Vol 51, No 1 (2025)

Phagocytes activation results in the production of reactive oxygen metabolites (ROM) exerting antimicrobial and host-damaging activity. Although the main pool of papers shows their potentiating action on a key humoral nexus of innate immunity, complement system, the data are controversial. Combined action of ROM with antimicrobial peptides of phagocytes also remains poorly characterized. We have investigated the influence of oxidative burst products on complement activation in different in vitro models. Hydrogen peroxide, including that in medium with Fe-EDTA did not affect parameters of complement activity in human blood serum. HOCl in millimolar concentrations stimulated production of C3a and C5a anaphylatoxins in 80% serum, the effect was inhibited by EDTA. We have identified bivalent ions-independent C5 cleavage in the presence of 16 mM HOCl. At the same time, HOCl served as an inhibitor of the alternative complement pathway in the model of surface-associated activation on rabbit erythrocytes in 5% serum. It inhibited production of C3a (IC₅₀ ~ 4 mM) and C5a as well as serum hemolytic activity (IC₅₀ ~ 0.2 mM); the inhibition of C5a generation was less pronounced in the presence of 4–16 mM HOCl. Decrease in anaphylatoxins generation was also observed in the system with zymosan in 5% serum. Under similar conditions but without activating surfaces, moderate HOCl concentrations enhanced C3a and C5a accumulation; EDTA inhibited this effect completely (C3a) or partially (C5a). Finally, in 70% serum, 16 mM HOCl enhanced the anaphylatoxins accumulation but in the presence of zymosan it inhibited this process almost completely. We hypothesize that HOCl can attack the thioester bond in C3 protein to form C3(HOCl) adduct which is capable of fluid-phase convertases formation; however, the attack of C3b can prevent its covalent fixation on membranes and blocks the complement amplification loop. Besides, we have demonstrated the additive character of the combined action of HOCl with antimicrobial peptides (LL-37 cathelicidin and α-defensins) towards Listeria monocytogenes and Escherichia coli. The data obtained precise the picture of the interaction between bactericidal factors of phagocytes and complement as key participants of the immune defense and host damage.

Pyrimidine base derivatives, having a wide spectrum of pharmacological activity along with low toxicity, are used as active ingredients of many drugs. Thus, many compounds of the uracil series have antitumor, anti-inflammatory, antiviral, immunomodulatory effects, in connection with which the synthesis of new biologically active derivatives of the pyrimidine series is relevant. It is known that the mechanism of hepatotoxicity of chemical compounds is largely associated with the activation of lipid peroxidation, therefore, uracil derivatives containing a proton-donor group in position C5 were chosen as objects of study, which significantly increases the antioxidant properties of the compound. For the synthesis of uracil derivatives at N¹, N³ positions, modification of 5-hydroxy- and 5-amino-6-methyluracils with pre-protected C5 functional groups with various alkyl substituents was carried out. The method of preliminary etching of cells with the hepatotoxicant tetrachloromethane and their treatment with the studied compounds was selected as a study of hepatoprotective activity. The introduction of various alkyl substituents at the N¹, N³ positions of 5-hydroxy and 5-amino-6-methyluracils leads to an increase in the solubility of these compounds, the hepatoprotective activity of the synthesized compounds was revealed. New di- and monoalkyl derivatives of 5-hydroxy- and 5-amino-6-methyluracil were obtained, their hepatoprotective activity was tested in vitro. According to the test results, five of the new 20 synthesized compounds promote cell survival when pre-treated with tetrachloromethane.

Studies of reaction ability fluorescent dyes for the production of labeled proteins and amino acids are important for the fields of bioengineering and biomedicine, in particular for obtaining bioimages for the purpose of cell visualization, studying the structure of labeled proteins by biophysical methods. Pyrylium dyes are able to interact with amino groups of proteins to form luminescent products, which allows them to be used in the field of proteomics. It is interesting to study the conjugation pyrylium dyes, containing both polyfluorinated fragment responsible to increased lipophilicity at proteins conjugation and pyrazoline fragment demonstrating anticancer activity. Pyrylium dyes containing a pyrazoline fragment and dialkylamino substituents (piperidino-, dibutylamino-, 4-hydroxypiperidino-) in a polyfluorinated aromatic ring in the donor part were synthesized by Knoevenagel condensation reaction. The reaction of pyrylium dyes with compounds containing a primary amino group was carried out to obtain a pyridinium dyes by the ANRORC mechanism (Addition of Nucleophiles, Ring Opening and Ring Closure). The ability of pyrуlium dyes to react with bovine serum albumin (BSA) and amino acids such as Lys, Arg, Cys, Phe to form pyridinium luminophore was shown. The spectral-luminescent properties of the resulting luminophores were investigated. The product of the reaction of pyrуlium dye (Е)-2,6-dimethyl-4-(4-{3-phenyl-5-[2,3,5,6-tetrafluorо-4-(piperidine-1-yl)phenyl]-4,5-dihydro-1Н-pуrazole-1-yl}-styryl)pyrylium tetrafluoroborate with Lys was isolated and its structure was confirmed by NMR spectroscopy. The binding site of pyrylium dyes with BSA – e-amino group of Lys was determined. Along with pyridinium luminophores, in aqueous solutions hydrolysis products are formed that are not bonded with protein and absorb in the short-wavelength region. The calculated amount of luminophore bound to BSA is two molecules of pyrylium dye per one molecule of BSA. The synthesized pyrylium dyes react with BSA in the mixture of phosphate buffer with methanol (pH 7.4) 3–4 orders of magnitude faster than the well-known julolidine dye Py-1. The relative reaction rates of (Е)-2,6-dimethyl-4-(4-{3-phenyl-5-[2,3,5,6-tetrafluorо-4-(4-hydroxypiperidine-1-yl)phenyl]-4,5-dihydro-1Н-pуrazole-1-yl}styryl) pyrуlium tetrafluoroborate with amino acids were determined as Lys > Cys >> Phe ≥ Arg. The obtained polyfluoro pyrylium-pyrazolinium dyes have the application perspective in the field of bioimaging, proteomic and biomedicine, due to high conjunction rate and efficiency with BSA and amino acids.

Epigenetic modifications of histones in human, animal, and other eukaryotic cells play a crucial role in regulating gene expression. Histones can undergo a variety of post-translational modifications in different combinations, including methylation, acetylation, phosphorylation, and others at various amino acid residues, which determine the functional state of a given chromatin locus. Changes in epigenetic modifications accompany all normal and pathological cellular processes, including proliferation, differentiation, cancer transformation, and more. Currently, the development and application of new methods for analyzing the epigenome at the single-cell level, including in live cells, are particularly relevant. In this study, new sensor systems were developed for visualizing epigenetic modifications H3K9me3 (trimethylated Lys9), H3K9ac (acetylated Lys9), and the spatial colocalization of H3K9me3 with H3K9ac, based on fluorogenic dyes. The creation of these sensors involved the use of splitFAST system as well as the histone natural reader domains MPP8 and AF9. Adding the fluorogens HMBR and N871b to the cell medium allowed for the detection of clearly distinguishable fluorescence patterns in the green and red channels, respectively. We also performed the analysis of the obtained fluorescent images using the LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscape) computational method. Clustering of the resulting data showed agreement with the expected class labels corresponding to the presence of H3K9me3, H3K9ac, and the spatial colocalization of H3K9me3 and H3K9ac in the nucleus. The developed sensors can be effectively used to study histone modifications in various cellular processes, as well as in investigating disease development mechanisms.

In order to understand whether the lipid composition of the plasma membrane is related to life expectancy, in this work we conducted a comparative study of the profiles of molecular species of four main classes of plasma membrane phospholipids (phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS) and phosphatidylinositols (PI)) for the long-lived mussel Crenomytilus grayanus and the long-lived sea urchin Mesocentrotus nudus and the short-lived mussel Mytilus trossulus and the sea urchin Strongylocentrotus intermedius. Molecular profiles of these membrane lipids were determined using high-performance liquid chromatography in combination with high-resolution mass spectrometry. In this work it was shown that the profile of PI molecular species is not related to the lifespan of mussels and hedgehogs, in contrast to the profile of PC, PE, and PS molecular species. Sea urchins M. nudus and mussel C. grayanus with a longer lifespan were characterized by an increased content of PC, PE and PS with odd numbered alkyl/acyl chains and molecular species with arachidonic acid (20:4n-6), a higher content of which can contribute to a better adaptation of the mussel of C. grayanus and the sea urchin M. nudus and thus contribute to a longer lifespan. The lipidomic approach to studying the gerontological problem using sea urchin and bivalves as an example has shown a clear relationship between the profile of molecular types of membrane lipids and lifespan. The exact mechanisms of this need to be clarified further.

Combination of boron neutron capture therapy and chemotherapy can provide good efficacy in a cancer treatment. Development of therapeutic constructs that combine these two functions, the possibility of in vitro and in vivo visualization and a convenient platform for selective delivery to the tumor is of great relevance today. In this study, we focused on human serum albumin, a well-known drug delivery platform. We developed constructs based on albumin functionalized with boron clusters, analogues of the chemotherapeutic molecule gemcitabine and signaling molecules. To create the constructs, we developed new analogues of homocysteine thiolactone containing closo-dodecaborate or cobalt bis(dicarbollide) and a gemcitabine analogue containing closo-dodecaborate attached to the C5 carbon atom of the nitrogenous base. We have demonstrated that addition of the gemcitabine analogue to the conjugate structure increases its cytotoxicity towards human glioblastoma cell lines. Among the final conjugates, the highest cytotoxicity is demonstrated by the structure containing cobalt bis(dicarbollide). The final structures accumulate well in the cytoplasm of cancer cells. The albumin conjugate containing cobalt bis(dicarbollide) and a boron-containing gemcitabine analogue is capable of accumulating in the nuclei of T98G cell lines. Thus, both final albumin-based constructs showed sufficient efficacy against the human glioma cell line in vitro. We expect that the therapeutic conjugates we have constructed will significantly increase cytotoxicity against cancer cells when irradiated with epithermal neutrons. Combining a chemotherapeutic residue and a boron-containing group in a single construct provides the potential for more effective glioma therapy.

We propose a new fluorogen-activating protein nano-frFAST with only 98 amino acids based on a combination of the previously created fluorogen-activating proteins nanoFAST and frFAST. A series of fluorogens with an increased system of conjugated bonds, which are potentially capable of binding to this protein, were synthesized. Based on the results of the study, a promising fluorogen which is (Z)-5-((E)-3-(4-hydroxy-2,5-dimethoxyphenyl)allylidene)-2-thioxothiazolidin-4-one (HPAR-DOM) was identified. We demonstrate that the nano-frFAST–HPAR-DOM complex can be used as a genetically encoded far-red fluorescent label for staining individual structures of living cells.