Effects of paricalcitol on hydrogen peroxide-induced oxidative damage in human umbilical vein endothelial cells through vitamin D receptor signaling

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POSTERS

About these abstracts

Abstracts submitted to the Congress are not peer-reviewed. In addition, abstracts are published as submitted and are not copyedited prior to publication.

We are unable to make corrections of any kind to the abstracts once they are published.

Indexing

Abstracts published in FEBS Open Bio Supplement for the 43rd FEBS Congress will be included individually in the Conference Proceedings Citation Index published by Web of Science.

How to cite these abstracts

AuthorOne, A., AuthorTwo, B. (2018). Abstract title. FEBS Open Bio, 8: Abstract number*. doi:10.1002/2211-5463.12453

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P.01 DNA, RNA and nucleotides

P.02 Chromosome structure and chromatin P.03 Genome dynamics and epigenetics P.04 The structural organization of the cell P.05 Stem cells

P.06 Photosynthesis and plant biochemistry P.07 Biotechnology

P.08 Host–pathogen interactions P.09 Biochemistry and medicine P.10 Cancer biology

P.11 Xenobiochemistry and drug metabolism P.12 Gaseous molecules

P.13 Redox biochemistry and signalling

P.14 Biochemical processes at cellular membranes

P.15 Signalling through membranes and receptors P.16 Extracellular matrix

P.17 Glycans

P.18 Protein – folding, dynamics, interaction and degradation P.19 Structural biology P.20 Systems biology P.21 Synthetic biology P.22 Computational biology P.23 Structural bioinformatics P.24 Omics technologies P.25 Nanoworld

P.26 Chemistry of food and environment P.27 Biochemical education

P.28 Miscellaneous

* Each poster has been given a unique number beginning with the letter P; the next part relates to the session in which the poster will be presented.

106 FEBS Open Bio 8 (Suppl.S1) (2018) 105–106 DOI: 10.1002/2211-5463.12453

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POSTERS

DNA, RNA and nucleotides

P.01-001-Mon

Escherichia coli single-stranded DNA binding

protein SSB binds to AlkB and promotes DNA

repair

R. Nigam, M. Mohan, A. Roy

Indian Institute of Technology Hyderabad, Hyderabad, India Repair of alkylation damage in DNA is essential for maintaining genome integrity. E. coli DNA repair enzyme AlkB removes vari-ous alkyl adducts present in DNA by oxidative demethylation. Previous studies showed that AlkB preferentially repairs single-stranded DNA (ssDNA). However, ssDNA remains bound to E. coli single-stranded DNA binding protein SSB. It is unclear whether AlkB can repair alkyl adduct present in SSB-coated ssDNA. We have addressed this question by studying AlkB-mediated DNA repair using SSB-bound DNA containing alkyl-adduct as substrate. We have also found AlkB interacts with SSB. Yeast two-hybrid and in vitro pull-down experiments showed that SSB interacts with AlkB via structurally disordered 17 amino acid residues located in the disordered CTD. With reconstituted in vitro repair reaction we observed that AlkB effi-ciently remove alkyl-adducts from shorter oligonucleotide sub-strate but the efficiency of AlkB catalyzed reaction was less compared to SSB-bound DNA substrate. Using 70-mer oligonu-cleotide containing single alkyl adduct, we demonstrate that SSB-AlkB interaction was dispensable for the repair of terminally located methyl adducts but crucial for the faster repair of inter-nally located adducts. When ssDNA was bound to truncated SSB lacking the interacting residues, stimulation of AlkB activity was partially abolished suggesting functional importance of SSB-AlkB interaction.

P.01-002-Tue

The effect of sperm DNA fragmentation on the

pregnancy rate in Intra Uterine Cycles (IUI)

S. Isik1, S. Ege2

1

Trakya University, Edirne, Turkey,2Near East University, Nicosia, Mersin 10, Turkey

The objective of this study was to detect the DNA damage ratio of sperm samples of infertile couples scheduled for intrauterine insemination (IUI) treatment with the method of terminal deoxynucleotidyl transferase-mediated dUTP- biotin end –label-ing (TUNEL) test and to detect DNA damage with semen analy-sis and pregnancy rates. The study was designed as a prospective randomized controlled follow up study for 81 infertile couples. Assessment of sperm DNA damage prior to their use in IUI is essential for use in assisted conception. Semen samples analyzed with WHO laboratory manual. The percentage of each sperm with DNA fragmentation was determined using the method of TUNEL. The duration was 18 months after approval from of the ethics committee. Standard semen qualities are not sufficient for the outcome of IUI. However, sperm DNA quality has been emphasized on its clinical significance and its relationship with infertility. Written consent for use of the sperm for research was obtained from the patients who were selected for the IUI treat-ment. Questionary was performed to obtain smoking and alcohol consumption and days of abstinence prior to production of the semen sample. Gradient and swim-up preparation techniques were used in IUI. The most motile sperms in the supernatant

after the swim-up test were used for IUI and the spare sperm sus-pension in the pellet was prepared for TUNEL staining. The staining was done according to the TUNEL staining kit proce-dure and the ratio of the defected DNA in the sperms were deter-mined by fluorescent microscopy. A positive control was done on positive slides supplied in the kit. There was no statistical differ-ence between the pregnancy and no pregnancy groups in terms of liquefaction time, volume, pH, sperm concentration, motility, and morphology. Although the DNA damage percentage mea-sured by TUNEL test was less in the pregnancy achieved group compared to no pregnancy group, it did not reach statistically significant levels.

P.01-003-Wed

Novel backbone modified antisense

oligonucleotides with improved potency

effectively inactivate oncogenic miRNA-21

comparing to phosphorothioate

oligonucleotides

S. Miroshnichenko, O. Patutina, E. Burakova, B. Chelobanov, A. Fokina, D. Stetsenko, V. Vlassov, M. Zenkova

Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia

Vast amount of earlier studies confirms successful application of antisense oligonucleotides targeted to oncogenic miRNAs for the suppression of various events of carcinogenesis. Despite signifi-cant achievements in the field of antisense technology, develop-ment of new oligonucleotide structures and chemical modifications to increase the effectiveness of anticancer therapy remains an urgent and promising task nowadays. In the present work, the new effective antisense oligonucleotides containing the newest mesyl phosphoramidate modification of internucleotide bonds were developed and comprehensively studied. Functional-ity and biological potency of designed antisense oligonucleotides were assayed with respect to highly oncogenic miRNA-21. The study of biological properties of the engineered nucleic acids has shown a number of advantageous properties such as high binding affinity to the target, tremendous stability in the biological media and RNase H-activating ability. For these properties, the devel-oped oligonucleotides significantly exceed the well-known phos-phorothioate (PS) modification. In comparison with PS analogs it was shown that transfection of mesyl phosphoramidate oligonucleotides to the melanoma B16 cells is accompanied by low unspecific cytotoxicity and leads to the more significant dose-dependent decrease in the level of miR-21 and reduction in migration activity of cancer cells. Thus, a novel backbone modi-fied antisense oligonucleotides were developed which are a wor-thy alternative to phosphorothioates and can become a new powerful tool in the development of antisense technology. This work is supported by Grants of Russian Science Foundation No. 14-44-00068 and No. 17-44-07003, Grant of Russian Foundation for Basic Research No. 16-03-01055 and State funded budget project (VI.62.1.3, 0309-2016-0005).

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P.01-004-Mon

Analysis of microRNA at endometriosis and

uterine endometrial cancer

Z. Biscakova, M. Ferencakova, M. Rabajdova, I. Spakova, M. Marekova

Pavol Jozef Safarik University in Kosice, Faculty of Medicine, Department of Medical and Clinical Biochemistry, Kosice, Slovakia Oncological diseases of the female reproductive system are cur-rently among most commonly occurring diseases and are world-wide the main cause of female mortality. Clinical diagnostics of many cancers are often successful in advanced stages of the tumour, therefore current research is still directed towards identi-fication and characterization new biochemical and molecular markers. Molecules of miRNA can regulate cell proliferation, dif-ferentiation, apoptosis and many other biological processes in the cells. Due to properties, miRNA could be diagnostic marker. The main aim of our analysis was detection of molecular expression level of miR-17-5p and miR-99 in selected diseases of the female reproductive system. Experimental groups consisted from patients with histologically diagnosed endometriosis and carcinoma of corpus uteri(CU) (n= 15). The obtained material was used for isolation of miRNA, transcription into cDNA and determination expression levels of miR-17-5p and miR-99 in patients. Quantifi-cation of expression changes of selected miRNAs was detected by qRT-PCR. In the experimental group of patients with endometriosis, was observed a tendency to increase the miR-17-5p level by 132% compared to the control group. On the other side, was observed a rapid down – regulation of miR-99 up to 53%. At miR-17-5p in patients with CU were observed compared with the control group a significant increase of 207%. In the case of miR-99, in patients with CU was observed increased level expression by 136% versus the control group. The results corre-spond with the results of other scientific groups dealing with these diseases, which obtained different levels of relative expres-sion of miR-17-5p and miR-99. The pilot study indicates the using of miR-17-5p and miR-99 as possible markers to differen-tial diagnostics possibility of women cancer.

P.01-005-Tue

Vitamin C restores TET proteins activity in

leukemic cells

M. Gawronski, M. Starczak, A. Labejszo, M. Modrzejewska, D. Gackowski

Nicolaus Copernicus University, Collegium Medicum, Faculty of Pharmacy, Department of Clinical Biochemistry, Bydgoszcz, Poland Epigenetic mechanisms that regulate gene expression (such as methylation and active demethylation of DNA) play a key role in the genomic imprinting, cells proliferation and differentiation and their abnormalities may be one of the early stages of carcinogene-sis. Patients with acute myeloid leukemias frequently harbor muta-tions in genes involved in the DNA demethylation pathway. Loss-of-function mutations in TET2 have been described in various hematological malignancies and also TET1 has contrasting roles in myeloid and lymphoid transformation. We have analyzed levels of epigenetic DNA modifications arising from TETs activity, namely 5-hydroxymethylcytosine _{– derivative of 5-methylcytosine,} 5-for-mylcytosine and 5-carboxylcytosine, by using stable isotope dilu-tion two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry in DNA isolated form peripheral blood nuclear cells of patients with acute myeloid leukemias. Our study showed significantly lower levels of hmCyt and other 5-mCyt derivatives in afflicted individuals when compared with con-trol group. In various studies vitamin C enhanced TET proteins activity, therefore we have developed in vitro model in which we

cultured leukemic cells with different concentrations of vitamin C and observed dose-dependent increase of 5-hydroxymethylcytosine and 5-formylcytosine levels in cellular DNA. This suggests that vitamin C, by restoring TETs activity, normalize aberrant pattern of epigenetic modifications in leukemic cells and may be used as an adjuvant treatment of acute myeloid leukemias. This work was supported by the Polish National Science Centre (grant number UMO-2015/19/B/NZ5/02208).

P.01-006-Wed

Determination of epigenetic DNA

modifications in Drosophila melanogaster

M. Starczak, M. Gawronski, D. Gackowski

Department of Clinical Biochemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland

The presence of 5-methylcytosine (5-mCyt) in the Drosophila gen-ome has been subject to long-standing debates. It was demon-strated that cytosine DNA methylation could be detected and quantified in embryo development and in adult flies. Another epi-genetic modification is 5-hydroxymethylcytosine (5-hmCyt), pro-duct of 5-methylcytosine oxidation by ten-eleven translocation (TET) enzymes. This mark was detectable in adult stages of D. mel-anogaster. Some evidence from experimental studies suggests that TET may be also involved in synthesis of 5-hydroxymethyluracil (5-hmUra), a compound with epigenetic function. To determine the levels of epigenetic DNA modifications in D. melanogaster we used 2 experimental models. In in vitro model S2 cells were cultured in control medium and in medium containing 1 mM ascorbic acid (AA) for 24 h, 48 h and 72 h. In in vivo model we measured levels of epigenetic modifications in DNA isolated from adult flies. Due to low content of analyzed compounds in Drosophila genome we optimized classical phenol extraction protocol to obtain large amount of high-polymerized DNA. Received genetic material were enzymatically hydrolysed to deoxynucleosides, spiked with stable-isotope labeled internal standards and analyzed using 2D-UPLC-MS/MS method. In S2 cells we were able to detected levels of ura-cil, 5-hmUra and 8-oxoguanine (8-oxoGua). In material isolated from adult D. melanogaster we identify and quantify levels of 5-mCyt, 5-h5-mCyt, uracil, 5-hmUra and 8-oxoGua. Incubation S2 cells with ascorbic acid caused time-dependent increase of 5-hmdC (10 folds) and independent increase of 8-oxoGua (1.7 folds) levels. This suggest that some portion of 5-hmUra observed in Drosophila melanogastermay be a product of enzymatic reaction. The work was supported by Polish National Science Centre grant No. 2016/21/N/NZ1/00563.

P.01-007-Mon

Evaluation of transcription factor 7-like 2 gene

polymorphism in patients with prediabetes

and type 2 diabetes mellitus

M. Kant1, M. Akıs1, M. Calan2, T. Arkan3, F. Bayraktar4, H. Islekel5

1

Department of Medical Biochemistry, Dokuz Eylul University, Izmir, Turkey,2Division of Endocrinology, Izmir Bozkaya Research and Education Hospital, Izmir, Turkey,3Division of Endocrinology, Derince Research and Education Hospital, Kocaeli, Turkey,

4

Department of Internal Medicine, Division of Endocrinology and Metabolism, School of Medicine, Dokuz Eylul University, Izmir, Turkey,5Department of Molecular Medicine and Department of Medical Biochemistry, Dokuz Eylul University, Izmir, Turkey Type 2 diabetes mellitus is an endemic chronic metabolic disease characterized by hyperglycemia. Prediabetes is the subclinical

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stage of T2DM with moderate hyperglycemia and blood glucose fluctuations. Transcription factor 7-like 2 (TCF7L2) polymor-phisms are the most important single nucleotide polymorpolymor-phisms associated with T2DM. However, the association of TCF7L2 polymorphisms with prediabetes remains to be clarified and must be thoroughly investigated for determination of its effects on metabolism. Therefore, the aim of this study were to investigate TCF7L2 rs7903146 polymorphisms in patients with prediabetes and type 2 diabetes mellitus. TCF7L2 rs7903146 SNPs were ana-lyzed with DNA sequencing method with genetic analyzer in blood samples collected from prediabetes, T2DM patients and control subjects. These variants were examined by using BLAST 2 Sequences. Obtained results were evaluated together with clini-cal and laboratory findings and demographic features. Statisticlini-cal analyses were performed by using SPSS 22.0 and GraphPad Prism 5, and P< 0.05 was considered significant. TCF7L2 rs7903146 SNPs, 7% of the prediabetes patients, 22% of the T2DM patients and 9% of the healthy control group had homozygote TT allele and 50% of the prediabetes patients, 50% of the T2DM patients and 52% of the control group had heterozygote CT allele. The T2DM patients had homozygote TT allele minimum two times as frequently as the prediabetes and healthy control groups. There was a significant positive correla-tion between TCF7L2 SNPs and waist circumference in the T2DM patients (r_{= 0.479, P = 0.007). In conclusion our results} indicate that TCF7L2 rs7903146 variants were not significantly associated with prediabetes and T2DM but may be an important risk factor.

P.01-008-Tue

Transfer RNA-derived fragments target and

regulate ribosome-associated

aminoacyl-transfer RNA synthetases

A. M. Mleczko1, P. Celichowski1,2, K. Bakowska- _Zywicka1

1

Institute of Bioorganic Chemistry, PAS, Poznan, Poland,2Poznan University of Medical Sciences, Poznan, Poland

In the present study, we have functionally characterized molecular activity of Saccharomyces cerevisiae tRNA-derived fragments (tRFs) during protein biosynthesis. Ribosome-associated noncod-ing (ranc) RNAs are a novel class of short regulatory RNAs and the function as well as the trigger for the origin is not widely stud-ied. In order to gain insight into the tRF binding sites with the ribosome we performed the polysome gradient analyses followed by northern blot assays as well as in vitro binding assays. To study the role of tRFs in vivo, we performed metabolic labeling approach using yeast spheroplasts. To corroborate on in vivo assays, in vitro translation reactions were performed using S. cere-visiaecell-free extracts. Furthermore we investigated tRF’s influ-ence on the earliest step needed for the proper protein biosynthesis, namely the tRNA aminoacylation. Finally, to gain more insight into the mechanism of tRNA aminoacylation regula-tion by yeast tRFs, we set up a series of electrophoretic mobility shift assays aiming at identification of interaction partners for tRNA-derived fragments. Our results indicate regulatory potential during translation of not only ranc-50-tRFs (as reported previously) but also ranc-30-tRFs. We have demonstrated the asso-ciation of five ribosome-associated yeast 30-tRFs (as well as one 50-tRF) with small ribosomal subunit as well as aminoacyl-tRNA synthetases. Moreover, our proteomic analysis revealed that four yeast aa-tRNA synthetases directly interact with yeast ribosomes. The association of yeast aa-RSs to ribosomal particles, especially the small ribosomal subunit, is mediated by yeast tRFs. As a con-sequence, global tRNA aminoacylation is impaired.

P.01-009-Wed

Novel targets for the Orb2 CPEB protein in

nervous system of Drosophila

E. Kozlov1, R. Gilmutdinov1_{, K. Yakovlev}1_{, P. Schedl}2_,

Y. Shidlovskii1,3

1_{Institute of Gene Biology RAS, Moscow, Russia,}2_{Department of}

Molecular Biology, Princeton University, Princeton, United States of America,3_{I.M. Sechenov First Moscow State Medical}

University, Moscow, Russia

Gene expression is regulated at multiple levels. CPEB proteins bind special sequences in 30UTR and can promote both activa-tion or repression of translaactiva-tion through polyadenylaactiva-tion regula-tion. It helps to establish intracellular protein gradient and mediates cell polarization. Nervous system is characterized with high protein synthesis plasticity and specialization, that is why role of the CPEB proteins in such processes like postsynaptic potentiation is very important. One plausible model to study these phenomena is Drosophila CPEB protein Orb2 due to pow-erful genetic tools available for this model organism. Several Orb2 regulatory targets have been defined previously. Recently, based on cell culture model almost complete list of Orb2 regula-tory targets have been obtained. We proposed that using of native material, namely fly heads extracts, we will be able to describe the transcripts specific for nervous system. The fly stock carrying GFP protein fused to Orb2 was used. Immunoprecipita-tion against GFP antibodies was performed, the isolated RNA from precipitated RNA-protein complex was sequenced with help of NGS approach. Obtained results for several genes were veri-fied using real time PCR. We have obtained about 2000 tran-scripts enriched in our RIP-seq analysis. They were characterized with broad range of CPE- sequences. Transcripts with highest values of enrichment have elevated level in fly central nervous system. We identified near 100 RNA targets that previously were not described. Using RNA FISH, immunostaining and genetic tools we are investigating the role of novel Orb2-transcripts inter-actions in development and functioning of central nervous sys-tem. This study was supported by the Russian Foundation for Basic Research under Grant 16-34- 60214.

P.01-010-Mon

Experimental approaches for studying

degraded DNA with the use of Next

Generation Sequencing (NGS)

A. Matcvai1,2, I. Alborova1_{, E. Pimkina}2_{, K. Khafizov}1,2_,

K. Mustafin3

1_{Life Sciences Center, Moscow Institute of Physics and}

Technology, Dolgoprudny, Moscow Region, Russia,2_Central

Research Scientific Institute of Epidemiology, Moscow, Russia,

3_{Moscow Institute of Physics and Technology, Dolgoprudny, Russia}

The objectives of this study were to develop a protocol for sam-ple preparation of archaeological DNA for NGS and a software pipeline for the data analysis and interpretation. The materials for the study were the skull teeth found in excavations of burial sites dating the XVIIth century near Radonezh, provided by the Zagorsk state historical and art museum-reserve. The conditions of burial were typical for Central Russia, thus experience with these remains can be a base for working with other samples from this important historical region. The sample preparation was car-ried in a specially designed unit, provided with an evacuation sys-tem and the possibility of filling with high-purity nitrogen, minimizing intra-laboratory contamination. Low DNA concen-tration after isolation led to the impossibility of preparation the sample for NGS by standard protocols. A library of fragments

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suitable for sequencing was obtained only with the use of spe-cially designed modified adapters, which did not allow the forma-tion of adapter-dimeric structures. Sequencing was performed using the Illumina MiSeq platform. Identification of the organ-isms was carried out by aligning the individual reads, using the BLASTn program, to the NCBI Nucleotide Collection. About a third of all the reads could be identified. Overall, they were attributed to the 3,500 different species from 1,500 genera. About 22% of the identified reads belonged to organisms from the genus Streptomyces and Bradyrhizobium, which are soil bacteria. Homo sapiens took about 5% of raw data. The highest-quality alignment of the paired-end reads to the reference sequence of the human genome could be obtained using the algorithm BWA-backtrack. Sequencing of the negative control sample showed no contamination at the stage of sample preparation. Library enrich-ment application by DNA hybridization made it possible to sig-nificantly increase the concentration of individual fragments containing target loci for phylogenetic studies.

P.01-011-Tue

Defining the promoter sequence determinants

for NAD

+ -initiation by RNA polymerase

O. Ruiz-Larrabeiti, D. Pinkas, N. Panova, L. Krasny Institute of Microbiology, The Czech Academy of Sciences, Prague, Czech Republic

The status of the 50-end of RNA affects its stability, localization and translation efficiency, and it is considered as an epitranscrip-tomic regulatory element. A breakthrough discovery of the ubiq-uitous NAD+_{redox cofactor attached to the 5}₀_{-end of RNA in}

prokaryotic and eukaryotic cells revealed the existence of a new cap-like RNA modification. The biological significance of the NAD+_{-cap and its consequences for the RNA and the cell are}

unclear, but a number of mechanistic aspects of the NAD+

-cap-ping and its requirements are starting to be defined. It has been showed that NAD+ _{can be used by RNA polymerase (RNAP)}

as a non-canonical transcription initiation (di)nucleotide (NCIN) at promoters encoding +1A (+1 is the transcription start site). The efficiency NAD+-capping likely depends on promoter sequence. Here we present our study where we systematically investigated and defined the promoter sequence determinants for transcription initiation with NAD+. To achieve this, we created a series of chimeric promoters composed of the sequences of pro-moters that encode+1A and display different NAD+-initiating efficiencies, and used them as templates for in vitro transcription with Escherichia coli RNAP. These experiments identified a pro-moter region and its consensus sequence that maximizes utiliza-tion of NAD+ _{as the transcription initiating substrate. The}

results presented here thus constitute a step forward in the char-acterization of this newly found prokaryotic RNA-capping phe-nomenon. This work is supported by grant No. 17-03419S, from the Czech Science Foundation.

P.01-012-Wed

Epigenetic aspect of function of a promising

anticancer drug olivomycin A

A. Sergeev1, A. Tevyashova2,3, E. Gromova1

1

Moscow State University, Moscow, Russia,2Gause Institute of New Antibiotics, Moscow, Russia,3Moscow University of Chemical Technology of Russia, Moscow, Russia

Olivomycin A is a promising anticancer agent that belongs to a family of aureolic acid antibiotics. However, the mechanism of its action is not completely understood. The drug binds to the DNA minor groove in GC-rich regions as Mg2+-containing

complexes. Given that minor groove ligands are known to dis-rupt a key epigenetic process of DNA methylation, we aimed to investigate the impact of olivomycin A and its synthetic deriva-tive LCTA-1599 on the functioning of de novo murine DNA methyltransferase Dnmt3a. This enzyme establishes the DNA methylation pattern in eukaryotic cells. Using specially designed fluorescently labelled oligonucleotide substrates, we determined the optimal conditions for drug-DNA binding, including the con-centration of Mg2+_{ions that allowed for both olivomycin}

bind-ing and Dnmt3a functionbind-ing. The impact of olivomycin on the Dnmt3a-DNA complex formation under these conditions turned out to be minimal. The binding of olivomycin A and LCTA-1599 to Dnmt3a was not observed. We then examined the inhibitory effect of olivomycin A and LCTA-1599 on DNA methylation by Dnmt3a. Employing the efficient method for in vitro quantifica-tion of DNA methylaquantifica-tion that was recently developed in our lab-oratory, we have shown that both drugs can substantially inhibit the methylation reaction with IC50 values of 6 1 lM and

7.1 0.7 lM, respectively. Other minor groove ligands such as dimeric bisbenzimidazoles were found to have similar IC50values.

The inhibitory effect of olivomycin A and LCTA-1599 may be attributed to the disruption of movement of Dnmt3a catalytic loop toward DNA, which is necessary for the enzymatic activity of Dnmt3a and occurs via the DNA minor groove. Our results point at an epigenetic contribution to the mechanism of anti-cancer effect of aureolic acid family antibiotics. This work was supported by the RFBR grant 16-04-01087A.

P.01-013-Mon

Gre-like factors serve as lineage-specific

transcriptional regulators in Deinococcus

species

A. Agapov1,2, K. Azimov1,2, D. Esyunina1, A. Kulbachinskiy1,2

1

Institute of Molecular Genetics Russian Academy of Sciences, Moscow, Russia,2Molecular Biology Department, Biological Faculty, Moscow State University, Moscow, Russia

Transcription is a major step in the regulation of gene expression in bacteria. Among various proteins that interact with RNA polymerase (RNAP) and modulate its activity, a prominent group of factors can bind in the secondary channel of RNAP and affect catalysis by direct interactions with the active centre. While GreA is a universal bacterial factor that stimulates RNA cleavage, which is important for transcriptional proofreading, the functions of its homologues may greatly differ. The famously stress-resistant bacterium Deinococcus radiodurans encodes three factors from the Gre-family: GreA, Gfh1 and Gfh2. We previ-ously found that the Gfh factors from D. radiodurans strongly enhance site-specific pausing and intrinsic transcription termina-tion by RNAP. The pause-stimulatory activity of Gfh is greatly enhanced by manganese ions, which are accumulated in D. radio-duranscells under stress conditions. Uniquely, another bacterium from the Deinococcus genus, D. peraridilitoris encodes four differ-ent Gfh factors. In this work, we analyzed their activities in vari-ous in vitro transcription assays. The results show that one of the four proteins significantly inhibits transcription initiation, pro-longs transcriptional pausing and suppresses the RNA cleavage activity of RNAP. At the same time, the other three Gfh factors reveal much more moderate if any effects on different steps of transcription. We conclude that Gfh proteins serve as important transcriptional regulators in Deinococcus species and may poten-tially play a role in stress resistance, but the functions of their species-specific paralogues remain enigmatic. We speculate that in the case of D. peraridilitoris the Gfh proteins, though being homologous to transcription factors, may have additional func-tions in the cell that we will try to uncover in our future

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studies. This work was in part supported by Russian Science Foundation (grant 17-14-01393) and Russian Foundation for Basic Research (grant 18-34-00905).

P.01-014-Tue

Investigation of the argonaute protein from

Synechococcus elongatus

A. Olina, A. Kulbachinskiy, D. Esyunina

1_{Institute of Molecular Genetics Russian Academy of Sciences,}

Moscow, Russia

Argonaute proteins are key players in the diverse RNA interfer-ence pathways in eukaryotes. Eukaryotic argonautes bind short RNAs and use them as guides for target RNA recognition and cleavage. Argonaute proteins, often with unusual domain archi-tectures, are also found in many bacterial and archaeal species but their functions in prokaryotes remain largely obscure. Recent studies revealed that prokaryotic argonautes can bind short RNA or DNA molecules and act as endonucleases to protect the cells against foreign genetic elements. However, their cellular roles and the molecular mechanisms of action are poorly under-stood. In this work, we studied argonaute protein SynAgo from the blue-green algae Synechococcus elongatus. We used purified SynAgo protein for in vitro experiments and demonstrated that it is able to bind short DNA guides and cleave target DNA sub-strates of various structures. We further constructed S. elongatus strain with tagged-argonaute expression and used it for purifica-tion of SynAgo and associated nucleic acids. It was found that in S. elongatuscells SynAgo is bound with short DNAs correspond-ing to the whole genomic sequence. Similarly, SynAgo also asso-ciates with short DNAs when expressed in the heterologic Escherichia colisystem suggesting that no additional factors are required for short DNA processing. Thus, our experiments sug-gest that SynAgo is a DNA guided DNA nuclease that target various genomic sequences to perform its functions. To reveal these functions, we constructed a set of S. elongatus strains with various mutations in the argonaute gene and are now performing in vivoexperiments aimed at understanding the roles of SynAgo in genomic defense and cell physiology. This work was supported in part by the Russian Science Foundation (grant 16-14-10377).

P.01-015-Wed

1,N

6

-

a-hydroxypropanoadenine and 3,N

4

-

a-hydroxypropanocytosine - acrolein adducts to

adenine and cytosine, are substrates for AlkB

dioxygenase

M. Dylewska, J. Poznanski, J. T. Kusmierek, E. Grzesiuk, A. Maciejewska

Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland

1,N6_{-a-hydroxypropanoadenine (HPA) and 3,N}4_-

a-hydroxypro-panocytosine (HPC) are six-membered adducts formed in reac-tion of adenine and cytosine with acrolein (ACR). ACR is a mutagenic agent originated from different sources including cigarette smoke, exhaust fumes and overcooking. It is also gener-ated endogenously during oxidative stress as a by-product of lipid peroxidation. E. coli AlkB dioxygenase (EcAlkB) is DNA repair enzyme that remove alkyl lesions from bases via an oxida-tive mechanism restoring naoxida-tive DNA structure. It belongs to the superfamily of 2-a-ketoglutarate (aKG) and Fe(II) dependent dioxygenases. AlkB is induced within E. coli system of adaptive response to alkylating agents (Ada response). Our in vivo data show that HPA and HPC show mutagenic properties and, gener-ated in plasmids, causes (respectively) A?C and A?T

transversions, C?T transitions followed by C?A transversions. We established the optimal pH, Fe(II) and aKG concentrations for enzymatic reaction. Our data proved that the protonated form of HPA is preferentially repaired by EcAlkB, though the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited and reached 38 4 enzymatic cycles before its total inactivation. Molecular modeling of the AlkB/HPA complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S one seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning flu-orimetry allowing the identification of the active protein form with cofactor and cosubstrate bound and monitoring substrate binding.

P.01-016-Mon

The amount of humans with high or low

ribosomal repeat copy number in population

decreases with age

V. Sergeeva, E. Malinovskaya, E. Ershova, N. Lyapunova, V. Izevskaya, S. Kutsev, N. Veiko, S. K. Kostyuk Research Centre for Medical Genetics, Moscow, Russia

Ribosomal RNA genes (rDNA) encode the rRNA species that form the ribosomes. The diploid human genome contains several hundred copies of 43-kb rDNA units tandemly arrayed on five acrocentric chromosomes. Dysregulation of rRNA biogenesis has been implicated in some human diseases. One of the factors affecting rRNA biogenesis is the ribosomal genes copy number (CN). Some authors have shown that the rDNA CN in human genome reduces with age. However, other authors deny this phe-nomenon. It can be explained with high degree of rDNA damage in aged cells and low efficiency of qPCR. We compared two methods: qPCR and nonradioactive quantitative hybridization (NQH) in quantitative analysis of human rDNA. We have shown that NQH is less sensitive to severe DNA damage than qPCR and thus is more appropriate for damaged rDNA detection. We have shown earlier that rDNA CN is higher in patients with schizophrenia compared to healthy people of the same age. We assessed blood leukocyte rDNA CN of 250 subjects (group I, 17 -71 years) in comparison to 126 subjects (group II, 72–91 years) using NQH. The leukocyte rDNA CN showed no significant dif-ference in the median for group I and group II (median 419 vs 396, P= 0.05). However, the dispersion of CN for group I was much higher than for group II (200 -711 vs 252_{–541). In 80% of} group II subjects CN of rDNA had a very narrow dispersion_– from 300 to 450. Thus, the amount of humans with high or low rDNA CN decreases with age. This work was supported by RFBR grant No. 17-29-06017 ofi_m.

P.01-017-Tue

Ferritin2 RNA interference inhibits the

formation of iron granules in honey bees

(Apis mellifera)

C. Hsu

Chang Gung University; Chang Gung Memorial Hospital, Taoyuan, Taiwan

Iron granules containing superparamagnetic magnetite act as mag-netoreceptor for magnetoreception in honey bees. Biomineralization of iron granules occurs in the iron deposition vesicles of tropho-cytes and requires the participation of actin, myosin, ferritin2,

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and ATP synthase. If the formation of iron granules can be sup-pressed, it can help to understand the mechanism of magnetore-ception in honey bees. Whether ferritin RNA interference can inhibit the formation of iron granules is unknown. In this study, double-stranded RNA of ferritin2 was injected into hemolymph from the abdomen of newly emerged worker bees to knock down ferritin2. The results showed that ferritin2 RNA interference declined the mRNA and protein expression of ferritin2 and the formation and accumulation of 7.5 nm iron particles leading to immature iron granules. Superparamagnetic magnetite is formed in the center of mature iron granules. These findings can be used to further study the magnetoreception of honey bees.

P.01-018-Wed

DNA aptamers against procalcitonin as

emerging diagnostic molecules for sepsis

Y. Chen1, Y. Yeh1, L. Chau2, T. Hong1

1

National Cheng Kung University, Tainan, Taiwan,2National Chung Cheng University, Chiayi, Taiwan

Sepsis is the most common cause of death in Intensive Care Unit (ICU). Specific and rapid markers of bacterial infection have been sought for early diagnosis of sepsis. Procalcitonin (PCT), is the most promising sepsis markers, capable of complementing clinical signs and routine lab parameters suggestive of severe infection. The big issue in critical care patient is that lag time between sample collections and examining. Thus, real-time diag-nostic on the bedside is an urgent need for clinicians. The fiber optic particle plasmon resonance (FOPPR) biosensor has high potential to meet these requirements. Our study would like to develop an advanced in vitro diagnostic platform based on FOPPR biosensor as scaffold with targeting aptamers. In this study, we screened a 62-mers single chain DNA aptamer library against human PCT by using a nitrocellulose filter systematic evolution of ligands by exponential enrichment (SELEX). We identified two human PCT-specific aptamer, PCTA1 and PCTA3, bound to PCT with high specificity and affinity. The use of an aptamer-based FOPPR biosensor for detecting PCT were carried out using different concentrations of PCT solutions and clinical serum samples. The FOPPR biosensor with PCT-aptamers can detect very low concentrations of PCT. High selectivity of the biosensor against PCT was also demonstrated in the presence of competitive proteins such as human serum albumin. This approach will further evaluation for clinical use, the combination of a high-affinity aptamers and FOPPR detection has some bene-fits compared with other bioanalytical methods. Upon success of the clinical validation, our aptamer-FOPPR biosensor might be translated from benchtop research to commercialization in the future.

P.01-019-Mon

The screening and application of DNA

aptamers against neuropilin 1

T. Hong, Y. Tsai, Y. Wu, Y. Chen

National Cheng Kung University, Tainan, Taiwan

Aptamers are single-stranded DNA or RNA molecules which can bind to a target specifically by its special steric configuration. Because of its binding specificity, aptamers have a potential for diagnosis and treatment of cancers through binding to oncogenic molecules. Neuropilin 1 (NRP1) is a co-receptor for vascular endothelial growth factor 165 that promotes angiogenesis, tumor growth, tumor invasion and metastasis. High expression of NRP1 in non-small cell lung cancer (NSCLC) patients have been known to have low disease-free and overall survival rate.

Silencing of NRP1 significantly suppressed lung cancer cell migration, metastasis, and tumor angiogenesis. Thus, develop-ment of specific aptamers against NRP1 would be a promising therapy for NSCLC patients. Here, we screened a DNA aptamer library against NRP1 protein by using a nitrocellulose filter sys-tematic evolution of ligands by exponential enrichment (SELEX). Three NRP1-binding aptamers, AP5, AP15, and AP37, were iso-lated. We further screened a customized aptamer microarray, on which a short aptamer library was made based on the sequences of AP5, AP15, and AP37, and we got several short aptamers with high NRP1-binding affinity and specificity. We found that NRP1-specific aptamers decreased the migration ability of the highly invasive NSCLC cells, CL1-5, compared with that in cells treated with scramble-aptamers. Moreover, tube formation ability of HUVEC cells were also reduced while treating with NRP1-specific aptamers. These results showed that NRP1-NRP1-specific apta-mers decrease the migration ability of tumor cells and angiogene-sis. This study revealed that NRP1-specific aptamers might be developed a useful therapeutic strategy to lung cancer in the future.

P.01-020-Tue

Six-stacked G-quadruplexes as a preferential

target of p53

M. Bartas1, N. Mikyskova1_{, V. Br}_azda2_{, V. Karlick}_y3_,

J. Cerven1_{, V.}_Spunda3_{, P. Pe}_cinka1

1_{University of Ostrava, Ostrava, Czech Republic,}2_{Institute of}

Biophysics, Brno, Czech Republic,3_{Department of Physics,}

University of Ostrava, Ostrava, Czech Republic

Recently we have for the first time described more than four-stacked intramolecular G-quadruplex forming sequences. These sequences are very rare in human genome, but interestingly span important locations such promoters or sites of chromatin acetyla-tion in most cases. Also preferential binding of tumor suppressor protein p53 to quadruplex DNA structures was demonstrated previously in general. In this work we have inspected the differ-ences in p53 binding to the well known four-stacked intramolecu-lar G-quadruplex and newly described six-stacked intramolecuintramolecu-lar G-quadruplex. Using combination of wet-lab methods (elec-trophoretic mobility assay and circular dichroism spectroscopy), we have proven the preferential binding of wild type p53 to the six-stacked intramolecular G-quadruplexes in vitro on oligonu-cleotide quadruplex forming sequences, further indicating their substantial role in biological regulatory processes. Also we have compared the binding ability of wild type p53 (amino acid resi-dues 1 - 393) with the core domain of p53 (amino acid resiresi-dues 94 - 312) to G-quadruplex DNA. According to widely accepted hypothesis, isolated core domain of p53 have shown minimal binding activity in comparison to wild type p53, which further confirms the major role of intrinsically unstructured C-terminal tail of p53 in preferential binding to non canonical DNA struc-tures. Also we have observed temperature dependencies in p53 and six-stacked G-quadruplex interactions, indicating crucial role of physiological temperature achievement to the successful bind-ing. We have also demonstrated some important features of six-stacked quadruplexes distribution in the human and other sequenced mammalian genomes as well. Interestingly, in human genome, occurrence of six-stacked G-quadruplexes is strongly enriched in neurodegenerative diseases related genes so in the future, six-stacked G-quadruplexes could be a great target in sev-ere diseases treatment for their unique features.

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P.01-021-Wed

Prevalence of DNMT3B -149 C>>T and SYCP3

T657C polymorphisms among Russian women

N. N. Lapaev, A. A. Ahmed, M. M. Azova, O. O. Gigani, O. B. Gigani, A. V. Aghajanyan, S. P. Syatkin

1_{Peoples’ Friendship University of Russia (RUDN University) 6}

Miklukho-Maklaya St., Moscow, Russia

DNA methyltransferase 3b (DNMT3b) is an enzyme participat-ing in epigenetic modification and mediatparticipat-ing the transfer of the methyl group derived from folate to the 50position of a cytosine in embryonic stem cells and early post-implantation embryos. Synaptonemal complex protein 3 (SYCP3) is a DNA binding protein involved in the synaptonemal complex formation during meiosis I, and its activity leads to homologous chromosomes pairing and meiotic homologous recombination. Some studies reported variations in the frequency of both DNMT3b -149 (T>C) and SYCP3 T657C polymorphisms among populations and suggested that the mutations in the genes can be genetic risk factors causing reproductive disorders in women. To our knowl-edge, both gene polymorphisms have never been studied in Rus-sia before, thus the aim of our study was to investigate the polymorphism prevalence of these genes among the healthy women in Russia. 60 peripheral blood samples were collected from the healthy Russian women living in Central Russia. The extracted DNA was subjected to DNA genotyping using restric-tion fragment length polymorphism-PCR for DNMT3b and Allele-specific PCR for SYCP3 followed by agarose gel elec-trophoresis. The C allele and T allele frequencies were 72.5% and 27.5% while the CC, CT and TT genotype frequencies were 50%, 45% and 5% respectively for DNMT3b polymorphism. For SYCP3 polymorphism the T and C allele frequencies were 90.85% and 9.15%, while the TT, TC and CC genotype frequen-cies were 86.7%, 8.3%, 5% respectively. Consequently, we rec-ommend investigating both polymorphisms to determine whether there is a relationship between genetic changes in the DNMT3b and SYCP3 genes and reproductive problems in Russian women. The publication was prepared with the support of the “RUDN University Program 5-100”.

P.01-022-Mon

The SEPS1 G-105A polymorphism is

associated with risk of preterm birth in

Russian women

I. O. Musalaeva, E. V. Tarasenko, T. V. Galina,

G. I. Myandina, E. M. Zheludova, M. M. Azova, S. P. Syatkin Peoples’ Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya St., Moscow, Russia

Inflammation plays an important role in the initiation of preterm birth. At the current moment many genes and their products are considered as markers of inflammation, most of them are directly involved in the infectious process. This group includes more than 200 cytokines (interleukins (IL), tumor necrosis factors (TNF-a), interferons, growth factors), C-reactive protein, transforming growth factor (TGF-b), etc. The SEPS1 gene is regarded as a new marker of inflammation. Several polymorphisms have been detected in the SEPS1 gene, but they are insufficiently studied. In this study the correlation of the SEPS1 G-105A polymorphism (rs28665122) with the risk of premature birth in Russian popula-tion from Moscow region is shown. The SEPS1 G-105A poly-morphism was genotyped in 50 women with premature birth at terms from 23.5 to 37 weeks of pregnancy and 50 women with full-term pregnancy as a control group using the polymerase chain reaction-restriction fragment length polymorphism

(PCR-RFLP) analysis. According to our data, the frequency of allele A among healthy women is 14%. In the group of women with pre-term birth the frequency of allele A is 20% which is significantly higher than in the control group (v2= 5.0249, P = 0.02499). The frequency of heterozygotes is also significantly higher in the group of women with preterm birth (v2= 6.002; P = 0.01429). Thus, our findings suggest that the SEPS1 G-105A polymor-phism may be a potential gene marker for preterm birth. The publication was prepared with the support of the “RUDN University Program 5-100”.

P.01-023-Tue

Upstream (-31/-24) box in RNA polymerase III

promoters

K. Tatosyan, D. Stasenko, I. Gogolevskaya

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia

Most of the genes transcribed by RNA polymerase III (pol III) contain an internal promoter. Usually, such promotor consists of two 11-bp boxes (A and B) spaced by 30–40 bp. Apart from these boxes, tRNA genes of yeast, plants, and insects contain a TATA-box (TATAAAA or a similar heptamer) located in the upstream sequence between positions -24 and - 30. TATA-bind-ing protein (TBP) binds this box, which promotes transcription initiation. However, tRNA genes of mammals lack typical TATA-boxes. We studied the genes of two noncoding mouse RNAs, 4.5SH and 4.5SI. These genes contain A and B boxes but have no canonical TATA-box. We replaced each nucleotide of 4.5SH RNA gene upstream sequence -31/-24 with three other ones. HeLa cells were transfected with 24 constructs and the effi-ciency of 4.5SH RNA gene transcription was estimated. The con-structs containing substitutions T(-30) to G, C(-29) to A, A(-28) to C, and A(-27) to G or C demonstrated the strongest transcrip-tion decrease (up to 6-fold). The obtained data indicate that these nucleotides are important for the initiation of pol III transcrip-tion. In the case of 4.5SI RNA, the entire -31/-24 box (CTA-CATGA) was replaced with other nucleotide sequences. Certain sequences (for example, GCACTAGT) could successfully func-tion as a -31/-24 box. On the contrary, GC-rich sequence totally repressed the gene transcription. Mouse SINEs B1 and B2, which are also transcribed by pol III, were studied likewise. Most of the studied B1 and B2 copies could be transcribed effectively; how-ever, the replacement of the -31/-24 sequence with a TATA-box or the -31/-24 boxes of 4.5SH and 4.5SI RNA genes additionally increased B1 and B2 transcription. Thus, although various sequences can function as a -31/-24 box, the presence of certain nucleotides at critical positions is necessary for the maximum activity. This work was supported by the Program of Fundamen-tal Research for State Academies for 2013_{–2020 years (no.} 01201363821).

P.01-024-Wed

Affect of N6-methyladenosine mRNA

modification on translation rate of bacteria

I. Garanina, D. Evsutina, G. Fisunov

Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia N6-methylation of adenosine (m6A) is the most abundant mRNA modification in eukaryotes and viruses. Recently was shown the widespread occurrence of m6A modification in mRNA of bacteria. Modified adenosines consist of about 0.20% and 0.28% of all adenosines of Pseudomonas aeruginosa and Escheri-chia coli that comparable with eukaryotes. We used a

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bioinformatics approach to assess the effect of adenosine modifi-cations on mRNA abundance and protein production in bacteria. For every gene we collected published available data about its mRNA and protein levels in different conditions, absolute pro-tein number, translation efficiency, and adenosine modifications. We correlated mRNA and protein abundances for genes in each growth condition and compared mean correlation coefficients among all conditions between modified and unmodified genes. Modified genes have statistically significant lower correlation (P-value Mann-Whitney test _{<0.01) between mRNA and protein} abundances. Using ribosome profiling data we analyzed the impact of modifications on intermediate stages of cell informa-tion flow: from mRNA to ribosomes and from ribosomes to pro-teins. Modifications affect ribosome occupancy in such a way that translation efficiency only slightly correlates with mRNA level. Ribosome occupancy within modification sites is slightly lower than in other parts of the genes. We didn’t find any differ-ence in ribosome distribution upstream or downstream of modifi-cation sites. Was created the model that predicts protein abundance based on expression level, translation rate, and adeno-sine modification status. The model explains up to 63% of the variation in protein abundance, the model based on decision trees shows that m6A abundance has 15% contribution to protein level prediction. So, our calculations show statistically significant impact m6A modifications on information flow from mRNA to protein level. This work was supported by RSF grant No. 14-24-00159.

P.01-025-Mon

G-quadruplex RNA in Kirsten ras transcript as

promising target for anticancer therapy

A. Tikhomirov1,2, G. Miglietta3, S. Cogoi3, A. Shchekotikhin1,2, L. Xodo3

1

Gause Institute of New Antibiotics, Moscow, Russia,2D. I. Mendeleev University of Chemical Technology of Russia, Moscow, Russia,3Department of Medical and Biological Sciences, University of Udine, Udine, Italy

Quadruplex-forming motifs of promoters of some oncogenes are considered as prospective targets for cancer chemotherapy. The human Kirsten ras (KRAS) transcript is capable to form several putative G-quadruplexes (G4s) as predicted by methods of bioinformatics. Among the three expected G4 motifs only one was recognised by BG4, an antibody specific for G-quadru-plexes that confirms the initial assumption. Previously, it was demonstrated that heteroarene-fused anthraquinones have a strong affinity and selectivity to telomeric and Harley ras G-quadruplexes. Therefore, we investigated an ability of series ligands on heteroareneanthraquinones scaffolds to bind to G4s in the 50-UTR of mRNA and suppress the KRAS oncogene in pancreatic cancer cells. Found, that 4,11-bis(2-aminoethylamino) anthra[2,3-b]furan-5,10-dione (ATFD) stabilizes G4 in the KRAS transcript (DTM up to 32°C). Moreover, synthesis and

evaluation of biotinylated ATFD by means of biotin-streptavi-din pulldown assay revealed that ATFD binds to RNA G4s in the KRAS transcript under low-abundance cellular conditions. Dual-luciferase assays showed that ATFD repress translation in a dose-dependent manner. The new G4-ligand efficiently pene-trate into the Panc-1 cancer cells, suppressing protein p21KRAS to <10% of the control followed by apoptosis together with a dramatic reduction of cell growth at submicromolar concentra-tions. As the result one may conclude, a suppression of the KRAS oncogene via stabilization of G4s in the 50-UTR of mRNA by low molecular weight ligands can be considered as a new strategy in G4-oriented anticancer therapy. Acknowledg-ment: this work has been carried out in part with the financial

support of Italian association for cancer research (IG2013, pro-ject code 143, to Prof. L. Xodo) and the grant of the President of the Russian Federation for young Russian scientists (MK-2474.2018.3, to Dr. A. Tikhomirov).

P.01-026-Tue

C-reactive protein and renin-angiotensin

system gene polymorphisms in patients with

coronary artery stenosis

K. B. Bogatyreva, M. M. Azova, Z. K. Shugushev, A. V. Aghajanyan, L. V. Tskhovrebova, A. Ait Aissa, M. L. Blagonravov, S. P. Syatkin

Peoples’ Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya St., Moscow, Russia

The latest studies in the field of molecular cardiology demon-strate the important role of inflammation and renin-angiotensin system (RAS) in the pathogenesis of atherosclerosis. Gene poly-morphisms of their components have been described to be associ-ated with the development of coronary artery disease. Our work was aimed at studying the polymorphisms of the C-reactive pro-tein (CRP), angiotensin II receptor type 1 (AGTR1), and angio-tensin (AGT) genes in patients with coronary artery stenosis. 65 patients and 59 healthy individuals participated in the study. All of them were Russians. Total DNA was extracted from the blood and genotyped using the Real-time PCR. The CRP C1444T, AGT Thr174Met, AGT Met235Thr, and AGTR1 A1166C poly-morphisms were analyzed. The Chi-square test and Fisher’s exact test were used to estimate differences between groups. It was found that in patients the frequencies of most of minor alleles were significantly higher (AGT 174T 40.7% vs. 11%; AGTR1 1166C 39.2% vs. 19.5%; CRP 1444T 42.2% vs. 24.7%), which was accompanied by growth of the frequency of homozygous rare genotypes. Comparison of patients with multifocal atherosclerosis (n= 20) with other patients (n = 45) revealed that in this group the genotypic distributions of RAS genes were dif-ferent due to a significant increase in the frequency of heterozy-gotes (AGT Thr174Met 15.8% vs. 4.3%; AGT Met235Thr 21% vs. 10.9%; AGTR1 A1166C 52.6% vs. 15.2%). The obtained results may be of interest for personalized medicine because heterozygosity for the given polymorphisms may predispose to the multifocal atherosclerosis. To confirm this idea it is necessary to continue research in larger samples. The publication was pre-pared with the support of the “RUDN University Program 5– 100”.

P.01-027-Wed

The expression of a nested alternative open

reading frame may be a novel mechanism for

intronless gene expression control

T. V. Komarova1,2, E. V. Sheshukova1,2, N. M. Ershova1,2, Y. L. Dorokhov1,2

1

Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia,2Lomonosov Moscow State University, Moscow, Russia

The regulation of gene expression in animal and plant tissues at different stages of development and after exposure to exter-nal stimuli occurs at the transcriptioexter-nal, post-transcriptioexter-nal and post-translational levels. Recently, in the Solanaceae family of plants, we found an intronless gene for the Kunitz protease inhibitor-like protein (KPILP) with low mRNA levels in the leaves of an intact plant. However, these levels sharply increased after a 72-h incubation in the dark and under biotic stresses. To identify the mechanism for the regulation of

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Solanaceae KPILPs, we identified and examined the function of the KPILP nested alternative open reading frame (aORF) that controls the expression of maternal mRNA. Here, we investigated the regulation of KPILP under stress conditions at the level of transcription. We isolated a portion of Nicotiana benthamiana chromosomal DNA upstream of the KPILP cod-ing sequence (proKPILP) and showed that proKPILP directed the expression of the E. coli UidA reporter gene encoding b-D-glucuronidase (GUS) in plant. A bioinformatic analysis revealed potential light-responsive elements in the proKPILP sequence. To understand if these elements influence proKPILP activity, agroinjected plants were isolated for 3 days in com-plete darkness. Then, the level of GUS and corresponding mRNA content were assessed in the leaves and compared with the constitutive CaMV 35S promoter. The results showed that, as in the case of the 35S promoter, darkness had a negligible effect on the activity of the proKPILP. We concluded that the stress-dependent content of the KPILP mRNA in the leaves is determined mainly by the aORF-mediated mechanism of maternal mRNA control. Thus, Solanaceae KPILP is an intronless matryoshka gene and is the first example of a gene expression regulation mechanism that is probably characteristic for intronless genes. This study was performed with financial support from the Russian Foundation for Basic Research (pro-ject No. 17-29-08012).

P.01-028-Mon

Transcriptome analysis reveals influence of

RNases from toxin-antitoxin systems on gene

expression in Staphylococcus aureus

B. Wladyka1, A. Sabat2, M. Bukowski1, M. Hydzik1, K. Hyz1, K. Chlebicka1, V. Akkerboom2, A. Friedrich2, E. Bonar1

1

Jagiellonian University, Krakow, Poland,2University Medical Center Groningen, Groningen, Netherlands

Toxin-antitoxin (TA) systems are widespread in bacteria. A typi-cal TA system consists of a toxin, which is a stable protein, often with enzymatic activity, and an antitoxin, constituting a labile component with inhibitory properties towards the toxin. In case of plasmid-encoded TA systems a role in the maintenance of mobile genetic elements is attributed to these entities. However, localization of TA genes in chromosomal parts of bacterial gen-omes implies the existence of other functions. Staphylococcus aureus is a carrier of chromosomally-encoded MazEF TA system and less frequently of PemIK-Sa1, which is present in a number of staphylococcal plasmids. Toxins from these systems are sequence-specific RNases towards UACAU and UAUU, respec-tively. We hypothesize that the toxins, released from TA com-plexes upon activation, may degrade transcripts, and thus influence gene expression. To challenge this hypothesis we intro-duced plasmids carrying toxins’ genes under inducible promoter to S. aureus null mutants for the respective TA systems. Upon induction of toxins’ expression, RNA was isolated and RNA-seq was performed using Illumina platform. Interestingly, over-expression of MazF toxin resulted in rather moderate changes in gene expression. The number of genes with over two-fold differ-ence was below twenty, however slightly increased over the time. Conversely, in case of PemK-Sa1 toxin over 160 gene transcripts were affected. This clearly indicates that the length of the recog-nized target sequence correlates negatively with the number of altered transcripts. Moreover, among transcripts down-regulated by PemK-Sa1 was RNAIII, which is an important molecule in the accessory gene regulator system, which suggests the existence of a possible regulatory cascade/coupling. The presented data demonstrate that TA RNase toxins may modulate gene expres-sion in bacteria. The study was supported by the National

Science Centre, Poland, grant no.: UMO-2014/13/B/NZ1/00043 (to B.W.).

P.01-029-Tue

Eukaryotic ribosome discriminates 3’ context

and determines efficiency of stop codon

readthrough

E. Sokolova, T. Egorova, A. Schuvalov, E. Alkalaeva Engelhardt Institute of Molecular Biology, the Russian Academy of Sciences, Moscow, Russia

Nucleotide context, surrounding stop codons, significantly affects the efficiency of translation termination. To study the mechanism of its influence, we assessed properties of several 3’ contexts, unfavorable for translation termination, in the reconstituted mammalian translation system. Using pairs of stop codons divided by hexanucleotides we revealed, that the ribosome recog-nizes leaky and strong 3’ stop codon contexts by itself. Stop codon readthrough depends only on the presence of suppressor / near-cognate tRNA and leaky 3’ context. We estimated also the impact of 3’ contexts on translation termination activity of eRF1. All tested contexts demonstrated approximately equal translation termination efficiency in the presence of release fac-tors. However, comparison of basal readthrough levels showed the discriminate response among different weak context. It can reflect much more sophisticated mechanism of balancing between readthrough and termination, orchestrated by nucleotide sur-rounding of a stop codon that could be surmised on the first glance. The work was supported by the Russian Science Founda-tion (grant No. 14-14-00487) and by the Program of Fundamen-tal Research for State Academies for the years 2013 to 2020 (No. 1201363822).

P.01-031-Mon

2’-Modified RNA aptamers specific to

photoprotein obelin as a platform for new

bioluminescent biosensors

A. Davydova1, M. Vorobyeva1, V. Krasitskaya1,2,3, P. Vorobjev1, A. Tupikin1, M. Kabilov1, L. Frank2,3, A. Venyaminova1

1

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia,2Institute of Biophysics Siberian Branch of Russian Academy of Sciences, Krasnoyarsk, Russia,3Siberian Federal University, Krasnoyarsk, Russia

Aptamers are in vitro selected DNA or RNA molecules that can specifically bind a wide variety of targets from small molecules to proteins and living cells. Aptamers represent a high-potential alternative to monoclonal antibodies due to an opportunity to generate an aptamer to almost any desired target, longer shelf-life, cost-effective chemical synthesis, stability in a wide range of conditions, and low toxicity and immunogenicity. These unique properties determined aptamer applications as prospective thera-peutic agents as well as biosensors for diagnostic purposes. In this study we proposed a new design of bioluminescent aptasen-sor made of two different aptamer modules: one is responsible for specific binding of analyte, the other provides bioluminescent reporting. We suggested that aptamers to Ca2+_-regulated

photo-protein obelin could be used as universal reporting modules for bifunctional aptasensors. Due to the high quantum yield and low-level background, obelin established itself as a promising reporter molecule. We performed in vitro selection of 2’-F-modi-fied RNA aptamers against His-tagged obelin immobilized on Ni-NTA sepharose beads. After high-throughput sequencing and bioinformatical analysis, we obtained a series of individual

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aptamers. Full-length and truncated versions of candidate apta-mers were synthesized and screened for their affinity. As a proof of concept, the most prominent truncated aptamer was used as a reporting module of the structure-switching bifunctional aptamer for bioluminescent detection of human hemoglobin. Therefore, obtained 2’-modified RNA aptamers specific to obelin could be used as a platform for diagnostic bioluminescent aptasensors. Proposed bifunctional design of bioluminescent aptasensors could be adapted for a detection of wide range of molecular targets by varying an analyte binding aptamer. The work is supported by Russian Science Foundation (Grant No. 16-14-10296).

P.01-032-Tue

Chemo-enzymatic transglycosylation of

2-aminopurine derivatives bearing bulky

functional groups

D. A. Gruzdev1, B. Z. Eletskaya2, A. Y. Vigorov1, E. N. Chulakov1, G. L. Levit1, I. D. Konstantinova2, V. N. Charushin1, V. P. Krasnov1

1_{Postovsky Institute of Organic Synthesis, Russian Academy of}

Sciences (Ural Branch), Ekaterinburg, Russia,2_{Shemyakin and}

Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia

Biocatalytic method based on transglycosylation with purine nucleoside phosphorylases is an efficient approach to modified nucleosides. In the case of introduction of sugar residues into the purine position 9, the reaction, as a rule, proceeds stereo- and regioselectively. Typically, 2-aminopurine and purine derivatives without bulky groups at positions 2 and 6 are used as substrates. However, in cases where a further synthetic modification of sub-stituted purines is expected, there is a need for preliminary glyco-sylation of purine derivatives with bulky substituents. The purpose of this work was to study the possibility of obtaining modified glycosides as a result of chemo-enzymatic transglycosy-lation of 2-aminopurine derivatives containing substituents at positions 2 and 6, including bulky groups. We carried out a com-parative study of chemo-enzymatic transglycosylation of 2-ami-nopurine conjugates with amino acids and their protected derivatives in position 6. It has been shown that the 2-acetamido-purine derivatives containing tert-butyl glycinate, alaninate, S-or R-valinates, and S-phenylalaninate are good substrates fS-or the genetically engineered recombinant E. coli purine nucleoside phosphorylase; the derivative containing tert-butyl prolinate was not subjected to chemo-enzymatic transglycosylation. Effective-ness of the transglycosylation in the synthesis of 2-deoxyribosides of unsubstituted 2-aminopurine conjugates was about the same as the aforementioned derivatives containing bulky substituents. In the case of ribosides and arabinosides, the reaction rate was decreased by approximately 3_{–4 times when purine bases} contain-ing bulky substituents were used as substrates. The advantages of chemo-enzymatic transglycosylation compared with chemical introduction of sugar residues were demonstrated. The work was financially supported by the Russian Science Foundation (grant 14-13-01077).

P.01-033-Wed

Murine lncRNA LL35 as a probable homolog of

human lncRNA DEANR1

O. Sergeeva, S. Korinfskaya, I. Kurochkin, T. Zatsepin Skolkovo Institute of Science and Technology (Skoltech), Moscow, Russia

Non-coding RNAs (ncRNA) play a significant role in the regula-tion of many cellular processes, including transcripregula-tion, transla-tion and cell differentiatransla-tion. There are examples of ncRNA participation in the development of various diseases (C. Lin and L. Yang, Trends Cell. Biol. 2017), that allows to consider some ncRNAs as potential targets for therapy and diagnostics. It was demonstrated previously that DEANR1 ncRNA participates in the regulation of endoderm differentiation in human embryonic stem cells by cis-regulation of the Foxa2 transcription factor (W. Jiang et al., 2015). Foxa2 protein plays an important role in the glucose homeostasis in the liver, affects fatty acids catabolism under the stress conditions and regulates the biogenesis of high-density lipoproteins (M. Kanaki et al, 2017). Also DEANR1 influences on the epithelial-mesenchymal cell transition, which occurs not only during embryonic development but also in fibro-sis and during progression of cancer tumors (Y. Fan et al, 2016). We found a homologue of the ncRNA DEANR1 in mouse -LL35 ncRNA by bioinformatics. We demonstrated -LL35 expres-sion in the mouse cell lines and liver with preferable localization in cell nucleus. We used antisense oligonucleotides for efficient downregulation of ncRNA LL35 (85–90%) that was confirmed by qPCR and FISH techniques. We propose ncRNA LL35 as a homolog of human lncRNA DEANR1 that can be involved in the transcription regulation of the Foxa2 factor in the liver and affect metabolic processes and various liver diseases in mice. This work is supported by the Russian Science Foundation under grant 17-74-10140.

P.01-034-Mon

LC/MS analysis of viral RNA

A. Simonova, B. Svojanovska, H. Cahova IOCB CAS, Prague, Czech Republic

Viruses are a major force that shapes the evolution of both pro-and eukaryotic organisms. The mechanism of action of various viruses has been the primary focus of many studies, yet, there are surprisingly scarce data on RNA modifications or RNA conju-gates in any type of viruses. Development of vitally important methods for the sensitive analysis of RNA modification enabling detailed studies of the chemical structure of various RNA entities began only recently and they have never been applied to viral RNA. The simplicity of their genomes and well described struc-ture and machinery of various viruses make them a perfect model system for searching for new RNA modifications as well as for understanding of the role of already known RNA modifications and RNA conjugates. In this particular project, we focus on HIV-1 (representative of Retroviridae family) and some eukary-otic viruses from order of Picornavirales. We isolated RNA from viral particles and analysed by LC/MS technique. The surpris-ingly high level of various methylations led us to the development of LC/MS quantification techniques. To reveal the exact position of methylation we had to apply the methylation profiling tech-niques in combination with next-generation sequencing. In next part of our studies, we will concentrate on understanding of the role of these methylated nucleotides in viral RNA. The Ministry of Education, Youth and Sports supported this work from the programme ERC CZ (LL1603).

Effects of paricalcitol on hydrogen peroxide-induced oxidative damage in human umbilical vein endothelial cells through vitamin D receptor signaling

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DNA, RNA and nucleotides

P.01-001-Mon

Escherichia coli single-stranded DNA binding

protein SSB binds to AlkB and promotes DNA

repair

P.01-002-Tue

The effect of sperm DNA fragmentation on the

pregnancy rate in Intra Uterine Cycles (IUI)

P.01-003-Wed

Novel backbone modified antisense

oligonucleotides with improved potency

effectively inactivate oncogenic miRNA-21

comparing to phosphorothioate

oligonucleotides

P.01-004-Mon

Analysis of microRNA at endometriosis and

uterine endometrial cancer

P.01-005-Tue

Vitamin C restores TET proteins activity in

leukemic cells

P.01-006-Wed

Determination of epigenetic DNA

modifications in Drosophila melanogaster

P.01-007-Mon

Evaluation of transcription factor 7-like 2 gene

polymorphism in patients with prediabetes

and type 2 diabetes mellitus

P.01-008-Tue

Transfer RNA-derived fragments target and

regulate ribosome-associated

aminoacyl-transfer RNA synthetases

P.01-009-Wed

Novel targets for the Orb2 CPEB protein in

nervous system of Drosophila

P.01-010-Mon

Experimental approaches for studying

degraded DNA with the use of Next

Generation Sequencing (NGS)

P.01-011-Tue

Defining the promoter sequence determinants

for NAD

+ -initiation by RNA polymerase

P.01-012-Wed

Epigenetic aspect of function of a promising

anticancer drug olivomycin A

P.01-013-Mon

Gre-like factors serve as lineage-specific

transcriptional regulators in Deinococcus

species

P.01-014-Tue

Investigation of the argonaute protein from

Synechococcus elongatus

P.01-015-Wed

1,N

-

a-hydroxypropanoadenine and 3,N

-

a-hydroxypropanocytosine - acrolein adducts to

adenine and cytosine, are substrates for AlkB

dioxygenase

P.01-016-Mon

The amount of humans with high or low

ribosomal repeat copy number in population

decreases with age

P.01-017-Tue

Ferritin2 RNA interference inhibits the

formation of iron granules in honey bees

(Apis mellifera)

P.01-018-Wed

DNA aptamers against procalcitonin as

emerging diagnostic molecules for sepsis

P.01-019-Mon

The screening and application of DNA