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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Plotnikova MA, Bobkov DE, Klotchenko SA

Human MxA protein induced by type I and III interferons is an important innate immunity mediator, it shows antiviral activity against a broad spectrum of RNA and DNA viruses. According to the latest data, the MxA protein overexpression increases chemotherapy sensitivity and represents one of the favorable prognostic factors in patients with breast cancer. The exogenous mRNA capable of intracellular MxA protein production not only has the potential for treatment of viral respiratory infection, but also can become an important fundamental research tool. The study aimed to construct and produce the exogenous mRNA encoding the functional human cytoplasmic MxA protein by in vitro transcription (IVT); to study its translational properties; to assess and identify the patterns of the expression of some interferon system genes in response to introduction of this exogenous mRNA into cells. As a result of the study, the exogenous mRNAs capable of effective translation (up to 20 ng/mL of protein from 100 ng of mRNA per well of the 96-well plate) in the eukaryotic cell systems were successfully constructed and produced by IVT (in the amount of up to 200 µg); diffuse distribution of the MxA protein in the MDCK cells was confirmed; significant changes in the expression of the interferon-stimulated genes, such as OAS1, PKR (EIF2AK2), MDA5, RIG-I, were revealed. Our further research will be focused on assessing the developed exogenous mRNAs’ therapeutic potential against influenza A and B viruses, respiratory syncytial virus, and coronavirus SARS-CoV-2.

Received: 2024-09-30

Published online: 2024-10-30

DOI: 10.24075/brsmu.2024.048

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VIEWS 1155

Zlobovskaya OA, Kurnosov AS, Sheptulina AF, Glazunova EV

Recently, considerable commercialization of services for quantification of gut microbiota aimed to diagnose dysbiosis, the microbial imbalance, is observed. In the context of growing interest to the personalized approaches in medicine and preventive therapy, the diagnosis of dysbiosis is becoming increasingly important. The results of such screening are used to adjust guidelines on correction of the diet, lifestyle modification, or, where necessary, drug therapy prescription. Such assessment requires a reliable and accurate method for evaluation of microbiota, since validity of further recommendations and therapeutic interventions depends on the quality of the data obtained. The paper reports the main aspects of the two approaches used for microbiota quantification: 16S rRNA next-generation sequencing (16S NGS) and real-time PCR (qPCR). The strengths (from our perspective) and weaknesses of the approaches are also provided.

Received: 2024-10-15

Published online: 2024-10-30

DOI: 10.24075/brsmu.2024.047

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VIEWS 1261

Reshetnikov VV, Shepelkova GS, Rybakova AV, Trashkov AP, Yeremeev VV, Ivanov RA

mRNA vaccines turned out to be highly effective in combating the COVID-19 pandemic and other viral infections. Despite extensive study of mRNA vaccines in the last five years, the issue of safety of their use is still relevant. The study aimed to assess immunogenicity of two anti-tuberculosis mRNA vaccine doses in female and male rats 2 and 4 weeks after vaccination. Hematological and biochemical parameters of blood were determined within the same timeframe. The dose-dependent nature of mRNA vaccine immunogenicity was confirmed in both females and males. Vaccination led to moderate lymphopenia and neutrophilia in male rats, as well as to apparent dose-dependent and sex-related changes in blood biochemistry parameters at various time points. The findings suggest moderate toxicity of the anti-tuberculosis mRNA vaccine and the importance of assessing its toxic effects at various time points in animals of both sexes.

Received: 2024-09-16

Published online: 2024-10-30

DOI: 10.24075/brsmu.2024.045

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VIEWS 1606

Ponomarev GYu, Agdzhoyan AT, Potanina AYu, Adamov DS, Balanovska EV

Khanty and Mansi are small indigenous peoples of Western Siberia with the unique cultural, anthropological, and linguistic characteristics. The study of their gene pool will make it possible to reconstruct the genetic structure of the Ugric-speaking population, of which in modern times only Khanty and Mansi remain, along with Hungarians, in whose gene pool there are traces of medieval migration of the Ugric-speaking Magyars. The detailed characterization of the gene pool of Khanty and Mansi is important for reconstruction of Ugric populations and genetic history of the region. The study was aimed to assess representative samples of Khants (n = 83) and Mansi (n = 74) based on the standard panel of 60 SNP markers and the extended panel of 74 Y-chromosomal SNP markers by statistical and cartographic methods in the context of indigenous population of Urals and Western Siberia. The differences between the gene pools of Khanty and Mansi have been revealed based on both standard panel of Y chromosome haplogroups and branches of haplogroups N2 and N3a4. Most of the Khanty gene pool is evenly distributed between N2-Y3195 (26%), N2-VL67 (23%), and N3a4-Z1936 (23%). The “Western” branch N2-Y3195 predominates in the Mansi gene pool (69%). Mansi gravitate towards populations of the Urals-Volga region in the multidimensional genetic space. Based on the standard panel of Y haplogroups, Khanty are close to the populations of Western and South Siberia. However, the analysis of branches N3a4 has shown that Khanty are intermediate between the “Uralic” and “Siberian” clusters: when the ancestors of Khanty moved from the Ural region to the northeast, these acquired both genetic components. The gene geographic maps of 10 haplogroup N3a4 branches in the populations of Urals and Western Siberia reflect the dynamic changes of the gene pool that took place 4–2 kya.

Received: 2024-09-09

Published online: 2024-10-28

DOI: 10.24075/brsmu.2024.044

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VIEWS 1879

Sukhov DA, Kholoshenko IV, Petrova TV, Romanenko GA, Myshkin MYu, Kost VYu, Trofimov DYu, Usman NYu, Barsova EV

The novel ribonuclease inhibitor LoRI is a 63 kDa recombinant protein optimized for high-throughput expression in E. coli and purification by metal chelate affinity chromatography (IMAC). The product was obtained by N-terminal fusion of mouse placental RNase inhibitor polypeptide to a thioredoxin module. Advantage of the engineering strategy in terms of protein structure and function was predicted in silico. Under laboratory settings, the yield of purified soluble recombinant product was about 12 mg per 1 L of expression bacterial culture. By RNase inhibition capacity in vitro, the product is comparable or superior to a commercial reference. The kinetic data comply with Lineweaver-Burk model.

Received: 2024-09-02

Published online: 2024-10-28

DOI: 10.24075/brsmu.2024.043

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VIEWS 1379

Mayanskiy NA, Brzhozovskaya EA, Skvortsov-Igralov GA, Chausova SV, Chebotar IV

Pseudomonas aeruginosa, the opportunistic pathogen, occupies one of the leading places in the structure of pathogens causing nosocomial infections, which is due to high adaptive potential and the ability to quickly develop antimicrobial resistance. The study aimed to assess the influence of the P. aeruginosa adaptation to colistin on bacterial fitness. A total of nine isolates obtained during the experimental evolution of the P. aeruginosa strain (laboratory number 1202) under conditions of increasing colistin concentrations, the growth kinetics of which was compared to that of wild type strain, were included in the study; the whole genome sequencing of all isolates was performed, and the minimum inhibitory concentration of colistin was determined. Growth rate was estimated using the Varioskan LUX multimodal reader (Thermo Scientific, USA) throughout 18 h at 37 °С; optical density (OD) at λ = 600 nm was measured every 15 min. The maximum growth rate (GRmax, i.e. the maximum change in OD within 1h) and the time to reach 50% of the maximum OD reported when growing the wild type Ра_1202_0 strain (T_OD50%) were considered. Isolates of the clone carrying mutations of the genes phoQ, lptA, and prs showed low fitness values compared to wild type strains. For example, GRmax of the isolate Ра_1202_43 was 0.029 OD/h vs. 0.182 OD/h reported for the original isolate Ра_1202_0, and it reached OD50% 4.6 h later. The growth characteristics of the clone carrying mutations of lpxL and lptB, as well as the clone carrying mutant pmrB were generally comparable with the characteristics of the wild type strain. Thus, the genome modifications observed during the P. aeruginosa adaptation to colistin have an ambiguous effect on bacterial fitness.

Received: 2024-08-22

Published online: 2024-10-19

DOI: 10.24075/brsmu.2024.042

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VIEWS 1258

Zinchenko RA, Tebieva IS, Kadyshev VV, Murtazina AF, Borovikov AO, Marakhonov AV, Perepelov AV, Getoeva ZK, Kutsev SI

Currently, there is limited understanding about the cumulative prevalence, diversity, and frequency of distinct orphan hereditary diseases (OHDs) in the pediatric population, both within the Russian Federation and in the global literature. This gap exists despite a significant demand for such knowledge in healthcare and society. Variability and heterogeneity of the above indicators are common across different populations, reflecting significant genetic heterogeneity of OHDs. The study aimed to assess OHDs in the pediatric population of the Republic of North Ossetia – Alania (RNO-A). A total of 543,817 people were evaluated, including 145,560 children aged 0–18 years. The cumulative prevalence of autosomal recessive (AR), autosomal dominant (AD), and X-linked (XL) OHDs was determined. The findings indicate an overall prevalence of OHDs among children of the RNO-A of 1 : 119, meaning that approximately 1% of children are diagnosed with these conditions. Notably, the total burden in children of all types of OHDs in rural areas exceeds that in urban areas and district centers by more than twofold. We identified 1,241 patients from 1,037 families with 241 distinct OHDs (109 with AD inheritance, 102 with AR inheritance, and 30 with XL inheritance). Three diseases were particularly prevalent in this population and have not been documented in similar studies: congenital myasthenia type 12, a rare form of congenital adrenal cortex dysfunction (3-beta-hydroxysteroid dehydrogenase deficiency), and brachydactyly E — amelogenesis — mental retardation — nanism syndrome. Thus, the population of the RNO-A exhibits a unique spectrum of OHDs caused by rare mutations, some of which are infrequent in other populations of the world and the Russian Federation. The significantly higher prevalence of these disorders in rural populations is noteworthy, underscoring the need for tailored, region-specific programs aimed at preventing childhood disability and/or mortality.

Received: 2024-08-06

Published online: 2024-10-17

DOI: 10.24075/brsmu.2024.041

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VIEWS 1484

Shmitko AO, Bulusheva IA, Vasiliadis YuA, Suchalko ON, Syrko DS, Belova VA, Pavlova AS, Korostin DO

The MGI (MGI Tech Co. Ltd., China) next-generation sequencing platform, including the DNBSEQ-G50, -G400, and -T7 sequencers, is being actively adopted in research. Despite its widespread adoption, challenges persist in the form of limitations associated with the manufacturer's provided barcode set for library preparation. These limitations include constraints on the number of samples that can be concurrently sequenced, compatibility issues with barcodes from diverse or incomplete sets, and restrictions on the sample ratio. Purpose: to develop a universal method that allows sequencing of up to 252 samples simultaneously on a single sequencer lane, while eliminating barcode-related limitations. We proposed a “quad method” that provides 4 or 4n+2 equilibration of barcodes. This paper also delves into its comprehensive analysis, verification procedures, seamless integration into the sequencing process and validation of the method on the DNBSEQ G-400 platform. The quad method showed efficiency and reliability, allowing sequencing of up to 252 samples simultaneously without compromising data quality. The proposed method optimizes library preparation and improves the flexibility of sequencing on the MGI platform.

Received: 2024-08-15

Published online: 2024-10-10

DOI: 10.24075/brsmu.2024.040

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VIEWS 1672