International Journal of Biological Sciences

Japanese Encephalitis Virus (JEV) belongs to the Flavivirus family, and the RNA dependent RNA polymerase (RdRp) domain located at the C terminus of non structural protein 5 (NS5) regulates de novo viral genome synthesis. The conserved priming loop inserted in the thumb domain of RDRP initiates de novo genome synthesis. Interestingly, the reported replication process initiates with an interaction between NS5 (methyltransferase/RDRP) and NS3 (protease/helicase), and inhibiting this interaction directly correlates with the inhibition of viral replication. The function of the priming loop in the context of the NS5 NS3 interaction is not yet known. In this study, we studied the priming loop function in the NS5 NS3 interaction and viral replication. Using a structure based drug design approach, we screened the Maybridge compound library against the RdRp priming loop and identified 15 candidate compounds based on binding energy. Among them, DSHS00151 showed significant dose dependent inhibition of the NS5 NS3 interaction with an IC50 of 3.5 micromolar in the mammalian two hybrid assay, inhibited viral infectivity with an IC50 of 2.54 micromolar, and reduced viral RNA load with an IC50 of 2.9 micromolar, compared to CD10712. Molecular Dynamics (MD) simulations revealed that DSHS00151 exhibits stronger and more stable binding with the priming loop residues (790 to 812) compared to CD10712. The molecular mechanism suggested by the docking of the NS5 and NS3 proteins showed that the priming loop tends to rotate toward the NS3 protein to stabilize the complex, and compound binding restricts this loop rotation, thus compromising the stability of the NS5 and NS3 complex and affecting viral replication. Overall, our study validated the function of the NS5 priming loop as an allosteric site, and compounds binding to this allosteric site belong to the Non Nucleoside Reverse Transcriptase Inhibitors (NNRTI) class of inhibitors, which disrupt the NS5 and NS3 interaction and could be developed as novel anti JEV therapeutics.

Histone deacetylases (HDACs) are pivotal epigenetic regulators that modulate diverse cellular pathways by removing acetyl groups from lysine residues on both histone and non-histone proteins. Histone deacetylase 11 (HDAC11), the sole member of class IV HDACs, exhibits both deacetylation and fatty acid deacylation activities. Accumulating evidence implicates HDAC11 as a key epigenetic regulator of fundamental cellular processes, including metabolism, immune responses, and tissue development. Dysregulation of HDAC11 activity has been associated with inflammatory diseases, metabolic disorders, neurodegenerative conditions, and cancer, highlighting its potential as a therapeutic target. Although several HDAC11-specific inhibitors have been identified, none have progressed to clinical development. In this study, we aimed to discover HDAC11-selective inhibitors by integrating in silico and in vitro validation approaches. Homology modelling of the HDAC11 structure was conducted, followed by model validation, structure-based virtual screening, molecular dynamics (MD) simulations, and binding free energy calculations. We identified and validated three lead compounds and their intermediates using biochemical and cell-based assays. Fluorescence-based and HPLC-based enzymatic assays demonstrated potent inhibition of both the deacetylase and deacylase activities of HDAC11, with Inhibitor 6 and Inhibitor 3 exhibiting the strongest effects among the six compounds tested. Further, a decrease in lipid accumulation, reduced stability of the HDAC11 substrate SHMT2, as determined by immunoblot analysis and decreased cell viability, as assessed by MTT assay, confirmed HDAC11 inhibition in cellular models. The study shows that new HDAC11 inhibitors significantly reduce the viability of breast cancer cells and induce apoptosis; inhibitor 6, in particular, showed high potency, similar to the reference compound SIS-17. Flow cytometry showed that treated MDA-MB-231 cells exhibited cell-cycle arrest and increased apoptosis, a finding further confirmed by Annexin V/PI staining. Molecular analysis showed that BAX increased while BCL2 decreased, indicating that apoptotic pathways were activated in novel compound-treated MDA-MB-231 cells. The results suggest that inhibiting HDAC11 is an effective way to induce cancer cell death and provide a basis for further assessment of these compounds as potential treatments for breast cancer. Collectively, this study identifies novel zinc-chelating HDAC11 inhibitors containing a nitro-sp2 group, providing promising candidates for further therapeutic development.

A protein whose removal or deletion causes significant disruption or collapse of a protein-protein interaction (PPI) network is referred to as a vulnerable protein. Such proteins may serve as valuable therapeutic or diagnostic targets in disease-associated networks. In this study, two PPI networks were constructed, one for HPV-positive and the other for HPV-negative head and neck squamous cell carcinoma (HNSCC), and the vulnerable proteins of these networks were identified by the node deletion approach. After analyzing the networks, 27 unique vulnerable proteins in HPV-positive and 72 unique vulnerable proteins in HPV-negative HNSCC were identified. Among them, one HPV-positive and seven HPV-negative HNSCC vulnerable proteins were further chosen by integrating multi-omics data. To exploit the vulnerabilities of these proteins, candidate synthetic lethal (SL) partners were predicted whose inhibition may selectively impair tumor survival. Subsequently, drug-gene interaction analysis was performed to identify inhibitors targeting the SL partners of these vulnerable proteins. Notably, in HPV-positive HNSCC, TOP2A, CHEK1, and CHEK2 genes were identified as SL partners of TTN, and their inhibitors were already clinically approved. While in HPV-negative HNSCC, ADA and MMP19 were identified as an SL partner of LMO7; TMEM45B, CDH3, and ELF3 genes were identified as an SL partner of CGN; and ZNF433 was identified as an SL partner of FLNC. However, MMP19, ZNF433, and TMEM45B inhibitors were not reported. Thus, these vulnerable proteins, including their SL partners, provide novel avenues to explore and develop more efficient and precise therapeutic and diagnostic strategies.

Marine sponges are known sources of bioactive natural products (NPs), many of which are produced by associated bacterial symbionts via encoded biosynthetic gene clusters (BGCs). A particularly interesting subclass of sponge-derived NPs is comprised of small, brominated alkaloids, which are recovered from diverse habitats and host sponge taxonomies. Despite having been described decades ago, most of these NPs do not have an elucidated biosynthetic origin. We queried metagenomes of several sponge species by making use of a minimal set of core enzymes that we postulate to be necessary to produce these small peptidic NPs: an FADH2-dependent halogenase and an AMP-binding adenylation enzyme. This revealed a variety of novel BGC architectures, many of which showed conservation among sponge host phylogenies and were encoded in the genomes of diverse sponge-associated bacteria. Furthermore, we identified a BGC in the sponge G. barretti that is potentially linked to the production of the iconic barettins, given its enzymatic machinery and specific acidobacterial origin. The present work contributes to the challenging quest to link orphan brominated NPs to their parent BGCs in the sponge holobiont and beyond.

Prostate cancer progression is accompanied by dynamic remodeling of malignant cell states, whose metastatic potential and regulatory programs remain poorly defined. Here, we integrated single-cell, spatial, bulk transcriptomic, epigenomic, and experimental data to characterize malignant cell state transitions and identify candidate regulators associated with metastatic progression. A distinct intermediate state exhibits increased copy-number alterations, stemness, proliferation, and MYC activity, together with the highest transcriptional similarity to metastatic tumor cells, and is therefore designated as a metastasis-initiating-like (MIC-like) state. Transcriptional and regulon dynamics along the malignant cell state trajectory highlight FEV as a prominent candidate associated with the MIC-like state, with concordant expression and regulon activity patterns. Functionally, FEV is associated with a hybrid epithelial-mesenchymal phenotype, and its perturbation disrupts this phenotype, accompanied by reduced epithelial and mesenchymal features as well as impaired prostate cancer cell migration and invasion. Beyond this tumor-intrinsic feature, FEV is also associated with reduced cytotoxicity and interferon responses in the tumor microenvironment through its regulation of MIF-family genes. In addition to its potential roles in tumor cell plasticity and immune remodeling, FEV shows a reciprocal association with androgen signaling and distinct expression patterns across treatment response groups, highlighting its potential relevance in therapeutic contexts.

Objective White smoke inhalation injury (WSI) causes severe acute lung damage with no specific therapy currently available. Sphingolipid metabolism is implicated in pulmonary inflammation, but its transcriptional regulatory landscape in WSI remains unexplored. This study aimed to identify key sphingolipid metabolism related genes and evaluate their regulatory roles and therapeutic potential in WSI. Methods We established a rat model of WSI and performed integrated bulk RNA sequencing, weighted gene coexpression network analysis (WGCNA), and single-cell RNA sequencing (scRNAseq) to screen for differentially expressed sphingolipid metabolism-related genes (DESRGs). Protein-protein interaction (PPI) network with four centrality algorithms was used to prioritize hub genes. In silico gene knockout and molecular docking were conducted to assess regulatory functions and identify potential drug candidates. Results We identified 22 DESRGs that were predominantly enriched in DNA replication and cell cycle pathways rather than canonical sphingolipid metabolic processes. PPI consensus prioritized three hub genes--Top2a, Ttk, and Ccna2--with Top2a exhibiting the highest expression in epithelial cells and significant downregulation after smoke exposure. ScRNAseq revealed immune cell infiltration and epithelial differentiation trajectories. Virtual knockout showed that Top2a depletion affected the largest transcriptomic fraction (~0.4%) and was enriched in lysosome biogenesis, innate immunity, phagocytosis, and lipid catabolism. Molecular docking identified thalidomide as a high affinity ligand for Top2a (Vina score: -8.5 kcal/mol). Conclusion Our multiomics integrative framework identifies Top2a as a central regulatory hub linking sphingolipid associated inflammation to epithelial responses in WSI, and nominates thalidomide as a potential drug repurposing candidate. These findings provide prioritized targets for future translational investigation.

Neurotrophins regulate neuronal survival, differentiation, and synaptic plasticity through activation of tropomyosin receptor kinase (Trk) receptors, and their dysregulation is strongly implicated in neurodegenerative disorders, including Alzheimer's disease. However, the therapeutic application of recombinant neurotrophins remains limited by poor bioavailability, rapid degradation, and inadequate blood brain barrier permeability. Here, we report the discovery of Bavachin as a dual Trk-A/Trk-B neurotrophin mimetic via an integrated computational screening and experimental validation approach. Structure-based virtual screening of a bioactive natural product library derived from Traditional Chinese Medicine, combined with a novel consensus-ranking strategy, enabled the identification of flavonoid scaffolds targeting neurotrophin-binding sites of Trk receptors. Among these, Bavachin demonstrated robust, dose-dependent activation of both Trk-A and Trk-B receptors, but not Trk-C, indicating receptor selectivity. Induced-fit docking and molecular dynamics simulations revealed stable binding of Bavachin within conserved and receptor-specific ligand-binding pockets, consistent with its dual agonist activity. Collectively, this study identifies Bavachin as a putative small-molecule neurotrophin mimetic and establishes an integrated computational and experimental framework for the discovery of dual Trk-A/Trk-B agonists for neurodegenerative disease therapeutics.

Viral infections have long been proposed as environmental contributors to neurodegenerative diseases, including Parkinson's disease (PD), yet the molecular mechanisms linking infection and neurodegeneration are not well defined. Neuroinflammation and disruption of central nervous system (CNS) homeostasis have emerged as potential mediators. In this study, we used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, as a model pathogen to investigate convergent molecular pathways between viral infection and PD. Single-nucleus RNA sequencing (snRNA-seq) was performed on post-mortem striatal tissue from 14 individuals stratified into four groups: COVID-19 only (COVID-19), PD only (PD), comorbid PD with COVID-19 (PD/COVID-19), and controls (Control). The PD/COVID-19 group exhibited an expanded astrocytic population and a pronounced interferon-associated molecular signature characterized by increased expression of canonical interferon-stimulated genes, including IFI44L (average log2FC= 3.9; adjusted p=2.3 x 10-373), IFI44 (average log2FC=2.9; adjusted p=8.0 x 10-266), ISG15 (average log2FC=3.1; adjusted p=1.2 x 10-197), and RSAD2 (average log2FC= 3.5; adjusted p=8.6 x 10-111). Pathway analyses demonstrated activation of innate immune and antiviral signaling pathways, particularly within microglia and astrocytes, including interferon signaling, pattern-recognition receptor pathways, and complement-associated responses. In parallel, genes involved in lipid metabolism, cholesterol homeostasis, synaptic maintenance, and neuronal signaling were reduced across disease groups. Proteomic analyses independently confirmed enrichment of antiviral and interferon-associated pathways and identified convergent suppression of sterol, cholesterol, and lipid metabolic processes. Our findings identify a convergent molecular signature linking PD and COVID-19, pronounced in comorbid individuals and characterized by interferon-driven innate immune activation, glial inflammatory responses, and dysregulation of lipid metabolic homeostasis. Collectively, the data support a model in which severe viral infection amplifies biological pathways already implicated in PD pathogenesis.

Melittin, a highly active natural antimicrobial peptide derived from honeybee venom, holds immense pharmacological potential against solid tumors. However, its specific anti-tumor efficacy and transcriptomic dynamics in cervical carcinoma remain to be systematically characterized. This study evaluated the anti-cancer properties of melittin on murine U14 cervical cancer cells following by transcriptomic investigation of the underlying mechanism. Phenotypic evaluations revealed that melittin potently inhibited U14 cell viability, while wound healing assays demonstrated a profound, dose-dependent suppression of cellular migration, culminating in near-complete migratory arrest at high concentrations. Furthermore, flow cytometry quantified a dramatic, dose-dependent surge predominantly in late apoptotic cell populations. These apoptotic events were structurally corroborated by scanning electron microscopy (SEM), which revealed severe plasma membrane perforation and morphological exhaustion. Enrichment analyses indicated that the physical membrane disruption inflicted by melittin translated into a severe metabolic crisis, marked by a global suppression of ribosomal biogenesis and mitochondrial oxidative phosphorylation. Furthermore, melittin profoundly repressed the Tie2-mediated angiogenic pathway (Etv2 down-regulation) while triggering the lethal hyperactivation of the AP-1 transcriptional stress complex (Jun, Fos, Fosb). Collectively, these findings elucidate the pharmacological networks underlying melittins cytotoxicity, providing solid molecular evidence for its development as a natural therapeutic agent against cervical cancer.

Kaposi's sarcoma (KS) is an angioproliferative tumor associated with infection by human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). Among the viral genes involved in KS pathogenesis, the viral G protein-coupled receptor (vGPCR) plays a key role in oncogenic transformation and angiogenesis, and its sustained activity contributes to tumor development and maintenance. Quercetin (QUE), a naturally occurring flavonoid with well-recognized anticancer properties, has been shown to reduce cell viability and proliferation and to induce apoptosis in vGPCR-transformed endothelial cells. This study further investigated the mechanisms underlying the antineoplastic effects of QUE in vGPCR cells, an in vitro model of Kaposi's sarcoma. Initially, QUE promoted the activation of MAPK signaling pathways in vGPCR cells, increasing the phosphorylation levels of ERK1/2, JNK1/2, and p38, without affecting AKT phosphorylation. In parallel, QUE increased intracellular oxidant levels, followed by lipid peroxidation and DNA damage, supporting a link between oxidative imbalance and genotoxicity. However, increased total glutathione content, and unaltered Gpx4 mRNA expression, suggest the induction of oxidative stress and compensatory antioxidant responses. Furthermore, increased intracellular oxidant levels were associated with MAPK signaling activation, particularly JNK1/2, whereas ERK1/2 and p38 activation may reflect a compensatory response that is insufficient to overcome QUE-induced cytotoxicity. Taken together, these findings indicate that QUE exerts antiproliferative effects in vGPCR cells through a mechanism involving oxidative stress, MAPK activation, and DNA damage, supporting its potential relevance in the study of therapeutic strategies for virus-associated malignancies.

Near-infrared (NIR) spectroscopy is widely used for the rapid, non-destructive determination of constituents such as protein and moisture in cereal grains, but model comparisons in this field are often confounded by differences in the inputs received by each model. We benchmark a compact one-dimensional CNN derived by one-factor-at-a-time ablation (CNN-Baseline) and a randomly searched CNN (CNN-RS) against PLSR, SVR, XGBoost on six cereal-grain datasets (n=500-5046) under three input conditions: raw spectra, optimal preprocessing, and preprocessing plus wavelength selection. A single-filter with a kernel size of 11 of convolution and batch normalization proved sufficient. Given a unified raw input, CNN-RS was most accurate on five of the six datasets, while CNN-Baseline came within 0.003-0.017 in test R2 on the four larger datasets. PLSR led only on the smallest protein dataset (XDS, n=500) and was the most stable model. Preprocessing had almost no effect on CNN accuracy on the four larger datasets, but improved accuracy by 0.034-0.047 (CNN-Baseline) and 0.017-0.055 (CNN-RS) on the two smallest, and brought the networks to their optimum in a median of 49% fewer epochs. Therefore, replacing explicit preprocessing with learned filters was only justified when sufficient training data were available. Once each model received its own preprocessing and wavelength subset, SVR became most accurate on four of six datasets and XGBoost rose from the weakest model to within 0.01-0.03 of the best model on four of the six datasets, suggesting that the principal advantage of CNNs lied in robustness to raw input data rather than in attainable accuracy. Overall, model selection for NIR calibration of cereal grains depends more on sample size and the input preparation than on network architecture.

The Dengue virus (DENV), a major global health concern, causes dengue fever, predominantly affecting tropical and subtropical regions. The NS2B-NS3 protease complex of DENV is critical for viral replication, making it a promising target for antiviral drug development. This study investigates the structural dynamics of the NS2B-NS3 protease under varying pH conditions, integrating experimental and computational approaches. The recombinant NS2B-NS3 protease was expressed in E. coli, purified using affinity chromatography, and analysed for purity via SDS-PAGE. Circular dichroism (CD) spectroscopy revealed pH-dependent secondary structural changes, indicating stability at neutral to slightly basic pH and destabilization under acidic and highly basic conditions. Dynamic light scattering (DLS) analysis demonstrated protein aggregation and structural heterogeneity under extreme pH levels. Complementary in silico techniques, including homology modelling and molecular dynamics (MD) simulations, provided detailed insights into the conformational changes of the protease. The modelled structure, validated and refined through computational tools, revealed structural stability at physiological pH, with notable disruptions at pH extremes. DSSP (Dictionary of Secondary Structure of Proteins) and principal component analyses highlighted significant secondary structural transitions, especially at acidic pH where -helices and {beta}-sheets transformed into random coils Docking and MD simulation was carried out with the small molecule for therapeutic analysis between Dengue NS2bNS3 and small molecule. This study emphasizes the pH-dependent conformational plasticity of the NS2B-NS3 protease, contributing to the understanding of its functional mechanisms and providing a foundation for the rational design of pH-specific inhibitors. These findings underscore the importance of structural biology in advancing therapeutic strategies against dengue fever.

Immune checkpoint blockade has revolutionized cancer therapy, but the therapeutic efficacy is limited. Clinical trials on blockade of newly identified immune checkpoints didn't show promising result, suggesting that it might be insufficient to understand the function of immune checkpoints in cancer merely in the context of immunity. Here, we found mutually exclusive expression patterns of the immune checkpoint VISTA (or VSIR) and the neural stemness factor SETDB1, an oncoprotein that promotes immunoevasion, in xenograft tumors, suggesting that cells with high VISTA expression represents a differentiated, and hence, less or non-malignant state in tumor. Non-neural differentiation factors HHEX, MYOD1 and PPARG promote, whereas oncoproteins KRAS (and the mutant KRAS(G12D)) and SOX2, both being embryonic neural factors, repress VISTA expression. This tendency can be inferred from the finding that neural stemness is the core property of cancer cell. Manipulated expression of VISTA in cancer cells generated no significant effect on cell tumorigenicity and differentiation state, but led to change in cell morphology and actin cytoskeleton. Mechanistically, VISTA regulates a key cytoskeleton regulator, WASF2, leading to the change in cell morphology, which might interfere with signal transduction of immune response. The results suggest that 1) high expression of a protein in tumor might represent a less or non-malignant state, targeting of which would leave malignant cells intact, and consequently, leading to weak or even no therapeutic efficacy, a key factor worth considering for target selection; 2) immune checkpoints might play other roles in cells that interfere with regulation of anti-tumor immunity.

Alcohol-associated liver disease (ALD) is a leading cause of liver-related morbidity and mortality, yet its pathogenesis remains incompletely understood. While neural regulation is critical for hepatic homeostasis, how autonomic and neurotrophic signaling is regulated in the liver during ALD progression remains largely unexplored. Here, we performed an integrative analysis of bulk and single-cell transcriptomic datasets of human livers across the spectrum of ALD. We identified stage-dependent remodeling and coordinated expression patterns of autonomic and neurotrophic genes. Autonomic signaling exhibited a consistent decline, with reduced expression of sympathetic (DBH, TH) and the parasympathetic (ChAT) markers. In contrast, pan-neuronal and neuroplasticity-associated genes (UCHL1, TUBB3, STMN2) were upregulated, indicating active neural remodeling. In parallel, factors such as MANF, GDNF, and NRG2, with the receptor NTRK1, were upregulated and positively correlated with clinical indicators of disease severity, inflammation and fibrosis. In contrast, trophic factors including NTF3 and CDNF, and receptors such as GFRA1 and GFRA3, were downregulated and inversely associated with these parameters. Single-cell analyses revealed that neurotrophic factors are predominantly expressed by hepatic stellate cells, supporting their role as resident neuroglia-like cells in the liver. In another independent cohort, we observed a coordinated downregulation of NTF3 and sympathetic nerve markers in alcohol-associated hepatitis (AH), suggesting impaired NTF3-mediated neurotrophic support may contribute to sympathetic dysfunction in AH. Collectively, these findings uncover previously unrecognized neuro-regulatory dimensions in ALD and suggest that dysregulated autonomic and neurotrophic signaling may contribute to disease progression. Conflict of interestThe authors have declared that no conflict of interest exists.

Cancer is a serious public health problem, and is becoming more common, with a projected increase in deaths and more than 25 million new cases in coming decades. A number of molecular mechanisms are involved in the tumoral process, with one of them, never in mitosis A-related kinase 2 (NEK2), a serine/threonine protein kinase, being a frequent target of amplification in various malignancies that is responsible for chromosomal instability, aneuploidy and activation of several oncogenic pathways. Available kinase inhibitors are not yet optimized in terms of their pharmacokinetic properties for clinical use, and current therapies, such as chemotherapeutic agents or immunotherapies are often limited by their resistance. In silico methods represent an effective tool to search for novel potent inhibitors, before testing in animals, with time constraints and limited resources. To find new inhibitors of NEK2, we used E pharmacophore-based modeling and structure based virtual screening in this study. NEK2 was chosen as the target for therapeutic intervention and an energy optimized pharmacophore model was employed to screen the Enamine REAL library of millions of compounds. Pharmacodynamic and Pharmacokinetic properties of the Top hits were tested using ADMET profiling. These were further screened using molecular docking (standard precision and extra precision) and virtual screening to obtain three lead compounds 1, 2, and 3 which have docking score of -7.414, -8.037 and -7.562 respectively. MM-GBSA calculations were used to estimate the binding free energies for these complexes, which were determined to be -54.92, -54.18 and -49.23 kcal/mol. Lastly, 100 ns molecular dynamics simulations have been run to evaluate complex stability in dynamic situations. The overall results of the MD showed the overall stability of the NEK2-ligand complexes, and thus these three compounds are promising NEK2 inhibitor candidates and could be further validated in vitro and in vivo for clinical application. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/742111v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@14e9cfeorg.highwire.dtl.DTLVardef@24ec78org.highwire.dtl.DTLVardef@20be75org.highwire.dtl.DTLVardef@1b80c02_HPS_FORMAT_FIGEXP M_FIG C_FIG

Psychoneuroimmunology suggests that positive physiological states, including laughter, could affect anti-tumor immunity, but the underlying mechanisms remain unclear. Here, we investigated whether {beta}-endorphin (BE), an endogenous opioid peptide associated with positive physiological stimuli, modulates Natural Killer (NK) cell cytotoxicity and the anti-tumor activity of NK-derived extracellular vesicles (EVs). Using NK-92 cells, we assessed cytotoxicity against JIMT1 breast cancer cells, CD107a mobilization, cytotoxic activity of conditioned medium (CM), and EV yield, cargo, and function. BE enhanced NK-92-mediated killing of JIMT1 cells without increasing CD107a mobilization, suggesting that improved cytotoxicity was not driven by classical degranulation. Consistently, CM from BE-treated NK cells retained contact-independent cytotoxicity. NK-EVs were enriched in granzyme B and perforin following BE treatment exhibiting enhanced cytotoxicity against JIMT1 and BW tumor cells. BE also increased the cytotoxic activity of primary human NK cells, and BE-conditioned NK-EVs primed naive NK-92 cells for enhanced tumor killing. These findings indicate that BE enhances NK anti-tumor immunity by remodeling the cytotoxic secretome and generating EVs that act as both direct cytotoxic effectors and mediators of NK cell priming. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=119 HEIGHT=200 SRC="FIGDIR/small/741417v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16d4026org.highwire.dtl.DTLVardef@18e91fcorg.highwire.dtl.DTLVardef@1124baeorg.highwire.dtl.DTLVardef@268d0e_HPS_FORMAT_FIGEXP M_FIG C_FIG Proposed neuroendocrine-immune model linking positive physiological stimuli, NK cell-derived extracellular vesicles (EVs), and anti-tumor activity. Laughter is depicted as a conceptual upstream trigger of hypothalamic-pituitary signaling leading to {beta}-endorphin (BE) release. BE conditioning enhanced NK-92 cytotoxicity and the anti-tumor activity of NK-derived EVs, consistent with granzyme B and perforin enrichment and supporting EV-mediated contact-independent cancer cell killing.

Extracellular nucleotides regulate diverse inflammatory responses through the activation of P2 receptors. Here, we show that nucleotides regulate TLR3-induced chemokine responses in murine bone marrow-derived macrophages (BMDMs). Stimulation with the TLR3 agonist, poly(I:C), induced CCL2 and CXCL1 secretion, whereas degradation of extracellular nucleotides with apyrase reduced CCL2 but enhanced CXCL1 release. In agreement, the non-selective P2 receptor antagonists suramin and RB2 reduced poly(I:C)-induced CCL2 production. Among the highly expressed P2 receptors examined, selective inhibition of P2Y6 with MRS2578 reproduced the effects of apyrase, decreasing CCL2 while increasing CXCL1 secretion. The absence of effects of MRS2578 in P2ry6-deficient BMDMs confirmed the specificity of this antagonist. Transcriptional analysis further revealed that P2Y6 inhibition reduced early poly(I:C)-induced Ccl2 and Cxcl10 expression while enhancing later Cxcl1 and Cxcl2 expression. In line with our previous works on other pattern-recognition receptors pathways and innate immune cells, this study further supports a role for extracellular nucleotides in shaping TLR-induced chemokine responses in macrophages.

Leuprolide, a widely used androgen-deprivation therapy (ADT) for prostate cancer (PC), has been associated with increased cardiovascular events (CVE), although the underlying mechanisms remain unclear. Through integrated spatial transcriptomic analyses of murine heart and immune-stromal profiling in patient blood, we identified immune programs linked to leuprolide-associated CVE risk. Spatial analyses revealed the distinct organization of lymphoid (T+B cells) aggregates and inflammatory macrophages in the plaque following leuprolide exposure that accompanied by ventricular hypertrophy, relative to normal vasculature. Parallel profiling of circulatory soluble and cellular components in patients demonstrated CD8+T cell enriched pattern in ~44% of patients, including memory and type 1 cytotoxic subsets that linked to treatment-induced CVE, such as ventricular dysfunctions, but no contribution of adipocytokines. These translational findings highlight the stratification of leuprolide-associated CVE risk in PC patients based on circulating T cell profiling and could guide the risk mitigation with alternative ADT agents.

Mitochondrial RNA (mtRNA) released into the cytosol functions as a damage associated molecular pattern that activates pattern-recognition receptor (PRR)-mediated inflammation, yet its release mechanisms and cytoplasmic fate remain poorly understood. Here we report that chemical Abt-373-treatment and Vesicular stomatitis virus (VSV) infection induce mtRNA extrusion through Bax/Bak and VDAC1 channels, accompanied by mtDNA release. Extruded mtRNA in A549 cells activates multiple cytosolic PRRs, including RIG-I, MDA5, TLR3/7/8, and PKR, each contributing differentially to the innate immune signaling. Analysis of GEO datasets and methylated RNA immunoprecipitation (MeRIP) assays further reveals that mtRNA carries methyladenosine (m6A) modification. m6A machinery proteins are involved in the cytoplasmic retention time of mtRNA and its interaction with RIG-I, thereby modulating mtRNA-induced innate immunity. Thus, our work establishes in vitro models of mtRNA extrusion, and highlights m6A-dependent modulation as a potential therapeutic target for mtRNA-driven inflammation.

PACS1 syndrome is a rare neurodevelopmental disorder caused by a recurrent de novo missense variant (p.R203W) in the PACS1 protein. However, it remains unclear whether the p.R203W variant acts through a loss-of-function or alternative mechanism. Here, we used isogenic iPSC-derived neurons (iNs) to directly compare the effects of PACS1 p.R203W to complete loss of PACS1 function. Using a combination of proteomic, biochemical and electrophysiological approaches, we identified molecular and functional phenotypes associated with each genotype. While PACS1(+/R203W) and PACS1(-/-) iNs shared phenotypic abnormalities, the overall molecular and functional consequences of the p.R203W variant were distinct from those caused by PACS1 deficiency. Notably, PACS1(+/R203W) presented with unique proteomic and kinase signaling signatures and a shift in stimulus dependent excitability. These findings demonstrate that PACS1 syndrome is not caused by a simple loss of function and instead support a non-loss-of-function mechanism. Lastly, our interactome analysis suggests that the p.R203W variant retains aspects of canonical PACS1 function while acquiring novel molecular interactions that could contribute to PACS1 syndrome pathogenesis. Altogether, these findings provide a framework for future mechanistic studies and therapeutic development in PACS1 syndrome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/747101v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@d1522corg.highwire.dtl.DTLVardef@69e4dforg.highwire.dtl.DTLVardef@30eebcorg.highwire.dtl.DTLVardef@899b9d_HPS_FORMAT_FIGEXP M_FIG C_FIG