Cell Communication and Signaling

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by high metastatic dissemination, therapy resistance, and poor clinical outcome. Inhibitor of differentiation 1 or ID1, is frequently overexpressed in PDAC and is associated with tumour progression and adverse clinical outcome. However, the mechanisms governing its post-transcriptional regulation remain insufficiently characterized. Here, we identify tumour-suppressive miR-615-5p as a regulator of ID1 expression in PDAC. Integrative in-silico target prediction prioritized miR-615-5p based on seed complementarity and thermodynamic stability with the ID1 3' -UTR. Expression analysis of available PDAC clinical datasets revealed reduced miR-615-5p expression associated with increased ID1 expression. Direct association was validated using luciferase reporter assays, where miR-615-5p suppressed 3' -UTR reporter activity of ID1 in a sequence dependent manner, while mutation of the predicted binding site attenuated this effect. Further biotinylated-RIP and AGO2-RIP assays demonstrated the co-enrichment of ID1 transcripts and miR-615-5p with AGO2 associated RISC complexes, while AntimiR mediated inhibition of miR-615-5p perturbs association between miR/ID1 to AGO2, supporting interaction specificity. Functionally, modulation of miR-615-5p altered ID1 expression and impacted PDAC cell migration in vitro. Mechanistic analyses further indicated that the miR-615-5p/ID1 axis influences autophagic flux where miR-615-5p mediated inhibition of autophagy suppresses ID1 dependent cellular migration. Collectively, these findings define a previously uncharacterized miRNA-dependent regulation of ID1 expression and link this axis to autophagy-associated migratory responses in PDAC cells. The study expands the post-transcriptional regulatory landscape of ID1 and provides a possible mechanism where suppression of miR-615-5p leads to ID1 overexpression and subsequent poor clinical outcome in PDAC cells.

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.

ZNF217 is an established oncogenic transcription factor that promotes cancer progression and therapeutic resistance; however, the mechanisms regulating ZNF217 protein abundance remain poorly understood. Here, we identify ubiquitin-specific peptidase 15 (USP15) as a critical regulator of ZNF217 stability and define a reciprocal USP15-ZNF217 signaling loop that sustains malignant phenotypes in ovarian cancer. Stable overexpression of ZNF217 in OVCA420 ovarian cancer cells enhanced proliferation, epithelial-mesenchymal transition, migration, invasion, and extracellular matrix adhesion. Notably, ZNF217 overexpression increased USP15 protein abundance without altering USP15 mRNA levels, whereas ZNF217 depletion reduced USP15 protein levels, suggesting post-transcriptional regulation. Conversely, USP15 depletion markedly reduced ZNF217 protein abundance while increasing ZNF217 mRNA levels, indicating that USP15 regulates ZNF217 predominantly at the post-transcriptional level. Proteasome inhibition restored ZNF217 protein levels following USP15 depletion, further demonstrating that USP15 promotes ZNF217 protein stability. Functionally, USP15 depletion in ZNF217-overexpressing ovarian cancer cells suppressed proliferation and multiple metastatic phenotypes, including migration, invasion, extracellular matrix adhesion, anoikis resistance, and multicellular aggregate formation. In vivo, USP15 depletion significantly reduced tumor progression and metastatic burden and prolonged survival in mice bearing ZNF217-driven ovarian tumors. Furthermore, USP15 depletion enhanced the sensitivity of ZNF217-overexpressing cells to carboplatin, paclitaxel, and doxorubicin. Collectively, these findings identify USP15 as an upstream regulator of ZNF217 protein stability and reveal a positive-feedback loop between USP15 and ZNF217 that reinforces oncogenic signaling. Targeting USP15 may therefore represent an indirect therapeutic strategy for suppressing ZNF217-driven ovarian cancer, particularly given the challenges associated with directly targeting oncogenic transcription factors.

Background: Spatial organization is increasingly recognized as a key determinant of tumor-immune interactions in head and neck squamous cell carcinoma (HNSCC). The GSE300147 Xenium spatial transcriptomic resource generated by McCord and colleagues established a framework for mapping spatially coordinated T-cell states in HNSCC. However, how tumor-enriched epithelial immune states relate to metabolic, redox, and stress-adaptive transcript programs remains incompletely defined. Methods: A secondary, data-driven reanalysis of GSE300147 was performed, focusing on 17 confirmed HNSCC Xenium sections after exclusion of a non-HNSCC ameloblastoma specimen. A total of 1,148,244 cells were analyzed, including 558,867 EpCAM+ tumor-enriched epithelial cells. Tumor-enriched epithelial cells were classified into Hot, Intermediate, and Cold states using a Composite Hotness framework integrating T-cell inflammatory signature score, checkpoint-associated signaling, CD274 expression, IFN/antigen-presentation signature score (IFN/AP), and tumor-immune proximity. Six metabolic ecosystem states, neighborhood profiling, spatial permutation testing, and an integrated Immune-Metabolic-Redox Ecosystem Score (IMRES) were then applied. Results: Immune activation was spatially heterogeneous across HNSCC sections. Immune-hot tumor-enriched epithelial regions showed not only inflammatory, checkpoint-associated, and antigen-presentation signature scores, but also coordinated metabolic, oxidative-redox, and stress-response transcript programs. IMRES, derived from available immune, metabolic, redox, and stress-response transcript components represented in the Xenium panel, increased progressively from Cold to Intermediate to Hot tumor-enriched epithelial states and was associated with NFE2L2, GDF15, HLA-DRA, CD274, KEAP1, and MDM2. Integrating IMRES with Composite Hotness identified a distinct Hot+IMREShigh ecosystem comprising 106,874 tumor-enriched epithelial cells. This state showed the strongest immune-active and stress-adaptive features and was positioned closer to immune populations than expected by random assignment. An alternative rank-based robustness analysis reproduced the IMRES-associated ecosystem axis and correlated with the original module-based score (Spearman r = 0.597). Conclusions: This secondary reanalysis extends the original spatial T-cell framework by defining a complementary tumor-centered immune-metabolic-redox ecosystem in HNSCC. IMRES provides a transcript-derived framework for identifying Hot+IMREShigh neighborhoods where immune activation, checkpoint signaling, metabolic remodeling, and stress adaptation converge, providing a hypothesis-generating framework for studying immune resistance and therapeutic vulnerability.

Lysophosphatidic acid (LPA) is widely recognized as an extracellular lipid mediator; however, the functional significance of intracellularly produced LPA remains poorly understood. Here, we investigated the regulatory mechanism and functional role of a LPA-producing lysophospholipase D GDE4, also known as GDPD1, in prostate cancer cells. GDE4 expression is induced under ER stress conditions in a PERK-dependent manner and requires the transcription factor ATF3. Disruption of GDE4 expression resulted in altered intracellular levels of LPA and LPA precursor lysophosphatidylethanolamine, accompanied by reduced cell proliferation. RNA sequencing and subsequent validation identified a set of genes downregulated in GDE4-depleted cells. Pharmacological inhibition experiments indicated that peroxisome proliferator-activated receptor and {gamma} (PPAR and PPAR{gamma}) signaling pathways contribute to the regulation of these GDE4-dependent genes. Collectively, our findings suggest that GDE4-dependent lipid remodeling is associated with PPAR/{gamma}-mediated transcriptional regulation under ER stress conditions. These results provide a potential framework for understanding the link between intracellular lipid metabolism and stress-responsive gene regulation.

The immune checkpoint protein PD-L1 plays a pivotal role in tumor immune evasion by binding to PD-1 on immune cells, including T lymphocytes. While the expression and function of PD-L1 have been well studied, the importance of its spatial organization on the cell surface of tumor cells remains poorly understood. In this study, we used super-resolution microscopy combined with biochemical perturbations to investigate the factors regulating PD-L1 clustering and its effects on PD-1 binding and T cell inhibition. We found that PD-L1 is organized into nanoscale clusters at the plasma membrane, with distinct regulatory roles for the actin cytoskeleton, galectin-3, and cholesterol. Disruption of cortical actin increased PD-L1 cluster size, while galectin-3 promoted smaller, denser clusters and increased PD-L1 lateral mobility. Cholesterol depletion reduced PD-L1 cluster size and number and impaired PD-1 binding. These findings indicate that PD-L1 surface organization is collectively regulated by the actin cytoskeleton, galectin-3, and membrane cholesterol within the plasma membrane of tumour cells. Our results provide new insights into the dynamic regulation of PD-L1 and its potential as a therapeutic target in cancer immunotherapy.

The role of UBE2N in myeloid cell-mediated immune suppression in cancer remains undefined. Here, we examined the function of UBE2N in myeloid cell-mediated tumor progression using a temporally inducible myeloid-specific knockout model (LysMCreERUbe2nfl/fl). Temporally induced deletion of Ube2n in myeloid cells (Ube2nMyeKO) significantly hindered growth of YUMM1.7 melanoma. This was accompanied by reduced myeloid cell burden within the tumor microenvironment. We observed altered abundance of PD-1, PD-L1, and SPP1 in the Ube2nMyeKO tumor microenvironment at the tissue level. In vitro analysis showed that knock-in expression of a catalytically deficient UBE2NC87S mutant in bone marrow-derived macrophages (BMDMs) markedly decreased expression of Spp1. We observed decreased SPP1 secretion in Ube2nMyeKO BMDM-conditioned media (CM). Treatment with Ube2nMyeKO BMDM-CM decreased co-expression of PD-1, TIM-3, and LAG-3 on chronically stimulated T cells. Antibody-mediated neutralization of SPP1 in Ube2nWT BMDM-CM decreased PD-1 expression on CD8+ T cells. Together, these findings suggest a role for myeloid UBE2N in YUMM1.7 progression.

Abstract Background: Cytolytic activity (CYT), a widely used transcriptomic surrogate of anti-tumor immune cytotoxicity derived from GZMA (granzyme A) and PRF1 (Perforin 1) expression, is associated with clinical outcomes across cancers. MicroRNAs (miRNAs) are key post-transcriptional regulators of tumor immunity, yet their pan-cancer roles in modulating cytolytic activity remain incompletely understood. Objective: This study aimed to identify conserved miRNA regulators of tumor cytolytic activity and their downstream gene-mediated networks across diverse cancer types, while evaluating their clinical and therapeutic relevance. Methods: Matched miRNA and mRNA expression profiles from 9,288 primary tumors across 31 TCGA cancer types were analyzed. A multi-stage framework was applied: per-cancer Spearman correlations (|{rho}| >= 0.30, FDR < 0.05) identified recurrent CYT-associated miRNAs (at least 3 cancer types); these were integrated with TargetScan-predicted targets and subjected to pan-cancer and cross-cancer triple filtering (miRNA-gene and gene-CYT associations). All associations underwent tumor purity adjustment using Consensus Purity Estimate (CPE), with LUMP (Leukocytes Unmethylation for Purity) as sensitivity analysis. Candidates were further prioritized by random forest modeling with bootstrap stability, cancer-type-adjusted Cox regression, mediation analysis, immune cell deconvolution, k-means molecular subtyping, pathway enrichment, and DGIdb-based drug-target prioritization. Results: The analysis converged on 38 high-confidence miRNA-gene-CYT regulatory triplets involving 9 conserved miRNAs and 31 target genes after stringent purity adjustment and multi-layer validation. All nine miRNAs exhibited complete bootstrap stability. Mediation analysis confirmed significant gene-level mediation in 37 of 38 triplets (FDR < 0.01), with mediated proportions up to 94%. The final miRNA signature defined two distinct pan-cancer immune subtypes (immune-hot vs. immune-cold) with significantly different cytolytic activity and overall survival (OS) (HR = 0.754, FDR = 1.12 x 10^-4). The network was enriched for T-cell activation and lymphocyte differentiation pathways and highlighted multiple druggable targets, including CTLA4 and CD274 (PD-L1), nominating 124 candidate compounds. Conclusions: In conclusion, this tumor purity-adjusted pan-cancer study defines a compact, reproducible, and clinically relevant miRNA network that regulates cytolytic activity across diverse malignancies. By linking miRNA biology to immune subtyping and actionable therapeutic targets, the present work provides a valuable foundation for advancing precision immuno-oncology.

KRAS is one of the most frequently mutated oncoproteins in cancer. Its ability to induce malignant transformation relies on metabolic reprogramming that causes cells to become dependent on aerobic glycolysis as a primary source of energy and for generating biological building blocks. Thus far, the signaling mechanism used by oncogenic KRAS to promote these changes in cancer cell metabolism has not been fully elucidated. However, through studies in pancreatic ductal adenocarcinoma (PDAC) cell lines and patient-derived organoids, we now demonstrate how oncogenic KRAS triggers an increase in glycolytic activity and identify Survivin as a newly discovered and critical KRAS-signaling partner essential for promoting these metabolic changes. We show that oncogenic KRAS potently upregulates the expression of Survivin in PDAC cells and patient-derived organoids undergoing increased glycolysis, whereas depleting Survivin expression inhibits their glycolytic activity and growth. Through a combination of cellular, biochemical, and imaging approaches, we further show that Survivin promotes the formation of unique microtubule-based structures that resemble invadosome rosettes, allowing for the recruitment of the glycolytic enzymes triose phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to these super-structures which drives the increases in glycolysis. These findings demonstrate that by directing the assembly of a microtubule-based complex of metabolic enzymes, Survivin serves as a vital link in a KRAS signaling pathway responsible for promoting the metabolic changes necessary for the accelerated growth of PDAC cells, and thus potentially highlight new therapeutic strategies for treating KRAS-dependent cancers.

Proteins that distinguish quiescent cells from other non-proliferative states and actively regulate their return to proliferation remain poorly understood. Here, we combined quantitative proteomics with functional image-based screening to identify regulators of the quiescence-to-proliferation transition. Amongst the functional quiescence signature proteins we identified, we focussed on integrin 11 (ITGA11) which is induced across multiple models of reversible quiescence in distinct cell types and that has low expression in proliferating and senescent cells. Although ITGA11 is dispensable for proliferation of asynchronously cycling cells, it is required for efficient cell-cycle re-entry from quiescence. Mechanistically, ITGA11 promotes YAP accumulation and nuclear localization, thereby sustaining SKP2 expression and p27 degradation during cell cycle re-entry. Depletion of p27, or pharmacological activation of YAP signalling rescues the cell-cycle re-entry defect caused by ITGA11 depletion. Together, these findings identify ITGA11 as a functional quiescence signature protein that couples extracellular matrix sensing to YAP-dependent regulation of the Skp2-p27 axis, revealing a mechanism that controls the transition from quiescence to proliferation.

MYCN amplification drives replication stress in high-risk neuroblastoma, yet how MYCN-amplified tumour cells tolerate this stress to sustain proliferation remains poorly understood. Here we show that FBXL12, an SCF ubiquitin ligase substrate receptor that targets the Fanconi anaemia protein FANCD2 for degradation at replication forks, as well as the broader Fanconi anaemia and replication stress transcriptional program are elevated in high-risk and MYCN-amplified neuroblastoma. High FBXL12 expression independently predicts poor survival across neuroblastoma patient cohorts. FBXL12 loss stabilizes FANCD2 on chromatin, elevates ATR-dependent replication stress signalling and DNA damage during S phase, and impairs proliferation of MYCN-amplified neuroblastoma cells in vitro and in vivo. Mechanistically, MYCN directly engages the FBXL12-FANCD2 complex and antagonises FBXL12-mediated degradation of FANCD2 at replication forks, revealing that the oncogenic driver of replication stress also actively preserves the chromatin-bound FANCD2 pool required to tolerate it. Beyond S phase, FBXL12 loss disrupts FANCD2-dependent mitotic DNA synthesis and transmits unresolved replication intermediates into daughter cells. FBXL12-deficient cells consequently show transcriptional activation of MYC target gene, ATR, and mTOR signalling programs, and this pathway-concordant state confers differential sensitivity to ATR, and mTOR-targeting compounds, nominating candidate therapeutic strategies for this disease subset. Together, these findings define a MYCN-FBXL12-FANCD2 axis as a clinically relevant vulnerability in high-risk neuroblastoma.

Skin, the bodys largest mechanosensitive organ, relies on a tension gradient across epidermal layers to maintain structure and function, but how mechanical force contributes to epidermal development and disease pathogenesis is poorly understood. By anchoring intermediate filaments (IF) to the plasma membrane, desmosomes, the most abundant intercellular junctions in the epidermis, help create a supracellular scaffolding that provides mechanical resilience to the tissue. However, the contribution of the desmosome-IF network to the epidermal response to mechanical strain remains unknown. Here we show that the desmosome-IF connection is not only required to induce a proper cellular mechano-response but is actively strengthened in response to stretch through the PP2A-mediated phospho-regulation of the cytoskeletal linker protein desmoplakin (DP). Additionally, we show in human skin dephosphorylated DP localizes to high tension layers, suggesting this mechano-response mechanism is coordinated with the epidermal tension gradient. Furthermore, in models of Carvajal syndrome, a cardio-cutaneous disorder caused by truncating DP mutations, cells lose mechano-responsive behavior and exhibit abnormal morphology in high-tension epidermal layers. Together, these findings identify the DP-IF network as a key component of the response to mechanical strain and show that its disruption compromises epidermal homeostasis and contributes to disease pathogenesis.

Introduction: Gliomas integrate into neural circuits and heighten neuronal excitability, engaging in bidirectional communication whereby neuronal activity promotes tumor growth and proliferation. Aging reshapes the brain microenvironment through extracellular matrix changes, altered secretory factors, and immune dysfunction, creating conditions permissive to tumorigenesis and limiting immunotherapy efficacy in glioblastoma. However, its effect on neuronal excitability and signaling in glioblastoma remains poorly understood. Methods: We developed a novel classification system for glioblastoma by leveraging three classes of DNA methylation-based aging biomarkers: chronological, biological, and mitotic clocks. This approach stratified tumors into accelerated and decelerated epigenetic aging subtypes, which we then characterized at the molecular, functional, and clinical levels using multimodal analyses. Guided by these profiles, we evaluated the in vitro effects of the FDA-approved agents levetiracetam and riluzole, alone and in combination with temozolomide, on U87MG and A172 cell lines. Specifically, we assessed changes in cell viability, apoptosis, and the expression of marker genes related to stemness, neuronal hyperexcitability, and immunosuppression. Results: Tumors with decelerated epigenetic aging showed expression modules and CpG hypomethylation associated with neuronal activity and stemness, and carried significantly worse prognosis. Single-cell and spatial multi-omics analyses revealed enrichment for neurons and malignant neural stem-like cells in these tumors. They also displayed enhanced intercellular communication, driven predominantly by glutamate signaling across the malignant, neuronal, and immune compartments of the tumor microenvironment. In vitro pharmacological inhibition of glutamatergic signaling with levetiracetam and riluzole reduced cell viability, induced apoptosis, and suppressed expression of stemness, neuronal hyperexcitability, and immunosuppression markers. Both agents potentiated the cytotoxic and apoptotic effects of temozolomide, supporting glutamatergic inhibition as a strategy for improving chemosensitivity. Conclusion: By establishing a framework for decoding glioblastoma heterogeneity through epigenetic aging, we identified the glutamatergic pathway as a clinically actionable vulnerability. Our findings suggest that combining anti-glutamatergic therapies with temozolomide exerts synergistic antitumor effects while mitigating adverse chemotherapy-induced phenotypes, such as increased stemness, neuronal hyperexcitability, and immunosuppression, thereby laying the groundwork for novel therapeutic strategies.

Hirschsprung disease (HSCR) is a severe birth defect where ganglia of the enteric nervous system (ENS) are missing from distal bowel. The aganglionic segment is also characterized by increased epithelial permeability and pro-inflammatory immune activation. These problems may sequentially lead to translocation of gut microbes into the colon wall and systemic circulation, resulting in enterocolitis and sepsis. Current HSCR treatment via surgical resection of the aganglionic segment is lifesaving but not curative, often leaving patients with persistent gastrointestinal complications including recurrent risk of enterocolitis. As alternative, we are developing a regenerative medicine strategy based on in situ stimulation of tissue-resident ENS progenitors via rectal administration of the neurotrophic factor GDNF. Here, we report that GDNF-based therapy has pleiotropic gastrointestinal effects in a mouse model of short-segment HSCR, beyond its role in ENS regeneration. Interestingly, we found that these protective effects are not restricted to the aganglionic distal colon, also positively impacting the ENS-containing proximal colon. GDNF treatment reduces bacterial translocation both locally and in peripheral organs, and this is associated with recovery of the key epithelial junction proteins CLDN3, ZO1 and DSG2. Furthermore, multiparameter flow cytometry-based analysis of 55 lymphoid and 17 myeloid cell subtypes revealed that GDNF treatment has global anti-inflammatory effects, preferentially affecting innate over adaptive immunity. Overall, these findings highlight a critical role for GDNF treatment in reestablishing proper epithelial and immune cell homeostasis, offering promising therapeutic avenues not only for HSCR but also potentially for other intestinal disorders with overlapping pathophysiology.

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown great promise in treating bladder cancer and are now part of the standard treatment for advanced disease. However, many patients still fail to respond to treatment and at present many biomarkers are assessed but have yet shown only limited results. Therefore, with the advent of combination treatments and the increase of immune related adverse event, the search for reliable predictive biomarkers is paramount. Using a multiplexed enhanced FRET-FLIM based technique (QF-Pro) we quantified the interaction of PD-1/PD-L1, CTLA-4/CD80 and TIGIT/CD155 immune checkpoints in a pre-treatment TMA of 46 patients treated with atezolizumab. The association between higher PD-1/PD-L1 ICP interaction state and treatment efficacy was demonstrated in the male sample cohort, where it identified patients with better PFS. Conversely, patients exhibiting higher CTLA-4/CD80 engagement had a worse response to atezolizumab. Remarkably, the dual assessment of patients with high PD-1/PD-L1 and low CTLA-4/CD80 allowed to identify the best responders. These results indicate that the monitoring of patients immune profile in urothelial carcinoma might be critical in identifying patients who may benefit from combination therapy.

The cell-intrinsic complement system has emerged as an important orchestrator of a variety of cell-physiological processes, with complement components interacting with intracellular effector systems to regulate cellular responses to pathogens or noxious stimuli. For instance, intracellular C5 signaling through a mitochondrial C5a receptor (C5aR1) controls IL-1{beta} production in human monocytes and macrophages. Here, we investigated whether cell-intrinsic C3 similarly regulates inflammatory responses in macrophages. In LPS-stimulated C3 knockout THP-1-derived macrophages, interferon (IFN)-{beta} production was increased, accompanied by elevated expression of interferon-stimulated genes and enhanced secretion of IFN-induced cytokines and chemokines. C3-deficient cells showed increased phosphorylation of IRF3 at Ser396 and a stabilization of the interaction between IRF3 and TBK1, along with enhanced IRF3 dimerization and nuclear translocation. TBK1 phosphorylation was unaffected, indicating that C3 limits IRF3-TBK1 complex formation rather than upstream TBK1 activation. Small-molecule inhibitors of complement factors B and D restored full-length C3 abundance in LPS-stimulated primary human macrophages, consistent with inhibition of the C3 convertase. It also reduced LPS-induced IFN-{beta} production in primary human macrophages and THP-1 cells, suggesting that full-length, uncleaved C3 suppresses IFN-{beta} production. Collectively, these findings identify cell-intrinsic C3 as a suppressor of IFN-{beta} production in human macrophages, highlighting the importance of the cell-intrinsic complement system in fine-tuning inflammatory responses to pathogens.

Background: Transplant arterial injury is an underlying feature of acute organ transplant rejection and is a main cause of late heart transplant failure. The role of the gut microbiota, and especially specific microbial components of this community, in controlling immune responses that cause this aspect of rejection is poorly understood. Methods: We utilized a murine aortic interposition model of transplant arterial injury to investigate the role of the gut commensal bacteria, Akkermansia muciniphila, in controlling immune responses in transplant arteries. Results: Early life treatment of female mice with broad spectrum antibiotics, which delayed colonization of the intestinal tract with bacteria until after weaning, led to the development of dysbiosis in adults that was characterized by the absence of A. muciniphila. This was related to an elevation in systemic levels of CCL2 and a reduction in the immunomodulatory short-chain fatty acid, propionate. When transplant arterial injury was examined, there was more arterial injury indicative of acute rejection and increased intimal thickening reflective of transplant arteriosclerosis in grafts from dysbiotic mice compared to controls. Dysbiosis also increased macrophage accumulation early after transplantation in dysbiotic mice. Notably, restoring A. muciniphila in the gut microbiota of dysbiotic mice through voluntary oral administration in infants ameliorated macrophage-mediated transplant arterial injury. Conclusions: A. muciniphila is an immunomodulatory component of the gut microbiota that protects against vascular injury and pathology in organ transplantation.

The lack of effective anti-JEV therapy possesses significant challenge to control JEV. {beta}-catenin, a key mediator of Wnt signaling pathway regulates different viral replication and host immune responses. However, its role in JEV infection remains to be elucidated. Thus, the current study focused on evaluating iCRT-14, a specific {beta}-catenin inhibitor, against JEV. Treatment with iCRT-14 following JEV infection resulted efficient reduction in viral progeny release, viral RNA and protein levels in Huh7 and HEK293T cells. Further, active and total {beta}-catenin, Cyclin D-1 and GSK3-{beta}, the other key pathway players were also modulated in infected and inhibitor treated cells. Moreover, iCRT-14 showed an IC of 4.56 in Huh7 cell and maximal inhibition at the early stages of the JEV life cycle. Interestingly, the overexpression of {beta}-catenin in both the cells and siRNA-mediated {beta}-catenin knockdown (in Huh7 cells) significantly abrogated JEV replication, as evidenced by decreased viral titers, viral protein expression, and viral as well as total RNA levels. Moreover, the reduction in extracellular (84%) and intracellular (60%) viral titers following iCRT-14 treatment highlights its role in impairing JEV infection. Further, in silico molecular docking and co-immunoprecipitation studies demonstrated interactions between {beta}-catenin and the JEV NS5 and E proteins. Collectively, these findings suggest that optimum level of {beta}-catenin is required for efficient JEV infection, highlighting its potential as a target for designing host-directed control strategies to regulate viral infection.

Pancreatic ductal adenocarcinoma (PDAC) exhibits profound metabolic rewiring and strong resistance to DNA-damaging therapies, yet how metabolic pathways regulate genome maintenance remains poorly understood. The hexosamine biosynthetic pathway (HBP) integrates nutrient availability with protein glycosylation through production of UDP-GlcNAc, but its role in DNA damage response (DDR) regulation is unclear. Here we show that inhibition of the HBP enzyme phosphoglucomutase-3 (PGM3) reduces DNA repair capacity in pancreatic cancer cells. Transcriptomic and functional analyses reveal that the selective PGM3 inhibitor FR054 amplifies gemcitabine-induced replication stress, disrupts ATR-CHK1 and ATM-CHK2 checkpoint signaling, and selectively impairs homologous recombination. Glycoproteomic profiling identifies the AAA+ ATPase RUVBL2 as a key metabolic-DDR node. Gemcitabine increases RUVBL2 O-GlcNAcylation, with Thr81 identified as a modified residue within the Walker A nucleotide-binding motif. Structural modelling predicts that Thr81 O-GlcNAcylation stabilizes the RUVBL1-RUVBL2 complex without compromising ATP-Mg engagement. PGM3 inhibition and Thr81 mutation similarly reduced ATR and ATM abundance and promoted persistent DNA damage, supporting a role for RUVBL2 Thr81 O-GlcNAcylation in sustaining checkpoint signalling and genome stability. Consequently, PGM3 inhibition induces a BRCAness-like state that sensitizes pancreatic cancer cells to PARP inhibition, both in vitro and in vivo, as well as to ionizing radiation. These findings reveal a nutrient-sensitive mechanism linking protein glycosylation to genome maintenance and identify HBP-dependent DNA repair as a potentially actionable vulnerability in pancreatic cancer.

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