The evolution of ideas on the biological role of 5-methylcytosine oxidative derivatives in the mammalian genome

Cover Page


Summary: In this review, we summarize data on 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine – cytosine modifications which are produced by TET-mediated oxidation of 5-methylcytosine in DNA. We show the biochemistry of modified cytosine as well as methods for its global and location analysis. We also highlight the milestones in the evolution of ideas on the biological role of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine in the mammalian genome since their discovery in 2009 till present.

Olga A Efimova

Author for correspondence.
D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology
Russian Federation

Researcher, PhD, Laboratory for Prenatal Diagnosis of human inherited and inborn disorders

Anna A Pendina
D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology
Russian Federation

Researcher, PhD, Laboratory for Prenatal Diagnosis of human inherited and inborn disorders

Andrei V Tikhonov
D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology
Russian Federation

assistant researcher, Laboratory for Prenatal Diagnosis of human inherited and inborn disorders

Vladislav S Baranov
D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology
Russian Federation

Head of the laboratory, prof, Laboratory for Prenatal Diagnosis of human inherited and inborn disorders

  • Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science. 2009;324:929-930. doi: 10.1126/science.1169786.
  • Tahiliani M, Koh KP, Shen Y, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324:930-935. doi: 10.1126/science.1170116.
  • He YF, Li BZ, Li Z, et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science. 2011;333:1303-1307. doi: 10.1126/science.1210944.
  • Maiti A, Drohat AC. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J Biol Chem. 2011;286(41):35334-35338. doi: 10.1074/jbc.C111.284620.
  • Ефимова О.А., Пендина А.А., Тихонов А.В., и др. Метилирование ДНК — основной механизм репрограммирования и регуляции генома человека // Медицинская генетика. – 2012. – Т. 11. – № 4. – С. 10–18. [Efimova OA, Pendina AA, Tikhonov AV, et al. DNA methylation — a major mechanism of human genome reprogramming and regulation. Meditsinskaya genetika. 2012;11(4):10-18. (In Russ.)]
  • Kishikawa S, Murata T, Ugai H, et al. Control elements of Dnmt1 gene are regulated in cell-cycle dependent manner. Nucleic Acids Res Suppl. 2003;3:307-309. doi: 10.1093/nass/3.1.307.
  • Ko YG, Nishino K, Hattori N, et al. Stage-by-stage change in DNA methylation status of Dnmt1 locus during mouse early development. J Biol Chem. 2005;280(10):9627-9634. doi: 10.1074/jbc.M413822200.
  • Trasler JM, Alcivar AA, Hake LE, et al. DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells. Nucleic Acids Res. 1992;20(10):2541-2545.
  • Mertineit C, Yoder JA, Taketo T, et al. Sex-specific exons control DNA methyltransferase in mammalian germ cells. Development. 1998;125:889-897.
  • Chen T, Li E. Establishment and maintenance of DNA methylation patterns in mammals. Curr Top Microbiol Immunol. 2006;301:179-201.
  • Arand J, Spieler D, Karius T, et al. In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases. PLoS Genet. 2012;8(6):e1002750. doi: 10.1371/journal.pgen.1002750.
  • Ramsahoye BH, Biniszkiewicz D, Lyko F, et al. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci USA. 2000;97(10):5237-5242.
  • Shirane K, Toh H, Kobayashi H, et al. Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG methylation and role of DNA methyltransferases. PLoS Genet. 2013;9:e1003439. doi: 10.1371/journal.pgen.1003439.
  • Watanabe D, Suetake I, Tada T, et al. Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis. Mech Dev. 2002;118(1-2):187-190.
  • Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 1999;99:247-257.
  • Kato Y, Kaneda M, Hata K, et al. Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse. Hum Mol Genet. 2007;16:2272-2280. doi: 10.1093/hmg/ddm179.
  • Bourc’his D, Xu GL, Lin CS, et al. Dnmt3L and the establishment of maternal genomic imprints. Science. 2001;294(5551):2536-2539. doi: 10.1126/science.1065848.
  • Hata K, Okano M, Lei H, Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development. 2002;129(8):1983-1993.
  • Turek-Plewa J, Jagodzinski PP. The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett. 2005;10:631-647.
  • Goll MG, Kirpekar F, Maggert KA, et al. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science. 2006;311:395-398. doi: 10.1126/science.1120976.
  • Zhao H, Chen T. Tet family of 5-methylcytosine dioxygenases in mammalian development. J Hum Genet. 2013;58(7):421-427. doi: 10.1038/jhg.2013.63.
  • Baccarelli A, Bollati V. Epigenetics and environmental chemicals. Curr Opin Pediatr. 2009;21(2):243-251.
  • Dickson KM, Gustafson CB, Young JI, et al. Ascorbate-induced generation of 5-hydroxymethylcytosine is unaffected by varying levels of iron and 2-oxoglutarate. Biochem Biophys Res Commun. 2013;439:522-527. doi: 10.1016/j.bbrc.2013.09.010.
  • Li W, Liu M. Distribution of 5-hydroxymethylcytosine in different human tissues. J Nucleic Acids. 2011;2011:870726. doi: 10.4061/2011/870726.
  • Gustafson CB, Yang C, Dickson KM, et al. Epigenetic reprogramming of melanoma cells by vitamin C treatment. Clin Epigenetics. 2015;7:51. doi: 10.1186/s13148-015-0087-z.
  • Yuan BF. 5-methylcytosine and its derivatives. Adv Clin Chem. 2014;67:151-87. doi: 10.1016/bs.acc.2014.09.003.
  • Li M, Hu SL, Shen ZJ, et al. High-performance capillary electrophoretic method for the quantification of global DNA methylation: application to methotrexate-resistant cells. Anal Biochem. 2009;387:71-75. doi: 10.1016/j.ab.2008.12.033.
  • Fraga MF, Uriol E, Borja Diego L, et al. High-performance capillary electrophoretic method for the quantification of 5-methyl 2’-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues. Electrophoresis. 2002;23:1677-1681. doi: 10.1002/1522-2683(200206)23:11<1677::. AID-ELPS1677>3.0.CO;2-Z.
  • Stach D, Schmitz OJ, Stilgenbauer S, et al. Capillary electrophoretic analysis of genomic DNA methylation levels. Nucleic Acids Res. 2003;31:E2.
  • Fraga MF, Rodriguez R, Canal MJ. Rapid quantification of DNA methylation by high performance capillary electrophoresis. Electrophoresis. 2000;21:2990-2994. doi: 10.1002/1522-2683(20000801)21:14<2990::. AID-ELPS2990>3.0.CO;2-I.
  • Zinellu A, Sotgia S, De Murtas V, et al. Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field-amplified sample injection capillary electrophoresis with UV detection. Anal Bioanal Chem. 2011;399:1181-1186. doi: 10.1007/s00216-010-4417-x.
  • Wirtz M, Stach D, Kliem HC, et al. Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser-induced fluorescence detection. Electrophoresis. 2004;25:839-845. doi: 10.1002/elps.200305761.
  • Wang X, Song Y, Song M, et al. Fluorescence polarization combined capillary electrophoresis immunoassay for the sensitive detection of genomic DNA methylation. Anal Chem. 2009;81:7885-7891.
  • Motorin Y, Lyko F, Helm M. 5-methylcytosine in RNA: detection, enzymatic formation and biological functions. Nucleic Acids Res. 2010;38:1415-1430. doi: 10.1093/nar/gkp1117.
  • Yin R, Mao SQ, Zhao B, et al. Ascorbic Acid enhances tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals. J Am Chem Soc. 2013;135:10396-10403. doi: 10.1021/ja4028346.
  • Yang I, Fortin MC, Richardson JR, Buckley B. Fused-core silica column ultra-performance liquid chromatography-ion trap tandem mass spectrometry for determination of global DNA methylation status. Anal Biochem. 2011;409:138-143. doi: 10.1016/j.ab.2010.10.012.
  • Wang X, Suo Y, Yin R, et al. Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879:1647-1652. doi: 10.1016/j.jchromb.2011.04.002.
  • Kok RM, Smith DE, Barto R, et al. Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects. Clin Chem Lab Med. 2007;45:903-911. doi: 10.1515/CCLM.2007.137.
  • Romerio AS, Fiorillo G, Terruzzi I, et al. Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry. Anal Biochem. 2005;336:158-163. doi: 10.1016/j.ab.2004.09.034.
  • Rossella F, Polledri E, Bollati V, et al. Development and validation of a gas chromatography/mass spectrometry method for the assessment. Rapid Commun Mass Spectrom. 2009;23(17):2637-2646. doi: 10.1002/rcm.4166.
  • Tang Y, Gao XD, Wang Y, et al. Widespread existence of cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry. Anal Chem. 2012;84:7249-7255. doi: 10.1021/ac301727c.
  • Leonard SA, Wong SC, Nyce JW. Quantitation of 5-methylcytosine by onedimensional high-performance thin-layer chromatography. J Chromatogr. 1993;645:189-192.
  • Barciszewska MZ, Barciszewska AM, Rattan SI. TLC-based detection of methylated cytosine: application to aging epigenetics. Biogerontology. 2007;8:673-678. doi: 10.1007/s10522-007-9109-3.
  • Ito S, Shen L, Dai Q, et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011;333:1300-1303. doi: 10.1126/science.1210597.
  • Oakeley EJ, Schmitt F, Jost JP. Quantification of 5-methylcytosine in DNA by the chloroacetaldehyde reaction. Biotechniques. 1999;27:744-746,748-750,752.
  • Frommer M, McDonald LE, Millar DS, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA. 1992;89:1827-1831.
  • Hu J, Xing X, Xu X, et al. Selective chemical labelling of 5-formylcytosine in DNA by fluorescent dyes. Chemistry. 2013;19:5836-5840. doi: 10.1002/chem.201300082.
  • Пендина А.А., Ефимова О.А., Каминская А.Н., и др. Иммуноцитохимический анализ статуса метилирования метафазных хромосом человека // Цитология. – 2005. – Т. 47. – № 8. – С. 731–737. [Pendina AA, Efimova OA, Kaminskaya AN, et al. Immunocytochemical analysis of human metaphase chromosome methylation status. Tsitologiia. 2005;47(8):731-737. (In Russ.)]
  • Ефимова О.А., Пендина А.А., Тихонов А.В., и др. Сравнительный иммуноцитохимический анализ профилей метилирования ДНК метафазных хромосом из лимфоцитов взрослых индивидов и плодов человека // Молекулярная медицина. – 2015. – № 3. – С. 17–21. [Efimova OA, Pendina AA, Tikhonov AV, et al. A comparative immunocytochemical analysis of DNA methylation patterns in human metaphase chromosomes of adults and fetuses. Molekulyarnaya meditsina. 2015;(3):17-21. (In Russ.)]
  • Kokalj-Vokac N, Zagorac A, Pristovnik M, et al. DNA methylation of the extraembryonic tissues: an in situ study on human metaphase chromosomes. Chromosome Res. 1998;6(3):161-166.
  • Баранов В.С., Пендина А.А., Кузнецова Т.В., и др. Некоторые особенности статуса метилирования метафазных хромосом у зародышей человека доимплантационных стадий развития // Цитология. – 2005. – Т. 47. – № 8. – С. 723–730. [Baranov VS, Pendina AA, Kuznetsova TV, et al. Peculiarities of metaphase chromosome methylation pattern in preimplantation human embryos. Tsitologiia. 2005;47(8):723-730. (In Russ.)]
  • Pendina AA, Efimova OA, Fedorova ID, et al. DNA methylation patterns of metaphase chromosomes in human preimplantation embryos. Cytogenet Genome Res. 2011;132(1-2):1-7. doi: 10.1159/000318673.
  • Efimova OA, Pendina AA, Tikhonov AV, et al. Chromosome hydroxymethylation patterns in human zygotes and cleavage-stage embryos. Reproduction. 2015;149(3):223-233. doi: 10.1530/REP-14-0343.
  • Nestor C, Ruzov A, Meehan R, Dunican D. Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA. Biotechniques. 2010;48(4):317-319. doi: 10.2144/000113403.
  • Kinney SM, Chin HG, Vaisvila R, et al. Tissue-specific distribution and dynamic changes of 5-hydroxymethylcytosine in mammalian genomes. J Biol Chem. 2011;286:24685-24693. doi: 10.1074/jbc.M110.217083.
  • Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994;22:2990-2997.
  • Booth MJ, Branco MR, Ficz G, et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science. 2012;336:934-937. doi: 10.1126/science.1220671.
  • Yu M, Hon GC, Szulwach KE, et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell. 2012;149:1368-1380. doi: 10.1016/j.cell.2012.04.027.
  • Booth MJ, Marsico G, Bachman M, et al. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat Chem. 2014;6(5):435-440. doi: 10.1038/nchem.1893.
  • Thu KL, Pikor LA, Kennett JY, et al. Methylation analysis by DNA immunoprecipitation. J Cell Physiol. 2010;222:522-531. doi: 10.1002/jcp.22009.
  • Robertson AB, Dahl JA, Vagbo CB, et al. A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 2011;39: e55. doi: 10.1093/nar/gkr051.
  • Raiber EA, Beraldi D, Ficz G, et al. Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase. Genome Biol. 2012;13:R69. doi: 10.1186/gb-2012-13-8-r69.
  • Lu X, Song CX, Szulwach K, et al. Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA. J Am Chem Soc. 2013;135:9315-9317. doi: 10.1021/ja4044856.
  • Korlach J, Turner SW. Going beyond five bases in DNA sequencing. Curr Opin Struct Biol. 2012;22:251-261. doi: 10.1016/
  • Flusberg BA, Webster DR, Lee JH, et al. Direct detection of DNA methylation during singlemolecule, real-time sequencing. Nat Methods. 2010;7:461-465. doi: 10.1038/nmeth.1459.
  • Song CX, Clark TA, Lu XY, et al. Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nat Methods. 2012;9:75-77. doi: 10.1038/ncomms1237.
  • Globisch D, Munzel M, Muller M, et al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS One. 2010;5(12): e15367. doi: 10.1371/journal.pone.0015367.
  • Pfaffeneder T, Spada F, Wagner M, et al. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat Chem Biol. 2014;10:574-581. doi: 10.1038/nchembio.1532.
  • Song J, Pfeifer GP. Are there specific readers of oxidized 5-methylcytosine bases? Bioessays. 2016;38:1038-1047. doi: 10.1002/bies.201600126.
  • Stroud H, Feng S, Morey Kinney S, et al. 5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol. 2011;12: R54. doi: 10.1186/gb-2011-12-6-r54.
  • Hon GC, Song CX, Du T, et al. 5mC oxidation by Tet2 modulates enhancer activity and timing of transcriptome reprogramming during differentiation. Mol Cell. 2014;56:286-297. doi: 10.1016/j.molcel.2014.08.026.
  • Lu F, Liu Y, Jiang L, et al. Role of Tet proteins in enhancer activity and telomere elongation. Genes Dev. 2014;28:2103-2119. doi: 10.1101/gad.248005.114.
  • Song CX, Szulwach KE, Dai Q, et al. Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell. 2013;153:678-691. doi: 10.1016/j.cell.2013.04.001.
  • Shen L, Wu H, Diep D, et al. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell. 2013;153:692-706. doi: 10.1016/j.cell.2013.04.002.
  • Williams K, Christensen J, Helin K. DNA methylation: TET proteins-guardians of CpG islands? EMBO Rep. 2012;13:28-35. doi: 10.1038/embor.2011.233.
  • Rauch TA, Wu X, Zhong X, et al. A human B cell methylome at 100-base pair resolution. Proc Natl Acad Sci U S A. 2009;106(3):671-678. doi: 10.1073/pnas.0812399106.
  • Song CX, Szulwach KE, Fu Y, et al. Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol. 2011;29:68-72. doi: 10.1038/nbt.1732.
  • Spruijt CG, Gnerlich F, Smits AH, et al. Dynamic readers for 5-(hydroxy) methylcytosine and its oxidized derivatives. Cell. 2013;152:1146-1159. doi: 10.1016/j.cell.2013.02.004.
  • Iurlaro M, Ficz G, Oxley D, et al. A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol. 2013;14:R119. doi: 10.1186/gb-2013-14-10-r119.
  • Zhou T, Xiong J, Wang M, et al. Structural basis for hydroxymethylcytosine recognition by the SRA domain of UHRF2. Mol Cell. 2014;54:879-886. doi: 10.1016/j.molcel.2014.04.003.
  • Iurlaro M, McInroy GR, Burgess HE, et al. In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine. Genome Biol. 2016;17(1):141. doi: 10.1186/s13059-016-1001-5.
  • Bachman M, Uribe-Lewis S, Yang X, et al. 5-Formylcytosine can be a stable DNA modification in mammals. Nat Chem Biol. 2015;11:555-557. doi: 10.1038/nchembio.1848.
  • Raiber EA, Murat P, Chirgadze DY, et al. 5-Formylcytosine alters the structure of the DNA double helix. Nat Struct Mol Biol. 2015;22:44-49. doi: 10.1038/nsmb.2936.
  • Hashimoto H, Olanrewaju YO, Zheng Y, et al. Wilms tumor protein recognizes 5-carboxylcytosine within a specific DNA sequence. Genes Dev. 2014;28:2304-2313. doi: 10.1101/gad.250746.114.
  • Jin SG, Zhang ZM, Dunwell TL, et al. Tet3 reads 5-carboxylcytosine through its CXXC domain and is a potential guardian against neurodegeneration. Cell Rep. 2016;14:493-505. doi: 10.1016/j.celrep.2015.12.044.
  • Wang L, Zhou Y, Xu L, et al. Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex. Nature. 2015;523:621-625. doi: 10.1038/nature14482.
  • Hashimoto H, Zhang X, Cheng X. Activity and crystal structure of human thymine DNA glycosylase mutant N140A with 5-carboxylcytosine DNA at low pH. DNA Repair (Amst). 2013;12:535-540. doi: 10.1016/j.dnarep.2013.04.003.
  • Zhang L, Lu X, Lu J, et al. Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA. Nat Chem Biol. 2012;8:328-330. doi: 10.1038/nchembio.914.
  • Xu Y, Xu C, Kato A, et al. Tet3 CXXC domain and dioxygenase activity cooperatively regulate key genes for Xenopus eye and neural development. Cell. 2012;151:1200-1213. doi: 10.1016/j.cell.2012.11.014.
  • Allen MD, Grummitt CG, Hilcenko C, et al. Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase. EMBO J. 2006;25:4503-4512. doi: 10.1038/sj.emboj.7601340.
  • Cierpicki T, Risner LE, Grembecka J, et al. Structure of the MLL CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia. Nat Struct Mol Biol. 2010;17:62-68. doi: 10.1038/nsmb.1714.
  • Song J, Rechkoblit O, Bestor TH, Patel DJ. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science. 2011;331:1036-1040. doi: 10.1126/science.1195380.
  • Xu C, Bian C, Lam R, et al. The structural basis for selective binding of non-methylated CpG islands by the CFP1 CXXC domain. Nat Commun. 2011;2:227. doi: 10.1038/ncomms1237.
  • Branco MR, Ficz G, Reik W. Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat Rev Genet. 2011;13:7-13. doi: 10.1038/nrg3080.
  • Efimova OA, Pendina AA, Tikhonov AV, et al. Oxidized form of 5-methylcytosine — 5-hydroxymethylcytosine: a new insight into the biological significance in the mammalian genome. Russian Journal of Genetics: Applied Research. 2015;5:75-81.
  • Dean W. DNA methylation and demethylation: a pathway to gametogenesis and development. Mol Reprod Dev. 2014;81(2):113-125. doi: 10.1002/mrd.22280.
  • Amouroux R, Nashun B, Shirane K, et al. De novo DNA methylation drives 5hmC accumulation in mouse zygotes. Nat Cell Biol. 2016;18(2):225-233. doi: 10.1038/ncb3296.


Abstract - 86

PDF (Russian) - 42

Copyright (c) 2016 Efimova O.A., Pendina A.A., Tikhonov A.V., Baranov V.S.

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.