Bulletin of RSMU

ISSN Print 2500–1094 ISSN Online 2542–1204

Bulletin of RSMU

BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)

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Kozhin PM, Romashin DD, Rusanov AL, Luzgina NG

The HaCaT cell line represents the spontaneously immortalized non-carcinogenic human keratinocytes that are used as a model for studying the function of normal human keratinocytes. There are two TP53 alleles in the HaCaT cell genome, which comprise two gain-of-function (GOF) mutations acquired through spontaneous immortalization (mutTP53). Mutations result in the increased proliferation rate and violation of the stratification program. The study was aimed to assess the effects of the mutTP53 gene knockout on the HaCaT keratinocytes capability of proliferation and migration in the in vitro model of epidermal injury and regeneration (scratch test), and on the ability to form stratified epithelium in the organotypic epidermal model. To perform the scratch-test, cells were cultured until monolayer was formed, then the standardized injury was created. The organotypic model was obtained by growing keratinocytes in the polycarbonate membrane inserts with the pore size of 0.4 μm at the interface between the phases (air-liquid). It has been shown that the mutant TP53 gene knockout results in the increased migration capability of the HaCaT keratinocytes: in the HaCaT with the mutTP53 knockout, the defect closure occurred faster than in the appropriate group of the WT HaCaT (p < 0.05), on day three the defect size was 12% ± 3% and 66% ± 5% of the initial size. There is evidence that mutant TP53 in the HaCaT cells is a negative regulator of the laminin 5 expression (LAMC2 expression was 9.96 ± 1.92 times higher in the cells with the mutTP53 knockout, p < 0.05), however, this does not promote normalization of the program of epithelial differentiation and stratification followed by formation of the stratum corneum in the organotypic model.

Received: 2022-12-12

Published online: 2022-12-30

DOI: 10.24075/brsmu.2022.070

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VIEWS 1004

Shmygarev VI, Prokopenko YuA, Terekhov SS, Zakharova MYu, Dubinny MA, Smirnov IV, Yampolsky IV, Tsarkova AS

Coronavirus disease COVID-19, caused by the SARS-CoV-2 virus, is highly contagious and has a severe morbidity. Providing care to patients with COVID-19 requires the development of new types of antiviral drugs. The aim of this work is to develop a prodrug for the treatment of coronavirus disease using the antibiotic Amicoumacin A (Ami), the mechanism of action of which is based on translation inhibition. Enzymatic hydrolysis of an inactivated prodrug by the SARS-CoV-2 main protease can lead to the release of the active Ami molecule and, as a consequence, the suppression of protein biosynthesis in infected cells. To test the proposed hypothesis, a five-stage synthesis of an inactivated analogue of Amicoumacin A was carried out. Its in vitro testing with the SARS-CoV-2 recombinant protease MPro showed a low percentage of hydrolysis. Further optimization of the peptide fragment of the inactivated analog recognized by the SARS-CoV-2 MPro protease may lead to an increase in proteolysis and the release of Amicoumacin A.

Received: 2022-11-15

Published online: 2022-12-30

DOI: 10.24075/brsmu.2022.073

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VIEWS 1259

Kirshina AS, Kazakova AA, Kolosova ES, Imasheva EA, Vasileva OO, Zaborova OV, Terenin IM, Muslimov AR, Reshetnikov VV

It has been proven that mRNA vaccines are highly effective against the COVID-19 outbreak, and low prevalence of side effects has been shown. However, there are still many gaps in our understanding of the biology and biosafety of nucleic acids as components of lipid nanoparticles (LNPs) most often used as a system for inctracellular delivery of mRNA-based vaccines. It is known that LNPs cause severe injection site inflammation, have broad biodistribution profiles, and are found in multiple tissues of the body, including the brain, after administration. The role of new medications with such pharmacokinetics in inflammation developing in inaccessible organs is poorly understood. The study was aimed to assess the effects of various doses of mRNA-LNP expressing the reporter protein (0, 5, 10, and 20 μg of mRNA encoding the firefly luciferase) on the expression of neuroinflammation markers (Tnfα, Il1β, Gfap, Aif1) in the prefrontal cortex and hypothalamus of laboratory animals 4, 8, and 30 h after the intramuscular injection of LNP nanoemulsion. It was shown that mRNA-LNP vaccines in a dose of 10–20 μg of mRNA could enhance Aif1 expression in the hypothalamus 8 h after vaccination, however, no such differences were observed after 30 h. It was found that the Gfap, l11β, Tnfα expression levels in the hypothalamus observed at different times in the experimental groups were different. According to the results, mRNA-LNPs administered by the parenteral route can stimulate temporary activation of microglia in certain time intervals in the dose-dependent and site specific manner.

Received: 2022-12-01

Published online: 2022-12-30

DOI: 10.24075/brsmu.2022.068

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VIEWS 1229

Novoselova AV, Yushina MN, Patysheva MR, Prostakishina EA, Bragina OD, Garbukov EYu, Kzhyshkowska JG

Monocytes are large circulating white blood cells that are the main precursors of tissue macrophages as well as tumor-associated macrophages in the adult body. Different types of monocytes have multidirectional effects on the growth and metastatic spread of cancer cells, both activating and inhibiting these processes. Tumor progression is associated with the triggering of a whole cascade of inflammatory and immune reactions. These pathological processes are associated with changes in the amino acid content of monocytes, which can lead to disruption of their function, in particular their migration, division and maturation. The aim of the work was to profile the amino acids of monocytes, followed by a study of the amino acid composition of monocytes from patients with breast cancer using liquid chromatography with mass spectrometric detection. Significant differences in metabolite levels in monocytes of breast cancer patients and monocytes of healthy donors were found for glycine (p-value = 0.0127), asparagine (p-value = 0.0197), proline (p-value = 0.0159), methionine (p-value = 0.0357), tryptophan (p-value = 0.0028), tyrosine (p-value = 0.0127). In the study, we identified biological networks that could potentially be involved in altering the phenotype of monocytes affected by breast cancer (BC), using bioinformatic analysis of metabolic pathways involving the discovered amino acids. Mathematical models based on amino acid combinations with 100% sensitivity and specificity have been developed. Features of immune system cell metabolism in BC have been identified and potential diagnostic biomarkers have been proposed.

Received: 2022-12-08

Published online: 2022-12-28

DOI: 10.24075/brsmu.2022.064

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VIEWS 1054

Chebotar IV, Bocharova YuA, Chaplin AV, Savinova TA, Vasiliadis YuA, Mayanskiy NA

The carbapenem-resistant strains of Pseudomonas aeruginosa are considered as the dangerous pathogens of critical priority. Deciphering the mechanisms underlying the development of carbopenem resistance is an urgent challenge faced by modern medical science. The study was aimed to describe the diversity and fixation of mutations associated with the development of carbapenem resistance during the P. aeruginosa adaptation to the increasing meropenem concentrations. The objects of the study were P. aeruginosa isolates obtained by growing the ATCC 27853 P. aeruginosa reference strain exposed to increasing concentrations of meropenem. The isolates were tested for meropenem susceptibility using E-tests (Epsilometer tests) and by the agar dilution method. Genomes of the isolates were sequenced in the MGISEQ-2000 whole-genome sequencer. The findings show that in experimental settings P. aeruginosa develops high meropenem resistance very quickly (in 6 days). Evolution of resistance is associated with cloning involving the emergence of multiple clones with various genotypes. Mutagenesis that involves 11 genes, including oprD, pbuE, nalD, nalC, spoT, mlaA, mexD, mexR, oprM, mraY, pbp3, provides the basis for cloning. Regardless of the levels of their meropenem resistance, some of the emerging clones do not progressively develop and are replaced by more successful clones.

Received: 2022-11-25

Published online: 2022-12-28

DOI: 10.24075/brsmu.2022.063

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VIEWS 1206

Terekhov SS, Shmygarev VI, Purtov KV, Smirnov IV, Yampolsky IV, Tsarkova AS

The increasing size and density of the human population is leading to an increasing risk of infectious diseases that threaten to spread yet another pandemics. The widespread use of vaccination has reduced morbidity and mortality associated with viral infections and in some cases eradicated the virus from the population entirely. Regrettably, some virus species retain the ability to mutate rapidly and thus evade the vaccine-induced immune response. New antiviral drugs are therefore needed for the treatment and prevention of viral diseases. Modern research into the structures and properties of viral proteases, which are of key importance in the life cycle of viruses, makes it possible, in our opinion, to turn these enzymes into promising targets for the development of effective viral disease control methods.

Received: 2022-12-05

Published online: 2022-12-28

DOI: 10.24075/brsmu.2022.067

Comments 0

VIEWS 1669

Timofeeva AV, Fedorov IS, Tarasova AM, Gorina KA, Suhova YuV, Gusar VA, Ivanets TYu

Preeclampsia (PE) occurs in 2–8% of pregnancies. It is one of the leading causes of maternal and perinatal morbidity and mortality. Today, there are no tests adopted by the practitioners that enable accurate prediction of early (weeks 20 through 34) or late (after week 34) onset of PE when the pregnancy is in its 11th to 14th week. This study aimed to evaluate the feasibility of using secretory clusterin quantification to predict early or late PE during the first trimester of pregnancy. The choice of this protein is determined, on the one hand, by the specificity of its expression for cytotrophoblast, syncytiotrophoblast, and extracellular trophoblast cells, and, on the other hand, by the proven negative effect of clusterin on the invasive properties of trophoblastic cells and gestational transformations of uterine vessels, which play a key role in the pathogenesis of PE. The study included 40 pregnant women aged 27–40 years who underwent a comprehensive screening examination in the first trimester of pregnancy. Western blotting revealed a significant increase in the level of secretory clusterin (40 kDa) in the blood serum of pregnant women in the case of PE compared to physiological pregnancy: in early-onset PE, a twofold increase in the level of clusterin in the vesicular and extravesicular fractions of blood serum (p = 0.03 and p = 0.004, respectively), with late-onset PE — a threefold increase only in the extravesicular fraction of blood serum (p = 0.002). According to logistic regression models, the level of secretory clusterin in the extravesicular fraction of blood serum of pregnant women in the first trimester has prognostic significance in assessing the likelihood of developing early-onset PE (AUC = 0.97, Se = 1, Sp = 0.875, cutoff = 0.3877) and late-onset PE ( AUC = 1, Se = 1, Sp = 1, cutoff = 0.5).

Received: 2022-11-23

Published online: 2022-12-28

DOI: 10.24075/brsmu.2022.061

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VIEWS 1050

Stavskaya NI, Ilchuk LA, Okulova YuD, Kubekina MV, Varlamova EA, Silaeva YY, Bruter AV

The CDK8 cyclin-dependent transcription-associated kinase and its less studied paralog, CDK19, regulate the expression of the dependant genes via several mechanisms. CDK8/19 can directly phosphorylate some transcription factors (ICN, STAT1), but at the same time these kinases being a component of the mediator complex regulate transcrition via interaction with chromatin in the promoter and enhancer regions of appropriate genes. Recently the papers have appeared showing that CDK8/19 has kinase-independent mechanisms of action through comparison of the effects of the kinase activity genetic inactivation and chemical inhibition. The study was aimed to generate transgenic mice capable of the induced and tissue-specific expression of the kinase-negative (showing no phosphorylation activity) form of CDK8, CDK8 (D173A), which could be later used to study the CDK8 kinase-independent mechanisms of action in vivo. We obtained four F0 transgenic animals by microinjection of linear DNA into the pronucleus, two of these animals became the ancestors of two distinct lines. The copy number of the integrated construct was measured for all F0 and the lines generated. This model may be used to study the kinase-independent properties of the CDK8/19 proteins.

Received: 2022-11-22

Published online: 2022-12-28

DOI: 10.24075/brsmu.2022.066

Comments 0

VIEWS 1176