noncondensable inhibitory A-clade PP2C phosphatases. This straightforward osmosensing and relaying module can be fully reconstituted in Escherichia coli by coexpressing three components or in solution in a test tube using recombinant proteins. Our findings identify B-RAFs as the chief cellular osmosensors that detect low water potential by cocondensation, forming a signal hub with SnRK2s to orchestrate adaptive responses in plants, and represent an evolutionarily conserved osmosensing mechanism across kingdoms.

Thyroid hormones (THs) initiate metamorphosis in vertebrates, although the evolutionary origins of this process are uncertain. Here, we show that most TH processing genes are present in the proto-vertebrate model Ciona and play an instructive role in initiating metamorphosis, whereby swimming tadpoles with a chordate body plan are transformed into sessile filter feeders. Exogenous thyroxine (T4) accelerates metamorphosis, whereas TH inhibitors delay onset. Most notably, we present evidence that TH activates Opsin2 (Opsin1/2b) in a subset of photoreceptor cells in the simple brain of swimming tadpoles to initiate attachment, the first step in metamorphosis. Our findings suggest a deep evolutionary origin of TH-driven visual plasticity in vertebrates. We highlight the parallels between attachment of Ciona tadpoles and smoltification, whereby young salmonid fishes switch from UV to blue opsins for their transition from fresh to saltwater.

Some sacoglossan sea slugs (Gastropoda:Heterobranchia) possess the remarkable ability to sequester functional chloroplasts into digestive cells. Elysia atroviridis harvests chloroplasts from its algal prey, which are maintained for a relative long period in cells of the digestive tract. We successfully assembled a high-quality chromosome-level genome of E. atroviridis using PacBio and Hi-C sequencing technologies. The final assembly spans 829.0 Mb with contig and scaffold N50 length of 2.0 Mb and 43.9 Mb, respectively, 89.3% of the assembled sequences were anchored to 15 pseudochromosomes. We found no evidence for horizontal gene transfer (HGT), specifically, no photosynthetic genes encoded in the E. atroviridis nucleus genome. A total of 16,472 protein-coding genes were predicted, of which 96.0% were functionally annotated. Phylogenetic analysis indicated E. atroviridis and E. timida formed a clade and their divergence time was estimated approximately 33 million years ago (Mya). Collinearity analysis revealed a high level of synteny with the genome of both species. This genome provides a valuable resource for further investigation into the evolution and mechanisms of kleptoplasty in sacoglossa.

Gap junction plaques (GJPs) enable direct intercellular communication and consist of connexin channels arranged into two-dimensional lattices. While structures of purified connexin channels have informed models of gating, they omit key intracellular regions and lack native context. Here, we use cryo-electron tomography and focused ion beam milling to determine the in situ structure of human connexin 43 (Cx43) GJPs in HEK293 cells at 14-Å resolution. We reveal a previously unresolved structural contribution of the large carboxyl-terminal domain to lateral channel-channel interactions that appear critical for plaque assembly. Coarse-grained molecular dynamics simulations suggest how lipids and cholesterol occupy the space between adjacent connexins. These findings resolve a decades-old question regarding gap junction organization and highlight a mechanistic function for the carboxyl-terminal domain, likely regulated by a helix-loop-helix motif. Our study provides a structural blueprint for understanding how connexin diversity and regulation shape tissue-level communication in health and disease.

Retinitis pigmentosa (RP) is a leading cause of inherited blindness, yet current gene supplementation strategies are limited by heterogeneous responses, with more than 40% of patients showing insufficient rescue. Moreover, oxidative stress constitutes a defining pathological feature of RP and critically impairs the efficacy of gene therapy. Consistently, transcriptomic and ultrastructural analyses of Pde6brd10/rd10 (rd10) retinas revealed early and progressive dysregulation of oxidative stress-related pathways and photoreceptor degeneration. To overcome this barrier, we engineered an adeno-associated virus (AAV) vector covalently conjugated with a catalytic G-quadruplex-hemin DNAzyme (CoG4) via genetic code expansion and click chemistry. This design enables synchronized delivery of CoG4 and therapeutic Pde6b into photoreceptors, where CoG4 directly scavenges excess ROS and restores mitochondrial homeostasis, thereby creating a favorable microenvironment for gene supplementation. In rd10 mice, AAV-CoG4 treatment resulted in sustained expression of Pde6b, preservation of photoreceptor morphology, restoration of rod and cone function as evidenced by electroretinogram, and improved visual behavior, outperforming AAV or CoG4 monotherapies. Our findings establish oxidative stress as a major barrier to retinal gene therapy and demonstrate a dual-function platform that couples microenvironment modulation with genetic correction, offering a broadly applicable strategy for treating degenerative retinal diseases.