Search Results (134)

Extracellular vesicles (EVs) secreted by Fusarium oxysporum play an important role in the process of its infestation of the host, but the in vitro research system for EVs of F. oxysporum (Fo-EVs) has not yet been improved, and the mechanism of its action remains unclear. In this study, particle size distribution, particle concentration, number of particles per unit of protein, number of particles per unit of mycelial biomass, and concentration of contaminated proteins were used as indicators to evaluate the yield and purity of Fo-EVs. The optimal method for Fo-EV preparation and extraction was screened by comparing liquid culture, solid culture, and solid culture with enzymatic cell wall hydrolysis. The optimal system for Fo-EVs separation and purification was screened by a pairwise combination of three primary methods (Ultracentrifugation (UC), Ultrafiltration (UF), and Polyethylene glycol precipitation method (PEG)) and two secondary methods (Size-exclusion chromatography (SEC) and Aqueous two-phase system (ATPS)), respectively. The protein composition was identified via mass spectrometry technology, followed by GO annotation and GO enrichment analysis using whole-genome proteins as the background. Based on these steps, a Fo-EV protein library was constructed to reveal Fo-EV’s most active biological functions. The results showed that solid culture combined with the UC-SEC method could effectively enrich Fo-EVs with a typical cup-shaped membrane structure. The obtained Fo-EVs had an average particle size of 253.50 nm, a main peak value of 200.60 nm, a particle concentration of 2.04 × 10¹⁰ particles/mL, and a particle number per unit protein of 1.09 × 10⁸ particles/μg, which were significantly superior to those of other combined methods. Through proteomic analysis, 1931 proteins enriched in Fo-EVs were identified, among which 350 contained signal peptides and 375 had transmembrane domains. GO enrichment analysis revealed that these proteins were mainly involved in cell wall synthesis, vesicle transport, and pathogenicity-related metabolic pathways. Additionally, 9 potential fungal EV markers, including Hsp70, Rho GTPase family, and SNARE proteins, were screened. This study constructed an isolation system and a marker database for Fo-EVs, providing a methodological and theoretical basis for in-depth analysis of the biological functions of Fo-EVs. Full article

Apyrase (nucleotide triphosphate diphosphohydrolase, NTPDase; EC 3.6.1.5) functions in a variety of plant growth and developmental processes, as well as responses to pathogens, in part, by regulating extracellular ATP (eATP) concentrations. In this study, we investigated potential roles of apyrase in the recruitment of phosphate (Pi) from extracellular nucleotides in Arabidopsis thaliana seedlings that constitutively overexpress apyrase 1 (APY1). Under Pi limitation, both WT and APY1 seedlings had decreased Pi contents and a characteristic remodeling of root system architecture (RSA). This phosphate starvation response (PSR) was prevented by the uptake of Pi released through the metabolism of extracellular NTP, which occurred at a higher rate in APY1 seedlings. APY1 seedlings had higher Pi contents than WT seedlings on Pi-sufficient media supplemented with NTP and exhibited markedly increased LR and root hair (RH) formation. Genome-wide expression profiling revealed that this expanded RSA of APY1 seedlings was correlated with the induction of >100 genes involved in regulation of auxin homeostasis, signaling, and transport, which previous studies have shown to be increased when APY1 is overexpressed. APY1 regulation of [eNTP] and purinergic signaling may thus contribute to modulation of auxin responses, resulting in enhanced uptake of Pi from the medium, including Pi released via eNTP metabolism. Full article

Among the microorganisms present in the microbiome of Camellia japonica flowers, extracellular vesicles (EVs) derived from Leuconostoc mesenteroides were isolated to investigate their modulatory effects on moisturizing and barrier-related molecular markers. To identify the function of major proteins in L. mesenteroides DB-21-derived extracellular vesicles (LEVs), Gene Ontology (GO) analysis was performed, revealing ATP binding, ribosomal structural proteins, and metal ion binding as predominant molecular-function categories. These proteomic characteristics provide a molecular context that supports the interpretation of the moisturizing and barrier-related responses observed in this study. To further verify new findings, we performed functional evaluations using in vitro and 3D skin models. LEVs increased the mRNA expression level of HAS3, which encodes hyaluronic acid synthase. In addition, the expression levels of filaggrin and involucrin, key proteins involved in skin barrier formation, increased, and these markers were determined a concentration-dependent increase in a 3D artificial skin model. Also, we confirmed that the expression levels of filaggrin and involucrin, which were reduced by UVB damage, were restored when LEVs were applied. In conclusion, LEVs are effective in enhancing various molecular markers related to the skin barrier function, and these results reveal that they hold promise as next-generation microbiome-based functional ingredients. Full article

Perlecan, a major heparan sulfate proteoglycan in the vascular basement membrane, plays an essential role in maintaining endothelial barrier integrity, regulating fibroblast growth factor-2 signaling, and exerting anticoagulant activity. Although alterations in perlecan expression are implicated in the initiation and progression of atherosclerosis, the upstream regulatory mechanisms remain unclear. In this study, we investigated the effects of extracellular ATP on perlecan expression in vascular endothelial cells. ATP, but not ADP or adenosine, suppressed perlecan expression at both mRNA and protein levels in a time- and concentration-dependent manner. This suppression was recovered by knockdown of P2Y2 receptor (P2Y2R), but not by P2X4 receptor, P2X7 receptor, or P2Y1 receptor knockdown, indicating the selective involvement of P2Y2R. Mechanistically, ATP reduced Akt phosphorylation mediated by P2Y2R, and inhibition of Akt by inhibitors decreased perlecan expression, whereas inhibitors of phosphoinositide 3-kinase, mammalian target of rapamycin complex 1, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, c-Jun N-terminal kinases did not exhibit this recovery effect. These results suggest that ATP downregulates perlecan synthesis via the P2Y2R-mediated inhibition of Akt signaling. Given that ATP is markedly elevated under pathological conditions, such as inflammation and platelet activation, suppression of perlecan synthesis is an important mechanism by which ATP promotes vascular disease progression. Full article

In this review we propose the hypothesis that an energy-dissipating process precedes the continuous stimulation of insulin secretion by glucose. This process is mediated by connexin 36 hemichannels (Cx36H), or Cx36 connexons. Cx36H oligomers are expressed at the plasma membrane, and their gating activity (opening) is activated by plasma membrane depolarization after the closure of K⁺ATP channels by glucose (>5 mM) metabolism. This initial depolarization (1st step) might be responsible for the first phase of insulin secretion, with the subsequent opening of Cx36H increasing β-cell plasma membrane permeability, allowing for the efflux of metabolites (less than 1KD) (GABA, adenine nucleotides) and K⁺ (2nd step). This provokes a breakdown of oxidative glucose metabolism and the repolarization of the plasma membrane. As the extracellular glucose concentration increases further (>>5 mM), it exerts a progressive inhibition effect on Cx36H opening, allowing for the continuous stimulation of insulin secretion (3d step, second phase,). The glucose feature of regulating Cx36H closing with sigmoidal kinetics (8 mM IC₅₀ and around 20 mM at maximum) has been confirmed in mouse Cx36 connexin expression in Xenopus oocytes and in mouse islets stimulated by a range of glucose concentrations in the presence of 70 mM KCl. This gating activity was also inhibited by some non-metabolized glucose analogs. Glucose inhibition of Cx3H opening might not only contribute to making the insulin secretory response more specific for glucose but might also play a role in the pulsatility of sustained insulin secretion. Cx36H opening also offers the opportunity to potentiate the secretory effect in vivo by, permeant or not, metabolic stimuli. Confirmation of this novel physiological role for Cx36H in β-cells would place them as new susceptibility locus for type 1 and type 2 diabetes, whose physiological implication in the mechanism of insulin secretion regulation should be evaluated by in vivo studies in diabetic patients. Full article

Ozone (O₃) has re-emerged in periodontology for its antimicrobial, oxygenating, and immunomodulatory actions, yet its role in regeneration remains contentious. This narrative review synthesizes current evidence on adjunctive ozone use in periodontal therapy, delineates cellular constraints—especially in periodontal ligament fibroblasts (PDLFs)—and explores mitigation strategies using bioactive compounds and advanced delivery platforms. Two recent meta-analyses indicate that adjunctive ozone with scaling and root planing yields statistically significant reductions in probing depth and gingival inflammation, with no significant effects on bleeding on probing, plaque control, or clinical attachment level; interpretation is limited by heterogeneity of formulations, concentrations, and delivery methods. Mechanistically, ozone imposes a dose-dependent oxidative burden that depletes glutathione and inhibits glutathione peroxidase and superoxide dismutase, precipitating lipid peroxidation, mitochondrial dysfunction, ATP depletion, and PDLF apoptosis. Concurrent activation of NF-κB and upregulation of IL-6/TNF-α, together with matrix metalloproteinase-mediated extracellular matrix degradation and tissue dehydration (notably with gaseous applications), further impairs fibroblast migration, adhesion, and ECM remodeling, constraining regenerative potential. Emerging countermeasures include co-administration of polyphenols (epigallocatechin-3-gallate, resveratrol, curcumin, quercetin), coenzyme Q10, vitamin C, and hyaluronic acid to restore redox balance, stabilize mitochondria, down-modulate inflammatory cascades, and preserve ECM integrity. Nanocarrier-based platforms (nanoemulsions, polymeric nanoparticles, liposomes, hydrogels, bioadhesive films) offer controlled ozone release and co-delivery of protectants, potentially widening the therapeutic window while minimizing cytotoxicity. Overall, current evidence supports ozone as an experimental adjunct rather than a routine regenerative modality. Priority research needs include protocol standardization, dose–response definition, long-term safety, and rigorously powered randomized trials evaluating bioactive-ozone combinations and nanocarrier systems in clinically relevant periodontal endpoints. Full article

Background/Objectives: Glucagon-like peptide-1 (GLP-1) is an endogenous hormone with receptors widely expressed across multiple organs. GLP-1 receptor agonists (GLP-1RAs), primarily used for diabetes management, have demonstrated anti-inflammatory and antioxidant properties beyond glucose regulation. This study explores the protective effect of semaglutide, a GLP-1RA, in reducing oxidative stress and promoting wound healing in human dermal fibroblasts. Additionally, it assesses whether semaglutide offers the direct protection of retinal endothelial cells under oxidative stress. Methods: Human dermal fibroblasts and retinal endothelial cells were treated with semaglutide at concentrations ranging from 0 to 45 pg/mL for 24 h under oxidative stress induced by hydrogen peroxide (H₂O₂). Cell viability and ATP levels were measured via MTT and ATP assays. Apoptosis was evaluated using propidium iodide staining. Intracellular reactive oxygen species (ROS) and mitochondrial superoxide were assessed through confocal microscopy with specific fluorescent probes. Wound healing was tested using scratch assays, with closure monitored over time and quantified with ImageJ (version 1.51). Gene expression levels of antioxidants, extracellular matrix components, inflammatory cytokines, and MMPs (MMP3, MMP9) were determined via real-time PCR. Results: Semaglutide significantly improved cell viability and ATP production under oxidative stress (p < 0.001), while reducing apoptosis and intracellular ROS levels. It notably accelerated fibroblast wound closure, achieving near-complete restoration. Gene analysis revealed increased expression of antioxidant and ECM-related genes, along with decreased pro-inflammatory cytokines and MMPs, indicating reduced inflammation and enhanced tissue remodeling. Conclusions: Semaglutide offers robust antioxidative and cytoprotective effects in dermal fibroblasts and retinal endothelial cells, promoting wound healing. These findings highlight its therapeutic potential for diabetic foot ulcers and diabetic retinopathy, supporting further in vivo investigation. Full article

The administration of isolated mitochondria is a promising strategy for protecting cells from oxidative damage. This study aimed to identify mitochondrial characteristics that contribute to stronger protective effects. We compared two types of mitochondria isolated from C6 cells with similar ATP-producing capacity but differing in outer membrane integrity. To evaluate their stability in extracellular conditions, we examined their behavior in serum. Both types underwent mitochondrial permeability transition to a similar extent; however, under intracellular-like conditions after serum incubation, mitochondria with intact membranes retained more polarized mitochondria. Notably, mitochondria with intact outer membranes were internalized more efficiently than those with damaged membranes. In H9c2 cells, both types of mitochondria similarly increased intracellular ATP levels 1 h after administration under all tested conditions. When co-administered with H₂O₂, both suppressed oxidative damage to a comparable degree, as indicated by similar H₂O₂-scavenging activity in solution, comparable intracellular ROS levels, and equivalent preservation of electron transport chain activity. However, at higher H₂O₂ concentrations, cells treated with mitochondria possessing intact outer membranes exhibited greater survival 24 h after co-administration. Furthermore, when mitochondria were added after H₂O₂-induced damage and their removal, intact mitochondria conferred superior cell survival compared to damaged ones. These findings suggest that while both mitochondrial types exert comparable antioxidant effects, outer membrane integrity prior to administration plays a critical role in enhancing cell survival under conditions of oxidative stress. Full article

Background: Astrocytes play a key role in the inflammatory process accompanying various neurological diseases. Extracellular ATP accompanies inflammatory processes in the brain, but its effect on lipid mediators (oxylipins) in astrocytes remains elusive. Metformin is a hypoglycemic drug with an anti-inflammatory effect that has been actively investigated in the context of therapy for neuroinflammation, but its mechanisms of action are not fully elucidated. Therefore, we aimed to characterize the effects of ATP on inflammatory markers and oxylipin profiles; determine the dependence of these effects on the adaptation of astrocytes to high glucose levels; and evaluate the possibility of modulating ATP effects using metformin. Methods: We estimated the ATP-mediated response of primary rat astrocytes cultured at normal (NG, 5 mM) and high (HG, 22.5 mM) glucose concentrations for 48 h before stimulation. Cell responses were assessed by monitoring changes in the expression of inflammatory markers (TNFα, IL-6, IL-10, IL-1β, iNOS, and COX-2) and the synthesis of oxylipins (41 compounds), assayed with ultra-high-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). Intracellular pathways were assessed by analyzing the phosphorylation of p38; ERK MAPK; transcription factors STAT3 and NF-κB; and the enzymes mediating oxylipin synthesis, COX-1 and cPLA2. Results: The stimulation of cells with ATP does not affect the expression of pro-inflammatory markers, increases the activities of p38 and ERK MAPKs, and activates oxylipin synthesis, shifting the profiles toward an increase in anti-inflammatory compounds (PGD2, PGA2, 12-HHT, and 18-HEPE). The ATP effects are reduced in HG astrocytes. Metformin potentiated ATP-induced oxylipin synthesis (11-HETE, PGD2, 12-HHT, 15-HETE, 13-HDoHE, and 15-HETrE), which was predominantly evident in NG cells. Conclusions: Our data provide new evidence showing that ATP induces the release of anti-inflammatory oxylipins, and metformin enhances these effects. These results should be considered in the development of anti-inflammatory therapeutic approaches aimed at modulating astrocyte function in various pathologies. Full article

Acne is a very common skin condition that causes pimples in 80% of adolescents despite the many effective treatments developed. Various compounds have been employed in the treatment of acne, including erythromycin ointments and antiseptics, yielding mixed results. The rise in erythromycin-resistant C. acnes strains has driven the pursuit of new antimicrobial agents, especially those obtained from natural sources. Propolis that was collected in Rwanda was extracted, fractioned, and analyzed for its activity against C. acnes growth in accordance with NCLSI guidelines. Our work revealed that linoleic acid has a significant effect on C. acnes growth at a low concentration (16 µg/mL). A comparison of the antimicrobial activities of a broad panel of well-known fatty acids revealed a specific mode of action for linoleic acid, characterized by a significant membranotropic effect on Bacillus cereus established by measuring extracellular ATP levels as an indicator of membrane permeability. Our data suggest that linoleic acid is effective against C. acnes and could be a promising candidate for developing a propolis-based ointment for acne treatment. Full article

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the most prevalent foodborne pathogens worldwide. Hexanal is a natural aldehyde derived from plants. In this study, the antimicrobial and antibiofilm activities of hexanal against V. parahaemolyticus were investigated. Hexanal inhibited V. parahaemolyticus growth with a minimum inhibitory concentration (MIC) of 0.4 mg/mL. Hexanal (2 MIC and 4 MIC) increased the leakage of protein and lactic dehydrogenase, reduced intracellular ATP concentration, damaged membrane integrity, and induced abnormal V. parahaemolyticus morphology and ultrastructure. The results of colony enumeration suggested that hexanal exhibited bactericidal action against V. parahaemolyticus in different culture mediums and food systems (Spanish mackerel meat and shrimp paste). At 1/8 MIC and 1/4 MIC, hexanal inhibited biofilm formation of V. parahaemolyticus, as evidenced by crystal violet staining assay and scanning electron microscope (SEM) observation. Moreover, hexanal reduced the levels of extracellular polysaccharide, extracellular protein, and cyclic di-guanosine monophosphate (c-di-GMP) in V. parahaemolyticus. The result of real-time quantitative polymerase chain reaction (RT-qPCR) indicated that hexanal downregulated the expression of genes critical to V. parahaemolyticus biofilm development. This study provides a promising alternative for V. parahaemolyticus control and is conducive to promoting the application of hexanal in the food field. Full article

Nitrogen source is a necessary nutrient factor in the process of mycelial growth and metabolite synthesis. To improve the yield of ergosterol, in the previous study, we used transcriptome technology to explore the difference in gene expression of Cordyceps cicadae by CO(NH₂)₂ (urea) in the process of synthesizing ergosterol. In the present study, CO(NH₂)₂ was used to examine its effects on cell membrane permeability and metabolic flow in the process of C. cicadae fermentation and ergosterol synthesis. Metabonomic results showed that CO(NH₂)₂ supplementation caused significant changes in five aspects: (1) CO(NH₂)₂ notably increased biomass growth and extracellular ergosterol, and decreased intracellular ergosterol concentration; (2) CO(NH₂)₂ boosted the level of inositol 1,4-bisphosphate, which implied the acceleration of cell membrane decomposition, the weakening of integrity, and the increase in permeability and change in metabolic regionalization; (3) CO(NH₂)₂ changed the metabolic fluxes and metabolic speed, including increasing the levels of amino acids, vitamins, hormones, and nucleotides, which exhibited an elevated biomass growth, promoting the synthesis of intracellular flavonoids, alkaloids, and terpenes, facilitating extracellular ergosterol synthesis and decreasing the degradation of ergosterol; (4) in the fermentation anaphase, CO(NH₂)₂ caused the inhibition of 6-phosphogluconate dehydrogenase and α-ketoglutarate dehydrogenase activities, decreased NADPH, NADH, and ATP synthesis, and finally inhibited biomass growth and ergosterol synthesis. Collectively, metabonomics was a valuable strategy to study the regulatory effects of medium composition and incubation conditions on ergosterol synthesis by C. cicadae. Full article

Adenosine monophosphate (AMP) is a hydrolysis product of adenosine triphosphate (ATP) and adenosine diphosphate (ADP). In mammalian cells, extracellular AMP functions as a signaling molecule by binding to adenosine A1 receptors, thereby activating various intracellular signaling pathways. However, the role of extracellular AMP in plant cells remains largely unclear, and homologs of A1 receptors have not been identified. Our previous studies have demonstrated that extracellular ATP (eATP) is crucial for the normal germination and growth of Picea meyeri pollen tubes. In the present study, we observed that the exogenous addition of ATP to a pollen culture medium could be degraded into AMP and adenosine. Furthermore, the addition of AMP and adenosine to the culture medium was found to inhibit pollen germination and tube elongation. Notably, the addition of an AMP receptor inhibitor into the culture medium mitigated the inhibitory effects of AMP on pollen tube growth. Through intracellular staining for Ca²⁺ and microfilaments, we discovered that high concentrations of AMP disrupt the Ca²⁺ concentration gradient and impair microfilament organization, ultimately resulting in inhibited pollen tube elongation. In conclusion, we propose that extracellular AMP, as a hydrolysis product of eATP, also plays a significant role in regulating P. meyeri pollen germination and tube growth in vitro. Full article

The urothelium and lamina propria (LP) contribute to sensations of bladder fullness by releasing multiple mediators, including prostaglandins (PGs) and adenosine 5′-triphosphate (ATP), that activate or modulate functions of cells throughout the bladder wall. Mediators that are simultaneously released in response to bladder distention likely influence each other’s mechanisms of release and action. This study investigated whether PGs could alter the extracellular hydrolysis of ATP by soluble nucleotidases (s-NTDs) released in the LP of nondistended or distended bladders. Using an ex vivo murine detrusor-free bladder model to access the LP during bladder filling and a sensitive HPLC-FLD detection methodology, we evaluated the decrease in ATP and the increase in adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), and adenosine by s-NTDs released in the LP. Endogenous PGE₂ increased the spontaneous but not the distention-induced release of s-NTD via EP2 and EP3 prostanoid receptors, whereas exogenous PGE₂ increased the spontaneous s-NTD release via EP3, EP4, and FP receptors and the distention-induced s-NTD release via EP1-4 and FP receptors. Endogenous PGF_2α, PGD₂, and PGI₂ did not change the s-NTD release. Exogenous PGD₂ increased the spontaneous s-NTD release via DP2 receptors and the distention-induced s-NTD release via DP1 and DP2 receptors. Exogenous PGF_2α increased the spontaneous but not the distention-induced release of s-NTD via FP receptors. It is possible that higher concentrations of PGE₂, PGF_2α, and PGD₂ (as expected in inflammation, bladder pain syndrome, or overactive bladder) potentiate the release of s-NTDs and the consecutive degradation of ATP as a safeguard mechanism to prevent the development of excessive bladder excitability and overactivity by high amounts of extracellular ATP. Full article

4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) is a nicotinamide derivative, considered a new oncometabolite. 4PYR formation induced a cytotoxic effect on the endothelium. Elevated blood 4PYR concentration was observed in patients with cancer. Still, little is known about the metabolic and functional effects of 4PYR in this pathology. The study aimed to investigate whether this toxic accumulation of 4PYR may affect the activity of anticancer therapy with cyclophosphamide in the orthotropic model of breast cancer. Female Balb/c mice were injected with 4T1 breast cancer cells and assigned into three groups: treated with PBS (Control), cyclophosphamide-treated (+CP), 4PYR-treated (+4PYR), and mice treated with both 4PYR and CP(+4PYR+CP) for 28 days. Afterward, blood and serum samples, liver, muscle, spleen, heart, lungs, aortas, and tumor tissue were collected for analysis of concentrations of nucleotides, nicotinamide metabolites, and 4PYR with its metabolites, as well as the liver level of cytochrome P450 enzymes. 4PYR treatment caused elevation of blood 4PYR, its monophosphate and a nicotinamide adenine dinucleotide (NAD+) analog—4PYRAD. Blood 4PYRAD concentration in the +4PYR+CP was reduced in comparison to +4PYR. Tumor growth and final tumor mass were significantly decreased in +CP and did not differ in +4PYR in comparison to Control. However, we observed a substantial increase in these parameters in +4PYR+CP as compared to +CP. The extracellular adenosine deamination rate was measured to assess vascular inflammation, and it was higher in +4PYR than the Control. Treatment with 4PYR and CP caused the highest vascular ATP hydrolysis and adenosine deamination rate. 4PYR administration caused significant elevation of CYP2C9 and reduction in CYP3A4 liver concentrations in both +4PYR and +4PYR+CP as compared to Control and +CP. In additional experiments, we compared healthy mice without cancer, treated with 4PYR (4PYR w/o cancer) and PBS (Control w/o cancer), where 4PYR treatment caused an increase in the serum proinflammatory cytokine expression as compared to Control w/o cancer. 4PYR accumulation in the blood interferes with cyclophosphamide anticancer activity and induces a pro-inflammatory shift of endothelial extracellular enzymes, probably by affecting its metabolism by cytochrome P450 enzymes. This observation may have crucial implications for the activity of various anticancer drugs metabolized by cytochrome P450. Full article