International conference on bioinformatics of genome regulation
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Key words: oxidized lipoproteins, rare variant association analysis, APOB Motivation and Aim: Atherosclerosis represents one of the major problems in the mod- ern medicine and public health. Cardiovascular disorders, which result from atheroscle- rosis, are the leading cause of mortality in developed countries. Oxidized LDL (oxLDL) plays an important role in atherogenesis by promoting an inflammatory environment and lipid deposition in the arterial wall. The detection of mutations affecting the level of oxLDL is relevant. Our work aims to investigate the functional effect of genetic markers affecting oxLDL levels, major risk factor for atherosclerosis, on incidence/onset of atherosclerosis and atherosclerotic phenotypes in a cohort of 725 patients. Methods and Algorithms: Genotyping of 725 patients was performed using Cardio- Metabo Chip (Illumina) which allows to genotype 196 000 SNPs. Targeted sequencing of genomic region 2p24-p23 was performed with the TargetSeq™ Custom Enrichment Kit (Applied Biosystems, USA) using the SOLiD 5500W system (Applied Biosystems, USA). Alignment and search SNPs were implemented by data analysis tools with the LifeScope™ Genomic Analysis Software. Results: We performed the genome-wide association study (GWAS) using microarrays Cardio-Metabo Chip (Illumina). We found genetic locus, 2p24-p23, capturing a total number of 14 SNPs in the APOB gene or near it, significantly associated (after adjust- ment for multiple testing) with levels of oxLDL. We conduct sequencing of APOB gene and surrounding areas (locus of about 500 000 bp, Chr2: 20996301-21494945) in 96 patients (48 with high levels and 48 with low lev- els of oxLDL). ApoB locus was analyzed. For analysis we used data for 725 patients from Cardio- Metabo Chip (Illumina) and for 96 patients from targeted sequencing. We identified single-nucleotide polymorphisms (SNPs) and filtered them to narrow the search for the causal variant. It seems possible that much of the genetic control of common diseases is due to rare and generally deleterious variants that have a strong impact on the risk of disease in individual patients. It is also likely that the variants with the largest effect sizes will be those that have obvious functional consequences. So we focused only on nonsynonymous (protein-altering) changes, other variants have been removed from fur- ther consideration. To analyze the impact of the cumulative effect of various rare alleles to the level of oxidized LDL we used R package and statistical tests for rare mutations - CMC (Combined Multivariate and Collapsing Methods) and SKAT (Sequence kernel association tests). Conclusion: Our results may indicate the influence of functional mutations at the level of oxidized LDL. It is supported by RFBR #13-04-01259 123 THE TENTH INTERNATIONAL CONFERENCE ON BIOINFORMATICS OF GENOME REGULATION AND STRUCTURE\SYSTEMS BIOLOGY WHAT WE USUALLY STUDY WHEN WE THINK WE STUDY AGING A.N. Khokhlov Evolutionary Cytogerontology Sector, School of Biology, Lomonosov Moscow State University, Moscow, Russia * Corresponding author: khokhlov@mail.bio.msu.ru Key words: aging, gerontology, death probability, life span, non-aging organisms, cell cultures, systems approach The recent considerable increase in interest in experimental gerontological research has led to a paradoxical situation: although the number of studies in this field is ever increas- ing, only a small part of them actually deals with aging mechanisms. This situation is determined, among others, by the following circumstances. (1) As a rule, the classic defi- nition of aging as a set of age-related changes leading to an increase in the probability of death is ignored. (2) The focus in these studies is on an increase or a decrease in the lifespan, although this had nothing to do with modification of aging (in particular, it is possible to successfully extend the lifespan for non-aging organisms; on the other hand, the very existence of aging does not necessarily suggest a short lifespan). (3) The animals with certain abnormalities (such as genetic diseases) are often used as a control; thus, any favorable impact on the corresponding pathological processes leads to an increase in lifes- pan. (4) Too much importance is attached to an increase or a decrease in the AVERAGE lifespan, which is in many respects determined by the factors that are in no way associated with aging. (5) The ever increasing number of gerontological experiments involves the model systems that give only indirect information about the aging mechanisms and whose interpretation, in many respects, depends on the basic concept shared by the correspond- ing researchers. In particular, this refers to the situation with the term “cell senescence.” Initially, this term was introduced to denote various adverse changes in normal cells RE- SULTING from depletion of their mitotic potential. On the contrary, this term now is ever more frequently used to denote the inhibition of cell proliferation (including cancer cell proliferation) accompanied by a certain cascade of intracellular reactions and caused by various DNA-damaging factors. (6) Finally, there is the issue that may be referred to as the “reductionism problem.” The overwhelming majority of gerontological theories that have appeared during the last decades reduced all the mechanisms underlying both “normal” and modified (accelerated or slowed down) aging of multicellular organisms to certain macromolecular alterations (it is not important whether they are stochastic or programmed) in the constituent cells. This has given rise to numerous cytogerontological model systems for studying the “age-related” changes in the cells freed from a “body- level noise” associated with the functioning of the neurohumoral system. However, many of the conclusions reached earlier on the basis of the results of experiments conducted on Hayflick's model (aging in vitro), were subsequently found to be wrong. In addition, our cytogerontological studies of various anti-aging factors with the help of the "stationary phase aging" model, the cell kinetics model and assessment of colony-forming ability, have shown that in very many cases the factors studied have no beneficial effect on the viability of cultured cells, although they prolong life in experimental animals and increase the well-being of humans. This allowed us to assume that, in many cases, the anti-aging agent action appears only at the organism level, and is not limited to just improving the viability of some of its constituent cells. 124 THE TENTH INTERNATIONAL CONFERENCE ON BIOINFORMATICS OF GENOME REGULATION AND STRUCTURE\SYSTEMS BIOLOGY THE FIRST EDITION OF MUTAGENESIS BY CRISPR/CAS IN THE EXTREME DESICCATION TOLERANT CULTURED CELL T. Kikawada 1, 2 *, Y. Miyata 1, 3 , Y. Sogame 1, 4 , T. Furusawa 1 , S. Kikuta 5 , R. Cornette 1 , O. Gusev 6, 7 1 Institute of Agrobiological Sciences, NARO, Japan 2 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Japan 3 Center for Biological Resources and Informatics, Tokyo Institute of Technology 4 JSPS Research Fellow 5 Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan 6 Institute of Fundamental Medicine and Biology, Kazan Federal University,Russia 7 Preventive Medicine & Diagnosis Innovation Program (PMI), RIKEN, Japan * Corresponding author: kikawada@affrc.go.jp Key words: anhydrobiosis, cell culture, genome editing, CRISPR-Cas Motivation and Aim: We have accomplished draft-genome analysis of the anhydrobi- otic midge Polypedilum vanderplanki. From the comparative genomics, several specific genomic regions are likely to be involved in evolutional acquisition of the anhydrobiosis in P. vanderplanki; however, the molecular mechanisms underlying the anhydrobiosis remains to be elucidated. As with the larvae of P. vanderplanki, Pv11 cells, a cultured cell line derived from embryo of P. vanderplanki possesses a capability of the desiccation tolerance. To efficiently screen the responsible genes to the anhydrobiosis, we attempt to optimize the genome editing system, CRISPR-Cas for Pv11 cells. Methods and Algorithms: Using effective promoters isolated from genome sequence of P. vanderplanki, Cas9 and single guide RNA (sgRNA) expression vectors were con- structed. After transfection of the vectors, their expressions were evaluated with Western blot and Real-Time PCR. Finally, we checked phenotypes of the in-del mutated Pv11 cells after sorting the mutant cells. Results: We have already established a stable Pv11 cells expressing GFP (Pv-KH cells). Exogenous Cas9 and sgRNA expressions in Pv-KH cells were confirmed. To validate knock-out efficiency of P. vanderplanki-optimized CRISPR/Cas9 system, we observed the loss of function in the Pv-KH cells co-transfected with expression vectors for Cas9 and sgRNA that recognized GFP gene. As a result, a small percent of the cells was com- pletely loss of their fluorescence, indicating that CRISPR/Cas9 system could be worked in the anhydrobiotic midge. Conclusion: The genome editing, CRISPR/Cas9 system can be applicable in P. vander- planki. Acknowledgements: This work was financially supported by JSPS KAKENHI (16K18827, 16K15073 and 25252060). 125 THE TENTH INTERNATIONAL CONFERENCE ON BIOINFORMATICS OF GENOME REGULATION AND STRUCTURE\SYSTEMS BIOLOGY ANHYDRO-PRESERVATION OF EXOGENOUSLY-EXPRESSED DESICCATION-SENSITIVE ENZYME LUCIFERASE USING INSECT CELLS S. Kikuta 1 *, S. Watanabe 1 , O. Gusev 2, 3 , Y. Sogame 4 , R. Cornette 4 , T. Kikawada 4 1 Tokyo University of Agriculture and Technology, Tokyo, Japan 2 Kazan Federal University, Kazan, Russia 3 Riken Division of Genomic Technologies, Japan 4 National Agriculture and Food Research Organization, Japan * Corresponding author: singo@cc.tuat.ac.jp Key words: dehydration, trehalose, luminescence, dry-preservation, anhydrobiosis Motivation and Aim: An insect cell line (Pv11) derived from the desiccation tolerant insect, Polypedilum venderplanki can withstand without water. Upon rehydration, the desiccated cells begin to re-start their metabolisms and proliferate. The results indicate that the intracellular enzymes involved in the metabolism should be preserved under the dry state. We would like to know whether Pv11 can preserve exogenously expressed enzymes in dehydrated state., i.e. be a model of new generation of dry preservation technology for enzymes. Methods: As a representative exogenous enzyme, luciferase in firefly is suitable to ex- amine the activity without taking account of intrinsic proteins. We established Emerald Luciferase-expressing Pv11 (ELuc-Pv11) by DNA electroporation and antibiotics treat- ment. We measured the luminescence by luciferase of dehydrated ELuc-Pv11 at 1h after rehydration. Results: The luminescence by luciferase was clearly shown at 1h after rehydration. We concerned about the luciferase activities after rehydration. The activities were a poten- tial to show de novo synthesis in rehydrated Pv11 re-started metabolism. Using protein translation inhibitors, the luminescence was also detected after rehydration. These re- sults indicate that the conformation of the enzyme in the cells would be stably kept under the dehydrated state. Conclusion: Pv11 can preserve the exogenously-expressed enzyme under dehydration. Availability: Using this system, we can keep enzymes of interest without a deep freezer. This work was partially supported by Ministry of Science and Education of RF, research project identification number: RFMEFI58414X0002. 126 THE TENTH INTERNATIONAL CONFERENCE ON BIOINFORMATICS OF GENOME REGULATION AND STRUCTURE\SYSTEMS BIOLOGY MOLECULAR DYNAMICS CHARACTERIZATION OF GLYCYRRHIZIN INTERACTION WITH LIPID MEMBRANES A.V. Kim*, E.A. Shelepova, N.N. Medvedev Institute of Chemical Kinetics and Combustion SB RAS, Novosibirsk, Russia * Corresponding author kim@kinetics.nsc.ru Key words: glycyrrhizic acid, lipid bilayer, potential of mean force, molecular dynamics Motivation and Aim: Glycyrrhizic acid (GA) is a triterpene glycoside extracted from licorice root. It has a wide range of therapeutic activity with a minor amount of side effects. There are also many experimental evidences of its ability to enhance the bio- availability of another drug molecules when used together. But the mechanism of GA biological activity remains unknown. Computer simulation can shed the light on under- standing the processes considered, at a molecular level. Methods and Algorithms: All simulations were performed using the GROMACS 5.0 [1] molecular dynamics package. We used Berger’s lipids model for DOPC, POPC and DPPC bilayers. GA parameters were generated by ATBuilder on the basis of gro- mos53a6 forcefield. Free energy calculations were performed using umbrella sampling approach for potential of mean force (PMF) estimation. 30 windows, spaced by 0.2 nm were employed; each window contained 150 ns of production run, covering a total of 4.5 μs per system. Results: The series of 10 independent simulations of 200 ns for each of the three lipids were performed. The characteristic behavior of GA nearby and inside the model mem- brane was revealed. It was found, that GA being placed in the water in random orienta- tion, first diffuses to the membrane surface. Then after moving over the surface for 20- 80 ns it meets a suitable cavity and penetrates into the membrane, occupying the region under the lipid heads. In the case of DOPC bilayer GA stays in its first half-layer, not passing through the midplane. To estimate the energy barrier, the PMF calculation was performed for the process of GA penetration through the lipid bilayer. There are two energy barriers observed: the one is at the membrane surface and the other one is in the middle of the bilayer. The midplane barrier is about 3 Kcal/mol, which is approximate- ly 5 times RT. The thermal energy is not enough for GA to pass to the next half-layer of membrane. The partial density profile of GA in DOPC bilayer shows a good agree- ment with PMF. A steep energy downhill from water to bilayer surface is about 8 Kcal/ mol, so GA readily attaches to the membrane and penetrates in it, but does not able to escape it. Conclusion: GA easily incorporates in membranes and locates under its surface near central parts of lipids tails. It is in a good agreement with experimental NMR studies, where GA demonstrates an influence on the mobility of both the polar heads and the central parts of lipid’s hydrophobic tails. PMF shows a preference of GA to stay in the first half-layer of DOPC membrane due to an energy barrier of about 8 Kcal/mol. This fact is also in a good agreement with NMR results. References: 1. S. Pronk et al. (2014) GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit, Bioinformatics, 29(7):845–854. 127 THE TENTH INTERNATIONAL CONFERENCE ON BIOINFORMATICS OF GENOME REGULATION AND STRUCTURE\SYSTEMS BIOLOGY GENOMEASIA 100K INITIATIVE ANNOUNCED TO SEQUENCE 100,000 GENOMES IN SOUTH, NORTH AND EAST ASIA Hie Lim Kim, Elena S. Gusareva*, S.C. Schuster Singapore Centre on Environmental Life Sciences Engineering, Singapore Nanyang Technological University (NTU), Singapore * Corresponding author: egusareva@ntu.edu.sg Key words: GenomeAsia 100K, genetic diversity, Asian populations With recent insights into the genome diversity of Asian ethnicities, it is very important to understand the biology of disease in the currently under-studied Asian populations that represent 40% of mankind. The unique genetic diversity prevalent in South, North and East Asia can potentially provide a valuable source of clinical insights that should enhance our understanding of several rare and inherited diseases, as well as complex diseases such as cancer, diabetes and cardiovascular disease. The non-profit consortium, GenomeAsia 100K, announced an ambitious plan to se- quence 100,000 individuals from Afghanistan, Bangladesh, Bhutan, Burma, Brunei, Cambodia, China, India, Indonesia, Japan, Kazakhstan, Korea, Kyrgyzstan, Laos, Mal- dives, Malaysia, Mongolia, Pakistan, Philippines, Russia, Sri Lanka, Taiwan, Tajikistan, Thailand, Timor-Leste, Turkmenistan, Uzbekistan, and Vietnam. In the first phase, the project will focus on creating reference genomes for each of diverse Asian ethnic groups representing a major step forward in understanding the population history and substruc- ture of the region. The initiative aims 1) to generate ethnicity-specific reference genomes to comprehend Asian genomes and identify rare genetic variants and structural varia- tions with high accuracy in the populations, 2) to develop a database of allele frequency with defined ethnicities to share with academic communities, and 3) to develop insights into disease and human health by combining information from genome, clinical history and population substructure. Focus on inherited diseases, common diseases, oncology, metabolic disorders, neurology, and aging to promote precision medicine research. The GenomeAsia 100K hosted at Nanyang Technological University in Singapore. To date, 50,000 DNA samples have been collected through blood or saliva samples from a network of clinics across Asia with the help of two of the consortium’s founding mem- bers, genomics companies Macrogen in South Korea and MedGenome in India. The GenomeAsia 100K is looking for significant partners and supporters in Asian coun- tries and beyond. References: 1. GenomeAsia 100K Initiative Announced to Sequence 100,000 Genomes in South, North and East Asia. Feb 11, 2016 News from Emerge Ventures. 128 THE TENTH INTERNATIONAL CONFERENCE ON BIOINFORMATICS OF GENOME REGULATION AND STRUCTURE\SYSTEMS BIOLOGY THE SIGNIFICANCE OF DISSOCIATIVE NUCLEOTIDE CHANGES ACCUMULATION RATE IN THE GENOTYPE VARIABILITY OF TICK-BORNE ENCEPHALITIS VIRUS FOR GENE E D.O. Kiselev 1 *, S.Ju. Bukin 2 , A.I. Paramonov 3 , Ju.P. Dzhioev 1 , V.I. Zlobin 1 1 Irkutsk State Medical University, Irkutsk, Russia 2 Limnological Institute, Irkutsk, Russia 3 Scientific Centre for Human Reproduction and family health problems, Irkutsk, Russia * Corresponding author: Rancevich89@mail.ru Key words: tick-borne encephalitis virus, molecular epidemiology, molecular clock Motivation and Aim: The mechanism of genetic molecular clock forms structural and functional features of all living systems, including viruses. The aim of research is the study of molecular clock in the evolutionary variability of the tick-borne encephalitis virus (TBEV) for gene E by methods of molecular genetics and bioinformatics that can be used to obtain the important information about the variability and evolution of vi- rus. The material of the study is based on data about sequences of gene E of TBEV (55 strains), most of which were isolated in the Siberian region, as well as strains isolated in other regions. Methods and Algorithms: MEGA6 – algorithms (neighbor-joining trees, Tajima Relative Rate Test of Molecular Clock) [1,2] Results: Two phylogenetic trees are presented in the study (for 1-3 and 1-2 positions of codons of gene E). Significant differences of phylogeny structure have been found between two trees. The verification results of the strict molecular clock hypothesis ef- ficiency are presented in the second part of study for selected TBEV strains. Selected strains have been divided into 2 subgroups with different geographical origin of strains according to the rate of nucleotide substitutions accumulation. Conclusion: Results of the study demonstrate the high significance of the nucleotide substitutions accumulation in the 3-rd position of codons in the evolutionary history of TBEV, making significant adjustments to the process of studying the phylogeny and phylogeography of the pathogen. References: 1. Tamura K. et al. (2013) Molecular Biology and Evolution 30:2725-2729 2. Tajima, F. (1993) Simple methods for testing molecular clock hypothesis. Genetics, 135, 599–607. 129 THE TENTH INTERNATIONAL CONFERENCE ON BIOINFORMATICS OF GENOME REGULATION AND STRUCTURE\SYSTEMS BIOLOGY FUNCTIONAL AND STRUCTURAL CHARACTERISATION OF PPD-B1 PHOTOPERIOD INSENSITIVE ALLELE A.A. Kiseleva*, E.A. Salina Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia * Corresponding author: antkiseleva@bionet.nsc.ru Key words: wheat, Triticum aestivum, photoperiod sensitivity, heading date, Ppd-B1 Motivation and Aim: Photoperiod sensitivity is an important agronomic trait that influences wheat heading date. Ppd-1 genes are significant regulators of this process. Ppd-1 photoperiod insensitive alleles induce early heading of wheat. Ppd-B1 is the only Ppd-1 gene which dominant photoperiod insensitive allele is determined by the copy number variation. However, little is known about mechanisms determining its misexpression. The aim of our investigation was to reveal possible mechanisms of the photoperiod pathways involved Ppd-B1, to characterize functional specifications of Ppd-B1 photoperiod insensitive allele and its interaction with other photoperiod genes. Methods and Algorithms: PlantPAN 2.0 database (http://plantpan2.itps.ncku.edu.tw) was used to determine putative plant transcription factor binding sites. PCR analysis, molecular cloning and sequencing were used for the characterization of Ppd-B1 sequence. To analyze diurnal expression of genes regulating heading date Real-time PCR was performed. Results: We identified probable transcription factors involved in Ppd-B1 regulation and factors common for the promoters of all Ppd-1 homeologous genes. Promoter regions of such important genes regulating heading date as TaFT1, PhyC and Vrn-1 were analyzed too. Using two pairs of Near Isogenic Lines (NILs) with dominant or recessive allele of Ppd-B1 and different in their photoperiod sensitivity we investigated structure of Ppd-B1 distinct copies and detected some SNPs confirmed difference between NILs and their sibs, the indel in promoter region distinguished the lines under investigation from other alleles with copy number increment, but revealed no polymorphisms between Ppd-B1 gene copies. Then we analyzed diurnal expression of Ppd-B1, Ppd-D1, Ppd-A1, Vrn-A1, TaFT1, PhyC and some other genes important for the heading date. Conclusion: We detected some TFBSs specific to the Ppd-B1 promoter region but not Ppd-D1, Ppd-A1 allows suggesting different regulation of this genes. Taken together our data about transcription factor binding sites in the promoter regions of genes controlled heading date and the analysis of their diurnal expression suggest hypothetic scheme of their interaction and the impact of Ppd-B1 photoperiod allele on heading promotion. Acknowledgements: This study is supported by Russian Scientific Foundation (14-14- 00161) 130 THE TENTH INTERNATIONAL CONFERENCE ON BIOINFORMATICS OF GENOME REGULATION AND STRUCTURE\SYSTEMS BIOLOGY PHAGE INFECTION SLOWS DOWN SPECIATION CAUSED BY GENE LOSS AND HORIZONTAL GENE TRANSFER OF METABOLIC GENES IN MODELS OF SPATIALLY DISTRIBUTED BACTERIAL COMMUNITIES A.I. Klimenko*, Yu.G. Matushkin, N.A. Kolchanov, S.A. Lashin Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia Novosibirsk State University, Novosibirsk, Russia * Corresponding author: klimenko@bionet.nsc.ru Download 3.91 Kb. Do'stlaringiz bilan baham: |
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