Search Results (8,761)

Chronic exposure to benzo[a]pyrene (BaP), a Group 1 IARC carcinogen, is a major driver of lung carcinogenesis; however, how sustained subcytotoxic exposure reprograms bronchial epithelium toward preneoplastic states remains poorly defined. Here, we subjected BEAS-2B human bronchial epithelial cells to 12 weeks of continuous BaP at environmentally relevant concentrations (0.1 and 1.0 µM) and interrogated the resulting phenotypes using an integrated multi-scale framework encompassing functional toxicology, RT-qPCR, RNA-seq, phospho-kinase/NF-κB arrays, and organotypic air–liquid interface (ALI) cultures. Cells maintained metabolic competence throughout, evidenced by sustained CYP1A1 and CYP1B1 induction at both acute (4 h) and chronic (12-week) timepoints, while accumulating genotoxic stress as demonstrated by dose-dependent nuclear γ-H2AX foci formation and ATM phosphorylation (Ser1981). RNA-seq revealed a dose-dependent transcriptional shift: 0.1 µM BaP yielded 119 differentially expressed genes (DEGs; |log2FC| ≥ 1, FDR < 0.05), whereas 1.0 µM generated 255 DEGs. Downregulated transcripts were enriched for extracellular matrix and cell-adhesion programs (COL14A1, ADAMTS2, CSMD3, CADM3), while upregulated genes encompassed inflammatory, calcium-signaling, and vesicle-trafficking modules (NFATC4, CSF2RA, SYT1, PCLO). Phospho-kinase/NF-κB arrays confirmed a p53/NF-κB signaling nexus, with concurrent activation of MAPK/ERK (Thr202/Tyr204) and PI3K/Akt (Ser473) pathways. Despite persistent genotoxic stress, cells did not acquire anchorage-independent growth and remained non-tumorigenic in vivo. Critically, ALI organotypic cultures derived from BaP-exposed cells exhibited histological dysplasia, nuclear pleomorphism, and disrupted apical-basal polarity. These findings mechanistically link chronic BaP exposure to an initiation-like preneoplastic state and establish a validated 2D/3D multi-omics platform for PAH-driven lung carcinogenesis research. Full article

To improve the vitrification efficiency of bovine germinal vesicle (GV) oocytes, the use of hydroxyapatite (HA) nanoparticles as a novel cryopreservation additive represents a promising approach. This study aimed to investigate the effects of HA nanoparticles and permeable cryoprotective agents (CPAs) on the ultrastructure, developmental competence, and gene expression of bovine GV oocytes following vitrification. Oocytes were vitrified in vitrification solutions containing HA nanoparticles of different sizes (20, 40, or 60 nm) and concentrations (0.01%, 0.05%, or 0.1%) to determine the optimal conditions based on survival rate, mitochondrial membrane potential (MMP) level, and developmental competence. Subsequently, the synergistic effects of HA nanoparticles and permeable CPAs (VS: 20% EG + 20% DMSO; VS1: 17.5% EG + 17.5% DMSO) were further evaluated. The optimal treatment (40 nm 0.05% HA nanoparticles) significantly increased MMP level, and improved developmental competence compared with the vitrified control group (p < 0.05). Among the vitrified groups, vitrified oocytes in the VS1-HA group (combining HA nanoparticles with reduced concentrations of permeable CPAs) exhibited the highest MMP level (1.89), maturation rate (50.39%), cleavage rate (27.07%), and blastocyst rate (10.53%) (p < 0.05). Ultrastructural analysis further revealed that the VS1-HA group maintained more intact zona pellucida structures and showed reduced mitochondrial swelling compared with the vitrified control group. Moreover, the expression levels of genes associated with zona pellucida formation (ZP3), mitochondrial fusion (MFN1), and chromatin remodeling (NPM2) were significantly upregulated in the VS1-HA group relative to the vitrified control group. Overall, these findings indicate that the combination of HA nanoparticles with lower concentrations of permeable CPAs enhances MMP level, alleviates vitrification-induced ultrastructural damage, and upregulates the expression of key developmental genes, thereby improving the developmental competence of vitrified bovine GV oocytes. Full article

This study leverages surface plasmon resonance (SPR) Biacore^TM technology to unveil the diagnostic potential of detecting CXCR4 on extracellular vesicles (EVs). Despite its recognized potential as a cancer biomarker, the presence of CXCR4 on EVs remains underexplored for diagnostic purposes. Using reference material (rEVs), a standardized label-free and real-time SPR biosensor is established to molecularly profile CXCR4-positive EVs. The binding interactions between immobilized antibodies and EVs isolated from different cancer cell lines revealed a unique SPR molecular fingerprint (SPR-MFP) consisting of varying expression levels of the CD9, CD63 and CD81 EV biomarkers, as well as CXCR4. There was a strong correlation between CXCR4 expression on the cellular membrane measured by flow cytometry (FCM) and the CXCR4 SPR signal of purified EVs, indicating that the chemokine receptor is actively transferred to the extracellular space. The Biacore^TM biosensor is able to directly detect and molecularly profile EVs in buffer and spiked in cell culture supernatant supplemented with 10% EV-depleted serum. Altogether, our findings illuminate the potential of SPR Biacore^TM technology in EV-related research as well as reveal the diagnostic potential of EV-associated CXCR4, offering valuable insights and paving the way for medical applications in diseases associated with aberrant CXCR4 expression. Full article

The predatory bug Arma custos (Hemiptera: Pentatomidae) is a natural enemy insect capable of preying on over 40 types of agricultural and forestry pests. Here, we describe the characteristics of the morphology and ultrastructure of its venom apparatus visualized using light and electron microscopy. Light microscopy revealed that the venom apparatus of A. custos consists of a pair of main gland and tubular accessory gland. The main gland consist of two lobes, the anterior main gland (AMG) and posterior main gland (PMG). Between the two lobes of the main gland, there is a strong constriction, characterizing a hilum (Hi) where two separate ducts, the venom duct of the main gland (VD) and the duct connecting the accessory gland to the main gland (AMD), are inserted. The VD extends toward the head and connects to the venom pump (VP), while the AMD extends toward the thorax and connects to the accessory gland (AG). Ultrastructural examination of the venom glands reveals that the AMG and PMG consist of a layer of cubic or spherical glandular cells forming a large circular lumen, while the AG exhibits two narrow lumens. The secretory cytoplasm of AMG, PMG, and AG contains a well-developed rough endoplasmic reticulum, along with mitochondria, nuclei, secretory vesicles, autophagosomes, and secretory granules. However, significant differences exist in the ultrastructural characteristics among the three glands. Unlike glandular secretory cells in the venom glands, the ultrastructure of VD, and AMD reveals only well-developed nuclei, mitochondria, and elaborate plasma membrane folds. These results indicate that venom proteins are synthesized and stored by the AMG, PMG, and AG, while the VD and AMD ducts are responsible for transporting the venom. Full article

Cardiovascular disease remains the leading global cause of mortality, largely due to the limited regenerative capacity of adult human myocardium. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer a scalable platform for cardiac repair and disease modeling; however, their persistent metabolic immaturity—characterized by reliance on glycolysis, reduced oxidative phosphorylation (OXPHOS), and structurally underdeveloped mitochondria—limits functional integration and long-term therapeutic efficacy. Recent advances indicate that targeted metabolic reprogramming can enhance mitochondrial biogenesis, increase ATP production, and improve stress resilience in iPSC-CMs. This review examines the complementary integration of CRISPR-based metabolic engineering and extracellular vesicle (EV)-mediated metabolic modulation as a systems-level strategy for cardiac maturation. We discuss CRISPR activation, interference, and epigenome-editing approaches targeting regulators such as PGC-1α, TFAM, and PPARs to promote stable enhancement of mitochondrial networks and respiratory capacity. In parallel, engineered EVs delivering miRNAs, metabolic enzymes, and redox modulators provide non-genomic mechanisms to optimize bioenergetic function and mitigate oxidative stress. By synthesizing mechanistic insights, quantitative bioenergetic metrics, and translational considerations, we propose CRISPR-EV synergy as a precision framework for durable metabolic maturation of iPSC-CMs, with implications for regenerative therapy, pharmacologic screening, and myocardial repair. Full article

Extracellular vesicles (EVs), particularly exosomes, have emerged as critical mediators of intercellular communication, yet the metabolite fraction of their cargo remains substantially underexplored relative to proteins and nucleic acids. This review synthesizes current knowledge on the exosomal metabolome as a functionally distinct intercellular signaling system with unique biophysical properties. We review the mechanisms proposed to govern metabolite encapsulation into exosomes, encompassing membrane transporter involvement, lipid raft partitioning, and binding to luminal proteins, and discuss the unresolved question of whether metabolite loading is selective or stochastic. Critically, we present a quantitative framework evaluating whether delivered metabolite quantities are sufficient to alter recipient cell metabolic pools, distinguishing receptor-mediated signaling from bulk substrate delivery. We also address methodological considerations including contamination artifacts and isolation-method biases that complicate interpretation of EV metabolomics data. Exosomal metabolites are reviewed across four functional categories: energy substrates (ATP, lactate, amino acids), signaling molecules (TCA cycle intermediates, eicosanoids, nucleotides), redox cofactors and antioxidants (NADH, glutathione), and oncometabolites. For each category, available evidence is critically appraised, distinguishing metabolites with direct mass spectrometric detection from those whose roles are inferred from parent-cell biology. The review examines the roles of exosomal metabolites in tumor-stroma metabolic symbiosis, immunometabolic regulation, inter-organ crosstalk in metabolic diseases including type 2 diabetes and non-alcoholic fatty liver disease, cancer metastasis, viral infections, and immune evasion. A quantitative framework is discussed to evaluate whether delivered metabolite quantities are sufficient to alter recipient cell metabolic pools, distinguishing receptor-mediated signaling from bulk substrate delivery. Technical challenges in exosomal metabolomics are reviewed, including the impact of isolation method on data quality, contamination artifacts, and current standardization gaps. Therapeutic implications of exosomal metabolite signaling are discussed, encompassing metabolite-loaded exosomes as therapeutic vehicles and exosomal metabolite loading as a pharmacological target. Integration of single-vesicle technologies with systems biology approaches is highlighted as a promising direction for advancing this field toward precision medicine applications in oncological and metabolic disorders. Full article

Background: Extracellular vesicles (EVs) are increasingly explored as nanocarriers in drug delivery; however, selecting an appropriate loading strategy for a given small-molecule cargo still relies largely on empirical, resource-intensive parallel screening within EV formulation workflows. Despite the widespread application of passive incubation, electroporation, saponin-mediated permeabilization, freeze–thaw cycling, and sonication, there is currently no mechanistically grounded, descriptor-informed framework that enables rational prioritization of loading methods during the early design stage of EV-based dosage forms, leading to inefficient trial-and-error experimentation. Methods: We assembled a chemically diverse dataset of 21 compounds with experimentally determined loading efficiencies across five EV loading methods and calculated seven mechanistically motivated physicochemical descriptors (LogP, molecular weight, aqueous solubility, hydrogen bond donors/acceptors, polar surface area, and formal charge) for each drug. Separate Elastic Net regression models were trained for each loading strategy. Model performance was evaluated using leave-one-out cross-validation, a predefined external validation set (n = 4), and 50 repeated random train–test splits. The analysis emphasized decision-level ranking of loading methods rather than the precise prediction of absolute efficiencies. The applicability domain was assessed via leverage analysis to define the supported chemical space for prospective implementation in EV-based formulation development. Results: As anticipated for biologically heterogeneous EV systems, continuous regression performance remained modest (LOOCV R² = 0.06–0.41). In contrast, decision-level accuracy for identifying the experimentally optimal loading method was consistently high across validation schemes (internal: 76.5%; predefined external: 75%; repeated random validation: 80.5 ± 16.8%). Mechanical disruption methods (freeze–thaw and sonication) demonstrated comparatively greater predictive stability, while misclassification patterns suggested potential nonlinear behavior for highly polar, ionizable cargos. All compounds resided within the leverage-defined applicability domain, confirming adequate descriptor-space representation. Conclusions: This study establishes a mechanistically interpretable, descriptor-based decision-support framework capable of reliably prioritizing EV loading strategies for small-molecule cargos beyond empirical chance without altering standard protocols. By reframing the modeling objective from high-precision efficiency prediction to robust ranking of candidate methods, the approach offers a practical tool to triage between commonly used techniques, thereby reducing experimental burden in early-stage EV formulation development. The framework provides a quantitative basis for integrating molecular-descriptor-guided method selection into rational EV-based drug delivery design and can be expanded with membrane-specific descriptors and larger datasets. Full article

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, a genome-editing technology pioneered in 2012, enables the precise correction of deleterious mutations or disruption of disease-causing genes through targeted double-strand breaks (DSBs), offering potential for treating genetic diseases. However, CRISPR/Cas9 can cause off-target cleavage at non-specific DNA sites, leading to unintended insertions or deletions (indels), which limit its safety and applicability despite ongoing improvements in specificity. Recently, prime editing (PE), an advanced CRISPR-derived technology, has been employed with a Cas9 nickase (Cas9n) fused with a reverse transcriptase and a prime editing guide RNA (pegRNA) to enable precise insertions, deletions, and transversions without inducing DSBs, thus reducing risks of indels and chromosomal aberrations. Furthermore, ongoing optimizations, such as improved pegRNA design and enhanced editing efficiency, have expanded the applications of PE in medical therapeutics, agriculture, and fundamental research. This review summarizes recent advancements in the PE system, including optimized pegRNA designs and enzyme engineering for enhanced efficiency and specificity, alongside novel delivery methods. It also evaluates cutting-edge delivery strategies, such as adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs) and novel extracellular vesicle (EV)-based systems, and explores PE applications in vitro and in vivo, including disease modeling and therapeutic gene correction. Full article

Osteoarthritis (OA) is a multifactorial disease characterized by inflammation, extracellular matrix remodeling, and joint degeneration, and it still lacks disease-modifying treatments. Here, we applied an ex vivo OA model based on transwell co-cultures of cartilage and synovial membrane explants harvested from OA patients to compare the effects of adipose-derived stem/stromal cell (ASC) conditioned medium (CM) with dexamethasone (DEX), a clinically used corticosteroid. Explants were treated for 48 h with 100 nM DEX, CM derived from 5 × 10⁵ ASCs, or left untreated. Outcomes included gene and protein expression of key mediators, metalloprotease and aggrecanase activities, and nitric oxide release. DEX significantly reduced inflammatory markers (e.g., PTGS, IL-1β, and IDO) and VEGF expression in both tissues, while CM did not elicit consistent anti-inflammatory effects. Regarding matrix remodeling, both treatments reduced metalloprotease activity, with DEX modulating MMP3 and MMP13 expression in both tissues and CM reducing only MMP3 expression in cartilage while presenting high levels of TIMP-1. These results confirm the robustness of the model, demonstrated by reproducible responses to DEX and its high-throughput potential, and underscore the need for mechanistic studies to optimize novel biotherapeutics. Full article

Background/Objectives: Individuals with diabetes often experience difficulties in the healing of their alveolar sockets. Furthermore, obesity is strongly associated with the development and progression of type 2 diabetes through complex metabolic and inflammatory mechanisms. The current study provides new insights into the use of Luteolin (LU) and/or chitosan vesicles (CHV) to accelerate bone regeneration, highlighting a biologically and clinically relevant approach that leverages implants as a clinical solution. Methods: Sixty rats were randomly categorized into five groups: Group I (negative control); Group II (positive control), diabetic and obese rats; Group III (LU-treated), diabetic and obese rats with an extraction socket loaded with LU; Group IV (CHV-treated), diabetic and obese rats with an extraction socket loaded with CHV; and Group V (LU-CHV), diabetic and obese rats with an extraction socket loaded with LU-CHV. After 2 and 6 weeks, rats’ mandibles underwent histological, histomorphometric, biochemical, and statistical analyses. Results: The results demonstrated significant differences among the experimental groups. The LU-CHV formulation showed superior therapeutic performance compared with free luteolin and the untreated control group. In vitro release studies revealed sustained, controlled release from LU-CHV, whereas free luteolin exhibited rapid drug release. Additionally, LU-CHV significantly enhanced biological activity, as evidenced by improved anti-inflammatory and/or therapeutic markers compared to the other groups. These findings indicate that encapsulation within chitosan vesicles improved drug stability, bioavailability, and overall therapeutic efficiency. Conclusions: LU-CHV demonstrated superior efficacy compared to free luteolin, highlighting the advantage of chitosan-based vesicular delivery systems. LU-CHV not only enhanced controlled drug release and therapeutic outcomes but also presents a promising platform that could significantly advance targeted drug delivery strategies in inflammatory and metabolic disorders. The findings suggest that LU-CHV represents a transformative approach in improving treatment effectiveness and patient outcomes. Full article

Triple-negative breast cancer (TNBC) is an aggressive subtype lacking targeted therapies and characterized by pronounced heterogeneity and widespread dysregulation of microRNAs (miRNAs) that influence epithelial-to-mesenchymal transition (EMT) and metastasis. Tumor-derived extracellular vesicles (tEVs) further contribute to TNBC progression by transporting oncogenic cargo that can enhance pro-inflammatory signaling. The synthetic SMRwt peptide has been suggested to modulate oncogenic pathways; however, its effects on EV miRNA composition and inflammatory transcript profiles in TNBC remain unclear. Here, we investigated whether SMRwt alters tEV-associated miRNAs and cytokine transcript signatures relevant to EMT and inflammasome-linked pathways. Extracellular vesicles were isolated from SMR-treated and untreated MDA-MB-231 cells, followed by nanoparticle tracking analysis and small RNA sequencing. SMRwt treatment enriched 11 tumor-suppressive miRNAs (including Let-7a-5p, Let-7b-5p, miR-24-3p, miR-26b-5p, miR-92a-3p, miR-93-5p, and miR-496) previously associated with the regulation of proliferation, EMT, migration, and metastasis. We also observed modest, non-significant decreases (1.01–1.27-fold) in oncogenic miR-1200, miR-374a-5p, and miR-937-3p, which have been implicated in the progression of breast, lung, and bone malignancies. Complementary transcriptomic profiling using the NanoString nCounter Breast Cancer 360 Gene Expression Panel (NanoString Technologies, Inc., Seattle, CA, USA) demonstrated reduced expression of inflammasome-associated cytokines in TNBC cells relative to non-tumorigenic controls, including a log₂ fold change of −1.15 for IL 1β (MDA-MB-231 vs. MCF10A). These transcript-level changes suggest potential modulation. Additionally, SMRwt suppresses ASC-mediated caspase-1 activation and reduces IL-1β secretion, thereby inhibiting NLRP3 inflammasome signaling. Therefore, we infer that SMRwt simultaneously restores tumor-suppressive miRNA networks and suppresses inflammasome-driven inflammation, supporting its potential as a dual-target therapeutic strategy for TNBC. Full article

Extracellular vesicles (EVs) are lipid-enclosed particles secreted from a wide variety of cells, with the ability to transfer biologically active content from parent to recipient cells. Lung epithelial-derived EVs (LE-EVs) play an important role in the progression of pulmonary disease, but there is limited evidence regarding the impact of cigarette smoke (CS) and electronic cigarette aerosol (ECA) on epithelial-derived EVs. The aim of this systematic review was to evaluate the current published literature on the impact of cigarette smoke and electronic cigarette aerosol on LE-EVs. Original research studies and clinical data were included, but research involving microparticles or non-epithelial-derived EVs was excluded. A total of 29 articles were identified from three databases (EMBASE, Web of Science and PubMed), of which nine demonstrated that CS exposure leads to molecular changes in epithelial-derived EVs, whereas 21 reported that CS-induced LE-EVs can deliver their cargo to neighbouring cells. The results highlighted that LE-EVs secreted in response to cigarette or e-cigarette exposure presented altered EV cargo, associated with increased cellular damage, inflammation and disease development. The current literature suggests that conventional and electronic cigarettes can influence the secretion of EVs from lung epithelial cells, with these EVs potentially playing a role in the development of lung inflammation. Nonetheless, there is limited research studying the impact of ECA on LE-EVS. Further research examining the impact of electronic cigarettes on lung epithelial-derived EVs, using robust human in vitro models coupled with clinical studies, is required. Full article

Protein glycosylation plays a pivotal role in breast cancer biology, influencing cell proliferation, adhesion, migration, and immune evasion. Aberrant N- and O-glycosylation are hallmarks of neoplastic transformation and serve as sensitive indicators of disease progression. This review aims to characterize glycoprotein biomarkers in breast cancer identified using Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry. We examine specific glycosylation alterations—including hypersialylation, fucosylation, and truncated O-glycans—across different molecular subtypes (Luminal A/B, HER2-positive, TNBC) and assess their diagnostic and prognostic potential. Methodologically, the review contrasts MALDI-based profiling and Imaging Mass Spectrometry (MALDI-IMS) with other proteomic techniques, highlighting MALDI’s advantages in throughput and spatial resolution alongside its technical limitations. Furthermore, we discuss emerging frontiers in the field, such as the shift from whole-serum analysis to “liquid biopsy” components (e.g., extracellular vesicles). Ultimately, we argue that implementing quantitative glycoproteomics is essential for advancing personalized oncology. Full article

The genus Parabacteroides comprises widespread gastrointestinal commensals, known to produce immunomodulatory molecules and extracellular vesicles, yet its full diversity is incompletely cataloged. This study describes strain ASD2025^T, isolated from healthy child feces, using a polyphasic taxonomic approach including phenotypic profiling, chemotaxonomy, and comparative genomics. Cells were non-motile, polymorphic rods that produced extracellular vesicles. Phylogenomic analysis placed ASD2025^T within the genus Parabacteroides within a species complex consisting of P. acidifaciens, P. hominis, “P. massiliensis”, P. merdae, and P. johnsonii, with average nucleotide identities to the type strains of 85.5–89.9%. The large genome (5.16 Mbp, 46.2% GC content) contained integrative conjugative elements harboring antibiotic resistance genes and hankyphage-related prophage. The strain produced succinate as the major metabolic end product, and its major fatty acids were anteiso-C_15:0, iso-C_17:0 3-OH, and C_15:0. Conditioned medium from ASD2025^T antagonized the interleukin-8 response caused by E. coli lipopolysaccharide in HT29 cells. The majority of related metagenome-assembled genomes originate from mouse microbiomes. Based on these distinct characteristics, strain ASD2025^T (=VKM B-3926^T = JCM 37967^T) represents a novel species of the genus Parabacteroides, for which the name Parabacteroides vesiculifaciens sp. nov. is proposed. Full article

The interaction between probiotic bacteria and the innate immune system is of increasing interest due to its capacity to modulate inflammatory and antimicrobial responses. The murine macrophage cell line RAW 264.7 is widely used to investigate the immunomodulatory effects of probiotic bacteria and their cell-free derivatives, such as membrane vesicles (MVs). In this study, we evaluated whether MVs derived from Lactobacillus acidophilus promote superior modulation of cytokine production and antimicrobial signaling in RAW 264.7 macrophages compared with whole cells (WCs). Our results show that L. acidophilus MVs exhibited direct bactericidal activity against Escherichia coli and induced a more selective and balanced cytokine profile than whole cells. These findings highlight the potential of probiotic-derived membrane vesicles as acellular immunomodulatory effectors for the development of novel cell-free biotherapeutic strategies. Full article