Search Results (186)

Background: Prostate cancer (PC) progression is strongly driven by dysregulated signaling pathways, with NF-κB playing a central role. Sesquiterpene lactones have been reported to modulate this pathway. This study evaluated and compared the cytotoxic effects of two structurally distinct sesquiterpene lactones: Tatridin A, a germacranolide, and desacetyl-β-cyclopyrethrosin, a eudesmanolide derivative. Their mechanisms of action were also examined, focusing on oxidative stress induction and NF-κB modulation. Methods: Chemical structures were confirmed by NMR and X-ray crystallography. Cytotoxicity was assessed in DU-145 and 22Rv1 PC cells using real-time cell analysis. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were measured with fluorometric assays. NF-κB activity was determined in THP-1 reporter cells and by Western blot of IκBα phosphorylation. Results: Tatridin A markedly reduced viability, showing lower IC₅₀ values (81.4 ± 2.7 µM in DU-145 and 50.7 ± 1.9 µM in 22Rv1 cells) than desacetyl-β-cyclopyrethrosin (166.9 ± 3.2 µM and 290.3 ± 8.3 µM, respectively). It also inhibited proliferation at markedly lower concentrations, with clonogenic IC₅₀ values of 7.7 µM in DU-145 and 5.24 µM in 22Rv1cells. Both compounds increased ROS, but tatridin A induced earlier and stronger responses and ΔΨm loss. Furthermore, tatridin A more effectively inhibited NF-κB signaling than classical inhibitors. Conclusions: Tatridin A exerts cytotoxic effects through oxidative stress, mitochondrial impairment, and NF-κB inhibition, supporting the therapeutic potential of germacranolides for the treatment of advanced PC. Full article

Pulmonary neuroendocrine tumors (PULMONARY NETs) are heterogeneous tumors ranging from well-differentiated to highly aggressive neoplasms. The aim of this study is to prospectively test pre-operative circulating free DNA (cfDNA) in PULMONARY NET patients undergoing surgery and evaluate its relationship to clinicopathological features. From February to December 2024, 136 patients with suspected primary lung cancer underwent pre-operative blood sampling, of whom 21 were diagnosed with PULMONARY NETs. Total cell-free nucleic acid extraction was performed using the Genexus Purification System (Thermofisher). cfDNA was quantified using a fluorometric assay with the Qubit dsDNA HS Assay kit (Thermofisher) and a capillary electrophoresis-based assay (cell-free DNA ScreenTape kit) on the Tape Station 4200 systems (Agilent). A cfDNA quality assessment was also obtained (cfDNA sizing and % cfDNA). Most patients had Stage I (18/21.85.7%) typical carcinoids (16/21.76.2%). Nodal involvement was detected in one patient (0.5%). Six months after surgery, all patients were alive without recurrence. Larger tumors presented higher levels of cfDNA. The mean tumor size in patients with cfDNA > 40 ng was 266 mm (±16.7 mm), compared to 13.2 mm (±7.3 mm) for cfDNA < 40 ng (p-value = 0.018). Higher levels of cfDNA were observed in patients with pStages greater than IA (p-value = 0.007). Although limited by a small sample group and biases of a surgical series, we observed that larger/advanced PULMONARY NETs presented higher cfDNA levels pre-operatively. Full article

Addressing the critical challenge in the differential diagnosis of severe inflammatory lung diseases, we propose a novel methodology for the analysis of macrophage surface receptors, CD68 and CD206, using specific non-antibody ligands. We developed a non-antibody alternative for the fluorometric detection of CD68+ cells, focusing on macrophages as key functional markers in inflammatory processes. Our marker based on dioleylphosphatidylserine (DOPS), a specific ligand to CD68, was incorporated into a liposomal delivery system. The specificity of this DOPS-based ligand can be precisely modulated by the liposome’s composition and the polyvalent presentation of the ligand. We synthesized a series of fluorescently-labeled DOPS-based ligands and developed a liposome-based sandwich fluorometric assay. This assay enables the isolation and quantification of CD68 receptor presence from bronchoalveolar lavage fluid (BALF). The results confirmed the specific binding of DOPS/lecithin liposomes to CD68+ cells compared to control lecithin systems. Furthermore, the incorporation of PEGylated ‘stealth’ liposomes significantly enhanced binding specificity and facilitated the generation of distinct binding profiles, which proved valuable in differentiating various inflammatory conditions. This approach yielded unique binding profiles of PS-based ligands to CD68+ cells, which varied significantly among a broad range of respiratory conditions, including primary ciliary dyskinesia, bronchial asthma, bronchitis, bacterial infection, pneumonia, and bronchiectasis. Confocal Laser Scanning Microscopy demonstrated selective binding and intracellular localization of the DOPS-based marker within CD68+ macrophages from BALF samples of patients with bronchitis or asthma. The binding parameters of this multivalent composite ligand with the CD68 receptor are comparable to those of antibodies. The inherent binding specificity of phosphatidylserine may offer a sufficient and viable alternative to conventional antibodies. Our results demonstrate the remarkable potential of this novel DOPS-based assay as a complementary tool for the developing non-antibody-based systems for the differential diagnosis of the respiratory diseases, warranting further investigation in larger clinical studies. Full article

Postmenopausal osteoporosis is characterized by progressive bone loss and deterioration of trabecular microarchitecture, yet safe and effective nutritional interventions remain limited. This study investigated the skeletal effects of whole sericin compared to isolated L-serine and calcium-only formulations in an ovariectomized (OVX) rat model. Forty female Sprague–Dawley rats underwent either sham surgery or OVX, followed by 8 weeks of daily oral administration with vehicle (calcium + vitamin D, NS), sericin formulation (S55), or L-serine. Sham and untreated OVX groups served as controls. Serum D-serine concentrations and femoral trabecular microarchitecture were assessed using fluorometric assays and micro-computed tomography (μCT), respectively. OVX significantly decreased bone volume fraction (BV/TV), bone mineral density (BMD), and trabecular number, while increasing trabecular separation. Sericin supplementation markedly improved BV/TV, BMD, trabecular thickness, and trabecular number, and reduced trabecular separation compared to both vehicle and untreated OVX controls. Sericin improved multiple trabecular parameters compared with L-serine. Serum D-serine levels were significantly elevated in the sericin group relative to calcium-only controls, though comparable to sham. These findings suggest that whole sericin exerts skeletal benefits beyond those attributable to its primary amino acid component, supporting its potential as a functional food ingredient for enhancing postmenopausal bone health. 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

Candida species commonly secrete aspartic peptidases (Saps), which are virulence factors involved in nutrient acquisition, colonization, tissue invasion, immune evasion and host adaptation. However, the regulation of Sap production remains poorly characterized in emerging, widespread and multidrug-resistant members of the Candida haemulonii clade (C. auris, C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii). This study investigated the influence of temperature, pH and protein substrate on Sap production using bloodstream isolates of the C. haemulonii clade. Sap activity was initially assessed using the enzyme coefficient (Pz) in fungal cells grown on yeast carbon base (YCB) agar supplemented with bovine serum albumin (BSA) to determine optimal conditions for enzymatic production. C. auris and C. duobushaemulonii exhibited the highest Sap activity at 96 h, pH 4.0–5.0, and 37 °C, whereas C. haemulonii and C. haemulonii var. vulnera displayed more variable and isolate-dependent profiles. Sap production was markedly suppressed at pH 6.0. The addition of pepstatin A, an inhibitor of aspartic peptidases, abolished Sap activity and impaired fungal growth in a dose-dependent manner, confirming both the enzymatic identity and its critical role in nitrogen acquisition. Conversely, YCB supplemented with an inorganic nitrogen source (ammonium sulfate) supported fungal growth but did not induce Sap production. To explore substrate specificity, YCB was supplemented with a panel of proteins. Serum albumins (bovine and human) induced the highest Sap production, followed by globulin, gelatin, hemoglobin, collagen and immunoglobulin G, while elastin and mucin elicited the lowest Sap production. Isolate-specific preferences for protein substrates were observed. Finally, fluorometric assays using a Sap-specific fluorogenic peptide substrate confirmed the presence of Sap activity in cell-free supernatants, which was consistently and entirely blocked by pepstatin A. These findings highlight inter- and intraspecies variability in Sap regulation among C. haemulonii clade, stressing the critical roles of substrate availability, pH and temperature in shaping fungal adaptation to host environments. Full article

Background: Histone deacetylase 6 (HDAC6) is a unique class IIb HDAC isozyme characterized by two catalytic domains and a zinc finger ubiquitin-binding domain. It plays critical roles in various cellular processes, including protein degradation, autophagy, immune regulation, and cytoskeletal dynamics. Due to its multifunctional nature and overexpression in several cancer types, HDAC6 has emerged as a promising therapeutic target. Methods: In this study, we employed a ligand-based pharmacophore modeling approach using a structurally diverse set of known HDAC6 inhibitors. This was followed by the virtual screening of over 140,000 commercially available compounds from both the MolPort and Asinex databases. The screening workflow incorporated pharmacophore filtering, molecular docking, and molecular dynamic (MD) simulations. Binding free energies were estimated using Molecular Mechanics Generalized Born Surface Area (MM-GBSA) analysis to prioritize top candidates. A fluorometric enzymatic assay was used to measure HDAC6 activity, while cell viability assay by Cell Titer Glo was used to assess the anti-tumor activity against drug-sensitive and -resistant multiple myeloma (MM) cells. Western blotting was used to evaluate the acetylation of tubulin or histone H4 after treatment with selected compounds. Results: Three promising compounds were identified based on stable binding conformations and favorable interactions within the HDAC6 catalytic pocket. Among them, Molecular Mechanics Generalized Born Surface Area (MM-GBSA) analysis identified Compound 10 (AKOS030273637) as the top theoretical binder, with a ΔG_bind value of −45.41 kcal/mol. In vitro enzymatic assays confirmed its binding to the HDAC6 catalytic domain and inhibitory activity. Functional studies on MM cell lines, including drug-resistant variants, showed that Compound 10 reduced cell viability. Increased acetylation of α-tubulin, a substrate of HDAC6, likely suggested on-target mechanism of action. Conclusions: Compound 10, featuring a benzyl 4-[4-(hydroxyamino)-4-oxobutylidene] piperidine-1-carboxylate scaffold, demonstrates potential drug-like properties and a predicted bidentate zinc ion coordination, supporting its potential as an HDAC6 inhibitor for further development in hematologic malignancies. Full article

Nonspecific toxicity to normal and malignant cells restricts the clinical utility of many anticancer drugs. In particular, anemia in cancer patients develops due to drug-induced toxicity to red blood cells (RBCs). The anticancer alkaloid, cepharanthine (CEP), elicits distinct forms of cell death including apoptosis and autophagy, but its cytotoxicity to RBCs has not been investigated. Colorimetric and fluorometric techniques were used to assess eryptosis and hemolysis in control and CEP-treated RBCs. Cells were labeled with Fluo4/AM and annexin-V-FITC to measure Ca²⁺ and phosphatidylserine (PS) exposure, respectively. Forward scatter (FSC) was detected to estimate cell size, and extracellular hemoglobin along with lactate dehydrogenase and aspartate transaminase activities were assayed to quantify hemolysis. Physiological manipulation of the extracellular milieu and various signaling inhibitors were tested to dissect the underlying mechanisms of CEP-induced RBC death. CEP increased PS exposure and hemolysis indices and decreased FSC in a concentration-dependent manner with prominent membrane blebbing. Although no Ca²⁺ elevation was detected, chelation of intracellular Ca²⁺ by BAPTA-AM reduced hemolysis. Whereas SB203580, D4476, acetylsalicylic acid, necrosulfonamide, and melatonin inhibited both PS exposure and hemolysis, staurosporin, L-NAME, ascorbate, caffeine, adenine, and guanosine only prevented hemolysis. Interestingly, sucrose had a unique dual effect by exacerbating PS exposure and reversing hemolysis. Of note, blocking KCl efflux augmented PS exposure while aggravating hemolysis only under Ca²⁺-depleted conditions. CEP activates Ca²⁺-independent pathways to promote eryptosis and hemolysis. The complex cytotoxic profile of CEP can be mitigated by targeting the identified modulatory pathways to potentiate its anticancer efficacy. Full article

Emerging evidence suggests a bidirectional relationship between gut microbiota, melatonin synthesis, and breast cancer (BC) development in hormone receptor-positive patients (HR+HER2+ and HR+HER2-). This study investigated alterations in gut microbiota composition, the serum serotonin–N-acetylserotonin (NAS)–melatonin axis, fecal short-chain fatty acids (SCFAs) and beta-glucuronidase (βGD) activity, and serum zonulin in HR+ BC patients compared to healthy controls. Blood and fecal samples were analyzed using mass spectrometry for serotonin, NAS, melatonin, and SCFAs; ELISA for AANAT, ASMT, 14-3-3 protein, and zonulin; fluorometric assay for βGD activity; and 16S rRNA sequencing for gut microbiota composition. HR+ BC patients exhibited gut dysbiosis with reduced Bifidobacterium longum and increased Bacteroides eggerthii, alongside elevated fecal βGD activity, SCFA levels (e.g., isovaleric acid), and serum zonulin, indicating increased intestinal permeability. Serum serotonin and N-acetylserotonin (NAS) levels were elevated, while melatonin levels were reduced, with a higher NAS/melatonin ratio in BC patients. AANAT levels were increased, and ASMT levels were decreased, suggesting disrupted melatonin synthesis. Bifidobacterium longum positively correlated with melatonin and negatively with βGD activity, while Bacteroides eggerthii showed a positive correlation with βGD activity. These findings suggested that gut microbiota alterations, disrupted melatonin synthesis, microbial metabolism, and intestinal permeability may contribute to BC pathophysiology. The NAS/melatonin ratio could represent a potential biomarker, necessitating further mechanistic studies to confirm causality and explore therapeutic interventions. Full article

Sperm membrane potential (Em) hyperpolarization during capacitation is a functional hallmark of fertilizing ability and has been proposed as a predictive biomarker for conventional in vitro fertilization (IVF) success. However, it is unclear whether Em remains stable across ejaculates over time and can reliably guide assisted reproductive technology (ART) decisions in advance. Thus, we aimed to evaluate the temporal consistency of human sperm Em within individuals and assess its utility as a prognostic marker when measured days or weeks prior to IVF procedures. Em was assessed in capacitated and non-capacitated sperm from normospermic donors at three time points over 28 days, using a fluorometric assay. Capacitated values were compared to a −48.6 mV threshold previously associated with successful fertilization. Intra-donor Em variability and coefficients of variation (CV) were analyzed statistically. Our results showed that Em values exhibited significant intra-donor variability over time (p = 0.007), with approximately half of the donors crossing the −48.6 mV functional threshold across sessions. Capacitated sperm samples showed significantly greater variability than non-capacitated ones, with several donors exceeding a 30% CV cutoff. No consistent correlation was found between CV and mean Em values. While Em remains a promising functional marker when assessed on the day of IVF, its temporal variability undermines its reliability as a predictive tool for ART decisions made in advance. These findings underscore the importance of timing in functional sperm assessments and call for further studies to identify the physiological factors influencing Em stability. Full article

Epithelial–mesenchymal transition (EMT) is one of the key steps in cutaneous carcinogenesis. At the molecular level, this cellular dedifferentiation is modulated by the interaction of signalling pathways that favour basement membrane degradation under the influence of proinflammatory cytokines and matrix metalloproteinases (MMPs). Given the intricate role of these endopeptidases in modulating extracellular matrix turnover, the present study aimed primarily to identify the MMP-2 expression profile during the early stages of cutaneous malignant transformation. Forty-eight lesions with malignant transformation potential were excised in healthy tissue. Following the histopathological diagnosis of keratoacanthoma, Bowen’s disease and actinic keratosis, the biological preparations were deparaffinised and homogenised in order to perform the FRET technique using the “MMP-2 Assay Kit Fluorometric”. The results of the previous part of this research indicate that MMP-2 expression is more intense in lesions of actinic keratosis compared to normal tissues and to keratoacanthoma or Bowen’s disease lesions, inversely proportional to the histopathological degree of dysplasia. Monitoring metalloproteinase activity in dysplastic epithelium may improve the detection of malignant transformation and guide treatment decisions. Full article

The genus Marrubium (Lamiaceae) is widely used in traditional medicine. While some representatives of the genus have been well investigated, the biological activity of others remains largely unknown. The aim of the current study was to assess the in vitro antioxidant potential and the effect on the reactivity of isolated rat gastric smooth muscles (SM) of aqueous extracts of Marrubium friwaldskyanum inflorescences, stems and leaves, and Marrubium peregrinum as a whole herb. The antioxidant activity was analyzed through multiple spectrophotometric and fluorometric assays. The effect on SM reactivity was determined by the treatment of excised gastric muscles of 10 male Whistar rats with the plant extracts alone or successive to 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide, ketanserin, verapamil, and acetylcholine. According to the obtained results, the M. friwaldskyanum leaf extract exhibited the greatest antioxidant potential, followed by the M. peregrinum one. The SM reactivity analysis revealed that the treatment with all four extracts induced a dose-dependent contractile response with predominant cholinergic character. However, activation of serotoninergic and/or dopaminergic pathways was also observed. Furthermore, when applied after verapamil, the extracts showed a SM relaxant effect. The discovered biological activity will serve as a basis for future analyses through which the therapeutic effect of the plants will be investigated. Full article

Majority of commercial L-asparaginase (L-ASNase) activity assays are based on coupled enzymatic reaction, which converts aspartate into pyruvate, subsequently reacting with the probe to form a stable chromophore, which can be detected spectrophotometrically. However, in complex biological samples this method can be inaccurate due to poor optical transparency or presence of compounds interfering with the coupled enzyme reaction–for this kind of cases alternative methods have been suggested. Here we suggest a strategy to rationally pick a method of choice in a variety of situations, taking into consideration the upsides and downsides of each method. A high-throughput fluorometric assay employing the substrate Asp-AMC was rigorously validated for L-ASPNase activity screening. Aassay performance is evaluated in complex biological matrices, including bovine serum, whole and diluted human blood, and finally the mouse blood and liver homogenates samples obtained from pharmacokinetic studies. This comprehensive validation process ensures the reliability and applicability of the assay for assessing L-asparaginase activity in diverse and physiologically relevant environments. Potential interfering factors and matrix effects were addressed, and assay conditions were optimized for each matrix. The optimized assay was employed to screen various L-asparaginase types (intracellular L-ASNases type I RrA, periplasmic L-ASNases type II EcA and EwA) and ASPNase formulations (conjugates with polyamines or polyelectrolyte complexes), comparing their kinetic parameters and stability. Fourier-transform infrared (FTIR) spectroscopy was further employed to investigate the fine features of molecular mechanisms of L-asparaginase catalysis. FTIR spectra of Asn during hydrolysis were analyzed in buffer solutions and in complex biological matrices, such as blood sample or liver homogenates which is crucial in the context of pharmacokinetic research. This combined fluorometric and FTIR approach provides a powerful platform for optimizing L-ASNase formulations and therapeutic strategies for ALL. Based on the results obtained we have developed a strategy to choose an approach for L-Asparaginase activity assessment for a variety of difficult situations when dealing with complex biological samples. Full article

Objectives: To evaluate how a 10-day multi-stressor field-training course—combining high physical and psycho-emotional demands, caloric restriction, and severe sleep deprivation—affects systemic oxidative/antioxidative status and biomarkers of nucleic-acid and skeletal-muscle damage in trained military cadets. Methods: Seventy-five healthy cadets (8 women, 67 men; 22–34 y) completed the course. Standardised operational rations (700–800 kcal day⁻¹) and two 20 min tactical naps per 24 h were enforced. Pre- and post-course venous blood was collected after an overnight fast. Plasma superoxide-dismutase activity (SOD), reduced and oxidised glutathione (GSH, GSSG), malondialdehyde (MDA), and hydrogen peroxide (H₂O₂) were quantified by colourimetric/fluorometric assays; 8-hydroxy-2-deoxyguanosine (8-OHdG) and myoglobin were measured by ELISA. The oxidative-stress index (OSI) was calculated as GSSG·GSH⁻¹. Within-subject differences were assessed with Wilcoxon signed-rank tests; associations between biomarker changes were explored by Spearman correlation. Results: After training, GSH (+175%, p < 0.001) and GSSG (+32%, p < 0.001) rose significantly, whereas SOD (−19%, p = 0.002), H₂O₂ (−20%, p = 0.015), MDA (−50%, p < 0.001), 8-OHdG (−23%, p < 0.001), and OSI (−47%, p < 0.001) declined. Myoglobin remained unchanged (p = 0.603). Reductions in MDA correlated inversely with increases in GSSG (rₛ = −0.25, p = 0.041), while H₂O₂ changes correlated positively with GSSG (rₛ = 0.25, p = 0.046), indicating a glutathione-driven adaptive response. Conclusions: Ten consecutive days of vigorous, calorie- and sleep-restricted field training elicited a favourable redox adaptation characterised by enhanced glutathione-mediated antioxidant capacity and lower circulating oxidant concentrations, without evidence of DNA or skeletal-muscle damage. The data suggest that, in physically prepared individuals, prolonged multi-stressor exposure can strengthen endogenous antioxidant defences rather than precipitate oxidative injury. Full article

Cholinesterases, specifically acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), play critical roles in neurotransmission and are key targets for inhibitors with therapeutic and toxicological significance. This review focuses on the development and application of fluorometric and colorimetric biosensors for the detection of cholinesterase inhibitors. These biosensors take advantage of the unique properties of AChE and BChE to provide sensitive and selective detection methods essential for environmental monitoring, food safety, and clinical diagnostics. Recent advances in assay techniques, including the use of gold nanoparticles, pseudoperoxidase nanomaterials, and innovative enzyme–substrate interactions, are highlighted. This review also discusses challenges and future directions for optimizing these biosensors for practical applications, emphasizing their potential to enhance public health and safety. Full article