Diana Andreevna Reznikova

DNA sequencing technologies play a key role in modern soil microbiome research, providing a deep understanding of its structure and functional role in ecosystems. 16S rRNA gene, region of 18S-ITS-28S sequencing and shotgun sequencing using modern sequencing technologies (Illumina, Pacific Biosciences (PacBio), Oxford Nanopore Technologies (ONT)) allow us to identify the diversity and dynamics of microbial communities with high accuracy and resolution, which significantly expands our knowledge of biological processes and interactions between microorganisms in the soil. Soil microbiome analysis using sequencing contributes to the development of innovative methods for sustainable agricultural land management, improved fertility, plant disease management, and increased crop yields. Despite its significant potential, each sequencing technology has its own advantages and limitations related to accuracy, depth of coverage, cost, and data analysis complexity. Understanding these characteristics is crucial for selecting the optimal methods depending on the research objectives and available resources. This review systematizes modern sequencing methods, their technical capabilities and limitations, bioinformatics tools for sequencing data analysis, considers examples of successful applications in the study of soil microbiome in various ecosystems, and emphasizes new trends in metagenomics. In-depth study and development of soil microbiome sequencing technologies contributes to more sustainable and resource-efficient agriculture, emphasizing the need for a comprehensive and informed approach to the analysis of microbial communities.

Since there is currently no cure for Parkinson’s disease, pharmacobiotic approaches based on gut microbiota—capable of producing pharmacologically active compounds—are under development. In this study, we propose LfU21, derived from the strain Limosilactobacillus fermentum U-21, as a candidate pharmacobiotic. To evaluate its efficacy, a combined LPS- and lactacystin (LAC)-induced Parkinson’s disease model was established in Wistar rats. Effects were assessed using behavioral, biochemical, immunomodulatory, and transcriptomic biomarkers. LfU21 administration reduced α-synuclein levels, altered motor performance in the “Rung ladder” test, and modulated bdnf gene expression in the right and left striata. Under LPS exposure, LfU21 prevented alterations in immune response markers, GSH levels, drd2 and bdnf gene expression, and intestinal goblet cell counts. In LAC and LAC + LPS groups, LfU21 mitigated the rise in α-synuclein, the decline in bdnf expression, and behavioral deficits in the “Open Field” and “Rung ladder” tests, respectively. The multifunctional activity of LfU21 in a combined Parkinson’s disease model underscores its therapeutic potential and helps identify a target patient cohort for future clinical trials.

Since there is no cure for Parkinson's disease yet, pharmacobiotic drugs based on the gut microbiota capable of producing the necessary pharmacologically active compo-nents are being developed. The drug LfU21 based on the strain of Limosilactobacillus fermentum U-21 is proposed as a candidate for pharmacobiotics in this work. To char-acterize the effectiveness of the studied drug, a combined LPS and lactacystin (LAC) induced PD model in the Wistar rat line was used. The analysis was performed using behavioral, biochemical, immune, and transcriptomic biomarkers. LfU21 reduces the level of α-synuclein, changes motor activity in the “Rung ladder” test and the expres-sion of the bdnf gene in both hemispheres of the brain. When exposed to LPS, LfU21 prevents changes in the level of the immune response, GSH, expression of the drd2 and bdnf genes, and the number of goblet cells in the intestine. When exposed to LAC and LAC+LPS, LfU21 prevents an increase in α-synuclein, a decrease in bdnf expression, and indicators in the “Open Field” and “Rung ladder” tests, respectively. The effect of LfU21 in a combined model of Parkinson's disease confirms its multifunctionality. This allows us to determine the cohort of patients for future clinical trials of LfU21.

Bifidobacteria, a genus of obligate anaerobes, comprise a major component of the intestinal microbiota. Importantly, bifidobacteria participate in immune reactions. These bacteria carry a species-specific operon in which the fn3 gene encodes a multifunctional protein FN3 that mediates bacterial adhesion to the intestinal epithelium and is capable of binding individual cytokines. Bioinformatics and biochemical approaches were used to study the possible interaction of recombinant ∆FN3 fragments of B. longum and B. bifidum strains with cytokines TNF-α, IL-6, IL-8, and IL-10. De novo molecular modeling generated, for the first time, the structural models of species-derived ∆FN3 proteins and revealed new tentative regions for differential cytokine binding. Combined treatment with ∆FN3 and TNF-α induced TNF-α mRNA abundance in the human monocytic cell line. Altogether, these findings provide structural evidence for the regulation of immune reactions by microbiota-derived proteins.

Bifidobacteria, a genus of obligate anaerobes, comprise a major component of the intestinal microbiota. Importantly, bifidobacteria participate in the immune reactions. These bacteria carry a species-specific operon in which the fn3 gene encodes a multifunctional protein FN3 that mediates the bacterial adhesion to the intestinal epithelium and is capable of binding individual cytokines. Bioinformatics and biochemical approaches were used to study the possible interaction of recombinant protein fragments ∆FN3 of B. longum and B. bifidum strains with cytokines TNF-α, IL-6, IL-8 and IL-10. De novo molecular modeling generated, for the first time, the structural models of species-derived ∆FN3 proteins and revealed new tentative regions for differential cytokine binding. Combined treatment with ∆FN3 and TNF-α induced TNF-α mRNA abundance in the human monocytic cell line. Altogether, these findings provide structural evidence for regulation of immune reactions by microbiota-derived proteins.

In 2022, the World Health Organization reported that tuberculosis (TB) was the second leading cause of death globally from a single infectious agent following COVID-19. The development of new antitubercular agents with novel mechanisms of action for use in complex TB therapy is considered a key approach to combating TB. In this study, we examined the gene expression profile of M. smegmatis when exposed to a promising antituberculosis agent, quinoxaline 1,4-dioxide (QdNO) 7-chloro-2-(ethoxycarbonyl)-3-methyl-6-(piperazin-1-yl)quinoxaline-1,4-dioxide-1 (LCTA-3368). We investigated how the bacterial response changed with different minimum inhibitory concentrations (MIC) (1/4 × MIC, 1/2 × MIC, and 1 × MIC) and durations (30 min and 90 min) of treatment with the drug. Our analysis revealed significant upregulation in genes involved in DNA repair and replication processes, as well as changes in the expression of 95 genes encoding proteins with oxidoreductase activity. We additionally showed that the concentration of reactive oxygen species increases in a dose-dependent manner upon exposure of M. smegmatis to LCTA-3368. These findings support the proposed mechanism of antibacterial action of QdNOs, which is associated with the formation of free radicals leading to DNA damage.

In 2022, the World Health Organization reported that tuberculosis (TB) was the second leading cause of death globally from a single infectious agent, following COVID-19. The development of new antitubercular agents with novel mechanisms of action for use in complex TB therapy is considered a key approach to combating TB. In this study, we examined the gene expression profile of M. smegmatis when exposed to a promising antituberculosis agent, quinoxaline 1,4-dioxide (QdNO) 7-chloro-2-(ethoxycarbonyl)-3-methyl-6-(piperazin-1-yl)quinoxaline-1,4-dioxide-1 (LCTA-3368). We investigated how the bacterial response changed with different doses (1/4 × MIC, 1/2 × MIC, and 1 × MIC) and durations (30 minutes and 90 minutes) of treatment with the drug. Our analysis revealed significant upregulation of genes involved in DNA repair and replication processes, as well as changes in the expression of 95 genes encoding proteins with oxidoreductase activity. These findings support the proposed mechanism of antibacterial action of QdNOs, which is associated with the formation of free radicals leading to DNA damage.

In 2022, the World Health Organization reported that tuberculosis (TB) was the second highest cause of death globally from a single infectious agent, following COVID-19. Development of new antitubercular agents with a novel mechanism of action for use in the complex TB therapy is considered one of the key approaches for fighting TB. In this study we examined the gene expression profile of M. smegmatis when exposed to a promising antituberculosis agent quinoxaline 1,4-dioxide (QdNO) 7-chloro-2-(ethoxycarbonyl)-3-methyl-6-(piperazin-1-yl)quinoxaline-1,4-dioxide-1 (LCTA-3368). We looked at how the bacterial response changed with different doses (1/4 × MIC, 1/2 × MIC, and 1 x MIC) and durations (30 min and 90 min) of treatment with the drug. Our analysis revealed significant upregulation of the expression of genes involved in DNA repair and replication processes, and changes in the expression of 95 genes encoding proteins with oxidoreductase activity. This confirmed the assumption about the mechanism of antibacterial action of QdNOs associated with the formation of free radicals leading to DNA damage.

Agricultural and industrial activities are increasing pollution of water bodies with low doses of xenobiotics that have detrimental effects on aquaculture. The aim of this work was to determine the possibility of using Levilactobacillus brevis 47f culture in fish aquaculture under the influence of low doses of xenobiotics as an adaptogen. An increase in the survival of Danio rerio individuals exposed to the xenobiotic bisphenol A solution and fed with the L. brevis 47f was shown compared to control groups and, at the same time, the cytokine profile in the intestinal tissues of Danio rerio was also investigated. Analysis of differential gene expression of the L. brevis 47f grown under the action of high concentrations of bisphenol A showed changes in mRNA levels of a number of genes, including genes of various transport proteins, genes involved in fatty acid synthesis, genes of transcriptional regulators, genes of the arabinose operon, and the oppA gene. The identification of L. brevis 47f proteins from polyacrylamide gel by mass spectrometry revealed L-arabinose isomerase, Clp chaperone subunit, ATP synthase subunits, pentose phosphate pathway and glycolysis enzyme proteins, which are likely part of the L. brevis 47f strain’s anti-stress response, but probably do not affect its adaptogenic activity toward Danio rerio.

In this study, the results of evaluating the acute toxicity of Bisphenol A on Danio rerio are presented, encompassing peripheral blood parameters, the composition of hematopoietic cells of erythroid and myeloid lines in the head kidney, and data from histological studies. The LC50 values of Bisphenol A for adult zebrafish individuals for 12, 24, and 48–96 h were determined, which were 18.04, 7.55, and 6.22 mg/L, respectively. The study includes data on the morphology and quantitative frequency of specific cells in the hematopoietic tissue of the head kidney, along with the consideration of adaptive mechanisms in hematopoiesis under BPA exposure. The application of polynomial regression analysis to reveal the concentration–effect relationship for some hematological and histological parameters was demonstrated. Significant increases in the frequency of erythrocyte nuclear abnormalities were observed at BPA concentrations of 6 and 8 mg/L, which indicates a genotoxic effect. BPA’s impact on fish peripheral blood parameters manifested as an increase in the number of erythrocytes (RBC) and immature erythrocytes, as well as a decrease in the number of lymphocytes. The most notable pathological changes in the head kidney’s hematopoietic tissue included circulatory disturbances and the formation of inflammation/degradation foci, as confirmed by histopathologic indices. At BPA concentrations of 2 and 4 mg/L, the observed changes were compensated for by hematopoietic adaptation mechanisms; however, at concentrations of 6 and 8 mg/L, acute systemic toxicity was evident.