Ribosomal protein S6 (RPS6) is the only phosphorylatable protein of the eukaryotic 40S ribosomal subunit. Ribosomes with phosphorylated RPS6 can selectively translate 5’TOP-(5’-terminal oligopyrimidine)-containing mRNAs that encode most proteins of the translation apparatus. The study of translational control of 5’TOP-mRNAs, which are preferentially translated when RPS6 is phosphorylated and cease to be translated when RPS6 is de-phosphorylated, is particularly important. In Arabidopsis thaliana, AtRPS6 is phosphorylated by kinase AtRPS6K2, which should in turn be phosphorylated by upper level kinases (AtPDK1 – at serine (S) 296, AtTOR – at threonine (T) 455 and S437) for full activation. We have cloned AtRPS6K2 cDNA gene and carried out in vitro mutagenesis replacing codons encoding S296, S437 and T455 by triplets of phosphomimetic glutamic acid (E). After the expression of both natural and mutated cDNAs in Escherichia coli cells, two recombinant proteins were isolated: native AtRPS6K2 and presumably constitutively active AtRPS6K2(S296E, S437E, T455E). The activity of these variants was tested in vitro. Both kinases could phosphorylate wheat (Triticum aestivum L.) TaRPS6 as part of 40S ribosomal subunits isolated from wheat embryos, though the non-mutated variant had less activity than phosphomimetic one. The ability of recombinant non-mutated kinase to phosphorylate TaRPS6 can be explained by its phosphorylation by bacterial kinases during the expression and isolation steps. The phosphomimetically mutated AtRPS6K2(S296E, S437E, T455E) can serve as a tool to investigate preferential translation of 5’TOP-mRNAs in wheat germ cell-free system, in which most of 40S ribosomal subunits have phosphorylated TaRPS6. Besides, such an approach has a biotechnological application in producing genetically modified plants with increased biomass and productivity through stimulation of cell growth and division.

Tumor necrosis factor (TNF) is one among the key cytokines that mediate the immune system to protect humans against viral infections. Throughout evolution, anthropogenic Variola virus (VARV) has developed effective mechanisms to overcome human defense reactions. The viral genome encodes soluble proteins imitating the structure of cellular cytokine receptors. These proteins compete with cellular receptors for cytokine binding, thus blocking the antiviral immune response. In particular, the G2R gene of VARV encodes the TNF decoy receptor, VARV-CrmB protein. This protein consists of N-ended TNF-biding (TNF-BD) and C-ended chemokine binding (Ch-BD) domains. Recombinant VARV-CrmB protein has been produced in insect cells using molecular cloning methods and its TNF neutralizing activity has been shown in vitro and in vivo. To decrease the immunogenicity of this protein, a recombinant plasmid coding for shortened TNF-BD protein of VARV in Escherichia coli cells has been constructed. Using the method of immobilized metal affinity chromatography, recombinant TNF-BD protein corresponding to the TNF-biding domain of VARV-CrmB protein was purified from E. coli cells. The therapeutic potential of TNF-BD was studied using an experimental model of LPS-induced septic shock. After septic shock induction, several doses of recombinant TNF-BD were injected and the mortality of experimental animals was observed during 7 days. All mice not injected with TNF-BD had been dead by day 3 of the experiment, but 30, 40 and 60 % of the experimental animals, who received different TNF-BD doses, survived in a dose-dependent manner. Data obtained demonstrate that recombinant TNF-BD protein is pharmacologically active in the experimental model of LPS-induced septic shock.

Assessment of the genetic resources of chickpea (Cicer arietinum L.) in a zone that is atypical for its cultivation (eastern forest-steppe of Ukraine) gives an opportunity to identify valuable starting material for priority breeding areas. The article presents the results of a cluster analysis on chickpea accessions from the National Center for Plant Genetic Resources of Ukraine (NCPGRU) for a set of agronomic characteristics. In 2005-2017, 653 chickpea accessions from the NCPGRU's core collection were studied: 369 kabuli accessions and 284 desi accessions. One hundred and fifty two sources of valuable traits were identified for 11 parameters: drought tolerance, resistance to Ascochyta leaf and pod spot, early ripening (vegetation period length), yield, performance, number of productive pods and seed number per plant, response to nitrogenization, protein content, seed size, and cooking quality. These accessions (77 kabuli accessions are light-colored and 75 desi ones are dark-colored) were grouped by a set of valuable economic characteristics using cluster analysis with the Euclidean distance as a measure. The study showed that this sample consisted of 4 clusters. Cluster 1 contained mainly kabuli accessions with optimal combinations of valuable traits: drought tolerance, resistance to Ascochyta leaf and pod spot, large seeds, high yield capacity and performance, pod and seed numbers as well as protein content in seeds. This cluster includes standards and most of reference varieties, which are well-adapted to the conditions of the eastern forest-steppe of Ukraine. The accessions of cluster 2 are characterized by high resistance to Ascochyta leaf and pod spot, late ripening, small seeds, low protein content, moderate response to nitrogenization, high performance attributed to a large number of productive pods and seeds per plant. Most of the accessions of this cluster are small-seeded late-ripening kabuli accessions. Cluster 3 consists of 3 accessions, which have large seeds and high protein content in them, give moderate yields, are highly responsive to nitrogenization and poorly resistant to Ascochyta leaf and pod spot. Cluster 4 comprises mainly desi accessions (63 %), which are mid-ripening, with small seeds, low performance, moderate yield capacity, medium protein content, poor cooking quality, moderate resistance to Ascochyta leaf and pod spot, and low drought tolerance. Representatives of this cluster are predominantly sources of one trait and may have restricted application in specialized breeding programs. Based on the data obtained, we concluded that the accessions of cluster 1 were preferable in breeding programs to develop chickpea varieties for the forest-steppe zone.

Broadening of the genetic diversity of donors of resistance to biotic environmental factors is a challenging problem concerning Triticum L., which can be solved by using wild relatives of wheat, in particular, Aegilops tauschii Coss., in breeding programs. This species, believed to be the donor of D genome of common wheat (T. aestivum L.), is a source of some traits important for breeding. This greatly facilitates the possibility of crossing Ae. tauschii with common wheat. Aegilops L. species are donors of effective genes for resistance to fungal diseases in wheat. For instance, genes that determine resistance to rust agents in common wheat were successfully introgressed from Ae. tauschii into the genome of T. aestivum L. The aim of our study was to identify differences in metabolomic profiles of Ae. tauschii forms (genotypes), resistant or susceptible to such fungal pathogens as Puccinia triticina f. sp. tritici and Erysiphegraminis f. sp. tritici.These indicators may be used as biochemical markers of resistance. A comparative analysis of groups of Ae. tauschii accessions showed that metabolomic profiles of the forms with or without resistance to fungal pathogens differed significantly in the contents of nonproteinogenic amino acids, polyols, phytosterols, acylglycerols, mono- and oligosaccharides, glycosides, phenolic compounds (hydroquinone, kempferol), etc. This fact was consistent with the previously obtained data on the relationship between Fusarium resistance in oats (Avenasativa L.) and certain components of the metabolomic profile, such as acylglycerols, nonproteinogenic amino acids, galactinol, etc. Thus, our studies once again confirmed the possibility and effectiveness of the use of metabolomic analysis for screening the genetic diversity of accessions in the VIR collection, of Ae. tauschii in particular, in order to identify forms with a set of compounds in their metabolomic profile, which characterize them as resistant. Ae. tauschii accessions with a high content of pipecolic acids, acylglycerols, galactinol, stigmasterol, glycerol, azelaic and pyrogallic acids, campesterol, hydroquinone, etc., can be used for creating wheat and triticale cultivars with high resistance to fungal pathogens causing powdery mildew, brown rust, and yellow rust.

Remote sensing using hyperspectral cameras is an important technology for non-destructive monitoring of plant pigment composition, which is closely related to their physiological state or infection with pathogens. The paper presents the experience of using Specim IQ, a mobile hyperspectral camera, to study common root rot (the pathogen is the fungus Bipolaris sorokiniana Shoem.) affecting the seedlings of four wheat varieties and to analyze the pulp of potato tubers of 82 lines and varieties. Spectral characteristics were obtained for seedlings and the most informative spectral features (indices) for root rot detection were determined based on the data obtained. Seedlings of control variants in the visible part of the spectrum show an increase in reflectance with a small peak in the green area (about 550 nm), then a decrease due to light absorption by plant pigments with an extremum at a wavelength of about 680 nm. Analysis of histograms of vegetation index values demonstrated that the TVI and MCARI indices are the most informative for detecting the pathogen on wheat seedlings according to hyperspectral survey data. For potato samples, regions of the spectrum were found that correspond to local maxima and minima of reflection. It was shown that the spectra of potato varieties have the greatest differences within wavelength ranges of 900-1000 nm and 400-450 nm, which in the former case may be associated with the level of water content, and in the latter, with the formation of melanin in the tubers. It was shown that according to the characteristics of the spectrum, the samples studied are divided into three groups, each characterized by increased or reduced intensity levels for the specified parts of the spectrum. In addition, minima in the reflection spectra corresponding to chlorophyll a were found for a number of varieties. The results demonstrate the capabilities of the Specim IQ camera for conducting hyperspectral analyses of plant objects.

The multitude of existing methods for assessing the phenotypic stability of plants makes breeders be faced with the problem of choosing an appropriate variant. The purpose of this study was to compare different methods of analyzing the genotype x environment interaction and, on their basis, assess the stability of the yield of 7 varieties of winter wheat. The article compares 17 stability statistics by applying them to data obtained from agrotechnical experiments carried in 2009-2011 for evaluating the grain yield of 7 varieties of winter common wheat of Siberian selection (Novosibirskaya 32, Novosibirskaya 40, Novosibirskaya 51, Novosibirskaya 3, Novosibirskaya 2, Obskaya winter, Omskaya 6). Analysis of variance revealed a significant (p < 0.001) genotype x environment interaction in the experiments, which indicates a different reaction of genotypes to changes in environmental conditions. Genotypes were ranked according to the level of stability. Based on the analysis of the rank correlation matrix, the stability statistics were categorized in five groups. Recommendations were made on which group of methods to use depending on the objectives of the study. In the case when the goal of breeding research is the selection of the most biologically stable varieties with the minimum variance across a range of environments, one should use the methods of the static concept. If it is necessary to choose a genotype with a predictable reaction to changes of environmental conditions, corresponding to the calculated level or forecast, the regression approach is the most appropriate. The stability statistics generally identified Novosibirskaya 32 as the most stable variety from a biological point of view. The regression approach showed that Novosibirskaya 3 was the genotype with the smallest deviation from mean yield in all environments, while methods accessing the contribution of each genotype to the genotype x environment interaction defined Novosibirskaya 51 as the most stable variety.

Nearly all lethal viral outbreaks in the past two decades were caused by newly emerging viruses. Viruses are often studied by electron microscopy (EM), which provides new high-resolution data on the structure of viral particles relevant to both fundamental virology and practical pharmaceutical nanobiotechnology. Electron microscopy is also applied to ecological studies to detect viruses in the environment, to analysis of technological processes in the production of vaccines and other biotechnological components, and to diagnostics. Despite the advances in more sensitive methods, electron microscopy is still in active use for diagnostics. The main advantage of EM is the lack of specificity to any group of viruses, which allows working with unknown materials. However, the main limitation of the method is the relatively high detection limit (107 particles/mL), requiring viral material to be concentrated. There is no most effective universal method to concentrate viruses. Various combinations of methods and approaches are used depending on the virus and the goal. A modern virus concentration protocol involves precipitation, centrifugation, filtration, and chromatography. Here we describe the main concentrating techniques exemplified for different viruses. Effective elution techniques are required to disrupt the bonds between filter media and viruses in order to increase recovery. The paper reviews studies on unique traps, magnetic beads, and composite polyaniline and carbon nanotubes, including those of changeable size to concentrate viral particles. It also describes centrifugal concentrators to concentrate viruses on a polyethersulfone membrane. Our review suggests that the method to concentrate viruses and other nanoparticles should be chosen with regard to objectives of the study and the equipment status of the laboratory.

The article reports an original method for producing vitamin D3-binding protein (DBP) and its conversion into macrophage-activating factor GcMAF-RF. According to an original protocol, DBPs were obtained from human blood plasma using affinity chromatography, purified and modified to GcMAF-RF using cytoimmobilized glycosidases (beta-galactosidase and neuraminidase). The presence of the polypeptide obtained in the Gc group of blood plasma globulins was confirmed by Western blot using specific antibodies. The molecular properties of this polypeptide put it in correspondence with the GcMAF protein described in the literature, which is undergoing clinical trials in the USA, Britain, Israel and Japan (at Saisei Mirai; Reno Integrative Medical Center; Immuno Biotech Ltd; Efranat; and Catalytic Longevity). The biological activity of the GcMAF-RF preparation was detected by the induction of phagocytic activity of macrophages and their ability to produce nitrogen monoxide (NO) in vitro.The phagocytic activity of macrophages was evaluated by their ability to uptake magnetic beads. The degree of activation of macrophages was calculated by the ratio of trapped beads to the total number of macrophages. The level of NO production was estimated by the accumulation of nitrogen monoxide in the culture supernatants of peritoneal macrophages by the colorimetric method using the Griess reagent. It was shown that GcMAF-RF multiplies the phagocytic activity of macrophages and significantly increases their production of nitrogen monoxide. The macrophage activator GcMAF-RF, according to its characteristics, corresponds to similar preparations which are made available to the market by foreign companies, and can be considered as a new biologically active preparation with a wide spectrum of action. Of greatest interest is its ability - through the activation of macrophages - to enhance the adaptive immunity. In this regard, two areas of therapeutic use of the GcMAF-RF are proposed. The preparation will be in demand in the field of cancer treatment, and, in addition, it can be used in the treatment of a number of neurodegenerative pathologies.

The TRPM8 gene encodes the ion channel, which is a cold receptor in afferent neurons of the mammalian somatosensory system. We studied the frequency of haplotype distribution from six SNPs in the TRPM8 gene in Eurasian human populations, including Russians, Kazakhs and Chukchi. Four of the six SNPs are located in exon 7 (rs13004520, rs28901637, rs11562975, rs17868387), rs7593557 is in exon 11. These exons encode parts of the N-terminus, which is necessary for channel functioning in the plasma membrane of neurons. The rs11563071 is in exon 23 encoding part of the C-terminus. The primary difference in population distribution of haplotypes determines the SNP from exon 11 which leads to Ser419Asn substitution in protein. The most pronounced dif­ferences in the patterns of diversity and frequencies of haplotypes were observed between Chukchi and Russians. The frequency of major H1 haplotype encompassing the 419Ser gene variant differs in examined populations; 0.738 (Russians), 0.507 (Kazakhs) and 0.337 (Chukchi), p < 0.001. The TRPM8 gene variants encoding 419Asn and carrying the minor alleles of rs28901637 (P249P) and rs11562975 (L250L) in exon 7 are characteristic of Asian populations. The frequency of all 419Asn variants in Chukchi is comparable to that in Africans, however, the minor allele frequencies of rs28901637, rs11562975 in Africans is low. Apparently in the process of human colonization of Eurasia, minor alleles of these SNPs diverged depending on rs7593557 structure in exon 11. We analyzed sequences of five TRPM8 mRNA isoforms extracted by researchers from different tissues. Sequence analysis demonstrates that they are transcribed from major H1 variant of the TRPM8 gene but contain different translation start codons, which are generated by alternative splicing from pro-mRNA.

The article presents a variant of maturity onset diabetes of the young type 2, caused by a rare mutation in the GCK gene. Maturity onset diabetes of the young (MODY) is a hereditary form of diabetes with an autosomal dominant type of inheritance, an onset at a young age, and a primary defect in pancreatic β-cell function. This type of diabetes is different from classical types of diabetes mellitus (DM1 and DM2) in its clinical course, treatment strategies, and prognosis. Clinical manifestations of MODY are heterogeneous and may vary even among members of the same family, i. e., carriers of identical mutations. This phenotypic variation is due to the interaction of mutations with different genetic backgrounds and the influence of environmental factors (e. g., lifestyle). Using next-generation sequencing technology, the c.580–1G>A substitution (IVS5 –1G>A, rs1554335421) located in an acceptor splice site of intron 5 of the GCK gene was found in a proband. The identified variant cosegregated with a pathological phenotype in the examined family members. The GCK gene encodes glucokinase (hexokinase 4), which catalyzes the first step in a large number of glucose metabolic pathways such as glycolysis. Mutations in this gene are the cause of MODY2. The illness is characterized by an insignificant increase in the fasting glucose level, is a well-controlled disease without medication, and has a low prevalence of micro- and macrovascular complications of diabetes. The presented case of MODY2 reveals the clinical significance of a mutation in the splice site of the GCK gene. When nonclassical diabetes mellitus is being diagnosed in young people and pregnant women, genetic testing is needed to verify the diagnosis and to select the optimal treatment method.

Plant genetic resources (PGR) are the foundation of agriculture as well as food and nutritional security. The ICAR-NBPGR is the nodal institution at national level for management of PGR in India under the umbrella of Indian Council of Agricultural Research (ICAR), New Delhi. India being one of the gene-rich countries faces a unique challenge of protecting its natural heritage while evolving mutually beneficial strategies for germplasm exchange with other countries. The Bureaus activities include PGR exploration, collection, exchange, characterization, evaluation, conservation and documentation. It also has the responsibility to carry out quarantine of all imported PGR including transgenics meant for research purposes. The multifarious activities are carried out from ICAR-NBPGR headquarters and its 10 regional stations located in different agro-climatic zones of India. It has linkages with international organizations of the Consultative Group on International Agricultural Research (CGIAR) and national crop-based institutes to accomplish its mandated activities. NBPGR collects and acquires germplasm from various sources, conserves it in the Genebank, characterizes and evaluates it for different traits and provides ready material for breeders to develop varieties for farmers. ICAR-NBPGR encompasses the National Genebank Network and at present, the National Genebank conserves more than 0.40 million accessions. NBPGR works in service-mode for effective utilization of PGR in crop improvement programmes which depends mainly on its systematic characterization and evaluation, and identification of potentially useful germplasm. NBPGR is responsible for identifying trait-specific pre-adapted climate resilient genotypes, promising material with disease resistance and quality traits which the breeders use for various crop improvement programmes. The system has contributed immensely towards safeguarding the indigenous and introducing useful exotic PGR for enhancing the agricultural production. Presently, our focus is on characterization of ex situ conserved germplasm and detailed evaluation of prioritized crops for enhanced utilization; assessment of impact of on-farm conservation practices on genetic diversity; genome-wide association mapping for identification of novel genes and alleles for enhanced utilization of PGR; identification and deployment of germplasm/landraces using climate analog data; validation of trait-specific introduced germplasm for enhanced utilization.

Microbial culture collections are very important components of biological science. They provide researchers with material for studies and preserve biological resources. One such collection is the Collection of Rhizosphere Microorganisms, kept at the Institute of Biochemistry and Physiology of Plants and Microorganisms of the Russian Academy of Sciences, Saratov (IBPPM). Its activity is primarily directed toward the isolation and preservation of microorganisms from the plant root zone. The international research interest in microorganisms from this ecological niche is not waning, because they are very important for plant growth and development and, consequently, for plant breeding. The group of bacteria with properties of significance for plants has been given the name "plant-growth-promoting rhizobacteria” (PGPR). This group includes nitrogen-fixing soil alpha-proteobacte-ria of the genus Azospirillum, which form the core of the IBPPM collection. First discovered by Brazilian scientists in the 1970s, azospirilla are now a universally recognized model object for studying the molecular mechanisms underlying plant-bacterium interactions. The broad range of useful properties found in these microorganisms, including the fixation of atmospheric nitrogen, production of phytohormones, solubilization of phosphates, control of pathogens, and formation of induced systemic resistance in the colonized plants, make these bacteria an all-purpose tool that has been used for several decades in basic and applied research. This article reviews the current state of Azospirillum research, with emphasis on the results obtained at the IBPPM. Scientific expeditions across the Saratov region undertaken by IBPPM microbiologists in the early 1980s formed the basis for the unique collection of members of this bacterial taxon. Currently, the collection has more than 160 Azospirillum strains and is one of the largest collections in Europe. The research conducted at the IBPPM is centered mostly on the Azospirillum structures involved in associative symbiosis with plants, primarily extracellular polysaccharide-containing complexes and lectins. The development of immunochemical methods contributed much to our understanding of the overall organization of the surface of rhizosphere bacteria. The extensive studies of the Azospirillum genome largely deepened our understanding of the role of the aforesaid bacterial structures, motility, and biofilms in the colonization of host plant roots. Of interest are also applied studies focusing on agricultural and environmental technologies and on the "green” synthesis of Au, Ag, and Se nanoparticles. The Collection of Rhizosphere Microorganisms continues to grow, being continually supplemented with newly isolated strains. The data presented in this article show the great importance of specialized microbial culture repositories, such as the IBPPM collection, for the development and maintenance of the microbial research base and for the effective solution of basic and applied tasks in microbiology.