Kinetic Potentiometry as a Method for Studying the Interactions of Antioxidants with Peroxyl Radicals.

This work presents a new method using kinetic potentiometry to study the thermodynamic and kinetic parameters of the reactions of antioxidants with peroxyl radicals. The rate constants of the reaction of antioxidants with radicals have been determined, and the groups of "fast" and "slow" antioxidants have been conventionally distinguished. Fast antioxidants include ascorbic, uric, gallic, chlorogenic, caffeic acids, glutathione, L-cysteine, and catechol with constant values from (1.05-9.25) × 103 M·s-1; "slow" antioxidants are α-tocopherol (in aqueous media), ionol, 2,6-ditretbutylphenol, and compounds of the azoloazine series, modified with polyphenolic fragments, with constant values from (4.00-8.50) × 102 M·s-1. It is shown that the value of the rate constant is directly related to the type of kinetic dependence of the potential recorded when an antioxidant is introduced into the solution of the radical initiator. It is shown that the method with the determination of the induction period is difficult in the study of "slow" antioxidants. It has been established that the area above the curve of the kinetic dependence Exp(∆E) is directly related to the amount of inhibited peroxyl radicals and can be used to assess the inhibitory properties of an antioxidant from a thermodynamic point of view. "Fixed time method" and "Initial rate method" were used. Positive correlations between the described method have been established. The utility of the parameter of the area above the curve of the kinetic dependence Exp(∆E) in the study of objects of complex composition is shown.

Unexpected ubiquity of heart-shaped scale morphotype in Centroplasthelida (Haptista): Ancestral trait or multiple acquisitions?

Centrohelids (Haptista: Centroplasthelida) are axopodial protists with a remarkable diversity of external siliceous scale morphologies. It is believed that the last common ancestor of centrohelids had a double layer of siliceous scales composed of plate scales closer to a cell surface and spine scales radiating outwards. The characteristic morphotype of spine scales with a heart-shaped base was once believed to be a unique feature of the genus Choanocystis, as it was defined by Siemensma and Roijackers (1988). Further research revealed that this morphology is present in different and sometimes distantly related lineages: Ozanamiidae, Meringosphaeridae, and Marophryidae. Here, we report the fourth clade, Pterocystidae, which is also revealed to contain representatives having this phenotype. Cernunnos gen. nov. is erected here to place Cernunnos uralica sp. nov., Cernunnos arctica sp. nov., Cernunnos america sp. nov., and Cernunnos antarctica Tikhonenkov et Mylnikov, 2010, Gerasimova comb. nov. C. uralica was studied with scanning electron microscopy and SSU rDNA sequencing. Molecular phylogenetic analysis placed it into marine environmental clade P within Pterocystida. The ubiquity of spine scales with heart-shaped bases could be an example of parallel evolution, but taking into account the considerable similarity it is likely an ancestral trait, acquired from the last common ancestor of centrohelids.

Behavioral profile of adult zebrafish acutely exposed to a selective dopamine uptake inhibitor, GBR 12909.

BACKGROUND: The dopamine transporter (DAT) is the main regulator of dopamine concentration in the extrasynaptic space. The pharmacological inhibition of the DAT results in a wide spectrum of behavioral manifestations, which have been identified so far in a limited number of species, mostly in rodents. AIM: Here, we used another well-recognized model organism, the zebrafish (Danio rerio), to explore the behavioral effects of GBR 12909, a highly-affine selective DAT blocker. METHODS: We evaluated zebrafish locomotion, novelty-related exploration, spatial cognition, and social phenotypes in the novel tank, habituation and shoaling tests, following acute 20-min water immersion in GBR 12909. RESULTS: Our findings show hypolocomotion, anxiety-like state, and impaired spatial cognition in fish acutely treated with GBR 12909. This behavioral profile generally parallels that of the DAT knockout rodents and zebrafish, and it overlaps with behavioral effects of other DAT-inhibiting drugs of abuse, such as cocaine and D-amphetamine. CONCLUSION: Collectively, our data support the utility of zebrafish in translational studies on DAT targeting neuropharmacology and strongly implicate DAT aberration as an important mechanisms involved in neurological and psychiatric diseases.

Theoretical simulation and experimental design of selenium and gold incorporated polymer-based microcarriers for ROS-mediated combined photothermal therapy.

Recently, multi-modal combined photothermal therapy (PTT) with the use of photo-active materials has attracted significant attention for cancer treatment. However, drug carriers enabling efficient heating at the tumor site are yet to be designed: this is a fundamental requirement for broad implementation of PTT in clinics. In this work, we design and develop hybrid carriers based on multilayer capsules integrated with selenium nanoparticles (Se NPs) and gold nanorods (Au NRs) to realize reactive oxygen species (ROS)-mediated combined PTT. We show theoretically and experimentally that cooperative interaction of Se NPs with Au NRs improves the heat release efficiency of the developed capsules. In addition, after uptake by tumor cells, intracellular ROS level amplified by Se NPs inhibits the tumor growth. As a consequence, the synergy between Se NPs and Au NRs exhibits the advantages of hybrid carriers such as (i) improved photothermal conversion efficiency and (ii) dual-therapeutic effect. The results of in vitro and in vivo experiments demonstrate that the combination of ROS-mediated therapy and PTT has a higher tumor inhibition efficiency compared to the single-agent treatment (using only Se-loaded or Au-loaded capsules). Furthermore, the developed hybrid carriers show negligible in vivo toxicity towards major organs such as the heart, lungs, liver, kidneys and spleen. This study not only provides a potential strategy for the design of multifunctional "all-in-one" carriers, but also contributes to the development of combined PTT in clinical practice.

Subclinical Carotid Atherosclerosis in Patients with Rheumatoid Arthritis at Low Cardiovascular Risk.

OBJECTIVE: To evaluate the rate of subclinical carotid atherosclerosis and clinical significance of immunoinflammatory markers in patients with rheumatoid arthritis (RA) at low cardiovascular risk. MATERIALS AND METHODS: The study included 275 RA patients and a control group of 100 participants without autoimmune diseases. All study participants were at low cardiovascular risk, calculated by the QRISK3 scale (<20%), and free of cardiovascular disease. Ultrasound examination of carotid arteries was performed to measure cIMT and to detect atherosclerotic plaques (ASP) in carotid arteries. sIСАМ-1, sVСАМ, and sCD40L levels were determined by enzyme immunoassay. RESULTS: Carotid ASP was observed more frequently in RA patients (27%) than in the control group (17%), p = 0.03. The frequency of ASP in RA patients did not depend on the disease's stage or activity. There was a significant correlation between cIMT and age, cardiovascular risk determined by QRISK3, level of total cholesterol, LDL, and blood pressure in RA patients, p < 0.05 in all cases. No correlation between cIMT and blood levels of sCD40L, sVCAM, and sICAM was found. In RA patients, a higher concentration of sVCAM was detected in the carotid ASP group compared to the non-atherosclerotic group. sCD40L was associated with cIMT and total cholesterol in the ASP group and with total cholesterol and blood pressure in non-atherosclerotic patients. CONCLUSIONS: Subclinical atherosclerotic lesions of the carotid arteries were observed significantly more frequently in RA patients with low cardiovascular risk than in the control group. The results of the study demonstrate the association between cIMT, traditional cardiovascular risk factors, and immunoinflammatory markers in RA patients.

Mitochondrial DNA copy number in patients with systemic sclerosis.

Introduction: Systemic scleroderma (SSc) is a chronic autoimmune disease of inflammatory origin. Mitochondrial dysfunction is considered as an important mechanism in the pathogenesis of SSc. Currently mitochondrial DNA (mtDNA) copy number is used as a surrogate marker of mitochondrial dysfunction. Previous studies demonstrate that innate immune cells are important participants in inflammatory and fibrotic processes in SSc. The aim of the study was to evaluate the number of mtDNA copies in CD14+ monocytes and whole blood of patients with SSc in comparison with healthy individuals. Methods: Absolute mtDNA copy number was measured using digital PCR. It was found that the number of mtDNA copies in CD14+ monocytes was significantly higher in patients with SSc compared to control, while the number of mtDNA copies in the whole blood did not have significant differences. Results: The correlation analysis revealed an inverse association of mtDNA copy number with disease duration and the relationship between pro-inflammatory activation of CD14+ monocytes in terms of LPS-stimulated IL-6 secretion and mtDNA copy number. At the same time, basal and LPS-stimulated secretion of IL-6 by cultured CD+ monocytes were significantly higher in SSc group in comparison with control. Discussion: The study results suggest that increase of mtDNA copy number in CD14+ monocytes is a possible mechanism to maintain the reduced function of defective mitochondria in monocytes from patients with SSc associated with the development and progression of SSc.

Systemic Sclerosis and Atherosclerosis: Potential Cellular Biomarkers and Mechanisms.

Systemic sclerosis (SSc) is a rare systemic autoimmune disease of unknown etiology, which is characterized by endothelial dysfunction, pathologic vasculopathy, and increased tissue fibrosis. Traditionally, SSc has been regarded as a prototypical fibrotic disease in the family of systemic autoimmune diseases. Traditionally, emphasis has been placed on the three components of the pathogenesis of SSc: vascular, immune, and mesenchymal. Microvascular lesions, including endothelial dysfunction and smooth muscle cell migration into the intima of vessels in SSc, resemble the atherosclerotic process. Although microvascular disease is a hallmark of SSc, understanding the role of atherosclerotic vascular lesions in patients with SSc remains limited. It is still unknown whether the increased cardiovascular risk in SSc is related to specific cardiac complications (such as myocardial fibrosis) or the accelerated development of atherosclerosis. Different immune cell types appear to be involved in the immunopathogenesis of SSc via the activation of other immune cells, fibrosis, or vascular damage. Macrophages, B cells, T cells, dendritic cells, neutrophils, and endothelial cells have been reported to play the most important role in the pathogenesis of SSc and atherosclerosis. In our article, we reviewed the most significant and recent studies on the pathogenetic links between the development of SSc and the atherosclerotic process.

Towards Zebrafish Models of CNS Channelopathies.

Channelopathies are a large group of systemic disorders whose pathogenesis is associated with dysfunctional ion channels. Aberrant transmembrane transport of K+, Na+, Ca2+ and Cl- by these channels in the brain induces central nervous system (CNS) channelopathies, most commonly including epilepsy, but also migraine, as well as various movement and psychiatric disorders. Animal models are a useful tool for studying pathogenesis of a wide range of brain disorders, including channelopathies. Complementing multiple well-established rodent models, the zebrafish (Danio rerio) has become a popular translational model organism for neurobiology, psychopharmacology and toxicology research, and for probing mechanisms underlying CNS pathogenesis. Here, we discuss current prospects and challenges of developing genetic, pharmacological and other experimental models of major CNS channelopathies based on zebrafish.

Novel Chromone-Containing Allylmorpholines Induce Anxiolytic-like and Sedative Effects in Adult Zebrafish.

Chromone-containing allylmorpholines (CCAMs) are a novel class of compounds that have demonstrated acetyl- and butyryl-cholinesterase-inhibiting and N-methyl-D-aspartate (NMDA) receptor-blocking properties in vitro, but their in vivo pharmacological activity remains underexplored. In this work, we evaluated the psychotropic activity of five different CCAMs (1 (9a), 2 (9j), 3 (9l), 4 (33a), and 5 (33b)) using the novel tank test (NTT) and light/dark box (LDB) test in adult zebrafish. The CCAMs were screened in the NTT at a range of concentrations, and they were found to induce a dose-dependent sedative effect. Compound 4 (33a) was also evaluated using the LDB test, and it was found to have anxiolytic-like properties at low concentrations. To assess the potential contribution of the glutamate and cholinergic mechanisms in the effects of the CCAMs, we conducted experiments with pre-exposure to putative antagonists, NMDA and biperiden. Neither biperiden nor NMDA were able to diminish or cancel the effects of the CCAMs, countering the in vitro data obtained in previous studies. The apparent discrepancy could be related to the specifics of CCAM metabolism or to the interspecies differences between the putative target proteins, possibly due to the relatively low identity percentage of their sequences. Although further research in mammals is required in order to establish their pharmacological properties, novel CCAMs may represent an appealing group of psychoactive drug candidates.

The Novel Potentiometric Approach to Antioxidant Capacity Assay Based on the Reaction with Stable Radical 2,2'-diphenyl-1-picrylhydrazyl.

For the first time, new possibilities of using the DPPH• as a signal-forming oxidant molecule with potentiometric detection are shown. The CV method confirmed the presence of a quasi-reversible potential-determining system DPPH•/DPPH-H under experimental conditions. This fact makes it possible to use DPPH• as the model of the oxidizing agent for obtaining an analytical signal by the potentiometry method. The potentiometric approach makes it possible to obtain the value of the Nernst slope and the antioxidant capacity in one experiment. It consists of an antioxidant supplement and two consecutive DPPH• supplements. In this case, the calculation of the Nernst slope is carried out by introducing the second addition of the oxidizing agent and constructing a calibration curve against the reaction background with an antioxidant. Solutions of individual antioxidants α-tocopherol, quercetin, (±)-catechin hydrate, and α-lipoic acid were studied by the developed approach. A high correlation with the results of spectrophotometric measurements is shown. At the same time, the potentiometry method is devoid of the concentration limitations of the spectrophotometric method, which was confirmed. In the study of plant materials extracts, a high correlation of antioxidant capacity, obtained by potentiometric and spectrophotometric methods, was shown only for objects whose color did not contribute to the DPPH• absorption. The versatility of the potentiometric method for studying objects of any color was shown.

Activation Markers on B and T Cells and Immune Checkpoints in Autoimmune Rheumatic Diseases.

In addition to identifying the major B- and T-cell subpopulations involved in autoimmune rheumatic diseases (ARDs), in recent years special attention has been paid to studying the expression of their activation markers and immune checkpoints (ICPs). The activation markers on B and T cells are a consequence of the immune response, and these molecules are considered as sensitive specific markers of ARD activity and as promising targets for immunotherapy. ICPs regulate the activation of the immune response by preventing the initiation of autoimmune processes, and they modulate it by reducing immune cell-induced organ and tissue damage. The article considers the possible correlation of ICPs with the activity of ARDs, the efficacy of specific ARD treatments, and the prospects for the use of activation molecules and activation/blocking ICPs for the treatment of ARDs.

Hyperhomocysteinemia Increases Cortical Excitability and Aggravates Mechanical Hyperalgesia and Anxiety in a Nitroglycerine-Induced Migraine Model in Rats.

Homocysteine is a sulfur-containing endogenous amino acid leading to neurotoxic effects at high concentrations. Population studies suggest an association between plasma homocysteine levels and the risk of migraine headaches. The aim of this study was to analyze the sensitivity of rats with prenatal hyperhomocysteinemia (hHCY) in respect of the development of behavioral correlates of headache and spreading cortical depolarization (CSD) in a migraine model induced by the administration of the nitric oxide (NO) donor nitroglycerin. Animals with hHCY were characterized by migraine-related symptoms such as mechanical hyperalgesia, high-level anxiety, photophobia, as well as an enhanced level of neuronal activity in the somatosensory cortex along with a lower threshold of CSD generation. Likewise, acute or chronic intermittent administration of nitroglycerin also induced the development of mechanical allodynia, photophobia and anxiety in control groups. However, these symptoms were more pronounced in rats with hHCY. Unlike hHCY, nitroglycerin administration did not affect the threshold of CSD generation, but like hHCY, increased the background neuronal activity in layers 2/3 and 4 of the cerebral cortex. The latter was more pronounced in animals with hHCY. Thus, the migraine profile associated with hHCY can be further exaggerated in conditions with enhanced levels of migraine triggering the gaseous transmitter NO. Our data are consistent with the view that high levels of plasma homocysteine can act as a risk factor for the development of migraine.

Macrophage Dysfunction in Autoimmune Rheumatic Diseases and Atherosclerosis.

One of the problems of modern medical science is cardiovascular pathology caused by atherosclerotic vascular lesions in patients with autoimmune rheumatic diseases (ARDs). The similarity between the mechanisms of the immunopathogenesis of ARD and chronic low-grade inflammation in atherosclerosis draws attention. According to modern concepts, chronic inflammation associated with uncontrolled activation of both innate and acquired immunity plays a fundamental role in all stages of ARDs and atherosclerotic processes. Macrophage monocytes play an important role among the numerous immune cells and mediators involved in the immunopathogenesis of both ARDs and atherosclerosis. An imbalance between M1-like and M2-like macrophages is considered one of the causes of ARDs. The study of a key pathogenetic factor in the development of autoimmune and atherosclerotic inflammation-activated monocyte/macrophages will deepen the knowledge of chronic inflammation pathogenesis.

Applying a Fast-Scan Cyclic Voltammetry to Explore Dopamine Dynamics in Animal Models of Neuropsychiatric Disorders.

Progress in the development of technologies for the real-time monitoring of neurotransmitter dynamics has provided researchers with effective tools for the exploration of etiology and molecular mechanisms of neuropsychiatric disorders. One of these powerful tools is fast-scan cyclic voltammetry (FSCV), a technique which has progressively been used in animal models of diverse pathological conditions associated with alterations in dopamine transmission. Indeed, for several decades FSCV studies have provided substantial insights into our understanding of the role of abnormal dopaminergic transmission in pathogenetic mechanisms of drug and alcohol addiction, Parkinson's disease, schizophrenia, etc. Here we review the applications of FSCV to research neuropsychiatric disorders with particular attention to recent technological advances.

Morphology and spicules elemental composition of Marophrys nikolaevi spec. nov. (Haptista: Centroplasthelida).

Marophrys is a genus of spicules-bearing centrohelids belonging to Heterophrys-like organisms (HLO's). Here Marophrys nikolaevi spec. nov. is described. Four strains were isolated from brackish waters (16-22 ppt) of the Tuzlukkol' River, the Tuzluchnoe Lake (South Urals, Russia) and the Black Sea. All the strains were characterised with light microscopy and electron microscopic study of the whole mount preparations. Molecular phylogenetic analysis has put SSU rDNA sequences, obtained for all strains, inside Marophryidae clade sister to M. marina. The organisms have a cell diameter of 4-11 µm and are surrounded with organic spicules of two types. Short (0.6-0.7 µm) and thin (0.01-0.02 µm) mostly tangentially oriented spicules form a lax sheath, surrounding the cell. Longer (3-6 µm) and thicker (0.04-0.05 µm) spicules are embedded in this sheath and are radially or obliquely oriented. Energy-dispersive X-ray analysis has shown that the spicules are purely organic. The taxonomy of marine Heterophrys-like organisms is discussed.

Design and Antioxidant Properties of Bifunctional 2H-Imidazole-Derived Phenolic Compounds-A New Family of Effective Inhibitors for Oxidative Stress-Associated Destructive Processes.

The synthesis of inhibitors for oxidative stress-associated destructive processes based on 2H-imidazole-derived phenolic compounds affording the bifunctional 2H-imidazole-derived phenolic compounds in good-to-excellent yields was reported. In particular, a series of bifunctional organic molecules of the 5-aryl-2H-imidazole family of various architectures bearing both electron-donating and electron-withdrawing substituents in the aryl fragment along with the different arrangements of the hydroxy groups in the polyphenol moiety, namely derivatives of phloroglucinol, pyrogallol, hydroxyquinol, including previously unknown water-soluble molecules, were studied. The structural and antioxidant properties of these bifunctional 5-aryl-2H-imidazoles were comprehensively studied. The redox transformations of the synthesized compounds were carried out. The integrated approach based on single and mixed mechanisms of antioxidant action, namely the AOC, ARC, Folin, and DPPH assays, were applied to estimate antioxidant activities. The relationship "structure-antioxidant properties" was established for each of the antioxidant action mechanisms. The conjugation effect was shown to result in a decrease in the mobility of the hydrogen atom, thus complicating the process of electron transfer in nearly all cases. On the contrary, the conjugation in imidazolyl substituted phloroglucinols was found to enhance their activity through the hydrogen transfer mechanism. Imidazole-derived polyphenolic compounds bearing the most electron-withdrawing functionality, namely the nitro group, were established to possess the higher values for both antioxidant and antiradical capacities. It was demonstrated that in the case of phloroglucinol derivatives, the conjugation effect resulted in a significant increase in the antiradical capacity (ARC) for a whole family of the considered 2H-imidazole-derived phenolic compounds in comparison with the corresponding unsubstituted phenols. Particularly, conjugation of the polyphenolic subunit with 2,2-dimethyl-5-(4-nitrophenyl)-2H-imidazol-4-yl fragment was shown to increase ARC from 2.26 to 5.16 (104 mol-eq/L). This means that the considered family of compounds is capable of exhibiting an antioxidant activity via transferring a hydrogen atom, exceeding the activity of known natural polyphenolic compounds.

Real-Time Temperature Monitoring of Photoinduced Cargo Release inside Living Cells Using Hybrid Capsules Decorated with Gold Nanoparticles and Fluorescent Nanodiamonds.

Real-time temperature monitoring within biological objects is a key fundamental issue for understanding the heating process and performing remote-controlled release of bioactive compounds upon laser irradiation. The lack of accurate thermal control significantly limits the translation of optical laser techniques into nanomedicine. Here, we design and develop hybrid (complex) carriers based on multilayered capsules combined with nanodiamonds (NV centers) as nanothermometers and gold nanoparticles (Au NPs) as nanoheaters to estimate an effective laser-induced temperature rise required for capsule rupture and further release of cargo molecules outside and inside cancerous (B16-F10) cells. We integrate both elements (NV centers and Au NPs) in the capsule structure using two strategies: (i) loading inside the capsule's cavity (CORE) and incorporating them inside the capsule's wall (WALL). Theoretically and experimentally, we show the highest and lowest heat release from capsule samples (CORE or WALL) under laser irradiation depending on the Au NP arrangement within the capsule. Applying NV centers, we measure the local temperature of capsule rupture inside and outside the cells, which is determined to be 128 ± 1.12 °C. Finally, the developed hybrid containers can be used to perform the photoinduced release of cargo molecules with simultaneous real-time temperature monitoring inside the cells.

Hyperhomocysteinemia increases susceptibility to cortical spreading depression associated with photophobia, mechanical allodynia, and anxiety in rats.

Epidemiological data suggest that elevated homocysteine is associated with migraine with aura. However, how homocysteine contributes to migraine is still unclear. Here, we tested whether hyperhomocysteinemia (hHCY) promotes cortical spreading depression (CSD), a phenomenon underlying migraine with aura, and whether hHCY contributes to pain behavior. hHCY was induced by dietary methionine in female rats while the testing was performed on their 6-8week-old offspring. CSD and multiple unit activity (MUA) induced by KCl were recorded from the primary somatosensory cortex, S1, using multichannel electrodes. In hHCY rats, compared to control, we found: i) higher probability of CSD occurrence; ii) induction of CSD by lower concentrations of KCl; iii) faster horizontal propagation of CSD; iv) smaller CSD with longer duration; v) higher frequency of MUA at CSD onset along with slower reappearance. Rats with hHCY demonstrated high level of locomotor activity and grooming while spent less time in the central area of the open field, indicating anxiety. These animals showed light sensitivity and facial mechanical allodinia. Thus, hHCY acquired at birth promotes multiple features of migraine such as higher cortical excitability, mechanical allodynia, photophobia, and anxiety. Our results provide the first experimental explanation for the higher occurrence of migraine with aura in patients with hHCY.

Do Mitochondrial DNA Mutations Play a Key Role in the Chronification of Sterile Inflammation? Special Focus on Atherosclerosis.

BACKGROUND: The aim of the elucidation of mechanisms implicated in the chronification of inflammation is to shed light on the pathogenesis of disorders that are responsible for the majority of the incidences of diseases and deaths, and also causes of ageing. Atherosclerosis is an example of the most significant inflammatory pathology. The inflammatory response of innate immunity is implicated in the development of atherosclerosis arising locally or focally. Modified low-density lipoprotein (LDL) was regarded as the trigger for this response. No atherosclerotic changes in the arterial wall occur due to the quick decrease in inflammation rate. Nonetheless, the atherosclerotic lesion formation can be a result of the chronification of local inflammation, which, in turn, is caused by alteration of the response of innate immunity. OBJECTIVE: In this review, we discussed potential mechanisms of the altered response of the immunity in atherosclerosis with a particular emphasis on mitochondrial dysfunctions. CONCLUSION: A few mitochondrial dysfunctions can be caused by the mitochondrial DNA (mtDNA) mutations. Moreover, mtDNA mutations were found to affect the development of defective mitophagy. Modern investigations have demonstrated the controlling mitophagy function in response to the immune system. Therefore, we hypothesized that impaired mitophagy, as a consequence of mutations in mtDNA, can raise a disturbed innate immunity response, resulting in the chronification of inflammation in atherosclerosis.