Search Results (804)

The galactose/N-acetyl-D-galactosamine (Gal/GalNAc)-inhibitable lectin of Entamoeba histolytica plays essential roles in host cell adhesion and cytotoxicity. The intermediate subunit lectin-1 (Igl1) contributes to these functions, but its molecular state under different environmental conditions remains unclear. In this study, we found that Igl1 is present as multiple fragments in the culture supernatant of trophozoites, whereas a single major species corresponding to intact Igl1 was detected in cell lysates. Notably, the molecular sizes of both intact Igl1 and its extracellular fragments differed depending on culture conditions, with larger apparent sizes observed under co-culture with Caco-2 cells. These differences were not explained by changes in transcript levels, protein folding, or N-terminal truncation. Fragmentation of Igl1 was suppressed by a cysteine protease inhibitor, indicating extracellular generation. These findings demonstrate that host-cell-associated conditions alter the molecular size of Igl1 and that extracellular protease-dependent processing generates multiple Igl1 fragments, providing new insights into the regulation of this key virulence factor. The presence of extracellular fragments further suggests a potential contribution to host tissue damage during amoebiasis. Full article

Botrytis cinerea is a major phytopathogenic fungus responsible for substantial economic losses in horticultural crops, underscoring the need for sustainable alternatives to synthetic fungicides. This study investigated the influence of physical, chemical and biological culture parameters on the antifungal activity of culture filtrates produced by Trichoderma harzianum BOL-12QD. Culture conditions were sequentially optimised by evaluating light-filter exposure, carbon and nitrogen source composition, potato ecotype selection, co-cultivation with Botrytis cinerea, and volatile-mediated interactions. Antifungal activity was assessed using mycelial growth inhibition assays against Botrytis cinerea. Among the individual factors, violet-filter illumination, a medium containing 5 g L⁻¹ glucose and 250 g L⁻¹ potato extract, the Leke Pek’e potato ecotype, ammonium nitrate as nitrogen source, and co-cultivation with Botrytis cinerea at 10⁴ conidia mL⁻¹ produced the highest inhibitory effects. Sequential integration of these optimised conditions resulted in enhanced antifungal activity, reaching up to 62% inhibition. Volatile organic compounds produced by Trichoderma harzianum BOL-12QD exhibited only minimal antifungal activity under the conditions tested, suggesting that volatile-mediated antagonism plays a limited role in this system. In contrast, culture-dependent modulation of extracellular metabolite profiles was evidenced by comparative ¹H NMR fingerprinting, which revealed condition-specific spectral differences, with the optimised treatment displaying a distinct metabolic signature relative to all other conditions. Cytotoxicity assays in murine peritoneal macrophages showed no significant reduction in cell viability at concentrations up to 200 μg mL⁻¹. In vivo exposure to the optimised culture filtrate (250 mg kg⁻¹ d⁻¹ for 10 days) induced transient treatment-related clinical observations without mortality, indicating a need for further detailed toxicological characterisation. Overall, these findings demonstrate that the antifungal activity of Trichoderma harzianum BOL-12QD is strongly modulated by interacting environmental, nutritional and biological culture parameters. The results support the potential of optimised culture filtrates as a source of bioactive metabolites for biocontrol applications, while highlighting the importance of integrated biochemical and toxicological evaluation. Full article

Three-dimensional (3D) cell culture models more faithfully reproduce native tissue organization and function than conventional two-dimensional systems, yet many existing bioprinting methods depend on scaffolds, complex instrumentation, or limited control over cell positioning. This review examines magnetic bioinks as a versatile platform for contactless 3D cell manipulation and biofabrication. It first outlines the fundamentals of magnetophoresis and defines magnetic bioinks as combinations of magnetic agents, including magnetic nanoparticles or paramagnetic salts, with biological components such as cells, proteins, or fluids. The review then compares label-based strategies, in which cells are magnetized and guided by positive magnetophoresis, with label-free approaches that exploit magnetic susceptibility differences to position diamagnetic cells through negative magnetophoresis. Across these methods, magnetic bioinks have enabled single-cell sorting, spatial patterning, spheroid and co-culture assembly, multilayer tissue formation, and hydrogel-integrated printing. These capabilities support applications in disease modeling, drug screening, biosensing, regenerative medicine, and emerging biofabrication under microgravity conditions. The paper also highlights key limitations, including nanoparticle biocompatibility, paramagnetic salt toxicity, osmotic stress, and the need for better assay standardization and translational validation. Overall, magnetic bioinks represent a promising scaffold-free approach for rapidly producing physiologically relevant 3D biological constructs for research and clinical innovation. Full article

The intracellular accumulation of amyloid beta 42 (iAβ42) has been proposed as an early pathological indicator of familial Alzheimer’s disease (FAD). DJ-1 is a multifunctional protein sensitive to oxidative stress (OS) that has been associated with neurodegeneration; however, its role in iAβ42 pathology is unclear. In this study, we examined whether oxidized (sulfonic) DJ-1 (Cys¹⁰⁶-SO₃H) drives iAβ42 accumulation using postmortem brain samples and in vitro 3D iPSC-derived cerebral organoids (COs) or 2D induced pluripotent stem cells (iPSC)-derived ChLNs (cholinergic-like neurons) models from a PSEN1 E280A patient and a healthy volunteer (as a control sample). Post-mortem analyses of the temporal and frontal cortices and hippocampus from FAD PSEN1 E280A patients revealed strong intracellular co-localization of sulfonic DJ-1 and iAβ42, which was absent in control samples. To validate these findings, we generated COs from an iPSC PSEN1 E280A FAD patient and a healthy donor. In these organoids, we observed the co-localization of oxidized DJ-1 and Aβ42 in the absence of extracellular fibrils or plaques, as confirmed by BTA-1 staining. To further support these observations, 2D iPSC PSEN1 E280A-derived ChLNs cultures showed that intracellular Aβ42 accumulates progressively in direct correlation with increasing DJ-1 oxidation, as demonstrated by immunofluorescence microscopy and Western blotting analysis. These results indicate that DJ-1 oxidation accompanies the earliest intracellular stages of Aβ42 pathology. Furthermore, complementary in silico molecular docking analysis revealed a higher affinity between Aβ42 and oxidized sulfonic DJ-1 (DJ-1 Cys¹⁰⁶-SO₃H) compared to sulfenic (DJ-1 Cys¹⁰⁶-SOH) or sulfinic acid (DJ-1 Cys¹⁰⁶-SO₂H) forms. Likewise, ELISA tests and seeding assays confirmed that oxidized DJ-1 binds to and decelerates Aβ42 aggregation kinetics. Together, our results identify DJ-1 oxidation as a critical molecular event in the accumulation of iAβ42 in FAD. These findings suggest that oxidized DJ-1 represents not only a potential early biomarker of intracellular pathology but also a pharmacological target. Preventing the oxidation of DJ-1 or its pathological aggregation could provide new biomarkers and therapeutic strategies for reducing the intracellular accumulation of Aβ42 and neurodegeneration in FAD. Full article

Background: Diffuse Large B-cell Lymphoma (DLBCL) is a biologically heterogeneous subtype of non-Hodgkin’s lymphoma (NHL), accounting for 30–40% of cases worldwide. Despite the incorporation of rituximab into standard chemo-immunotherapy regimen, approximately one-third of patients present with relapsed or refractory disease, implicating the need for improved prognostic markers and therapeutic targets. Gene expression profiling successfully classified DLBCL into Germinal Center B-cell-like (GCB) and non-GCB subtypes, which differ in genetic alterations, response to therapy, and clinical outcome. While intrinsic tumor biology has been extensively studied, the contribution of the tumor microenvironment (TME) to disease progression and therapeutic resistance still remains incompletely understood. Methods: In this study, we investigated the mutational landscape of stromal-related genes in DLBCL and evaluated their impact on gene expression, downstream signaling pathways, and tumor progression. Results: A total of 176 DLBCL patients were screened, of which 113 were enrolled based on availability of complete clinical data. The cohort demonstrated male predominance (male:female ratio: 2.1:1), advanced disease stage in 72.6% of patients, and elevated serum lactate dehydrogenase levels in 57.5%. Based on immunohistochemistry, 43.4% cases were classified as GCB-DLBCL and 56.6% as non-GCB DLBCL. Although the International Prognostic Index (IPI) retained prognostic significance for event-free survival (EFS) and overall survival (OS), considerable heterogeneity was observed within similar risk groups. Whole-exome sequencing (WES) uncovered recurrent somatic mutations in key oncogenic and epigenetic regulators, including TNFAIP3, NFIB, NOTCH1, TSC2, EZH2, EP300, KMT2D, and B2M, with subtype-specific distribution. Pathway enrichment analysis implicated role of Notch, Wnt, mTOR, JAK-STAT, TGF-β, and antigen-presentation pathways. Comprehensive WES analysis identified multiple novel mutations in genes associated with the stromal/extracellular matrix with distinct patterns in GCB and non-GCB DLBCL, accompanied by concordant alterations in gene expression profiles, suggesting functional relevance within the TME. Functional validation through primary cell culture demonstrated significantly elevated Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) cytokines in co-cultures containing both neoplastic cells and stromal components, underscoring the role of TME in DLBCL progression. Conclusions: Taken together, this study provides novel insights into stromal mutational signatures and cytokine-mediated tumor–stroma interactions, offering potential prognostic biomarkers and therapeutic targets for the improved management of DLBCL. Full article

Cellular senescence and polyploidy are fundamental stress responses that shape cancer progression and therapeutic outcomes. While senescence initially suppresses tumor growth, senescent cells accumulate in aging and therapy-exposed tissues and actively remodel the tumor microenvironment through the senescence-associated secretory phenotype (SASP) and extracellular matrix (ECM) reorganization. Senescent stromal cells increase collagen deposition and generate disordered matrix architectures, as evidenced by enhanced second harmonic generation (SHG) signal and increased anisotropic variation across in vitro systems, 3D co-culture models, and fibrotic lung tissues. These biochemical and mechanical alterations promote cancer cell plasticity and create conditions permissive for disease progression. Polyploid giant cancer cells (PGCCs) are a rare but highly resilient cancer cell population enriched under genotoxic stress. PGCCs arise through mitotic failure, including mitotic slippage and cytokinesis defects, and can survive chemotherapy and radiation due to their altered cell-cycle regulation. Emerging evidence indicates that senescence-driven microenvironments promote the formation of PGCCs and multinucleated cells, linking ECM remodeling and mechanical stress to polyploidization. Functionally, PGCCs exhibit abnormal cytoskeletal and nuclear mechanics that support migratory persistence and enable survival within hostile tumor environments. In addition, PGCCs can promote the survival of neighboring cancer cells during treatment, suggesting a stromal-like role in establishing therapy-resistant niches. These cells can persist in a dormant state and later generate proliferative progeny, contributing to tumor recurrence and metastasis. Together, these findings support a model in which senescent niches may promote PGCC formation, persistence, and tumor repopulation. Targeting both senescence-associated microenvironments and PGCC-specific survival mechanisms may improve long-term therapeutic outcomes. Full article

Anthracyclines such as doxorubicin (DOX) are widely used in cancer treatment, but their benefits are offset by dose-related cardiotoxicity. Neuregulin-1β (NRG1) has been studied as a cardioprotective factor, yet its mechanisms during DOX treatment, particularly in the presence of cancer, are not well understood. This study evaluated daily recombinant NRG1 co-administered with DOX in 4T1-tumor-bearing female BALB/c mice. The mice were randomized to saline, DOX (3 mg/kg i.p. on days 0, 3, 6, 9; cumulatively 12 mg/kg) or DOX + NRG1 (20 µg/kg i.p. daily, starting one day before DOX). Body weight and tumor growth were monitored throughout treatment. Cardiac structure and function were assessed by transthoracic echocardiography at baseline and before sacrifice. Mechanistic studies included left ventricular proteomics and single-cell RNA-seq. We also used human 3D cardiac microtissues and 2D primary cardiac fibroblast-enriched cultures under defined experimental conditions, with targeted fibroblast gene perturbations. We found that early DOX exposure induced systolic dysfunction and pathological remodeling, while daily NRG1 preserved the ejection fraction and attenuated structural changes without impairing anti-tumor efficacy. Proteomic analysis identified Serping1 as one of the most strongly upregulated proteins soon after DOX exposure, an effect that was reversed by NRG1. Notably, Serping1 has not previously been implicated in anthracycline cardiotoxicity or NRG1-mediated protection. Single-cell RNA sequencing localized Serping1 expression to cardiac fibroblasts. Mechanistically, we found that Serping1 modulation was associated with altered Igfbp5 processing and fibroblast survival under DOX-induced stress; its suppression by NRG1 was linked to reduced fibroblast apoptosis and a shift toward a pro-survival-associated state. In human cardiac microtissues, NRG1 treatment or fibroblast-specific Serping1 knockdown accelerated cardiomyocyte contraction dynamics. These changes occurred without an increase in apoptosis and point to a paracrine effect of fibroblasts on cardiomyocyte function. Additionally, scRNA-seq revealed an Erbb4+ fibroblast subpopulation associated with early pro-fibrotic activation that expanded after DOX but was reduced by NRG1. Taken together, NRG1 preserved cardiac function during anthracycline treatment while maintaining anti-tumor efficacy. Our data identify fibroblast-associated signaling, particularly through Serping1, as a potential contributor to the early protective effects of NRG1. These findings add a new dimension to the understanding of NRG1 cardioprotection and suggest that fibroblast–myocyte interactions may contribute to the early cardiac response to DOX. Full article

Anterior cruciate ligament reconstruction relies on interference screw fixation, yet insufficient graft osseointegration remains a critical clinical challenge. This study aimed to develop and characterize a 3D-printed polymeric–hydroxyapatite composite interference screw with an osteoinductive surface to enhance localized osteogenic responses. Screws were designed, modeled, and fabricated using fused deposition modeling 3D printing with a polycaprolactone-poly(lactic-co-glycolic acid)-hydroxyapatite composite. Physico-chemical characterization was performed using scanning electron microscopy. Biocompatibility was assessed through mesenchymal stem cell metabolic activity assays and morphological analysis. Osteogenic gene expression was quantified by RT-qPCR following culture in osteogenic differentiation medium. In vivo osseointegration was evaluated histologically at five and nine weeks following implantation in the proximal tibial epiphysis of a rat model. 3D printing successfully produced screws with consistent geometry and surface characteristics. The composite material supported robust mesenchymal stem cell proliferation without cytotoxicity or morphological abnormalities. Histological examination revealed progressive bone formation with no adverse tissue reactions, including the absence of cyst formation, osteolysis, or excessive fibrosis. RT-qPCR revealed upregulation of osteogenic markers in those enhanced screws. These results indicate that the 3D-printed polymeric–hydroxyapatite composite screws are biocompatible and capable of stimulating localized osteogenic activity, supporting their potential as a biological foundation for future evaluation in anterior cruciate ligament reconstruction applications. Full article

Epithelial ovarian cancer (EOC) remains a lethal malignancy requiring novel therapeutic strategies due to high recurrence and chemoresistance. This study evaluated the combined antitumor effect of metformin and the co-transfection of tumor-suppressor microRNAs miR-145 and miR-23b in A2780 and OV90 EOC cell lines using both 2D and 3D models. In monolayer cultures, our approach significantly reduced the expression of proliferation markers Ki-67 and c-MYC, and decreased cell migration and invasion in both cell lines compared to controls. In 3D spheroid models, the treatment reduced VEGF secretion and relative spheroid area in A2780 cells, significantly increasing cytotoxicity; however, OV90 spheroids exhibited marked resistance. Fluorescent miRNA tracking revealed that this resistance occurs despite successful intracellular delivery, indicating an intrinsic biological resistance conferred by the 3D microenvironment. Overall, these findings suggest that the combined administration of metformin and miRs effectively limits tumor progression, but also strongly underscore the importance of using complex 3D models to accurately evaluate therapeutic efficacy and intrinsic resistance mechanisms. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a widespread condition with a complex pathogenesis. Cell-based models are important tools for studying the mechanisms underlying its development and progression. The aim of this review is to analyze the HepaRG, Huh-7, immortalized human hepatocyte (IHH), and primary human hepatocyte (PHH) cell lines for modeling and studying MASLD. HepaRG represents the most metabolically competent immortalized hepatocyte model with preserved biotransformation activity and a physiological bioenergetic response to lipid loading, making it valuable for pharmacological and toxicological studies. Huh-7 is distinguished by its accessibility and suitability for studying steatosis, lipotoxicity, insulin resistance, and paracrine mechanisms of fibrogenesis; however, its use is limited by its tumor origin, impaired carbohydrate metabolism, and low activity of xenobiotic-metabolizing enzymes. The IHH model occupies an intermediate position because of its non-tumor origin and is of interest for studies of senescence, epigenetic regulation, and signaling pathways involved in steatosis, although interpretation of results requires consideration of immortalization-related effects and specific metabolic limitations. PHH remains the most physiologically relevant platform for MASLD modeling, particularly in three-dimensional (3D) and microphysiological formats; however, its use is limited by high cost, interindividual variability, and the limited duration of the differentiated phenotype. Increasing model complexity—from two-dimensional (2D) monocultures to co-cultures, spheroids, and organ-on-chip systems—enhances physiological relevance and enables reproduction not only of steatosis but also of the inflammatory and fibrogenic components of MASLD progression, yet it reduces reproducibility and complicates standardization. Overall, none of the existing models is universal, and the optimal strategy is to select models according to the specific research question. A key direction for future research is the standardization of steatosis induction protocols and the unification of criteria for evaluating results. Full article

Objective: Developing foundational biomaterial platforms for cultured meat research requires 3D co-culture systems capable of supporting multiple relevant cell types in a spatially organized manner. This study aimed to establish a compartmentalized tri-culture hydrogel disc incorporating a lipid-containing barrier phase as a proof-of-concept in vitro model. Methods: Beeswax/alginate (Bw/Algi) hydrogels were fabricated and evaluated for morphology and cytocompatibility as a lipid-containing scaffold component. Fucoidan/alginate (Fu/Algi) hydrogels were prepared at varying fucoidan concentrations and screened to identify conditions compatible with C2C12 viability and early-stage differentiation. A composite beeswax/fucoidan/alginate disc (Bw/Fu/Algi) was then assembled by casting cell-laden Fu/Algi regions (myoblasts, fibroblasts, and endothelial cells), separated by Bw/Algi barrier layers and ionically crosslinked with CaCl₂. Scaffold performance was assessed using standard assays for morphology, cytocompatibility, myogenic marker expression, protein production, and thermal stability. Results: Bw/Algi supported cytocompatible C2C12 attachment and growth, while Fu/Algi exhibited concentration-dependent effects on myogenic marker expression, enabling selection of an optimized fucoidan concentration for 3D assembly. The final Bw/Fu/Algi disc maintained viable compartmentalized tri-culture and supported indirect co-culture through spatial separation by the Bw barrier. Myogenic regions exhibited myogenic marker expression with measurable protein production, and differential scanning calorimetry confirmed structural stability under heating. Conclusion: This work establishes a Bw/Fu/Algi tri-culture disc integrating a lipid-containing barrier component with hydrogel-based myogenic compartments, providing a preliminary platform for multicellular in vitro modeling and scaffold design relevant to cultured meat research. Full article

Multifunctional scaffolds that combine structural support with the controlled delivery of bioactive agents remain a major challenge in tissue engineering. To extend the use of these devices in biomedicine, 3D printing is presented as an alternative that enables the manufacture of complex devices tailored to each patient, thereby solving specific problems in a timely and efficient manner. In this study, porous 3D scaffolds were fabricated via digital light processing (DLP) using a PET-RAFT resin composed of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and poly(ethylene glycol) diacrylate (PEGDA₅₇₅). Sodium chloride (NaCl) was incorporated as a porogen, while insulin-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were embedded as osteoinductive agents. The printed constructs exhibited high-resolution, reproducible trabecular-like architectures, as confirmed by micro-computed tomography (micro-CT), with interconnected pores averaging 70.7 ± 24.7 μm and a total porosity of 57.0 ± 6.98%. Thermal and chemical analyses confirmed scaffold stability and controlled degradability. Cytocompatibility assays using MC3T3-E1, C2C12, hGMSCs, and C166-GFP cells showed viability above 80% after 7 days (ISO 10993-5). Insulin-loaded nanoparticles enabled sustained release, characterized by an initial burst followed by gradual release up to 72 h. Dynamic bioreactor culture enhanced cell adhesion and RUNX2 expression, confirming the osteoinductive potential of the hybrid scaffold for advanced BTE applications. This study introduces an innovative PET-RAFT-derived resin that combines structural reinforcement with spatiotemporal regulation of insulin release, offering a potential strategy for enhanced biomaterial tissue engineering and tailored therapeutic interventions. Full article

Vasculogenic mimicry (VM) is the ability of cancer stem cells (CSCs) to express an endothelial-like phenotype and participate in tumor neovascularization via the formation of a blood-conducting, matrix-rich network. We previously reported that pancreatic ductal adenocarcinoma (PDAC) CSCs develop their VM phenotype via two interacting and coordinated factors that support the formation of the VM network: (i) the overexpression of genes for endothelial factors and vascular receptors and (ii) the very high secretion of numerous pro-angiogenic/growth factors. While microenvironmental acidosis (low pHe) is an important driver of tumor metastasis, especially in PDAC, and is a component of the CSC niche, its role in VM and the ion transporters involved remains unknown. As normal stem cell differentiation is regulated by Na⁺/H⁺ exchanger 1 (NHE1)-driven pH, we investigated the role of NHE1 and the intracellular signaling involved in the acidosis-induced VM using a platform of 3D organotypic cultures composed of Matrigel with increasing concentrations of Collagen I. VM was highest on 90% Matrigel:10% Collagen I, representative of an early tumor ECM, and it decreased with increasing concentrations of Collagen I, representative of advanced tumors. In all ECM compositions, VM capacity increased stepwise with pHe acidification, and both basal and acid-stimulated VM were dependent on NHE1 activity. Acidification also decreased resting pHi and increased NHE1 proton extrusion activity, NHE1/ß1 integrin co-expression, and intracellular Ca²⁺. The stimulation of VM by extracellular acidosis depended on the transport of extracellular Ca²⁺ into the cell and the consequent increase in intracellular Ca²⁺. Altogether, these data demonstrate that extracellular acidification triggers cellular mechanisms that upregulate VM to overcome the constraints imposed by ECM composition, thereby permitting VM in ECMs where this phenotype is not expressed and extending the VM phenotype towards the tumor center to further drive metastasis. Full article

Owing to the multifactorial nature of inflammatory bowel disease (IBD) pathogenesis, conventional two-dimensional (2D) models inadequately recapitulate the complex in vivo microenvironment. This study sought to develop an immune-microenvironment-integrated intestinal-on-a-chip model to overcome these limitations. A microfluidic chip was engineered to co-culture intestinal epithelial (Caco-2) cells and macrophages, facilitating the simulation of IBD pathological conditions for mechanistic investigations. Following inflammatory stimulation, M0 macrophages polarized into the M1 phenotype, concomitant with the upregulation of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). This induction disrupted the expression of tight junction proteins (e.g., zonula occludens-1 [ZO-1]) in Caco-2 cells, thereby compromising epithelial barrier integrity. Infliximab was used as a model drug to inhibit TNF-α and modulate macrophage polarization within the chip, effectively rescuing impaired epithelial barrier integrity. This study establishes a reliable intestinal-on-a-chip model that recapitulates macrophage–epithelial interactions in IBD, providing a robust platform for elucidating the mechanisms underlying intestinal barrier dysfunction and developing targeted therapeutic strategies. Full article

The co-culture between Trametes sp. D and Aspergillus niger L14 resulted in a distinct orange-brown antagonistic band at their interface. Direct hyphal contact was associated with markedly enhanced production of numerous secondary metabolites (SMs), some of which were absent or decreased in monocultures. T. sp. D induced indolic compounds and cyclic dipeptides, such as Indole-3-acetamide and Cyclo-(Pro-Phe), whereas A. niger L14 overproduced polyketide-derived pigments and organic acids, such as Fonsecin and Kojic acid. These SMs did not inhibit their producer but suppressed the opponent’s growth, indicating reciprocal chemical antagonism. Transcriptomic analysis revealed upregulation of stress-related and metabolic genes, consistent with each fungus activating defense pathways. Biochemical assays showed that the confrontation zone had the highest oxidative stress markers, cell wall-degrading enzyme activity, and acidification (notably by A. niger L14), reflecting intense interfungal antagonism. The stress-response mitogen-activated protein kinase (MAPK) pathway was also activated in both fungi. Our findings supported a mechanistic model of fungal competition involving direct contact, chemical exchange, enzymatic attack, and stress signaling, highlighting that physical interactions likely contributed to triggering cryptic secondary metabolism and robust defense responses. Full article