Development of the cell-ELISA test for the subtype identification of circulating influenza A(H1) and A(H3) viruses

The sensitive version of cell-ELISA was developed for the subtype-specific differentiation of current influenza A(H1N1)pdm09 and A(H3N2) viruses that are circulating in the human population. This method is based on the estimation of virus reproduction in infected MDCK cells. The detection step of this method is an interaction of the subtype-specific monoclonal antibodies (mAbs) with the viral hemagglutinin (НА) molecule. The influenza A virus strains, isolated in the 2014 epidemic season, were used to validate this method.It was shown that when using mAbs # 1/ # 2 or # 4 at a concentration of 10-15 µg/ml, the developed variant of cell-ELISA was able to detect НА protein synthesized in the infected cells of influenza A(H3N2) and A(H1N1)pdm09 viruses, respectively.The developed method can be used for the identification of modern influenza A viruses with low hemagglutination activity, which is not possible by the conventional hemagglutination inhibition test.The sensitive version of cell-ELISA was developed for the subtype-specific differentiation of current influenza A(H1N1)pdm09 and A(H3N2) viruses that are circulating in the human population. This method is based on the estimation of virus reproduction in infected MDCK cells. The detection step of this method is an interaction of the subtype-specific monoclonal antibodies (mAbs) with the viral hemagglutinin (НА) molecule. The influenza A virus strains, isolated in the 2014 epidemic season, were used to validate this method. It was shown that when using mAbs # 1/ # 2 or # 4 at a concentration of 10-15 µg/ml, the developed variant of cell-ELISA was able to detect НА protein synthesized in the infected cells of influenza A(H3N2) and A(H1N1)pdm09 viruses, respectively. The developed method can be used for the identification of modern influenza A viruses with low hemagglutination activity, which is not possible by the conventional hemagglutination inhibition test

Influence of single amino acid substitutions in the hemagglutinin on the antigenic and receptor-binding properties of influenza virus B/Florida/04/2006 of Yamagata-like evolutionary lineage

Influenza A and B viruses use sialylated oligosaccharide chains expressed on the surface of a host cell as the cell entry receptors. The type of the bond between sialic acid (SA) and the neighboring galactose residue (Gal) is one of the main characteristics that define the type of receptor. Influenza viruses recognize SAα2-3Gal- or SAα2-6Gal-structures on the surface of the cells. Influenza A viruses of avian origin bind α2-3-sialylated glycans, while the human strains bind preferentially α2-6-sialylated ones. However, the receptor-binding specificity of influenza B viruses has not been characterized sufficiently so far. In this study, we selected the escape mutants of influenza B/Florida/04/2006 strain (Yamagata-like lineage) using monoclonal antibodies (mAb) to hemagglutinin (HA). The analysis of the amino acid sequences of mAb-induced escape mutants revealed the single amino acid substitutions 40Tyr→His, 85His→Tyr, 202Asn→Lys and 242Ser→Arg in 10F4-, 8Н11-, 8Н3- and 9А3-induced HA variants, correspondingly. It was shown that the single amino acid substitutions 202Asn→Lys and 242Ser→Arg alter the receptor-binding specificity of the influenza B virus. These findings are important for the understanding of the influence of individual amino acid residues in HA on the receptor-binding properties of influenza B Yamagata-like lineage viruses and allow us to predict the possible ways of their evolution.Influenza A and B viruses use sialylated oligosaccharide chains expressed on the surface of a host cell as the cell entry receptors. The type of the bond between sialic acid (SA) and the neighboring galactose residue (Gal) is one of the main characteristics that define the type of receptor. Influenza viruses recognize SAα2-3Gal- or SAα2-6Gal-structures on the surface of the cells. Influenza A viruses of avian origin bind α2-3-sialylated glycans, while the human strains bind preferentially α2-6-sialylated ones. However, the receptor-binding specificity of influenza B viruses has not been characterized sufficiently so far. In this study, we selected the escape mutants of influenza B/Florida/04/2006 strain (Yamagata-like lineage) using monoclonal antibodies (mAb) to hemagglutinin (HA). The analysis of the amino acid sequences of mAb-induced escape mutants revealed the single amino acid substitutions 40Tyr→His, 85His→Tyr, 202Asn→Lys and 242Ser→Arg in 10F4-, 8Н11-, 8Н3- and 9А3-induced HA variants, correspondingly. It was shown that the single amino acid substitutions 202Asn→Lys and 242Ser→Arg alter the receptor-binding specificity of the influenza B virus. These findings are important for the understanding of the influence of individual amino acid residues in HA on the receptor-binding properties of influenza B Yamagata-like lineage viruses and allow us to predict the possible ways of their evolution

Spectral Studies of Rat Bone Tissue in Modeling Osteoporosis and Effectiveness of Treatment By Hydroxyapatite

Presents the result of experiments on the study of the model of osteoporosis in rats using Raman spectroscopy and the effectiveness of its treatment with hydroxyapatite. Were revealed spectral differences between groups of samples (control group, group with the model of osteoporosis and a group with the model of osteoporosis after treatment with hydroxyapatite). In addition, optical coefficients were introduced to evaluate the effectiveness of treatment. Keywords: Raman spectroscopy, optical coefficients, osteoporosis, hydroxyapatite, collagen matri

Changes in the antigenic and genetic structure of influenza viruses: analysis of surveillance data of influenza A and B in Russia in 2006-2013

The goal of this research project was to study the natural variability of human influenza A and B viruses based on the analysis of the population structure of influenza viruses, circulating in Russia in 2006-2013, in order to determine the direction of their genetic and antigenic drift by comparison to the WHO reference strains. Our results proved that during that period significant changes occurred in the genetic structure of influenza viruses, their phylogenetic affiliation, as well as their sensitivity to antiviral drugs. According to the surveillance data, the percentage of influenza A(H1N1) viruses among patients with influenza-like illness or acute respiratory infection gradually decreased from 42% of the total number of influenza viruses in 2006-2007 to 19% in 2008- 2009. Influenza A(H1N1) viruses are characterized by «silent» variability that manifests in the gradual accumulation of amino acid substitutions in the minor undetectable group of viruses.The share of influenza A(H3N2) viruses varied from 10% in the 1st post pandemic year to approx. 60% in 2008-2009 and 2011- 2012 epidemic seasons. All of the influenza A strains isolated during the last years of the period, covered in this study, were found to be susceptible to neuraminidase inhibitors and resistant to adamantane antivirals.Influenza B viruses of both Yamagata and Victoria lineages circulated in Russia in the period from 2006 to 2013. The vast majority of these influenza B viruses belonged to the Victoria lineage. Phylogenetic and antigenic analyses of influenza B viruses have demonstrated a gradual drift of Russian isolates from the reference strains. No changes leading to resistance to oseltamivir or zanamivir were found in influenza B strains isolated until 2013.The goal of this research project was to study the natural variability of human influenza A and B viruses based on the analysis of the population structure of influenza viruses, circulating in Russia in 2006-2013, in order to determine the direction of their genetic and antigenic drift by comparison to the WHO reference strains. Our results proved that during that period significant changes occurred in the genetic structure of influenza viruses, their phylogenetic affiliation, as well as their sensitivity to antiviral drugs. According to the surveillance data, the percentage of influenza A(H1N1) viruses among patients with influenza-like illness or acute respiratory infection gradually decreased from 42% of the total number of influenza viruses in 2006-2007 to 19% in 2008- 2009. Influenza A(H1N1) viruses are characterized by «silent» variability that manifests in the gradual accumulation of amino acid substitutions in the minor undetectable group of viruses. The share of influenza A(H3N2) viruses varied from 10% in the 1st post pandemic year to approx. 60% in 2008-2009 and 2011- 2012 epidemic seasons. All of the influenza A strains isolated during the last years of the period, covered in this study, were found to be susceptible to neuraminidase inhibitors and resistant to adamantane antivirals. Influenza B viruses of both Yamagata and Victoria lineages circulated in Russia in the period from 2006 to 2013. The vast majority of these influenza B viruses belonged to the Victoria lineage. Phylogenetic and antigenic analyses of influenza B viruses have demonstrated a gradual drift of Russian isolates from the reference strains. No changes leading to resistance to oseltamivir or zanamivir were found in influenza B strains isolated until 2013

Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNAiMet attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNAiMet, but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNAiMet reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNAiMet, consistent with its suggested role in promoting the ‘closed’ conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the ‘closed’ complex and is likely ejected from the P-site upon start codon recognition

Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNAiMet attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNAiMet, but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNAiMet reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNAiMet, consistent with its suggested role in promoting the ‘closed’ conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the ‘closed’ complex and is likely ejected from the P-site upon start codon recognition

Метаболические и реологические нарушения в остром периоде ишемического инсульта

Objective: to study the specific features of metabolism and blood rheological properties in the acute period of ischemic stroke (IS) in patients aged less than 50 years.Subjects and methods. Thirty patients (mean age 45.1±1.1 years) having acute IS were examined. According to its severity, the patients were divided into 3 groups: 1) 8 patients with mild IS; 2) 11 patients with moderate IS; 3) 11 with severe IS. All Group 3 patients were treated at an intensive care unit. A control group comprised 20 healthy individuals (mean age 44.7±1.0 years). In all the patients, fasting blood homocysteine concentrations were measured on an IMMULITE One immunochemiluminescent analyzer (USA). The rheological properties of blood were examined, by measuring its viscosity on a rotary viscometer (Russia) at a shear rate of 10 to 200 sec-1. Fibrinogen concentrations were determined on an ACL-100 coagulograph.Results. The patients who had experienced ischemic stroke at the age of under 50 years were found to have atherogenic dyslipidemia, elevated homocysteine and fibrinogen levels and considerably increased blood viscosity, which correlated with the severity of their condition and the outcome of stroke. The highest values were noted in patients with severe ischemic stroke and a poor outcome.Conclusion. Studies of homocysteine and fibrinogen concentrations and blood viscosity may be used as additional criteria for evaluating the severity of ischemic stroke and predicting its outcome in patients aged less than 50 years. Цель исследования — изучить особенности метаболизма и реологические свойства крови в остром периоде ишемиче-ского инсульта (ИИ) у пациентов в возрасте до 50 лет.Материалы и методы. Обследовано 30 пациентов в остром периоде ИИ (средний возраст 45,1±1,1 лет). По степени тяжести пациентов распределили на три группы: 1-я группа — пациенты с ИИ легкой степени тяжести (n=8), 2-я — пациенты с ИИ средней степени тяжести (n=11), 3-я — пациенты с ИИ тяжелой степени тяжести (n=11). Все пациенты третьей группы находились на лечении в отделении реанимации. Контрольную группу составили 20 здоровых лиц (средний возраст 44,7±1,0 лет). У всех пациентов натощак определяли в крови концентрацию гомоцистеина на иммунохемилюминесцентном анализаторе «IMMULITE One» (США). Показатели липидного обмена (общий холестерин, холестерин липопротеидов различной плотности, тригли-цериды) определяли автоматизированными методами на анализаторе «HITACHI-912». Реологические свойства крови изучали, измеряя ее вязкость на ротационном вискозиметре (Россия) при скоростях сдвига в диапазоне от 10 до 200с-1. Концентрацию фибриногена определяли на коагулографе ACL-100.Результаты. У пациентов, перенесших ишемический инсульт в возрасте до 50 лет, обнаружено наличие атерогенной дислипидемии, а также коррелирующие со степенью тяжестью состояния и исходом инсульта повышенный уровень гомоцистеина, фибриногена и значительное увеличение вязкости крови. Наиболее высокие значения отмечены у больных с ишемическим инсультом тяжелой степени тяжести, с неблагоприятным исходом.Заключение. Исследования концентрации гомоцистеина, фибриногена и вязкости крови могут использоваться в качестве дополнительных критериев при оценке степени тяжести и прогнозирования исхода ишемического инсульта у пациентов в возрасте до 50 лет.

mRNA localization, reaction centre biogenesis and thylakoid membrane targeting in cyanobacteria

The thylakoid membranes of cyanobacteria form a complex intracellular membrane system with a distinctive proteome. The sites of biogenesis of thylakoid proteins remain uncertain, as do the signals that direct thylakoid membrane-integral proteins to the thylakoids rather than to the plasma membrane. Here, we address these questions by using fluorescence in situ hybridization to probe the subcellular location of messenger RNA molecules encoding core subunits of the photosystems in two cyanobacterial species. These mRNAs cluster at thylakoid surfaces mainly adjacent to the central cytoplasm and the nucleoid, in contrast to mRNAs encoding proteins with other locations. Ribosome association influences the distribution of the photosynthetic mRNAs on the thylakoid surface, but thylakoid affinity is retained in the absence of ribosome association. However, thylakoid association is disrupted in a mutant lacking two mRNA-binding proteins, which probably play roles in targeting photosynthetic proteins to the thylakoid membrane

Результаты 5-летнего мониторинга за циркуляцией сезонных коронавирусов у госпитализированных детей в препандемическом периоде

Coronaviruses can cause damage to various parts of the respiratory system, gastrointestinal tract, and other organs and systems.The aim of the study: to monitor the circulation of seasonal coronaviruses in hospitalized children in the pre-pandemic period.Materials and methods: real-time multiplex PCR was used to test samples of nasopharyngeal mucus from 2188 patients aged 1 monthto 17 years, hospitalized with acute respiratory infection in 2014—2018. The results are presented with the indication of the fractions (%) and the calculation of the 95% confidence interval according to Klopper-Pearson. The differences between the groups were evaluated using the Pearson χ2 test. The differences in the groups were considered statistically significant at the level of the criterion p< 0.05.Results: monitoring of the circulation of pathogens of acute respiratory viral infection (ARVI) during 5 epidemic seasons showed that the appearance of a new subtype of coronavirus in 2019 was preceded by a gradual displacement of influenza, RS-and bocavirus infections from the circulation due to a statistically significant increase in the proportion of seasonal coronaviruses from 3.6% in 2014—2015 to 10.8% in the prepandemic season 2018—2019 (p= 0.007). The circulation of seasonal coronaviruses had a distinct seasonality (november-april)with the peak of registration in february (28.4%) and march (36.7%). Seasonal coronaviruses were detected in 7.3% of hospitalized children with ARVI, with a predominance in the age groups under 2 years (58.2%) and 3—6 years (25.4%). Hospitalization was more often required for patients with lower respiratory tract lesions (58.2%), a fifth of which was pneumonia (21.8%). In most children, ARVI caused by coronaviruses occurred as a monoinfection (79.9%), combined infection with other pathogens was observed in 20.1% of cases with fluctuations from 18.2% to 28.6% in different epidemic seasons. Viral associations are most common in young children (85.2%).Коронавирусы могут вызвать поражение различных отделов дыхательной системы, желудочно-кишечного тракта, а также других органов и систем.Цель исследования: провести мониторинг за циркуляцией сезонных коронавирусов у госпитализированных детей в препандемическом периоде.Материалы и методы: методом мультиплексной ПЦР в реальном времени проведено тестирование образцов носоглоточной слизи 2188 пациентов в возрасте от 1 мес. до 17 лет, госпитализированных с острой респираторной инфекцией в 2014—2018 гг. Результаты представлены с указанием долей (%) и расчетом 95% доверительного интервала по Клопперу-Пирсону. Различия между группами оценивали с помощью критерия χ² Пирсона. Различия в группах считались статистически значимыми при уровне критерия p< 0,05.Результаты: мониторинг за циркуляцией возбудителей острой респираторной вирусной инфекции (ОРВИ) в течение 5-ти эпидемических сезонов показал, что появлению нового подтипа коронавируса в 2019 г. предшествовало постепенное вытеснение из циркуляции возбудителей гриппа, РС- и бокавирусной инфекции за счет статистически значимого увеличения доли сезонных коронавирусов с 3,6% в 2014—2015 гг. до 10,8% в препандемическом сезоне 2018—2019 гг. (р= 0,007). Циркуляция сезонных коронавирусов имела отчетливую сезонность (ноябрь-апрель) с пиком регистрации в феврале (28,4%) и марте (36,7%). Сезонные коронавирусы выявлялись у 7,3% госпитализированных детей с ОРВИ, с преобладанием в возрастных группах до 2 лет (58,2%) и 3—6 лет (25,4%).Госпитализация чаще требовалась пациентам с поражением нижних отделов респираторного тракта (58,2%), пятую часть из которых составляла пневмония (21,8%). У большинства детей ОРВИ, обусловленная коронавирусами, протекала в виде моноинфекции (79,9%), сочетанное инфицирование с другими возбудителями наблюдалось в 20,1% случаев с колебаниями от 18,2% до 28,6% в различные эпидемические сезоны. Вирусные ассоциации наиболее характерны для детей раннего возраста (85,2%