Electrochemical Impedance Spectroscopy for the Sensing of the Kinetic Parameters of Engineered Enzymes.

The study presents a promising approach to enzymatic kinetics using Electrochemical Impedance Spectroscopy (EIS) to assess fundamental parameters of modified enteropeptidases. Traditional methods for determining these parameters, while effective, often lack versatility and convenience, especially under varying environmental conditions. The use of EIS provides a novel approach that overcomes these limitations. The enteropeptidase underwent genetic modification through the introduction of single amino acid modifications to assess their effect on enzyme kinetics. However, according to the one-sample t-test results, the difference between the engineered enzymes and hEKL was not statistically significant by conventional criteria. The kinetic parameters were analyzed using fluorescence spectroscopy and EIS, which was found to be an effective tool for the real-time measurement of enzyme kinetics. The results obtained through EIS were not significantly different from those obtained through traditional fluorescence spectroscopy methods (p value >> 0.05). The study validates the use of EIS for measuring enzyme kinetics and provides insight into the effects of specific amino acid changes on enteropeptidase function. These findings have potential applications in biotechnology and biochemical research, suggesting a new method for rapidly assessing enzymatic activity.

The Influence of Mesotrione on Human Colorectal Adenocarcinoma Cells and Possibility of Its Toxicity Mitigation by Cichoric Acid.

Mesotrione, as a widely used herbicide, is present in the environment in detectable amounts, causing serious damage. Here, we aimed to investigate the effect of mesotrione on Caco-2 cells and the possibility of its toxicity mitigation by cichoric acid. Therefore, we analyzed the cytotoxicity of both these compounds and the selected oxidative stress parameters, apoptosis and interaction of both the tested compounds with the cell membrane and their accumulation within the cells. In cytotoxicity studies, the stimulating activity of mesotrione was observed, and simultaneously, the inhibitory effect of cichoric acid was noticed. This effect was related to the results of oxidative stress analysis and apoptosis measurements. The activity level of key enzymes (glutathione peroxidase, catalase and superoxide dismutase) in Caco-2 cells exposed to cichoric acid was higher as compared to that of the control. The treatment with mesotrione did not induce apoptosis in the Caco-2 cells. The penetration of the studied compounds into the Caco-2 cells was measured by using an HPLC methodology, and the results indicate mesotrione's high penetration capacity. The distribution of charge on the surface of the cell membranes changed under the influence of both compounds. Considering the mutual interactions of beneficial and potentially toxic food ingredients, it should be noted that, despite the observed favorable trend, cichoric acid is not able to overcome the toxic and cancer-stimulating effects of this pesticide.

Alteration of zeta potential and cell viability in rat-derived L6 skeletal muscle cells and H9c2 cardiomyocytes: A study with submicron polystyrene particles.

BACKGROUND: Microand nanoplastics pollution can cause substantial damage to ecosystems. Since scientists have focused mainly on their impact on aquatic environments, less attention has been paid to the accumulation of polymer particles in terrestrial organisms. OBJECTIVES: We checked if submicron (<5 mm) polystyrene (PS) particles, which can accumulate in living organisms, lead to changes in the physicochemical properties of mammalian cell membranes. MATERIAL AND METHODS: The influence of submicron PS particles on the properties of rat-derived L6 myocytes and H9c2 cardiomyocytes was analyzed. Non-functionalized and amine-functionalized PS particles of 100 nm and 200 nm in diameter were used. The MTT assay was performed to evaluate the viability of the polymers-treated cells. The effect of short (6 h) and prolonged (48 h) incubation with different concentrations of PS particles on the cell's zeta (ζ) potential was examined with the electrophoretic light scattering technique (ELS). Polystyrene particles' physicochemical characteristics (size and stability) were performed using dynamic light scattering (DLS) and electrophoretic light scattering methods. RESULTS: The results show that submicron PS particles affect cell viability and cause changes in the physiochemical parameters of rat cell membranes. Differences were observed depending on the origin of the cells. We observed doseand time-dependent alterations in the studied parameters after submicron PS particle incubation in L6 myotubes and H9c2 cardiomyocytes. CONCLUSIONS: The size and modification of PS particle surfaces determine the extent to which they affect the analyzed properties of rat cardiomyocytes and myocytes membranes.

Selected Fungicides as Potential EDC Estrogenic Micropollutants in the Environment.

An increasing level of pesticide exposition is being observed as a result of the consumption of large amounts of fruits, vegetables and grain products, which are key components of the vegetarian diet. Fungicides have been classified as endocrine-disrupting compounds, but their mechanisms of action have not yet been clarified. The effect of boscalid (B), cyprodinil (C) and iprodione (I) combined with Tamoxifen (T) and 17ß-estradiol (E2) on cell viability, cell proliferation, reporter gene expression, ROS content, the cell membrane's function, cell morphology and antioxidant enzymes gene expression in MCF-7 and T47D-KBluc cell lines were investigated. The cell lines were chosen due to their response to 17ß -estradiol. The selected fungicides are commonly used in Poland to protect crops against fungi. Our results revealed that the studied fungicides caused significant increases in cell viability and proliferation, and estrogenic activity was present in all studied compounds depending on their concentrations. Oxidative stress activated uncontrolled cancer cell proliferation by inducing ROS production and by inhibiting antioxidant defense. Our findings verify that the studied fungicides could possibly exhibit endocrine-disrupting properties and exposure should be avoided.

Structures, Antioxidant Properties, and Antimicrobial Properties of Eu(III), Gd(III), and Dy(III) Caffeinates and p-Coumarates.

In this study, we investigated the structures of lanthanide (Eu(III), Dy(III), and Gd(III)) complexes with p-coumaric (p-CAH2) and caffeic (CFAH3) acids using the FTIRKBr, FTIRATR, and Raman spectroscopic methods. The compositions of the solid phase caffeinates and p-coumarates were obtained on the basis of the amounts of hydrogen and carbon determined using an elemental analysis. The degree of hydration and the thermal decomposition of each compound were examined via a thermal analysis of TG, DTG, and DSC. Antioxidant spectroscopic tests were performed using the DPPH (1,1-diphenyl-2-picrylhydrazyl radical), FRAP (ferric reducing antioxidant activity), and ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (diammonium salt radical cation) methods. The antimicrobial activity of each compound against Escherichia coli, Bacillus subtilis, and Candida albicans was investigated. The electrical properties of the liposomes which mimicked the microbial surfaces formed in the electrolyte containing the tested compounds were also investigated. The above biological properties of the obtained complexes were compared with the activities of p-CAH2 and CFAH3. The obtained data suggest that lanthanide complexes are much more thermally stable and have higher antimicrobial and antioxidant properties than the ligands (with the exception of CFAH3 in the case of antioxidant activity tests). The Gd(III) complexes revealed the highest biological activity among the studied lanthanide complexes.

A Preliminary Study of the Effect of Quercetin on Cytotoxicity, Apoptosis, and Stress Responses in Glioblastoma Cell Lines.

A growing body of evidence indicates that dietary polyphenols show protective effects against various cancers. However, little is known yet about their activity in brain tumors. Here we investigated the interaction of dietary flavonoid quercetin (QCT) with the human glioblastoma A172 and LBC3 cell lines. We demonstrated that QCT evoked cytotoxic effect in both tested cell lines. Microscopic observations, Annexin V-FITC/PI staining, and elevated expression and activity of caspase 3/7 showed that QCT caused predominantly apoptotic death of A172 cells. Further analyses confirmed enhanced ROS generation, deregulated expression of SOD1 and SOD2, depletion of ATP levels, and an overexpression of CHOP, suggesting the activation of oxidative stress and ER stress upon QCT exposure. Finally, elevated expression and activity of caspase 9, indicative of a mitochondrial pathway of apoptosis, was detected. Conversely, in LBC3 cells the pro-apoptotic effect was observed only after 24 h incubation with QCT, and a shift towards necrotic cell death was observed after 48 h of treatment. Altogether, our data indicate that exposure to QCT evoked cell death via activation of intrinsic pathway of apoptosis in A172 cells. These findings suggest that QCT is worth further investigation as a potential pharmacological agent in therapy of brain tumors.

Caffeic Acid/Eu(III) Complexes: Solution Equilibrium Studies, Structure Characterization and Biological Activity.

Caffeic acid (CFA) is one of the various natural antioxidants and chemoprotective agents occurring in the human diet. In addition, its metal complexes play fundamental roles in biological systems. Nevertheless, research on the properties of CFA with lanthanide metals is very scarce, and little to no chemical or biological information is known about these particular systems. Most of their properties, including their biological activity and environmental impact, strictly depend on their structure, stability, and solution behaviour. In this work, a multi-analytical-technique approach was used to study these relationships for the Eu(III)/CFA complex. The synthesized metal complex was studied by FT-IR, FT-Raman, elemental, and thermal (TGA) analysis. In order to examine the chemical speciation of the Eu(III)/CFA system in an aqueous solution, several independent potentiometric and spectrophotometric UV-Vis titrations were performed at different M:L (metal:ligand) and pH ratios. The general molecular formula of the synthesized metal complex in the solid state was [Eu(CFA)3(H2O)3]∙2H2O (M:L ratio 1:3), while in aqueous solution the 1:1 species were observed at the optimum pH of 6 ≤ pH ≤ 10, ([Eu(CFA)] and [Eu(CFA)(OH)]-). These results were confirmed by 1H-NMR experiments and electrospray-ionization mass spectrometry (ESI-MS). To evaluate the interaction of Eu(III)/CFA and CFA alone with cell membranes, electrophoretic mobility assays were used. Various antioxidant tests have shown that Eu(III)/CFA exhibits lower antioxidant activity than the free CFA ligand. In addition, the antimicrobial properties of Eu(III)/CFA and CFA against Escherichia coli, Bacillus subtilis and Candida albicans were investigated by evaluation of the minimum inhibitory concentration (MIC). Eu(III)/CFA shows higher antibacterial activity against bacteria compared to CFA, which can be explained by the highly probable increased lipophilicity of the Eu(III) complex.

Effect of Selected Anionic and Cationic Drugs Affecting the Central Nervous System on Electrical Properties of Phosphatidylcholine Liposomes: Experiment and Theory.

Interactions between phospholipid membranes and selected drugs affecting the central nervous system (CNS) were investigated. Small, unilamellar liposomes were used as biomimetic cell membrane models. Microelectrophoretic experiments on two-component liposomes were performed using the electrophoretic light scattering technique (ELS). The effect of both positively (perphenazine, PF) and negatively (barbituric acid, BA) charged drugs on zwitterionic L-α-phosphatidylcholine (PC) membranes were analyzed. Experimental membrane surface charge density (δ) data were determined as a function of pH. Quantitative descriptions of the adsorption equilibria formed due to the binding of solution ions to analyzed two-component membranes are presented. Binding constants of the solution ions with perphenazine and barbituric acid-modified membranes were determined. The results of our research show that both charged drugs change surface charge density values of phosphatidylcholine membranes. It can be concluded that perphenazine and barbituric acid are located near the membrane surface, interacting electrostatically with phosphatidylcholine polar heads.

The Pro-Apoptotic Effect of Silica Nanoparticles Depends on Their Size and Dose, as Well as the Type of Glioblastoma Cells.

Despite intensive investigations, nanoparticle-induced cellular damage is an important problem that has not been fully elucidated yet. Here, we report that silica nanoparticles (SiNPs) demonstrated anticancer influence on glioblastoma cells by the induction of apoptosis or necrosis. These effects are highly cell type-specific, as well as dependent on the size and dose of applied nanoparticles. Exposure of LN-18 and LBC3 cells to different sizes of SiNPs-7 nm, 5-15 nm, or 10-20 nm-at dosages, ranging from 12.5 to 1000 µg/mL, for 24 and 48 h reduced the viability of these cells. Treatment of LN-18 and LBC3 cells with 7 nm or 10-20 nm SiNPs at doses ≥50 µg/mL caused a strong induction of apoptosis, which is connected with an increase of intracellular reactive oxygen species (ROS) production. The 5-15 nm SiNPs exhibited distinct behavior comparing to silica nanoparticles of other studied sizes. In contrast to LBC3, in LN-18 cells exposed to 5-15 nm SiNPs we did not observe any effect on apoptosis. These nanoparticles exerted only strong necrosis, which was connected with a reduction in ROS generation. This suggests that SiNPs can trigger different cellular/molecular effects, depending on the exposure conditions, the size and dose of nanoparticles, and cell type of glioblastoma.

Molecular and Cellular Effects of Chemical Chaperone-TUDCA on ER-Stressed NHAC-kn Human Articular Chondrocytes Cultured in Normoxic and Hypoxic Conditions.

Osteoarthritis (OA) is considered one of the most common arthritic diseases characterized by progressive degradation and abnormal remodeling of articular cartilage. Potential therapeutics for OA aim at restoring proper chondrocyte functioning and inhibiting apoptosis. Previous studies have demonstrated that tauroursodeoxycholic acid (TUDCA) showed anti-inflammatory and anti-apoptotic activity in many models of various diseases, acting mainly via alleviation of endoplasmic reticulum (ER) stress. However, little is known about cytoprotective effects of TUDCA on chondrocyte cells. The present study was designed to evaluate potential effects of TUDCA on interleukin-1ß (IL-1ß) and tunicamycin (TNC)-stimulated NHAC-kn chondrocytes cultured in normoxic and hypoxic conditions. Our results showed that TUDCA alleviated ER stress in TNC-treated chondrocytes, as demonstrated by reduced CHOP expression; however, it was not effective enough to prevent apoptosis of NHAC-kn cells in either normoxia nor hypoxia. However, co-treatment with TUDCA alleviated inflammatory response induced by IL-1ß, as shown by down regulation of Il-1ß, Il-6, Il-8 and Cox2, and increased the expression of antioxidant enzyme Sod2. Additionally, TUDCA enhanced Col IIα expression in IL-1ß- and TNC-stimulated cells, but only in normoxic conditions. Altogether, these results suggest that although TUDCA may display chondoprotective potential in ER-stressed cells, further analyses are still necessary to fully confirm its possible recommendation as potential candidate in OA therapy.

Monitoring of the Surface Charge Density Changes of Human Glioblastoma Cell Membranes upon Cinnamic and Ferulic Acids Treatment.

Cinnamic acid (CA) and ferulic acid (FA) are naturally occurring phenolic acids claimed to exert beneficial effects against disorders related to oxidative stress, including cancer. One such malignancy that still remains a therapeutic challenge mainly due to its heterogeneity and inaccessibility to therapeutic agents is Glioblastoma multiforme (GBM). Here, the influence of CA and FA on the surface charge density of human GBM cell line LN-229 was studied using the electrophoretic light scattering technique. Also, the cytotoxicity of both phenolic acids was determined by metabolic activity-assessing tetrazolium test (MTT) analysis after exposure to CA and FA for 24 h and 48 h. Results showed that both compounds reduced cell viability of LN-229 cells, with more pronounced effect evoked by CA as reflected in IC50 values. Further analyses demonstrated that, after treatment with both phenolic acids, the negative charge of membranes decreased at high pH values and the positive charge of the membranes increased at low pH values compared to the data obtained for untreated cells. Afterward, a four-equilibrium model was applied to estimate the total surface concentrations of both acidic and basic functional groups and their association constants with solution ions in order to calculate theoretical values of membrane surface charge densities. Then, the theoretical data were compared to the experimental data in order to verify the mathematical model. As such, our results indicate that application of electrochemical methods to determine specific drug-membrane interactions might be crucial for predicting their pharmacological activity and bioavailability.

The modulating effect of lipid bilayer/p-coumaric acid interactions on electrical properties of model lipid membranes and human glioblastoma cells.

Biological membranes are one of the most important elements of living cells determining their permeability to the active compounds. Still, little is known about the drug-membrane interactions in terms of pharmacological properties of potential drugs. Chemoprevention based on natural compounds is becoming a strong trend in modern oncopharmacology, and p-coumaric acid (p-CoA) is one such compound with tentative anticancer activity. The microelectrophoretic mobility measurements and electrochemical impedance spectroscopy were applied to study the effects of p-CoA on electrical properties of liposomes, spherical bilayers, and human glioblastoma cell membranes. Our results demonstrated that after treatment with p-CoA, the surface charge of LBC3, LN-229 and LN-18 cell lines was significantly changed in alkaline pH solutions, but not in acidic pH solutions. In contrast, no changes in surface charge density values were registered for phosphatidylethanolamine liposomal membranes and A172 cell membranes after p-CoA treatment. The impedance data showed an increase in values of both the electrical capacitance and the electrical resistance, indicating that p-CoA can be partially inserted into the phosphatidylcholine bilayers. The MTT assay showed cell line-dependent cytotoxic effect of p-CoA. Further molecular analyses revealed the ATP depletion and gene transcription modulation, which might indicate organelle membrane-crossing potential of p-CoA. These results suggest, that changes in surface charge of membranes of living cells not only might be potential predictor of membrane permeability, but also indicate differential composition of cell membranes in various cell lines. Thus further multidirectional analyses are required to implement electrochemical methods as standard testing procedures during drug development process.

The Modulating Effect of p-Coumaric Acid on The Surface Charge Density of Human Glioblastoma Cell Membranes.

p-Coumaric acid (p-CoA), a phenolic acid belonging to the hydroxycinnamic acids family, is a compound with tentative anticancer potential. Microelectrophoretic mobility measurements conducted at various pH values of electrolyte solution were applied to study p-CoA effects on electrical properties of human glioblastoma cell membranes. The obtained results demonstrated that after the p-CoA treatment, the surface charge density of cancer cells changed in alkaline pH solutions, while no noticeable changes were observed in cell membranes incubated with p-CoA compared to control at acidic pH solutions. A four-equilibrium model was used to describe the phenomena occurring on the cell membrane surface. The total surface concentrations of both acidic and basic functional groups and their association constants with solution ions were calculated and used to define theoretical curves of membrane surface charge density versus pH. The resulting theoretical curves and the experimental data were compared to verify the reliability and validity of the adopted model. The deviation of both kinds of data obtained at a higher pH may be caused by disregarding interactions between the functional groups of cancer cells. Processes occurring in the cell membranes after their incubation with p-CoA can lead to disorders of existing equilibria, which result in changes in values of the parameters describing these equilibria.

Theoretical Considerations and the Microelectrophoresis Experiment on the Influence of Selected Chaotropic Anions on Phosphatidylcholine Membrane Surface Charge Density.

Influence of sodium salts of selected chaotropic anions from the Hofmeister series (NaCl, NaBr, NaNO3, NaI) on the surface charge density of phosphatidylcholine membranes was studied. Small unilamellar lipid vesicles were used as a model system in the investigations. The theoretical and experimental approach to the interactions between inorganic anions and phosphatidylcholine membranes is presented. Experimental membrane surface charge densities data were determined as a function of pH of the aqueous electrolytes using microelectrophoresis method. The quantitative description of the interactions between zwitterionic phosphatidylcholine membrane and monovalent anions is presented. The equilibria constants of the binding of solution ions onto phospholipid surface were calculated. Knowledge of these parameters was essential to determine the theoretical membrane surface charge density values. The theoretical data were compared to the experimental ones in order to verify the mathematical model. Both approaches indicate that the anion-phosphatidylcholine membrane interaction increases with the size of the anion. The adsorption of chaotropic anions to membranes was found to follow the Hofmeister series I- > NO3- > Br- > Cl-.

The effect of divalent ions on L-α-phosphatidylcholine from egg yolk monolayers at the air/water interface.

The Langmuir monolayers of L-α-phosphatidylcholine from egg yolk were studied by Wilhelmy method. The surface pressure versus molecular area isotherm of lipid on pure water and different subphase (with a presence of divalent ions: Sr2+, Cd2+, Ba2+, Pb2+) was obtained. The limiting area of the isotherms depends on the presence of subphase ions. The addition of divalent ions to the subphase stabilized the monolayers and increased the limiting areas of the monolayer. During the compression in monolayer complexes of 1:1 and 2:1 stoichiometry between L-α-phosphatidylcholine from egg yolk and divalent ions are formed. We used the equilibrium theory to describe the behavior of monolayer components at the air/water interface. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants and area occupied by one molecule of LMe2+ or L2Me2+ complexes, and complex formation energy (Gibbs free energy) values. These mathematically derived and experimentally confirmed values are of great importance for the interpretation of phenomena occurring in lipid monolayers and bilayers.

Equilibria Between Cell Membranes and Electrolyte Solution: Effect of Fatal Accidental Hypothermia.

Equilibria between the membranes of erythrocytes as well as thrombocytes and solution ions in fatal accidental hypothermia were analyzed using a theoretical four-equilibria model. The model was developed to determinate parameters characterizing cell membrane-surrounding ion interactions: the total surface concentrations of both acidic and basic groups C A, C B, and association constants K AH, K BOH. Knowledge of these parameters was necessary to calculate the theoretical values of surface charge density. The model was validated by curve-fitting the experimental data points to simulated data generated by the model. The experimental and theoretical surface charge density values agree at pH 2-8, at higher pH the deviation was observed.

Changes in Surface Charge Density of Blood Cells in Fatal Accidental Hypothermia.

The objective of this research was to evaluate postmortem changes concerning electric charge of human erythrocytes and thrombocytes in fatal accidental hypothermia. The surface charge density values were determined on the basis of the electrophoretic mobility measurements of the cells conducted at various pH values of electrolyte solution. The surface charge of erythrocyte membranes after fatal accidental hypothermia increased compared to the control group within whole range of experimental pH values. Moreover, a slight shift of the isoelectric point of erythrocyte membranes towards high pH values was observed. The surface charge of thrombocyte membranes in fatal accidental hypothermia decreased at low pH compared to the control group. However, at pH range 4-9, the values increased compared to the control group. The isoelectric point of thrombocyte membranes after fatal accidental hypothermia was slightly shifted towards low pH values compared to the control group. The observed changes are probably connected with the partial destruction and functional changes of the blood cell structure.

The effect of pH on the electrical capacitance of phosphatidylcholine-phosphatidylserine system in bilayer lipid membrane.

This paper reports measurements on the pH dependence of the electrical capacitance of lipid membranes formed by 1:1 phosphatidylcholine-phosphatidylserine mixtures. A theoretical model was developed to describe this dependence, in which the contributions of functional groups (as the active centers of adsorption of the hydrogen and hydroxide ions) to the overall membrane capacitance were assumed to be additive. The proposed model was verified experimentally using electrochemical impedance spectroscopy. The theoretical predictions agreed with the experimental results over the measured pH range. A minimum corresponding to the isoelectric point appeared in both the theoretical equation and the experimental data.

Cellular stress responses as modulators of drug cytotoxicity in pharmacotherapy of glioblastoma.

Despite the extensive efforts to find effective therapeutic strategies, glioblastoma (GBM) remains a therapeutic challenge with dismal prognosis of survival. Over the last decade the role of stress responses in GBM therapy has gained a great deal of attention, since depending on the duration and intensity of these cellular programs they can be cytoprotective or promote cancer cell death. As such, initiation of the UPR, autophagy or oxidative stress may either impede or facilitate drug-mediated cell killing. In this review, we summarize the mechanisms that regulate ER stress, autophagy, and oxidative stress during GBM development and progression to later discuss the involvement of these stress pathways in the response to different treatments. We also discuss how a precise understanding of the molecular mechanisms regulating stress responses evoked by different pharmacological agents could decisively contribute to the design of novel and more effective combinational treatments against brain malignancies.