This review has considered the mechanisms of intracellular and intercellular signaling regulation by myeloperoxidase (MPO), an enzyme in neutrophil azurophilic granules, during oxidative/halogenative stress and inflammation development. The stages of enzyme functioning and the formation of reactive halogen species are described. The functioning of MPO and production of reactive halogen species are shown to depend on the activity of NADPH oxidase; the role of NADPH oxidase and reactive oxygen species in the regulation of MPO function is discussed. Particular attention is focused on the role of biological molecules modified by reactive halogen species in modulating NADPH oxidase activity, exocytosis of granular proteins, NETosis, and other neutrophil functions, based on the principle of positive feedback. A special feature of the review is the discussion of the non-canonical function of MPO, namely its signaling role in the regulation of cellular processes, which is not associated with the catalytic activity of the enzyme.

Activation of neutrophil membrane receptors initiates intracellular signal transduction cascades that orchestrate the cell's effector functions,including phagocytosis,production of reactive oxygen and halogen species,degranulation,and NETosis(formation of neutrophil extracellular traps[NETs]).NETs,which contain antimicrobial compounds such as myeloperoxidase(MPO),represent a strategy to combat infection.However,excessive production of NETs promotes thrombosis,diabetes mellitus,and other diseases.Therefore,investigations into the mechanisms of NETosis and the identification of modulators of this process are critical for developing strategies to address NETosis-related disorders.Here,we identified a novel NETosis inducer,human serum albumin(HSA)modified by the MPO product hypochlorous acid(HSAHOCl),whose accumulation in vivo was correlated with inflammatory processes.Using human blood neutrophils,we investigated HSAHOCl-induced NETosis and detected NET formation by flow cytometry.The results showed that the mechanism of HSAHOCl-induced NETosis involved MPO,NADPH oxidase,and phosphatidylinositol 3-kinases(PI3Ks),and that HSAHOCl activated a reactive oxygen species-dependent suicidal type of NETosis.Moreover,HSAHOCl-induced NETosis was inhibited by an anti-HSAHOCl monoclonal antibody.Thus,our findings may facilitate the development of strategies to modulate NETosis in inflammation associated with elevated MPO activity.

The influence of selenium (Se) nanoparticles in the form of rods (SeNrs) and spheres (SeSps), synthesized by laser ablation, on the structural and functional properties of human blood erythrocytes and neutrophils was studied for anticancer activity in vitro. SeNrs and SeSps do not have cytotoxicity towards neutrophils and do not cause hemolysis. The elastic modulus and resistance of erythrocytes to HOCl-induced hemolysis increased after binding of Se nanoparticles to the plasma membrane. The interaction of Se nanoparticles with neutrophils is accompanied by their actin-dependent macropinocytosis, triggering intracellular signaling processes leading to the assembly and activation of NADPH oxidase. Comparative analysis of the effects of SeNrs and SeSps on cells showed that they have similar effects. This may be due to the fact that SeNrs interact with the cell surface with their end faces, and, therefore, have the same initial contact with the plasma membrane as SeSps. However, SeSps and SeNrs showed chronic cytotoxicity after 48 h incubation, indicating the need to find ways to reduce their toxicity further. Further use of Se nanoparticles in anisotropic form in biomedical research for the development of therapeutic agents seems promising.

Fucoidan is of considerable interest for the development of drug carriers. The inclusion of fucoidan allows calcium carbonate microparticles in the form of vaterite to acquire new properties, enabling their use in the immobilization of protein preparations. In this work, we investigated the properties of hybrid vaterite microparticles with fucoidan from Fucus vesiculosus obtained by co-precipitation and loaded with recombinant human lactoferrin from goats. The hybrid microparticles had a smaller diameter (3–4 µm), larger surface area (35–36 m2g−1), smaller pore size (5–10 nm average), and more negative ζ-potential (−(11–13) mV) than the control vaterite microparticles. The incorporation of lactoferrin into the microparticles by co-precipitation in complex with fucoidan was greater than when the protein was adsorbed onto the hybrid microparticles. Microparticles with fucoidan and lactoferrin were stable in acidic environments, released both components over a prolonged period at pH 7.4, and possessed mucoadhesive properties and anticoagulant activity. The antibacterial properties of hybrid microparticles with fucoidan and lactoferrin against Bacillus subtilis were characterized. Microparticles of vaterite with fucoidan can serve as a platform for the microfabrication of effective means of delivering therapeutic proteins.

The detailed study of the different signal statistics in laser induced breakdown spectroscopy (LIBS) measurements has been carried out. It was demonstrated that the Nd:YAG laser pulse energy distribution function is not described by the Gaussian distribution function, but can follow the normal distribution if proper lasing conditions (pumping level, Q-switch type, etc.) are chosen. The time gated LIBS signals (atomic lines intensity, plasma background emission, acoustic signal) have been shown to be biased from a Gaussian distribution function. Alternatively, distribution function of the time and spectra integrated plasma emission corresponded to the normal distribution. The normalization procedure did not change the distribution functions for LIBS signals so the generalized extreme value distribution was a better choice. In case of the generalized extreme value distribution for the LIBS signals, the limits of detection should be defined by distribution functions rather than calibration curve and “3-sigma criteria” approach.

Vaterite microparticles, metastable form of calcium carbonate, are promising forms of delivery of medicinal compounds. For more efficient delivery of target molecules (increased incorporation and retention), vaterite microparticles must be functionalized with biopolymers. In this article the effect of polysaccharides, mucin and vaterite microparticles, as well as hybrid vaterite microparticles with the above-mentioned biopolymers was studied on platelet aggregation. It was found that fucoidan, heparin and dextran sulfate (when added to platelet-rich plasma) and mucin (when added to isolated platelets) initiated cell aggregation. Pectin and chondroitin sulfate inhibited ADP- and thrombin-induced aggregation in a dose-dependent manner, mucin suppressed ADP-induced, and dextran sulfate suppressed thrombin-induced platelet aggregation. Vaterite microparticles at a concentration of 100–1000 µg/mL did not affect the aggregation of isolated platelets, but caused 10–15 at a concentration of 1000 µg/mL vaterite microparticles prevented agonist-induced cell aggregation by 30 that hybrid vaterite microparticles with fucoidan or heparin, when added both to platelet-rich plasma and to isolated cells, are capable to initiate platelet aggregation. Vaterite microparticles functionalized with pectin or chondroitin sulfate had no effect on spontaneous cell aggregation, and did not affect (with chondroitin sulfate) or inhibit (with pectin) agonist-induced platelet aggregation. Thus, the use of hybrid vaterite microparticles with pectin or fucoidan/heparin may be promising for the delivery of drugs aimed at modulating (inhibition with pectin or activation with fucoidan/heparin) the platelet component of hemostasis.

Type 2 diabetes mellitus (T2DM) is accompanied by halogenative stress resulting from the excessive activation of neutrophils and neutrophilic myeloperoxidase (MPO) generating highly reactive hypochlorous acid (HOCl). HOCl in blood plasma modifies serum albumin (Cl-HSA). We studied the formation of neutrophil extracellular traps (NETs) in the whole blood and by isolated neutrophils under the action of Cl-HSA. It was found that Cl-HSA induces neutrophil priming and NETosis. MPO-containing as well as MPO-free NETs were found. These NETs with different composition can be a product of NETosis of one and the same neutrophil. NET formation in neutrophils with vacuolated cytoplasm was detected. In the presence of Cl-HSA, acceleration of NET degradation was observed. Accelerated NET degradation and neutrophil priming can be the factors contributing to the development of complications in T2DM.

A study was carried out on the spectralluminescent properties of fl uorescein after its reaction with various reactive oxygen and halogen species ( O_2^∙-, H2O2, HOCl, HOBr, HOSCN, N-chloramine, taurine N-chloramine, and taurine N-bromamine) as well as in the myeloperoxidase (MPO)–H2O2–Cl–/Br–/SCN– system. Reaction with only HOBr or with the MPO–H2O2–Br system turns fluorescein into a compound with an absorption maximum at 518 nm. The fluorescence maximum is recorded at 540 nm when excited at 520 nm, corresponding to eosin Y (brominated fluorescein). Conditions with phosphatebuffered saline (PBS) at pH 7.4 containing 137 mM NaCl, 5 mM fluorescein, 15–30 mM NaBr, and 25–50 mM H2O2 were found to be optimal for detecting HOBr in solution. A qualitative method for determining the brominating activity of MPO in vitro has been proposed. This method was used to study the effect of physiological and synthetic inhibitors as well as reactive oxygen and halogen species scavengers on the brominating activity of MPO. Our results indicate that fluorescein holds promise for use in a fluorescent method for detecting the brominating activity of mammalian hemecontaining peroxidases.

Co-precipitation of biopolymers into calcium carbonate crystals changes their physicochemical and biological properties. This work studies hybrid microcrystals of vaterite obtained in the presence of natural polysaccharides, as carriers for the delivery of proteins and enzymes. Hybrid microcrystals with dextran sulfate, chondroitin sulfate, heparin, fucoidan, and pectin were obtained and compared. The impact of polysaccharides on the morphology (particle diameter, surface area, nanocrystallite and pore size), polysaccharide content and surface charge of hybrid microcrystals was studied. Only microcrystals with fucoidan and heparin exhibited antioxidant activity against •ОН radical. The surface charge and pore size of the hybrid microcrystals affected the sorption of albumin, catalase, chymotrypsin, mucin. A decrease in the catalytic constant and Michaelis constant was observed for catalase sorbed on the hybrid crystals. The biocompatibility of microcrystals depended on the nature of the included polysaccharide: crystals with sulfated polysaccharides increased blood plasma coagulation but not platelet aggregation, and crystals with dextran sulfate had the greatest cytotoxicity against HT-29 cells but not erythrocytes. Hybrid microcrystals with all polysaccharides except chondroitin sulfate reduced erythrocyte lysis in vitro compared with vaterite crystals. The obtained results enable to create novel carriers based on hybrid vaterite crystals with polysaccharides, beneficial for the delivery of protein drugs.

The influence of the solution acidity on the interaction between bovine serum albumin (BSA) molecules and gold nanoparticles in solutions has been investigated by absorbance spectroscopy, fluorescence spectroscopy, and dynamic light scattering (DLS). The influence of pH on the processes of aggregation–disaggregation of gold nanoparticles with BSA and without it and on the denaturation of protein solution is demonstrated. It is also shown that BSA molecules can stabilize gold nanoparticles at acidic pH values of 2.0–4.0. The data obtained can be useful for physiologists studying the influence of nanoparticles on different biological media of the body.

This review provides an overview of molecular mechanisms of intracellular signal transduction involving free radicals. The structure and functions of enzymes that can produce superoxide anion-radical and hydrogen peroxide are considered in detail. The mechanisms of regulation of cell properties with the participation of specialized redox chains formed by a group of proteins interacting through electron transport processes are discussed. Genetically mediated mechanisms of regulation of cellular redox homeostasis are analyzed. Particular attention is paid to the issue concerning quantitative characterization of the network of interactions of oxidizing and reducing agents, which determines the species-specific and individual characteristics of redox homeostasis and the stress response of cells.

The effect of graphene oxide-based photoconversion covers on the growth and photosynthesis of tomatoes (Solanum lycopersicum) was investigated. Two types of photoconversion composite for covers were produced. In the first, only graphene oxide nanoparticles were used as a phosphor, and in the second, the graphene oxide nanoparticles were used jointly with europium oxide nanoparticles. The freshly prepared composites for covers had almost identical photoluminescence spectra: an intense peak in the red region and a minor peak in the blue region. It was revealed that during operation, luminescence in the red region decreased, while in the blue region it increased, probably due to the photothermal reduction of graphene oxide. It was shown that the photoconversion covers increased productivity (25%) and intensified photosynthesis (30–35%) in the tomato plants. It is suggested that the stimulation of plant growth is caused by changes in the light spectrum induced by the photoconversion covers.

The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP–luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands.

Optical methods (spectroscopy, spectrofluorometry, dynamic light scattering, and refractometry) were used to study the change in the state of hen egg-white lysozyme (HEWL), protein molecules, and gold nanoparticles (AuNPs) in aqueous colloids with changes in pH, and the interaction of protein molecules with nanoparticles was also studied. It was shown that changing pH may be the easiest way to control the protein corona on gold nanoparticles. In a colloid of nanoparticles, both in the presence and absence of protein, aggregation–deaggregation, and in a protein colloid, monomerization–dimerization–aggregation are the main processes when pH is changed. A specific point at pH 7.5, where a transition of the colloidal system from one state to another is observed, has been found using all the optical methods mentioned. It has been shown that gold nanoparticles can stabilize HEWL protein molecules at alkaline pH while maintaining enzymatic activity, which can be used in practice. The data obtained in this manuscript allow for the state of HEWL colloids and gold nanoparticles to be monitored using one or two simple and accessible optical methods.

The article is devoted to the study of melted ingots, plates rolled from them, and the resulting spherical powder made of corrosion-resistant 316L steel with the addition of 0.2 wt.% and 0.5 wt.% Ag. The study of antibacterial properties, microstructure, and distribution of silver concentrations, as well as qualitative analysis of silver content was carried out. The optimal mode of homogenization annealing of the ingot was 1050 °C for 9 h, which leads to the formation of an austenitic structure. It is shown that the addition of a small amount of silver does not affect the formation of the austenitic structure and silver is distributed evenly throughout the volume of the ingot. The austenitic structure also prevails in the plates after rolling. Silver is distributed evenly throughout the entire volume of the plate. It is noted that the addition of 0.2 wt.% Ag does not affect the strength, elongation, and microhardness of steel, and the addition of 0.5 wt.% Ag does not significantly reduce the strength of steel, however, all samples meet the mechanical characteristics according to the ASTM A240 standard. The qualitative chemical composition of samples made of corrosion-resistant steels was confirmed by X-ray fluorescence analysis methods. By the method of energy-dispersion analysis, the presence of a uniform distribution of silver over the entire volume of the powder particle was determined. The particles have a spherical shape with a minimum number of defects. The study of the antibacterial activity of plates and powder shows the presence of a clear antibacterial effect (bacteria of the genus Xanthomonas campestris, Erwinia carotovora, Pseudomonas marginalis, Clavibacter michiganensis) in samples No. 2 and No. 3 with the addition of 0.2 wt.% and 0.5 wt.% Ag.

BACKGROUND: The modern market of medical devices in Belarus and Russia is represented by a wide range of hemostatic agents, of which the most popular are local hemostatics of plant origin possessing the significant technological potential for renewal and improvement. A promising reserve for this may be biologically active compounds of mosses, which are characterized by anti-inflammatory, antibacterial and antifungal effects. However, their hemostatic effect is almost not studied, which determines the relevance of this work. AIM: The aim of this work is to study the effect of lectin-containing substances from mosses of three species collected in East Antarctica and Belarus on the parameters of human blood hemostasis in vitro. MATERIALS AND METHODS: We studied mosses of the genera Bryum, Ceratodon, and Coscinodon, collected in the area of the Belarusian Antarctic station Gora Vechernyaya in East Antarctica and in Belarus. Lectin-containing substances of mosses were obtained by extracting shoots in 0.05 M tris-HCl buffer (pH 8.0), centrifugation, filtration. The assessment of the biological activity of lectin-containing substances in mosses was carried out by the agglutination reaction of rabbit erythrocytes, as well as the effect on human platelet aggregation and in the test for activated partial thromboplastin time. RESULTS: It was established that lectin-containing substances of the studied moss species had agglutinating activity against erythrocytes in the range from 11708.28 (Belarusian samples) to 1333979.59 U/mg of protein (Antarctic samples) depending on the species and localization; initiated the aggregation of human platelets (25–80% of the effect of thrombin) regardless of blood group, Rh and gender of donors; influenced the plasma link of hemostasis, reducing activated partial thromboplastin time (by 15–18%). CONCLUSIONS: It was found that some species of mosses of the genera Bryum, Ceratodon and Coscinodon of Antarctica and Belarus had an agglutinating and hemostatic effect on erythrocytes and platelets, with the greatest activity noted for Antarctic species. A hypothesis has been put forward that the observed phenomenon is due to the structural features of proteins, including lectins. It is assumed that lectins are possible inducers of erythrocyte agglutination and platelet aggregation in mosses. It is shown that the moss species Bryum pseudotriquetrum and Ceratodon purpureus have a certain resource potential in Belarus for their annual harvest. The results obtained expand the list of moss species with hemostatic activity, and can be used to develop new hemostatics of plant origin for local use from Belarusian plant materials.

Hypochlorous acid (HOCl) derived from hydrogen peroxide and chloride anion by myeloperoxidase (MPO) plays a significant role in physiological and pathological processes. Herein we report a phenoxazine-based fluorescent probe Celestine Blue B (CB) that is applicable for HOCl detection in living cells and for assaying the chlorinating activity of MPO. A remarkable selectivity and sensitivity (limit of detection is 32 nM), along with a rapid "turn-on" response of CB to HOCl was demonstrated. Furthermore, the probe was able to detect endogenous HOCl and reactive halogenated species by fluorescence spectroscopy, confocal microscopy, and flow cytometry techniques. Hence, CB is a promising tool for investigating the role of HOCl in health and disease and for screening the drugs capable of regulating MPO activity.

A system of kinetic equations describing the changes in the concentration of reactive oxygen species (ROS) in aqueous solutions of proteins was obtained from the analysis of chemical reactions involving singlet oxygen. Applying the condition of the stationarity of the intermediate products to the system, we determined the functional dependence of the hydrogen peroxide concentration on the protein concentration under the action of thermal and laser radiation. An approximate analytical solution to the nonlinear system of differential equations that define the ROS concentration dynamics was found. For aqueous solutions of bovine serum albumin (BSA) and bovine gamma globulin (BGG), the orders and rate constants of the reactions describing the ROS conversions were determined by minimizing the sum of squared deviations of the functions found by solving both the static and dynamic problems from experimentally measured dependences. When solving the optimization problem, the Levenberg–Marquardt algorithm was used.

Hyperglycemia-induced protein glycation and formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of diabetic complications and pathological biomineralization. Receptors for AGEs (RAGEs) mediate the generation of reactive oxygen species (ROS) via activation of NADPH-oxidase. It is conceivable that binding of glycated proteins with biomineral particles composed mainly of calcium carbonate and/or phosphate enhances their neutrophil-activating capacity and hence their proinflammatory properties. Our research managed to confirm this hypothesis. Human serum albumin (HSA) was glycated with methylglyoxal (MG), and HSA-MG was adsorbed onto mineral microparticles composed of calcium carbonate nanocrystals (vaterite polymorph, CC) or hydroxyapatite nanowires (CP). As scopoletin fluorescence has shown, H2O2 generation by neutrophils stimulated with HSA-MG was inhibited with diphenyleneiodonium chloride, wortmannin, genistein and EDTA, indicating a key role for NADPH-oxidase, protein tyrosine kinase, phosphatidylinositol 3-kinase and divalent ions (presumably Ca2+) in HSA-MG-induced neutrophil respiratory burst. Superoxide anion generation assessed by lucigenin-enhanced chemiluminescence (Luc-CL) was significantly enhanced by free HSA-MG and by both CC-HSA-MG and CP-HSA-MG microparticles. Comparing the concentrations of CC-bound and free HSA-MG, one could see that adsorption enhanced the neutrophil-activating capacity of HSA-MG.

Hyperglycemia in diabetes mellitus induces modification of proteins by glucose and its derivative methylglyoxal (MG). Neutrophils perform their bactericidal activity mainly via reactive halogen (RHS) and oxygen (ROS) species generation catalyzed by myeloperoxidase (MPO) stored in neutrophil azurophilic granules (AGs) and membrane NADPH oxidase, respectively. Herein, we study the binding of human serum albumin (HSA) modified with MG (HSA-MG) to MPO and its effects on MPO activity and release by neutrophils. Peroxidase activity of MPO was registered by oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and chlorinating activity by decolorization of Celestine blue B dye. Binding of HSA-MG to MPO was studied by affinity chromatography, disc-electrophoresis, ligand Western blotting and enzyme-linked solid phase immunoassay using monoclonal antibodies (mAbs) to MPO. ROS and RHS generation were detected by lucigenin (Luc) and luminol (Lum) chemiluminescence (CL), respectively. Neutrophil degranulation was assessed by flow cytometry using fluorescent labeled antibodies to the marker proteins CD63 from AGs and CD11b from peroxidase-negative granules (PNGs). NETosis was assayed by quantifying DNA network-like structures (NET-like structures) in blood smears stained by Romanowsky. HSA-MG bound to MPO, giving a stable complex (Kd = 1.5 nM) and competing with mAbs, and non-competitively inhibited peroxidase and chlorinating MPO activity and induced degranulation of PNGs but not of AGs. HSA-MG enhanced Luc-CL per se or following PMA, unlike Lum-CL, and did not affect spontaneous or PMA-stimulated NETosis. Thus, HSA modified under hyperglycemia-like conditions stimulated NADPH oxidase of neutrophils but dampened their functions dependent on activity of MPO, with no effect on its release via degranulation or NETosis. This phenomenon could underlie the downregulation of bactericidal activity of MPO and neutrophils, and hence of innate immunity, giving rise to wound healing impairment and susceptibility to infection in patients with hyperglycemia.