Vol 20, No 4 (2022)

AIM: The aim of the study was to carry out species identification of the hybridogenic complex of European water frogs (Pelophylax esculentus complex) in the conditions of transformed biotopes of the south of the Central Russian Upland based on molecular genetic markers

MATERIALS AND METHODS: The study included 36 local populations (770 individuals) living in conditions of transformed biotopes of the south of the Central Russian Upland. Identification of cryptic forms was carried out by a Multiplex PCR. Two molecular genetic markers were used for amplification: intron 1 of the SAI-1 DNA serum albumin gene to determine hybrids and cryptic forms, and a fragment of the first subunit of the cytochrome oxidase COI mtDNA gene to determine maternal lines.

RESULTS: According to the data obtained, “pure” R-type population systems predominate (58.33%) in the study region. Mixed RE-type population systems were identified in 14 localities, REL-type in the region is extremely rare and is noted only in one locality. No pure L-type, E-type, or mixed LE-type population systems have been identified. The study revealed a statistically significant (p < 0.001) predominance of haplotypes of the “Western” form (Pelophylax ridibundus).

CONCLUSIONS: The data obtained indicate active adaptive changes in the population structure of European water frogs in the study area. Degradation of water bodies caused by abiotic and anthropogenic factors forces amphibians to migrate to neighboring reservoirs in which hybridization of representatives of this complex occurs. Introgressive and hybrid forms of the marsh frog, as well as hybrid edible individuals with greater ecological plasticity and tolerance to anthropogenic pressure, displace populations of Pelophylax lessonae. Based on the above, we consider it necessary to include the pond frog (P. lessonae) in the Red Book of the Belgorod region.

BACKGROUND: N.I. Vavilov Institute of Plant Genetic Resources (VIR) (Saint Petersburg, Russia) maintains a large collection of pea (Pisum sativum L.). Earlier, several growth and yield parameters were recorded for plants of 99 accessions grown under inoculation with nodule bacteria and arbuscular mycorrhizal fungi.

MATERIALS AND METHODS: Polymorphism of genes encoding symbiotic receptor kinase PsSym29 [participating in the autoregulation of nodulation (AON) system] and closely related receptor kinase PsNRLK1 (with yet unknown function in symbiosis) was assessed in 99 pea genotypes from the VIR collection. Nucleotide diversity, Tajima’s D, and Fay and Wu’s H statistics were calculated using DNAsp 5.0 software. The significance of associations of allelic state of the sequenced genes with the growth and yield parameters was tested by two-way ANOVA followed by FDR correction and by regression analysis.

RESULTS: Nucleotide diversity and the ratio of synonymous to non-synonymous substitutions was greater in PsNRLK1 as compared to PsSym29. The analysis of Fay and Wu’s H in sliding window revealed signatures of positive selection in one site of PsSym29 and in three sites of PsNRLK1 gene sequences located in 1^st exons encoding LRR (leucine rich repeat) domains. No significant associations of allelic state of neither PsSym29 nor PsNRLK1 genes was found with plant growth and yield parameters.

CONCLUSIONS: The sequences of both PsSym29 and PsNRLK1 genes undergo positive selection, but the conditions in which specific allelic states of the genes become adaptive are to be elucidated in future.

Based on the selected criteria data from studies of the genotoxic activity of antiepileptic drugs in eukaryotic test systems in vitro and in vivo, performed by DNA comet assay, chromosomal aberrations and micronuclei assays, published in the period 1995–2022, were selected and summarized.

Among the 20 drugs reviewed, for one drug (N03AA05 Benzobarbital), there are no data on studies of genotoxic activity; for 7 drugs information is presented only as a summary on the FDA and EMA websites without primary data and information on experimental designs. Among the remaining 12 drugs, only three drugs (phenobarbital, valproic acid and levetiracetam) have information on in vivo studies, both by DNA damage assay and by cytogenetic methods. Based on known publications, it is impossible to draw reasonable conclusions about the genotoxic potential of individual drugs. The available data are fragmentary, incomplete and contradictory. It remains to state the facts of detection of genotoxic effects in individual drugs in separate studies. In general, there is no doubt about the potential genotoxic hazard of this group the drugs in.

Additional studies are needed to clarify the data on the genotoxicity of antiepileptic drugs including beyond the standard protocols. In the course of their implementation, one should take into account the possible tissue-specific manifestation of antiepileptics genotoxicity, as indicated by the facts of genotoxic effects detection in tissue cells that are not targets in classical genotoxic studies. The expediency of objectifying approaches when choosing a drug for safe therapy, taking into account information about its genotoxicity, is emphasized, and the prospects for possible studies on antigenotoxic prophylaxis in patients with epilepsy are pointed out.

BACKGROUND: Recent studies show that the bacterial microbiome of the respiratory tract can influence the development of a number of diseases of the human respiratory system. Changes in the composition of the microbiome in patients are associated with dysbiosis, and in addition, many bacteria have a genotoxic potential and can directly or indirectly damage the genome in the cells of the host organism.

AIM: The aim of the study was to analyze the composition of the sputum microbiome and its relationship with chromosome damage in the blood leukocytes of patients with chronic dust bronchitis (CDB).

MATERIALS AND METHODS: The taxonomic composition of the sputum microbiome of 22 patients with CKD and 22 sputum donors from the control group was studied using next-generation sequencing (NGS) technology of 16S rRNA of bacterial genes. At the same time, the basic frequencies of chromosomal aberrations and micronuclei were determined in blood leukocytes.

RESULTS: The sputum microbiome of chronic dust bronchitis patients had a significant reduction in alpha and beta diversity parameters compared to healthy study participants. In addition, an increase in the relative abundance of the genus Streptococcus (29.97 ± 3.03 vs. 18.78 ± 2.47; p = 0.003) was found in the sputum of CP patients compared with the control. Thus, the results of metagenome sequencing indicate a common dysbiotic process with a predominance of one dominant genus of bacteria in this pulmonary pathology. The results of cytogenetic analysis of blood leukocytes showed a significant increase in the proportion of aberrant metaphases in CKD patients compared with healthy donors (3.41% vs. 1.84%; p < 0.01) and the absence of significant differences in frequency leukocytes with micronuclei between the compared groups (1.28% vs. 1.11%). Correlation analysis revealed the presence of significant direct relationships between the frequency of aberrant metaphases and the percentage of representatives of the genera Bacteroides in the sputum of patients with chronic dust bronchitis (r = 0.471; p = 0.031); Lachnoanaerobaculum (r = 0.446; p = 0.043) and Alloprevotella (r = 0.444; p = 0.044). Further studies should be devoted to the search for possible mechanisms of influence of these bacteria on clastogenic effects in the cells of the host organism.

BACKGROUND: RNA aptamers are short, single-stranded oligonucleotides, with remarkable binding ability to target molecules characterized by high specificity and affinity. Such targets are vastly diverse and range from specific ions to entire cells. RNA aptamers are widely used in biology and medicine for basic research, as well as for practical purposes as in therapy and diagnostics. At present, chemical or in vitro methods of synthesis are mainly used to obtain RNA aptamers. However, such methods are expensive and time-consuming with low productivity. Therefore, in vivo methods are becoming more attractive to researchers working on optimizing high-scale production of RNA aptamers.

AIM: The aim of this work is to develop a reporter system for optimizing the synthesis of small RNA molecules in Saccharomyces cerevisiae yeast cells.

MATERIALS AND METHODS: We used the Broccoli fluorescent RNA aptamer to develop a reporter system allowing us to optimize the conditions for in vivo short RNA synthesis in yeast cells. This aptamer is about 112 bp in size and binds to the fluorogenic dye DFHBI-1T. Only upon binding, the aptamer-dye complex exhibits fluorescence properties. After excitation using light with a wavelength of 482 nm, the aptamer-dye complex emission is observed with a peak at 505 nm.

RESULTS: We have designed a reporter system providing the synthesis of the fluorescent Broccoli RNA aptamer in S. cerevisiae yeast cells. Transcription of RNA molecules containing the aptamer is carried out by the regulated promoter of the GAL1 gene. The synthesized transcripts contain the HH and HDV ribozymes to ensure precise cleavage of the RNA aptamer sequences.

CONCLUSIONS: This reporter system is based on the Broccoli RNA aptamer, and it can be used to optimize the in vivo synthesis of RNA aptamers in S. cerevisiae yeast cells. This work serves an urgent task in connection with the active use of such aptamers in scientific research, biotechnology and medicine.

BACKGROUND: Yeast display is an effective technology for exposure target proteins to the cell surface by fusing them with cell wall proteins. This technique, among other things, makes it possible to obtain vaccine preparations based on yeast by exposing antigen proteins on their cell surface. Finding and selecting proteins that allow effective exposure of target proteins on the surface of yeast cells is an urgent task.

AIM: The aim of this work was to evaluate the efficiency of cell wall proteins ScAGα1p, KpCW51p, KpCW61p for displaying the reporter protein on the Komagataella phaffii cell surface, including the study of several variants of the ScAGα1 gene coding sequence.

MATERIALS AND METHODS: The studied gene sequences were cloned under the control of the AOX1 gene promoter in the same reading frame as the eGFP reporter protein gene and integrated into the genome of the K. Phaffii yeast strain X-33.

RESULTS: Cytoimmunochemical analysis and confocal microscopy of strains displaying the eGFP protein on their surface under conditions of induction of the AOX1 gene promoter made it possible to identify the most effective anchor protein. The best efficiency was demonstrated for the sequence of the ScAGα1 gene without the native 3' non-coding region.

CONCLUSIONS: The plasmids obtained in the work will make it possible to obtain a yeast strain K. phaffii that effectively exposure proteins, including antigens, on its surface, which can be used as a vaccine preparation.