Adaptation of the quantitative PCR method for the detection of the main representatives of cereal grain mycobiota

The content of fungal DNA and mycotoxins in cereal crops (31 varieties of wheat, oats, and barley) was quantitatively determined and used for comparative characterization of grains. The quantitative PCR has been adapted for the analysis of the target DNA of Alternaria spp., Bipolaris sorokiniana (B. sorokiniana), Fusarium graminearum (F. graminearum), F. culmorum, and F. sporotrichioides fungi, which are often present in mycobiota of small grain cereals. The content of DNA of aggressive pathogen B. sorokiniana was determined using quantitative PCR for the first time. The DNA of Alternaria fungi was found abundantly in all grain samples, but its content in the oat was significantly higher compared to barley and wheat (5 and 9 times higher, respectively). In barley grain, the content of B. sorokiniana DNA was on average significantly higher than in the grains of oats and wheat. The presence of F. graminearum DNA was established in all the analyzed grain samples while the F. culmorum DNA was found in 70% of the oat’s samples and in all samples of barley and wheat. Mycotoxin deoxynivalenol (DON) produced by these fungi was detected in all analyzed cereal grains in a range from 77 to 4133 μg/kg. The DNA of F. sporotrichioides was detected in 70% of oats and 50% of barley samples but was not found in wheat. The T-2 toxin produced by this fungus was detected in 45% of all samples within the range from 2 to 89 μg/kg. The statistically significant positive correlation with the Pearson correlation coefficient (r) equal to 0.49 (p<0.05) was observed between the amount of F. graminearum DNA and DON in the grain samples. Another significant positive correlation (r = 0.72, p<0.01) was found between DNA contents of Alternaria fungi and F. sporotrichioides in the grain samples. This leads to the suggestion that conditions for growth of these fungi in grain substrates are similar.The content of fungal DNA and mycotoxins in cereal crops (31 varieties of wheat, oats, and barley) was quantitatively determined and used for comparative characterization of grains. The quantitative PCR has been adapted for the analysis of the target DNA of Alternaria spp., Bipolaris sorokiniana (B. sorokiniana), Fusarium graminearum (F. graminearum), F. culmorum, and F. sporotrichioides fungi, which are often present in mycobiota of small grain cereals. The content of DNA of aggressive pathogen B. sorokiniana was determined using quantitative PCR for the first time. The DNA of Alternaria fungi was found abundantly in all grain samples, but its content in the oat was significantly higher compared to barley and wheat (5 and 9 times higher, respectively). In barley grain, the content of B. sorokiniana DNA was on average significantly higher than in the grains of oats and wheat. The presence of F. graminearum DNA was established in all the analyzed grain samples while the F. culmorum DNA was found in 70% of the oat’s samples and in all samples of barley and wheat. Mycotoxin deoxynivalenol (DON) produced by these fungi was detected in all analyzed cereal grains in a range from 77 to 4133 μg/kg. The DNA of F. sporotrichioides was detected in 70% of oats and 50% of barley samples but was not found in wheat. The T-2 toxin produced by this fungus was detected in 45% of all samples within the range from 2 to 89 μg/kg. The statistically significant positive correlation with the Pearson correlation coefficient (r) equal to 0.49 (p<0.05) was observed between the amount of F. graminearum DNA and DON in the grain samples. Another significant positive correlation (r = 0.72, p<0.01) was found between DNA contents of Alternaria fungi and F. sporotrichioides in the grain samples. This leads to the suggestion that conditions for growth of these fungi in grain substrates are similar

Vaccine interchangeability: problems and prospects

Vaccines by different manufacturers are available for most of the vaccine-preventable infections covered by the National Immunisation Schedule of the Russian Federation. Determination of the possibility of replacing a vaccine in the case of routine or emergency vaccination still remains a challenging issue. The aim of the study was to substantiate the problem of vaccine interchangeability, outline specific challenges and ways of solving them, analyse criteria underlying evaluation of vaccine interchangeability in Russia, and international experience in this area. The procedure for determining the interchangeability of biological products, including vaccines, was established in the Decree of the Government of the Russian Federation of 5 September 2020, No. 1360 “On the procedure for determination of interchangeability of medicinal products for human use”. The paper analyses the applicability of the official criteria for interchangeability of biological products, including vaccines. It outlines the main problems of performing evaluation of vaccine interchangeability in accordance with the established criteria. It is concluded that the vaccine interchangeability criteria need to be clarified in order to allow for assessment of comparability of active substances, and comparison of efficacy, safety, and immunogenicity of vaccines, taking into account vaccination schedules for different age groups. The possibility of evaluating interchangeability based on the results of post-authorisation studies also needs clarification. It is also necessary to align patient information leaflets for interchangeable vaccines

Characterization of resistance of winter wheat varieties to Fusarium head blight

In this study, naturally and artificially inoculated winter wheat varieties were studied with respect to their productivity and resistance to Fusarium head blight (FHB). We used the following set of disease assessment parameters: the percentage of visually and latent Fusarium-damaged grains (FDG); the DNA content of Fusarium fungi; the productivity of inoculated plants compared with non-inoculated plants; and the amount of mycotoxins in the grain. In case of naturally infected grains, the average FDG was found to be about 6.1 % (range of 0–15 %). The amount of DNA of Fusarium graminearum was found to be in the range of (1.1–42.7) × 10–5 ng/ng wheat DNA. The mycotoxin deoxynivalenol (DON) was detected in 15 samples of grain from plants that were grown under natural infection. The maximum DON amount was found to be 420 μg/kg. Fumonisin B1 (FB1) was not be detected in naturally infected grain. In case of artificially inoculated plants, the average FDG was found to be 25.8 % (2–54 %). The amount of F. graminearum DNA was found to be significantly higher (4.24– 49.8) × 10–3 ng/ng than it was detected in grain of non-inoculated plants. The wheat varieties inoculated with F. graminearum contained DON in high amounts from 20255 to 79245 μg/kg. Furthermore, a significant amount of FB1 was detected in all wheat varieties in the range of 980–20326 μg/kg. Among the analysed wheat varieties, Adel was characterized to be the most resistant to fungal infection as well as to the contamination by mycotoxins. Antonina, Lebed and Pamyat varieties were classified more relatively resistant than that of other varieties, and Utrish variety was found to be the most susceptible to FHB. The similar resistance of wheat varieties against F. graminearum and F. verticillioides infection was recorded, and the interactions between the fungi during the colonization of grain were shown

The kinetics of L10 superstructure formation in the Cu–56Au alloy (at. %): resistometric study

Due to the improved strength properties compared to the equiatomic Cu–50 at. % Au alloy, non-stoichiometric Cu–56 at. % Au alloy can be used both in dentistry and as a corrosion-resistant conductor of weak electrical signals in tool engineering. The work studies the kinetics of the disorder→order phase transformation in the Cu–56Au alloy, during which the disordered fcc lattice (A1-phase) is rearranged into an atomically ordered one with the L10 superstructure. The initial disordered state of the alloy was obtained in two ways: applying plastic deformation by 90 % or quenching at a temperature of above 600 °C (i. e., from the region of the A1-phase existence). To form the L10 superstructure, annealing was carried out at temperatures of 200, 225, and 250 °C. The annealing duration ranged from 1 h to 2 months. Resistometry was chosen as the main technique to study the kinetics of the disorder→order transformation. The temperature dependences of the electrical resistivity of the alloy in various structural states are obtained. The authors constructed the graphs of the electrical resistance dependence on the annealing time logarithm, based on which, the rate of the new phase formation was estimated. To evaluate the structural state of the alloy at various transformation stages, the authors used X-ray diffraction analysis (XRD). The crystal structure rearrangement during the transformation is shown by the example of splitting the initial cubic A1-phase peak (200) into two tetragonal ordered L10 phase peaks – (200) and (002). Based on the resistometry and X-ray diffraction analysis data, the authors carried out a quantitative assessment of the rate of the disorder→order phase transformation in the alloy under the study. It is established that the values of the converted volume fraction (resistometry) and the long-range order degree (X-ray diffraction analysis) are close. The study shows that in the temperature range of 200–250 °C, the rate of atomic ordering according to the L10 type in the nonstoichiometric alloy Cu–56 at. % Au is maximum at 250 °C. It is identified that the disorder→order transformation in the initially quenched specimens of the investigated alloy proceeds approximately an order of magnitude faster than in preliminarily deformed specimens

Cold-induced changes in gene expression in brown adipose tissue, white adipose tissue and liver

Cold exposure imposes a metabolic challenge to mammals that is met by a coordinated response in different tissues to prevent hypothermia. This study reports a transcriptomic analysis in brown adipose tissue (BAT), white adipose (WAT) and liver of mice in response to 24 h cold exposure at 8°C. Expression of 1895 genes were significantly (P<0.05) up- or down-regulated more than two fold by cold exposure in all tissues but only 5 of these genes were shared by all three tissues, and only 19, 14 and 134 genes were common between WAT and BAT, WAT and liver, and BAT and liver, respectively. We confirmed using qRT-PCR, the increased expression of a number of characteristic BAT genes during cold exposure. In both BAT and the liver, the most common direction of change in gene expression was suppression (496 genes in BAT and 590 genes in liver). Gene ontology analysis revealed for the first time significant (P<0.05) down regulation in response to cold, of genes involved in oxidoreductase activity, lipid metabolic processes and protease inhibitor activity, in both BAT and liver, but not WAT. The results reveal an unexpected importance of down regulation of cytochrome P450 gene expression and apolipoprotein, in both BAT and liver, but not WAT, in response to cold exposure. Pathway analysis suggests a model in which down regulation of the nuclear transcription factors HNF4α and PPARα in both BAT and liver may orchestrate the down regulation of genes involved in lipoprotein and steroid metabolism as well as Phase I enzymes belonging to the cytochrome P450 group in response to cold stress in mice. We propose that the response to cold stress involves decreased gene expression in a range of cellular processes in order to maximise pathways involved in heat production

Concerning the Application of Heterologous Preparations in Practical Healthcare

The paper presents the data on the assessment of the demand for heterologous drugs and highlights the need for their use in practical healthcare. Currently, 15 heterologous immunoglobulins and sera are registered in the Russian Federation. Nine of them are against bacterial and viral infections (anthrax, diphtheria, tetanus, botulism, rabies, anti-gangrenous), one – against snake venom, five other – anti-thymocyte immunoglobulins produced by domestic and foreign manufacturers. Analysis of the data has demonstrated that anti-rabies immunoglobulin, anti-diphtheria and anti-botulinic sera, A, B and E type are the most in-demand. Production of other therapeutic drugs from this group, for instance, anti-gangrenous serum, tends to decline. Adequate provision of medical curative and preventive institutions with these preparations is a significant element in timely treatment of infectious diseases such as anthrax, diphtheria, tetanus, botulism, rabies, as well as snake bites. In prophylaxis and treatment of certain diseases (emergency aid in case of anthrax, rabies, venomous snake bites) heterologous preparations still oftentimes do not have an alternative. The key problem in regulation of heterologous preparation circulation is absence of quality, efficacy and safety criteria applicable to other immunobiological preparations. Development of unified requirements to standardization of heterologous sera and immunoglobulins of various specificity, principles of efficacy and safety evaluation is necessary for harmonization of current guidelines on medical use. Earlier elaborated by WHO approaches to heterologous sera to venoms of snakes can be used for monitoring and harmonization of normative legal documents on the improvement, evaluation of quality, efficacy and safety of alternative drugs being in circulation in the pharmaceutical market of the Russian Federation

Diagnostic value of <i>Rf1</i> gene molecular markers in sunflower

Background. Modern production of sunflower seeds is currently based on the cultivation of high-yielding heterotic F1 hybrids from crossbreeding of lines with cytoplasmic male sterility (CMS) of PET1-type and fertility restorer lines. The paternal parent serves as a donor of the nuclear Rf1 gene functional allele, which is responsible for pollen fertility restoration in F1 plants. The detection of carriers of the Rf1 locus recessive and dominant alleles using diagnostic molecular markers accelerates breeding of female and male parental lines for creating hybrids.Materials and methods. The material for the study included 75 lines of various origins from the VIR sunflower genetic collection as well as hybrids from crosses of VIR 116A sterile line with fertile lines differing in the type of cytoplasm (fertile or sterile) and the presence of molecular markers, most of which were linked to the Rf1 locus. For marker validation, two different approaches were used: either by analyzing associations between the ability of a line to restore pollen fertility and the presence of molecular markers in its genotype, or by estimating recombination frequency between the Rf1 locus and marker loci in four segregating hybrid populations.Results. According to the obtained results, no markers demonstrated 100% efficiency in the analysis of the sample of genotypes. The ORS511 marker was most frequently observed among the lines presumably carrying the dominant allele Rf1. Pollen fertility of F1 hybrids from interline crossings was 89-99%. The segregation for fertility/sterility in F2 fitted the theoretical ratio of 3:1 expected in case of the monogenic control of the trait. The markers HRG01, HRG02 and ORS511 were linked to the fertility restoration trait, with recombination rates between Rf1 locus and markers varying in different cross combinations. The analysis of VIR 116А × VIR 740 and VIR 116А × RIL 130 hybrids showed that among the marker loci studied, the ORS511 was closest to the Rf1 locus Rf1 (recombination frequency of 2.2 and 3.3%, respectively). The recombination rate between the Rf1 and ORS511 loci equaled 7.5% in the cross VIR 116А × VIR 210 and 8.9% in VIR 116 × VIR 195.Conclusion. The markers ORS511, HRG01 and HRG02 are the most efficient for the identification of alleles of the Rf1 gene and for the marker assisted selection in hybrid populations produced involving sunflower lines from the VIR collection

Resistance of oat breeding lines to grain contamination with Fusarium langsethiae and T-2/HT-2 toxins

Fusarium disease of oats reduces yield quality due to decreasing germination that is caused by then contamination of grain with mycotoxins produced by Fusarium fungi. The aim of this study was to characterize the resistance of naked breeding lines of oats to fungal grain infection and to contamination with T-2 and HT-2 toxins. Thirteen naked oat breeding lines and two naked varieties, Nemchinovsky 61 and Vyatskiy, as well as a husked variety Yakov, were grown under natural conditions in the Nemchinovka Federal Research Center in 2019–2020. The contamination of grain with fungi was determined by the mycological method and real-time PCR. The analysis of mycotoxins was carried out by ELISA. In oats, Alternaria (the grain infection was 15–90 %), Cochliobolus (1–33 %), Cladosporium (1–19 %), Epicoccum (0–11 %), and Fusarium (3–17 %) fungi prevailed in the grain mycobiota. The predominant Fusarium species were F. poae (its proportion among Fusarium fungi was 49–68 %) and F. langsethiae (29–28 %). The highest amounts of F. langsethiae DNA ((27.9–71.9)×10–4 pg/ng) and T-2/HT-2 toxins (790–1230 μg/kg) were found in the grain of husked oat Yakov. Among the analysed naked oat lines, the amount of F. langsethiae DNA varied in the range of (1.2–42.7)×10–4 pg/ng, and the content of T-2/HT-2 toxins was in the range of 5–229 μg/kg. Two oat breeding lines, 54h2476 and 66h2618, as well as a new variety, Azil (57h2396), can be characterized as highly resistant to infection with Fusarium fungi and contamination with mycotoxins compared to the control variety Vyatskiy

Overview of the Situation on Highly Pathogenic Avian Influenza Virus H5 in Russia in 2016–2017

In 2016–2017, several outbreaks among wild and domestic birds caused by highly pathogenic influenza virus were recorded in the territory of the Russian Federation. After isolation of the highly pathogenic influenza A (H5N8) virus in the territory of the Republic of Tyva in May 2016, the virus was introduced into the west by wild birds, where it caused a massive epizootic. The death of agricultural poultry was noted in several regions of the European part of Russia and epizootics continued until the end of 2017. This paper presents the data on circulation of highly pathogenic influenza virus over the past two years. The current status regarding H5 influenza in the territory of the Russian Federation is also considered, where for the first time since 2005 a large-scale epizooty caused by the influenza virus subtype A (H5N8) was recorded. It is shown that the territory of Russia plays an important geographical role in the spread of influenza virus by wild birds from Southeast Asia to Europe and North America, and the research also suggests the possible continuation of circulation of highly pathogenic viruses in the territory of Russia

Влияние уровня минерального питания и комплекса агротехнических приемов на семенную продуктивность и качество новых сортов картофеля разных групп спелости в конкретных почвенно-климатических условиях

Relevance. The aim was to study the reaction of new potato varieties early ripening group Zabava and middle-ripening group Smolyanochka on the timing of planting and doses of mineral fertilizers for obtaining maximum yield of seed potatoes. As mineral fertilizers used azophoska, superphosphate and potassium chloride.Results. It was found that the variety Zabava compared to the variety Smolyanochka, had a higher stem-forming ability and exceeded the parameters of the stem in all variants of the experiment. This is due to the biological characteristics of the studied varieties, since the Zabava variety, unlike the Smolyanochka variety, had more multiple eyes on the surface of the tuber. Against the background of applying mineral fertilizers N60P72K90, the density of the stem was higher, compared with the background of applying N32P32K32 in both the Zabava variety and the Smolyanochka variety. It should also be noted that there was a tendency to reduce the stem at a later date of planting in all variants of the experiment. The percentage of viral diseases affected increased with later planting dates and was highest with the planting dates of 14 and 20 may. The highest yield of tubers of the seed fraction was obtained in the variant with a planting date of may 14 against the background of balanced doses of mineral fertilizers (N60P72K90) and amounted to 13.3 t/ha in the Zabava variety, 13.0 t/ha in the Smolyanochka variety. It should be noted that different backgrounds of mineral fertilizers did not have a significant impact on the percentage of potato tubers affected by diseases. At the same time, the incidence of tubers with diseases did not exceed the regulatory requirements of GOST R 53136 – 2008 "Seed potatoes. Technical conditions».Актуальность. В статье изложен экспериментальный материал по реакции новых сортов картофеля разных групп спелости Забава и Смоляночка на уровни минерального питания, сроки посадки, урожайность и качество продукции.Материал и методы. Цель исследований – изучить реакцию новых сортов картофеля ранней группы спелости Забава и среднеранней группы спелости Смоляночка на сроки посадки и дозы минеральных удобрений для получения максимального урожая семенного картофеля. В качестве минеральных удобрений использовали азофоску, суперфосфат и калий хлористый.Результаты. Установлено, что сорт Забава по сравнению с сортом Смоляночка, обладал более высокой стеблеобразующей способностью и превышал параметры стеблестоя во всех вариантах опыта, что связано с биологическими особенностями изучаемых сортов. Так сорт Забава, в отличие от сорта Смоляночка, обладал более множественными глазками на поверхности клубня. На фоне внесения минеральных удобрений N60P72K90 густота стеблестоя была выше, по сравнению с фоном внесения N32P32K32 как у сорта Забава, так и у сорта Смоляночка. Наблюдалась тенденция снижения стеблестоя при более поздних сроках посадки во всех вариантах опыта. Процент поражения вирусными болезнями также увеличивался при более поздних сроках посадки и наибольшим был при посадке 14 и 20 мая. Наибольший выход клубней семенной фракции был получен в варианте со сроком посадки 14 мая на фоне сбалансированных доз минеральных удобрений (N60Р72К90) и составил у сорта Забава 13,3 т/га, у сорта Смоляночка – 13,0 т/га. Разные фоны внесения минеральных удобрений не оказали существенного влияния на процент поражения клубней картофеля болезнями. При этом пораженность клубней болезнями не превышала нормативных требований ГОСТа Р 53136 – 2008 «Картофель семенной. Технические условия»