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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Rogovaya OS, Alpeeva EV, Ruchko ES, Eremeev AV, Vorotelyak EA

Tissue-engineered constructs (TECs), the dermal equivalent (DE) and the skin equivalent (SE), are allogenic equivalents of the skin and derm used to treat critical skin loss. Selection of storage conditions that contribute to longer shelf life, thereby expanding the possibilities of logistics and use, is one of the major issues related to the TECs development. The study was aimed to determine the shelf life of the DE and SE TECs stored in normal saline at room temperature by assessing morphology and viability of the cells on their surface, along with the levels of endothelial growth factor (VEGF) secreted by these cells. Using the MTT assay and staining with vital dye, we discovered the following: when TECs of both types were stored in normal saline, the cells viability and metabolic activity decreased by more than 50% by days 3–4 of storage. Furthermore, these decreased faster in DEs than in SEs. Morphology of the cells isolated from DEs and SEs after the 3-day storage remained unchanged. Mesenchymal stem cells on the surface of TECs kept producing VEGF after TECs culture medium was changed for saline solution (confirmed by immunofluorescence assay), which could indicate that the cells retained essential secretory activity.

Received: 2022-12-16

Published online: 2023-01-30

DOI: 10.24075/brsmu.2023.003

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VIEWS 2512

Sitnikov DS, Ilina IV, Filatov MA, Silaeva YY

The zona pellucida (ZP) is a dynamically changing object that plays an important role during the preimplantation stage of embryogenesis. The ZP thickness may affect the implantation success and pregnancy rate, it is considered as a prognostic factor in a number of studies. The study was aimed to assess the dynamic changes in the mouse embryonic ZP thickness after laser assisted hatching (LAH) that involved breaching the ZP integrity at the blastocyst stage. Femtosecond laser pulses were used to perform the zona microsurgery. The zona thickness was measured both at the stage of blastocyst microsurgery (~Е3.5, i.e. 3.5 days of embryogenesis) and at the hatching stage (~Е5). Significant differences in the ZP thickness were revealed in the control group of embryos: from 6.21 µm (Е3.5) to 5.4 µm (Е5). The changes in thickness from 6.6 µm (Е3.5) to 6.2 µm (Е5) observed in the group subjected to LAH were non-significant. Tracing the ZP thickness of a particular embryo from the blastocyst stage to the hatching stage made it possible to estimate the thinning coefficients in the experimental and control groups. The findings that indicate lower tensile strength of the zona in case of LAH can provide the basis for further research on the ZP properties in case of using the embryo cryopreservation protocols.

Received: 2022-11-09

Published online: 2023-01-27

DOI: 10.24075/brsmu.2023.002

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VIEWS 2555

Postnikov MA, Butvilovsky AV, Alsharifi A AM, Madatyan AV, Kopetskiy IS, Eremin DA

Anatomical features of the teeth should be accounted for dental treatment plans. The need for constant monitoring of changes in the dentition system determines the relevance of this research. The study aimed to establish the size of anterior teeth with the help of odontometry. We made bi-layer single stage impressions and cast diagnostic models of the anterior teeth of 50 male and 50 female participants aged 18–24 years. The absolute sizes of crowns of incisors and canines were established. To assess the reduction of lateral incisors, we calculated the interincisor index (Ii) of teeth 22 and 21; sexual dimorphism was determined using the Garn–Lewis formula. It was discovered that there are no differences in the mesiodistal widths of crowns of contralateral teeth on the right and left sides (p > 0.05). The mesiodistal width of crowns of anterior teeth decreases (significant changes) in the following order: maxillary central incisors → maxillary canines → mandibular canines and maxillary lateral incisors → mandibular lateral incisors → mandibular central incisors. The degree of reduction of lateral incisors is low (Ii = 74.9) and more prominent in males than in females. In the examined patients, the greatest mean length of crowns of anterior teeth is that of upper central incisors and lower canines, while upper canines are shorter in length and upper lateral incisors, lower central and lateral incisors have the shortest mean crown length. Males have longer (mean length) crowns of lower canines, upper incisors and canines than females, the difference being significant (p < 0.001). The parameters of the crowns determined in this study showed that they have sufficient height and mesiodistal width, which, together with the low degree of reduction of the lateral incisors, justifies the possibility of direct fabrication of orthodontic fixed retainers. The data can also be used at the stage of dental treatment planning.

Received: 2022-11-11

Published online: 2022-12-31

DOI: 10.24075/brsmu.2022.069

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VIEWS 2128

Siniavin AE, Russu LI, Vasina DV, Shidlovskaya EV, Kuznetsova NA, Gushchin VA, Gintsburg AL

The COVID-19 disease pandemic remains a significant global problem, resulting in hundreds of millions of cases and millions of deaths. The search for specific inhibitors of SARS-CoV-2 for the treatment of this infection remains relevant. Drugs such as Favipiravir and Molnupiravir, which exhibit specific antiviral activity against SARS-CoV-2, are already being used to treat patients. However, there is limited evidence of their effectiveness, especially against novel genetic variants of the COVID-19 pathogen. The aim of this study was to investigate the antiviral effect of these drugs using an in vitro experimental model of SARS-CoV-2 infection in Vero E6 cell culture and an animal model of infection using Syrian hamsters. It has been established that Molnupiravir has an inhibitory effect against variants of the SARS-CoV-2 with IC50 values from 16.51 to 7.88 μM in vitro, and reduces the infectious titer of the virus in the lungs of animals by ~1.5 Log10 in vivo, in while Favipiravir shows lower activity and severe toxicity. Dose selection and frequency of use remain unexplored.

Received: 2022-12-05

Published online: 2022-12-31

DOI: 10.24075/brsmu.2022.071

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VIEWS 2468

Bormotov DS, Shamraeva MA, Kuzin AA, Shamarina EV, Eliferov VA, Silkin SV, Zhdanova EV, Pekov SI, Popov IA

The primary method of initial treatment of meningiomas is radical neurosurgical intervention. Various methods of intraoperative diagnostics currently in development aim to improve resection efficiency; we focus on methods based on molecular profiling using ambient ionization mass spectrometry. Such methods have been proven effective on various tumors, but the specifics of the molecular structure and the mechanical properties of meningiomas raise the question of applicability of protocols developed for other conditions for this particular task. The study aimed to compare the potential clinical use of three methods of ambient ionization in meningioma sample analysis: spray from tissue, inline cartridge extraction, and touch spherical sampler probe spray. To this end, lipid and metabolic profiles of meningioma tissues removed in the course of planned neurosurgical intervention have been analyzed. It is shown that in clinical practice, the lipid components of the molecular profile are best analyzed using the inline cartridge extraction method, distinguished by its ease of implementation and highest informational value. Analysis of oncometabolites with low molecular mass is optimally performed with the touch spherical sampler probe spray method, which scores high in both sensitivity and mass-spectrometric complex productivity.

Received: 2022-11-27

Published online: 2022-12-30

DOI: 10.24075/brsmu.2022.072

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VIEWS 2120

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 2281

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 2492

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

Comments 0

VIEWS 2501