Day 2 :
- Track 15: Transcriptomics and Proteomics of Plants, Track 16: Transcriptome technologies market, Track 17: New Horizons to the Transcriptome
Chair
Patrick Xuechun Zhao
Samuel Roberts Noble Foundation, USA
Co-Chair
Zsolt Ponya
Kaposvar University, Hungary
Session Introduction
Patrick Xuechun Zhao
Samuel Roberts Noble Foundation, USA
Title: The LegumeIP (2015 release) - a web-based comparative genomics and gene expression atlas platform to study gene function and genome evolution in legumes
Time : 09:30-09:50
Biography:
Dr. Patrick Xuechun Zhao is an Associate Professor of Bioinformatics and Computational Biology at the Samuel Roberts Noble Foundation. Dr. Zhao received his Ph.D. degree in Communication and Information Systems from Shanghai Jiao Tong University in 2000. After his graduation, he joined the Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, as a research associate developing bioinformatics technologies to discover genes and biological networks that are essential to the determination of the normal aging process, in particular those related to successful aging in centenarians. In 2004, he joined the Samuel Roberts Noble Foundation as an Assistant Professor and Faculty Principle Investigator. Dr. Zhao’s current research centers on bioinformatics and computational biology, plant and soil microbe functional genomics, statistical machine learning for ‘omics’ data mining and biological knowledge discovery. Dr. Zhao has published over 60 papers in refereed scientific journals. He also published a series of software and bioinformatics resources including a dozen of databases and web servers for plant gene function and gene regulatory network analysis, which are publicly available and used by life scientists from around the world.
Abstract:
Legumes are one of the most evolutionarily well-adapted plants that play vital roles in ecology and agriculture. They have the unique ability to carry out symbiotic nitrogen fixation (SNF) through endosymbiotic interactions with bacteria. Aside from the root nodulation and nitrogen fixation symbiosis with rhizobia, legumes possess many unique features that are not found in the model plant Arabidopsis thaliana, such as mycorrhization, compound leaf development, protein-rich physiology, a profuse secondary metabolism, glandular trichome development and border cells in roots. We present LegumeIP1 (http://plantgrn.noble.org/LegumeIP/v2/) - a web-based comparative genomics and gene expression atlas platform that integrates large-scale genomics, transcriptomics data and bioinformatics tools to study gene function and genome evolution in legumes. The LegumeIP (2015 release) hosts 1) genomic data such as genomics sequences, Sanger sequencing- and RNA-seq-based expressed sequence tag (EST) sequences, gene models, and annotations that are cross-refereed to the UniProt TrEMBL, InterProScan, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes (KEGG) reference database for six model and crop legumes: Medicago truncatula, Glycine max (soybean), Lotus japonicus, Phaseolus vulgaris (common bean), Cicer arietinumx (chickpea) and Cajanus cajan (pigeon pea), and two reference model plants: A. thaliana and Poplar trichocarpa; 2) gene expression atlases that include 276 array hybridizations from L. japonicas, 1,915 array hybridizations from M. truncatula, 1,379 gene expression profiles of G. max, and 4,164 array hybridizations of A. thaliana; and 3) gene families, orthologous groups, gene family phylogenetic trees and syntenic regions across hosted species. LegumeIP also integrates a suite of bioinformatics tools for exploring and analyzing 1) whole-genome synteny and detailed syntenic gene alignments; 2) gene annotations, gene families and orthologous groups; and 3) gene expression patterns, clusters and co-expression networks.
Vishwanath Sollapura
Agriculture and Agri-Food, Canada
Title: Comparative transcriptome analysis between mature pollen and stigma of Brassicaceae and Triticeae crop species.
Time : 09:50-10:10
Biography:
Dr. Vishwanath Sollapura completed his PhD in plant molecular biology in 2013 from Carleton University, Ottawa, Canada, where he identified and characterized three alcohol-forming fatty acyl-CoA reductases associated with suberin biopolymer. Prior to that, he worked in India for the plant biotech company Avesthagen Ltd on a project to generate Poly Unsaturated Fatty Acids (PUFAs) in Sunflower and Mustard oilseed plants using recombinant approaches. Currently, he is working as a NSERC visiting post-doctoral fellow at Agriculture and Agrifood Canada in Dr. Laurian Robert’s laboratory focusing on the molecular genetics of crop flower development.
Abstract:
Pollination is an important step in Angiosperm sexual reproduction that involves pollen adhesion, recognition, rejection/acceptance, hydration, germination, and other cell-cell interactions with the stigma (the female receptive tissue). A detailed knowledge of the transcriptomes of the mature pollen grain and stigma will help in further understand these interactions and identify the various molecular participants. This fundamental knowledge will in turn provide tools to enable molecular interventions aimed at managing pollination and thereby leading to improved sustainable crop production. We are currently working towards assembling high quality pollen and stigma reference transcriptomes for both dicots (Brassicaceae) and monocots (Triticeae) crops using next-generation sequencing of transcriptomes (RNA-seq). We report the results of transcriptome analyses performed on the mature pollen and stigma of Brassica carinata and Triticale (x Triticosecale Wittmack), two crops with potential applications as bio-industrial platforms. Both Illumina and 454 sequencing were performed generating about 100 million (100 bp lengths) and 1 million (400 bp lengths) paired reads per sample respectively. The RNA-seq analysis was carried out using CLC genomics workbench 7.0 (Qiagen Inc.). Comparisons of transcript profiles between pollen and stigma of B carinata and Triticale and the contribution of polyploidy to reproductive gene expression will be presented. The reference transcriptome generated for B carinata and triticale will facilitate the study of other members of the Brassicaceae (e.g. B. napus, B. oleracea, B. rapa) and Triticeae (wheat, barley, rye) which together represent the most cultivated crops in Canada
Zsolt Ponya
Kaposvar University, Hungary
Title: How gamete micromanipulation and cell engineering can contribute to establishing a time scale for performing transcriptomics studies in wheat (Triticum aestivum, L.) during fertilisation and proembryo formation
Time : 10:10-10:30
Biography:
Zsolt Pónya has completed his PhD at the age of 32 years from the Eötvös Lóránd University of Arts and Sciences, Budapest, Hungary, and following obtaining his degree, he has launched his postdoctoral studies at the University of Siena, Italy, followed by a postdoc research fellowship at the Ben-Gurion University of the Negev, Israel. He is currently a senior scientist at the University of Kaposvár, Hungary. He has published a number of papers in reputed international journals and is a member of the editorial board of several prestigious scientific journals
Abstract:
Land plants are sessile organisms; consequently they had to evolve powerful genetic and epigenetic tools and strategies to ward off perils of their surroundings and to render them adaptive to environmental stress. Additionally, they have to be capable of fast tracking the adaptive optima imposed by transient selective forces while permanently tuning their body to cope with the challenges posed by the ever-changing environment. Thus, in terms of genetic plasticity, land plants are by far more flexible organisms as compared to animals as they have much more dynamic and plastic genome. Furthermore, unlike in mammals, in angiosperm plants germline cells are not set aside at an early stage during development so cells partaking of reproduction can be viewed as the depositories of “cell memories” which have accumulated due to survival/adaptation strategies having proven successful in the cells of the mother plant that, through numerous cell divisions in the sporophytic generation, ultimately serves as the source of progenitor cells for the gametes produced during the haploid developmental stage. This very fact is thought to be behind the evolutionary success of land plants and also makes them an attractive system to study genetic plasticity. In this sense angiosperm nucellar cells are particularly interesting as they reveal versatile developmental modalities; they can either use or avoid meiosis without compromising subsequent development. Normally, once the molecular “decision” is made, a highly conserved, modular developmental pathway unfolds producing the female gametophyte (FG) with a haploid egg. Occasionally, the egg cell can go through parthenogenesis which is an extraordinary phenomenon whereby the egg cell initiates cell division without paternal genetic contribution. Although the study of parthenogenesis/apomixis in angiosperms has recently leapt ahead, the cellular and molecular events of the activation of the egg foregoing sperm incorporation remains to be elucidated. To explore whether the cascade events of signal transduction triggered by sperm-egg fusion and leading to egg activation can be elicited in lieu of fertilisation, egg cells of wheat isolated at different stages of in situ development were microinjected with the DefH9-iaaM gene, which had been previously demonstrated to confer auxin synthesis specifically on ovules and derived tissues in other systems as well as induce parthenocarpy in several plant species (tobacco, egg plant, tomato). Furthermore, wheat zygotes were used to study the relationship between cell cycle and morphogenesis, as in the angiosperm zygote cellular differentiation and cell cycle control are closely linked because cytoplasmic reorganisation occurs concomitantly upon fertilisation leading to asymmetrical cell cleavage. By exploiting a protein delivery system facilitating functional studies in living cells, an antibody raised against the 17-amino acids at the C-terminus of a synthetic peptide based on the deduced protein encoded by the cdc2 gene, a key protein in cell cycle regulation isolated previously from wheat, was introduced into in vitro fertilized wheat egg cells at defined times permitting the chronological follow-up of the impact of blocking cdc2 activity on morphogenesis during wheat zygote/proembryo development. The findings of microinjecting DNA encoding Rho GTPases implicated in cellular response to extracellular signals by inducing coordinated changes in the organisation of the actin cytoskeleton and in transcription to drive a wide range of fundamental biological processes such as cell cycle and morphogenesis, will also be presented. In order to ensure the maintenance of in planta observed polarity of in vitro fertilised egg cells, which was found to be lost during in vitro culture, a micromanipulation-based technique enabling re-implantation of fusion products into maternal tissues (ovules) was elaborated. The relevance to stem cell research of our preliminary results of exploratory cell engineering experiments on the creation of half clones/cybrids via fusion of karyoplasts isolated from unfertilised wheat egg cells and cytoplasts stemming from in vitro fertilised female gametes, will be addressed. In practical terms research of this kind may ultimately have ramifications in achieving clonal propagation in agriculturally important plants through seeds hence contributing to efforts aimed at fixing desirable gene combinations/hybrid vigour in crops and to defining time windows for isolating egg cells/zygotes/proembryos for trancriptomics studies in wheat.
Jianbo Wang
Wuhan University, China
Title: Transcriptome and small RNA gene expression changes in synthetic allohexaploids of Brassica
Time : 10:45-11:05
Biography:
Jianbo Wang has completed his PhD from Wuhan University. He is interested in the research on plant molecular evolution and has published more than 30 papers in reputed journals.
Abstract:
Polyploidy played important roles in promoting plant genome evolution through genomic merging and doubling. We compared transcriptome and small RNA expression differences between a synthetic Brassica allohexaploid and its parents using RNA-seq approach. Total of 29260, 29060 and 29697 genes were expressed in B. rapa, B.carinata and the allohexaploid respectively. The majority of the 3184 DEGs between the allohexaploid and B. rapa were involved in photosynthesis, biosynthesis of secondary metabolites and circadian rhythm. For the 2233 DEGs between the hexaploid and B. carinata, many played roles in photosynthesis, plant hormone signal transduction, biosynthesis of secondary metabolites and limonene and pinene degradation. There were more differences between the allohexaploid and its paternal parent than that between it and its maternalparent in gene expression. Many methyltransferase genes and transcription factor genes showed differential expression between the hexaploid and its parents. On the other hand, we detected 613, 784 and 742 miRNAs expressed in B. rapa, B. carinata and the allohexaploid respectively and 618 miRNA genes were differentially expressed between Brassica hexaploid and its parents while 425 miRNA genes showed non-additive expression in the allohexaploid. Moreover, many non-additively expressed miRNAs were repressed in the allohexaploid and there was a bias towards repression of B. rapami RNA genes which is consistent with progenitor biased gene repression in these allopolyploids. Our study represented the comprehensive analysis of functional gene and small RNA gene expressions in allopolyploid which would lead to better understanding of plasticity of allopolyploid genomes.
Lerato BameTsalaemang Matsaunyane
Agricultural Research Council-Vegetable and Ornamental Plant Institute (ARC-VOPI), South Africa
Title: Molecular characterization of the unintended and unexpected effects of genetic engineering on the endogenous Solanum tuberosum genome
Time : 11:05-11:25
Biography:
Lerato Bame Tsalaemang Matsaunyane is an alumni of the University of Free State, and she has done her BSc in Biological Sciences and completed her Master’s degree from University of Pretoria. After completion of her postgraduate studies, she then joined the University of Johannesburg as a Doctorate student. She has presented her research findings from her postgraduate degrees in both national and international conferences and has used her skill to mentor and train students for internships as well as postgraduate qualifications. She is currently a Molecular Biologist at the Agricultural Research Council (ARC) at the Vegetable and Ornamental Plant Institute (South Africa) in the Crop Protection Division.
Abstract:
Genetically modified (GM) crops with diverse traits are currently developed and produced to generate social, economic and environmental benefits focused on amongst others, resource-poor farmers. However, current safety assessments of these GM crops are thought to be biased as they focus on potential hazards posed by the inserted transgene and its products. As a result, a project was initiated to study the unintended effects of the integration of the transgene within the host genome. The model plant for the study was a Malus domestica polygalacturonase inhibiting protein 1 (Mdpgip1) transgenic potato produced for enhanced resistance against Verticillium wilt. Genome walking revealed that the T-DNA containing the transgene was successfully inserted into the potato genome with no non-T-DNA sequences from the binary vector having been inserted into the potato genome. Furthermore, studies indicated the insertion site of the Mdpgip1 transgene as being adjacent to the photosystem QB gene on chromosome 1 of the potato genome. DNA fingerprinting analysis revealed unintended effects that resulted in alterations in the expression genes encoding the PsaC, AFG3, 25S rRNA, tryptophan/tyrosine permease, Ef-Tu domain, SKP1-like 1A, StPGIP1 and XTH proteins in the transgenic. It can thus be concluded that the insertion and expression of the Mdpgip1 transgene in the transgenic potato does not appear to have a major effect on the gene expression levels of the endogenous potato genes and that the transgenic potato is significantly equivalent to its traditional counterpart with a few quantifiable differences as determined by tools used for gene expression analysis.
Ozge Celik
Ä°stanbul Kultur University, Turkey
Title: Differential regulation of antioxidative gene expressions in response to salt stress in rice
Time : 11:25-11:45
Biography:
Özge Çelik has received her PhD degree from Istanbul University (Istanbul, Turkey) in 2010. She is working on mutation breeding, abiotic stress tolerance, molecular marker analysis and plant molecular biology. She is currently an Associate Professor of Molecular Biology and Genetics Department at the Istanbul Kultur University. She has authored more than 35 publications in the fields of plant stress tolerance and molecular biology and has been serving as an editorial board member of repute. She has published one scientific book and a chapter in a reputed book in the field of Plant Science.
Abstract:
Rice (Oryza sativa L.) is one of the most important crops because it is a nutritional source of more than one-third of the world population. Soil salinity is one of the most important abiotic stress factors that affects plant growth and productivity adversely. Rice growth and yield is also affected by salinity and at the seedling stage although it is known to be susceptible to salinity. Reactive oxygen species are induced by salt stress and some responsive mechanisms are evolved against to the detrimental effects caused by salt stress. Salinity response is especially controlled by obtaining homeostasis between antioxidative mechanisms and accumulation of reactive oxygen species (ROS) produced as a result of oxidative stress caused by salinity. It is known that combined expression profiles of antioxidative system enzymes may provide increased tolerance capacity. Therefore, the expression profiles of antioxidative enzymes in two different rice cultivars which have different salt sensitivities at four different salt stress conditions, were determined. Salinity responses of two different rice varieties were investigated at seedling stage. Four different salinity treatments were then applied using Yoshida solution containing 0, 30, 90, 150 and 210 mM NaCl to the nutrient solution for seven days. At the end of the seven days, the leaves were harvested and stored at −20oC for further experiments. The salt stress responsive gene specific primers were amplified by designed primers due to the 3’-UTR regions of each of the following genes by aligning all available related genes in the databases of NCBI and KOME: Mn-SOD, Cu/Zn-SOD, Fe-SOD, Cytosolic APX, Thylakoid-bound APX, stromal APX, Cytosolic GR and CatA. Mn-SOD was consisted with the trend of variation in SOD activities of rice varieties. The expression pattern of CAT A gene was markedly decreased compared to control in both rice varieties. Str-APX gene expression was up-regulated during salt stress treatments in both rice varieties. Transcript levels of Cyt-APX and Thy-APX were up-regulated in accordance with increasing salt stress in Osmancık-97 variety. The expression pattern of gene encoding enzyme Cyt-GR1 showed a gradual up-regulation as a response to subjected increasing NaCl stress in Mevlutbey variety while only after 90 mM treatment, an upregulation was observed for Osmancık variety. These data indicated that the antioxidative responses of salt tolerant and salt sensitive rice varieties are differentially regulated.
Monika Bansal
Lovely Professsional University, India
Title: Isolation and functional characterization of cold stress inducible promoter CAT01 from tomato
Time : 11:45-12:05
Biography:
Monika Bansal completed her PhD on the topic “Genetic transformation of Tomato for introduction of salinity tolerance. She worked as Senior Research Fellow at Haryana Agriculture University (June 2003-Dec 2003). Later she completed her Post-Doctoral fellow in the UGC funded project “Isolation and characterization of stress Inducible Promoter from Tomato" at Delhi University South Campus with Dr. Arun Sharma (Aug 2008 –Aug2011). She joined as Assistant Professor at Asian Institution Affiliated to Punjab University, Patiala during August 2011-July 2013). From August 2014 till date she is working as Assistant Professor at School of Agriculture, Lovely Professional University.
Abstract:
Environmental stress such as drought, salinity and extreme temperatures are the major factors limiting plant growth and productivity. Cold stress leads to diverse change in transcription and translation of genes involved in developmental and physiological metabolism of plants. Constitutive over expression of transgenes imparting stress tolerance may hamper plant growth and productivity. It is desirable to produce transgenic plants that accumulate transgenic products only under stress conditions. To identify stress related transcripts we had utilized cold subtracted cDNA library to monitor gene expression changes after 24 hours of cold stress in tomato seedlings. Relative mRNA levels of cold stress associated gene CAT01 (NAC type transcription factor) from cold treated sample was compared with wild types. Sequence upstream of start codon from this transcript was selected to design the primers, promoter sequence was fused with GUS reporter gene and the recombinant transgenes was introduced into tomato cv. Pusa Ruby with Agrobacterium tumefaciens mediated genetic transformation. Histo-chemical and florometric analyses revealed promoter is inducible under all abioticstress conditions. This promoter is effective and desirable for deriving a low constitutive transgene expression under normal condition and high induction in response to cold, salt and drought stress. This method shall be advantageous as it will not have a major impact on plant biomass and yield.
Closing Ceremony
Hetalkumar J Panchal
Navsari Agricultural University, India
Title: De novo RNA seq assembly and annotation of important legume-Vicia sativa L. (SRR403901)
Biography:
Hetalkumar J Panchal is currently working as an Associate Professor at Gujarat Agricultural Biotechnology Institute, Navsari Agricultural University (NAU), Surat, Gujarat (India). He has published more than 35 papers in national/international Journals and more than 30 papers in national and international conferences & seminars. He worked as Faculty of Bioinformatics (10 years) at MSc (Bioinformatics) course at Department of Computer Science, Sardar Patel University, Vallabh Vidyanagar, Gujarat (India). He is guiding 3 PhD students and has been amentor of the more than 25 dissertation thesis. He has delivered so many keynote lectures /expert lectures/invited talks. He serves as a visiting faculty/external examiner at various universities. He is associated with so many national and international journals as member of editorial board and as reviewer. He has received Best Paper Award in faculty category at National Conference on “Advanced Data Computing Communications & Security”. He is a member of Indian Science Congress Association (ISCA) and a Life Member of Computer Society of India.
Abstract:
Vicia sativa L. which is also known as common vetch; is a nitrogen fixing leguminous plant in the family Fabaceae. This study is focus on RNA-seq of Vicia sativa L. of SRR403901 from NCBI database for de novo Transcriptome analysis. A total of 12.4 million single reads were generated with N50 of 588 bp. Sequence assembly contained total 22748 contigs which is further search with known proteins, a total of 7652 genes were identified. Among these, only 500 unigenes were annotated with 18761 gene ontology (GO) functional categories and sequences mapped to 122 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). These data will be useful for gene discovery and functional studies and the large number of transcripts reported in the current study will serve as a valuable genetic resource of the Vicia sativa L.
- Track 2: RNA Editing and RNA Interference: Interplay, Track 3: Next Generation Sequencing (NGS) Technologies, Track 4: Disease Interrogation and Applications of RNA-Seq
Location: Continental Ball Room 5 & 7
Chair
Momiao Xiong
University of Texas Health Science Centre, USA
Co-Chair
Yongming Sang
Kansas State University, USA
Session Introduction
Momiao Xiong
University of Texas Health Science Centre, USA
Title: Integrative image and RNA-Seq data analysis
Time : 10:35-10:55
Biography:
Momiao Xiong, Ph. D, Professor, Division of Biostatistics and Human Genetics Center, School of Public Health, the University of Texas Health Science Center at Houston . He obtained his PhD degree from the Department of Statistics at the University of Georgia in 1993 and finished his postdoctoral training in computational biology from the University of Southern California in 1995. His research is in the area of statistical genetics, bioinformatics, machine learning and image analysis.
Abstract:
Emerging integrative analysis of increasingly larger and complex genomic and anatomical imaging data which has not been well developed, provides invaluable information for the holistic discovery of the genomic structure of disease and has the potential to open a new avenue for discovering novel genetic influence on the imaging variation and disease which cannot be identified if they are analyzed separately. Both imaging and genomics generate a huge amount of data that present critical bottleneck in the interactive analysis. A key issue to the success of imaging and genomic data analysis is how to reduce dimensions of both imaging and genomic data. Most previous investigated methods for imaging information extraction and RNA-seq data reduction do not explore imaging spatial information and often ignore gene expression variation at genomic positional level. To overcome these limitations, we extend one dimensional principle component analysis to two dimensional principle component analyses (2DFPCA) and use 2DFPCA scores to represent image data. Unlike the widely used linear regression for modeling association of microarray gene expressions with phenotypes which quantifies the expression level of a gene/transcript by a single number, we develop a multiple functional linear model (MFLM) in which functional principal scores of images are taken as multiple quantitative traits and RNA-seq profile across a gene is taken as a function predictor for assessing the association of gene expression with imaging signals which can take gene splicing and expression variation at genomic positional level into account. The developed method has been applied to image and RNA-seq data of ovarian cancer and KIRC studies. We Identified 24 and 84 genes whose expressions were associated with imaging variations in ovarian cancer and KIRC studies, respectively. Our results showed that many significantly associated genes with images were not differentially expressed, revealed their morphological and metabolic functions. The results also demonstrated that the peaks of the estimated regression coefficient function in the MFLM often allowed to discover splicing sites and multiple isoform of gene expressions.
Atsushi Shimizu
Iwate Medical University, Japan
Title: Reduction of systematic bias in transcriptome data from human peripheral blood mononuclear cells for transportation and biobanking
Time : 10:55-11:15
Biography:
Atsushi Shimizu graduated from Aoyama Gakuin University, Tokyo, Japan and obtained PhD degree in science from Aoyama Gakuin University in 1999. He then worked as a post-doc with Professor Nobuyoshi Shimizu at Keio University School of Medicine, Tokyo, Japan. He was an Associate Professor at the Keio University School of Medicine. He is now a Principal Investigator at Iwate Medical University. Over the last two years, he has developed a research project with his group focusing on the large-scale genome cohort study aiming to characterize the genome, epigenome and transcriptome analysis.
Abstract:
Transportation of samples is essential for large-scale biobank projects. However, RNA degradation during pre-analytical operations prior to transportation can cause systematic bias in transcriptome data, which may prevent subsequent biomarker identification. In this study, we examined the effectiveness of RNA-stabilizing reagents to prevent RNA degradation during pre-analytical operations with an emphasis on RNA from PBMCs to establish a protocol for reducing systematic bias. To this end, we obtained PBMCs from 11 healthy volunteers and analyzed the purity, yield, and integrity of extracted RNA after performing pre-analytical operations for freezing PBMCs at -80°C. We selected 7 samples from 11 healthy volunteers, and systematic bias in expression levels was examined by RNA-Seq experiments and data analysis. Our data demonstrated that omission of stabilizing reagents significantly lowered RNA integrity, suggesting substantial degradation of RNA molecules due to pre-analytical freezing. RNA-Seq for 25,223 transcripts suggested that about 40% of transcripts were systematically biased. These results indicated that appropriate reduction in systematic bias is essential in protocols for collection of RNA from PBMCs for large-scale biobank projects. Among the seven commercially available stabilizing reagents examined in this study, RNA-Seq experiments consistently suggested that RNALock, RNA/DNA Stabilization Reagent for Blood and Bone Marrow, and 1-Thioglycerol/Homogenization solution could reduce systematic bias. On the basis of the results of this study, we established a protocol to reduce systematic bias in the expression levels of RNA transcripts isolated from PBMCs. We believe that these data provide a novel methodology for collection of high-quality RNA from PBMCs for biobank researchers
Yongming Sang
Kansas State University, USA
Title: Genome-wide transcriptomes integrate animal intra- and inter-organism processes and evoke the ecoimmunological concept in animal health
Time : 11:15-11:35
Biography:
Yongming Sang has completed his DSc and PhD at Nanjing Agricultural University and Kansas State University, respectively. He is a principal investigator and an Assistant Professor currently at Kansas State University. He has published 40 papers in reputed journals and has been serving as a reviewer of multiple journals and grant panel of repute. Main research efforts in his lab focus on immunometabolic regulation underlying animal viral and obesity diseases
Abstract:
Whereas classic immunology deals with animal health primarily in the immune system of a laboratory-based animal species, the emerging discipline of ecoimmunology seeks to understand bona fide immune reactions integrating into intra-organismic systems and interacting with ecological and even evolutionary processes. In this context, non-bias transcriptomic analyses, such as RNA-Seq, provide genome-wide gene expression profiles underlying versatile interaction of the immune system with other systems at intra- and inter-organismic levels. For example, our transcriptomic analysis of porcine macrophages at different activation statuses in response to a viral infection, not only revealed differential expression of multiple immune gene families but also highlighted the involvement of lipid metabolism and even circadian rhythm genes linking to neuroimmune regulation. In addition, our RNA-Seq data in ileal samples of obese rats induced using high-fat diet plus an adenoviral infection showed that not only genes in lipid metabolic pathways but genes in endocrine and immune systems were significantly differentially regulated. Moreover, gene ontology (GO) analysis of the rat ileal RNA-Seq data also revealed that signaling pathways involved in multi-organism interaction were significantly altered in the obese animals, indicating that inter-organism processes (such as gut microbiota alteration) provoke the development of obesity. In sum, as multifaceted interactions centered on immune responses may be intuitive and highlighted in other related studies in animal health, genome-wide transcriptomic analyses provide a non-bias quantifying approach for studying multi-system integration, which may elicit mechanistic studies and therapeutic strategies pertinent to the discipline of ecoimmunology.
Bingsong Zheng
Zhejiang A & F University, China
Title: Identification by deep sequencing and profiling of conserved and novel hickory microRNAs involved in the graft process
Time : 11:35-11:55
Biography:
Bingsong Zheng has completed his PhD in 2003 from Zhejiang University and Postdoctoral studies from INRA. He is the Vice-Dean of School of Forestry & Biotechnology, Zhejiang A & F University. He has published more than 30 papers in reputed journals and has been serving as an Editorial Board Member of repute.
Abstract:
MicroRNAs (miRNAs) play a vital role in plant development and growth through negative regulation of post-transcriptional gene expression. Carya cathayensis (hickory) is an important species for dried nuts and oil in China with high nutritional and economic value. The graft technique is an important strategy for hickory cultivation. To understand the role of miRNAs involved in the hickory graft process, we constructed three small ribonucleic acid (RNA) libraries from hickory rootstock (2 years old) and scion (1 year old) at 0, 7 and 14 days postgrafting. Sequence analysis of the three libraries identified 21 conserved miRNAs belonging to 13 families and 10 novel and 8 potentially novel miRNAs belonging to 15 families. Among these miRNAs, 12 miRNAs were differentially expressed during the graft process in hickory and two-thirds were downregulated. Quantitative real-time polymerase chain reaction (qRT-PCR) validated that 14 miRNAs and their expression trends were similar to the results obtained by Solexa sequencing. Further, a total of 89 target genes for conserved and 26 target genes for novel miRNAs were predicted. This study will help in understanding the roles and regulatory modes of miRNAs involvement in the hickory graft process.
Michael Bonin
IMGM Laboratories GmbH, Germany
Title: RNA Sequencing in degraded tumor RNA samples: Two natural enemies finally reconciled
Time : 11:55-12:15
Biography:
Michael Bonin has completed his PhD in Genetics at the Kassel University. From 2001 to 2014 he served as Head of the Microarray Core Facility located at the Institute of Medical Genetics and Applied Genomics, Medical Faculty, University Tuebingen. Since August 2014 Dr. Bonin is the managing director of IMGM Laboratories, a leading genomic service provider in Europe. He has published more than 120 papers in reputed journals with an overall impact factor of more than 600.
Abstract:
Carved in stone, painted on canvas or scratched in vinyl expressions of human thoughts and feelings have been conserved over generations and been ever since an easily accessible treasure of knowledge and inspiration. When it comes to expression of genes conserved in FFPE or degraded RNA samples, things are different. Given the degradation of RNA due to fixation or other circumstances to shreds and pieces, the scientific treasure of potential insights and discoveries is everything but easy to unearth. Unfortunately, classic approaches often suffer from high sample amount input requirements and highly variable results. These hamper immensely the effective gene expression analysis of challenging FFPE or degraded but nevertheless precious and irreplaceable RNA samples. In the current study strongly degraded total RNA samples from stomach endoscopies of patients with gastric carcinomas should be analyzed for gene expression changes between tumor regions and normal tissue regions. To overcome the described problems, we established a new RNA Sequencing Service workflow. The library preparation with the Illumina TruSeq RNA Access kit served as a key step especially addressing low-quality RNA samples and requiring input amounts of as little as 20-100 ng. RNA was evaluated for usability by the newly established Illumina DV200 quality parameter. The subsequent stranded RNA Access library preparation included a sequence-specific enrichment technology. This enables reproducible transcript capture not compromised by poly-A bias using >4,25000 probes, interrogating >2,10000 targets of 21,415 genes of hg19 and thus covering 98.3% of RefSeq. By this approach, variability was diminished while required sequencing depth was greatly reduced. The IMGM´s FFPE RNA Sequencing Service was expanded by 2×75 bp paired-end sequencing on the NextSeq500 platform and a comprehensive data quality control and analysis on the CLC Bio Genomic Workbench. Sequencing resulted in a high amount of high quality paired end reads and a very high portion could be mapped to the human genome regardless the level of degradation of the underlying RNA samples. The comparison of the resulting data with data from two of the samples which underwent a classical mRNA library preparation protocol showed that the used TruSeq RNA Access protocol enhanced the amount of mappable reads and diminished sequencing of intergenic regions. Thereby paired-end reads could be concentrated on meaningful gene regions in high sequencing depth finally enhancing the focus for detection of significant differential expression levels and fusion genes. Thus, the used approach of the IMGM´s FFPE RNA Sequencing Service powered by the Illumina TruSeq RNA Access kit serves as an efficient step towards gaining so far hidden transcriptomic insights in difficult to access RNA samples.
Glen M Borchert
University of South Alabama, USA
Title: RNA Editing Alters MicroRNA Targeting in Human Breast Cancer
Time : 12:15-12:35
Biography:
Glen M Borchert has completed his PhD in Genetics from University of Iowa in the year 2006. He did his first postdoctorate in Structural Biology from University of California in the year 2008 and the second one in Immunology from Illinois State University in the year 2012. After which he joined as Assistant Professor in University of South Alabama and currently working as Assistant Professor, Pharmacology in USA College of Medicine. He has been honored by NIH with a Research award to study hypoxia induced mutation. Also, National Science Foundation (NSF) gave him the CAREER award of $533,000 to research miRNA targeting
Abstract:
RNA editing by RNA specific adenosine deaminase (ADAR) is increasingly being found to alter microRNA (miR) regulations. Editing of miR transcripts can affect their processing as well as what mRNAs they target. Further, editing of target mRNAs can also affect their complementarily to miRs. Notably, ADAR editing is often increased in malignancy with the effect of these RNA changes largely unclear. In addition, reports have also identified many miRs to be differentially expressed in cancer though the majority of their targets are also undefined. Here we propose that modulating the targets of miRs via mRNA editing can play a direct role in the pathology of many carcinomas. In order to more accurately characterize the relationship between these two regulatory processes this study examined RNA editing events within mRNAs sequences of two breast cancer cell lines (MCF-7 and MDA-MB-231) and determined whether or not these edits modulate miR associations. Computational analyses of RNA-Seq data from the two cell lines identified over 250,000 edit sites within mRNAs, many of which were located in 3’ UTR regions. When these locations were screened against the list of currently annotated miRs we discovered that editing caused a subset (~5%) to have significant alterations to mRNA complementarity. One miR in particular, miR-140-3p has been shown to be abhorrently expressed in many breast cancers. Interestingly, we found that mRNA editing made this specific miR able to target the apoptosis inducing gene DFFA in MCF-7 but not in MDA-MB-231 cells. As these two cell lines are known to have distinct characteristics in terms of morphology, invasiveness and physiological responses, it is feasible that RNA editing could contribute to the phenotypic differences observed between the two cell lines and help explain why an increased incidence of ADAR activity is detected in a number of malignancies. Broadly, these results suggest that the creation of miR targets may be an underappreciated function of ADAR and may help further elucidate the role of RNA editing in tumor pathogenicity.
Sang Ming WANG
University of Nebraska Medical Center
USA
Title: Genome-scale mapping promoter variation through exome sequencing
Time : 12:35-12:55
Biography:
San Ming Wang finished his Master of Medicine during September, 1983—July, 1986 from Shandong Medical University, Jinan, China. He pursued Doctor of Medicine December, during the year December 1989—June, 1994 at Genetic Unit, Swiss Institute for Experimental Cancer Research (ISREC) / University of Lausanne, Switzerland. He worked as Assistant Professor during January, 2004–October, 2009 at Northwestern University. Later he got appointed as Director for Center for Functional Genomics, ENH Research Institute (Now named NorthShore University HealthSystem Research Institute). From October, 2010 – till date, he is working as an Associate Professor at Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Center, Nebraska.
Abstract:
Promoter plays essential roles in regulating gene expression. Alteration in promoter sequences can change gene expression, leading to profound impact in evolution, physiology, and disease. Comprehensive analysis of promoter sequences is critical to understand gene expression regulation under biological and pathological conditions. Here we report the development of a method named EPA (Exome-based Promoter Analysis) for the purpose. The method takes the advantage of the random fragmentation used in exome sequencing to obtain the promoter-exon containing sequences. A unique feature of the method is that it preserves the original contents in promoters for sequencing, therefore, allows de novo detection of the changes in promoters without a priori knowledge. This overcomes the weakness in hybridization-based methods as they rely on the normal promoter sequences to design probes. Our evaluation of the method in an exome data set generated from human CD4+ T helper cells shows that the method provides high sensitivity to detect most of the gene promoters, and high specificity to detect the common, rare and novel variants in the detected promoters, TF binding motifs, and TATA boxes. EPA method provides a simple but effective means for genome-scale promoter study.
- Track 7: Epigenetics, Track 11: Clinical Applications and Related Disorders
Location: Continental Ball Room 5 & 7
Chair
Max H Garzon
The University of Memphis, USA
Co-Chair
Yingwei Mao
Pennsylvania State University, USA
Session Introduction
Yingwei Mao
Pennsylvania State University, USA
Title: A critical role of an exon junction complex (EJC) factor in regulation of embryonic neurodevelopment and implications in neurodevelopmental disorders
Time : 09:00-09:20
Biography:
Yingwei Mao has completed his PhD at University of Michigan School of Medicine and postdoctoral studies from MIT Picower Institute of Learning and Memory. He is the assistant professor of Penn State University. His lab studies molecular mechanism of neurodevelopment and the relationship to psychiatric disorders. He has published more than 25 papers in reputed.
Abstract:
Nonsense-mediated mRNA decay (NMD) is an RNA surveillance mechanism that degrade mRNAs carrying premature termination codons (PTCs). This mechanism requires several key EJC factors to distinguish PTC from the normal stop codon. But how this mechanism modulates the embryonic neurodevelopment and behaviors is largely unknown In this study, we demonstrated that RBM8a plays a key role in neural progenitor proliferation and differentiation. First, RBM8a is highly expressed in the subventricular zone of early embryonic cortex, suggesting that RBM8a may play a role in regulating neural progenitor cells (NPCs). To test this hypothesis, we used in utero electroporation to overexpress or knock down RBM8a in mouse brain at E14. RBM8a stimulates embryonic neural progenitor proliferation and suppresses neuronal differentiation, indicating that RBM8a positively regulates NPC proliferation. Conversely, knockdown of RBM8a in the neocortex reduces NPC proliferation and promote premature neuronal differentiation. Consistently, Nes-cre; RBM8afl/+ mice show severe developmental defects including microcephaly and postnatal lethality. When RBM8a is overexpressed in the dentate gyrus of adult brain using stereotactic viral injection, mice showed altered anxiety and depressive-like behaviors. To uncover the underlying mechanisms, genome-wide RNAseq identifies potential substrates of RBM8a in the brain, which have been implicated in neurogenesis and plasticity. Interestingly, autism- and schizophrenia-risk genes are highly representative in RBM8a-associate transcripts. Taken together, we identify a novel role of RBM8a in regulation of neurodevelopment and behaviors. Our studies provide a deeper insight on causes of mental illnesses and will facilitate the development of new therapeutic strategies for neurodevelopmental illnesses.
Zsolt Boldogkoi
University of Szeged, Hungary
Title: Transcription Interference Networks examined by Single Molecule Long Read Sequencing Technology
Time : 09:20-09:40
Biography:
Zsolt BoldogkÅ‘i received his PhD degree (1999) in molecular biology from Szent Istvan University at GödöllÅ‘. He also worked at the Whistar Institute (Philadelphia, PA, USA) as a PhD student then had post-doctoral training at University of Bonn. He received his DSc degree (2008) at University of Szeged. His primary field of interest is the molecular biology of herpes viruses with special emphasis on the regulation of gene expression analysis and utilization of herpesviruses as tools in various fields of biology including neurobiology and cardiology. He and his group currently also work in different fields of genomics, focusing on human diseases. He has published more than 70 papers in reputed journals. Currently, Zsolt BoldogkÅ‘i is the head of Department of Medical Biology at Faculty of Medicine of University of Szeged.
Abstract:
Introduction
Gene expression is mainly controlled at the level of transcription initiation through the interaction between cis- and trans-acting regulatory elements and epigenetic processes. The Transcriptional Interference Network (TIN) hypothesis assumes the existence of a new layer of gene regulation, based on the confrontations between the transcriptional machineries of functionally linked genes located in the same cluster. Pseudorabies virus (PRV) is a neurotropic herpesvirus of swine, which – due to the characteristic genomic features – is a perfect model organism for the study of TIN. PRV is a compact, double-stranded DNA virus; the viral genome is relatively large (~ 143 kb), and the known 70 protein coding genes and the non protein coding transcripts are located in – convergent, divergent and nested - clusters.
Methods
In this study we have used a third generation single molecule sequencer (Pacific Biosciences RS II) to analyze and characterize the PRV transcriptome and to find evidences for the existence of TIN. Immortalized porcine kidney 15 (PK-15) cells were infected with PRV. Cells were harvested after 1, 2, 4, 6, 8 and 12 hours after infection. Total RNA was isolated and converted to cDNAs, followed by PacBio SMRTBell Library preparation and sequencing.
Results
Transcriptional read-through was found between the convergently oriented genes (gene clusters). The read-through efficiency and its orientation shows time dependence. Our data show inverse expression dynamics between the convergent clusters. Previously unknown interactions were found between the nested clusters as well as between the divergently oriented genes. Temporal changes of these gene interactions were also characterized. New long non-coding and overlapping RNAs were found which might give rise to transcription interference and serve as an added layer of regulation
Krzysztof Wieczerzak
University Rostock, Germany
Title: A comparative transcriptome provides candidate genes for determination the cause of males infertility.
Time : 09:40-10:00
Biography:
Krzysztof Wieczerzak finished his M.Sc. in Department of Genetics at Jagiellonian University in Kraków, Poland. Then he worked as young scientist in Institute of Pharmacology, Polish Academy of Science. Next, he has studied and completed his PhD from Institute of Human Genetics at Georg-August-University in Göttingen, Germany. He is interested in molecular aspects of reproduction biology, gene therapy and cancer biology. Now he is working as a post-doc in Institute of Experimental Gene Therapy and Cancer Research at Rostock University, Germany. He is working with CRISPR-Cas9 system in cancer aspects
Abstract:
Since most cases of male infertility remain idiopathic, analysis of the mechanism in which genes are involved in spermatogenesis control might help to better understand the reasons. During years we tested several knockout mouse line with disruption of genes important for human fertility. Now, we have analyzed the phenotype and determined the cause of males infertility from a multiple knockout mouse line with knockouted six germ cell-specific genes. We observed in about 20% of mice infertility, but in both, fertile and infertile males, increased number of spermatozoa with abnormal head and also reduction of sperm motility were found. In order to examine more thoroughly which genes might be involved in the infertility of males, we have performed transcriptome assay and compared the expression of genes in the testes of fertile and infertile animals. We identified 55 candidates genes and analyzed a new gene 4933400A11Rik. In wild type mice, 4933400A11Rik is expressed in germ cells, detected from 16dpp, when primary spermatocytes are produced. 4933400A11RIK has sequence similar to the actin filament capping protein family, which control the process of cytoskeleton growth by the regulation of actin filaments reorganization. The 4933400A11RIK colocalises with Factin capping protein subunit alpha-1. Dysregulation of germ cells actin cytoskeleton reorganization may be the underlying cause of male infertility in 6xKO mice, because 4933400A11Rik gene is expressed in testis of fertile, but not present in infertile males. In conclusion, the data obtained from comparative transcriptome provides candidate genes for analysis the cause of human males infertility.
Biography:
Dr. Yunlong Qin works at Morehouse School of Medicine, Cancer Biology Program as a senior researcher.
Abstract:
Aging in humans is irreversible, characterized by a decline of physiological functions in various organs and tissues, leading to multidimensional process of physical, psychological, and social change, causing most human diseases and an increasing probability of death. At present, the exact mechanism and biological basis of aging is unknown. The antagonistic pleiotropy theory and competing model of senescence explain the aging occurrence of some physiological functions of human body from different angles. The single nucleotide polymorphisms (SNPs) of genome suggest the individual’s difference of aging process is associated with SNPs-related individual’s sensitivity to diseases. As the naturally occurring DNA damages and ageassociated accumulation of DNA damage as well as decline in gene expression regulation indicate individual life expectancy, whereas the shortening mechanism of telomeric terminals of chromosomes and Hayflick limit of cell division determine human life span. The genomic aging exerts impact on various physiological functions, also imprints individual’s appearance, such as wrinkled, thinning skin, graying and loss of hair. Feeling helpless before slowing down senescence of the genetic materials people turn to anti-aging products, spending billions of dollars each year on such creams and lotions for decreasing signs of aging and nutrients for postponing decline of physiological functions. Indeed, recently, scientists carried out reversing muscle aging in mice model through feeding an experimental compound and made a comment that for humans the effect would be similar to a 60-year-old feeling like a 20-year-old, but still powerless for reversing of genomic aging, even a little Is there a possibility of U-turn in the highway of life progress? Recently, the discovery that mature cells can be reprogrammed to become pluripotent and the development of rehabilitative and regenerative medicine rekindle spark in hope of humans against aging. The induced pluripotent stem cells (iPSCs) hold great promise in the field of regenerative medicine because each individual could have their own pluripotent stem cell line and these unlimited supplies of autologous cells bypass body’s immune defense system, immune rejection, this will be a new milestone in transplantation medicine, one of the most challenging and complex areas of modern medicine. Nowadays, iPSCs are mainly used in medical research, including organ synthesis, disease modeling of cellular basis and drug development, tissue repair in animal model. Here is an opinion regarding the possibility and feasibility of iPSCs application in gerontology. The gerontology is the study of the social, psychological and biological aspects of aging, itself is a youthful field, its progress having been slowed by aging's daunting complexity and a tendency among scientists and doctors to consider diseases as entirely separate, rather than as manifestations of a common origin. In comparison to the complexity and difficulty of synthesizing other organs or systems through iPSCs, there is great advantage for iPSCs being applied into the immune system, the functions of which are mainly carried out by immune cells, same as red blood cells, these immune cells travel to everywhere of human body. The application of cell levels of iPSCs is more reliable and easier in technology. The aging process accompanies gradual deterioration of the immune system. This age-associated immune deficiency is ubiquitous, decreasing host’s capacity to respond to infections, increasing individual’s sensitivity to most human diseases, including autoimmune disorders. The functional capacity of T-cells is most influenced by the effects of aging. Since all the blood cells come from pluripotent hematopoietic stem cells (HSCs) of bone marrow, no matter what age you are, but these freshly produced immune cells from aging body are still senescent. The raised question is when is best time period to collect adult cells to be reprogrammed genetically into iPSCs? Here is a suggestion with the best timing for preparing the individual specific iPSCs should be a period of time of strongest function of immune system, thinking roughly 30 years of age. Comparing to the cord blood stem cells, unlimited supplies of iPSCs will have more attractive application prospect once its safety is more reliable and accepted and more mature in technology. There is an expectation to help elderly people re-establish a strong immune system to resist the attack of pathogens including bacteria and viruses and make them live in a higher level of health state. Interestingly, recently, type O red blood cells were synthesized at the Scottish National Blood Transfusion Service from iPSCs. Type O can be transfused into all patients, this is unprecedented brilliance in the transfusion history considering more than 100 million blood donations are collected globally every year. Where do we go on the journey against aging? It seems we see some light we could reach from the rapid progress of life sciences and biotechnology today. Hopefully, it is not star light.
- Workshop: Impact of variation in regulatory region on gene expression
Session Introduction
San Ming Wang
University of Nebraska Medical Center, USA
Title: Impact of variation in regulatory region on gene expression
Time : 10:00-10:45
Biography:
San Ming Wang finished his Master of Medicine 1986 from Shandong Medical University, Jinan, China. He pursued Doctor of Medicine at Genetic Unit, Swiss Institute for Experimental Cancer Research (ISREC)/University of Lausanne, Switzerland. He worked as Assistant Professor from 2004-2009 at Northwestern University. Later he got appointed as Director for Center for Functional Genomics, ENH Research Institute (Now named NorthShore University Health System Research Institute). From October, 2010–till date, he is working as an Associate Professor at Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Center, Nebraska.
Abstract:
Promoter is the regulatory part of a gene. In promoters, the interactions between cis-regulatory elements, e.g., transcription-factor binding sites (TFBS), and trans-regulatory elements, e.g., transcription factors (TFs), spatially and temporally regulate gene expression to ensure homeostasis of biological functions. Alterations in promoter sequences can change cis-trans interactions resulting in modified gene expression, impacting downstream biological processes, and leading to the development of disease. In this workshop, I will give an introduction on the topic, show the data from my breast cancer studies, and then have a round table discussion/presentation for attendees to address the issue covering normal gene expression regulation, evolution conservation, alternation in disease, etc.
- Track 14: Transcriptomics and Proteomics of Microorganisms
Chair
Subha Bhassu
University of Malaya, Malaysia
Co-Chair
Ilana Kolodkin
Weizmann Institute of Science, Israel
Session Introduction
Claire Lemaire
National Center for Scientific Research, France
Title: Quantitative analysis of the mitochondrial proteome and phosphoproteome in the yeast Saccharomyces cerevisiae
Time : 11:00-11:20
Biography:
Claire Lemaire is biochemist specialized in membrane proteins. She began her career in the photosynthesis field on the assembly and regulation of photosynthetic complexes (I.B.P.C. ,Paris). She then joined the C.N.R.S. (French National Center for Scientific Research) where she acquired a solid expertise in the respiratory complexes. Over the last six years, she has developed a research project with her group focusing on the regulation of OXPHOS complexes by phosphorylation.
Abstract:
The mitochondrion is an organelle with multiple functions, the most important of these being to provide energy to the cell as a whole in the form of ATP, generated by oxidative phosphorylation catalyzed by the respiratory enzymes in the mitochondrial inner membrane. In humans, deregulation of mitochondrial functions, particularly with regard to the respiratory chain, is associated with several pathologies, including neurodegenerative diseases, neuromuscular diseases, type II diabetes and cancer. The activity of the respiratory enzymes may be modulated in response to metabolic demand and various types of stress. Several levels of regulation may be conceived, including changes in protein expression magnitudes, reversible interaction with effectors, and post-translational modifications, such as phosphorylation. The steadily increasing number of identified mitochondrial phosphoproteins, kinases and phosphatases suggests that reversible protein phosphorylation could be an important level of regulation in mitochondria. However, this hypothesis cannot be tested without quantitative data on variations in the abundance of mitochondrial proteins and their level of phosphorylation under different growth conditions. The yeast Saccharomyces cerevisiae is a powerful tool for studying various energetic and physiological states as it is a facultative aerobe which can grow on either fermentative or respiratory substrates. We present for the first time a quantitative study of both protein abundance and phosphorylation levels in isolated yeast mitochondria under respiratory and fermentative conditions. To focus our analysis specifically on mitochondrial proteins, we performed a subcellular fractionation and used LC–MS/MS to overcome the limitations of 2D gel electrophoresis. Protein abundances were quantified using a label-free method. The phosphoproteome was analyzed quantitatively using the multiplex stable isotope dimethyl labeling procedure. For all quantified phosphopeptides, protein abundance was determined, allowing normalization of the data and permitting analysis of the specific variation of phosphorylation status independent of changes in protein abundance. This study provided reliable information on how the yeast mitochondrial proteome and phosphoproteome adapt to different carbon sources.
Ilana Kolodkin-Gal
Weizmann Institute of Science, Israel
Title: Maintaining motile cells inside the biofilm through cell-to-cell signaling, transcription regulation and evolution
Time : 11:20-11:40
Biography:
Ilana Kolodkin Gal completed her PhD in the year 2009 form Department of Molecular Biology, The Hebrew University of Jerusalem, Israel. During 2009-2012 she was a post-doctoral research fellow under supervision of Prof. Richard Losick, department of molecular and cellular biology, Harvard and co-mentored by Prof. Kolter, department of microbiology and molecular genetics, Harvard Medical School, USA. Since 2012 till date, she is working as Senior Researcher, department of molecular genetics, Weizmann institute of Science, Israel.
Abstract:
Bacteria in nature are usually found in complex multicellular structures, termed biofilms, which protect them from numerous environmental threats. The biofilm structure is the end result of collective behavior of different cell types in a coordinated fashion, achieved by the accurate regulation of dedicated genetic programs. So far, it was well established that in the single-cell level once a bacterial cell commits to the biofilm state it represses motility both transcriptionally and post-translationally. However, at the population level, we found a motile cell sub-population that is tightly retained inside the biofilm of Bacillus subtilis, enabling fast occupation of new niches, as well as invasion of neighboring colonies of foreign bacterial species.This dogma-breaking phenomenon depends on a unique signal originating in the extracellular matrix (ECM) that surrounds the cells in the biofilm, maintaining a motile cell sub-population. This ECM-based signal acts to induce motility, as mutants deficient in ECM production show lower numbers of motile cells. When the ECM extracted from a wild type biofilm was externally added to the ECM mutants, motility level was restored. In particular, TasA, the proteinaceous component of B. subtilis biofilm, appears to be a specific chemical signal that strongly induces bacterial motility. Using evolutionary and genomics approaches we found striking evidence for the importance of maintaining motility in the biofilm. During the maturation of tasA mutant colonies hyper-motile suppressors appeared in high frequency. We suggest that in the absence of ECM-based induction of motility, a strong selective pressure acts to reinstate motility in the biofilm. We are currently using RNA-seq to compare between wild type and ECM mutants transcriptomes in order to identify the different genetic programs that are activated by ECM-derived signaling genome-wide.
Subha Bhassu
University of Malaya, Malaysia
Title: Transcriptomic and proteomic profiling of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in giant freshwater prawns
Time : 11:40-12:00
Biography:
Subha Bhassu completed her graduation BSc (Biomedical Sciences) in the year 1996 from University Kebangsaan Malaysia, Kualalumpur. Later she did her PhD in Molecular Biology from University Putra Malaysia. In the year 2010 she got appointed as CEBAR Biosafety Committee (CBC) Member.
Abstract:
Prawn shrimp aquaculture which captured billions of dollars often affected by viral and bacterial diseases which have led to new technologies such as expression and proteomic profiling using Next Generation Sequencing and 2D proteomic being used as an empowerment measures to tackle these problems in a more rapid, cost and effective way. With advancement of these technologies, will next generation sequencing and proteomic profiling be sufficient to explain the actual physiological changes in prawns that are affected from the Infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single stranded DNA virus which is found in host nucleus as membrane bound phagosomes that has cause slow growth in freshwater prawns, deformities in Peneaus monodon and death in Litopeneaus varnamei. This is the first presentation of the pond bred freshwater prawns that survived the disease outbreak and their bio-physiological changes were captured using Next Generation Sequencing and proteomic profiling. Using muscles of survived and and death prawn muscle transciptomes, there are five major pathways that are affected and showed significant differential expressed genes which are 312 genes in cellular macromolecular machines, 398 in motor neuronal related disease, 36 genes in antigen processing and presentations, 72 genes in viral myocardiatic viral diseases and 100 genes in muscle contraction and oxidative phosphorylation pathways. Protein profiling revealed that ten proteins were significantly up-regulated whereas ten other new proteins were significantly down regulated. The up-regulated protein plays an important role in innate immune system response to infection of M. rosenbergii whilst the down regulated protein indicated metabolite and cell function response to environmental changes. The given insights from transcriptome and proteomic results have shown the physiological changes at host level towards the viral infection which indicates why the post-larvae of freshwater prawns can face severe mortality due to these physiological changes that occur when they are young. Epizootic diseases have caused huge economical loses in shrimp and prawn culture. However, within the commercial fresh water prawn aquaculture industry, M. rosenbergii seems less susceptible to viral diseases in comparison to paneaids. This is a study that would lead future advance technologies such as CHIP-sequencing, immuno precipitation studies that can be conducted to elucidate the survival of these freshwater prawns in comparison to other peneaid species.
Dharmendra Kumar
Narendra Deva University of Agriculture and Technology, India
Title: Transcriptome analysis of nematode-trapping fungi during infection of plant-parasitic nematodes
Time : 12:00-12:20
Biography:
Dharmendra Kumar has completed his PhD from Banaras Hindu University, Varanasi, India in year 2003 on Predacity and bio-control potential of a nematode-trapping fungus Arthrobotrys dactyloides. He is working as Assistant Professor of Plant Pathology in Narendra Deva University of Agriculture and Technology, Kumarganj, Faizabad, India. He also worked as DST BOYSCAST Fellow at Microbial Ecology, Department of Biology, Lund University, Sweden under guidance of Professor Anders Tunlid and Dr Dag Ahrén in the year 2011. He has published 19 papers in refereed scientific journals.
Abstract:
Nematode-trapping fungi are a group of soil living carnivorous microorganisms that form unique infection structures called traps to capture and kill the vermiform nematodes. Majority of nematode- trapping fungi belong to a monophyletic group consisting of single family of the order Orbiliales (Ascomycota). We studied the transcriptome expressed by Arthrobotrys oligospora forming adhesive networks, Monacrosporiumcionopagum forming adhesive branches and constricting ring forming species Arthrobotrys dactyloides during infection of root-knot nematode Meloidogyne hapla and sugar beet cyst nematode Heterodera schachtii. Comparative transcriptome analysis during infection process including trapping, penetration and digestion of nematode hostby nematode-trapping fungi showed that the divergence in gene expression pattern associated with fungal species was significantly larger than that related to the host nematode species. Genes that were highly expressed in all nematode-trapping fungi encoded endopeptidases such as peptidase S8, peptidase M3 and aspartic proteases; cell-surface proteins containing the carbohydrate-binding domain WSC; stress response proteins; membrane transporters; transcription factors and cell singling genes containing the Ras domain. Transcripts containing the Ricin-B lectin and Atg8 domain were also highly expressed in all nematode- trapping fungi. Differentially expressed transcripts among the fungal species encoded various lectins such as the fungal fruit-body lectin and the D-mannose binding lectin; transcription factors; cell-signaling components; proteins containing a WSC domain and proteins containing a DUF3129 domain. Interestingly, DUF 3129 was highly expressed in M. cionopagum but not expressed at all in A. dactyloides. Differentially expressed transcripts during infection of different host nematodes; including peptidases, WSC domain proteins, tyrosinases and small secreted proteins with unknown function.
Dora Tombacz
University of Szeged, Hungary
Title: Characteristics of Pseudorabies virus transcripts analyzed by PacBio and Illumina sequencing techniques
Time : 12:20-12:40
Biography:
Dora Tombacz is MSc in Biology (Faculty of Sciences, University of Szeged - 2006) and PhD in Medical Sciences (Faculty of Medicine, University of Szeged - 2010). She works in the Department of Medical Biology as an assintant professor at the Faculty of Medicine at University of Szeged in the Boldogkoi’s group. Their primary field of interest is the analysis of herpesvirus gene expression and utilization of herpesviruses as tools in various fields of biology including neurobiology and cardiology. She currently works with next – and 3rd generation sequencing techniqes, focusing on virology and genomics of human diseases at the University of Szeged (Szeged, Hungary) and the Stanford University (Stanford, CA, USA).
Abstract:
Introduction
Pseudorabies virus (PRV) is an alpha-herpesvirus that causes Aujeszky’s disease of swine. The viral genome is double stranded DNA and contains protein-coding and non-coding genes. This virus is a widely used model for the study of neural circuits and it serves as gene delivery vector into mammalian cells. Thanks to its relatively small, compact genome, PRV is also a useful model organism for the analysis of gene expression regulation at different levels.
Materials and Methods
Immortalized porcine kidney 15 (PK-15) cells were infected with Kaplan strain (Ka) of Pseudorabies virus (PRV). For PacBio sequencing, infections were stopped after 1, 2, 4, 6, 8 or 12 hours after infection. Polyadenylated RNAs were isolated from the total RNA samples, then were converted to cDNAs with anchored Oligo(dT)20 primer. cDNAs were used for the SMRTBell library preparation. For Illumina sequencing, we mixed infected cells. Strand-specific total RNA libraries were prepared for random hexamer primed amplification and the sequencing of 2x100 bp fragments. Expression patterns were also examined by strand specific qPCR for validation.
Results
PacBio long read sequencing technology was used for the first time to analyze viral gene expression. With the PacBio sequencing technique, full-length transcripts of up to about 2 kb could readily be monitored with little sequence loss at the 5′ ends. We have detected 27 new transcript isoforms (alternative polyadenylation sites of protein coding genes). Previously unknown splicing sites (in the following genes: ep0; ul12; ul22; ul43; ul3.5), alternative splice variants of the genes (in: us1; ul15 genes) were also detected.
- Workshop: RNA editing and Next Generation Sequencing
Location: Continental Ball Room 5 & 7
Session Introduction
Glen M Borchert
University of South Albama, USA
Title: RNA editing and Next Generation Sequencing
Time : 13:45-14:30
Biography:
Glen Mark Borchert has completed his PhD in Genetics from University of Iowa in the year 2006. He did his first Postdoctorate in Structural Biology from University of California in the year 2008 and the second one in Immunology from Illinois State University in the year 2012. After which he joined as Assistant Professor in University of South Alabama and currently working as Assistant Professor, Pharmacology in USA College of Medicine. He has been honored by NIH with a Research award to study hypoxia induced mutation. Also, National Science Foundation (NSF) gave him the CAREER award of $533,000 to research miRNA targeting.
Abstract:
RNA editing by RNA specific adenosine deaminase (ADAR) is increasingly being found to alter microRNA (miR) regulations. Editing of miR transcripts can affect their processing as well as what mRNAs they target. Further, editing of target mRNAs can also affect their complementarity to miRs. Notably, ADAR editing is often increased in malignancy with the effect of these RNA changes largely unclear. In addition, reports have also identified many miRs to be differentially expressed in cancer though the majority of their targets are also undefined. We propose that modulating the targets of miRs via mRNA editing can play a direct role in the pathology of many carcinomas. To address this, we have recently developed new algorithms to identify and analyze RNA edit sites in RNA-Seq data and used these to identify microRNA target sites created and destroyed by deamination of mRNA adenosines. Initial computational analyses of some of our breast cancer RNA-Seq data identified over 250,000 A-to-I edit sites primarily located in mRNA 3’ UTRs. When these locations were screened against the list of currently annotated miRs we discovered that these A-to-I editing events caused a subset (~5%) of human miRs to have significantly altered mRNA complementarities leading us to propose that modulating the targets of miRs via mRNA editing plays a direct role in the pathology of many carcinomas. Broadly, these results suggest that the creation of miR targets may be an underappreciated function of ADAR and may help further elucidate the role of RNA editing in tumor pathogenicity. Furthermore, our results strongly indicate that the creation of miR regulatory sites is a novel (and surprisingly prevalent) function for ADAR activity, and consequently, many miR target sites are only identifiable through the examination of expressed sequences.
- Track 1: Transcriptome analysis and Gene Expression-An Overview, , Track 5: Exploring the complexity of the Transcriptome & Track 6: Refining expression analysis
Location: Continental Ball Room 5 & 7
Chair
Andrey S. Krasilnikov
Penn State University, USA
Co-Chair
Yan Li
The Fourth Military Medical University, China
Session Introduction
Andrey S. Krasilnikov
Penn State University, USA
Title: Transcripts to cleave transcripts: Structure and function of RNA-based Ribonucleases P and MRP
Time : 14:30-14:50
Biography:
Andrey S. Krasilnikov has received his PhD degree from the Institute of Chemical Physics (Moscow, Russia) in 1995. During his postdoctoral training at the UIC and Northwestern University (Chicago, USA) he became interested in enzymes that rely on their RNA moiety for catalysis. He is currently an Associate Professor of Biochemistry and Molecular Biology at the Penn State University. The main focus of his research is the structure and function of the multicomponent eukaryotic catalytic ribonucleoproteins of the RNase P/MRP family. He has authored more than 30 publications in the fields of biochemistry, molecular and structural biology, and physics.
Abstract:
RNA-based enzymes of the Ribonuclease (RNase) P/MRP family are large catalytic ribonucleoprotein complexes. These enzymes are highly unusual as they do not use proteins as their catalytic moiety, but rely on RNA for catalysis. The best known enzyme of the family, RNase P, is found in all three domains of life and is primarily responsible for the maturation of the 5’-end of tRNAs. Bacterial RNase P consists of an RNA component that is capable of cleaving substrates in vitro without any protein participation, and a small protein that is required for activity under physiological conditions. Archaeal and eukaryal RNases P have RNA components that are similar to that in the bacterial enzyme, although the catalytic ability of their RNA is severely diminished. At the same time, the essential auxiliary protein part of the archaeal and eukaryal RNases P grew considerably. The reasons for the increased reliance on proteins in the more evolutionarily advanced RNases P are not well understood. The other, much less studied enzyme of the RNase P/MRP family, is RNase MRP. RNase MRP is universally found in all eukaryotes. It is an essential enzyme that participates in the metabolism of a wide range of transcripts, from rRNA to some mRNAs. Mutations in RNase MRP result in a range of developmental disorders in humans. Structurally RNase MRP resembles eukaryotic RNase P, but it has evolved to have distinct specificity. We will discuss the recent advances in our understanding of the structure, function, and evolution of RNases P and MRP
Marcelo S. Ferro
University Sao Judas Tadeu, Brazil
Title: The mitochondrial transcription factor and the interaction on Cell Aging
Time : 14:50-15:10
Biography:
Marcelo S. Ferro is a nutritionist, former athlete, post graduate in clinical pharmacology, master in aging science (gerontology) in organelles of cardiomyocytes, nutritionist holder of the Paralympic judo team of Brazil, former university professor of biochemistry and nutrition, acts as technical coordinator companies processing of nutritional supplements in Brazil, worked in partnership with the Group of Pain in the Hospital das Clinicas (HC-USP) as Nutritionist for several years in São Paulo, Brazil.
Abstract:
The mitochondria are essential in numerous physiological processes, including energy production, redox potential, modulation of calcium and several metabolic pathways. When the number or mitochondrial activity is insufficient, the human body quickly goes into fatigue due to ATP deficiency. The oxidative capacity of muscle tissue and the preservation of mitochondria depends on the mitochondrial biogenesis that occurs through the transcription factor proliferator-activator receptor-γ coactivator1α (PGC-1α). The oxidative process and the progressive change in the biogenesis of mitochondria have direct influence on the aging of muscle tissue. The regulation of the biogenesis occurs through the PGC-1α combined with nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factors (TFAM). Abnormalities in mitochondria and mutagenesis in mitochondrial DNA (mtDNA) are tied to multi-system degeneration, as well as intolerance to stress, and decreased energy in aging in humans, rats and monkeys. The mitochondrial functions are dramatically altered in heart disease, demonstrating a decrease in expression of PGC-1α, which plays a key role in the coordination of energy metabolism. This process can be reversed by the PGC-1α itself. The identification of compounds capable of activating the transcription of PGC-1α could be part of future therapies to reverse pathologies associated with the decline of this organelle. Morphophysiological and biochemical changes of these organelles directly reflect the physiological performance of all body tissues. Evidence demonstrated that physical activity, both in young and aged is a major ally in mitochondrial biogenesis by activating the transcription of PGC - 1α and that future nutritional interventions may be of great aid in the health and performance of mitochondria. However, further studies are needed in order to understand and clarify this operation, since currently these mechanisms are only partially known.
Yan Li
The Fourth Military Medical University, China
Title: NDRG2, a new estrogen-targeted gene
Time : 15:10-15:30
Biography:
Yan Li has completed her PhD from the Fourth Military Medical University (China) in 2009 and she used to be a Visiting Scholar for Postdoctoral research in Purdue University (USA). Now she is an Associate Professor of the department of Biochemistry and Molecular Biology, the Fourth Military Medical University (China). She has published more than 20 papers in academic journals including J BiolChem, Mol Ther, Breast Cancer Res and Cell Death Dis.
Abstract:
HumanN-myc downstream regulated gene 2(NDRG2) has been proved to be a multifunctional protein associated with cell proliferation, differentiation, transmembrane transportation and stress response. The expression of NDRG2 is transcriptionally regulated by Myc, TNFαIGF-1, hypoxia, DNA damage and many hormones including dexamethasone, insulin, androgens and aldosterone. In this study, we analyzed the promoter region flanking 5’ of NDRG2 and found a potential ERE (estrogen response element). Moreover, we revealed that estrogencan up-regulate the expression of NDRG2 inboth dose and time-dependent manners. In addition, we demonstrated that ERβ but not ERα, bound specifically to the ERE at position of -1455 to -1131 bp of NDRG2 promoter and trans-activated NDRG2 promoter. These data mean that estrogen as an important circulating hormone also plays a regulative role in NDRG2 expression. In our previous studies we found that NDRG2 interacts with β1-subunit of Na+/K+-ATPase and is involved in estrogen-mediated Na+ and Cl− transport in some epithelial cells. Therefore, characterization of the novel estrogen/NDRG2/Na+/K+-ATPase β1 regulation pathway will broaden the understanding of the regulatory role of estrogen on Na+/K+-ATPase and distribution of this pathway may potentially provide a basis for the intervention of isohydria and internal environment homeostasis in some pathological conditions.
Sai Vishnu Priya
Centre for Cellular & Molecular Biology, India
Title: Expression analysis of candidate genes present in the QTL regions for both iron and zinc in the F7 RILs of Madhukarx Swarna
Time : 15:30-15:50
Biography:
Sai Vishnu Priya has completed her PhD in 2004 from Sri Venkateswara University; worked as post doctoral fellow at Directorate of Rice Research, and visiting scientist at International Crops Research Institute for Semi-Arid Tropics (ICRISAT), Hyderabad. Now, she is working as Project Investigator (PI) (DST, WOS A) at Centre for Cellular and Molecular Biology, Hyderabad.
Abstract:
Micronutrients are necessary for both plant and human survival. Rice is the staple food for fifty percent of the world’s population. However, the polished grain, also known as white rice, contains nutritionally insufficient concentrations of iron (Fe) and Zinc (Zn) to meet the daily requirements in diet leading to adverse effect on human health. Identification of candidate genes in major QTLs and their transcriptome analysis will be useful in gene discovery for biofortification. Genome- wide maps showed 14 QTLs for iron and zinc concentration in unpolished rice grains of F7 recombinant inbred lines from Madhukar x Swarna. Five candidate genes (OsNAS3, OsNRAMP1, OsHMA, OsAPRT and OsZIP8) which encode for both Fe and Zn and underlie four major QTLs on chromosomes 7 and 12 were used for expression analysis. Three leaf stage (32- days old) greenhouse grown plants showed ≥2.15 & ≥1.8 fold expressions of OsNAS3 and OsHMA respectively compared to Madhukar. When 7- days old seedlings were transferred to hoagland solution supplemented with Fe (0.18 mM) and Zn (0.26 mM) and grown for 25- days, all high iron zinc lines (HL) and 75 percent of low iron zinc lines (LL) showed increased expression of OsZIP8 (≥2.2 fold) while other genes were down regulated. Omission of Fe and Zn resulted in stunted growth with reduction in chlorophyll, primary root length and number of roots. In omission of Fe and Zn, showed high transcript levels in all HLs and one LL-270(M) with OsNRAMP1, OsHMA compare to sufficient conditions. Sequence analysis of OsHMA in HL and LL showed variations at 32nd and 37th positions where valine is replaced by alanine and arginine with cystein respectively in 75 percent LL.
- Workshop: On the way to disentangling the enigma of angiosperm egg activation
Session Introduction
Zsolt Ponya
Kaposvar University, Hungary
Title: The unique characteristics of the angiosperm plant life cycle and their potential consequences on the developmental plasticity of nucellar cells using wheat as a model system
Time : 13:25-14:10
Biography:
Zsolt Pónya has completed his PhD at the age of 32 years from the Eötvös Lóránd University of Arts and Sciences, Budapest, Hungary, and following obtaining his degree, he has launched his postdoctoral studies at the University of Siena, Italy, followed by a postdoc research fellowship at the Ben-Gurion University of the Negev, Israel. He is currently a senior scientist at the University of Kaposvár, Hungary. He has published a number of papers in reputed international journals and is a member of the editorial board of several prestigious scientific journals.
Abstract:
Angiosperm seed development is a crucial factor of agricultural production. Thus, a comprehensive understanding of the mechanisms governing it in cultivated plants is pivotal for crop productivity. The unique features of angiosperm life cycle such as continuous development, avoidance of a germline, flexible and reversible cellular differentiation, and the alteration of haploid and diploid generations are characteristics that confer a high level of plasticity upon flowering plants. Intriguingly, angiosperm nucellar cells can either engage in or forgo meiosis, a cell fate “decision” depending on the developmental context. Consequently, in angiosperms, two developmental pathways of reproduction can be followed: sexual or amphimictic, and asexual (apomictic); the latter remaining the holy grail of basic research initiatives due to its elusive nature. When paralleled, the cell fate decision of the initiatory cell being the depository of the information triggering incipient mechanisms eventually leading to embryo formation of a particular nucellar/sexual cell in the angiosperm ovule is in contrast with the conditions required in vitro either for a reconstituted oocyte exploited to produce clones by somatic cell nuclear transfer (SCNT), or for mammalian stem cells induced to undergo meiosis and produce synthetic gametes. Viewed from an evolutionary perspective, the genomes of angiosperm species are more dynamic and labile at both the sequence and chromosome level, whereas mammalian genomes are more stable mainly due to setting the germ line cells apart during an early stage of development. It appears that in attempts to unravel the molecular and cellular events leading to the “derailment” of the normal sexual pathway in the female gametophyte as it unfolds during the alternative developmental pathway during asexual seed formation, considering only gene-regulation networks in terms of the transcriptome may not supply us with a comprehensive picture pertaining to addressing the developmental flexibility of the nucellar tissues since an even greater complexity is conferred upon the angiosperm nucellus by the proteome. Furthermore, the wide range of plant transcription factors could be expounded by a unique feature of plants: their complex secondary metabolism providing a plethora of secondary metabolites; up to 25% of all plant genes are associated with a unique set of secondary metabolites not found in animals (the total number of plant secondary metabolites is close to 50,000, albeit it is estimated to be several hundred thousands) Essentially, transcriptomics studies launched in wheat, being one of the most staple food crops in the world, promise to be of use in disentangling the expression profile of genes that control many agriculturally important traits. However, the use of wheat microarrays for gene expression measurements in hexaploid wheat having an enormous genome may be deemed to be limited, and even when coupled with approaches based on powerful tools such as sequencing-based transcriptomics techniques may not be deemed to enable us to fully decipher the complexity of mechanisms involved in embryonic development ensuing either upon or without fertilisation in female gametes/nuclear cells. Elements of a complex system involving auxin gradient, positional information, maternal control end epigenetic factors will be touched upon in summarising the state of the art of endeavours addressing these issues of utmost importance in developmental biology.
- Track 10: Expression Profiling, Track 13: Genomics, Proteomics and Bioinformatics
Chair
Sang Ming WANG
University of Nebraska Medical Center, USA
Co-Chair
Punit Kaur
Morehouse School of Medicine, USA
Session Introduction
Max H Garzon
The University of Memphis, USA
Title: Genomic Positioning Systems for DNA and the Tree of Life
Time : 14:10-14:30
Biography:
Max H. Garzon is professor of computer science and bioinformatics. He has (co-)authored over 150 books, book chapters, journal and refereed conference publications. The main theme of his research is biomolecule-based computing and applications to areas such as bioinformatics, nanotechnology, self-assembly, semantic indexing and data mining. He is on the editorial board of several journals in these areas, including the International Journal of Genomic Medicine. He has served as TPC and organizer of many scientific conferences and professional meetings in these areas. He has been a visiting professor and guest scientist at a number of research institutions around the world.
Abstract:
Geographic information systems represent the modern version of the scientific revolution ushered in by the introduction of now standard Cartesian coordinates and analytic geometry in the 1600s. They provide a universal means of communication and computation in a unified system that makes possible identification, comparisons and validations of geographic position, previously unattainable on a global scale. In this talk, I propose an analogous coordinate system for the far more complex genomic biosphere based on the concept of “genomic” distance enjoying the geometric properties characteristic of geometric distance. The system has several properties: it is universal (applicable to any genome, known or unknown), feasible (computable for every given genome), zoomable (adaptable precision to various scales of resolution) and, more importantly, capable of revealing important functional and structural features of a given genome relative to other genomes. After a brief sketch of the foundations and contrast with earlier approaches, I illustrate how these two latter features make possible a number of applications in various omics areas, such as molecular phylogenetics, universal genetic marker generation and classification, and comparative whole-genomic analyses on a common framework that may reveal significant commonalities in hitherto disparate taxonomies and procedures. Time permitting, some potential applications will be sketched in some detail, including applications of interest in transcriptomics, such as molecular systematics, the Tree of Life, and Next-Generation Sequencing
Tao Liu
University of New South Wales, Australia
Title: The long noncoding RNA MALAT1 promotes tumour-driven angiogenesis by up-regulating pro-angiogenic gene expression
Time : 14:30-14:50
Biography:
Dr Tao Liu obtained his PhD from The University of New South Wales Australia in 2000. He then worked as a post-doc with Professor Samuel Breit at St. Vincent's Centre for Applied Medical Research Sydney. He is now Head of Histone Modification Group at Children’s Cancer Institute Australia, and an Australian Research Council Future Fellow at The University of New South Wales. In the last 5 years, Dr. Liu has been working on the roles of long noncoding RNAs, histone methyltransferases and histone deacetylases in modulating gene transcription, N-Myc-regulated malignant transformation, neuroblastoma initiation and progression in vitro and in vivo.
Abstract:
Neuroblastoma is the most common solid tumour during early childhood, and accounts for 15% of all childhood cancer death. One of the key features of neuroblastoma is disease progression due to tumour-driven angiogenesis. However, the mechanism through which neuroblastoma cells drive angiogenesis is unclear. Here we showed that the long noncoding RNA MALAT1 was over-expressed in human neuroblastoma cells under hypoxic condition, the condition which triggers pro-angiogenic switch and angiogenesis. In vitro angiogenesis assays demonstrated that conditioned medium from neuroblastoma cells transfected with MALAT1 siRNAs, compared with conditioned medium from neuroblastoma cells transfected with control siRNAs, induced considerably less endothelial cell migration, invasion and vascular sprouting under hypoxic condition. Affymetrix microarray differential gene expression study showed that one of the genes most significantly down-regulated by MALAT1 siRNAs in human neuroblastoma cells under hypoxic condition was fibroblast growth factor (FGF). RT-PCR and immunoblot analyses confirmed that MALAT1 siRNAs reduced FGF mRNA and protein expression, and in vitro angiogenesis assays demonstrated that forced over-expression of FGF in neuroblastoma cells blocked MALAT1-mediated endothelial cell migration, invasion and vascular sprouting. Taken together, our data suggest that over-expression of MALAT1 in human neuroblastoma cells plays an important role in tumour-driven angiogenesis by up-regulating FGF mRNA expression.
Bingsong Zheng
Zhejiang A & F University, China
Title: Studying the expression pattern of auxin-associated genes in Carya cathayensis (Hickory) during the grafting process
Time : 14:50-15:10
Biography:
Bingsong Zheng has completed his PhD in 2003 from Zhejiang University and Postdoctoral studies from INRA. He is the Vice-Dean of School of Forestry & Biotechnology, Zhejiang A & F University. He has published more than 30 papers in reputed journals and has been serving as an Editorial Board Member of repute.
Abstract:
Application of various hormones improves the grafting process and among which auxin plays a vital role for the formation of vascular reconnection. Auxin role in the grafting process was analyzed by studying the differential expression pattern of auxin-associated genes (ARF, GH3) and transporter genes (ABC and Aux: Hyd) under auxin- and NPA- (an auxin inhibitor) applied conditions at 0, 3, 7 and 14 days after grafting (dag). Analysis shows that the expression of GH3, ARF and Aux: Hyd genes were found to be low at the time of grafting but increased at 3 and 7 dag and again get reduced at 14 dag. While the expression of ABC gene was found to be high at 14 dag and got reduced at 3 and 7 dag. Further the application of IAA or NPA to the grafted sample is not influencing the gene expression in a concordant way. With the availability of rough draft unigene library for Hickory tree species to our group, 34 different ARF genes were identified and analyzed for their expression level at 0, 7 and 14 dag. Among the 23 genes analyzed, 15 genes expression level are not affected at various time of analysis and 5 of the genes expression were not detected in the grafted plants. While 3 of the gene expression level got drastically reduced at 7 and 14 dag when compared with 0 dag which shows that these ARF genes have specific role in the grafting process which has to be studied in detail in the future.
Punit Kaur
Morehouse School of Medicine, USA
Title: A Mouse Model for Triple-Negative Breast Cancer Stem Cells (TNBC-CSC) Exhibits an Aggressive Phenotype
Time : 15:10-15:30
Biography:
Punit Kaur finished her B.Sc. in 1999 with Botany, Zoology and Chemistry from Punjab University, Chandigarh, India. Then she went for Postgrad (M.Sc.) (1999-2001) in Microbiology from Guru Nanak Dev University, Amritsar, Punjab, India. Later she pursued for Ph.D (2001-2008) on the topic Experimental Medicine and Biotechnology from the Postgraduate Institute of Medical Education and Research, Chandigarh, India
Abstract:
There has been an increase of 40% in the number of Americans diagnosed with diabetes. Over that same period, the obesity rate has also increased by nearly 20%. Therefore, efforts to identify any biomarker(s) to assist with obesity and diabetes prevention are of paramount importance. This study was designed to address the link between obesity and diabetes using proteomic analysis of drawn blood and collected urine samples. Four of 14 primary care clinics of a large University-affiliated, multi-specialty group practice associated with an 186,000-member HMO in Central Texas. Six volunteer adult female Caucasian patients aged 35 years to 55 were identified with study inclusion criteria via Electronic Medical Record (EMR) search. Patients were divided into four groups on the basis of body mass index (BMI) and fasting blood glucose (FBG): group A presented with BMI>25, FBG<100; group B with BMI>25, FBG 110-125; group C with BMI>25, FBG>125; and group D were controls with BMI<25 and FBG<100. Plasma was separated from drawn blood, aliquoted and kept at -80°C until required. The plasma samples were analyzed using liquid chromatography mass spectrometry to measure changes in the protein profiles. Proteins including angiotensinogen protein, glycoprotein, apolipoproteins, complement proteins, hemopexin and vitronectin were found to be significantly increased in patients with obesity and diabetes. Importantly, angiotensinogen protein was significantly increased in group C considered as obese and diabetic individuals. Our results suggest that angiotensinogen protein, known to be linked to hypertension, might be an important early biomarker that links obesity and diabetes. Further studies using a larger sample size is required before concluding angiotensinogen as a potential biomarker that links obesity and diabetes.
Baris E. Suzek
Mugla Sitki Kocman University, Turkey
Title: Locating disease-associated single amino acid polymorphisms on proteins
Time : 15:30-15:50
Biography:
Baris E. Suzek has completed MS in Computer Science at Johns Hopkins University and PhD in Bioinformatics and Computational Biology at George Mason University. Currently, he is an Assistant Professor in Computer Engineering Department at Mugla Sitki Kocman University in Turkey and an Adjunct Assistant Professor at Georgetown University in USA. Before 2013, he served as a Research Assistant Professor in Department of Biohemistry and Bioinformatics Team Co-lead in Protein Information Resource at Georgetown University. He has published more than 20 papers in reputed journals, holds one patent and is recipient of several national awards in USA.
Abstract:
The next generation sequencing technologies are continuously improving and large sets of human mutations are becoming available within institutions and through public resources. A subset of these mutations lead to single amino acid polymorphisms (SAPs). Identifying SAPs’ disease-causing impact and developing methods to identify SAPs disease-associations are active research areas. In this work, we investigated the link between disease-associated SAPs and their protein sequence context (i.e. sequence features). We have integrated disease-associated SAPs and sequence features from several public bioinformatics resources. The SAPs and sequence features are then mapped to each other using protein resource UniProt Knowledgebase. Based on our initial results, over 39% of disease-associated SAPs are located on functional domains, topological domains and near post translational modifications. Over 19% of them are located on known secondary structures. Our findings will help in better understanding of SAPs’ disease associations. In the future, the protein sequence context for SAPs can be utilized in development of more accurate computational methods to predict disease-associated SAPs.
Babak Behnam
Iran University of Medical Sciences, Iran
Title: SLUG and SOX9 Cooperatively Regulate Tumor Initiating Niche Factors in Breast Cancer
Time : 15:50-16:10
Biography:
Babak Behnam has completed his MD degree at Iran University of Medical Sciences (IUMS), and his PhD degree at the University College London (UCL) in Human Genetics. Then he served as postdoctoral research fellow for 4 years at the University of Michigan School of Medicine and University of Central Florida. Then he has joined IUMS and directed medical genetics laboratory at IUMS-Children hospital in parallel to his translational research focused on molecular metastatic pathway and breast cancer stem cell. He has published more than 25 peer-reviewed papers and is also serving as an Editorial Board Member of scientific journals.
Abstract:
Background Presence of tumor initiating cells and a proper niche is essential for metastatic colonization. SLUG and SOX9 transcription factors play essential roles in induction and maintenance of tumor initiating capacity in breast cancer cells. On the other hand, Tenascin-C and Periostin are crucial factors in metastatic niche that support tumor initiating capability in breast cancer. Method In this study, regulatory effect of SLUG and SOX9 transcription factors on the expression of Tenascin-C and Periostin transcripts was examined. SLUG and SOX9 were overexpressed and knocked-down via lentivirus constructs in MCF7 in MDA-MB-231 cells, as non-invasive and invasive breast cancer-derived cells respectively. Then, Tenascin-C and Periostin expression levels were measured side by side of the SLUG and SOX9 via Real-Time PCR. Results Simultaneous overexpression of SLUG and SOX9 significantly induced Tenascin-C and Periostin Expression. SLUG and SOX9 knock-down also significantly reduced the expression of Tenascin-C and Periostin. In this analysis, Periostin showed the most deviation in both up- and down-regulation levels. Conclusion This regulatory effect might shed light to a crosstalk between factors involved in the tumor initiating capacity and metastatic niche of the breast cancer.
- Track 8: Functional Impact of Non-Coding RNA (ncRNA), Track 9: Cancer Genomics: Integrative And Computational Approaches, Track 12: Transcriptional Regulation and Transcriptional Attenuation
Location: Continental Ball Room 5 & 7
Chair
Harry Jarrett
University of Texas San Antonio, USA
Co-Chair
Ding-Gan Liu
Chinese Academy of Sciences, China
Session Introduction
Harry Jarrett
University of Texas San Antonio, USA
Title: New method for analyzing transcription factors and other proteins involved in transcription using MALDI-TOF mass spectrometry
Time : 16:05-16:25
Biography:
Jarrett received his Ph.D. from the Univ. North Carolina-Chapel Hill in 1976. After postdoctoral fellowships at the Mayo Clinic, UCSD, and U.GA , he began an assistant professorship at IN U-Purdue U at Indianapolis. After promotion he joined the faculty at the U. TN Health Science Center where he was promoted to professor and remained for 18 years. In 2006, he joined the faculty at U. TX San Antonio as the Lutcher Brown Distinguished Professor of Biochemistry where he remains. His research has involved the purification of transcription factors and the RNA pol 2 transcription complex. His discoveries have included coupling methods which led to the first DNA-silica HPLC columns and developed both oligonucleotide trapping and promoter trapping to purify transcription proteins for mass spectrometry characterization. Most recently, this research has involved coating MALDI plates to direct proteomic discovery to DNA-binding proteins for a new method called T3.
Abstract:
MALDI plates coated with polyvinylpyrrolidone do not bind protein but avidly bind DNA and DNA-protein complexes. The DNA sequence determines which proteins bind. When DNA, either oligonucleotides or longer DNAs such as core promoter sequences, is mixed with nuclear extract and spotted on the plate, DNA-protein complexes are bound while other proteins can be simply washed away. The samples are then digested with trypsin and matrix is added on the plate and the plate is then ready for mass spectrometry analysis. Highest signal-to-noise is obtained with core promoters (in this case a 300 bp human telomerase (hTERT) promoter) than with 20-mer duplex oligonucleotides but usable data is obtained with either. Using overlapping oligonucleotides representing the entire hTERT promoter, binding sites for TCF23 (an E-box binding protein), SP1, and AP2 were all localized to specific sequence regions. When the intact promoter is used, SP1 and AP2 are also found but instead of TCF23, USF2 is found. This suggests that USF2 binds to the E-boxes in the promoter context. Other components of the RNA polymerase 2 transcription complex such as TF2H were also characterized. The method allows as many as 384 samples to be analyzed on a single plate in a matter of three days and thus the method is capable of high-throughput analysis of DNA-binding proteins.
Ding-Gan Liu
Chinese Academy of Sciences, China
Title: Independent 3’untranslated region RNA: A novel non-coding regulator RNA
Time : 16:25-16:45
Biography:
Ding-Gan Liu has completed his M.S. from the Shanghai Institute of Biochemistry, Chinese Academy of Sciences (CAS) and gradually upgraded to research associate, associate professor and full professor in that Institute (now the Institute of Biochemistry and Cell Biology, Shanghai institutes for Biological Sciences, CAS). During 1980's he was a visiting scientist in the Institute for Physical and Chemical Research (RIKEN), Tokyo, Japan, and in the Tsukuba Life Science Center, RIKEN, Japan. He has been the first author, corresponding author or co-author of more than 50 research papers in Chinese and international scientific journals, such as Nucleic Acids Res, PloS One, BBRC, DNA Cell Biol, and Nature etc.
Abstract:
Classically, the 3’untranslated region (3’UTR) is that region in eukaryotic protein-coding genes from the translation termination codon to the polyA signal. It is transcribed as an integral part of the mRNA encoded by the gene. However, there exists another kind of RNA, which consists of the 3’UTR alone, without all other elements in mRNA such as 5’UTR and coding region. The importance of independent 3’UTR RNA (referred as I3’UTR) was prompted by results of artificially introducing such RNA species into malignant mammalian cells. Since 1991, we found that the middle part of the 3’UTR of the human nuclear factor for interleukin-6 (NF-IL6) or C/EBP gene exerted tumor suppression effect in vivo. Our subsequent studies showed that transfection of C/EBP 3’UTR led to down-regulation of several genes favorable for malignancy and to up-regulation of some genes favorable for phenotypic reversion. Also, it was shown that the sequences near the termini of the C/EBP 3’UTR were important for its tumor suppression activity. Then, the C/EBP 3’UTR was found to directly inhibit the phosphorylation activity of protein kinase CPKC in SMMC-7721, a hepatocarcinoma cell line. Recently, an AU-rich region in the C/EBP 3’UTR was found also to be responsible for its tumor suppression. Recently we have also found evidence that the independent C/EBP 3’UTR RNA is actually exists in human tissues, such as fetal liver and heart, pregnant uterus, senescent fibroblasts etc. Through 1990’s to 2000’s, world scientists found several 3’UTR RNAs that functioned as artificial independent RNAs in cancer cells and resulted in tumor suppression. Interestingly, majority of genes for these RNAs have promoter-like structures in their 3’UTR regions, although the existence of their transcribed products as independent 3’UTR RNAs is still to be confirmed. Our studies indicate that the independent 3’UTR RNA is a novel non-coding RNA species whose function should be the regulation not of the expression of their original mRNA, but of some essential life activities of the cell as a whole.
Yosef Yarden
Weizmann Institute of Science, Israel
Title: Transcription-based chronobiology of receptor tyrosine kinases: Relevance to cancer progression
Time : 16:45-17:05
Biography:
Yosef Yarden completed his PhD from The Weizmann Institute of Science; Rehovot, Israel in the year 1985. Later he went for his postdocs and got appointed as a Senior Scientist in Department of Immunology in The Weizmann Institute in 1988. Currently he is working as a Professor in Department of Molecular Cell Biology in the same institute. He has many awards on his name mentioning some the EMET prize in Biochemistry in 2007 and the MERIT award of the U.S. National Cancer Institute in 2005.
Abstract:
Tumor-specific combinations of oncogenic mutations often free cancer cells from their reliance on growth factors. One important example comprises the epidermal growth factor receptor (EGFR) and its kin, HER2. In tumors, both EGFR and HER2 frequently display overexpression, internal deletions and point mutations. Accordingly, monoclonal antibodies and kinase inhibitors specific to these receptors have been approved for clinical application. The lecture will introduce efforts to resolve the logic underlying gene expression programs activated by growth factors and relationships to tumor progression. Wave-like induction of defined groups of transcripts follows receptor activation, and a similar pattern is displayed by microRNAs. Interestingly, the earliest event we detect is a concerted downregulation of microRNAs that collectively suppress the earliest wave of up-regulated genes, a group of transcription factors that includes FOS and JUN. Importantly, the waves of mRNAs and microRNAs are reflected in breast and other tumors, implying that growth factors play vital roles in tumor progression. The lecture will focus on two emerging features of gene expression programs, which are initiated by activated receptors for growth factors. The first feature is a seemingly pulsatile mode of signals that regulates cell cycle progression. Accordingly, commitment to S-phase entry depends on a fruitful second pulse of signaling, which removes inhibition by the wild type form of p53. The other feature relates to an apparent diurnal regulation of growth factor signaling. This requires signaling by steroid hormones and entails a crosstalk between nuclear receptors and receptor tyrosine kinases, such as EGFR. The implications of both pulsatile regulation of transcription and its diurnal control will be discussed in the context of tumor progression.
Nancy Amaral Reboucas
University of Sao Paulo, Brazil
Title: Parathyroid hormone regulates Nhe3 gene core promoter: EGR1 and Sp3 might regulate the RNA polymerase II pause?
Time : 17:05-17:25
Biography:
Nancy Amaral Reboucas graduated as a medical doctor in 1976 at Federal University of Goiás, Brazil. She concluded my medical residence in Nephrology in 1979, and my PhD in Human Physiology at the Institute of Biomedical Science, Department of Physiology and Biophysics, University of São Paulo, in 1983. Then, she went on to do post-doctoral fellowship at Yale University for two years, from 1989 to 1991, in the Peter Aronson and Peter Igarashi laboratory, Department of Internal Medicine, section of nephrology. Since then, she coordinates her own laboratory at University of São Paulo, in the Department of Physiology and Biophysics, where she teaches renal physiology and membrane physiology to medical students. Her main line of research is transcriptional and functional regulation of the sodium-hydrogen exchanger NHE3 in renal tubules. She is also responsible for a course on Molecular Biology since 1998 that is open to the community. The purpose of this course is introduce graduate students and medical and biomedical professionals to the Molecular Biology of the cell and to the fundamental methods used in a Molecular Biology laboratory.
Abstract:
The main Na+ reabsorption mechanism in the renal proximal tubules is the Na+/H+ exchanger 3 (NHE3) which is acutely and chronically down regulated by parathyroid hormone (PTH). In rats, continuous administration of PTH to induce hyperparathyroidism reduces the expression of NHE3 both at RNA and protein level with clear decrease of NHE3 at the apical membrane of proximal tubules. Analysis with reporter gene of the rat Nhe3 gene promoter in Opossum Kidney Proximal Tubule (OKP) cells indicated inhibition of transcription by PTH associated to a decrease in NHE3-mRNA stability. We demonstrated that PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways changed PTH from an inhibitor into an activator of promoter activity as did point mutations in the EGR1, Sp1 and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1-mRNA and of some Sp3-protein isoforms. Electrophoretic mobility shift assay showed a super shift of the -61 to -42bp probe with an anti-EGR1 antibody in PTH-treated cells suggesting the EGR1 binding is relevant for the inhibitory activity of PTH. Our results suggest that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression to EGR1 binding to the proximal and core promoters and to PKA and JAK/STAT pathway activation. The higher expression of the long isoform (Sp3-li, 90 kDa) and of the short isoform (Sp3-si3) of Sp3 in addition to higher sumoylation of one of the short isoforms of Sp3 induced by PTH could also be related to repression of Nhe3gene expression. As Sp3 was reported to promote RNA polymerase II pause by recruiting phosphatases to the p21CIP1 gene promoter, we hypothesize the PTH might increase RNA Pol II pause at the Nhe3 gene promoter.
Wei Zhang
The University of Texas M.D. Anderson Cancer Center, USA
Title: Interrogating Cancer Transcriptome reprogramming
Biography:
Wei Zhang is a Professor in Pathology and Cancer Biology in the Department of Pathology and the director of the Cancer Genomics Laboratory at MD Anderson Cancer Center. Dr. Zhang has published more than close to 300 peer-reviewed papers and 21 book chapters. Dr. Zhang co-edited two books (Statistical and Computational Approaches to Genomics, 1st and 2nd edition, and Genomic and Molecular Neuro-Oncology) and co-authored one book (Microarray Quality Control). Dr. Zhang is a co-director of one of seven Genome Data Analysis Centers (GDAC) under the Cancer Genome Atlas (TCGA) project funded by NCI.
Abstract:
Recent high throughput interrogations of cancer landscape through programs such as The Cancer Genome Atlas (TCGA) have gained deeper understanding of cancer as a disease of heterogeneity of different stages of cell fate evolution and dedifferentiation. Epithelial tumors often exhibit mesenchymal features as a result of epithelial to mesenchymal transition (EMT), a process that can be driven by both genetic and epigenetic alterations in a form of network regulation. In ovarian cancer, loss of epithelial features and gain of a mesenchymal phenotype is associated with more aggressive tumor features. We performed an integrated genomic analysis, which revealed a miRNA-regulatory network that further defined a robust integrated mesenchymal subtype associated with poor overall survival in 459 cases of serous ovarian cancer from TCGA and 560 cases from independent cohorts. Eight key miRNAs, including miR-506, miR-141, and miR-200a, were predicted to regulate 89% of the targets in this network. Follow-up functional experiments illustrate that miR-506 inhibited EMT by targeting SNAI2, a transcriptional repressor of E-cadherin, as well as mesenchymal regulators vimentin and N-cadherin. MiR-506 also induced senescence by blocking CDK4/6-FoxM1 axis. We used our well-characterized nanoparticle platforms for systemic delivery of miR-506 in orthotopic OvCa mouse models and found such delivery to be highly effective for reduced tumor growth. Interestingly, mesenchymal tumors such as leiomyosarcoma and synovial sarcoma often exhibit epithelial features in a process of mesenchymal to epithelial transition (MET). MET in sarcoma is associated with improved prognosis. Therefore, reprogramming is an intrinsic hallmark of cancer. Understanding of this reprogramming promises to lead to intervention that program cancer into an attractor stage that is more similar to normal cells or a stage that leads to irreversible cell death.
- Young Researchers Forum
Session Introduction
Yu Zhou
Tulane University, USA
Title: Pathway analysis for female osteoporosis
Time : 16:25-16:45
Biography:
Yu Zhou is from Tulane University, USA. His research interests reflect in his wide range of publications in various national and international journals.
Abstract:
Osteoporosis is a prevalent bone metabolic disease characterized by bone fragility. As a key pathophysiological mechanism, the disease is caused by excessive bone resorption (by osteoclasts) over bone formation (by osteoblasts). Peripheral blood monocytes represent a major systemic cell type for bone metabolism by serving as progenitors of osteoclasts and producing cytokines important for osteoclastogenesis. To identify the key dysfunctional pathways in osteoporosis, we performed pathway analysis on microarray data of monocytes from 73 Caucasian females with extremely high or low hip BMD (bone mineral density). In current major approaches, pathways are treated as independent, but the genes overlap among them will cause “crosstalk” phenomenon and interfere the results severely. So, we firstly performed a traditional pathway analysis (Fisher’s exact test using kegg database) on our microarray data. Then we proposed a novel approach which considers the correlation among genes in the same pathway based on the real experiment data to correct the crosstalk effects in the analysis. We also applied a correction technique, MIE (maximum impact estimation) which has been reported, to our study. In traditional analysis, 10 pathways were found to be significantly associated with BMD variation. After correction by our methods, four of them (hsa05010, hsa00601, hsa01212, hsa04622) were still significant. Thus, they were considered as independent and important pathways in bone mechanism. Moreover, one pathway, MAPK signaling pathway (hsa04010) which has been proved to be important for osteoclastogenesis, became significant. Comparing with the available correction technique (MIE), all the findings in our method were biologically meaningful for osteoporosis. Because it was based on the correlation among genes in real experiment data, our method was available to reduce the false positive results and get better understanding of biological networks in the disease. In summary, we described a novel method to correct the crosstalk effect in pathway analysis and found five key independent pathways involved in BMD regulation.
Peter Olah
University of Szeged, Hungary
Title: Characterization of 5-methylcytosine patterns in Pseudorabies virus
Time : 16:45-17:05
Biography:
Peter Olah is a graduate student at the Department of Medical Biology on the University Of Szeged, Hungary. He obtained his MSc in Biotechnology from the University of Debrecen, Hungary, followed by work as a sequencing laboratory assistant. His main area is bioinformatics and sequencing data analysis related to the life cycle and gene expression regulation of herpesviruses.
Abstract:
Introduction: Pseudorabies Virus (PRV or Aujeszky's disease virus) is a neurotrophic herpes virus with a broad host range consisting of mammalian species which causes fatalities in swine populations prenatally and in young animals. It is also a widely used model organism for studies of gene expression, neuronal tracing and the viral life cycle of Alpha herpesviridae. A main characteristic of alphaherpes viruses is the strictly regulated, cascade-like gene expression pattern during lytic infection. In addition, viral genes are arranged in tightly packed functional clusters in various overlapping orientations. In order to assess whether 5-methylcytosine is present and occurs at specific loci in relation to the gene clusters in the PRV genome, wild-type and mutant PRV strains were subjected to bisulfite sequencing (BS-Seq). Materials & Methods: PRV virions of the wild-type PRV strain Kaplan and mutant viruses lacking key trans-activators (US1, UL54, EP0 and IE180 null-mutants) were harvested 12 hours post infection from PK-15 cells by ultracentrifugation followed by DNA isolation. BS-Seq libraries were constructed for paired-end 100 bp sequencing on an Illumina Hiseq 2000. Alignments were generated using Bowtie2 following strict adapter and quality trimming; the resulting alignments were processed in Bismark for methylation calling. Results: In PRV, interpreting CpG methylation is complicated by the extremely high GC-content of the virus. The number of methylated cytosine residues varied slightly (4-7%) between samples at lower amounts than the average methylation density in mammalian CpG islands. The intra-sample distribution of methylated residues was however non-random, indicating preferred sites of base modification and was consistent between samples which suggests that methylation at intergenic boundaries might serve important roles in viral gene expression regulation.
Saurabh Chaudhary
Guru Gobind Singh Indraprastha University, India
Title: De novo transcriptome assembly and identification of cold and freeze responsive genes in sea buckthorn
Time : 17:05-17:25
Biography:
Saurabh Chaudhary is presently pursuing his PhD under the guidance of Prof P C Sharma, Dean, University School of Biotechnology, GGSIPU, New Delhi, India. His research area includes transcriptome analysis of sea buckthorn, a medicinally and ecologically important plant. He has studied digital gene expression profiling under cold and freeze stress using DeepSAGE in sea buckthorn. The major achievements of his work are: comprehensive study of sea buckthorn transcriptome, assembly of 88,297 putative uni-genes of sea buckthorn and identification of 11,922 differentially expressed genes under cold and freeze stress in seabuckthorn. He has three research publications in peer reviewed international journals.
Abstract:
Sea buckthorn (Hippophae rhamnoides L.) is well known for its immense medicinal, nutritional and ecological value, and also for its ability to grow in extreme environments. We used next generation Illumina sequencing to gain a comprehensive view of the seabuckthorn transcriptome. A total of 86,253,874 high quality short reads were assembled using different assembly tools and parameters. The use of short read assembler ABySS with an additive k-mer approach followed by TGICL suite was found most promising for de novo transcriptome assembly. Finally, 88,297 transcripts (>100 bp) were generated representing 53 Mb transcriptome. The average transcript length and N50 length remained 610 bp and 1,193 bp, respectively and 91% of the short reads uniquely mapped back to the seabuckthorn transcriptome. Further, 41,340 (46.8%) transcripts showed significant similarity with the sequences present in nr protein databases of NCBI (E-value <1E-06). Assembled transcripts were also screened for the presence of transcription factors and simple sequence repeats. Next, DeepSAGE, a tag based approach was also followed to identify differentially expressed genes under cold and freeze stress. In all, 11,922 differentially expressed genes (DEGs) were identified including 6,539 up regulated and 5,383 down regulated genes. Gene ontology and KEGG pathway analysis was performed to assign gene ontology term to DEGs and ascertain their biological functions. Expression of selected 22 DEGs was validated using qRT-PCR.A combination of de novo transcriptome assembly and DeepSAGE analysis proved to be a powerful method for transcriptome analysis and identifying abiotic stress responsive genes in seabuckthorn.