Bridging the buzz: In vivo EPR imaging unlocking the secrets of honey bee health.

Honey bees play a pivotal role in shaping ecosystems and sustaining human health as both pollinators and producers of health-promoting products. However, honey bee colony mortality is on the rise globally, driven by various factors, including parasites, pesticides, habitat loss, poor nutrition, and climate change. This has far-reaching consequences for the environment, economy, and human welfare. While efforts to address these issues are underway, the current progress in electron paramagnetic resonance (EPR) instrumentation affords using the immense potential of this magnetic resonance technique to study small samples such as honey bees. This paper presents the pioneering 2D in vivo EPR imaging experiment on a honey bee, revealing the ongoing redox-status of bees' intestines. This way, by monitoring the spatio-temporal changes of the redox-active spin-probes' EPR signal, it is possible to gain access to valuable information on the course of ongoing bees' pathologies and the prospect of following-up on the efficiency of applied therapies. Employing a selection of diverse spin-probes could further reveal pH levels and oxygen concentrations in bee tissues, allowing a noninvasive assessment of bee physiology. This approach offers promising strategies for safeguarding pollinators and understanding their biology, fostering their well-being and ecological harmony.

Solanum dulcamara L. Berries: A Convenient Model System to Study Redox Processes in Relation to Fruit Ripening.

The present study provides, for the first time, a physicochemical and biochemical characterization of the redox processes associated with the ripening of Solanum dulcamara L. (bittersweet) berries. Electron Paramagnetic Resonance Spectroscopy (EPRS) and Imaging (EPRI) measurements of reactive oxygen species (ROS) were performed in parallel with the tissue-specific metabolic profiling of major antioxidants and assessment of antioxidant enzymes activity. Fruit transition from the mature green (MG) to ripe red (RR) stage involved changes in the qualitative and quantitative content of antioxidants and the associated cellular oxidation and peroxidation processes. The skin of bittersweet berries, which was the major source of antioxidants, exhibited the highest antioxidant potential against DPPH radicals and nitroxyl spin probe 3CP. The efficient enzymatic antioxidant system played a critical protective role against the deleterious effects of progressive oxidative stress during ripening. Here, we present the EPRI methodology to assess the redox status of fruits and to discriminate between the redox states of different tissues. Interestingly, the intracellular reoxidation of cell-permeable nitroxide probe 3CP was observed for the first time in fruits or any other plant tissue, and its intensity is herein proposed as a reliable indicator of oxidative stress during ripening. The described noninvasive EPRI technique has the potential to have broader application in the study of redox processes associated with the development, senescence, and postharvest storage of fruits, as well as other circumstances in which oxidative stress is implicated.

Rich in Phenolics-Strong Antioxidant Fruit? Comparative Study of 25 Strawberry Cultivars.

Phenolic compounds of 25 newly introduced strawberry cultivars were profiled using spectrophotometry, electron paramagnetic resonance (EPR) spectroscopy, and high-performance liquid chromatography-mass spectrometry. Total phenolic and anthocyanin content (TPC and TACY, respectively), as well as vitamin C, and concentrations of individual phenolic compounds in fruits were evaluated to identify the most promising cultivars according to their phenolic profile. The highest values of TPC, TACY, and vitamin C were recorded in 'Premy' (1.53 mg eq GA g-1 FW), 'Sandra' (30.60 mg eq Pg-3-g 100 g-1 FW), and 'Laetitia' (56.32 mg 100 g-1 FW), respectively. The DPPH and •OH radicals scavenging activity of fruit methanolic extracts was estimated using EPR spectroscopy. All cultivars are almost uniformly effective in the scavenging of •OH radical, while 'Tea', 'Premy', and 'Joly' were marked as highly potent cultivars (over 70%) in terms of DPPH-antiradical activity. Specific peroxidase activities were the highest in 'Garda', 'Federica', and 'Rumba' (0.11, 0.08, and 0.06 U mg-1 prot, respectively). 'Laetitia', 'Joly', 'Arianna', 'Tea', and 'Mila' cultivars were distinguished from others as the richest concerning almost all flavonoids and phenolic acids, including some other parameters of bioactivity. These cultivars could be recommended to consumers as functional fruit foods.

Lignin and organic free radicals in maize (Zea mays L.) seeds in response to aflatoxin B1 contamination: an optical and EPR spectroscopic study.

BACKGROUND: Aflatoxin B1 (AFB1 ) is the most dangerous of the mycotoxins that contaminate cereal seeds naturally. A stress lignin formation is linked with the accumulation of reactive oxygen species causing a change in the redox status and formation of stable organic radicals, constituting the first layer of defense. The relationship between AFB1 and changes in lignin organic free radicals in seeds is not known, nor is the part of the seed that is more targeted. Using optical and electron paramagnetic resonance spectroscopy, we investigated AFB1 -induced changes in lignin and organic free radicals in seeds, and whether the inner and outer seed fractions differ in response to increasing AFB1 . RESULTS: Different changes in the content of lignin and free radicals with increasing AFB1 concentrations were observed in the two seed fractions. There was a significant positive linear correlation (R = 0.9923, P = 0.00005) between lignin content and AFB1 concentration in the outer fraction, and no correlation between the lignin content and the AFB1 concentration in the inner fraction. We found a positive correlation between the area of the green spectral emission component (C4) and the AFB1 concentration in the outer fraction. CONCLUSIONS: To the best of our knowledge, the results showed, for the first time, that maize seed fractions respond differently to aflatoxin with regard to their lignin and organic free radical content. Lignin content and (C4) area may be reliable indicators for the screening of lignin changes against AFB1 content in the seeds, and thus for seed protection capacity. © 2021 Society of Chemical Industry.

Graphene quantum dot antioxidant and proautophagic actions protect SH-SY5Y neuroblastoma cells from oxidative stress-mediated apoptotic death.

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical (•OH), superoxide anion (O2•-), and lipid peroxidation. Nonselective antioxidants, •OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing •OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy-limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proautophagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NH4Cl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both •OH/NO scavenging and induction of cytoprotective autophagy.

Controlled killing of human cervical cancer cells by combined action of blue light and C-doped TiO2 nanoparticles.

In this study, C-doped TiO2 nanoparticles (C-TiO2) were prepared and tested as a photosensitizer for visible-light-driven photodynamic therapy against cervical cancer cells (HeLa). X-ray diffraction and Transmission Electron Microscopy confirmed the anatase form of nanoparticles, spherical shape, and size distribution from 5 to 15 nm. Ultraviolet-visible light spectroscopy showed that C doping of TiO2 enhances the optical absorption in the visible light range caused by a bandgap narrowing. The photo-cytotoxic activity of C-TiO2 was investigated in vitro against HeLa cells. The lack of dark cytotoxicity indicates good biocompatibility of C-TiO2. In contrast, a combination with blue light significantly reduced the survival of HeLa cells: illumination only decreased cell viability by 30% (15 min of illumination, 120 µW power), and 60% when HeLa cells were preincubated with C-TiO2. We have also confirmed blue light-induced C-TiO2-catalyzed generation of reactive oxygen species in vitro and intracellularly. Oxidative stress triggered by C-TiO2/blue light was the leading cause of HeLa cell death. Fluorescent labeling of treated HeLa cells showed distinct morphological changes after the C-TiO2/blue light treatment. Unlike blue light illumination, which caused the appearance of large necrotic cells with deformed nuclei, cytoplasm swelling, and membrane blebbing, a combination of C-TiO2/blue light leads to controlled cell death, thus providing a better outcome of local anticancer therapy.

Spatial distribution of apoplastic antioxidative constituents in maize root.

Apoplastic antioxidative constituents (enzymes, primary and secondary metabolites, ROS) from different root zones of hydroponically grown maize (Zea mays L.) were investigated using a noninvasive isolation procedure: filter strip method. Filter strips were placed at specific positions on the root surface: apical zone (tip) and basal zone (base) to absorb apoplastic fluid. Three major classes of low-weight metabolites (organic acids, sugars, and phenolics) have been identified by HPLC-ECD. The longitudinal distribution of sugars and organic acids had the same pattern: higher concentration in the tip than the base, while it was vice versa for phenolics. The specific activities of guaiacol peroxidase, superoxide dismutase, and ascorbate peroxidase were higher in the apoplastic fluid from the root base than the tip, and their different isoforms were separated by isoelectric focusing. Electron paramagnetic resonance (EPR) spectroscopy coupled with the spin-trapping method using DEPMPO showed a persistent generation of hydroxyl radical in the root tip. In vivo EPR imaging of the whole maize root with membrane-permeable and impermeable aminoxyl spin-probes, enabling real-time detection of ROS formation within and outside the membranes, demonstrated ROS accumulation on the root surface, while endodermis and central cylinder were ROS free. For the first time in plant research, 2D EPR images enabled the direct demonstration of site-specific free radical production along the root. Highly sensitive analytical techniques combined with the filter strips, as a non-invasive tool, have increased our knowledge of metabolic processes occurring in the apoplast and their spatial-temporal changes in small regions of the intact root tissue.

In Vivo/Ex Vivo EPR Investigation of the Brain Redox Status and Blood-Brain Barrier Integrity in the 5xFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by cognitive decline and total brain atrophy. Despite the substantial scientific effort, the pathological mechanisms underlying neurodegeneration in AD are currently unknown. In most studies, amyloid ß peptide has been considered the key pathological change in AD. However, numerous Aß-targeting treatments have failed in clinical trials. This implies the need to shift the research focus from Aß to other pathological features of the disease. OBJECTIVE: The aim of this study was to examine the interplay between mitochondrial dysfunction, oxidative stress and blood-brain barrier (BBB) disruption in AD pathology, using a novel approach that involves the application of electron paramagnetic resonance (EPR) spectroscopy. METHODS: In vivo and ex vivo EPR spectroscopy using two spin probes (aminoxyl radicals) exhibiting different cell-membrane and BBB permeability were employed to assess BBB integrity and brain tissue redox status in the 5xFAD mouse model of AD. In vivo spin probe reduction decay was analyzed using a two-compartment pharmacokinetic model. Furthermore, 15 K EPR spectroscopy was employed to investigate the brain metal content. RESULTS: This study has revealed an altered brain redox state, BBB breakdown, as well as ROS-mediated damage to mitochondrial iron-sulfur clusters, and up-regulation of MnSOD in the 5xFAD model. CONCLUSION: The EPR spin probes were shown to be excellent in vivo reporters of the 5xFAD neuronal tissue redox state, as well as the BBB integrity, indicating the importance of in vivo EPR spectroscopy application in preclinical studies of neurodegenerative diseases.

Bioevaluation of glucose-modified liposomes as a potential drug delivery system for cancer treatment using 177-Lu radiotracking.

Liposomes are promising drug's delivery systems due to decreased toxicity of the liposome-encapsulated drug, but wider clinical application requires their more efficient tumor targeting with uptake, controlled drug release and higher shelf life. The unique metabolic characteristics of cancer cells based on higher demand for energy and therefore increased glucose utilization were exploited in the design of glucose modified liposomes (GML) with the aim to provide increased tumor targeting via glucose transporters and increased ability of drug delivery into tumor cells. Tumor accumulating potential of GML and non-glucose liposomes (NGL) were investigated on CT26 and LS174T tumor-bearing mice by simple and reliable radiotracer method using 177Lu as radioactive marker. Both liposomes, GML and NGL were radiolabeled in high radiolabeling yield, showing high in vitro stability in biological media, as the main prerequisite for the biodistribution studies. Tumors displayed significantly better accumulation of 177Lu-GML with the maximum uptake 6 h post-injection (5.8 ± 0.2%/g in LS174T tumor and 5.1 ± 0.5%/g in CT26 tumor), compared to negligible uptake of 177Lu-NGL (0.6 ± 0.1%/g in LS174T tumor and 0.9 ± 0.2%/g in CT26 tumor). Results of comparative biodistribution studies of 177Lu-NGL and 177Lu-GML indicate that increased accumulation of GML is enabled by glucose transporters and subsequent endocytosis, resulting in their prolonged retention in tumor tissues (up to 72 h). Direct radiolabeling of liposomes with 177Lu may be used not only for biodistribution studies using radiotracking, but also for cancer treatment.

Electrophilic characteristics and aqueous behavior of fatty acid nitroalkenes.

Fatty acid nitroalkenes (NO2-FA) are endogenously-generated products of the reaction of metabolic and inflammatory-derived nitrogen dioxide (.NO2) with unsaturated fatty acids. These species mediate signaling actions and induce adaptive responses in preclinical models of inflammatory and metabolic diseases. The nitroalkene substituent possesses an electrophilic nature, resulting in rapid and reversible reactions with biological nucleophiles such as cysteine, thus supporting post-translational modifications (PTM) of proteins having susceptible nucleophilic centers. These reactions contribute to enzyme regulation, modulation of inflammation and cell proliferation and the regulation of gene expression responses. Herein, focus is placed on the reduction-oxidation (redox) characteristics and stability of specific NO2-FA regioisomers having biological and clinical relevance; nitro-oleic acid (NO2-OA), bis-allylic nitro-linoleic acid (NO2-LA) and the conjugated diene-containing nitro-conjugated linoleic acid (NO2-cLA). Cyclic and alternating-current voltammetry and chronopotentiometry were used to the study of reduction potentials of these NO2-FA. R-NO2 reduction was observed around -0.8 V (vs. Ag/AgCl/3 M KCl) and is related to relative NO2-FA electrophilicity. This reduction process could be utilized for the evaluation of NO2-FA stability in aqueous milieu, shown herein to be pH dependent. In addition, electron paramagnetic resonance (EPR) spectroscopy was used to define the stability of the nitroalkene moiety under aqueous conditions, specifically under conditions where nitric oxide (.NO) release could be detected. The experimental data were supported by density functional theory calculations using 6-311++G (d,p) basis set and B3LYP functional. Based on experimental and computational approaches, the relative electrophilicities of these NO2-FA are NO2-cLA >> NO2-LA > NO2-OA. Micellarization and vesiculation largely define these biophysical characteristics in aqueous, nucleophile-free conditions. At concentrations below the critical micellar concentration (CMC), monomeric NO2-FA predominate, while at greater concentrations a micellar phase consisting of self-assembled lipid structures predominates. The CMC, determined by dynamic light scattering in 0.1 M phosphate buffer (pH 7.4) at 25 °C, was 6.9 (NO2-LA) 10.6 (NO2-OA) and 42.3 µM (NO2-cLA), respectively. In aggregate, this study provides new insight into the biophysical properties of NO2-FA that are important for better understanding the cell signaling and pharmacological potential of this class of mediators.

Bifunctional catalytic activity of Zn1-xFexO toward the OER/ORR: seeking an optimal stoichiometry.

Eco-friendly and rapid microwave processing of a precipitate was used to produce Fe-doped zinc oxide (Zn1-xFexO, x = 0, 0.05, 0.1, 0.15 and 0.20; ZnO:Fe) nanoparticles, which were tested as catalysts toward the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) in a moderately alkaline solution. The phase composition, crystal structure, morphology, textural properties, surface chemistry, optical properties and band structure were examined to comprehend the influence of Zn2+ partial substitution with Fe3+ on the catalytic activity of ZnO:Fe. Linear sweep voltammetry showed an improved catalytic activity of ZnO:5Fe toward the ORR, compared to pure ZnO, while with increased amounts of the Fe-dopant the activity decreased. The improvement was suggested by a more positive onset potential (0.394 V vs. RHE), current density (0.231 mA cm-2 at 0.150 V vs. RHE), and faster kinetics (Tafel slope, b = 248 mV dec-1), and it may be due to the synergistic effect of (1) a sufficient amount of surface oxygen vacancies, and (2) a certain amount of plate-like particles composed of crystallites with well developed (0001) and (0001[combining macron]) facets. Quite the contrary, the OER study showed that the introduction of Fe3+ ions into the ZnO crystal structure resulted in enhanced catalytic activity of all ZnO:Fe samples, compared to pure ZnO, probably due to the modified binding energy and an optimized band structure. With the maximal current density of 1.066 mA cm-2 at 2.216 V vs. RHE, an onset potential of 1.856 V vs. RHE, and the smallest potential difference between the OER and ORR (ΔE = 1.58 V), ZnO:10Fe may be considered a promising bifunctional catalyst toward the OER/ORR in moderately alkaline solution. This study demonstrates that the electrocatalytic activity of ZnO:Fe strongly depends on the defect chemistry and consequently the band structure. Along with providing fundamental insight into the electrocatalytic activity of ZnO:Fe, the study also indicates an optimal stoichiometry for enhanced bifunctional activity toward the OER/ORR, compared to pure ZnO.

Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods.

Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O2•-) formation rates (measured by dihydroethidium-based HPLC assay), pointing to a self-amplification mechanism of oxidative stress. Based on our ex vivo data, the CoulArray quantification method for 3-NT seems to have some advantages regarding sensitivity and selectivity. Establishing a reliable automated HPLC assay for the routine quantification of 3-NT in biological samples of cell culture, of animal and human origin seems to be more sophisticated than expected.

Liposomal integration method for assessing antioxidative activity of water insoluble compounds towards biologically relevant free radicals: example of avarol.

The liposomal integration method, in conjunction with electron paramagnetic resonance (EPR) spectroscopy, has been presented for the investigation of antioxidant activity of selected water-insoluble compound towards biologically relevant free radicals. This method was applied to avarol, a sesquiterpenoid hydroquinone isolated from the marine sponge Dysidea avara. The antioxidant activity of water-insoluble avarol towards •OH, O2•- and NO• radicals was attained by its incorporation into the DPPC liposomes bilayer, and towards ascorbyl radicals in the organic solvent. Avarol's activity towards •OH, O2•-, NO• and ascorbyl radicals was 86.2%, 50.9%, 23.6% and 61.8%, respectively, showing its significant radical scavenging potential.

Nitrate inhibits primary root growth by reducing accumulation of reactive oxygen species in the root tip in Medicago truncatula.

In Medicago truncatula, nitrate, acting as a signal perceived by NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY 6.8 (MtNPF6.8), inhibits primary root growth through a reduction of root cell elongation. Since reactive oxygen species (ROS) produced and converted in root tip (O2•- → H2O2 → •OH) have been reported to control cell elongation, the impact of nitrate on the distribution of these ROS in the primary root of M. truncatula was analyzed. We found that nitrate reduced the content of O2•-, H2O2 and •OH in the root tip of three wild type genotypes sensitive to nitrate (R108, DZA, A17), inhibition of root growth and O2•- accumulation being highly correlated. Nitrate also modified the capacity of R108 root tip to produce or remove ROS. The ROS content decrease observed in R108 in response to nitrate is linked to changes in peroxidase activity (EC1.11.1.7) with an increase in peroxidative activity that scavenge H2O2 and a decrease in hydroxylic activity that converts H2O2 into •OH. These changes impair the accumulation of H2O2 and then the accumulation of •OH, the species responsible for cell wall loosening and cell elongation. Accordingly, nitrate inhibitory effect was abolished by externally added H2O2 or mimicked by KI, an H2O2 scavenger. In contrast, nitrate has no effect on ROS production or removal capacities in npf6.8-2, a knockdown line insensitive to nitrate, affected in the nitrate transporter MtNPF6.8 (in R108 background) by RNAi. Altogether, our data show that ROS are mediators acting downstream of MtNPF6.8 in the nitrate signaling pathway.

Redox properties and human serum albumin binding of nitro-oleic acid.

Nitro-fatty acids modulate inflammatory and metabolic stress responses, thus displaying potential as new drug candidates. Herein, we evaluate the redox behavior of nitro-oleic acid (NO2-OA) and its ability to bind to the fatty acid transporter human serum albumin (HSA). The nitro group of NO2-OA underwent electrochemical reduction at -0.75 V at pH 7.4 in an aqueous milieu. Based on observations of the R-NO2 reduction process, the stability and reactivity of NO2-OA was measured in comparison to oleic acid (OA) as the negative control. These electrochemically-based results were reinforced by computational quantum mechanical modeling. DFT calculations indicated that both the C9-NO2 and C10-NO2 positional isomers of NO2-OA occurred in two conformers with different internal angles (69° and 110°) between the methyl- and carboxylate termini. Both NO2-OA positional isomers have LUMO energies of around -0.7 eV, affirming the electrophilic properties of fatty acid nitroalkenes. In addition, the binding of NO2-OA and OA with HSA revealed a molar ratio of ~7:1 [NO2-OA]:[HSA]. These binding experiments were performed using both an electrocatalytic approach and electron paramagnetic resonance (EPR) spectroscopy using 16-doxyl stearic acid. Using a Fe(DTCS)2 spin-trap, EPR studies also showed that the release of the nitro moiety of NO2-OA resulted in the formation of nitric oxide radical. Finally, the interaction of NO2-OA with HSA was monitored via Tyr and Trp residue electro-oxidation. The results indicate that not only non-covalent binding but also NO2-OA-HSA adduction mechanisms should be taken into consideration. This study of the redox properties of NO2-OA is applicable to the characterization of other electrophilic mediators of biological and pharmacological relevance.

Impact of Differences in Economic Development and Socioeconomic Stability on Benzodiazepine Exposure between the Three Balkans Countries.

INTRODUCTION: Anxiety disorders are among the most common mental disorders. Benzodiazepines belong to the group of anxiolytic sedatives and the most prescribed drugs in the world. The aim in ours study was to evaluate the differences in the exposure of the population to benzodiazepines (in period from 2014-2018) between Serbia, Slovenia and Croatia, the three countries of the Southwestern Balkans with varying degrees of socioeconomic development. STUDY DESIGN: A academic investigator initiated, pharmacoepidemiological difference-in-difference time series analysis of population exposure to benzodiazepines between the three, geographically close Balkans countries (Slovenia, Serbia, Croatia) with varying degrees of socioeconomic development has been carried out. Study was conducted as academic investigator initiated, in a retrospective manner on monthly basis international data set from January 2014 to December 2018. RESULTS: At the annual level, during the study period from January 2014 to December 2018, compared to Slovenia, Serbia and Croatia had higher DIDs, from 5 fold (Croatia) to 6 fold (Serbia), for all benzodiazepines in total. By analyzing the differences-in-difference, we have shown that influence of both time (month) and country on DIDs is significant as well as their mutual interaction (the country x month) for all benzodiazepines in total. CONCLUSION: Serbia and Croatia must implement explicit measures of reducing benzodiazepine prescription in health primary care based on evidence-based recommendations in the indications where general medicine practitioners/family doctors most commonly prescribe these medicines. Without providing a realistic supplement/alternative to benzodiazepines such as increasing the availability of psychotherapy and improving the structure of psychiatric professionals in healthcare settings, implicit measures are not recommended for reducing prescription, implementing accountability measures for prolonged prescription of benzodiazepines, and in particular for "masked" somatic diseases. All this comes to the fore by raising economic development and socioeconomic stability.

Changes of the peripheral blood mononuclear cells membrane fluidity from type 1 Gaucher disease patients: an electron paramagnetic resonance study.

Gaucher disease (GD) is a lysosomal storage disorder, caused by an impaired function of ß-glucocerebrosidase, which results in accumulation of glucocerebroside in cells, and altered membrane ordering. Using electron paramagnetic resonance spin labeling, a statistically significant difference in the order parameter between the peripheral blood mononuclear cell membranes of GD patients and healthy controls was observed. Moreover, the results show that the introduction of the enzyme replacement therapy leads to the restoration of the physiological membrane fluidity. Accordingly, this simple method could serve as a preliminary test for GD diagnosis and therapy efficiency.

Electrochemistry and electron paramagnetic resonance spectroscopy of cytochrome c and its heme-disrupted analogs.

Cytochrome c (cyt c) is one of the most studied conjugated proteins due to its electron-transfer properties and ability to regulate the processes involved in homeostasis or apoptosis. Here we report an electrochemical strategy for investigating the electroactivity of cyt c and its analogs with a disrupted heme moiety, i.e. apocytochrome c (acyt c) and porphyrin cytochrome c (pcyt c). The electrochemical data are supplemented with low-temperature and spin-probe electron paramagnetic resonance (EPR) spectroscopy. The main contribution of this report is a complex evaluation of cyt c reduction and oxidation at the level of surface-localized amino acid residues and the heme moiety in a single electrochemical scan. The electrochemical pattern of cyt c is substantially different to both analogs acyt c and pcyt c, which could be applicable in further studies on the redox properties and structural stability of cytochromes and other hemeproteins.

Maleimido-proxyl as an EPR spin label for the evaluation of conformational changes of albumin.

Albumin is the most abundant plasma protein and as such has been the subject of many studies using a variety of techniques. One of them, capable of monitoring the conformational changes and the binding capacity of proteins, is electron paramagnetic resonance spectroscopy (EPR) spin labeling. To date, albumin has been investigated using a number of different spin labels, mostly spin-labeled fatty acids (SLFAs). However, albumin can bind up to seven equivalents of fatty acids, making it difficult to determine which parts of the molecule undergo conformational changes. To obtain information from a specific site on a protein, spin labels that bind to free cysteine residues may be used. In this work, the applicability of such a label, 3-maleimido proxyl (5-MSL), was evaluated for monitoring conformational changes of bovine serum albumin (BSA) at different temperatures and pH values. Also, the effect of ethanol, reactive oxygen species (hydrogen peroxide and superoxide radical), and the binding of ligands specific for albumin, namely fatty acids, and several drugs were evaluated. The results indicate that the labeling of albumin at its free cysteine residue (Cys-34) using 5-MSL may successfully be used for the detection of conformational changes, even in the case of the subtle alterations induced by ligand binding.

Antioxidative mechanisms in chlorogenic acid.

Although chlorogenic acid (5CQA) is an important ingredient of various foods and beverages, mechanisms of its antioxidative action have not been fully clarified. Besides electron spin resonance experiment, this study includes thermodynamic and mechanistic investigations of the hydrogen atom transfer (HAT), radical adduct formation (RAF), sequential proton loss electron transfer (SPLET), and single electron transfer - proton transfer (SET-PT) mechanisms of 5CQA in benzene, ethanol, and water solutions. The calculations were performed using the M06-2X/6-311++G(d,p) level of theory and CPCM solvation model. It was found that SET-PT is not a plausible antioxidative mechanism of 5CQA. RAF pathways are faster, but HAT yields thermodynamically more stable radical products, indicating that in acidic and neutral media 5CQA can take either HAT or RAF pathways. In basic environment (e.g. at physiological pH) SPLET is the likely antioxidative mechanism of 5CQA with extremely high rate.