Search Results (28,595)

Background/Objectives: Methylglyoxal-derived hydroimidazolone-1 (MG-H1), an advanced glycation end product, has implications in the pathogenesis of diabetic kidney disease (DKD). Although liquid chromatography–mass spectrometry is the current gold standard for quantifying MG-H1, its overall complexity limits its utility. We developed an ELISA to measure MG-H1 using a specific monoclonal antibody. Methods: Competitive ELISA was used to quantify total, high-molecular-weight (HMW), and low-molecular-weight (LMW) MG-H1 in serum. The assay’s specificity was validated against structurally related compounds. Spike-and-recovery experiments were conducted to assess accuracy and precision. Serum samples from healthy controls, diabetic patients without kidney disease, and patients with DKD were analyzed (n = 10, 23, and 19, respectively). MG-H1’s correlation with renal biomarkers and diagnostic performance was assessed using receiver operating characteristic analyses. Results: The ELISA exhibited preferential reactivity toward MG-H1 compared with structurally related compounds. Spike-and-recovery experiments resulted in recovery rates ranging 108–119%. MG-H1 levels were increased in patients with DKD, although the magnitude of the changes varied among the MG-H1 forms. All MG-H1 forms correlated positively with serum creatinine and blood urea nitrogen, and negatively with estimated glomerular filtration rate. No significant correlations were observed with glycoalbumin, and only a modest association was observed between LMW MG-H1 and HbA1c. Exploratory ROC analyses suggested that all MG-H1 forms could discriminate DKD from DM, with total and HMW MG-H1 showing performance comparable to that of conventional renal function markers. Conclusions: This competitive ELISA enables high-throughput quantification of MG-H1 in serum and demonstrates analytical feasibility; further multicenter validation is required before clinical implementation. Full article

The bacterial Quorum Sensing system refers to the recognition of signaling molecules called autoinducers produced by bacteria when a certain cell density is reached in the environment. Those cell-density-dependent autoinducers regulate and coordinate diverse functional processes, such as bioluminescence, biofilm production, sporulation, and even the expression of some virulence factors, among others. There is a wide variety of autoinducers, and for Gram-negative bacteria, the canonical autoinducers are the N-acyl-homoserine lactones (AI-1). Presently, the production of autoinducers in Leptospira has not been described; therefore, the objective of this study was to detect and identify autoinducers in this bacterial genus. We report here the expression of AI-1 in cultures ≥2.4 × 10⁸ of Leptospira meyeri. Ethyl acetate extracts of Leptospira culture supernatants were capable of activating the β-galactosidase system in the biosensor Agrobacterium tumefaciens strain NTL4. Partial identification of the leptospiral supernatant extracts was done by thin-layer chromatography (TLC), showing a similar retention factor to the synthetic standard N-Octanoyl-DL-homoserine lactone (C8-AHL) in the Leptospira supernatant extracts. In addition, infrared spectroscopy (IR) analysis showed peaks corresponding to the lactone and amide groups in both the C8-AHL standard and the Leptospira meyeri culture extracts. Moreover, High-Performance Liquid Chromatography–Mass Spectrometry (HPLC-MS/MS) confirmed the same retention time (10.7 ± 0.1 min) in both the Leptospira meyeri supernatant extracts and the C8-AHL standard. These results show that Leptospira meyeri synthesizes N-acyl homoserine lactone family autoinducers, particularly the N-Octanoyl-DL-homoserine lactone, and lay the groundwork for future research on Quorum Sensing systems in Leptospira. Full article

Given the strong association between major histocompatibility complex (MHC) class I and psoriatic arthritis (PsA), and the role of the MHC in antigen presentation potentially driving immunopathology, we performed mass spectrometry-based immunopeptidome analysis to identify MHC class I-bound peptides in PsA. MHC class I expression on circulating immune cells was determined by flow cytometric analysis. MHC class I-bound peptides were isolated via immunoaffinity purification and identified by mass spectrometry in PsA patients and healthy controls. MHC class I expression was increased on circulating immune cells in PsA. MHC class I immunopeptidome analysis revealed a high prevalence of peptides of nine-amino-acid length originating from intracellular source proteins, typical for MHC class I antigen presentation. Analysis of the MHC class I immunopeptidome showed a greater relative abundance of glutamic acid at position 2 in nonamers from PsA patients, but not controls. This finding was further corroborated by an unsupervised clustering analysis showing five different clusters in healthy individuals and six in PsA, with one featuring higher abundance of glutamic acid at position 2. The glutamic acid-containing PEQLRKLFI peptide from the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was exclusively identified in PsA patients both via MHC class I immunopeptidome analysis and via PEPperMap epitope mapping. In PsA, MHC class I expression is increased on circulating immune cells, and the MHC class I immunopeptidome is perturbed, with disease-related skewing towards greater relative abundance of glutamic acid at position 2 in nonamers and presentation of a unique peptide motif from the putative autoantigen hnRNP A1. Full article

Abdominal aortic aneurysm (AAA) is a progressive and often asymptomatic vascular disease associated with high mortality after rupture, but reliable circulating biomarkers for noninvasive detection remain limited. We aimed to identify and validate plasma protein biomarkers for AAA using an integrated proteomics-based approach. Plasma samples from 22 patients with AAA and 22 healthy controls were analyzed through data-independent acquisition (DIA) mass spectrometry. Differentially expressed proteins were subjected to bioinformatic analyses, including Gene Ontology enrichment, Kyoto Encyclopedia of Genes and Genomes pathway analysis, protein–protein interaction, and weighted gene coexpression network analyses. Candidate biomarkers were selected on the basis of differential abundance, diagnostic performance, and biological relevance and subsequently validated by enzyme-linked immunosorbent assay in an independent cohort comprising 93 patients with AAA and 83 non-AAA controls. DIA proteomics identified 111 differentially abundant proteins, revealing enrichment of pathways related to mitochondrial respiration, oxidative stress, inflammation, extracellular matrix remodeling, and proteostasis. Among the candidates, plasma CHRDL1 levels were significantly reduced, whereas OGN and CCL18 levels were significantly elevated in patients with AAA; these findings were consistently confirmed in the validation cohort. A combined three-protein model demonstrated strong diagnostic performance, with an area under the receiver operating characteristic curve of 0.890. These findings identify CHRDL1, OGN, and CCL18 as promising plasma biomarkers for AAA detection and further highlight mitochondrial dysfunction, chronic inflammation, ECM remodeling, and dysregulated proteostasis as key molecular features of AAA. Full article

Mango (Mangifera indica L.), particularly the ‘Nam Dok Mai’ cultivar, is an economically important fruit crop in Thailand and is widely produced in the Lower Northern Region for domestic consumption and export. However, pesticide residues in mango remain a major food safety concern because of their potential effects on consumer health and compliance with national and international maximum residue limits (MRLs). This study investigated pesticide residue contamination and assessed the dietary health risks associated with mango consumption among different age groups. A total of 255 composite mango samples were collected from production areas in Phichit, Phitsanulok, and Phetchabun provinces, Thailand. Pesticide residues were analyzed using a validated modified QuEChERS extraction method combined with gas chromatography–tandem mass spectrometry (GC–MS/MS). Multiple pesticide residues were frequently detected in the samples analyzed. Of the detected residues, 22 pesticides exceeded Thai MRL standards and 14 exceeded Codex Alimentarius MRLs. Dietary exposure was evaluated using acute and chronic hazard quotients (HQa and HQc) and cumulative hazard indices (HIa and HIc). The results showed that acute exposure to several insecticides, particularly carbofuran, lambda-cyhalothrin, and fenpropathrin, exceeded acceptable risk thresholds in all age groups. At the pesticide-class level, cumulative acute exposure to organophosphates, carbamates, and pyrethroids showed HIa values greater than 1 in several age groups, suggesting potential short-term health concerns, especially among children. In contrast, chronic exposure to individual pesticides and cumulative pesticide groups remained below the level of concern for all age groups, with HQc and HIc values below 1. These findings indicate that although long-term dietary risk from mango consumption is within acceptable levels, acute exposure to certain pesticide residues may pose non-negligible health risks. Continuous residue monitoring, stricter enforcement of good agricultural practices, and evidence-based risk management are essential to ensure mango safety and protect consumers. Full article

To investigate the differences in pulp metabolites among four varieties of Pouteria caimito fruit (JZL1, JZL3, JZL5, and JZL6), ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed for the separation and identification of metabolites. A total of 1285 metabolites were identified, including 648 in positive ion mode and 637 in negative ion mode. At the class level, carboxylic acids and derivatives (14.32%) and organooxygen compounds (11.83%) accounted for the highest proportions; at the subclass level, amino acids, peptides, and analogues (12.53%) as well as carbohydrates and carbohydrate conjugates (9.49%) were the main categories. PLS-DA analysis revealed significant differences in metabolite profiles among the four varieties, with numerous up-regulated and down-regulated metabolites in each comparison group, and some metabolites showed fold changes > 10 or <0.1. KEGG pathway enrichment analysis further indicated that differential metabolites were primarily enriched in pathways such as ABC transporters, citrate cycle (TCA cycle), starch and sucrose metabolism, and linoleic acid metabolism. This study provides an important metabolomic basis for variety identification, nutritional quality evaluation, and functional component development of Pouteria caimito fruit. Full article

Background/Objectives: Essential oils (EOs) are complex mixtures extracted from plants. The aim of this study was to evaluate the chemical composition, antioxidant and antimicrobial properties, as well as in vivo toxicity against Drosophila melanogaster of the EOs extracted from Achillea millefolium L. (AM), Mentha longifolia L. (ML), and Thymus serpyllum L. (TS) from the mountains in Sibiu County, Romania. Methods: EOs were extracted by hydrodistillation. Their phytocompounds were determined by Gas Chromatography–Mass Spectrometry. Antioxidant potential was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), and ferric-reducing antioxidant power (FRAP) assays. Antibacterial action was measured against eight pathogenic bacteria. An in vivo D. melanogaster diet-dependent viability assay was conducted to assess the toxicity of the EOs. Results: Chamazulene (49.72%), pulegone (36.20%), and thymol (42.11%) were the most abundant constituents of the AM, ML, and TS EOs, respectively. All tested EOs exerted antioxidant activity, with TS EO being the most efficient in the DPPH (98.19% inhibition) and ABTS (1.82 mmol Trolox equivalent (TE)/mL EO) scavenging assays, while AM EO performed best in the FRAP assay (66.70 μmol TE/mL EO). Against TS EO, Bacillus subtilis was the most sensitive, with an inhibition zone of 37 mm. A dose-dependent decrease in the viability of D. melanogaster L. was observed when their diet was supplemented with AM, ML, and TS EOs. Conclusions: The analyzed EOs demonstrated antioxidant and antimicrobial effects, suggesting their potential applications in various fields. However, due to their toxicity, an in-depth analysis is required before introducing such products in therapy. Full article

To reveal the chemical composition and concentration distribution, spatial distribution characteristics, source composition, and health impacts of volatile organic compounds (VOCs) in the two multi-industry industrial parks in Tianjin, 57 VOC species were determined by using SUMMA canister sampling with preconcentration gas chromatography/mass spectrometry. Based on these measurements, the Positive Matrix Factorization (PMF) model was used for source analysis to achieve quantitative identification and contribution analysis of different source factors. The health risks of VOC concentrations were analyzed based on the assessment framework recommended by the United States Environmental Protection Agency (USEPA). The results showed that the average concentrations of TVOCs in A-1 (Industrial Park and Cluster A, 5 m), A-2 (Industrial Park and Cluster A, 10 m), B-1 (Industrial Park and Cluster B, 5 m), and B-2 (Industrial Park and Cluster B, 10 m) were 59.72 ppbv, 45.35 ppbv, 32.44 ppbv, and 21.65 ppbv, respectively. TVOC concentrations were higher at A-1 and B-1 than at A-2 and B-2, and were generally higher at site A than at site B. The VOC components were mainly alkanes (53.5%–71.4%), followed by aromatic hydrocarbons (15.6%–36.2%), alkenes (4.6%–9.9%), and alkynes (3.1%–7.6%). The proportion of aromatic hydrocarbons in A was higher (22.13% and 36.22%), while alkanes dominated absolutely in B (71.4% and 66.9%). n-Hexane was the key species driving the spatial differences (a typical source of VOCs in solvent usage). The concentration in A-1 was 3.1 times that of A-2, and B-1 was 3.3 times that of B-2. It was mainly controlled by local solvent unorganized emissions. The differences in species between the two points at site A for toluene, xylene, etc., were relatively gentle, while at site B, the concentration of the same aromatic hydrocarbons at B-1 was significantly higher than that at B-2, presenting a clearer spatial characteristic, indicating differences in the spatial distribution of organic solvent-related industrial activities in different sites. The source analysis showed that A-1 was dominated by solvent usage (38.64%) and natural gas/LPG sources (36.96%); A-2 by vehicle exhaust (40.20%) and natural gas/LPG sources (36.15%); B-1 by cleaning-agent usage (38.81%) and fuel combustion (30.33%); and B-2 by plastic and rubber production (36.90%) and fuel combustion (31.84%). The health risk assessment showed that benzene, n-hexane, and xylene dominated the non-carcinogenic risks, but the overall non-carcinogenic (HI < 1) and carcinogenic (CR < 1 × 10⁻⁶) risks were both below the threshold. This study reveals inter-site differences in VOC concentrations, compositions, and source contributions across two mixed industrial parks, providing a basis for site-specific VOC control priorities, source-targeted monitoring strategies, and risk management in mixed industrial areas. Full article

Honey is a natural food product highly valued for its nutritional and biological properties. Its quality and safety are increasingly affected by environmental contamination and apicultural practices. Among the most relevant contaminants, pesticide residues, veterinary antibiotics, and heavy metals represent major concerns due to their potential impact on human health. This review provides a comprehensive overview of the occurrence, sources, and distribution of these contaminants in honey, with particular emphasis on their relationship with agricultural activities, environmental pollution, and beekeeping treatments. Advanced analytical techniques, including liquid chromatography–tandem mass spectrometry (LC-MS/MS), gas chromatography–mass spectrometry (GC-MS), and inductively coupled plasma–mass spectrometry (ICP-MS), are highlighted for their ability to enable sensitive multi-residue and trace-level detection. The application of chemometric tools for data analysis and sample classification is also addressed, supporting the identification of contamination patterns and origin-related differences. In addition, recent developments in rapid screening methods and environmentally sustainable analytical approaches are discussed. Overall, this review emphasizes the importance of continuous monitoring and the integration of advanced analytical strategies to ensure honey safety, support regulatory compliance, and protect consumers. Full article

Lonicerae japonicae flos (LJ), derived from Lonicera japonica Thunb., is a classic medicinal and edible plant traditionally used to clear heat, detoxify, and disperse wind-heat. It is widely consumed as a health tea or food additive and played a significant role during the COVID-19 pandemic. Its closely related species, Lonicerae flos (LFS), derived from L. macranthoides, L. hypoglauca, L. confusa, and L. fulvotomentosa, share similar functions. Distinguishing between these two is crucial to minimizing medication-related risks; however, studies on their differences in inorganic elements are currently insufficient. This study investigated the content differences in ten inorganic elements in 97 samples using inductively coupled plasma mass spectrometry and inductively coupled plasma atomic emission spectrometry. LJ exhibited significantly higher iron (Fe) content and considerably lower manganese (Mn) content than LFS. Fe, Mn and Na were identified as key discriminatory elements through orthogonal partial least squares-discriminant analysis, and cluster analysis based on the Fe/Mn ratio also achieved clear separation between the two groups. Network pharmacology and pathway enrichment analyses were subsequently conducted to explore the potential biological relevance of Fe- and Mn-associated differences. Additionally, molecular docking provided exploratory computational assessments of theoretical interactions between Fe/Mn and selected target proteins under simplified conditions. It is important to note that these computational results should not be interpreted as direct evidence of biological activity, since no in vitro or in vivo validation was performed. Overall, this study provides elemental profiling data to differentiate between LJ and LFS and highlights the potential value of the Fe/Mn ratio as a complementary chemical indicator for the authentication and quality evaluation of Lonicera medicinal materials. Full article

This study evaluated potentially toxic element (PTEs) contamination (As, Cd, Co, Cr, Cu, Ni, Pb, and Zn) in soil and grapevine leaves across 36 sites in a historical vineyard near Iași, Romania, transitioning toward residential development. Soil samples were analyzed using energy-dispersive X-ray fluorescence (ED-XRF), while leaf tissues collected during the véraison stage (mid-August) underwent microwave-assisted acid digestion followed by inductively coupled plasma mass spectrometry (ICP-MS). Soil analyses revealed that average concentrations of Cr, Ni, and Pb exceeded national normal legal thresholds, while Cu and As surpassed alert permissible limits. Contamination factor ($CF$) and Geoaccumulation Index ($I_{geo}$) metrics confirmed significant anthropogenic Cu enrichment from historical viticultural treatments. Soil-to-leaf transfer factors ($TF$) approached or exceeded 1 for Cu and Zn, though grapevine was not identified as a hyperaccumulator. Spearman correlation modeling demonstrated a statistical decoupling between soil concentrations and foliar tissue levels for Cu ($r_s=-0.21$) and Zn ($r_s=0.075$), confirming that canopy accumulation is governed by historical agrochemical spraying and atmospheric deposition rather than root-driven uptake. Conversely, Pb ($r_s=0.43$) exhibited a moderate direct soil-to-leaf pathway. The potential ecological risk ($PER$) index indicated moderate-to-considerable cumulative risk, primarily driven by Cu ($E_{Cu}=68.64$) and Cd ($E_{Cd}=59.17$). Human health risk assessments established direct ingestion as the primary exposure pathway, with current non-carcinogenic hazards remaining within tolerable limits ($HI<1$). However, the proposed residential conversion increases exposure risks for vulnerable cohorts, underscoring the necessity of systematic soil screening and remediation prior to land redevelopment. Full article

Background/Objectives: Niaprazine is widely used for the management of sleep disorders in pediatric and geriatric patients; however, no commercially available oral liquid formulation is currently available in Italy, making extemporaneous compounding necessary. In routine practice, the beyond-use date (BUD) is often limited to 30 days, potentially affecting therapeutic continuity. This study aimed to evaluate the chemical, physical, and microbiological stability of a compounded niaprazine syrup to support evidence-based BUD and to assess the suitability of selected ready-to-use compounding vehicles for preparing alternative niaprazine oral liquid formulations. Methods: A niaprazine syrup (3 mg·mL⁻¹) was prepared in a sucrose-based vehicle acidified with tartaric acid and preserved with potassium sorbate. Chemical stability of niaprazine was evaluated by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD), whose specificity was confirmed by forced degradation studies. Stability was monitored for up to 9 months at 4–8, 25, and 40 °C and confirmed after 12 months by HPLC-DAD and mass spectrometry (MS). Physical stability of the formulation was monitored by pH and visual inspection up to 12 months. Microbiological quality was assessed for 2 months at 4–8 and 25 °C according to the European Pharmacopoeia. In parallel, four selected ready-to-use compounding vehicles were evaluated for their suitability to prepare stable niaprazine oral liquid formulations. Results: Niaprazine concentrations in the syrup remained within pharmacopeial acceptance limits (±10%) at all temperatures, although a decrease was observed under accelerated conditions (40 °C). pH remained stable (≤0.5-unit variation) and the formulation stayed clear and homogeneous throughout the study, with only minor visual changes after prolonged storage at 40 °C. Statistically significant differences (p < 0.05) were observed in both the HPLC-DAD and HPLC-MS datasets. Microbiological testing confirmed compliance up to 2 months at 4–8 and 25 °C (TAMC ≤ 10³ CFU/mL; TYMC ≤ 10² CFU/mL; Escherichia coli absent). Comparable chemical and physical stability was observed for three of the four formulations prepared with the ready-to-use vehicles over at least two months. Conclusions: The compounded niaprazine syrup demonstrated chemical, physical, and microbiological stability under refrigerated and room-temperature storage, supporting evidence-based beyond-use dating of up to two months under the tested conditions. Ready-to-use vehicles may represent a practical complementary approach, offering standardized alternatives for the preparation of niaprazine oral liquid formulations. Full article

Herein, we report an unexpected pseudo-multicomponent transformation discovered during attempts to selectively nitrate the pyridine core of 3-acetyl-2,4,6-trimethylpyridine (3). Despite employing standard nitration conditions, including KNO₃–H₂SO₄ and HNO₃–H₂SO₄ mixtures, electrophilic substitution of the aromatic ring did not occur. Instead, the reaction sequence promoted an in situ nitrozation, dehydration to nitrile oxide intermediates, and subsequent [3+2]-cycloaddition involving two substrate molecules. This process yielded a novel, highly functionalized furoxan derivative, precisely identified as 3,4-bis(2,4,6-trimethylnicotinoyl)-1,2,5-oxadiazole 2-oxide (5). The molecular architecture of compound 5 was established by ¹H and ¹³C NMR spectroscopy, mass spectrometry, elemental analysis, and single-crystal X-ray diffraction (XRD) analysis. To elucidate the stereochemical and electronic features governing compound 5, DFT calculations were performed at the ωB97X-D/6-311++G(d,p) level of theory. The experimental crystallographic disorder of the N-oxide oxygen atom was computationally rationalized by the thermodynamic near-degeneracy (ΔG < 0.63 kcal/mol) of two orientational isomers (5a and 5b). Furthermore, frontier molecular orbital analysis within the framework of perturbation theory accounted for the head-to-tail regioselectivity during cyclization, while wide energy gaps (ΔE = 8.13–8.27 eV) and high chemical hardness (η = 4.07–4.14 eV) underscored the kinetic stability of the heterocycle. Phenotypic and target-specific in silico profiling using PASS Online identified Matrix Metalloproteinase-9 (MMP-9) as a relevant target for potential hemorheological and cardioprotective applications. Validated molecular docking simulations across three human MMP-9 crystallographic domains (PDB: 8K5Y, 6ESM, 4XCT) demonstrated competitive binding affinities and balanced Ligand Efficiency metrics (LE = 0.26–0.29 kcal/mol/heavy atom), anchoring compound 5 within the catalytic pocket via conventional hydrogen bonds and π-mediated interactions. Finally, in vitro evaluations using a blood hyperviscosity model confirmed significant hemorheological efficacy, as compound 5 effectively prevented the rise in blood viscosity, outperforming the reference drug pentoxifylline. The convergence of computational insights and experimental functional activity establishes this novel bis(nicotinoyl)furoxan framework as a promising candidate for further hemorheological and cardioprotective applications. Full article

Proteomics has emerged as a powerful tool for advancing our understanding of brain disorders by enabling large-scale characterization of protein expression, post-translational modifications, and interaction networks. Neurological conditions are often characterized by complex and dynamic molecular changes that are not fully captured by traditional diagnostic approaches. Proteomic technologies, particularly mass spectrometry-based and affinity-based methods, offer the ability to identify disease-specific protein signatures and elucidate underlying pathophysiological mechanisms, including neurodegeneration, neurodevelopment and neuroinflammation and alterations happening to the extracellular matrix and body fluid homeostasis. In recent years, artificial intelligence has emerged as a powerful tool to proteomics, enabling improved analysis of complex biological datasets. This integration has significantly enhanced the discovery of biomarkers for early diagnosis, disease stratification, and monitoring of therapeutic responses. Thus, cerebrospinal fluid and blood-based proteomic analyses have revealed promising candidates for neurological diseases. This review summarizes current advances in proteomics across a range of brain disorders, highlighting key molecular pathways, biomarker discovery efforts, and evolving clinical applications. Furthermore, it outlines future directions, including the application of machine learning for improved biomarker identification and precision medicine. Full article

Umbilical cord mesenchymal stem cell-derived extracellular vesicles (UCMSC-EVs) hold strong promise for regenerative medicine, yet their intrinsic size heterogeneity remains a critical barrier to clinical translation, as it obscures molecular and functional specialization within bulk EV preparations. Here, we pioneer the application of the automated EXODUS platform to directly fractionate EVs from cell culture supernatants, resolving bulk UCMSC-EVs into three size-defined subpopulations. By integrating this platform with high-resolution mass spectrometry, we systematically characterize the molecular and functional landscapes of these UCMSC-EV size subpopulations. We demonstrate that EV size is tightly linked to distinct biogenetic origins, biomolecular corona composition, and functional programs: smaller EVs are enriched in exosome-associated proteins, ECM–glycan interfaces, and corona-associated molecules, and preferentially engage endocytosis- and phagosome-related pathways, whereas larger EVs exhibit ectosomal signatures. These findings identify EV size as a critical determinant of molecular architecture and biological function, providing insight into size-dependent EV heterogeneity and informing the rational design and optimization of UCMSC-EV-based therapeutic strategies. Full article