International Journal of Biological Sciences

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.

Non-small cell lung cancer (NSCLC) is the most common form of lung cancer accounting for most cancer-related deaths worldwide. Despite substantial recent advances in targeted therapies and immunotherapy, the prognosis for advanced-stage disease remains comparably poor, which is why the identification of novel molecular biomarkers as well as therapeutic targets influencing tumor development, progression, and metastasis remain important. This study focusses on SERPINB13, a serine-protease inhibitor expressed in selected tissues that is dysregulated in several tumor entities. However, its role in NSCLC still remains largely unclear. We analyzed SERPINB13 transcription in a cohort of non-small cell lung cancer (NSCLC) cases including both lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) by transcriptome profiling. Epigenetic modifications were assessed via Methylation BeadChips. Additionally, SERPINB13 protein expression was assessed by immunohistochemistry (IHC) in an independent cohort of NSCLC comprising 126 LUSC patients. Correlation analyses were performed to associate SERPINB13 expression with key clinico-pathological parameters, including overall survival and extent of tumor-infiltrating immune cells. To functionally investigate the regulatory influence of peripheral blood mononuclear cells (PBMCs) on SERPINB13 expression in LUSC tumor cells in vitro, we utilized the SERPINB13-expressing LUSC cell line LUDLU-1. Here, gene transcription was analyzed by quantitative real-time PCR (RT-qPCR), confirmed by Western blot on the protein level. Transcriptome analysis revealed a significant upregulation of SERPINB13 in lung squamous cell carcinoma (LUSC) compared to lung adenocarcinoma (LUAD), highlighting a subtype-specific expression pattern. This differential expression was further associated with a distinct epigenetic DNA methylation signature at the SERPINB13 loci in LUSC, suggesting transcriptional regulation via hypomethylation. IHC analysis demonstrated that high SERPINB13 protein expression is significantly associated with prolonged overall survival in LUSC. Notably, SERPINB13 expression was enriched in immune-inflamed ("hot") tumors, characterized by elevated infiltrating lymphocytes and immune activation. Mechanistically, co-culture experiments with PBMCs induced SERPINB13 expression in a LUSC cell line in a dose- and time-dependent manner in the absence of direct cell contact. This suggests that soluble factors secreted by immune cells might play a key role in regulating SERPINB13 expression in the tumor microenvironment. Taken together, SERPINB13 is a novel prognostic indicator in LUSC that is modulated by Immune cells. Further studies are necessary to decipher the crosstalk of Immune cells on the Serpin B13 expressing tumor cells in depth, with regard to a possible interventional strategy. immunomodulatory potential strategies and personalized therapeutic approaches in NSCLC.

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.

Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder caused by pathogenic variants in members of the BMP9/ALK1 signaling hub. In the present study we show that, regardless of whether the alterations are caused by reduced BMP9/ALK1 signaling (pathogenic variants in the ENG or ALK1 genes) or by overactivation of this pathway (such as the SMAD6 pathogenic variants), all are associated with increased endothelial cell (EC) proliferation and high levels of ERK MAPK activation in patient biopsies. We reproduced this phenotype in vitro in ECs lacking SMAD6 or after SMAD1 knockdown using siRNA. Loss of SMAD6 leads to dysregulation of the Notch pathway, with downregulation of phosphatases and consequent overstimulation of ERK. In normal ECs, BMP9 and Notch signaling inhibit ERK activity by upregulating PPP1R3C, a regulatory subunit of the PP1 phosphatase. Notably, BMP9-mediated inhibition of ERK is abolished when cells are transfected with siRNA targeting PPP1R3C. ERK hyperactivation was also observed in an HHT2 mouse model (ALK1-2loxP;Cdh5-CreERT2). Loss of both ALK1 alleles in adult mice leads to vascular failure and hemorrhages in the lung and intestine; these injuries are significantly reduced by treatment with the MEK/ERK inhibitor selumetinib. Overall, our work identifies a key role for ERK activation involved in HHT pathogenesis, suggesting that ERK inhibition may represent a promising therapeutic strategy for these patients. Translational PerspectiveHereditary hemorrhagic telangiectasia (HHTs) is a rare vascular disorder caused by mutations in members of the BMP9/ALK1 signaling hub. In the present study we show that all different forms of HHTs are associated with increased endothelial cell (EC) proliferation, which correlates with high levels of ERK activation in patient biopsies. ERK hyperactivation is also observed in an HHT2 mouse model in which loss of both ALK1 alleles in adult mice leads to vascular failure and hemorrhages in the lung and intestine. These injuries are significantly reduced by treatment with the MEK/ERK inhibitor selumetinib. Overall, our work identifies a key role for ERK activation in HHT pathogenesis, suggesting that ERK/MEK inhibitors may represent a promising therapeutic strategy for these patients.

SMAD4 is a central transcriptional effector of the TGF-{beta} signaling pathway and a frequently inactivated tumor suppressor gene in various cancers. Missense mutations in its MH2 domain are among the most prevalent somatic alterations in colorectal cancer (CRC). These mutations are associated with disease progression and poor prognosis, yet their precise mechanistic consequences have remained incompletely characterized. Here, we show that CRC-derived SMAD4 MH2 hotspot mutations (D351H, S357P, R361C, and R361H) selectively impair co-activator recruitment without disrupting chromatin occupancy. RNA-seq profiling demonstrated broad suppression of TGF-{beta} target gene expression across all mutants. Notably, the mutations confer distinct degrees of TGF-{beta} pathway unresponsiveness: R361H is completely refractory to TGF-{beta} stimulation, whereas R361C and S357P retain partial transcriptional responsiveness suggesting allele-specific differences in the severity of co-activator interface disruption. Genome-wide chromatin binding analysis by greenCUT&RUN confirmed that all mutants maintain wild-type-like genomic occupancy, as expected given that the MH1 DNA-binding domain is intact in each case. Proximity-dependent biotinylation mass spectrometry in COLO205 cells revealed that all four mutants exhibit markedly reduced interactions with the CREBBP/EP300 histone acetyltransferase complex and BRD4 relative to wild-type SMAD4 identifying disrupted co-activator engagement. Collectively, our findings establish that SMAD4 MH2 mutations impair TGF-{beta}-induced transcription by selectively reducing CREBBP/EP300 recruitment, which provides a molecular mechanism for the loss-of-function SMAD4 phenotype in CRC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/735541v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@14f542eorg.highwire.dtl.DTLVardef@11fd220org.highwire.dtl.DTLVardef@1c3aa1org.highwire.dtl.DTLVardef@14d5a8e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Objectives; The role of nuclear receptor subfamily 4, group A, member 3 (NR4A3) in hepatic steatosis, inflammation, and insulin resistance (IR) within the context of metabolic dysfunction-associated steatotic liver disease (MASLD) remains largely underexplored. Consequently, this study aimed to examine NR4A3's impact on MASLD and the potential underlying mechanisms. Methods; We aimed to elucidate the functional role of NR4A3 in MASLD through its knockdown in cell culture and animal models. To establish the cell culture model of MASLD, LO2 cells were treated with free fatty acids (FFAs), while male C57BL/6 mice were fed a high-fat diet (HFD) to create the animal model. NR4A3 knockdown was achieved using specific short hairpin RNA (NR4A3-shRNA) in the mice model and three small interfering RNAs (NR4A3-siRNAs) in the cell culture model. The lipids content, fatty acid synthesis, inflammatory factors, and IR were then assessed with and without NR4A3 knockdown. Furthermore, the underlying mechanism through which NR4A3 exerts its influence was explored by analyzing the interaction between NR4A3 and activating transcription factor 3 (ATF3). Results: In the cell culture experiments, the knockdown of NR4A3 significantly decreased the lipids content, fatty acid synthesis, and inflammatory factors in the LO2 cells treated with FFAs in the NR4A3-shRNA group compared with those in the NC-shRNA control group. In the animal model experiments, NR4A3 knockdown in the HFD male C57BL/6 mice significantly ameliorated HFD-induced hepatic steatosis, inflammation, and IR. Mechanistically, the knockdown of NR4A3 downregulated the expression and transcriptional activity of ATF3, resulting in an impaired ATF3 function. ATF3 overexpression significantly reversed lipid accumulation decline and reduced inflammation after NR4A3 knockdown. Conclusion: The downregulation of NR4A3 alleviates MASLD by modulating ATF3, suggesting this may be a promising therapeutic target.

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.

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.

Mucopolysaccharidosis III type C (MPS IIIC) is a rare neurological lysosomal storage disorder caused by genetic deficiency of the lysosomal membrane enzyme, heparan--glucosaminide N-acetyltransferase (HGSNAT). To assess the feasibility of therapeutic strategies based on cross-correction of neurons by HGSNAT secreted from transplanted cells overexpressing the enzyme, we generated induced cortical neurons (iCN) from induced pluripotent stem cells (iPSCs) derived from MPS IIIC patients. The neurons were treated with extracellular vesicles (EV) purified from the culture medium conditioned by human endothelial cells transduced with a lentiviral vector encoding EGFP-tagged HGSNAT (LV-HGSNAT-EGFP). The isolated EV showed supraphysiologic HGSNAT activity levels and efficiently delivered the enzyme to the lysosomes of MPS IIIC iCN reducing lysosomal size and restoring normal synaptic protein levels. EV-mediated delivery of HGSNAT to neurons was further confirmed by the analysis of MPS IIIC iCN either co-cultured with iPSC-derived MPS IIIC microglia (iMGL) transduced with LV-HGSNAT-EGFP or treated with the iMGL conditioned medium. MPS IIIC iCN co-cultured with iMGL overexpressing HGSNAT achieved a complete phenotypic rescue, including normalization of lysosomal size, and the levels of heparan sulfate, GM2-ganglioside, synaptic proteins and brain-derived neurotropic factor. Treatment of MPS IIIC iCNs with conditioned medium led to a partial defects correction. Our findings reveal the translational potential of EV-mediated enzyme delivery in MPS IIIC patients treated with LV-mediated haematopoietic progenitor stem cell gene therapy.

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

Malignant melanoma is frequently driven by constitutive activation of the RAS-RAF-MEK1/2-ERK1/2 pathway, yet adaptive signaling limits the long-term efficacy of MAPK-targeted therapies. Although activation of the MEK5-ERK5 pathway has emerged as a mechanism of resistance to RAF-MEK1/2-ERK1/2 inhibition, whether ERK5 inhibition reciprocally activates the canonical MAPK cascade and the molecular basis of this crosstalk remain unknown. Here, we show that genetic and pharmacological inhibition of ERK5 induces further activation of the MEK1/2-ERK1/2 pathway in BRAFV600E melanoma cells. Based on our previous transcriptomic analyses, we investigated the role of the small GTPase R-Ras, identified among the genes upregulated following ERK5 silencing. Accordingly, R-Ras mRNA and protein levels increased upon both genetic and pharmacological ERK5 inhibition, whereas R-Ras silencing abolished ERK1/2 hyperactivation and potentiated the anti-proliferative and pro-apoptotic effects of ERK5 targeting. Conversely, inhibition of the RAF-MEK1/2-ERK1/2 pathway increased R-Ras expression and ERK5 activation, both of which were prevented by R-Ras depletion. Besides ERK1/2, overexpression of a constitutively active mutant of R-Ras promoted ERK5 activation, placing R-Ras upstream of both signaling cascades. Finally, the pan-Ras inhibitor RMC-6236 potentiated the antitumor activity of either ERK5- or RAF-MEK1/2-ERK1/2-targeted therapies in either two-dimensional cultures or melanoma spheroids. Collectively, these findings identify R-Ras as a central regulator of reciprocal rewiring between ERK1/2 and ERK5 pathways under targeted MAPK inhibition. Functional disruption of this signaling circuit enhances melanoma cell death, providing a mechanistic rationale for co-targeting R-Ras together with MAPK signaling to limit adaptive responses to targeted therapy in BRAFV600E melanoma.

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.

The identification of small molecule modulators of immune checkpoint proteins remains a significant challenge in drug discovery due to the flat, featureless nature of protein-protein interaction interfaces and the characteristically low hit rates observed in conventional high-throughput screening campaigns. Here we report OracleScreen-LILRB4, an ensemble machine learning framework trained on quantitative biophysical screening data from two structurally diverse compound libraries (19,800 compounds total) screened against the myeloid immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3). By formulating binding prediction as a regression task targeting continuous {Delta}Fnorm values rather than binary hit classifications, OracleScreen-LILRB4 achieved a mean Spearman R of 0.61 and ROC-AUC of 0.86 under scaffold-aware cross-validation. Prospective virtual screening of a 45,760-member compound library and experimental validation of the top 200 predictions yielded a 28.5% hit rate, representing a 15.0-fold enrichment over baseline, with 16 compounds demonstrating nanomolar-affinity LILRB4 (ILT3) engagement. Lead compounds ORS-22 and ORS-14 restored anti-tumor immune activity across patient-derived colorectal cancer and acute myeloid leukemia co-culture systems, reversing SCG2-mediated immunosuppression and recovering cytotoxic T-cell function. These findings establish OracleScreen-LILRB4 as an effective computational framework for accelerating small molecule discovery against non-enzymatic immune checkpoint targets. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/732859v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1ef70a9org.highwire.dtl.DTLVardef@cd976dorg.highwire.dtl.DTLVardef@1907ebforg.highwire.dtl.DTLVardef@1716aec_HPS_FORMAT_FIGEXP M_FIG C_FIG

Despite significant advancements in microsurgical techniques in recent years, the treatment and prognosis of craniopharyngiomas remain unsatisfactory. As a central nervous system tumor located adjacent to important brain structures such as the hypothalamus-pituitary axis and accompanied by a highly inflammatory microenvironment, the tumor heterogeneity and tumor microenvironment characteristics of papillary craniopharyngiomas (PCPs) remain unclear. In this study, we integrated multimodal single-cell and spatial profiling from PCP tissue and peripheral blood mononuclear cells (PBMCs) to elucidate the tumor heterogeneity and microenvironment characteristics of PCP. Our single-cell and spatial analyses defined four specific tumor cell states in PCP, representing specific transcriptional regulatory programs and spatial heterogeneity characteristics during tumor progression. By constructing a spatial niche composed of tumor, immune, and stromal cells, we analyzed the cellular and spatial ecosystem of PCP at multiple levels to further assess the communication relationships between different tumor cell states and microenvironment cells. This study established a multidimensional molecular atlas of PCP from the perspectives of cell state, spatial structure, and microenvironment interactions, providing a foundation for understanding its biological behavior and exploring new intervention strategies.

Interleukin 27 (IL-27) is an anti-HIV cytokine that induces 14 novel microRNAs (miRNAs) in T cells. We previously reported that transfection of two of these miRNA mimics, miRTC10 and miRTC14, differentially induced interferons (IFN)A2, A8, A13, and L1 expression in human primary macrophages. However, the mechanism underlying this activation remains unclear. Here, we show that miRTC14 does not directly target IFN-regulatory genes but instead engages cytosolic RNA-sensing proteins. Using miRNA pull-down coupled with mass spectrometry and immunoblotting, we identified a metabolic enzyme, pyruvate carboxylase (PC) and laboratory of genetics and physiology 2 (LGP2/DHX58) as direct binding partners of miRTC14. Functional analyses revealed that miRTC14 induces IFN expression by more than100-fold (p < 0.001), whereas PC and LGP2 depletion markedly attenuated this response (50-100 fold reduction, p < 0.01). Reconstitution of PC and LGP2 in deficient HEK293 cells restored miRTC14-driven IFN induction. We found that miRTC14-induced IFN activation depends on sequence features at the duplex termini and is unlikely to arise from canonical miRNA-mediated gene silencing. These findings establish PC as a novel miRNA-binding protein and define a previously unrecognized RNA-sensing mechanism by which miRTC14 drives IFN production, linking metabolic enzymes to RNA sequence-dependent innate immunity.

Lebers Hereditary Optic Neuropathy (LHON) is a maternally inherited mitochondrial disorder characterised by painless, progressive, and sequential visual failure. Most cases of LHON are driven by mitochondrial DNA mutations which cause dysfunction of respiratory Complex I, triggering retinal ganglion cell loss. Retinal ganglion cell degeneration in LHON is thought to be linked to reduced production of metabolic intermediates and adenosine triphosphate, and enhanced reactive oxygen species production. Thus, decades of research have focussed on LHON as a disease of the retinal ganglion cells, which has considerably improved our understanding of the pathology but has yielded few therapeutic interventions. In addition, some LHON-associated phenomena remain unclear. In particular, we still do not fully understand the mechanisms underlying the recorded phenomenon of spontaneous visual recovery, in which patients experience measurable increases in visual acuity following onset of LHON vision loss. Understanding this phenomenon may be critical for developing new therapeutic approaches for LHON. Moreover, the contribution of non-neuronal cell populations to LHON pathology remains poorly understood despite a growing appreciation for the roles played by these cells in other neurodegenerative conditions. Astrocytes are a highly heterogeneous group of glial cells, found throughout the central nervous system including the retina and optic nerve, and are well-known for their role as key homeostatic mediators. In recent years, our appreciation for the role played by astrocytes in neurodegenerative diseases has expanded considerably, and we are now aware that astrocytes undergo significant loss of their homeostatic functions in neurodegenerative disease, acting as key mediators of neuronal loss. Importantly, the contributions astrocytes make toward mediating LHON pathology and visual recovery remain unclear, and provide promising ground for potentially novel therapeutic angles and enhanced understanding of this complex pathology. Here, we leverage human iPSC-derived astrocytes from patients with the LHON m.14484T>C genotype, to explore the role astrocytes play in LHON pathology, stratifying cells by their visual recovery status. We report that astrocytes undergo significant morphological and bioenergetic compromise in LHON, and that differences between recovery and non-recovery astrocytes may explain individual capacity for visual recovery, potentially opening novel therapeutic approaches.

STRUCTURED ABSTRACT Purpose of Research: The generation of iPSC lines expressing 21, 56 and 79 glutamine repeats within the HTT protein and homozygous KO of HTT in the KOLF2.1J background as an additional disease series within the iPSC Neurodegenerative Disease Initiative (iNDI) collection. Major Findings: All iPSCs, even those expressing long repeats of 79Q or HTT KO, were capable of differentiating to striatal and cortical neurons, astrocytes and microglia using established protocols. General quality control stains and morphological analyses are described for each differentiation. A selected set of assays were carried out on differentiated cells; expanded repeat expressing astrocytes showed altered expression of astrocyte protein markers and morphological characteristics, and striatal neurons showed altered DARPP-32/CTIP2 colocalization. mRNAseq carried out for striatal neurons showed high similarities in gene expression changes between 79Q and KO lines compared to the unexpanded repeat. Conclusions: The KOLF2.1J isogenic CAG repeat series serves as a community resource to study HD mechanisms with the potential for direct comparison across other neurodegenerative diseases through the iNDI collection.

BackgroundInduced pluripotent stem cell (iPSC)-derived brain cells are widely utilized as in vitro models for several neuropsychiatric disorders, as they retain the donors genetic profile, offering a unique opportunity to study living human brain cells and perform controlled experimental manipulations. In this study, we conducted whole transcriptome sequencing of cortical networks (co-cultures of neurons and astrocytes) derived from 12 participants with bipolar disorder (BD) and 12 participants without a history of mental health disorders. We aimed to identify new molecular mechanisms underlying the pathophysiology of bipolar disorder. MethodsiPSCs were generated by reprogramming peripheral blood mononuclear cells using episomal vectors. They were then differentiated into neural progenitor cells and matured into cortical networks that express markers of neurons and astrocytes. Whole transcriptome data were obtained using the Illumina NovaSeq X sequencing platform. ResultsDifferential expression analysis was performed using DESeq2 in R, and the identified genes were used for gene set enrichment analysis, which identified 191 enriched pathways in BD. Of these, the toll-like signalling pathway, which is downregulated in BD, was further investigated. ConclusionOur results suggest a profound immune dysregulation in BD, particularly highlighting the immune systems role as a complex signalling network.