XIII IMBG All-Ukrainian Conference of Young Scientists

Abstract

Background. The influenza A(H1N1)pdm2009 (H1N1/2009) virus, that emerged during March and early April 2009, spread rapidly among humans to develop into the first human influenza pandemic over 40 years. Hemagglutinin is known to be a major target region of neutralizing antibodies, which inhibit binding with sialic acid receptors effectively. The virus evades these antibodies primarily by accumulating amino-acid substitutions in the HA’s antigenic sites. It is known that the H1 molecule has five antigenic sites: Sa, Sb, Ca1, Ca2, and Cb. The antigenic sites Sa and Sb, which contain the largest number of amino acid residues, are key in neutralizing epitopes that are adjacent to the receptor-binding pocket. The aim of our work was to analyze variability of [the] influenza viruses A(H1N1)pdm09 amino acid substitutions in antigenic sites of hemagglutinin. Methods. Nasal-throat swabs taken from influenza-affect-ed patients from different regions of Ukraine, collected during 2009-2017, were used in the study. The samples were analyzed using real-time polymerase chain reaction (RT-PCR). Influenza viruses were isolated in MDCK and MDCK-SIAT cell culture. Sequencing of influenza viruses genes, isolated in our laboratory, was performed in the World Influenza Center in London using the technology of RNA-SEQ, which allows sequencing coding and noncod-ing mRNA. [The] Nucleotide sequences were translated into [the] amino acid sequences using MEGA 6 software. Results. Ukrainian isolates between 2009-2017 years clustered in the influenza genetic groups 2, 6, 7, and 8. Genetic changes were observed in each of the antigenic sites: Sa – S162T, K163Q, K163I; Sb – S185T, A186T, S190G, S190R; Ca1 – S203T, R205K, E235V, E235D, S236P; Ca2 – P137H, H138R, A141T, D222G, D222N; Cb – A73S, S74R, S74N. The greatest number was detected on the sites Ca1 and Ca2. The smallest number of amino acid substitutions was detected in the antigenic site Cb. In spite of detected mutations in antigenic sites, Ukrainian isolates retained the similarity to the vaccine strain A/California/07/09 during 2009-2017. Conclusions. Information about the changes in antigenic sites is very important for prediction of the next dominant strains. It is well-documented that antigenic changes of HA occasionally result in the acquisition of carbohydrate side chains on the HA molecule. Since the carbohydrate side chains in the vicinity of antigenic sites mask the neutralizing epitopes on the HA surface, the amino acid substitutions associated with the acquisition of carbohydrate chains are believed to efficiently generate antigenic variants. Background. Swiss cheese (sws) is a Drosophila melanogaster ortholog of human Neuropathy target esterase (NTE or PLPLA6), a molecular target for the organophosphorus compound-induced delayed neuropathy (OPIDN). SWS is a transmembrane protein, loss of its function causes age-dependent neurodegeneration, glial hyperwrapping, and neuronal apoptosis. As shown previously, the sws mutants have decreased lifespan. This phenotype is accelerated even further when animals are starved or fed with organophosphates. Our aim was to find genes-partners of sws in order to clarify in which cell the signaling SWS is involved and to understand better the cellular mechanism of SWS/NTE-dependent neuropathy formation. Methods. Based on in-silico predictions and theoretical searching we selected 25 candidate genes that can potentially interact with SWS. To verify their functional interactions with SWS in vivo , we analyzed heterozygous animals with a reduction by one copy of the candidate gene in the sws mutant background (sws1). We analyzed three sws–related phenotypes: sensitivity to starvation, viability after organophosphate Diazinon treatment and brain tissue degeneration. To verify the sensitivity to starvation, 4–6 day-old flies were collected and transferred to vials with [the] control solution (10 % sucrose, 1 % yeast extract) or only with water; during next 6 days we counted dead flies. After 24-hour exposition to 0.0015 mg/ml Diazinon[, the] flies were transferred to a standard food vial, and their survival during next 14 days was estimated using GraphPad Prism 6 (Graphpad Inc., La Jolla, CA, USA). Log-rank test was used to estimate statistically significant difference. For brain tissue analysis we made paraffin sections of Background. To date long non-coding RNAs (lncRNAs) are actively studied since their regulatory potential in different biological processes was established, particularly, in transcription, translation, imprinting, cellular differentiation and development. The lncRNA HOTAIR (HOX antisense intergenic RNA) epigenetically affects the activity of cells cycle genes and its overexpression correlates with enhanced proliferation. Therefore, HOTAIR could be involved in [the] oncological transformation process, for instance, bladder cancer (BC) development. The aim of the research was to study the association between rs1899663 HOTAIR single nucleotide polymorphism (SNP) and BC metastasis in Ukrainian patients. Materials and methods. Venous blood of 141 patients with BC (68 patients with metastasis and 73 subjects without metastatic foci) was collected for genotyping. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was carried out in Thermocycler GeneAmp PCR System 2700 (Thermo Fisher Scientific, USA) for rs1899663 HOTAIR allele’s discrimination. The statistical processing of obtained results was done in the Statistical Package for Social Science software (SPSS, version 17.0, Chicago, IL, USA) and P < 0.05 was accepted as significant. Results. It was found the protective effect of TT-genotype against BC metastasis in both recessive (Pc = 0.047; ORc = 0.334, 95 % CI = 0.113-0.986) and additive (Pc = 0.04; ORc = 0.301, 95 % CI = 0.096-0.944) models of inheritance. Background. HA, NA, and NP genes are potential candidates as markers of genetic variability in avian influenza virus in birds, animals, and humans. Aim. The purpose of this study was to extract the potential of the HA, NA, and NP genes as markers of the polymorphism of avian influenza A viruses in domestic animals and humans with respect to the subtype H7N9 birds. Methods. The study used the MEGA6 and VectorNTI-11 Smith-Waterman algorithm nucleotide sequences of the hemaglutinin (HA), neurominidase (NA) and nucleoprotein (NP) genes taken from the virus database. A common region at the 3’-end of the segments was used to select the reverse primer sequences for amplifying the HA-NA and NP genes. To select direct primers, the multiple alignment of all the HA, NA, and NP gene sequences deposited in the ISD database (www.flu.lanl.gov) was performed. Then, the consensus sequences were formed for the HA and NA-ta NP genes; the common conservative areas (motifs) and selected sequences of direct primers were found for the first and second stages of amplification in compliance with the general principles; the primers were chosen. The following algorithm was used to select oligonucleotide markers capable of specifically detecting of a specific subtype of AIV: all HA, NA and NP sequences in the database were divided into groups according to certain variants of hemagglutinin (H1 and H7), neuraminidase (N1 and N9) and nucleoprotein (NP). 40 resulting groups were divided into subgroups according to the origin of the virus (isolated from humans, birds, etc. ), which formed a consensus sequence. Then, the most conservative motifs, limited by the primers of the second amplification stage, of the genomic segment, were determined. When it was impossible to identify a rather conservative phylogenetic analysis of the sequences was performed within the subgroup with its division into smaller subgroups. Then a consensus sequence was formed and a search for conservative areas was conducted. Results. A total of 3500 sequences of hemagglutinin, neuraminidase, and AIV nucleoproein segments were analyzed. The variable structural regions of the nucleotide sequences of genes were determined. In all genes, single nucleotide substitutions are acquired, and the most polymorphic loci are located at the 3 ‘and 5’ ends of the sequences. The number of oligonucleotides selected to determine a specific variant of HA, NA and NP depended on the number of identified conservative regions in the amplified region and on the degree of variability within the region. Based on the analysis, the primers were selected (4 for each gene of two subtypes) for an isothermal loop amplification reaction of viral genes, the set was constructed consisting of 24 discriminating oligonucleotides for specific analysis of AIV subtypes. Conclusions. The results of the analysis showed that the identification of RNA mutations and the most variable gene loci could be used for the genotyping of influenza A viruses. Aim. Development of an easy-to-use smartphone-based fluorescent sensor system using molecularly imprinted polymer (MIP) membranes for rapid and accurate detection of widespread food mycotoxin — aflatoxin B1; optimisation of composition of the MIP membranes used as recognition elements of the sensor systems, and application of the sensor system for analysis of both model and real samples (food products and feeding stuff). Methods. The technique of molecular imprinting and method of in situ polymerization were used for molecularly imprinted polymer membranes synthesis. The MIP membranes were synthesized using nontoxic and non-fluorescent close structural analogue of aflatoxin B1 (ethyl-2-oxocyclopentanecar-boxylate) as a dummy template. Triethyleneglycoldimethacrylate was used as a cross-linker. Compositions of MIP membranes were optimized using the method of computational model-ling. Results. The quantitative detection of aflatoxin B1, selectively adsorbed by the MIP membranes, is based on its natural fluorescence. Sensor responses were registered after selective adsorption of mycotoxins by the nanostructured rec