Ganglioside GM3, a fundamental glycosphingolipid on the mammalian cell surface, is a key regulator of transmembrane signaling and cellular recognition. In oncology, GM3 acts as a tumor suppressor by modulating the activity of various receptor tyrosine kinases (RTKs) and their downstream pathways. Recent studies highlight its function in the tumor microenvironment (TME), specifically its ability to impede pathological angiogenesis. This review summarizes the molecular mechanisms by which GM3 interferes with pro-angiogenic signaling, such as the VEGF/VEGFR axis, and discusses how this inhibition can be used for therapy. We explore the clinical potential of GM3-based strategies, including monoclonal antibodies and cancer vaccines, discussing the potential of targeting GM3 to reshape the TME and suppress tumor-associated vascularization.

BackgroundLAMA2-related congenital muscular dystrophy (LAMA2-MD) is a genetically heterogeneous disorder defined by progressive muscle weakness, brain structural abnormalities, epilepsy, and multisystem involvement. The primary goal of this study was to characterize the clinical features, temporal progression, and genotype-phenotype correlations of LAMA2-MD.MethodsMedical records of patients with genetically confirmed LAMA2-MD were extracted from a clinical data repository and analyzed retrospectively. Clinical manifestations, laboratory findings, and neuroimaging features were systematically reviewed and compared across different age groups. Variant data were retrieved from public databases to perform comprehensive genetic analyses.ResultsA total of five patients (two males and three females) were enrolled, delayed motor milestones and varying degrees of ankle contractures and persistent motor impairment in all patients were the initial presenting symptom at diagnosis in all cases, and two patients also exhibited cognitive delays. Laboratory analysis of muscle enzymes showed varying degrees of abnormalities, with creatine kinase (CK) levels displaying the most significant elevation. Cranial magnetic resonance imaging (MRI) revealed symmetrical white matter abnormalities in four patients. Seizures were documented in three school-aged patients. All patients carried compound heterozygous variants in the LAMA2 gene. A literature review indicated that the most common variant types were stop-gain and missense variants: stop-gain variants were predominantly associated with complete merosin deficiency (MDC1A), whereas missense variants typically correlated with late-onset limb-girdle muscular dystrophy.ConclusionLAMA2-MD exhibits a broad phenotypic spectrum and a progressive disease course. Early manifestations include muscle weakness, delayed achievement of developmental milestones, joint contractures, seizures and characteristic intracranial abnormalities.

Apolipoprotein B-100 (apoB-100) is the main structural protein of apoB-containing lipoproteins including low-density lipoprotein (LDL). Its organization or lipidation process in an apoB-containing lipoprotein particle is still unclear. To understand its organization in a LDL particle, the combination of atomic force microscopy (AFM) with lipid depletion by Nonidet P-40 (NP-40) or methyl-β-cyclodextrin (MβCD) was utilized for the first time to in situ visualize LDL delipidation process and lipid-poor/-free apoB-100 at a physiological condition. During LDL delipidation process, different morphologies/structures were visualized successively including spheroidal structure with a smaller size than native LDL, spheroidal structure with one or more holes, closed annular/circular structure, opened annular/circular structure, C/U-shaped (or horseshoe-shaped) structure, and V/S/I-shaped structure. Based on the concentration-dependent structural distributions, these structures probably reflect 5 stages of LDL delipidation (e.g., a slightly delipidated LDL stage, a partially delipidated LDL stage, a neutral lipid-poor/-free apoB-100 stage, a lipid-poor apoB-100 stage, and a lipid-free apoB-100 stage, respectively). Our findings could provide structural evidence to reconcile the previous controversy and provide potential evidence/clues/implications for understanding apoB-100 lipidation and the organization of apoB-100 in apoB-containing lipoprotein particles. Potentially, this study also can provide new structural insights into the design of food-grade lipid carriers. Moreover, the combination of AFM with lipid depletion, which has many advantages over traditional electron microscopy (e.g., label-free, in situ, and real-time imaging under physiological conditions, etc.), is a potentially ideal novel strategy for studying the structure of apolipoproteins or lipoproteins.

BackgroundInfantile epileptic spasm syndrome (IESS) is a severe age-dependent epileptic encephalopathy in infancy with poor prognosis and unclear pathogenesis. Neuroinflammation plays a pivotal role in epileptogenesis, and the high-mobility group box 1 protein (HMGB1)-Toll-like receptor 4 (TLR4) axis acts as a core mediator of neuroinflammation. However, its specific role in IESS remains elusive.ObjectiveThis study aimed to explore the HMGB1-TLR4-mediated neuroinflammatory mechanism in a rat model of IESS induced by prenatal stress combined with NMDA, and to evaluate the effects of anti-HMGB1 neutralizing antibody and adrenocorticotropic hormone (ACTH) on epileptic seizures and neuroinflammation, so as to provide novel therapeutic targets for clinical practice.MethodsPregnant Sprague-Dawley rats were randomly divided into prenatal stress (PS) and non-prenatal stress (NPS) groups. PS rats received cold water immersion and hot air drying, while NPS rats were reared normally. On postnatal day 12 (P12), offspring in the PS group were intraperitoneally injected with NMDA to establish the IESS model, and the NPS group was assigned to blank control (BC) and negative control (NC) subgroups. Model rats were randomly divided into ACTH, anti-HMGB1, ACTH+anti-HMGB1, normal saline, and untreated groups. After intervention on P13, NMDA was re-administered, and seizure latency and severity score were recorded. At the end of the experiment, the expression of HMGB1 and TLR4 in brain tissue was detected, HMGB1 co-localization was observed, and the levels of iNOS, Arg1 and cytokines (IL-1β, IL-2R, IL-8, TNF-α) were measured.ResultsPrenatal stress combined with NMDA successfully established a stable IESS model in young rats. The expression of HMGB1, TLR4, iNOS, IL-1β, IL-2R, IL-8 and TNF-α was significantly upregulated, while Arg1 was markedly downregulated. Treatment with ACTH, anti-HMGB1, and their combination prolonged seizure latency, reduced seizure severity, downregulated HMGB1 and TLR4 expression, suppressed HMGB1 levels in neurons, astrocytes and activated microglia, inhibited iNOS and proinflammatory cytokines, and promoted Arg1 expression, with the combined intervention showing the optimal efficacy.ConclusionPrenatal stress combined with NMDA activates the HMGB1/TLR4 pathway and neuroinflammation in IESS rats. ACTH and anti-HMGB1, alone or in combination, alleviate neuroinflammation by inhibiting this pathway to ameliorate IESS, and the combined therapy yields the best therapeutic effect.

BackgroundInfantile epileptic spasms syndrome (IESS) is a severe age-specific epileptic encephalopathy with unclear pathogenesis, and neuroinflammation is involved in its progression. The HMGB1-TLR4 signaling pathway, a key neuroinflammatory mediator in various epilepsies, has not been studied for its role and clinical biomarker potential in IESS.MethodsA retrospective study included 66 IESS patients treated with a modified prednisone regimen and 53 age-matched healthy controls. Serum HMGB1, TLR4, IL-1β, IL-2, IL-2R, IL-8 and TNF-α were detected by ELISA/chemiluminescent immunoassay in IESS patients (pre- and 2-week post-treatment) and controls; clinical data were collected via electronic medical records and follow-up.ResultsIESS patients had significantly higher serum HMGB1, TLR4, IL-2, IL-2R, IL-8 and TNF-α than controls (P < 0.05; no IL-1β difference, P>0.05), and these elevated indicators decreased markedly post-treatment (P < 0.05). Logistic regression showed identified etiology and focal seizures were risk factors for short-term prednisone ineffectiveness, while ΔPre-Post HMGB1 was a protective factor (P < 0.05). Long-term follow-up (≥18 months) found identified etiology to be a risk factor for uncontrolled epilepsy and poor neurodevelopment (P < 0.05); early spasm remission and long-term seizure control were protective factors for neurodevelopment (P < 0.05).ConclusionsThe HMGB1-TLR4 pathway mediates neuroinflammation in IESS pathogenesis and may serve as a therapeutic target. A high ΔPre-Post HMGB1 level was identified as a protective factor against short-term treatment failure of prednisone, indicating that dynamic monitoring of HMGB1 has clinical value for predicting short-term treatment responses to prednisone, though it does not predict long-term seizure control or neurodevelopmental outcomes. Identified etiology is a common risk factor for poor IESS outcomes, highlighting the importance of early etiological screening and sustained seizure control for IESS management.

Yiqi Huoxue Compound (YQHX), a traditional Chinese medicine formulation, has demonstrated therapeutic potential for ischemic stroke (IS). However, its underlying mechanisms are not fully understood. This study aimed to elucidate the neuroprotective effects and mechanisms of YQHX using an integrated strategy encompassing UHPLC-Q-Orbitrap HRMS-based chemical profiling, in vivo pharmacodynamic evaluation, transcriptomics, network analysis, molecular docking, and in vitro validation. Chemical analysis identified 196 constituents in YQHX, mainly terpenoids, saccharides, and polyphenols. In a mouse model of middle cerebral artery occlusion (MCAO), treatment with low, medium, or high doses of YQHX significantly improved neurological function scores, ameliorated cerebral histopathological damage, increased Nissl bodies, and reduced neuronal apoptosis. This was evidenced by decreased TUNEL-positive cells and regulated expression of apoptosis-related proteins (Bax, Bcl-2, cleaved-caspase 3). Transcriptomic analysis of brain tissue highlighted the PI3K-AKT signaling pathway as a key target, which was further supported by molecular docking showing strong binding affinities between core YQHX components and pathway targets. Importantly, YQHX administration upregulated the p-PI3K/PI3K and p-AKT/AKT ratios and attenuated mitochondrial ultrastructural damage in MCAO mice. In vitro, using HT22 cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), YQHX treatment suppressed inflammatory cytokine release (TNF-α, IL-6, IL-1β), inhibited apoptosis, and restored the balance of apoptosis-related and PI3K/AKT pathway proteins. Collectively, these results demonstrate that YQHX protects against ischemic brain injury by activating the PI3K/AKT pathway, thereby reducing inflammation and apoptosis. This study provides a mechanistic foundation for the clinical application of YQHX in IS.

The rational design of cellular interfaces is essential for advancing biocatalytic systems. White rot fungi are promising biodegraders of pharmaceutically active compounds (PhACs), but they are readily inactivated in wastewater treatment, while conventional carriers cannot simultaneously immobilize cells and retain extracellular enzymes. In this study, a stable fungus-enzyme synergistic interface was constructed by assembling Trametes versicolor with a positively charged porous thienyl cyclodextrin polymer (Th-CDP) to form Tv@Th-CDP. This design simultaneously immobilized fungal cells and retained their secreted laccases within the carrier, enhancing PhAC removal in continuous-flow reactors treating secondary municipal effluent to 93%. The system demonstrated sustained 30-d performance and reduced effluent toxicity by 58%. This performance was associated with the high positive charge density of Th-CDP, which enhanced mycelial attachment and laccase retention (48.2 U kg-1). Furthermore, the hydrophobic cavities of cyclodextrin, coupled with electrostatic interactions, enhanced PhAC adsorption and enriched Trametes sp. (46%). This immobilization strategy improved the fungus-enzyme interactions, with enhanced laccase-related catalytic currents observed in electrochemical assays. Metaproteomics analysis and transformation pathway identification supported a degradation model mediated by electrostatic interactions between the fungus and the retained enzymes. A total of 305 proteins associated with oxidoreductase activity were enriched, while the key enzymes aldehyde dehydrogenase (ALDH) and epoxide hydrolase (EPHX2) were upregulated. These changes coincided with enhanced C-N bond cleavage, indicating activation of associated metabolic pathways. Overall, this study provides mechanistic understanding of immobilization mediated by electrostatic interactions and demonstrates its potential to improve the biodegradation of emerging contaminants in engineered biocatalytic systems.

Mussel foot proteins (Mfps) are a class of specialized adhesive proteins synthesized by marine mussels. Among these, type 3 mussel foot protein (Mfp-3) has tremendous application potential as a cosmetic and medical raw material, owing to its excellent antioxidant and anti-inflammatory properties. However, the low efficiency of the natural extraction method (only 1 mg of protein can be obtained from 10,000 mussels) has hindered the industrial application. Heterologous expression via genetic recombination offers a reliable alternative. However, a major challenge is that recombinant expression frequently results in misfolded, biologically inactive protein aggregates. In this study, we identified endogenous molecular chaperones in Mytilus galloprovincialis and successfully constructed a strain that co-expresses Mfp-3 and these chaperone proteins, thereby achieving the soluble expression of Mfp-3. Furthermore, through optimization of fermentation conditions, the soluble yield of Mfp-3 in high-density fermentation using a 5-L fermenter reached 713 mg/L. Meanwhile, following in vitro tyrosinase modification, the L-3,4-dihydroxyphenylalanine (DOPA) content of Mfp-3 reached 4.45%, corresponding to a modification rate of 22.26%. On this basis, this study also demonstrated that soluble Mfp-3 possesses excellent cell migration-promoting ability. Moreover, it exhibited superior biological activity in the treatment of atopic dermatitis by reducing levels of pro-inflammatory cytokines and immunoglobulins. This research provides a new strategy for the production of soluble Mfp-3 in Escherichia coli and offers a reference for the large-scale production of soluble Mfps.

Spinal cord injury (SCI) is a severe neurotraumatic condition for which effective therapeutic options remain limited, and advances in clinical pharmacological research have been slow. The diverse pathophysiological alterations that occur after SCI initiate cellular pyroptosis, which in turn exacerbates tissue damage, impedes neuronal functional recovery, and connects multiple pathological processes involved in SCI. In recent years, research on natural bioactive compounds has made substantial progress in the field of neurotrauma, including SCI, leading to the identification of several compounds capable of effectively modulating pyroptosis and promoting functional recovery. Therefore, a comprehensive synthesis of the mechanisms underlying pyroptosis during SCI pathophysiology, along with an overview of natural bioactive constituents with the potential to modulate SCI-related pyroptosis, may provide useful insights for future pharmacological studies, mechanistic investigations, and clinical management of SCI.

Background: Atherosclerosis (AS) remains a leading cause of cardiovascular mortality worldwide and is significantly driven by hyperlipidemia. While ganglioside GM3 is known to regulate cellular lipid metabolism, its systemic pharmacological effects on atherosclerosis remain unclear. This study aims to evaluate the anti-atherosclerotic efficacy of exogenous GM3 and elucidate its underlying systemic mechanisms. Methods: C57BL/6N ApoE-/- mice fed a high-fat diet were intravenously treated with exogenous GM3 (1 or 4 mg/kg) every three days for 12 weeks. Atherosclerotic progression and lipid profiles were evaluated through histological analyses of the aortic arch and aortic sinus, alongside biochemical and molecular assessments of plasma, hepatic, and intestinal tissues. Results: GM3 treatment significantly reduced plaque formation in the aortic arch and aortic sinus, along with decreased plasma levels of triglycerides, total cholesterol, and LDL-C. Mechanistically, GM3 suppressed hepatic VLDL secretion by downregulating ApoB100 and MTTP expression. Concurrently, hepatic lipid clearance was enhanced via the upregulation of Ldlr, Scarb1, and Lrp1. GM3 also lowered circulating PCSK9 levels and reduced intestinal cholesterol absorption by decreasing NPC1L1 expression. Although GM3 promoted lipid accumulation in the liver, no evidence of liver dysfunction or systemic toxicity was observed. Conclusions: Exogenous GM3 acts as a potent multi-target modulator that attenuates atherosclerosis by coordinating hepatic lipoprotein metabolism and restricting intestinal cholesterol uptake. This multi-organ metabolic partitioning strategy highlights the potential of GM3-based therapeutics for managing complex dyslipidemia.

Extracellular vesicles (EVs) are nanoscale lipid-bilayer-enclosed particles released by most cell types, serving as pivotal mediators of intercellular communication, cargo transport, and immune regulation. Owing to their intrinsic biological functions and biocompatibility, EVs demonstrate tremendous potential in medical applications. However, a major challenge in EV research is the coisolation of lipoprotein (LP) contaminants, particularly plasma lipoproteins, during the purification of biofluids (e.g., plasma or serum) or cell culture supernatants. LPs and EVs exhibit substantial overlap in physicochemical properties, including particle size and density, which likely contributes to their coisolation. Notably, mutual-contamination between these two particle populations can significantly interfere with downstream analyses, leading to misinterpretation of their respective compositions and biological functions. Therefore, obtaining high-purity EV and LP isolates free from mutual contamination is crucial. To address this technical challenge, there is an urgent need to establish robust isolation methods and standardized characterization systems. This review systematically evaluates current EV/LP isolation technologies with varying separation specificities, while innovatively proposing characterization strategies capable of distinguishing EVs, LPs, and potential EV-LP complexes. By elucidating the "mutual-contamination" issues between these particles, it is aimed to promote and call for the establishment of stricter methodological standards in this field.

Ovarian cancer (OC) disseminates via ascites and interaction with peritoneal extracellular matrix (ECM). To dissect the crosstalk between ECM and ascites in OC cell migration, we developed an ovarian tumor-on-chip integrating OC tumor spheroids, perfusion-induced shear stress of fluid supplemented with key components of ascites or patient-derived ascites, and biomimetic ECMs mimicking either early-stage basement membrane or late-stage connective tissues. We evaluated the individual and combined effects of two key ascitic components, fibronectin and TGF-β, and compared these results with the perfusion of patient ascites. Results showed that cell migration, morphology, and epithelial-to-mesenchymal transition (EMT) markers such as the reorganization of vimentin cytoskeleton depend strongly on ECM composition, regardless of biochemical cues. Fibronectin and TGF-β synergistically enhanced migration and EMT signatures, especially on basement membrane-rich ECM. Patient ascites further promoted migration but did not override ECM-driven migration patterns. Our findings show that clinical ascites perfusion exemplifies the ECM-dependence of cancer cell migration. This highlights that ECM protein composition is a dominant regulator of OC cell migration, providing key insights for in vitro tumor modeling and therapeutic strategies targeting the metastatic microenvironment. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-11-EQPX-0029 Morphoscope2, ANR-10-INBS-04FBI, ANR-21-CE19-0006

During the process of acute lung injury (ALI) associated with sepsis, the α7nAChR in the cholinergic anti-inflammatory pathway (CAP) plays a crucial role. However, the roles of electroacupuncture (EA) and specialized pro-resolving mediators (SPMs) in this context remain unclear. In this study, we demonstrated that EA activates CAP via α7nAChR, reducing lung permeability and inflammatory cytokine release. Our results highlighted lipoxin A4 (LXA4) as a crucial SPM in this process. EA was shown to enhance LXA4 synthesis and alleviate symptoms in patients with sepsis-related acute respiratory distress syndrome (ARDS). Studies using α7nAChR-deficient mice confirmed its essential role in LXA4 regulation. Macrophages in bronchoalveolar lavage fluid (BALF) were identified as key contributors to the protective effects of LXA4, further supported by experiments involving pulmonary macrophage depletion. In summary, we discovered a novel anti-inflammatory pathway where EA activates α7nAChR, leading to increased LXA4 production and lung protection.

Hepatocellular carcinoma (HCC) represents a significant global health challenge, characterized by a high incidence rate. Mitochondria have emerged as an important therapeutic target for HCC. Donafenib, a multi-receptor tyrosine kinase inhibitor, has been approved for the treatment of advanced HCC. However, the underlying mechanisms remain to be elucidated. In this study, we aim to investigate the effects of Donafenib on mitochondrial function in HCC cells. Firstly, we show that Donafenib induces mitochondrial oxidative stress in SNU-449 liver cancer cells by increasing mitochondrial ROS while reducing glutathione peroxidase (GPx) activity and the expression of Mn-SOD. We also demonstrate that Donafenib decreases mitochondrial membrane potential (MMP) and induces the opening of the mitochondrial permeability transition pore (mPTP). Furthermore, Donafenib reduces mitochondrial respiratory rate, COX IV activity, and ATP production. Notably, Donafenib induces mitochondrial fragmentation and reduces mitochondrial length by increasing the expression of DRP1, without affecting Mfn1 or Mfn2. Silencing of DRP1 protects against mitochondrial dysfunction induced by Donafenib, indicating that DRP1 plays a key role in mediating Donafenib's effects on mitochondrial function in HCC cells.

Objectives:To study the characteristic factors associated with the occurrence of malignant nodules in patients presenting with pulmonary nodules, develop a predictive model, and evaluate its diagnostic performance. Methods:This study analyzed the clinical and imaging data of 830 patients with pulmonary nodules from the Affiliated Hospital of North Sichuan Medical College. The Least Absolute Shrinkage and Selection Operator (LASSO) and multivariate logistic regression analysis were utilized to identify characteristic predictors. Multiple machine learning classification models were employed for analysis, with the optimal model ultimately selected. A Shapley Additive Explanations (SHAP) framework was developed for personalized risk assessment. Finally, external testing was performed using data from 330 pulmonary nodule patients at Guang'an People's Hospital. Results:The predictive factors for malignant pulmonary nodules included: age, gender, nodule diameter, spiculation, lobulation, calcification, vacuole, vascular convergence sign, air bronchogram sign, pleural traction, and density of the nodule. The Gradient Boosting Decision Tree (GBDT) classification model demonstrated optimal performance, with an area under the curve (AUC) of 0.873 (95% confidence interval [CI]: 0.840-0.906) on the internal test set and 0.726 (95% CI: 0.668-0.784) on the external test set. Both the calibration curve and clinical decision curve analysis (DCA) indicated excellent model calibration and substantial clinical benefits. Conclusions:We developed a GBDT model that provides a basis for differentiating malignant pulmonary nodules, which may assist in the diagnosis and treatment of patients with pulmonary nodules.

Aggregated low-density lipoprotein (agLDL) has long been regarded as an atherogenic factor and was recently supposed to play a driving force role in atherogenesis. However, the underlying mechanisms for its atherogenicity remain poorly understood. Here, agLDL was successfully induced by physical (vortexing) or biological (alpha-chymotrypsin or α-CT and sphingomyelinase or SMase, a protease and a phospholipase, respectively) methods, and verified by turbidimetry, size distribution, and relatively low-resolution morphology. Moreover, the produced agLDL had the aggregation degree-dependent atherogenic property according to macrophage lipid deposition data. Then, the single-aggregate morphological and biomechanical (stiffness and stickiness) properties of agLDL at early stages of aggregation (e.g., LDL dimer, trimer, and multimer) were detected by atomic force microscopy (AFM). Interestingly, intra-aggregate LDL of agLDL had a relatively larger average size than native LDL (i.e., LDL monomer). Most importantly, we revealed that agLDL has lower average Young's modulus and stronger average adhesion force than native LDL (i.e., agLDL is softer and stickier than native LDL). The softer and stickier properties of agLDL than native LDL probably are responsible for the atherogenicity of agLDL according to its aggregation degree-dependent atherogenic property. Targeting the biomechanical properties (e.g., stiffness and stickiness) of agLDL or other LDL derivatives may be a potential strategy for preventing atherosclerosis.