�����ƽ��

Glioblastoma (GBM) remains a formidable challenge in clinical settings due to limited treatments available. The surface protein CD133 marks glioblastoma stem cells (GSCs), cells capable of overcoming therapeutic pressures and correlate with more aggressiveness tumor phenotypes. In this study, we employed a CRISPR-Cas9 functional screen to deconvolute CD133 dynamics in tumors. This led us to establish that SOX2 is a key player in controlling the PROM1 gene, which in turn influences how cells react to stress factors, including those induced by chemoradiation treatment. The discoveries in this study shed light on the complex web of mechanisms that control the survival and resistance of GSCs, offering promising new avenues for targeting and potentially overcoming therapy resistance.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20183-7.

Mare endometrosis is a chronic degenerative condition of the endometrium, primarily characterized by fibrosis, involving interactions among fibroblasts, immune cells, and epithelial cells regulated by cytokines and growth factors. T helper (Th)1 and Th2 cells seem to play a pivotal role in fibrosis. However, their roles in equine endometrial fibrosis remain unknown. This study explores Th1 and Th2 cell distribution across different stages of endometrium histopathological Kenney and Doig categories; and evaluated their secretome effects on non-fibrotic endometrium derived fibroblast functional characteristics, extracellular matrix (ECM)-associated mRNA transcription, and transcriptomic profiles. Th1 and Th2 cells, along with cytokines (IFN-γ, IL-4, IL-13) and their receptors, were present in mare endometria at all endometrium stages. Th1 secretome influenced genes enriched in metabolism, cell cycle, and ECM-related pathways, while Th2 secretome regulated genes enriched in tissue remodeling and signaling pathways, suggesting their role in the development of fibrosis in the endometrosis progression.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20152-0.

Erythroblast enucleation is a unique process during mammalian erythropoiesis, yet its regulatory mechanisms remain largely elusive. Here, we demonstrate the specific regulatory role of the oncogene PIM1, the most highly expressed protein kinase in orthochromatic erythroblasts, in enucleation. Unlike its well-established roles in cancer cell proliferation and survival, knockdown of PIM1 in human erythroid cells does not affect cell growth or apoptosis, but specifically inhibits erythroblast enucleation without altering differentiation. To elucidate the functional conservation of PIM1 in mammalian erythropoiesis, we generate Pim1fl/flEpoRCre mice in which Pim1 is deleted in erythroid cells. Consistent with human erythropoiesis, deletion of Pim1 in mice has no detectable effect on apoptosis or differentiation of erythroid cells, but specifically inhibits erythroblast enucleation. Phosphoproteomic analysis reveals that PIM1 deficiency causes a pronounced decrease in phosphorylation of GTPase-associated proteins involved in actin assembly and vesicle trafficking. Functionally, this perturbation results in an aberrant distribution of F-actin and endocytic vesicles within enucleating cells. These findings reveal the unexpected role of PIM1 in normal erythropoiesis and enhance our understanding of mammalian erythroblast enucleation. The oncogene PIM1 regulates erythroblast enucleation via GTPase-dependent cytoskeletal remodeling and vesicle trafficking, independent of its canonical roles in cell proliferation and apoptosis.

The use of blood vessel organoids (BVOs), or vascular organoids derived from human induced pluripotent stem cells (hiPSC), provides a valuable tool for research on vascular regeneration. Vascular organoid culture relies on the use of Matrigel, a murine sarcoma-derived matrix, which limits its translational potential and reproducibility. This study evaluated the potential alternatives to Matrigel for both 2D hiPSC and 3D vascular organoid culture to enhance the clinical applicability of hiPSC-derived vascular organoids. Vitronectin is a suitable replacement for Matrigel in hiPSC culture and expansion, maintaining pluripotency and facilitating subsequent differentiation into vascular organoids. Additionally, we demonstrated that fibrin-based hydrogels effectively support vascular organoid differentiation, promoting vascular network formation and endothelial cell sprouting comparable to Matrigel-based cultures. Through gene expression analysis, surface area quantification, and immunohistochemistry, we validated the efficacy of Vitronectin and fibrin in supporting hiPSC-derived vascular organoid differentiation. This Matrigel-free protocol not only enhances reproducibility but is a step forward in xeno-free culture conditions, offering a versatile platform for disease modelling, vascular tissue engineering, and patient-specific therapeutic screening.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20091-w.

The development of stem-cell-derived models of mammalian embryogenesis has provided invaluable tools for investigating embryo development. However, constructing embryo models that can continuously recapitulate the developmental trajectory, from zygotic genome activation to gastrulation, remains challenging. Here we report the development of a chemical cocktail to induce totipotent-like cells with robust proliferative ability and leverage these cells to establish a stepwise protocol for generating a continuous embryo model. This model sequentially mimics mouse embryogenesis from embryonic day 1.5 to 7.5. It recapitulates key developmental milestones, including zygotic genome activation in 2-cell embryos, the diversification of embryonic and extraembryonic lineages from 4-cell to 64-cell stages, the formation of blastocysts and the subsequent development into post-implantation egg cylinders. Notably, these structures undergo gastrulation, as indicated by the formation of a primitive streak-like structure and the subsequent emergence of several early organogenesis hallmarks. Our study opens avenues for modelling mammalian embryogenesis in vitro. The authors identify a chemical cocktail to generate totipotent-like cells, which they then use to build an embryo model. This model captures a developmental spectrum from early embryogenesis to post-implantation events.

Cancer immunotherapy using dendritic cells (DC) pulsed ex vivo with tumour antigens is considered safe, but its clinical efficacy is generally modest. Here we engineer DC progenitors (DCP), which can replenish conventional type 1 DCs (cDC1) in mice, to constitutively express IL-12 together with a non-signalling chimeric receptor, termed extracellular vesicle-internalizing receptor (EVIR). By binding to a bait molecule (GD2 disialoganglioside) expressed on cancer cells and their EVs, the EVIR enforces EV internalization by cDC1 to promote their cross-dressing with preformed, tumour-derived MHCI-peptide complexes. Upon systemic deployment to mice, the engineered DCPs cause only mild and transient elevation of liver enzymes, acquire tumour-derived material, engage tumour-specific T cells, and enhance the efficacy of PD-1 blockade in an immunotherapy-resistant melanoma model comprising both GD2-positive and -negative cancer cells, without the need for ex vivo antigen pulsing. These results indicate that EVIR-engineered DCPs may avert the positive selection of antigen-negative cancer cells, potentially addressing a critical limitation of immunotherapies targeting defined tumour antigens. Tumour-antigen-pulsed mature dendritic cells (DC) have not been as efficient for cancer therapy as hoped to be, due to their sub-optimal antigen-presentation and migration capacities. Here the authors utilise DC progenitors, constitutively expressing IL-12 and an engineered extracellular vesicle-internalizing receptor (EVIR), which give rise to mature conventional type 1 DCs with improved antigen presenting capacities, resulting in improved anti-tumour immunity in a mouse model of melanoma.

ABSTRACTDysregulated coagulation and platelet activation contribute to liver dysfunction and fibrosis, but mechanisms initiating these events are undefined. The hepatic stellate cell (HSC) agonist Concanavalin A (ConA) rapidly induces hepatitis, which progresses to hepatic fibrosis after serial exposure for 8 weeks. Extravascular platelets were intercalated throughout normal liver parenchyma, with ConA treatment activating resident platelets to degranulate and display P‐selectin. HSCs matured from activated human induced pluripotent stem cells or the human LX‐2 cell line released extracellular vesicles (EVs) that stimulated platelet aggregation, yet these particles lacked known platelet agonists, and this response differed from established behaviors. Enzymatic assays, biologic, chemical and aptamer inhibitors, immunohistochemistry, qPCR, REN siRNA, and western blotting elucidate a novel HSC‐EV mediated pathway to platelet activation. The aspartyl protease inhibitor pepstatin, or specific inhibitors Aliskiren and VTP23999 of the aspartyl protease renin suppressed HSC EV‐induced platelet activation, as did siRNA knockdown of prorenin. HSC maturation from mesenchymal cells increased prorenin transcripts, and HSC‐EV contained surface‐associated prorenin. Platelets expressed the prorenin receptor that overcomes prorenin auto‐inhibition, and HSC EV interacting with platelets generated renin peptidolytic activity. This interaction stimulated extrinsic and common coagulation cascades, formation of thrombin over time, activated platelet PAR‐1 thrombin receptors, and induced robust TGF‐β release. This TGF‐β stimulated the LX‐2 cell TGFbR1 receptor, Smad phosphorylation, and profibrotic protein expression. We identify hitherto undiscovered pathways by which platelet and HSC interaction stimulates thrombosis through prorenin activation and show intrahepatic platelets are positioned to stimulate fibrotic protein deposition in a model of hepatic fibrosis. Platelets interact with extracellular vesicles shed by hepatic stellate cells to activate both cell types. Extracellular vesicles shed by activated hepatic stellate cells contain inactive prorenin. HSC‐EV interact with quiescent platelets to non‐proteolytically activate ligated prorenin. This enables time‐dependent organization of coagulation protease complexes on platelets that generate active thrombin. Thrombin activates platelet PAR‐1, induces robust platelet activation and aggregation with TGF‐β release. Platelet‐derived TGF‐β stimulates HSC fibrotic protein expression and deposition.

Sepsis is a life-threatening condition arising from immune dysregulation, particularly in patients with underlying diseases like rheumatoid arthritis (RA). However, experimental data on this phenomenon are lacking. Using flow cytometry, we investigated immune responses in mice with or without collagen-induced arthritis (CIA) following Streptococcus infection. Mice without CIA effectively cleared the infection, maintained hematopoiesis, and mobilized lymphocytes. In contrast, CIA mice exhibited impaired bacterial clearance, leukopenia, and sepsis. Emergency hematopoiesis in CIA mice led to depletion of lineage-committed progenitor cells, correlating with an accumulation of immature neutrophils that exhibited diminished cytokinogenesis. Notably, immune dysregulation in CIA mice appeared before sepsis onset. We detected an increase in neutrophils and monocytes in draining lymph nodes and joints. Importantly, lymphadenopathy and hyperactivated synovial fibroblasts, along with articular immune cell infiltration, drove excessive cytokine production, increasing sepsis risk. Our findings emphasize the importance of rigorous medical management of RA to mitigate infection-related complications. Graphical Abstract Diagram illustrating the relationship between blood infection and sepsis risk, with a gradient from resilience to sepsis risk. On the left, under “Healthy,” qualities include mature leukocyte persistence and stable hematopoiesis. On the right, under “Arthritis,” conditions include leukopenia and cytokine hyperproduction. A mouse represents blood infection at the top.

IntroductionIn human neurocysticercosis (NCC), the cellular and molecular mechanisms of host-parasite interactions triggering brain inflammation and epileptic seizures in Sub-Saharan Africa are poorly understood. Emerging evidence indicates that the viability of the cyst of the pork tapeworm Taenia solium determines brain inflammation and, thus, symptom development and disease severity. We have previously shown that while viable cyst-released molecules promote immune regulation and often asymptomatic disease, the fluid from degenerating cysts causes inflammation in microglia and peripheral immune cells, potentially driving immune-mediated pathology. This study aims to elucidate the apoptotic signaling pathways underlying this process and their relevance for symptomatic disease in NCC patients.Materials and methodsHuman and porcine peripheral immune cells, as well as murine microglia, were exposed to T. solium cyst vesicular fluid (CVF). Apoptosis signaling pathways were analysed using flow cytometric FLICA (fluorochrome-labeled inhibitors of caspases) caspase 8 and 9 assays, while mitochondrial dysfunction was assessed via TMRE and MitoTracker Deep Red and Green fluorescent probes. Apoptosis-inducing CVF molecules were identified by differential mass spectrometry and functionally tested using specific inhibitors. Caspase activity and soluble mediators (FasL, ROS, TNFα) were measured in NCC asymptomatic and symptomatic patients’ sera, and inflammatory T cell infiltrates expressing caspases near viable and degenerating cysts in naturally infected pig brain slices were examined via immunohistology.ResultsWe found that vesicular fluid derived from cysts primarily induced apoptosis and caspase 3 and 9 activity, and only minimal necrosis, in a dose-dependent manner across central and peripheral immune cells. This effect was prominent in CD16+ monocytes, microglia, and in CD3+ T cell-expressing caspase 3 near degenerating brain cysts. Apoptotic signaling was predominantly mediated by a dynamic remodeling of caspase 9 pathway, accompanied by a significant loss of mitochondrial potential and a sharp decrease in Bid and Bcl2 transcription, favoring the intrinsic over the FasL-dependent extrinsic pathway and mechanisms. This process is primarily mediated by small molecules (< 30 kDa), and remained unaffected by heat and proteinase treatment. Notably, symptomatic NCC patients exhibited elevated FasL levels correlating with increased caspase activity, underscoring the potential contribution of apoptosis to disease pathogenesis.ConclusionsThis study identifies caspase 9-mediated apoptosis as a mechanism of helminth-induced brain inflammation and implicates FasL in symptomatic disease progression. These insights enhance our understanding of NCC immunopathogenesis and may inform future therapeutic strategies targeting apoptotic pathways.

Rapid and uniform labeling of plasma membrane proteins is essential for high-resolution imaging of dynamic membrane topologies and intercellular communication in live mammalian cells. Existing strategies for labeling live cell membranes, such as fluorescent fusion proteins, enzyme-mediated tags, metabolic bioorthogonal labeling, and lipophilic dyes, face trade-offs in the requirement of genetic manipulation, the presence of non-uniform labeling, the need for extensive preparation times, and limited choices of fluorophores. Here, we present a streamlined protocol that leverages N-hydroxysuccinimide (NHS)-ester chemistry to achieve rapid (≤5 min), covalent conjugation of synthetic small-molecule dyes to surface-exposed primary amines, enabling pan-membrane-protein labeling. This workflow covers dye stock preparation, labeling for suspension and adherent cells, multiplex live-cell imaging, fusion protein co-staining (including insulin-triggered receptor endocytosis), 3D membrane visualization, and in vivo assays for visualizing membrane-derived material transfers between donor and recipient cells using a lymphoma T-cell mouse model. This high-density labeling approach is compatible with various cell types across diverse imaging platforms. Its speed, versatility, and stability make it a broadly applicable tool for studying plasma membrane dynamics and intercellular membrane trafficking. Key features • Rapid high-density membrane labeling with small-molecule fluorescent dyes.• Enables live-cell multiplexed imaging, amenable to primary cells and cells expressing fluorescent fusion proteins, and supports in vivo studies of membrane-associated cell–cell communications.• Compatible with various fluorescence imaging modalities.

The current understanding of the avian immune system primarily stems from research conducted in chickens, given their economic significance as a food source. Extending the research to other avian species like Psittaciformes requires the use of label‐free techniques. Therefore, budgerigar (Melopsittacus undulatus) splenic leukocytes were characterized in this study, with the help of an immunological toolbox, integrating single‐cell transcriptomics and multiplex RNA in situ hybridization (ISH). Twenty‐four distinct psittacine splenic leukocyte populations were identified and characterized, including, amongst others, germinal center (GC) B cells and regulatory T cells (Tregs). For each of these 24 populations, markers were defined for subsequent use in immunological assays. To further examine splenic organization, multiplex RNA ISH was applied, successfully characterizing six out of the nine selected markers. This study showed that the psittacine immune system closely mirrors that of chickens. However, a detailed, comprehensive examination was hindered by the lack of a complete sequenced and annotated budgerigar genome and the limited number of replicates. Consequently, further investigation is imperative to advance our understanding of the avian immune system.

Highlights•The effect of NSAIDs on equine TNF-α during endotoxemia remains poorly understood.•Effects of flunixin meglumine (FM) and meloxicam on TNF-α were compared in vitro.•FM and meloxicam did not inhibit TNF-α production in LPS-stimulated PBMCs.•FM significantly inhibited TNF-α levels in LPS-stimulated neutrophils.•FM may play a key role in early endotoxemia when neutrophils are mobilized. Systemic inflammatory response syndrome (SIRS) induced by endotoxemia is usually secondary to colitis and is a major cause of high morbidity and mortality in horses. Non-steroidal anti-inflammatory drugs (NSAIDs), such as flunixin meglumine (FM) and meloxicam (MX), are used to improve clinical outcomes in SIRS/endotoxemia. These NSAIDs suppress tumor necrosis factor-alpha (TNF-α) levels; however, the underlying mechanisms remain unclear. The aim of this study was to investigate the inhibitory effects of FM and MX on TNF-α in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) and neutrophils in vitro. Blood samples were collected from three healthy thoroughbred horses, and PBMCs and neutrophils were isolated using density gradient centrifugation. Cells were cultured with LPS (0.3 ng/mL) and FM or MX (5, 10, or 50 μM). TNF-α gene expression was analyzed using quantitative real-time PCR, and protein levels were measured using ELISA. No significant inhibitory effects of FM or MX on TNF-α gene or protein expression were observed in LPS-stimulated PBMCs. However, FM significantly inhibited the increase in TNF-α protein levels in LPS-stimulated neutrophils in a concentration-dependent manner (p < 0.05). MX showed a similar tendency in LPS-stimulated neutrophils, but the differences were not significant. The regulation of neutrophil-derived TNF-α by FM administration could be a promising therapeutic strategy for equine SIRS/endotoxemia, providing mechanistic insight for optimizing anti-inflammatory therapy.