Search Results (233)

Cold atmospheric plasma (CAP) combined with pulsed electric fields (PEF) demonstrates synergistic cytotoxicity against HeLa cells; however, the differential contributions of short-lived versus long-lived reactive oxygen and nitrogen species (RONS) remain unclear. This study compared direct CAP treatment with indirect CAP-treated liquid treatment, both followed by PEF, to elucidate underlying mechanisms. Direct CAP + PEF treatment resulted in significantly greater cell death than indirect CAP + PEF, with both showing synergistic effects relative to single treatments. Analysis of intracellular RONS and membrane integrity revealed that direct CAP treatment enhanced intracellular RONS levels and PEF-induced membrane permeabilization immediately after treatment. Time-course analysis demonstrated that hydrogen peroxide specifically inhibits membrane resealing following PEF-induced electroporation, as evidenced by progressive calcein leakage over 20 min, while immediate pore formation remained unaffected. Catalase rescue experiments confirmed that hydrogen peroxide removal prevented progressive membrane damage without affecting immediate pore formation, thereby restoring cell viability. These findings identify hydrogen peroxide-mediated inhibition of membrane resealing as a novel mechanism underlying synergistic cytotoxicity, distinct from immediate membrane damage. This two-phase mechanism provides new insights for optimizing plasma-based cancer therapies. Full article

Liposomes are widely recognized as versatile nanocarriers in drug delivery due to their biocompatibility, tunable physicochemical properties, and ability to incorporate both hydrophilic and hydrophobic compounds. In this study, the encapsulation and release of diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), using lecithin–cholesterol liposomes are explored. Encapsulation parameters were first optimized with calcein as a model fluorophore, confirming that cholesterol addition enhances encapsulation efficiency by reducing membrane permeability. Guided by these results, liposomes containing equal weight fractions of lecithin and cholesterol were selected as an optimized formulation, providing calcein and diclofenac encapsulation efficiencies up to approximately 35% while maintaining hydrodynamic diameters below 300 nm with low polydispersity (PdI < 0.2), optimal for intravenous administration and prolonged systemic circulation. Release studies demonstrated sustained drug release over 15 days, with cumulative release exceeding 80%. Weibull modeling yielded ${\theta }_{\mathrm{\infty }}$ ≈ 1 and β values up to ~1.6 at higher loadings, with β > 1 indicating a complex, sigmoidal (non-Fickian) release mechanism. These findings support the potential of liposomes as delivery platforms for NSAIDs with solubility and bioavailability limitations. Full article

Background: Glioblastoma (GBM) is the most aggressive malignancy of the central nervous system (CNS) and is characterized by poor prognosis and significant resistance to available treatments. Surgery, radiation therapy, and chemotherapy are the standard treatments; however, their efficacy is often limited by resistance. Resveratrol (RES), a naturally occurring polyphenol with antioxidant properties, has shown significant anticancer effects through inhibition of multiple cellular pathways. However, our earlier research revealed that the LN428 cell exhibited resistance, while the U251 cell showed sensitivity to RES monotherapy. Hence, RES and AG490, a JAK2 inhibitor, were used to overcome GBM cell resistance, which might enhance therapeutic efficacy. Methods: Human GBM cell lines LN428 and U251 were used. CCK-8, H&E staining, transwell, wound healing, calcein AM/PI, and flow cytometry assays were performed to evaluate cell proliferation, migration, and apoptosis. Molecular docking was performed to analyze the binding energy. Western blot, immunocytochemistry (ICC), and immunofluorescence (IF) were used to assess protein expression following treatment with RES, AG490, and their combination. Results: The results revealed that U251 cells were more sensitive to RES, AG490, and RES + AG490 than LN428 cells. Additionally, the combination of both compounds significantly reduced cell viability, proliferation, and migration, while increasing apoptosis in the LN428 and U251 cell lines. Moreover, the combination of RES and AG490 led to increased BAX protein expression while decreasing BCL-2 expression in LN428 and U251 cell lines. Notably, the monotherapy administration of RES did not significantly inhibit STAT3 or pSTAT3 protein expression in LN428 cells, while combination therapy significantly inhibited the expression of these proteins in LN428 and U251 cell lines. Conclusions: The concurrent administration of RES and AG490 effectively inhibited the JAK2/STAT3 signalling pathway and enhanced antitumor effects in GBM cells, indicating their potential as a therapeutic strategy. Full article

Three-dimensional (3D) cell culture models provide physiologically relevant systems that mimic the native endometrial environment better than 2D models and offer reliable platforms to study embryo implantation and maternal–embryo interactions. One widely used 3D culture model is the generation of spheroids. However, standardized and reproducible methods for generating uniform spheroids from trophoblast and endometrial stromal cells are limited. In this study, we established and validated a robust protocol for spheroid formation using human trophoblast (HTR8/SVneo, JEG3) and endometrial stromal (St-T1b, tHESC) cell lines. The protocol was further extended to generate spheroids from decidualized tHESC, representing a novel approach that closely reflects the receptive endometrial environment. Key parameters, including cell concentration and methyl cellulose supplementation, were optimized to produce compact and homogeneous spheroids. Spheroid formation was monitored at defined intervals (0, 8, 24, 32, and 48 h), and decidualized spheroids were assessed up to 72 h. Long-term cryopreservation over 11 months demonstrated high post-thaw viability across all spheroid types, as confirmed by Calcein-AM staining. This standardized workflow provides a reliable 3D model incorporating hormonally primed stromal cells and offers a practical platform to investigate the mechanisms underlying normal and trophoblast invasion in vitro. Full article

Necrox-5 is an indole-derived antioxidant that inhibits necrotic cell death, likely through prevention of mitochondrial stress, oxidative stress, inflammation, and hypoxia/reoxygenation. However, its protective role against ferroptotic toxicity has not yet been studied. In this study, we induced ferroptosis in HT-22 cells, an immortalized hippocampal neuronal cell line, using ferroptosis-inducing agents. We also tested Necrox-5 against toxicity induced by propargite, a pesticide known to inhibit complex V (mitochondrial adenosine triphosphate [ATP] synthase) and induce necrosis. We evaluated cytotoxicity using calcein AM and lactate dehydrogenase (LDH) release assays. Additionally, we conducted intracellular and cell-free C11-BODIPY assays to assess the efficacy of Necrox-5 in inhibiting lipid peroxidation. Intracellular glutathione (GSH) levels were measured using the mBCI-GSH assay, while ATP levels were determined through bioluminescence assays. Our findings show that Necrox-5 is a potent inhibitor of ferroptosis induced by erastin, RSL3, FINO2, and iron plus arachidonic acid. Furthermore, we demonstrated that Necrox-5 protects against ferroptosis-like propargite toxicity, although it did not prevent propargite-induced depletion of GSH and ATP. We identified radical-scavenging antioxidant activity as the primary mechanism by which Necrox-5 protects from ferroptosis and propargite toxicity. In conclusion, Necrox-5 is a potent cytoprotective compound that warrants further study for its potential role in ferroptosis-associated complications such as neurodegenerative diseases. Full article

Fucoidans are natural compounds that exhibit bioactivity against age-related macular degeneration (AMD), the leading cause of central vision loss in industrialized nations. Pathological factors like oxidative stress and lipid peroxidation play vital roles in AMD pathogenesis. Lipid-induced alterations in the retinal pigment epithelium (RPE) contribute to AMD development. In this study, a commercial fucoidan from Fucus vesiculosus (FVs) was tested for its activity regarding lipid-peroxidation-related effects. The human RPE cell line ARPE-19, primary porcine RPE, and RPE/choroid explants were stimulated with erastin, acting as an inducer of lipid peroxidation, and treated with fucoidan. Effects on cell viability (tetrazolium bromide (MTT) or calcein staining), vascular endothelial growth factor (VEGF) and interleukin 8 (IL8) secretion (ELISA), reactive oxygen species (ROS), protein expression (glutathione peroxidase 4 (GPX4), CD59, and retinoid isomerohydrolase (RPE65), analyzed via Western blot), and gene expression (RT-qPCR) were investigated. FVs showed protective effects against erastin-induced reduction in viability (with a 12.7% increase in viability compared to erastin), RPE65 expression (with a 4.2-fold increase compared to erastin), and GPX4 expression (with a 2.3-fold increase compared to erastin) in primary RPE. Erastin-induced VEGF secretion was attenuated by FVs in ARPE-19 and primary RPE (with an up to 1.7-fold reduction compared to erastin). Elevated IL8 levels were reduced by FV treatment in primary RPE (with a 9.1-fold reduction compared to erastin). Induced VEGF in RPE/choroid explants was reduced by FVs (with an up to 2.9-fold reduction compared to erastin), and this reduction was correlated with slight improvements in viability. In conclusion, FVs exerted protective effects against lipid-induced stress. This study reveals further effects of fucoidans against AMD-related pathologies. Full article

Numerous modern scientific studies have demonstrated that animal venoms harbor a wealth of diverse anticancer active components, serving as a valuable resource for the development of natural antitumor drugs. AI-based computation and prediction models enable rapid screening of extensive active peptides. In this study, the anticancer activity of seven peptides was predicted using our previous deep learning model. Further verification experiments confirmed that Lpep3 can selectively and efficiently inhibit the growth of leukemia cells. Electron microscopy observations revealed cell shrinkage in morphology and honeycomb-like perforations on the cell membrane in the treated group. It is hypothesized that high-concentration peptides disrupt the cell membrane and increase cell permeability, which was confirmed by trypan blue staining and Calcein-AM/PI double-staining assays. Lpep3 induces the release of lactate dehydrogenase (LDH) and ATP in a concentration-dependent manner, further suggesting that this peptide disrupts the cell membrane. In addition, although Lpep3 does not affect the cell cycle of MV-4-11, it can induce cell apoptosis. Western blotting and RT-qPCR results showed that compared with the control group, the expression levels of Bax were upregulated, while the expression level of Bcl-2 protein was downregulated in the Lpep3 group. In vivo experiments demonstrated that Lpep3 has good biological safety, and compared with the control group, the Lpep3 group could inhibit the growth of tumor cells in mice. Collectively, Lpep3 is characterized by high potency and specificity and may serve as a promising lead compound for the development of anti-leukemia drugs. Full article

Background/Objectives: Melanoma is a highly malignant skin tumor with poor response to conventional chemotherapeutic regimens. Melanoma cells induce cytotoxic T cell-mediated immune responses, and immunotherapy has significantly improved survival rates for patients with advanced disease. Methods: Here, we investigate NK cell-mediated melanoma cell killing and its regulation by PD-L1/PD-1 blockade and IFNβ. Four melanoma cell lines were used in this study. To evaluate NK cell cytotoxicity, cells were exposed to NK cells with or without IFNβ. The calcein release assay was used to measure cell death, while specific inhibitors and siRNA silencing were applied to determine the contribution of individual effector pathways. Results: NK cells were able to kill melanoma cells with sensitivity to killing varying between different cell lines. Cytotoxic effects were mainly mediated through activation of the TRAIL signaling cascade. In cell lines with low sensitivity to NK cell killing, expression of PD-L1 was noted and killing by NK cells could be significantly increased by inhibition of the PD-L1/PD-1 checkpoint. Killing of melanoma cells could be further increased by incubation of NK cells with IFNβ. Conclusions: Our results point to a role of NK cells in the killing of melanoma cells and a potential clinical benefit of a combination therapy of IFNβ and anti-PD-1 antibody. Full article

Brassica yellows virus (BrYV) mainly infects cruciferous crops and has been widely prevalent across China. To develop a rapid and highly sensitive method for detecting BrYV in oilseed rape, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established. Four specific primers were designed to target the conserved gene of BrYV, with total RNA extracted from BrYV-infected oilseed rape leaves used as the template for the RT-LAMP assay. The optimal reaction conditions were determined, including a primer concentration ratio of 1:8, MgSO₄ concentration of 4 mM, reaction temperature of 64 °C, and a suitable reaction time of 60 min. Sensitivity analysis demonstrated that the RT-LAMP assay could detect total RNA at a concentration of 0.091 × 10⁻³ μg/μL, which was 100-fold more sensitive than conventional RT-PCR for BrYV detection. In addition to visualizing results by electrophoresis, the RT-LAMP assay could also be easily visualized using calcein-MnCl₂. These results indicate the potential of the established RT-LAMP assay for rapid BrYV detection in oilseed rape plants, which can provide better technical support for field diagnosis, disease forecasting, and the implementation of effective control strategies against the virus. Full article

To address the shortcomings of existing anti-VEGF monotherapy in neovascular age-related macular degeneration (nAMD), we investigated the therapeutic capabilities of exosomes obtained from human induced pluripotent stem cell (hiPSC)-derived retinal organoids in a mouse model of laser-induced choroidal neovascularization (CNV). To evaluate Retinal Organoid-derived exosome (RO-Exo) distribution after intravitreal (IVT) injection, calcein-labeled RO-Exo was observed using confocal microscopy. CNV was induced in C57BL/6 J mice by laser photocoagulation. RO-Exo was isolated from retinal organoids (differentiation days 55–65) and injected 5 days post-laser. Therapeutic efficacy was evaluated on day 12. Vascular leakage and CNV size were assessed by angiography and CD31 immunostaining. We also examined HIF-1α/VEGF-A expression (Western blotting), Retinal Pigment Epithelium (RPE) integrity markers (immunofluorescence staining for α-SMA, fibronectin, and ZO-1), and the activation of the Mitogen-Activated Protein Kinase (MAPK) pathway (phospho-ERK, -p38, -JNK) in CNV lesions. After IVT injection, RO-Exo migrated to the RPE layer, showing high retinotropic distribution. In the CNV model, RO-Exo significantly reduced vascular leakage and CNV size, with greater suppression of HIF-1α and VEGFA expression than aflibercept, the standard-of-care anti-VEGF drug. CD31-positive vasculature was decreased, accompanied by downregulation of fibronectin (a fibrotic marker) and restoration of RPE hexagonality and integrity. Furthermore, RO-Exo inhibited the activation of ERK, P38, and JNK in CNV lesions. Our study results demonstrate that RO-Exo exhibits multi-target therapeutic effects—including anti-angiogenic, anti-fibrotic, and neuroprotective actions—offering a promising alternative to conventional anti-VEGF therapy for nAMD. Full article

Avian influenza viruses (AIV) cause severe economic losses in poultry production and pose zoonotic threats, necessitating rapid, field-deployable diagnostics. While real-time PCR is the gold standard, its use is limited in resource-constrained settings. This study aimed to develop and validate optimized loop-mediated isothermal amplification (LAMP) protocols for AIV detection directly at sample collection sites. We optimized Real-Time RT-LAMP and colorimetric LAMP assays targeting the conserved M gene, using primers described in the literature. Analytical sensitivity was assessed with a plasmid standard (10⁶–10⁰ copies/μL); specificity was evaluated against 27 AIV strains (H1–H12) and heterologous avian viruses (Newcastle disease, infectious bronchitis, Gumboro, and laryngotracheitis viruses). Reverse transcription was integrated into the LAMP reaction. Real-Time LAMP with SYBR Green achieved 100% analytical sensitivity (95% CI: 80–100; detection limit: Ct = 38), while colorimetric LAMP (cresol red, malachite green, calcein) detected 10² plasmid copies (Ct = 32) with 91.67% sensitivity (95% CI: 76.1–100). No cross-reactivity occurred. These optimized LAMP protocols offer sensitivity and specificity comparable to PCR, require minimal equipment, and enable rapid AIV screening, significantly enhancing early detection and epidemiological surveillance in field conditions. Full article

Lung cancer remains a leading cause of cancer-related mortality worldwide, where conventional chemotherapy is often limited by severe side effects and drug resistance. Ginsenosides, the primary bioactive triterpenoid saponins isolated from the root of Panax ginseng C. A. Mey, have demonstrated potential in combating non-small-cell lung cancer (NSCLC). However, their efficacy under nutrient-deficient conditions remains unclear. This study aimed to investigate the effects of ginsenosides on the growth and death of lung cancer cells under low-nutrient conditions and to explore the underlying mechanisms. A549 cells were divided into two groups: one cultured in 10% serum and another under serum-free conditions, followed by treatment with ginsenosides CK, Rh2(S), and Rg3(S) for 24 h. Cell proliferation and apoptosis were evaluated using a CCK-8 assay, Calcein/PI fluorescence staining, Hoechst 33258 staining, and flow cytometry. Potential targets and signaling pathways of ginsenosides were predicted using network pharmacology and bioinformatics analyses. The mRNA expression of key genes was measured by qRT-PCR, and mitochondrial membrane potential was assessed using JC-1 staining. The results showed that ginsenosides induced dose-dependent apoptosis in serum-starved A549 cells. Bioinformatics analysis suggested the involvement of the PI3K/Akt/FoxO signaling pathway, which was supported by decreased Akt mRNA levels and increased FoxO mRNA expression. Furthermore, mRNA levels of Bim, Caspase-3, Caspase-8, and Caspase-9 were significantly upregulated, accompanied by a loss of mitochondrial membrane potential. These findings indicate that under serum deprivation, ginsenosides enhance apoptosis in A549 cells, likely through the regulation of the PI3K/Akt/FoxO pathway. Full article

This study investigates the scavenger activity of Polynucleotide High Purification Technology (PN HPT^TM), alone or in combination with hyaluronic acid (PN HPT^TM + HA) against oxidative stress induced by hydrogen peroxide (H₂O₂). Since oxidative stress is implicated in numerous pathological conditions, identifying effective antioxidants is crucial for therapeutic development. We employed a cell-free fluorometric assay based on Calcein-AM, a fluorescence probe whose signal increases proportionally to the generation of reactive oxygen species (ROS), to evaluate the ability to neutralize ROS under varying oxidative stress conditions and determine the dose- and time-dependent effects of these compounds. PN HPT^TM, HA, and PN HPT^TM + HA were tested at various concentrations over multiple time points. Our results demonstrated that all tested treatments significantly lowered ROS levels compared to the untreated control. Notably, the PN HPT^TM -based compounds exhibited robust scavenging activity, with PN HPT^TM + HA displaying the strongest and most consistent ROS-neutralizing effect across all concentrations and time points. This enhanced performance suggests a synergistic interaction between PN HPT^TM and HA, potentially due to complementary mechanisms of free radical scavenging and structural stabilization. These findings highlight the potential of PN HPT^TM and PN HPT^TM + HA as effective antioxidative agents, offering potential for therapeutic applications where oxidative stress is central, including wound healing and tissue regeneration. Full article

Quantitative determination of calcium content in milk and dairy products is an important analytical task due to the great importance of this element in the human diet. Calcium determination in milk and dairy products was performed using semi-automatic complexometric titration with a webcam as a color change detector. The color changes were registered directly in turbid dairy dispersions, creating a white background. The analytical signals tested were the color component (R, G, B) read from the titration beaker images and the calculated Hue parameter. For the calcein indicator, the optimal signals are Green and Hue. For the HNB indicator, the optimal signals are Red and Hue. The developed method of titration using an RGB camera detector is characterized by excellent linearity (R² = 0.9999) and accuracy. RSD values range from 0.3 to 2.9%. Recovery values range from 105 to 113%. Examples of calcium determination in commercial products include milk, fermented products, cream, and cottage cheese. Full article

Osteoporosis compromises bone quality and impairs implant osseointegration. Since an adequate bone bed is essential for implant stability and success, this study evaluated the effects of implant surface functionalization with zoledronic acid (ZOL), alone or combined with ruterpy (TERPY), on peri-implant bone healing in healthy (SHAM) and osteoporotic (OVX) rats. ZOL has antiresorptive properties, while TERPY exhibits osteoinductive potential. The hypothesis was that ZOL + TERPY would act synergistically by inhibiting bone resorption and promoting new bone formation. Sixty-six female Wistar rats (3 months old) were divided into six groups (n = 11) according to systemic condition (SHAM or OVX) and implant type: conventional (CONV), ZOL, or ZOL + TERPY. Surgeries (sham or bilateral ovariectomy) were performed on day 0, and implants were placed in the tibial metaphysis on day 90. Fluorochromes were administered on days 104 (calcein) and 114 (alizarin), and euthanasia was performed on day 118. Samples were analyzed histologically via confocal microscopy and micro-computed tomography (Micro-CT). The ZOL + TERPY groups demonstrated significantly accelerated peri-implant bone repair, showing greater bone formation and organization; improved BV/TV, Tb.N, and I.S.; and reduced Tb.Sp and Po.Tot compared to CONV and ZOL-alone groups. In conclusion, ZOL + TERPY enhances and speeds bone healing, even under osteoporotic conditions. Full article