Vavilovskii Zhurnal Genetiki i Selektsii最新文献

De novo motif search is the main approach for determining the nucleotide specificity of binding of the key regulators of gene transcription, transcription factors (TFs), based on data from massive genome-wide sequencing of their binding site regions in vivo, such as ChIP-seq. The number of motifs of known TF binding sites (TFBSs) has increased several times in recent years. Due to the similarity in the structure of the DNA-binding domains of TFs, many structurally cognate TFs have similar and sometimes almost indistinguishable binding site motifs. The classification of TFs by the structure of the DNA-binding domains from the TFClass database defines the top levels of the hierarchy (superclasses and classes of TFs) by the structure of these domains, and the next levels (families and subfamilies of TFs) by the alignments of amino acid sequences of domains. However, this classification does not take into account the similarity of TFBS motifs, whereas identification of valid TFs from massive sequencing data of TFBSs, such as ChIP- seq, requires working with TFBS motifs rather than TFs themselves. Therefore, in this study we extracted from the Hocomoco and Jaspar databases the TFBS motifs for human and fruit fly Drosophila melanogaster, and considered the pairwise similarity of binding site motifs of cognate TFs according to their classification from the TFClass database. We have shown that the common tree of the TF hierarchy by the structure of DNA-binding domains can be split into separate branches representing non-overlapping sets of TFs. Within each branch, the majority of TF pairs have significantly similar binding site motifs. Each branch can include one or more sister elementary units of the hierarchy and all its/their lower levels: one or more TFs of the same subfamily, or the whole subfamily, one or several subfamilies of the same family, an entire family, etc., up to the entire class. Analysis of the seven largest human and two largest Drosophila TF classes showed that the similarity of TFs in terms of TFBS motifs for different corresponding levels (classes, families) is noticeably different. Supplementing the hierarchical classification of TFs with branches combining significantly similar motifs of TFBSs can increase the efficiency of identifying involved TFs through enriched motifs detected by de novo motif search for massive sequencing data of TFBSs from the ChIP-seq technology.

Long non-coding RNAs (lncRNAs) play an important role in the regulation of gene expression, including interactions with microRNAs (miRNAs), acting as molecular "sponges". Bioinformatics methods are generally used to predict such interactions. To refine computational predictions, additional evidence based on the co-expression of miRNAs and lncRNAs can be incorporated. In the present study, we investigated potential interactions between lncRNAs and miRNAs in the maize mutant line fuzzy tassel (fzt), which is characterized by reduced expression of certain miRNAs due to a mutation in the Dicer-like1 (DCL1) gene in shoot and tassel tissues. Transcriptome assembly was performed based on RNA-seq data from maize shoot and tassel tissues of control and mutant lines, with data obtained from the NCBI SRA archive. In the shoot, 10 lncRNAs with significantly altered expression levels between control and mutant groups were identified, 9 of which were upregulated in the mutant plants. In the tassel, 34 differentially expressed lncRNAs were identified, with 20 showing increased expression in the mutant line. For lncRNAs with increased expression and miRNAs with decreased expression in the mutant line, potential interactions were predicted using the machine learning algorithm PmliPred. The IntaRNA program was used to confirm possible complementary binding for the identified miRNA-lncRNA pairs, which enabled the construction of competing endogenous RNA (ceRNA) networks. Structural analysis of these networks revealed that certain lncRNAs are capable of binding multiple miRNAs simultaneously, supporting their regulatory role as "sponges" for miRNAs. The results obtained deepen our understanding of post-transcriptional regulation in maize and open new perspectives for breeding strategies aimed at improving stress tolerance and crop productivity.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized primarily by joint involvement with progressive destruction of cartilage and bone tissue. To date, RA remains an incurable disease that leads to a significant deterioration in quality of life and patient disability. Despite a wide arsenal of disease-modifying antirheumatic drugs, approximately 40 % of patients show an insufficient response to standard treatment, highlighting the urgent need to identify new pharmacological targets. The aim of this study was to search for novel biological processes that could serve as promising targets for the targeted therapy of RA. To achieve this goal, we employed an approach based on the automated extraction of knowledge from scientific publications and biomedical databases using the ANDSystem software. This approach involved the reconstruction and subsequent analysis of two types of associative gene networks: a) gene networks describing genes and proteins associated with the development of RA, and b) gene networks describing genes and proteins involved in the functional responses to drugs used for the disease's therapy. The analysis of the reconstructed networks identified 11 biological processes that play a significant role in the pathogenesis of RA but are not yet direct targets of existing disease-modifying antirheumatic drugs. The most promising of these, described by Gene Ontology terms, include: a) the Toll-like receptor signaling pathway; b) neutrophil activation; c) regulation of osteoblast differentiation; d) regulation of osteoclast differentiation; e) the prostaglandin biosynthetic process, and f) the canonical Wnt signaling pathway. The identified biological processes and their key regulators represent promising targets for the development of new drugs capable of improving the efficacy of RA therapy, particularly in patients resistant to existing treatments. The developed approach can also be successfully applied to the search for new targeted therapy targets for other diseases.

Reconstruction and analysis of gene networks regulating biological processes are among the modern methodological approaches for studying complex biological systems that ensure the vital activity of organisms. Thermoregulation is an important evolutionary acquisition of warm-blooded animals. Multiple physiological systems (nervous, cardiovascular, endocrine, respiratory, muscular, etc.) are involved in this process, maintaining stable body temperature despite changes in ambient temperature. This study aims to perform a computer reconstruction of the human thermoregulation gene network and present the results in the Termo_Reg_Human 1.0 knowledge base. The gene network was reconstructed using the ANDSystem software and information system, designed for the automated extraction of knowledge and facts from scientific publications and biomedical databases based on machine learning and artificial intelligence methods. The Termo_Reg_Human 1.0 knowledge base (https://www.sysbio.ru/ThermoReg_Human/) contains information about the human thermoregulation gene network, including a description of 469 genes, 473 proteins, and 265 microRNAs important for its functioning, interactions between these objects, and the evolutionary characteristics of the genes. Using the ANDVisio software tool (a module of ANDSystem), each gene, protein, and microRNA involved in the thermoregulation of the human body was prioritized according to its functional significance, i. e., the number of interactions with other objects in the reconstructed gene network. It was found that the key objects with the largest number of functional interactions in the human thermoregulation gene network included the UCP1, VEGFA, PPARG and DDIT3 genes; STAT3, JUN, VEGFA, TLR4 and TNFA proteins; and the microRNAs hsa-mir-335 and hsa-mir-26b. We revealed that the set of 469 human genes from the network was enriched with genes whose ancestral forms originated at an early evolutionary stage (Unicellular organisms, the root of the phylostratigraphic tree) and at the stage of Vertebrata divergence.

Identification of the connections between the various functional components of the immune system is a crucial task in modern immunology. It is key to implementing the systems biology approach to understand the mechanisms of dynamic changes and outcomes of infectious and oncological diseases. The data characterizing an individual's immune status typically have a high-dimensional state space and a small sample size. To study the network topology of the immune system, we utilized previously published original data from Toptygina et al. (2023), which included measurements of the immune status in 19 healthy individuals (children, 9 boys and 10 girls, aged 1 to 2 years), i. e., the immune cells (42 subpopulations) obtained by flow cytometry; cytokine levels (13 types) obtained by multiplex analysis; and antibody levels (4 types) determined by using enzyme immunoassay. To correctly identify statistically significant correlations between the measured variables and construct the respective network graph, it is necessary to use an approach that takes into account the small size of the dataset. In this study, we implemented and analyzed an approach based on the regularized debiased sparse partial correlation (DSPC) algorithm to evaluate sparse partial correlations and identify the network structure of relationships in the immune system of healthy individuals (children) based on immune status data, which includes a set of indicators for subpopulations of immune cells, cytokine levels, and antibodies. For different levels of statistical significance, heatmaps of the partial correlations were constructed. The graph visualization of the DSPC networks was performed, and their topological characteristics were analyzed. It is found that with a limited measurements sample, the choice of a statistical significance threshold critically affects the structure of the partial correlations matrix. The final verification of the immunologically correct structure of the correlation-based network requires both an increase in the sample size and consideration of a priori mechanistic views and models of the functioning of the immune system components. The results of this analysis can be used to select the therapy targets and design combination therapies.

Oncological diseases remain a leading cause of pathological mortality worldwide, making the development of anticancer drugs a critical focus in medicinal chemistry. A promising strategy to enhance therapeutic efficacy and reduce chemotherapy-induced toxicity involves the combined inhibition of DNA repair enzymes and topoisomerases. Of particular interest are minor-groove DNA ligands, which exhibit potent inhibition of DNA-dependent enzymes while having low toxicity and mutagenicity. A number of research groups, including ours, are developing inhibitors of DNA repair enzymes that act simultaneously on several targets: tyrosyl-DNA phosphodiesterase 1/2 (TDP1/TDP2), poly(ADP-ribose) polymerase 1 (PARP1)/TDP1, topoisomerase 1 (TOP1)/TDP1. Such bifunctional inhibitors are designed to resolve the problem of tumor cell resistance to known chemotherapy drugs and increase the effectiveness of the latter. In this study, we evaluated the inhibitory activity of 22 minor-groove DNA ligands - bis- and trisbenzimidazoles against four key repair enzymes: TDP1, TDP2, PARP1, and PARP2. Four series of dimeric compounds and their monomeric units were studied. The difference in inhibitory activity of dimeric bisbenzimidazoles depending on the structure of the compound and the enzyme is shown. Our findings reveal distinct structure-activity relationships, with monomeric and dimeric ligands exhibiting potent TDP1 inhibition at micromolar to submicromolar IC50 values (half-maximal inhibitory concentration). Notably, dimeric compounds from the DB2Py(n) and DB3P(n) series demonstrated superior TDP1 inhibition compared to their monomers. In contrast, all tested compounds showed negligible activity against the other three repair enzymes; so, the compounds demonstrate specificity to TDP1. It should be noted that in this work, in the experiments with TDP1 and TDP2, the effect of the tested compounds as narrow-groove ligands binding to DNA was excluded, and their direct effect on the enzyme was investigated. The results of molecular docking suggest the possibility of direct interaction of active compounds with the active center of TDP1. According to the results of modeling, the inhibitors are located in the binding region of the 3'-end of DNA in the active site of TDP1 and could form stable bonds with the catalytically significant TDP1 residues His263 and His493. These interactions probably provide the high inhibitory activity of the compounds observed in biochemical experiments.

Stem rust, caused by the fungus Puccinia graminis f. sp. tritici (Pgt), is a harmful disease affecting grain crops. The traditional way to combat this and other infectious plant diseases is to use chemical pesticides. Biopesticides, as well as plant disease resistance inducers - in particular those based on chitosan, a derivative of chitin - are increasingly being considered as an effective and safe alternative. Recently, a globular form of chitosan, Novochizol, has been developed, which has a number of advantages and has shown its effectiveness in preliminary field and laboratory experiments. However, there are no works devoted to the effect of this preparation on the expression of defense genes. Therefore, the aim of this work was to search for genes involved in the response of common wheat (Triticum aestivum L.) to stem rust infection and to evaluate the effect of Novochizol treatment on their transcription during the infection process. The wheat line ISr6-Ra with the stem rust resistance gene Sr6 and two Pgt isolates - an avirulent one, Avr6, and a virulent one, vr6 - were used as a model, allowing us to compare the effects of Novochizol depending on the genetic compatibility in the plant-pathogen pathosystem. To analyze the transcription level of defense genes, leaf material was collected at different time points from 3 to 144 h after inoculation of plants with the pathogen. Quantitative PCR analysis showed an increase in the transcription levels of the CERK1, PR3, PR4, PR5, PR6 and PR9 genes in plants treated with Novochizol and infected with various Pgt isolates compared to untreated infected plants. Pgt isolate Avr6 induced the highest expression of some defense genes (primarily CERK1), which is consistent with the phytopathology data showing the maximum degree of resistance (IT1) to stem rust in Novochizol-treated plants with a combination of Sr6-Avr6 genes. The data obtained confirm that one of the optimal strategies for increasing the resistance of grain crops to fungal pathogens is a combination of selection for specific resistance genes with the use of biological control agents.

Hypertension is among the major risk factors of many cardiovascular diseases. Chronic psychoemotional stress is one of its key causes. Studies of molecular mechanisms of human hypertension development are conducted in animals, including artificial rat strains that model various forms of the disease. The RatDEGdb database, used in our work, includes 144 hypothalamic genes that represent the common response to single short-term restraint stress in hypertensive ISIAH and normotensive WAG rats. These rat genes were annotated with changes in the expression of the human orthologs using data on 17,458 differentially expressed genes (DEGs) from patients with hypertension compared to normotensive subjects. We applied principal component analysis to orthologous pairs of DEGs identified in hypertensive patients and rat hypothalamic DEGs upon single short-term restraint stress. Two principal components, corresponding to a linear combination of log2 expression changes associated with the similarity (PC1) and difference (PC2) in the response to psychoemotional stress in two rat strains, on the one hand, and different forms of human hypertension, on the other, explained 64 % and 33 % of the variance in differential gene expression, respectively. The significant correlation revealed between PC1 and PC2 values for the group of DEGs with stress-induced downregulation indicates that psychoemotional stress and hypertension share a common molecular mechanism. Functional annotation suggests that stress-induced downregulation of genes involved in the plasma membrane function and, simultaneously, interactions with the extracellular matrix is the most likely contribution of psychoemotional stress to the development of the hypertensive status in patients, and the SMARCA4 transcription factor is the most likely mediator in the epigenetic modification affecting gene expression under chronic stress. Peripheral blood markers for the diagnosis of psychoemotional stress are proposed.

Site-directed mutagenesis using genetic constructs carrying the CRISPR/Cas system is an effective technology that is actively used to solve a variety of problems in plant genetics and breeding. One of these problems is to improve the nutritional value of grain sorghum, a high-yielding heat- and drought-tolerant cereal crop that is becoming increasingly important in the conditions of climate aridization. The main reason for the relatively low nutritional value of sorghum grain is the resistance of its storage proteins, kafirins, to proteolytic digestion. We have previously obtained mutants with improved kafirin in vitro digestibility using the CRISPR/Cas technology in grain sorghum variety Avance. The nucleotide sequence of one of the genes (k1C5) of the gene family encoding the signal polypeptide of 22 kDa α-kafirin was used as a target. The aim of this study was to investigate the manifestation of the main agronomically-important traits in the progeny of these mutants and inheritance of high in vitro protein digestibility, and also sequencing nucleotide sequences encoding the 22 kDa α-kafirin signal polypeptide in a number of plants from the T0 generation and their T1 progeny. It was revealed that four of the six studied T0 plants, as well as their progeny, had the same mutation: a T→C substitution in the 23rd position of the nucleotide sequence of the k1C5 gene encoding the signal polypeptide, which led to a substitution of the coding triplet CTC→CCC (Leu→Pro). This mutation is located off-target, 3' from the PAM sequence. It is suggested that this mutation may have arisen as a result of Cas9 nuclease errors caused by the presence of multiple PAM sequences located close to each other. It was found that the progeny of two of the three studied mutants (T2 and T3 families), grown in the experimental field conditions, differed from the original variety by a reduced plant height (by 12.4-15.5 %). The peduncle length, 1,000-grain mass, and grain mass per panicle did not differ from the original variety, with the exception of the progeny of the 2C-1.2.5b mutant, which had a reduced grain yield per panicle. Unlike the original variety, plants from the T2 and T3 generations had kernels with a modified type of endosperm (completely floury, or floury with inclusions of vitreous endosperm, or with a thin vitreous layer). The level of grain protein digestibility in the progeny of mutants 2C-2.1.1 #13 and 2C-1.2.5a #14 varied from 77 to 84 %, significantly exceeding the original variety (63.4 ± 2.3 %, p < 0.05). The level of protein digestibility from kernels with modified endosperm was higher than that of kernels with normal vitreous endosperm (84-93 %, p <0.05). The reasons for the variation in endosperm texture in the progeny of the mutants and its relationship with the high digestibility of kafirins are discussed.

Phenolic compounds constitute a significant group of secondary metabolites in barley grain and influence its technological qualities when used in brewing, feed production, and food manufacturing. Proanthocyanidins - polymeric flavonoids localized in the seed coat - play a particularly important role among them. These compounds are responsible for several production issues, such as colloidal haze in beer and browning of groats after heat treatment. Although proanthocyanidins possess health-beneficial properties based on their antioxidant activity, they can act as antinutritional factors due to their ability to bind proteins. In this regard, the breeding of barley varieties completely lacking proanthocyanidins in the grain was initiated, primarily for use in the brewing industry. Initially, it was assumed that their absence would not be critical for the plant, since wheat, corn, and rice varieties lacking proanthocyanidins in the grain had been identified. However, accumulated evidence indicates that proanthocyanidins perform important physiological functions: they contribute to the maintenance of seed dormancy, provide protection against fungal and bacterial pathogens and pests, and their absence negatively affects agronomic traits. For instance, proanthocyanidin-free barley mutants obtained through induced mutagenesis exhibit reduced productivity and pathogen resistance, an increased risk of pre-harvest sprouting, and deterioration of several technologically important properties. Nevertheless, these mutant lines are actively used in breeding programs to develop varieties for various purposes. This review aims to systematize and analyze global experience in breeding proanthocyanidin-free barley varieties, describing achieved results to identify the most successful approaches and define future research directions. The work examines challenges faced by breeders when using mutant lines, as well as strategies that have helped minimize negative side effects. It is demonstrated that through targeted crossing and optimal selection of mutant alleles, competitive varieties have been developed that combine the required technological qualities with satisfactory agronomic performance, meeting the demands of both the brewing and food industries.