Vol 57, No 1 (2023)

The study of the role of cytokines in various pathological conditions of the body is one of the topical areas of modern biomedicine. Interleukin 11 (IL-11) was discovered in 1990 in fibrocyte-like bone marrow stromal cells, but there has been an increased interest in the cytokine in recent years. The cytokine plays a significant role in the central nervous system; local expression of this cytokine by nerve cells has been shown. Studies show the involvement of IL-11 in the mechanisms of development of a number of pathologies of the nervous system. The review summarizes information that shows the involvement of IL-11 in the mechanisms of development of brain pathologies. Probably, in the near future, this cytokine will be able to find clinical application in the correction of mechanisms that are involved in the formation of pathological conditions of the nervous system.

In non-coding regions of the genome, the widest range of SNP markers associated with human diseases and petrogenetically significant features were identified. This raised the critical question of identifying the mechanisms that explain these associations. Previously, we identified a number of associations of polymorphic variants of genes encoding DNA repair proteins with multifactorial diseases. To clarify the possible mechanisms underlying established associations, we carried out a detailed annotation of the regulatory potential of the studied markers using a number of on-line resources (GTXPortal, VannoPortal, Ensemble, RegulomeDB, Polympact, UCSC, GnomAD, ENCODE, GeneHancer, EpiMap Epigenomics 2021, HaploReg, GWAS4D, JASPAR, ORegAnno, DisGeNet, OMIM). The article characterizes the regulatory potential of polymorphic variants rs560191 (in the TP53BP1 gene), rs1805800 and rs709816 (in the NBN gene), rs473297 (MRE11), rs189037 and rs1801516 (ATM), rs1799977 (MLH1), rs1805321 (PMS2), rs20579 (LIG1). Both the general characteristics of the studied markers and information on their influence on the expression of “own” and co-regulated genes, on changes in binding affinity of transcription factors are given. Known data on both adaptogenic and pathogenicity potential of these SNPs and on histone modifications co-localized with them are presented. The potential involvement in regulatory function of not only genes that contain SNPs studied but also nearby genes may explain the association of the markers with diseases and their clinical phenotypes.

Dopamine, serotonin and glutamate systems are jointly involved in the pathogenesis and pharmacotherapy of schizophrenia. We formulated a hypothesis that polymorphic variants of the GRIN2A, GRM3, and GRM7 genes may be associated with the development of hyperprolactinemia in patients with schizophrenia taking conventional and atypical antipsychotics as basic treatment. 432 Caucasian patients diagnosed with schizophrenia were examined. DNA was isolated from peripheral blood leukocytes by the standard phenol-chloroform method. For pilot genotyping, 12 SNPs in the GRIN2A gene, 4 SNPs in the GRM3 gene, and 6 SNPs in the GRM7 gene were selected. Allelic variants of the studied polymorphisms were determined by real-time PCR. The level of prolactin was determined by enzyme immunoassay. Among persons taking conventional antipsychotics, there were statistically significant differences in the distribution of genotype and allele frequencies in groups of patients with normal and elevated prolactin levels for the GRIN2A rs9989388 and GRIN2A rs7192557 polymorphic variants, as well as differences in serum prolactin levels depending on the genotypes of the GRM7 rs3749380 polymorphic variant. Among persons taking atypical antipsychotics, statistically significant differences were found in the frequencies of genotypes and alleles of the GRM3 rs6465084 polymorphic variant. For the first time, an association of polymorphic variants of the GRIN2A, GRM3, and GRM7 genes with the development of hyperprolactinemia in patients with schizophrenia taking conventional and atypical antipsychotics has been established. They not only confirm the close connection of the dopaminergic, serotonergic, and glutamatergic systems in the development of schizophrenia, but also demonstrate the potential of taking into account the genetic component for its therapy.

Personalization of gastric cancer treatment is an urgent problem due to clinical heterogeneity and aggressive course of the disease. In 2014, Cancer Genome Atlas researchers divided gastric cancers into four subtypes based on molecular characteristics: Epstein–Barr virus positive (EBV+), microsatellite instability (MSI), chromosomal instability, genomically stable. To date, there is no single method for detecting chromosomal instability and genomically stable subtypes, while MSI analysis and EBV assessment are used in routine practice and are of the greatest clinical importance. We analyzed 159 gastric cancer samples for the presence of MSI, EBV DNA, and somatic mutations in codons 12–13 (exon 2), 61 (exon 3), and 146 (exon 4) of the KRAS gene, codons 597–601 (exon 15) of the BRAF gene and codons 542–546 (exon 9), 1047–1049 (exon 20) of the PIK3CA gene. As a result, the EBV+ gastric cancer was detected in 8.2% of samples, MSI – in 13.2%. MSI and EBV+ were found to be mutually exclusive. The mean age of patients with EBV+ and MSI cancers was 54.8 and 62.1 years, respectively. In 92.3% of EBV+ cancer was detected in men, of which 76.2% were older than 50 years. diffuse and intestinal adenocarcinomas in EBV+ cancer accounted for 6 (46.2%) and 5 (38.5%) cases, respectively. MSI occurred in almost equal proportions in men and women (n = 10; 47.6%, n = 11; 52.4%), with a predominance of intestinal histological type (71.4%) and lesion of the lesser curvature (28.6%). One case of EBV+ cancer was diagnosed with the E545K variant in the PIK3CA gene. A combination of variants in the KRAS and PIK3CA genes was found in all MSI cases. The EBV+ subtype was associated with a better prognosis. Overall five-year survival rates for MSI and EBV+ cancers were 100.0 and 54.7%, respectively.

The expression level of heterologous genes in transgenic plants serves as an important indicator of gene efficiency. The small number of currently known effective promoters, limits the possibilities in fine-tuning the expression of transgenes. We cloned and characterized a tissue-specific promoter fragment of the soybean chitinase class I gene (GmChi1). The GmChi1 promoter (GmChi1P) was cloned from Jungery soybean. The promoter sequence contains a number of putative cis-acting elements, including tissue-specific and stress-regulated motifs. By histochemical analysis, the GmChi1P-controlled β-glucuronidase (GUS) reporter enzyme activity was shown to be highest in the roots of transgenic Nicotiana tabacum cv. NC89 at the four-leaf sprout formation stage. Interestingly, the high GUS activity in transgenic tobacco roots was effectively suppressed by salicylic acid (SA) treatment. Deletion analysis of GmChi1P revealed that the sequences located between positions ‒719 and ‒382 contain key cis-elements responsible for the reporter uidA gene expression (encoding GUS) in leaves, roots, and wounds of Nicotiana tabacum. In addition, fluorometric analysis showed that the activity of the shortened ChiP(‒1292) to ChiP(‒719) promoters in the roots of transgenic tobacco was significantly suppressed by abscisic acid and completely suppressed by SA. The ChiP(‒382) promoter was also found to be expressed exclusively in the stigma of transgenic tobacco flowers. Using the GUS reporter enzyme, no staining was detected in other flower organs in transgenic Nicotiana tabacum, including sepals, petals, anthers, filaments, and ovaries, or in any vegetative tissues. The results indicate that the promoter fragment ChiP(‒382) can be used in tissue-specific regulation of gene expression and plant genetic engineering.

ArdB proteins are known to inhibit the activity of type I restriction–modification (RM-I) system, in particular EcoKI (family IA). The mechanism of ArdB’s activity still remains unknown; the spectrum of targets inhibited by them has been poorly studied. In this work, it was shown that the presence of the ardB gene from R64 plasmid could suppress the activity of EcoAI endonuclease (IB family) in Escherichia coli TG1 cells. The absence of specificity of ArdB to a certain RM-I system (it inhibits both the IA- and IB-family), it can be a-ssumed that the mechanism of the anti-restriction activity of this protein does not depend on both the s-equence DNA at the recognition site and the structure of the restrictase of the RM-I systems.

CP190 protein is one of the key components of Drosophila insulator complexes, and its study is important for understanding the mechanisms of gene regulation during cell differentiation. However, Cp190 mutants die before reaching adulthood, which significantly complicates the study of its functions in imago. To overcome this problem and to investigate the regulatory effects of CP190 in adult tissues development, we have designed a conditional rescue system for Cp190 mutants. Using Cre/loxP-mediated recombination, the rescue construct containing Cp190 coding sequence is effectively eliminated specifically in spermatocyte, allowing us to study the effect of the mutation in male germ cells. Using high-throughput transcriptome analysis we determined the function of CP190 on gene expression in germline cells. Cp190 mutation was found to have opposite effects on tissue-specific genes, which expression is repressed by CP190, and housekeeping genes, that require CP190 for activation. Mutation of Cp190 also promoted expression of a set of spermatocyte differentiation genes that are regulated by tMAC transcriptional complex. Our results indicate that the main function of CP190 in the process of spermatogenesis is the coordination of interactions between differentiation genes and their specific transcriptional activators.

Liquid–liquid phase separation of proteins occur in a number of biological processes, such as regulation of transcription, processing, and RNA maturation. Sm-like protein 4 (LSM4) is involved in multiple processes, including pre-mRNA splicing and P-bodies assembly. Before investigating the involvement of LSM4 in the separation of the two liquid phases during RNA processing or maturation, the separation of the liquid phases in an in vitro preparation of LSM4 protein should be first be detected. The mCherry-LSM4 plasmid was derived from pET30a and used to isolate mCherry-LSM4 protein from prokaryotic cells (Escherichia coli strain BL21). The mCherry-LSM4 protein was purified using Ni-NTA resin. The protein was further purified by fast protein liquid chromatography. Delta-Vision wide-field fluorescence microscopy was used to observe the dynamic liquid–liquid phase separation of the LSM4 protein in vitro. Analysis of the LSM4 protein structure using the Predictor of Natural Disordered Regions database revealed that its C-terminus contains a low complexity domain. A purified preparation of full-length human LSM4 protein was obtained from E. coli. Human LSM4 was shown to provide concentration-dependent separation of liquid–liquid phases in vitro in buffer with crowding reagents. Salts in high concentration and 1,6-hexanediol block the LSM4-induced separation of the two liquid phases. In addition, in vitro fusion of LSM4 protein droplets is observed. These results indicate that the full-length human LSM4 protein has the ability to form liquid inclusions and induce liquid–liquid phase separation in vitro.

Recently, the prediction of protein structure and function from its sequence underwent a rapid increase in performance. It is primarily due to the application of machine learning methods, many of which rely on the predictive features supplied to them. It is thus crucial to retrieve the information encoded in the amino acid sequence of a protein. Here, we propose a method to generate a set of complex yet interpretable predictors, which aids in revealing factors that influence protein conformation. The proposed method allows us to generate predictive features and test them for significance in two scenarios: for a general description of the protein structures and functions, as well as for highly specific predictive tasks. Having generated an exhaustive set of predictors, we narrow it down to a smaller curated set of informative features using feature selection methods, which increases the performance of subsequent predictive modelling. We illustrate the effectiveness of the proposed methodology by applying it in the context of local protein structure prediction, where the rate of correct prediction for DSSP Q3 (three-class classification) is 81.3%. The method is implemented in C++ for command line use and can be run on any operating system. The source code is released on GitHub: https://github.com/Milchevskiy/protein-encoding-projects.

The enzymes involved in the transsulfuration pathway and hydrogen sulfide production – cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) – play an important cytoprotective role in the functioning of the organism. Using CRISPER/Cas9 technology, we obtained Drosophila lines with deleted cbs, cse, and mst genes as well as with double deletion of cbs and cse genes. We analyzed the effect of these mutations on the pattern of protein synthesis in the salivary glands of third instar larvae and in the ovaries of mature flies. In the salivary glands of lines with cbs and cse deletions, a decrease was found in the accumulation of the FBP2 storage protein containing 20% methionine amino acid residues. In the ovaries, changes were detected in the level of expression and isofocusing points of proteins involved in cell protection against oxidative stress, hypoxia, and protein degradation. It was shown that in the lines with deletions of transsulfuration enzymes the proteins have a similar degree of oxidation to that of the control line. A decrease in the total number of proteasomes and their activity was found in the lines with deletions of the cbs and cse genes.