Search Results (10)

Background/Objectives: Doxorubicin-induced cardiotoxicity (DIC) is driven by oxidative stress and impaired oxidative phosphorylation (OXPHOS). Antioxidant properties of carotenoids in vivo depend on extraction, while their direct role in mitigating DIC remains undetermined. This study evaluated the cardioprotective potential of natural deep eutectic solvents (NADES)-derived pumpkin carotenoid extract in a rat model of DIC. Methods: NADES-derived pumpkin pulp extract was characterized by spectrophotometry and HPLC-DAD. Wistar rats (n = 30) were assigned to six groups: C (control), N (NADES, 1 mL, p.o.), P (extract, 900 µg/kg body weight/day of total carotenoids, p.o.), D (doxorubicin, four i.p. doses, 2 mg/kg), ND (NADES/doxorubicin) and PD (900 µg/kg body weight/day of total carotenoids/doxorubicin). The activity of antioxidant enzymes and mitochondrial respiration in saponin-permeabilized left ventricular fibers using a Clark-type oxygen electrode) was measured. Results: Compared to control, cardiac antioxidant enzyme activities, mitochondrial respiration in complex I–linked respiration, ADP-supported OXPHOS, maximal respiratory capacity, and complex II and IV-linked respiration were significantly impaired by doxorubicin and unaltered by NADES. Co-administration of the extract significantly improved antioxidant enzyme activities (GSH-Px: D group 45 vs. PD group 95 nmol/mg proteins; GR: D group 95 vs. PD group 145 nmol/mg proteins; GST: D group 15 vs. PD group 22 nmol/mg proteins; SOD: D group 9 vs. PD group 17 U/mg proteins) and attenuated mitochondrial respiratory dysfunction compared with doxorubicin-treated group, indicating partial preservation of electron transport system capacity. Conclusions: Despite limitations of the study (single sex, single dose), results suggest NADES-based carotenoid extracts have cardioprotective properties in DIC by enhancing antioxidant defenses and supporting mitochondrial respiration. Full article

Mitochondrial reactive oxygen species (ROS) play a central role in cardiac ischemia/reperfusion injury, heart failure, and arrhythmogenesis, while also serving essential signaling functions under physiological conditions. Among the eleven identified mitochondrial ROS-producing sites, complexes I and III are considered the major contributors, particularly under conditions of impaired electron flow. However, much of the existing knowledge comes from rodent models or cultured cells and is often assumed to apply to humans. Here, ROS production from complexes I and III was measured directly in human myocardial and skeletal muscle biopsies and compared with corresponding rat tissues under identical experimental conditions. Hydrogen peroxide generation was quantified using Amplex UltraRed, with simultaneous monitoring of mitochondrial respiration using a Clark-type oxygen electrode. Across all examined mechanisms—reverse and forward electron transport at complex I and the ubiquinol oxidation site of complex III, rat tissues produced more ROS than human tissues, consistent with their higher respiratory rates. However, the dominant ROS-producing sites differed: in rats, complex III was the primary source, whereas in human tissues the highest ROS production occurred during reverse electron transport at complex I. When normalized to respiration, human tissues showed relatively greater ROS generation at complex I but markedly lower production at complex III. These direct measurements of mitochondrial ROS production in human myocardium provide new insight into cardiac redox physiology and may explain the limited clinical translation of cardioprotective strategies targeting mitochondrial ROS production, such as interventions aimed at modulating reperfusion injury or preconditioning. Full article

ROS (i.e., reactive oxygen species) scavenging is a key function of various Mn-based enzymes, including superoxide dismutases (SODs) and catalases, which are actively linked to oxidative stress-related diseases. In this study, we synthesized and characterized two novel Mn(III)-based synzymes (i.e., synthetic enzymes), designated C1 ([MnL1Cl(H₂O)]Cl·3H₂O) and C2 ([MnL2Cl₂]·2H₂O), which differ in the presence of a bridging aliphatic or aromatic group in the chelator. Using a range of analytical techniques, we found that the aromatic C2 bridge significantly influences the Mn(III) center’s cis-β configuration, unlike C1, which adopts a trans configuration. We then thoroughly evaluated the oxidation-reduction properties of C1 and C2, including their redox potentials (by cyclic voltammetry) and capacity to consume various ROS species (using DPPH, hydroxyl radical, hydrogen peroxide, and superoxide UV–visible spectrophotometric assays). The specific kinetics of the H₂O₂ dismutation process, as measured by a Clark-type electrode and time-resolved ESI-MS, revealed that both synzymes possess catalytic activity. Toxicological experiments using the Galleria mellonella larval model demonstrated the compounds’ innocuous nature towards higher eukaryotic organisms, while cytotoxicity assays confirmed their selective efficacy against lung cancer cells. Additional cytological assays, such as the thiobarbituric acid reactive substances assay and caspase-3 activity and p53 expression analysis, reported that C1 and C2 induce cytotoxicity against cancer cells via apoptosis rather than necrosis and behave very differently towards redox substances and ROS-regulating enzymes in vivo. These findings suggest that the structural differences between C1 and C2 lead to distinct redox properties and biological activities, highlighting the potential of these novel Mn(III)-based synzymes as therapeutic agents for the treatment of oxidative stress-related diseases, particularly lung cancer. Further studies are warranted to elucidate the underlying mechanisms of action and explore their clinical applications. Full article

Express assessment of the biochemical activity of microorganisms is important in both applied and fundamental research. A laboratory model of a microbial electrochemical sensor formed on the basis of the culture of interest is a device that provides rapidly information about the culture and is cost effective, simple to fabricate and easy to use. This paper describes the application of laboratory models of microbial sensors in which the Clark-type oxygen electrode was used as a transducer. The formation of the models of the reactor microbial sensor (RMS) and the membrane microbial sensor (MMS) and the formation of the response of biosensors are compared. RMS and MMS are based on intact or immobilized microbial cells, respectively. For MMS, the response of biosensor is caused both by the process of transport of substrate into microbial cells and by the process of the initial metabolism of substrate; and only initial substrate metabolism triggers the RMS response. The details of the application of biosensors for the study of allosteric enzymes and inhibition by substrate are discussed. For inducible enzymes, special attention is paid to the induction of microbial cells. This article addresses current problems related to implementation of the biosensor approach and discusses the ways how to overcome these problems. Full article

An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O₂ reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O₂ scavenging membrane. The TMAO sensor operates at a potential of −0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 µM and 15 mM, with a sensitivity of 2.75 ± 1.7 µA/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 µM TMAO. Full article

Oxygen levels in plant tissues may vary, depending on metabolism, diffusion barriers, and environmental availability. Current techniques to assess the oxic status of plant cells rely primarily on invasive microoptodes or Clark-type electrodes, which are not optimally suited for experiments that require high spatial and temporal resolution. In this case, a genetically encoded oxygen biosensor is required instead. This article reports the design, test, and optimization of a hypoxia-signaling reporter, based on five-time repeated hypoxia-responsive promoter elements (HRPE) driving the expression of different reporter proteins. Specifically, this study aimed to improve its performance as a reporter of hypoxic conditions by testing the effect of different untranslated regions (UTRs) at the 5′ end of the reporter coding sequence. Next, we characterized an optimized version of the HRPE promoter (HRPE-Ω) in terms of hypoxia sensitivity and time responsiveness. We also observed that severe oxygen deficiency counteracted the reporter activity due to inhibition of GFP maturation, which requires molecular oxygen. To overcome this limitation, we therefore employed an oxygen-independent UnaG fluorescent protein-coupled to an O₂-dependent mCherry fluorophore under the control of the optimized HRPE-Ω promoter. Remarkably, this sensor, provided a different mCherry/UnaG ratiometric output depending on the externally imposed oxygen concentration, providing a solution to distinguish between different degrees of tissue hypoxia. Moreover, a ubiquitously expressed UnaG-mCherry fusion could be used to image oxygen concentrations directly, albeit at a narrow range. The luminescent and fluorescent hypoxia-reporters described here can readily be used to conduct studies that involve anaerobiosis in plants. Full article

This paper considers the effect of multiwalled carbon nanotubes (MWCNTs) on the parameters of Gluconobacter oxydans microbial biosensors. MWCNTs were shown not to affect the structural integrity of microbial cells and their respiratory activity. The positive results from using MWCNTs were due to a decrease in the impedance of the electrode. The total impedance of the system decreased significantly, from 9000 kOhm (G. oxydans/chitosan composite) to 600 kOhm (G. oxydans/MWCNTs/chitosan). Modification of the amperometric biosensor with nanotubes led to an increase in the maximal signal from 65 to 869 nA for glucose and from 181 to 1048 nA for ethanol. The biosensor sensitivity also increased 4- and 5-fold, respectively, for each of the substrates. However, the addition of MWCNTs reduced the affinity of respiratory chain enzymes to their substrates (both sugars and alcohols). Moreover, the minimal detection limits were not reduced despite a sensitivity increase. The use of MWCNTs thus improved only some microbial biosensor parameters. Full article

The electrochemical reactor microbial sensor with the Clark oxygen electrode as the transducer was used for investigation of the competition between 3-chlorobenzoate (3-CBA) and its analogues, 2- and 4-chlorobenzoate (2-CBA and 4-CBA), for 3-chlorobenzoate-1,2-dioxygenase (3-CBDO) of Rhodococcus opacus 1CP cells. The change in respiration of freshly harvested R. opacus 1CP cells in response to 3-CBA served as an indicator of 3-CBDO activity. The results obtained confirmed inducibility of 3-CBDO. Sigmoidal dependency of the rate of the enzymatic reaction on the concentration of 3-CBA was obtained and positive kinetic cooperativity by a substrate was shown for 3-CBDO. The Hill concentration constant, S_0.5, and the constant of catalytic activity, V_max, were determined. Inhibition of the rate of enzymatic reaction by excess substrate, 3-CBA, was observed. Associative (competitive inhibition according to classic classification) and transient types of the 3-CBA-1,2-DO inhibition by 2-CBA and 4-CBA, respectively, were found. The kinetic parameters such as S_0.5ⁱ and V_maxⁱ were also estimated for 2-CBA and 4-CBA. The disappearance of the S-shape of the curve of the V versus S dependence for 3-CBDO in the presence of 4-CBA was assumed to imply that 4-chlorobenzoate had no capability to be catalytically transformed by 3-chlorobenzoate-1,2-dioxygenase of Rhodococcus opacus 1CP cells. Full article

We developed different types of glass cell-culture chips (GC³s) for culturing cells for microscopic observation in open media-containing troughs or in microfluidic structures. Platinum sensor and manipulation structures were used to monitor physiological parameters and to allocate and permeabilize cells. Electro-thermal micro pumps distributed chemical compounds in the microfluidic systems. The integrated temperature sensors showed a linear, Pt1000-like behavior. Cell adhesion and proliferation were monitored using interdigitated electrode structures (IDESs). The cell-doubling times of primary murine embryonic neuronal cells (PNCs) were determined based on the IDES capacitance-peak shifts. The electrical activity of PNC networks was detected using multi-electrode arrays (MEAs). During seeding, the cells were dielectrophoretically allocated to individual MEAs to improve network structures. MEA pads with diameters of 15, 20, 25, and 35 µm were tested. After 3 weeks, the magnitudes of the determined action potentials were highest for pads of 25 µm in diameter and did not differ when the inter-pad distances were 100 or 170 µm. Using 25-µm diameter circular oxygen electrodes, the signal currents in the cell-culture media were found to range from approximately −0.08 nA (0% O₂) to −2.35 nA (21% O₂). It was observed that 60-nm thick silicon nitride-sensor layers were stable potentiometric pH sensors under cell-culture conditions for periods of days. Their sensitivity between pH 5 and 9 was as high as 45 mV per pH step. We concluded that sensorized GC³s are potential animal replacement systems for purposes such as toxicity pre-screening. For example, the effect of mefloquine, a medication used to treat malaria, on the electrical activity of neuronal cells was determined in this study using a GC³ system. Full article

Oxygen (O₂) concentration inside the substrate is an important measurement for silage-research and-practical management. In the laboratory gas chromatography is commonly employed for O₂ measurement. Among sensor-based techniques, accurate and reliable in situ measurement is rare because of high levels of carbon dioxide (CO₂) generated by the introduction of O₂ in the silage. The presented study focused on assessing three types of commercial O₂ sensors, including Clark oxygen electrodes (COE), galvanic oxygen cell (GOC) sensors and the Dräger chip measurement system (DCMS). Laboratory cross calibration of O₂ versus CO₂ (each 0–15 vol.%) was made for the COE and the GOC sensors. All calibration results verified that O₂ measurements for both sensors were insensitive to CO₂. For the O₂ in situ measurement in silage, all O₂ sensors were first tested in two sealed barrels (diameter 35.7 cm; height: 60 cm) to monitor the O₂ depletion with respect to the ensiling process (Test-A). The second test (Test-B) simulated the silage unloading process by recording the O₂ penetration dynamics in three additional barrels, two covered by dry ice (0.6 kg or 1.2 kg of each) on the top surface and one without. Based on a general comparison of the experimental data, we conclude that each of these in situ sensor monitoring techniques for O₂ concentration in silage exhibit individual advantages and limitations. Full article