Search Results (595)

Objectives: To investigate the efficacy and underlying mechanisms of IBCar’s biological activity in breast cancer models, both in cell culture and in mice, and to compare its effects on cancer versus normal cells. Methods: The cytotoxicity of IBCar was evaluated using the MTS assay to assess metabolic activity and the clonogenic assay to determine reproductive integrity. The impact of IBCar on microtubule integrity, mitochondrial function, and multiple signaling pathways was analyzed using Western blotting, microarray analysis, and live cell imaging. The therapeutic effectiveness of orally administered IBCar was assessed in a transgenic mouse model of Luminal B breast cancer and in mice implanted with subcutaneous triple-negative breast cancer xenografts. Results: IBCar demonstrated potent cytotoxicity across a diverse panel of breast cancer cell lines, including those with mutant or wild-type TP53, and cell lines with short and long doubling times. Comparative analysis revealed distinct responses between normal and cancer cells, including differences in IBCar’s effects on the mitochondrial membrane potential, endoplasmic reticulum stress and activation of cell death pathways. In breast cancer cells, IBCar was cytotoxic at nanomolar concentrations, caused irreversible microtubule depolymerization leading to sustained mitochondrial dysfunction, endoplasmic reticulum stress, and induced apoptosis. In normal cells, protective mechanisms included reversible microtubule depolymerization and activation of pro-survival signaling via the caspase-8 and riptosome pathways. The therapeutic potential of IBCar was confirmed in mouse models of Luminal B and triple negative BC, where it exhibited strong antitumor activity without detectable toxicity. Conclusions: These findings collectively support IBCar as a promising, effective, and safe therapeutic candidate for breast cancer treatment. Full article

Background/Objectives: The gene pool of the Apennine wolf is affected by admixture with domestic variants due to anthropogenic hybridisation with dogs. Genetic monitoring at the population level involves assessing the extent of admixture in single individuals, ranging from pure wolves to recent hybrids or wolf backcrosses, through the analysis of nuclear and mitochondrial DNA (mtDNA) markers. Although individually non-diagnostic, mtDNA is nevertheless essential for completing the final diagnosis of genetic admixture. Typically, the identification of wolf mtDNA haplotypes is carried out via sequencing of coding genes and non-coding DNA stretches. Our objective was to develop a fast real-time PCR assay to detect the mtDNA haplotypes that occur exclusively in the Apennine wolf population, as a valuable alternative to the demanding sequence-based typing. Methods: We validated a qualitative duplex real-time PCR that exploits the combined presence of diagnostic point mutations in two mtDNA segments, the NDH-4 gene and the control region, and is performed in a single-tube step through TaqMan-MGB chemistry. The aim was to detect mtDNA multi-fragment haplotypes that are exclusive to the Apennine wolf, bypassing sequencing. Results: Basic validation of 149 field samples, consisting of pure Apennine wolves, dogs, wolf × dog hybrids, and Dinaric wolves, showed that the assay is highly specific and sensitive, with genomic DNA amounts as low as 10⁻⁵ ng still producing positive results. It also proved high repeatability and reproducibility, thereby enabling reliable high-throughput testing. Conclusions: The results indicate that the assay presented here provides a valuable alternative method to the time- and cost-consuming sequencing procedure to reliably diagnose the maternal lineage of the still-threatened Apennine wolf, and it covers a wide range of applications, from scientific research to conservation, diagnostics, and forensics. Full article

Strobilurins are a prominent class of fungicides capable of entering aquatic environments via runoff and leaching from the soil. Findings from previous studies suggest that strobilurins are highly toxic in aquatic environments, and evidence of acute developmental toxicity and altered behavioral responses have been emphasized. The objective here was to determine the effects of a new strobilurin, metyltetraprole (MTP), on zebrafish using developmental endpoints, gene expression, and behavioral locomotor assays. We hypothesized that MTP would cause developmental toxicity and induce hyperactivity in zebrafish (Danio rerio). To test this, developing zebrafish embryos/larvae were exposed to environmentally relevant levels of MTP (0.1, 1, 10, and 100 µg/L) until 7 days post-fertilization. Survival percentages did not differ among the treatment groups. No change in reactive oxygen species production was detected, but two genes involved in the mitochondrial electron transport chain (mt-nd3 and uqcrc2) were altered in abundance following MTP exposure. Moreover, the highest concentration (100 µg/L) of MTP caused notable hyperactivity in the zebrafish in the visual motor response test. Overall, results from this study increase our knowledge regarding sub-lethal effects of MTP, helping inform risk assessment for aquatic environments. Full article

Background: Obesity is associated with insulin resistance (IR) and characterized by impaired activation of the PI3K/AKT route and glucose uptake. Elevated plasma levels of palmitic acid (PA) diminish insulin signaling in vitro and in vivo. Origanum vulgare L. essential oil (OVEO) is rich in monoterpenes with protective effects against IR. Objective: The study aimed to assess total phenols content and antioxidant activity of OVEO and its cytotoxicity, as well as its effect on insulin signaling and glucose uptake in PA-treated adipocytes. Methods: The quantification of total phenolic content was determined using the Folin–Ciocalteu method, while the antioxidant capacity of OVEO was assessed by DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing antioxidant power) methods. The cytotoxicity of OVEO (0.1–10 µg/mL) was assessed using the MTS assay. SW872 adipocytes were incubated with 0.4 mM PA for 24 h, with or without a 2 h preincubation of OVEO, and then stimulated with insulin (100 nM, 10 min) or a vehicle. Phosphorylation of Tyr-IRS-1, Ser-AKT, and Thr-AS160 was analyzed by Western blot, and glucose uptake was measured using 2-NBDG. Results: OVEO contained phenols and exhibits antioxidant capacity. All the concentrations of OVEO assessed were not cytotoxic on SW872 adipocytes. PA decreased basal phospho-AS160 as well as insulin-stimulated phospho-IRS1, phospho-AKT, phospho-AS160 and glucose uptake, while OVEO co-treatment enhanced these markers. Conclusions: These findings suggest a beneficial effect of OVEO on the PA-impaired insulin pathway and glucose uptake, which might be explained by its phenolic content and antioxidant capacity, highlighting its potential as a complementary therapeutic agent for IR and related metabolic disorders. Full article

Background and Objectives: Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies worldwide. Although chemotherapy is an effective treatment for colorectal cancer (CRC), its effectiveness is frequently hindered by the emergence of resistant cancer cells. Studies have demonstrated a linkage between drug resistance and the pregnane X receptor (PXR), which influences the metabolism and the transport of chemotherapeutic agents. Likewise, autophagy is also a well-established mechanism that contributes to chemotherapy resistance, and it is closely tied to tumor progression. This pre-clinical study aims to investigate the role of mtKRAS-dependent autophagy with PXR expression after treatment with Irinotecan in colorectal cancer. Methods: CRC lines were treated with specific inhibitors, such as 3-methyladeninee, hydroxychloroquine PI-103, and irinotecan hydrochloride, and subjected to various assays, including MTT for cell viability, Western blot for protein expression, siRNA-mediated PXR knock-out, and confocal microscopy for autophagic vacuole visualization. Protein quantification, gene knockdown, and subcellular localization studies were performed under standardized conditions to investigate treatment effects on autophagy and apoptosis pathways. Conclusions: Our experiments showed that PXR knockdown does not alter autophagy levels following Irinotecan treatment, but it promotes apoptotic cell death despite elevated autophagy. Moreover, late-stage autophagy inhibition reduces PXR expression, whereas induction through PI3K/AKT/mTOR inhibition leads to increased expression of PXR. Our experiments uncover a mechanism by which autophagy facilitates the nuclear translocation of the PXR, thereby promoting resistance to Irinotecan across multiple cell lines. Full article

Tendinopathies are a significant challenge in musculoskeletal medicine, with current treatments showing variable efficacy. Electromagnetic transduction therapy (EMTT) has emerged as a promising therapeutic approach, but its biological effects on tendon cells remain largely unexplored. Here, we investigated the effects of EMTT on primary cultured human tenocytes’ behavior and functions in vitro, focusing on cellular responses, senescence-related pathways, and molecular mechanisms. Primary cultures of human tenocytes were established from semitendinosus tendon biopsies of patients undergoing anterior cruciate ligament (ACL) reconstruction (n = 6, males aged 17–37 years). Cells were exposed to EMTT at different intensities (40 and 80 mT) and impulse numbers (1000–10,500). Cell viability (MTT assay), proliferation (Ki67), senescence markers (CDKN2a/INK4a), migration (scratch test), cytoskeleton organization (immunofluorescence), and gene expression (RT-PCR) were analyzed. A 40 mT exposure elicited minimal effects, whereas 80 mT treatments induced significant cellular responses. Repeated 80 mT exposure demonstrated a dual effect: despite a moderate decrease in overall cell vitality, increased Ki67 expression (+7%, p ≤ 0.05) and significant downregulation of senescence marker CDKN2a/INK4a were observed, suggesting potential senolytic-like activity. EMTT significantly enhanced cell migration (p < 0.001) and triggered cytoskeletal remodeling, with amplified stress fiber formation and paxillin redistribution. Molecular analysis revealed upregulation of tenogenic markers (Scleraxis, Tenomodulin) and enhanced Collagen I and III expressions, particularly with treatments at 80 mT, indicating improved matrix remodeling capacity. EMTT significantly promotes tenocyte proliferation, migration, and matrix production, while simultaneously exhibiting senolytic-like effects through downregulation of senescence-associated markers. These results support EMTT as a promising therapeutic approach for the management of tendinopathies through multiple regenerative mechanisms, though further studies are needed to validate these effects in vivo. Full article

The Internet of Medical Things (IoMT) is revolutionizing healthcare by integrating smart diagnostic devices with cloud computing and real-time data analytics. The emergence of infectious diseases, including COVID-19, underscores the need for rapid and decentralized diagnostics to facilitate early intervention. Traditional centralized laboratory testing introduces delays, limiting timely medical responses. While point-of-care molecular diagnostic (POC-MD) systems offer an alternative, challenges remain in cost, accessibility, and network inefficiencies. This study proposes an IoMT-based architecture for fully automated POC-MD devices, leveraging WebSockets for optimized communication, enhancing microfluidic cartridge efficiency, and integrating a hardware-based emulator for real-time validation. The system incorporates DNA extraction and real-time polymerase chain reaction functionalities into modular, networked components, improving flexibility and scalability. Although the system itself has not yet undergone clinical validation, it builds upon the core cartridge and detection architecture of a previously validated cartridge-based platform for Chlamydia trachomatis and Neisseria gonorrhoeae (CT/NG). These pathogens were selected due to their global prevalence, high asymptomatic transmission rates, and clinical importance in reproductive health. In a previous clinical study involving 510 patient specimens, the system demonstrated high concordance with a commercial assay with limits of detection below 10 copies/μL, supporting the feasibility of this architecture for point-of-care molecular diagnostics. By addressing existing limitations, this system establishes a new standard for next-generation diagnostics, ensuring rapid, reliable, and accessible disease detection. Full article

Background/Objectives: Prostate cancer is the second most common type of cancer diagnosed in men. Various treatments for this cancer, such as radiation therapy, surgery, and systemic therapy, can cause side effects in patients; therefore, there is a need to develop new treatment alternatives. One promising approach is virotherapy, which involves using oncolytic viruses (OVs), such as the recombinant Newcastle disease virus (rNDV). Methods: We used the lentogenic rNDV rLS1 strain (the control virus) as our backbone to develop two highly fusogenic rNDVs: rFLCF5nt (the parental virus) and rFLCF5nt-IFN-γ (rFLCF5nt expressing human interferon-gamma (IFN-γ)). We evaluated their oncolytic properties in a prostate cancer cell line (DU145). Results: The results showed the expression and stability of the IFN-γ protein, as confirmed using Western blotting after ten passages in specific pathogen-free chicken embryo eggs using the IFN-γ-expressing virus. Additionally, we detected a significantly high oncolytic activity in DU145 cells infected with the parental virus or the IFN-γ-expressing virus using MTS (a cell viability assay) and Annexin V-PE assays compared with the control virus (p < 0.0001 for both). Conclusions: In conclusion, our data show that IFN-γ-expressing virus can decrease cell viability and induce apoptosis in human prostate cancer in vitro. Full article

Fuchs endothelial corneal dystrophy (FECD) is a progressive corneal disease characterized by corneal endothelial cell (CEC) loss and guttae formation. Elevated levels of Transforming Growth Factor-Beta 1 and 2 (TGF-β1/-β2) have been reported in the aqueous humor (AH) of FECD patients and have been implicated with abnormal extracellular matrix (ECM) production, endothelial-to-mesenchymal transition (EndoMT), the unfolded protein response, and cell death. However, how TGF-β signaling affects cell migration in FECD remains to be elucidated. In this study, we found that TGF-β2 levels were significantly elevated in the AH of FECD patients compared to controls. We performed bulk RNA sequencing on FECD CECs treated with TGF-β1 or TGF-β2 and identified the epithelial-to-mesenchymal (EMT) pathway as one of the top dysregulated pathways. We found that TGF-β1 and TGF-β2 increased EMT markers, filamentous-actin (F-actin) expression and produced more EMT-like phenotype in FECD and control CECs. We also observed that TGF-β1 and TGF-β2 significantly increased FECD CEC migration speed as detected by scratch assay and individual cell tracking and promoted individual cellular migration behavior. This study provides novel insight into FECD pathogenesis and how increased TGF-β signaling promotes EndoMT and alters cellular migration in FECD CECs. Full article

Lipophenols, combining phenolic and lipid characteristics in an amphiphilic molecule, offer unique bioactive properties with therapeutic potential, including anti-inflammatory and anti-oxidant effects. Thus, palmitoyl-epigallocatechin gallate (PEGCG), a lipophilic derivative of the extensively studied (poly)phenol epigallocatechin gallate (EGCG), has been stressed concerning enhanced stability in lipid-rich environments and bioavailability due to improved cellular uptake. Nonetheless, the effect of lipophilic esterification on some cellular processes, particularly at the mitochondrial level, remains underexplored. According to this knowledge gap, the present study uncovered the cytotoxic and mitochondrial effects of PEGCG, in vitro, upon the liver hepatocarcinoma cell line HepG2. The range of determinations developed, including the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, flow cytometry, and electron microscopy, allowed describing the distinct biological potential for both EGCG and PEGCG. Thus, while EGCG exhibited minimal cytotoxicity and apoptosis induction, PEGCG reduced cell viability dose-dependently at 24 h and triggered significant mitochondrial damage, including fragmentation and cristae loss, at 1 µmol/L. However, at 48 h, PEGCG-treated cells recovered viability and mitochondrial structure, suggesting the activation of adaptive mechanisms for the molecular changes induced by PEGCG. These findings underscore the dynamic interplay between lipophilic catechins and cellular stress responses, offering valuable insights into the PEGCG’s potential as a therapeutic agent and laying a foundation for further exploration of its biological power. Full article

Heterocyclic compounds have shown that they hold significant therapeutic activities, highlighting the importance of discovering and developing novel candidates against cancers, infections, and oxidative stress-associated disorders. In this study, we demonstrated the biological activity of our previously synthesized thiazolidinone derivatives (TZDs-1, 6, and 7). Furthermore, we synthesized and structurally characterized a new derivative (TZD-5) using IR, ¹H NMR, and ¹³C NMR spectroscopy, confirming the presence of its key functional groups, namely, carbonyl and imine. Their antioxidant activity was assessed through the DPPH assay, with TZD-5 showing the most potent effect (IC₅₀ = 24.4 µg/mL), comparable to ascorbic acid, an effect attributed to the methoxy group introduced via N-alkylation. Cytotoxicity was evaluated using the MTS assay on normal (HFF-1) and cancerous (HepG2 and A549) cell lines at two time points: 24- and 48 h exposure. Our findings highlight clear differences in cytotoxicity and selectivity among the tested thiazolidinone derivatives. TZD-1 and TZD-6 demonstrated significant, dose-dependent cytotoxic effects on both cancerous (HepG2 and A549) and normal (HFF-1) cell lines, thus limiting their therapeutic potential due to insufficient selectivity. TZD-5 exhibited moderate selectivity with higher susceptibility for HepG2 cells compared to normal cells. Notably, TZD-7 showed the most favorable cytotoxic profile, demonstrating strong selective cytotoxicity toward cancer cell lines with minimal adverse effects on normal fibroblasts. Overall, the results highlight TZD-5 and TZD-7 as promising candidates for antioxidant and selective anticancer therapies. Full article

Background: Lung cancer, predominantly NSCLC (80%), has a poor prognosis due to late diagnosis and limited treatment efficacy. DMRTA2 (DMRT5), a transcription factor linked to neural/germ cell development, is overexpressed in NSCLC per TCGA data, indicating its potential role in tumorigenesis and as a therapeutic target. Methods: Conduct a comprehensive search of the relevant theoretical foundations. Based on this, differential expression analysis will be performed using the DESeq2 package in R on RNA-seq data from lung adenocarcinoma and lung squamous cell carcinoma in the TCGA database. The research will then employ various methods, including CRISPR genome editing, MTS assay, flow cytometry, Western blot, co-immunoprecipitation, immunofluorescence, and qRT-PCR. Results: Through experimental validation, we found that DMRTA2 mRNA is highly expressed in non-small-cell lung cancer (NSCLC) tissues and is negatively correlated with poor prognosis. DMRTA2 binds to HSP90β, inhibiting the interaction between HSP90β and p53, thereby suppressing p53 ubiquitination and nuclear export. This activates the p53 pathway, inhibiting the proliferation and invasion of lung cancer cells. Conclusions: In NSCLC, DMRTA2 acts as a context-dependent regulator, stabilizing wild-type p53 through competitive HSP90β binding to suppress tumors, while in p53-compromised cells, potentially engaging HSP90β or alternative pathways to promote malignancy. Its dual localization and transport interactions reveal multifunctional, stress-responsive roles beyond transcription. Full article

Background: This study developed and characterized a novel drug delivery system (DDS) for potential use in oral surgery, combining poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with chlorhexidine (MS-CHX) and a polyethylene glycol (PEG)-based hydrogel containing dexketoprofen (HG-DXT). Methods: MS-CHX was synthesized using a double emulsion evaporation method, while HG-DXT was formulated from a PEG blend. The components were combined in a 2:1 ratio to create the MS-CHX/HG-DXT DDS. Characterization techniques included differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and energy-dispersive X-ray spectroscopy (EDS). Antibacterial activity was evaluated using disk diffusion assays against E. faecalis, E. coli, S. aureus, and C. albicans. Biocompatibility was assessed with MTS, and drug release was measured via high-performance liquid chromatography (HPLC) in vitro. Results: CHX-loaded microspheres showed spherical morphology, stability above 37 °C, and antimicrobial efficacy. HG-DXT demonstrated good biocompatibility (80% of cell viability) and stable physicochemical properties (stability at 50-day storage). The DDS exhibited a biphasic release: an initial burst of dexketoprofen for analgesia, followed by sustained release of chlorhexidine for antimicrobial protection. Conclusions: This novel dual-action DDS showed promising characteristics and a favorable release profile, supporting its potential as a therapeutic alternative for post-operative pain and infection control in oral surgical procedures. Full article

Ecuador, located in South America, ranks among the countries with the highest rates of pesticide use per unit of cropland. Pesticide exposure is linked to genotoxic effects and carcinogenicity. While most studies evaluating the effects of pesticides focus on the active ingredient, commercial formulations are complex mixtures of several components that may alter their toxicological profile. In this study, we analyzed four pesticides commonly used in corn cultivation, and their typical field mixtures, including the herbicides atrazine and pendimethalin, the insecticides chlorpyrifos and cypermethrin, and a fertilizer, to evaluate their genotoxic effects, oxidative status, and potential to induce cellular transformation. CHO-K1 cells were treated with subtoxic doses of these formulations. MTS, comet, micronucleus, H2AX expression, SOD and GPx activity, and wound healing assays were performed. The results showed these formulations induced genotoxicity, evidenced by the comet assay. Additionally, exposure activated cellular DNA repair mechanisms, evidenced by a 1.89- to 2.63-fold increase in H2AX expression across all treatments and mixtures after 10 h. Notably, pendimethalin was associated with signs of cellular transformation, as evidenced by a 1.4-times greater cell migration observed in the wound healing assay. These findings suggest that even at subtoxic concentrations, these pesticide formulations can cause genetic damage and potentially alter cellular control mechanisms. Full article

Aquaculture is one of the fastest-growing sectors in food production. The widespread use of antibiotics in fish farming has been identified as a driver for the development of antibiotic resistance. One of the promising approaches to solving this problem is the use of probiotics. There are many promising aquaculture probiotics in the Bacillus genus, which produces non-ribosomal peptides (NRPs). NRPs are known as antimicrobial agents, although evidence is gradually accumulating that they may have other effects, especially at lower (subinhibitory) concentrations. The mechanisms of action of many NRPs remain unexplored, and molecular docking and molecular dynamics studies are invaluable tools for studying such mechanisms. The purpose of this study was to investigate the in silico inhibition of crucial bacterial targets by NRPs. Molecular docking analyses were conducted to assess the binding affinities of the NRPs of Bacillus for protein targets. Among the complexes evaluated, bacillibactin with glutamine synthetase, dihydrofolate reductase, and proaerolysin exhibited the lowest docking scores. Consequently, these complexes were selected for further investigation through molecular dynamics simulations. As a result, three additional potential mechanisms of action for bacillibactin were identified through in silico analyses, including the inhibition of glutamine synthetase, dihydrofolate reductase, and proaerolysin, which are critical bacterial enzymes and considered as the potential antibacterial targets. These findings were further supported by in vitro antagonism assays using bacillibactin-producing Bacillus velezensis strains MT55 and MT155, which demonstrated strong inhibitory activity against Pseudomonas aeruginosa and Aeromonas veronii. Full article