Search Results (4,233)

Currently, poly- and monophenol antioxidants should be considered not only as inhibitors that interact with free radicals, but also take into account that they are biologically active substances that affect specific targets in cells and can induce the activity of certain genes or stimulate various signaling pathways. The phenols can directly influence different points of the apoptotic process, and/or the expression of regulatory proteins. In our present study the effect of two antioxidants, sterically hindered monophenols sodium anphene (ANa) and potassium phenosan (PhK), on cell apoptosis of splenocytes was studied by fluorescence and confocal microscopy. PhK has already been introduced into medical practice in the Russian Federation because it proved effective as an anticonvulsant and was useful in treating neonatal hypoxia. The study of ANa continues; it may be a promising anticancer drug for some types of tumors. The fluorescent and confocal microscopy methods demonstrate that ANa in combination with H₂O₂ enhances apoptosis in suspension of Lewis carcinoma cells and to a lesser extent in splenocyte culture. We also discovered that autofluorescence of FAD and immunofluorescence of NADPH enzymatic complexes (with the AV-FITC fluorophore) in splenocytes of normal cells increases symbatically. The autofluorescence of FAD in splenocytes of Lewis carcinoma cells significantly exceeded that of splenocytes of healthy animals. The exact distinctive result was obtained when using potassium phenozan. It turned out that PhK prevents the development of apoptosis in mouse splenocyte cell culture (F1(CBA×C57B)). The combined use of ANa and PhK had no effect on splenocyte apoptosis. We show that fluorescence and confocal microscopy allow observing and quantifying the apoptotic effect of ANa and hydrogen peroxide, and make it possible to visualize metabolic changes in the cell, increased FAD fluorescence in tumor cells and NADPH -oxidase complexes in splenocytes. The data obtained indicate the possibility of using ANa in combination with hydrogen peroxide as an antitumor drug acting on certain types of cells. The different effects of sterically hindered monophenols ANa and PhK on the level of the anti-apoptotic protein Bcl-2 in the cell were established. ANa acts to lower Bcl-2 levels, signaling apoptosis, while PhK prevents the development of apoptosis and induces repair processes. Full article

Pulse repetition frequency (PRF) controls pulse off-time and, therefore, the extent of thermal accumulation, melt expulsion, and dielectric recovery in finishing electrical discharge machining (EDM). This study clarifies how PRF modifies crater evolution and surface integrity in finishing EDM of 4Cr13 martensitic stainless steel, a corrosion-resistant mold steel used in precision dies and molds. A 2D axisymmetric electro-thermo-fluid model was established in COMSOL, where Gaussian current density, heat-flux, and plasma pressure were periodically imposed at PRFs of 25–100 kHz, while pulse-on time (6 μs) and peak current (8 A) were kept constant. The simulations tracked the transient pressure, heat-flux, velocity, and temperature fields over a common elapsed time of 25 μs. Finishing experiments were then carried out on flat 4Cr13 coupons at 50, 75, and 100 kHz using a copper electrode and deionized water, followed by characterization by laser confocal microscopy, SEM/EDS, and X-ray diffraction using the cosα method. Increasing PRF localized the coupled pressure-heat-flow fields near the crater rim, but shortened off-time and intensified inter-pulse heat accumulation. Accordingly, the surface roughness decreased from Ra = 1.18 μm at 50 kHz to 0.63 μm at 75 kHz, and then slightly increased to 0.71 μm at 100 kHz because of crater overlap, re-melting, and incomplete gap recovery. SEM observations confirmed large irregular craters with cracks at 50 kHz, more uniform fine craters at 75 kHz, and overlapping re-solidified traces at 100 kHz. The residual stress remained compressive for all tested conditions (−341 to −409 MPa). Overall, 75 kHz offers the best compromise between crater uniformity, roughness, and compressive stress for finishing EDM of 4Cr13 steel. Full article

This study investigated the biosynthesis of magnetite nanoparticles mediated by chia mucilage (CM-Fe₃O₄ NPs) and their application in the co-encapsulation of Lactobacillus rhamnosus GG (LGG) using spray drying. CM-Fe₃O₄ NPs were synthesized by combining CM extract with iron salts, in which hydroxyl and carbonyl groups of CM acted as natural ligands for Fe²⁺/Fe³⁺ ions. A response surface design was applied to optimize synthesis parameters, focusing on size distribution and zeta potential, and confirming the influence of pH on colloidal stability. Characterization by FE-SEM, DLS, XRD, UV-Vis, and FTIR revealed spherical particles with an inorganic core (50–300 nm) and a hydrated organic coating (600–900 nm), consistent with a spinel structure functionalized by CM. Spray-drying encapsulation tests showed that incorporating CM-Fe₃O₄ NPs did not compromise bacterial viability, maintaining optimal moisture content and survival. Growth curves and confocal microscopy corroborated the physiological compatibility of the nanoparticles, with no alterations in LGG morphology or growth dynamics. Under simulated gastrointestinal conditions, co-encapsulated microcapsules exhibited slightly improved survival in the gastric phase and significantly greater viability in the initial intestinal phase. These results suggest that CM-Fe₃O₄ NPs modulate matrix degradation and promote controlled release, ensuring therapeutic concentrations of LGG in the intestine. Overall, the CM-Fe₃O₄ nanocomposite system integrates the protective properties of biopolymers with the functional advantages of iron nanoparticles, offering dual functionality: probiotic stabilization and potential iron supplementation. This innovative, food-grade approach supports the development of next-generation functional foods with combined therapeutic and nutritional benefits. Full article

Bone formation requires a substantial energy supply to sustain extracellular matrix production and mineralization, yet the temporal contribution of lipid metabolism during osteoblast maturation remains incompletely characterized. This study investigated the molecular and transcriptional remodeling of lipid metabolism. Intracellular lipid distribution was analyzed by confocal microscopy using Nile Red staining. Transcriptional modulation of lipid synthesis, storage, lipolysis, genes associated with mitochondrial fatty acid oxidation, and osteogenic markers were assessed by quantitative real-time PCR, and the biochemical composition was evaluated by Raman spectroscopy. Early stages of spheroid development showed higher expression of genes involved in lipid synthesis and storage (FASN, DGAT2, and PLIN2) together with intracellular lipid accumulation, whereas later stages displayed increased expression of lipolytic and β-oxidation markers (PNPLA2/ATGL, CPT1A, and HADHA), accompanied by the redistribution of lipid droplets. The Raman analysis revealed a time-dependent variation of lipid-associated CH₂/CH₃ bands and modulation of protein-related Amide I–III signals, consistent with biochemical remodeling during maturation. Overall, the data indicate a coordinated transcriptional shift from lipid accumulation-associated pathways toward lipid mobilization during osteogenic progression in a 3D culture. This model provides a controlled experimental platform for investigating metabolic regulation during bone formation and for studying metabolic alterations associated with skeletal disorders. Full article

Graphene oxide (GO)–lipid hybrid nanostructures represent a promising class of multifunctional platforms for drug delivery and fluorescence-guided cellular imaging. In this study, we developed a graphene oxide-supported lipid bilayer system composed of rhodamine-labeled phosphatidylcholine (POPC-Rhod) for the delivery of the repurposed antispasmodic drug alverine citrate (ALV) to neuroblastoma cells. The hybrid nanostructures were assembled using two drug-loading strategies and characterized by UV–Vis spectroscopy, fluorescence analysis, dynamic light scattering, and atomic force microscopy to evaluate molecular interactions, vesicle size distribution, and nanomechanical properties. In vitro studies were performed using human neuroblastoma SH-SY5Y cells and their retinoic acid-differentiated neuronal-like counterparts. Confocal microscopy confirmed efficient cellular uptake of the fluorescent lipid–graphene hybrids, while viability and mitochondrial reactive oxygen species assays revealed differentiation-dependent cellular responses. ALV-loaded hybrids induced cytotoxic effects in proliferating neuroblastoma cells, whereas differentiated neuron-like cells exhibited greater tolerance and, at moderate concentrations, preserved viability despite increased oxidative stress. These findings demonstrate that graphene oxide–lipid hybrids can act as fluorescence-traceable drug delivery platforms and highlight the potential of alverine as a candidate for repurposing in neural cancer models. The system presented here provides a proof-of-concept framework for the development of multifunctional nanocarriers integrating therapeutic delivery with imaging capabilities. Full article

Streptococcus mutans (S. mutans), one of the main etiological pathogens of dental caries, forms dental plaque biofilms that drive tooth decay. Although bacterial membrane vesicles (MVs) are increasingly recognized as modulators of biofilm biology, little is known about MVs generated by S. mutans. The objective of this study is to investigate the role of S. mutans-derived MVs in the development of S. mutans biofilms formed under static conditions in plates or confocal dishes. Transmission electron microscopy and nanoparticle tracking analysis revealed that the MVs were cup-shaped with bilayered membranes and averaged 80.49 $\pm$ 32.24 nm in diameter. The addition of ≥5 µg/mL MVs enhanced biofilm formation during the initial adhesion stage (0 to 6 h), as demonstrated by crystal violet staining and XTT assays. Confocal laser scanning microscopy and scanning electron microscopy confirmed the incorporation of PKH26-labeled MVs into S. mutans biofilms and showed that supplemental MVs increased bacterial viability and extracellular polysaccharide biomass. Furthermore, RT-qPCR analysis revealed upregulated expression of genes related to adhesion and quorum-sensing systems in MV-treated biofilms. In conclusion, these findings indicate that S. muants MVs are integral biofilm components that promote biofilm establishment at the early stage of biofilm formation. Full article

Objective: The objective of this study is to evaluate the ability of silver oxide nanoparticle (Ag₂ONP)-functionalized high-efficiency particulate air (HEPA) filters and colloidal Ag₂ONP suspensions to inhibit biofilm formation by major respiratory pathogens causing infections at operating rooms. Background: Respiratory infections caused by bacterial pathogens such as Streptococcus pneumoniae, Pseudomonas aeruginosa and Staphylococcus species are often associated with the formation of biofilms, which confer increased resistance to antibiotics and host immune responses. Effective strategies to prevent biofilm formation on biological surfaces and in air filtration systems are urgently needed in clinical settings. Methods: The biofilm-forming ability of each bacterial strain was assessed by crystal violet microplate assay, viable count or confocal microscopy after prior incubation of the culture medium with Ag₂ONP-coated HEPA filter material or colloidal Ag₂ONP suspension. Results: Both silver-functionalized filters and silver nanoparticle suspensions significantly inhibited biofilm formation by S. pneumoniae and P. aeruginosa, with near-complete suppression observed. In the case of S. aureus and S. epidermidis, the silver nanoparticle suspension showed partial inhibition of biofilm development. Conclusions: Ag₂ONP-functionalized HEPA filters and colloidal Ag₂ONP suspensions effectively prevent biofilm formation by major respiratory pathogens, for both Gram-negative and Gram-positive bacteria. These materials show promise for integration with air filtration and surface coating systems to reduce microbial load and transmission in healthcare environments such as operating room facilities. Full article

Glioblastoma (GBM) remains the most aggressive primary malignant brain tumor in adults, with poor survival largely driven by diffuse cellular infiltration, profound heterogeneity, and near-universal recurrence following standard therapy. Although maximizing the extent of resection is a key determinant of patient outcome, current clinical imaging modalities lack the spatial resolution necessary to detect microscopic tumor invasion and therapy-resistant cell populations. Emerging in vivo imaging technologies capable of cellular and near-single-cell resolution have therefore become a major focus in preclinical neuro-oncology research, with growing relevance for surgical guidance, treatment adaptation, and translational discovery. This review evaluates multiple optical imaging modalities, including multi-photon microscopy, near-infrared II fluorescence imaging, bioluminescence imaging, photoacoustic imaging, optical coherence tomography, confocal laser endomicroscopy, Raman spectroscopy, autofluorescence microscopy, and fluorescence macroscopy with a focus on their ability to detect residual GBM cells. Despite significant advances, these approaches remain constrained by limitations in molecular target availability, probe delivery across the blood–brain barrier, and signal variability within heterogeneous tumor regions. The biological complexity of GBM further challenges detection, as residual tumor cells are spatially dispersed and phenotypically diverse, limiting the effectiveness of single-marker or single-modality strategies. Together, these findings highlight the need for integrated, biologically informed imaging approaches to improve detection of residual disease and guide surgical decision making. Full article

Temporomandibular disorder (TMD) is recognized as a major public health problem, causing pain and physiological and psychosocial limitations. In this context, the present in vitro study investigated the synthesis of a hyaluronic acid (HA) hydrogel with hydrocortisone (Hyd), designed to enhance joint lubrication by reducing mechanical friction and delivering the anti-inflammatory drug. The hydrogels were prepared with 3% HA (30 mg/mL) and Hyd—0.125% (1.25 mg/mL), 0.250% (2.5 mg/mL), 0.500% (5 mg/mL), or 1% (10 mg/mL). Physicochemical analyses included Fourier transform infrared spectroscopy (FTIR), thermogravimetry (TGA), rheological tests (frequency, amplitude, and temperature ramp scans), and field emission scanning electron microscopy (FESEM), performed before and after sterilization and cycling. In addition, cytocompatibility was evaluated by protocol OECD 129 and confocal microscopy, as well as genotoxicity (OECD487) in mouse macrophages (RAW 264.7 strain) per 24 h of exposure. FTIR demonstrated the spectral signatures of the compounds with no covalent interactions between the drugs, as well thermal stability on TGA. Rheology demonstrated that Hyd protected the HA structure after autoclaving, maintaining viscoelastic properties. SEM confirmed homogeneous porous morphology. Biological assays showed cell viability > 70%, but with a dose-dependent increase in genotoxicity (4–17 micronuclei). Confocal analysis revealed increasing cytotoxicity at high Hyd concentrations, indicating a balance between biocompatibility and adverse effects at concentrations ≤ 0.5%. Among the tested formulations, the 3% HA + 0.250% Hyd hydrogel provided the best balance of viscoelastic stability, cytocompatibility, and low genotoxicity, supporting its potential as a dual-function intra-articular candidate for TMD therapy. Full article

Liquid infant formula has garnered increasing attention due to its mild thermal processing and superior retention of bioactive nutrients. Within such matrices, the lipid source is a critical determinant of protein digestion behavior, yet its influence on peptide bioavailability and intestinal homeostasis remains undefined. Given that efficient peptide absorption is vital for the systemic delivery of bioactivity in infants, understanding the lipid–protein synergy is essential for formula optimization. Moreover, excessive oxidative stress is closely associated with impaired intestinal health and developmental disorders in infants, making the regulation of oxidative stress crucial for maintaining intestinal function. The present study evaluated the effects of three distinct lipid sources—soybean oil (SM), bovine milk fat (BM), and goat milk fat (GM)—on the physicochemical stability, proteolytic digestion, peptide release, intestinal absorption, and oxidative stress modulation of goat-milk-based infant formula. An integrated approach combining physicochemical characterization, in vitro simulated infant digestion, and a Caco-2 intestinal epithelial cell model was employed. we demonstrate that all three lipids (3% w/w) formed stable emulsions with uniform spherical structures and mean particle diameters of 117–300 nm, as visualized by laser confocal microscopy. Following in vitro simulation of infant gastrointestinal digestion, the SM group exhibited the most extensive protein hydrolysis, yielding the highest total peptide content (4.28 ± 0.10 mg/mL) and generated the highest number of peptides identified by LC-MS/MS (474 types). Bioinformatic analysis predicted that peptides from all groups possess potential antihypertensive, hypoglycemic, and immunomodulatory activities. The Caco-2 monolayer cell model demonstrated that although the GM group produced fewer identified peptide species than the SM group (365 types), it achieved significantly higher intestinal peptide absorption rate (55.34 ± 1.05%). Furthermore, the GM digests provided superior protection against H₂O₂-induced oxidative stress in Caco-2 cells, markedly reducing reactive oxygen species levels and suppressing the expression of pro-inflammatory cytokines TNF-α and IL-6. Collectively, these findings reveal that while soybean oil promotes more extensive proteolysis, the use of homologous goat milk lipid enhances peptide bioaccessibility and confers potential cytoprotective effects on intestinal epithelial cells, underscoring its potential as a preferred lipid source in infant formula formulations. Full article

Objectives: To analyze changes in ocular dendritic cells and their correlation with signs of keratitis in patients with herpes simplex stromal keratitis (HSK) using in vivo confocal microscopy (IVCM). Methods: This was a retrospective, cross-sectional, controlled, single-center study. Fifty-nine eyes from 59 patients with HSK and 40 eyes from 40 control subjects were studied. Each patient underwent IVCM and slit-lamp examinations. The density, area, size, and number of dendritic cells (DCs) in the corneas of both groups were analyzed. The severity of HSK was assessed, and the morphology and density of DCs in the cornea in the HSK group, categorized by ocular parameter severity levels, were compared with those in the control group. Results: DC density was significantly greater in patients with HSK than in controls. The DC field and size and the number of branches were also significantly greater in the HSK group. Furthermore, the DC density increased and morphological changes were exacerbated with increasing degree of corneal edema. The DC density was significantly increased and morphological changes were significantly exacerbated in the HSK group compared to the control group, even in those with the mildest cases of HSK. Conclusions: DC density and morphological changes correlate with the degree of corneal edema in patients with HSK. Changes in DC density and morphology can be observed even in mild cases of HSK. IVCM may be a powerful tool for monitoring ocular surface immune responses in patients with HSK, aiding in the clinical diagnosis and management of this disease. Full article

Oligomeric species of alpha-synuclein (α-syn) in saliva and phosphorylated α-syn deposits in the skin are established molecular biomarkers for Parkinson’s disease (PD). However, existing research has yet to fully explore the diagnostic potential of non-phosphorylated α-syn and other cutaneous morphometric parameters, such as variations in collagen type IV within the dermis and epidermis or α-syn expression in melanocytes. This study aims to evaluate and compare the diagnostic utility of these skin morphometric parameters in differentiating 32 PD patients from 19 healthy subjects (HSs), while also examining their correlation with salivary α-syn oligomer levels. Skin biopsies were analyzed via immunofluorescence and confocal microscopy, while salivary oligomeric α-syn was quantified through competitive ELISA. Results revealed a significant reduction in α-syn-positive fibres in PD patients compared to HSs (0.91; <0.0001). Conversely, the collagen staining area and the number of α-syn-positive melanocytes were significantly increased in the skin of PD patients. Specifically, the collagen type IV staining area was significantly higher in the dermis and surrounding the sweat glands of PD patients, demonstrating optimal diagnostic power (0.9448; <0.0001). Similarly, the increase in α-syn-positive melanocytes in PD patients showed robust diagnostic potential (0.84; <0.001). Salivary α-syn oligomers accurately discriminated between PD and HS groups. Furthermore, significant correlations were found between collagen type IV and melanocyte morphometric parameters and various clinical scores in PD. Our findings highlight how multimodal morphometric analysis of the skin can enhance diagnostic accuracy in PD, supporting the use of salivary and cutaneous biomarkers as complementary tools that may reflect distinct aspects of PD pathology. Full article

Graphene oxide (GO) is a promising nanocarrier for the delivery of oligonucleotides. It offers a high loading capacity, efficient cellular uptake, and surface functionalization. MicroRNA-21 (miR-21) is a well-characterized oncomiR commonly overexpressed in hepatocellular carcinoma (HCC). In HCC, miR-21 contributes to tumor progression, inflammation, and angiogenesis. In a previous in vitro study, we showed that GO alone induces the upregulation of pro-inflammatory and tumor-related genes in HepG2 cells. However, conjugation with an antisense miR-21 (GO-antisense miRNA 21) reverses this effect, suggesting a potential therapeutic application. This study aims to evaluate the antitumor and anti-angiogenic efficacy of the GO-antisense miR-21 nanosystem in ovo using the chick embryo chorioallantoic membrane (CAM) model. Fertilized chicken eggs (n = 4 per group) were randomized into untreated, GO-treated, and GO–antisense miR-21-treated cohorts. A dose of 200 μL (GO 10.0 µg/mL: antisense miR-21 5.0 pmol/mL) was administered intratumorally. Tumor size, volume, and vascularization were monitored through stereomicroscopy and histological analysis. The expression of inflammatory and tumor-associated genes (IL-8, MCP-1, TIMP-2, ICAM-1 and NF-kB) was assessed by quantitative PCR. Given its prominent response, IL-8 protein expression was further analyzed via immunofluorescence. To evaluate tumor-specific delivery, FITC-labeled GO was tracked by confocal microscopy. Our data revealed that treatment with unfunctionalized graphene oxide (GO) unexpectedly promoted tumor vascularization and led to a significant increase in tumor weight. This was accompanied by upregulation of inflammatory markers. In contrast, GO-antisense miR-21 significantly reduced the tumor volume and vessel density. It also successfully downregulated all target genes. Confocal imaging demonstrated preferential accumulation of the nanosystem within the tumor mass. Our results highlight the dual anti-inflammatory and anti-angiogenic effects of GO-antisense miRNA 21 in ovo and support its potential as a targeted nanoplatform for HCC treatment. Full article

The methodical work describes all the necessary steps for establishing a stable oral ex vivo biofilm using saliva and crevicular plaque samples from periodontal healthy donors. First, cover slips were preconditioned with saliva supernatants and subsequently inoculated with crevicular plaque suspensions. Ex vivo biofilm formation was characterized by confocal laser scanning microscopy (cLSM) after 1, 4, 24, 48 and 72 h of anaerobic cultivation. Exemplarily, the inhibitory characteristics of blackcurrant fruit extracts [all-fruit juice (AFJ); alcoholic fraction from berry skins (AFBS)] were observed on 1, 4 and 24 h-aged ex vivo biofilms. Chlorhexidine (CHX, 0.2%) served as positive control. After direct contact (3 min), biofilms were dispersed, plated onto agar and anaerobically cultivated for 24 h. Early ex vivo biofilms (1 h-biofilm) showed scattered microbial colonies. After 4 h of cultivation, a multilayered biofilm was formed. Biofilm mass gradually increased, displaying a complex polymicrobial structure after 24 h. At 72 h, the biofilms had a dense three-dimensional appearance. Treatment with AFJ and CHX was more efficient in inhibiting biofilm growth compared to AFBS. Early biofilms (1 h, 4 h) were more susceptible to AFJ and CHX compared to 24 h-biofilms. The introduced model can be recommended for testing the efficiency of plaque-controlling agents. Full article

Background/Objectives: The F3 peptide, a tumor-homing peptide known to bind cell-surface nucleolin, is frequently employed as a targeting vector in cancer research. However, the impact of the modification site on its cellular binding properties has not been investigated yet. In this work, we aimed to design an improved F3-based radioconjugate by identifying the optimal conjugation site and establishing a protocol for its biological evaluation in vitro. To achieve this, we compared F3 peptide derivatives modified at their N- or C-termini with DOTA for complexation of indium-111 (¹¹¹In) for SPECT or Auger electron therapy or a fluorophore (FITC) for optical imaging. Methods: N-and C-terminal DOTA-modified F3 peptides were radiolabeled with indium-111 and compared for their in vitro stability in different physiologically relevant media. Suitable nucleolin-positive cell lines for further in vitro studies were identified by confocal microscopy of a FITC-labeled F3 peptide derivative. The radioconjugates were then investigated on MDA-MB-231 (breast cancer) and PC-3 (prostate cancer) cells for nucleolin-specific cell binding and uptake, and several parameters of the in vitro assays were varied to establish a suitable protocol. Results: In general, in vitro assays with F3 peptide conjugates are challenging, as the outcome depends on a number of experimental parameters, leading, in some cases, to varying results. In particular, the presence of Ca²⁺ and Mg²⁺ had a decisive impact on the results, likely because the metal ions compete with the binding of F3 conjugates to nucleolin. The C-terminal modified, ¹¹¹In-labeled F3 radioconjugate performed better than the N-terminal modified analog. While several parameters of the in vitro experiments were optimized, the overall cell uptake in vitro of radioactivity was still low (<2% of applied radioactivity). Conclusions: A standardized in vitro protocol for evaluating F3 peptide conjugates on cancer cells was established, revealing that the C-terminus is the preferred site for modification. Because the cellular uptake of the radiotracer was shown to likely not be sufficient for radiotracer development, further studies on the optimization of the F3 peptide conjugates, including structural modifications, are required. Full article