She began her studies in the realm of plant molecular sciences three years ago at the University of Kaposvár, Hungary. As a research student she was involved in several projects led by Zsolt Pónya PhD, focusing on the cellular/molecular aspects in vitro versus in vivo zygotic embryo formation in angiosperms with special regard to comparing zygote formation and egg activation in the plant versus animal kingdom using micromanipulation techniques. She is co-author of the study by Zsolt Pónya, published in IJMS: “When Isolated a Full Receptivity, in Vitro Fertilized Wheat (Triticum aestivum, L.) Egg Cells Reveal [Ca2+]cyt Oscillation of Intracellular Origin”. She is currently the CEO of a software developing company: eSolution Ltd., Hungary, which deals with data processing and evaluation of results gathered in various research projects and in other versatile areas such as engineering.

Albeit Nawaschin discovered double fertilisation, during which two female reproductive cells are fertilised, well over a hundred years ago (1989), this unique reproduction strategy characteristic of flowering plants has remained elusive and defied attempts to disentangle the molecular and cellular mechanisms that ultimately lead to a life strategy fundamentally different from that of animals. The remarkable feature of this life cycle is the masterly treatment of the alternation of generations between a diploid sporophyte and a haploid gametophyte, the latter allowing for purging the population of deleterious mutations since the phenotype reflects the haploid genotype. Double fertilisation involves a complex series of interactions, a key step of which is the activation of the egg cell produced by the haploid female megagametophyte. The cascade events of the sperm-induced activation of the angiosperm egg and the concomitantly unfolding zygote development coupled with the switch from maternal to embryonic control of gene activation and resulting in asymmetrical cell cleavage that produces two daughter cells of different cell fate still await deciphering owing mainly to the inaccessibility of the female gametes/zygotes of higher plants as these develop deeply enclosed in surrounding maternal tissues hence rendering their isolation a deterring task. Thus, there is a considerable time lag in studying proembryo formation in plants as compared to animal embryology leaving a number of questions of overriding importance, such as: 1: how and when is cell polarity established in the angiosperm egg cell 2: how do molecular processes such as cell division, cell expansion, cell maturation and differentiation unfold which bring the zygote (the progenitor cell of the next sporophytic generation), from the unicellular to the multicellular stage 3: what genes are involved in triggering the first asymmetrical division of the zygote and how these genes are regulated, unanswered. Wheat is a staple crop and prevalent alimentation source in many parts of the world which warrants studies aimed at unravelling mechanisms ultimately responsible for seed set, the prerequisite of which is successful fertilisation of the egg as well as normal endosperm development. A micromanipulation technique based on gamete isolation, electrofusion performed at the gamete cell-pair level and microinjection of isolated wheat egg cells/fusion products was elaborated which permits a complex approach in addressing these issues. Capitalising on this method, one of the earliest marks of fertilisation-associated events, egg activation by the sperm cell through calcium signalling has been dissected together with the in vivo dynamics of the F-actin cytoskeleton shown to be involved in imparting spatial information to the egg cell concerning the micropylar-chalazal axis of the embryo sac. Furthermore, one of the main cell cycle protein kinases, cdc2 kinase, was localised in isolated and in vitro fertilised egg cells of wheat with double labelling the protein of interest and tubulin assisted by fluorescent and confocal microscopy. The importance of the pivotal role played by p34cdc2 in reorganising the intracellular structures of the wheat zygote for rendering it capable of preparing for mitosis that produces the proembryo possessing two cells with distinct cell fates will be discussed as well as the implications of this approach in transcript profiling of isolated wheat female gametes, zygotes, and two-celled embryos in the context of studying cell polarity/asymmetrical cell division.

Dianjing Guo is an associate professor at the Chinese University of Hong Kong. Prior to joining CUHK, she was a senior researcher at the Virginia Bioinformatics Institute. Dianjing received her Ph.D from the Chinese Academy of Science. Her primary research interests are in the field of genomics and computational biology. She has published many research articles in peer reviewed academic journals over the past 10 years.

H2O2 has been recognized as a signal molecule involved in gene expression regulation. Arabidopsis microarray data analysis revealed that about 1–2% of the transcriptome and one-third of the transcription factor mRNA are altered after exposure to H2O2). In the green plant, H2O2 is mainly produced during the process of photosynthesis, respiration, photorespiration, and wound/pathogen-triggered ROS burst. Apart from the ROS burst mediated by membrane-associated NADPH oxidases (rboh A∼F family) under wounding and/or pathogen invasion, the photorespiration and electro transfer during photosynthesis constitute more than 90% of the H2O2 production in healthy mesophyll cells. Both photorespiration and electro transfer during photosynthesis require light, which indicates major H2O2 production is light-dependent. Therefore, a high background level of H2O2 may exist in the light-grown plant, which may interfere with the observation of the H2O2effects in planta. The high background level of endogenous H2O2 in the light-grown seedlings may suggest that the oxidative effect has taken place and interfered with the observation of the H2O2 effects in planta, especially when investigating gene expression. In this study, the light-induced oxidative response was elucidated at the transcriptome level. The transcriptome profiles of plants grown under dark, light, and 5-mM H2O2 treatment under dark were investigated and compared. Each condition contained two biological replicates and total RNA extracted from 7-day-old seedlings were used for microarray analysis (Affymetrix Arabidopsis ATH1 Genome Array Microarray).. In total, 2050 and 8540 genes showed altered expression under H2O2 and light treatment, respectively (p < 0.01), accounting for about 10% and 37.5% of the Arabidopsistranscriptome, respectively. Interestingly, out of the 2050 H2O2-responsive genes, 1595 (77.8%) were also co-regulated by light. Among the 1595 genes, 1027 were up-regulated and 486 were down-regulated by both H2O2 and light, accounting for ∼95% of the co-regulated genes and 73.8% of H2O2-regulated genes. Co-expression was measured as Pearson’s correlation coefficient, which is ∼0.79 between the log2 transferred fold change in expression, indicating a high correlation between these two transcriptome profiles. The fact that a high percentage of H2O2-responsive genes are also regulated by light suggests the existence of H2O2 signaling under normal light conditions in Arabidopsis. The H2O2- and light-co-regulated genes were analyzed using the gene ontology (GO) enrichment plugin tool BiNGO. Over-represented GO categories were found enriched. Both the co-up- and down-regulated gene sets were analyzed for over-represented functions. It was found that co-up-regulated genes are highly related to plastid/nucleus functions and gene regulation function, such as nucleotide binding, protein binding, DNA binding, chromatin binding, transcription regulator activity, transcription process, and DNA metabolism. Other functions such as stress response-related, organelle organization, embryonic, and post-embryonic development were also enriched. Surprisingly, when the 486 co-down-regulated genes were analyzed, only the GO term of ‘response to biotic stress’ was enriched. The GO enrichment analysis indicated the genes co-up-regulated by light and H2O2 are involved in divergent biological processes and molecular functions, especially in gene expression regulation, but not restricted to stress-related. H2O2 is highly abundant in light-grown plants, indicating the active ROS signal pathways. Previous studies have implied the crosstalk between the light and H2O2 signal pathways. For example, Early Light Inducible Protein 2 (ELIP2) and Ascorbate Peroxidase 2 (APX2) genes were activated by both high light and H2O2 treatment, suggesting a H2O2-dependent pathway was involved in light stress signaling. Genome-wide expression study revealed that H2O2 plays a key role in the transcriptional up-regulation of small heat shock proteins during high-light stress. In addition, H2O2 was involved in high blue-light-induced chloroplast avoidance movements in Arabidopsis. These studies have revealed the ROS signal profiles in the high-light situation.

Artemisia annual L. is a common type of wormwood that grows throughout the world. Artemisinin, a terpene compound in Artemisia annua L, has recently been recognized as the most promising antimalaria drug. Artemisinin and other types of terpenoids are synthesized and accumulated in glandualr trichomes that appear on the surface of leaf, stem and flower bud. To understand the molecular basis of terpene biosynthesis in Artemisia annual L., a normalized glandular trichome cDNA collection was sequenced by Roche GS FLX pyrosequencing system. Two sequencing runs generated totally 85M nucleotides which were further assembled into 160000 unigenes (40000 contigs and 120000 sigletons). Putative functions were assigned to the unigenes based on Blast search against GeneBank database. Many terpene biosynthesis pathway genes were identified from the pyrosequencing ESTs, Among them, three putative sesquiterpene synthases SQS02039, SQS16267 and SQS14765 were identified and functional characterization are being carried out. Together with other identified Artemisia annual L. terpene pathway genes (Bouwmeester et al. 1999; Chang et al. 2000; Bertea et al. 2006; Teoh et al.2006), a global view of terpene biosynthsis in glandular trichomes of Artemisia annual L. were re-established. Meanwhile, two putative transcriptional factors, namely, bHLH35994 and WD24501 were identified. These two genes showed high sequence similarity at amino acid level with their Arabidopsis orthologs, which play key roles in determining trichome morphogenesis. Results from this research will shed lights on the molecular mechanism of glandular trichome morphogenesis and metabolic function in Artemisia annual L.

Dora Tombacz completed her PhD in Multidisciplinary Medicine, University of Szeged, Hungary, in the year 2009. Since then, she is working as an Assistant Professor in Department of Medical Biology at University of Szeged.

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 & 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 SMRT Bell 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.

Istvan Prazsak biologist received his diploma at the University of Szeged, Faculty of Sciences, Hungary. He worked at the Drosophila Stock Centre as student and as a PhD student at the Department of Genetics and Molecular Biology at the University of Szeged. He has worked at the Department of Medical Biology, Medical Faculty, University of Szeged as assistant research fellow as member of the Prof. Zsolt Boldogkői’s research group since 2010. His current research project is the generation of CTO mutant Pseudorabies virus with molecular cloning techniques. Beside his research Istvan is responsible for the German students at the institute.

Introduction: A newly discovered nearly 300 bp long RNA coding gene called CTO-S (“close to OriL-short”) was described in Pseudorabies virus (PRV) by analyzing PacBio and Illumina Hiseq full transcriptome sequencing data. CTO-S is situated between the ul22 and ul21 genes of the PRV genome and its putative promoter region is attached to the replication origin of the unique long (UL) viral genomic region. Our preliminary experiments show an abundant expression of the CTO-S transcripts which belong to the late kinetic group of PRV genes based on our Real-Time RT PCR data. To outline a concept about the function of CTO-S, we generated a mutant viral strain. Materials & Methods: 700 bp long viral sequences around the CTO-S gene were amplified by Pfu polymerase in a GC-rich buffer system (Roche). Primer pairs with recognition sequences of restriction modification enzymes (HindIII, EcoRI) were designed to generate flanking sequences of CTO-S for the nested PCR. The 5’ and 3’ flanking sequences were cloned into a pBSK-lacZ gene containing plasmid. To generate an insertion/deletion mutant virus strain, a lox-CMV-GFP-lox reporter gene cassette bracketed with EcoRI recognition sites was cloned between the 5’ and 3’ flanking sequences of CTO-S. Mutant PRV viruses were generated by homologous recombination transfecting PK-15 cells parallel with the wild type PRV (Kaplan strain) and the reporter gene cassette. Results: The two high GC-rich and repetitive sequence rich flanking regions of CTO-S were successfully amplified and cloned in a delivery pBSK plasmid vector. Both of them contain a HindIII linearizing restriction sites at the ends of the sequences and an insertion restriction site (EcoRI) at the middle of the construct. A CMV promoter driven GFP cassette embracing two tandem loxP sequences was inserted in two different orientations into the EcoRI site. Orientation of the reporter gene cassette was determined by traditional restriction digestion and PCR method. Activating loxP sequences by Crerecombinase produced a null mutant CTO-S PRV gene which could be further analyzed at transcriptomic level by Real Time RT PCR and sequencing.

Improvement of crop production will be required in order to feed the growing world population as the amount and quality of agricultural land decreases and salinity in soil increases. This has stimulated research to understand mechanisms of salt tolerance in plants. We have characterized the transcriptome of Suaeda fruticosa, an annual shrub in the family Chenopodiaceae that has the ability to sequester salts in its leaves. Twelve sequencing libraries prepared from control (0 mM NaCl treated) and optimum (300 mM NaCl treated) plants were sequenced using Illumina Hiseq 3000 to investigate differential gene expression between shoots and roots of Suaeda fruticosa. The reads were quality assessed, filtered and assembled de novo using Velvet and Oases k-45. We have clustered the transcripts using using CDHIT-EST. and these generated 65,069 transcripts. There are 475 downregulated transcripts and 44 upregulated transcripts when comparing the plants grown under optimal salt to those grown with no salt. BLAST analysis identified the differentially expressed genes and annotated with a cutoff E-value of 10-10. This work has identified those involved in mechanisms of salt tolerance in Suaeda fruticosa and has provided an outline of tools to use for de novo analysis of transcriptomes. The assembly has covered a considerable proportion of the transcriptome to analyze differential gene expression and identify specific genes that may be involved in salt tolerance in this plant. These data provide a genetic resource for discovery of potential genes for salt tolerance in this species and may serve as a reference sequence for study of other succulent halophytes.

Joseph A Piccirilli completed his Ph.D from Harvard University in 1989. Later he pursued Howard Hughes Postdoctoral research fellow from 1989-1993 from University of Colorado at Boulder. He joined as Assistant Professor at University of Chicago from 1993-2000. Currently he is working as an Investigator, Howard Hughes Medical Institute with joint appointment in the department of biochemistry and molecular biology.

Antibodies are powerful reagents for biomedical research, diagnostics, and therapeutics. The vast majority of antibody applications have focused on targeting features of the terrestrial proteome. Engineering antibodies to bind RNA directly offers a way to harness powerful technologies to study transcriptomes. We are developing phage display antibody libraries for engineering antibodies that recognize structured RNAs with high affinity and specificity. In one particular application, we are using the antibodies as chaperones for the crystallization and structure determination of RNAs.

As sessile organisms, plants are subjected to a wide range of abiotic and biotic stresses that affect their growth and development. Plants have evolved sophisticated adaptive responses involving reprogramming gene expression at the transcriptional, post-transcriptional and post-translational levels, and microRNAs in plants have firmly established as one of the master regulator of gene expression at the posttranscriptional level that control plant development, response to biotic and abiotic stress. The miR166 is one of the conserved miRNAs that exist in many dicot and monocot plant species including rice, wheat, Arapidopsis, soybean, etc. In this study, only Glyma09g02750 was found to be cleavage by gma-miR166 by 5’ RACE among 21 putative target genes under various stresses and subsequent sequence analysis of this gene Gyma09g02750 (named GmPHB), Class III Homeo Domain Leucine Zipper (HD-ZIP) family of transcription factors, is 88% similarity to Arabidopsis PHABULOSA. We investigated the effect of various stress on the expression of gma-miRNA166a and GmPHB. Gma-mIR166a was induced in various stress of different soybean genotype. Quantitative real-time PCR showed that the expression of GmPHB showed a significantly inverse correlation with corresponding gma-miR166a at different time courses. In addition, there are two DREs (dehydration-responsive element) in the upstream of the gma-miR166a by sequence analysis and suggested that miR-166a expression may be regulated by CBF/DREBs. These differentially expressed miRNAs can help in further study into the role of transcriptome homeostasis in the adaptation responses of soybean resistance to abiotic stresses.

Decapentaplegic (Dpp) is the homolog of vertebrate BMP-2 and BMP-4 and functionally interchangeable BMPs. The Bombyx mori (B. mori) and Bombyx mandarina (B. mandarina) Dpp genes share genetic homology with human BMPs and drosophila Dpp, but few studies have been executed to examine the functions of B. mori and B. mandarina Dpp and its function is not well understood. To date, there was also no reported splicing form of Dpp in silkworm. In this study, we investigated Dpp expression using synthesized cDNA from midgut tissue of B. mandarina by RT-PCR. Interestingly, lower band was discovered with band of full-length Dpp cDNA and it was identified as novel splicing form that a part (333 bp) of B. mandarina Dpp was deleted through DNA sequencing analysis. In addition, we found that the delated part in the variant was a portion of proprotein region compared to human BMP-2 and 7 candidate single nucleotide variants (SNVs) were able to affect formation of novel splicing form using variant calling analysis. To the best of our knowledge, this is the first approach to address the novel splicing form of Dpp in B. mandarina, which have been not found in B. mori. These results suggest that discovered splicing form of B. mandarina was degenerated in evolution process toward more advanced and domesticated B. mori.

Blood transcriptome reflects the status of diseases, and characteristic molecular signature provides a novel window on gene expression preceding acute coronary events. We aim to determine blood transcriptome-based molecular signature of acute coronary syndrome (ACS), and to identify novel serum biomarkers for early stage ST-segment-elevation myocardial infarction (STEMI). We obtained peripheral blood from the patients with ACS who visited emergency department within 4 hours after the onset of chest pain: STEMI (n=10), Non-ST-segment-elevation MI (NSTEMI, n=10) and unstable angina (UA, n=11). Blood transcriptome scans revealed that a characteristic gene expression change exists in STEMI, resulting in 531 outlier genes as STEMI molecular signature (Welch’s t test, P<0.05). Another analysis with a set of blood samples of patients with STEMI (n=7) before and 7 days after the primary percutaneous coronary intervention (n=7) and normal control (n=10) evidenced that STEMI molecular signature directly reflects the onset of STEMI pathogenesis. From the two sets of transcriptome-based STEMI signatures, we identified 10 genes encoding transmembrane or secretory proteins that are highly expressed in STEMI. We validated blood protein expression levels of these 10 putative biomarkers in 40 STEMI and 32 healthy subjects by ELISA. Data suggested that PGLYRP1, IRAK3 and VNN3 are more specific and sensitive diagnostic biomarkers for STEMI than traditional CK-MB or troponin. Blood transcriptome scans of ACS evidenced early stage molecular markers for STEMI. Here, we report novel biomarkers to diagnose STEMI at emergency department in hospitals by a simple ELISA method.

Sunghwa Choe has earned his PhD from University of Arizona and postdoctoral studies from Ceres, Inc., a California-based plant biotech company. He is a professor at Seoul National University, a premier University in South Korea. He has published more than 50 papers in reputed journals and has been serving as an editorial board member of repute journals including Molecules and Cells

Light plays critical roles in plant development. Depending on the light conditions, plants flexibly elongate or shorten their hypocotyls and leaves. Plant hormones including auxins, gibberellins, and brassinosteroids (BRs) regulate these processes acting alone or in concert. To understand the molecular signature of gene expression under the control of both light and BRs, we performed an Affymetrix genome-wide transcriptome analysis using total RNAs prepared from the four genotypes of Arabidopsis seedlings, Ws-2 wild type, phyB-77, bri1-5, and phyB-77 bri1-5 double. Using the list of genes filtered through the criteria of p < 0.01 and fold change > 1.5, we made three comparisons: phyB-77 vs. Ws-2; bri1-5 vs. Ws-2; and phyB-77 bri1-5 vs. Ws-2. Comparisons enabled us to find list of genes under epistatic interactions; bri1-5 was epistatic to phyB-77. The genes were further validated through quantitative RT-PCR and transgenic analysis. When At5g53870 was overexpressed in the bri1-5 mutant, the dwarf phenotype of bri1-5 was suppressed. Furthermore, we expressed two PEROXIDASEs (PRX2 and PRX73) that were downregulated in phyB-77 but were upregulated in bri1-5 in the antisense orientation; both PRX2 RNAi and PRX73 RNAi transgenic plants exhibited elongated hypocotyls, and the expression of the target genes was greatly reduced. Our transcriptome analysis followed by qRT-PCR and transgenic expression identified the genes that are critical in light-dependent growth of Arabidopsis.

The NIH (National Institutes of Health) Molecular Libraries and Imaging (MLI) initiative was funded as a cross-institutional effort to identify novel chemical compounds-probes that can interact with a cell or cell byproduct with precision. Between 2004 and 2013, MLI funded ten high-throughput Screening Centers resulting in the deposition of 5677 assays into PubChem and the nomination of 240 publicly available chemical probes. About 25% of these probes were found to be undesirable by a crowd-sourcing effort suggesting more rigorous probe evaluation methods are appropriate. In this study, we offer two in silico evaluation methods for probe quality assessment. In the first method, we annotated the 149 NIH probes and the 81 Bioassays with biomedical concepts using their probe synonyms and assay text descriptions. 8 of the 149 MLP probes are confirmed by this biomedical term matching method. For these 8 probes, overlapping concept identifiers might point to an agonist or antagonist related information revealing potential target protein-ligand information through the semantic ties. 4 of these 8 probes have been assessed medium-high reliable in an earlier crowd-sourced study. This is a promising support for text-mining based probe filtering as an in silico tool to include in future drug discovery initiatives. In the second part, we computed the percentage of probes that targets the same gene with an RNAi assay. An earlier study reported that 40% of the targets had no active probe compounds with potency better than 10 mM. When no such potent chemical exist, these targets could be alternatively silenced by RNA interference suggesting a potential to develop more potent drugs through the combined efforts of medicinal chemistry and siRNA therapeutics approaches. Among 240 existing chemical probes in PubChem, 8 of the targets were found active in an RNAi screening Bioassay.

Żaneta Matuszek is a graduate student of the College of Inter-faculty Individual Studies in Science and Mathematics at the University of Warsaw (Poland). She is involved in two majors: biotechnology and chemistry. Since 2011 she has been working in the Institute of Genetics and Biotechnology, University of Warsaw, in a Prof. Kufel’s lab of RNA metabolism in Eukaryotic cells. Żaneta is the organizer of the International Conference Aspects of Neuroscience held annually in Warsaw. Since January 2014 she has been a leader of her own research project granted by “Generacja Przyszłości” Polish Ministry of Science and Higher Education Funding Programme

Small nucleolar RNAs (snoRNAs) are the best characterized non-coding RNAs transcribed by RNA polymerase II. They are produced as precursors, whose extended 3’ ends are trimmed exonucleolytically, whereas 5’ ends either undergo combined endo- and exonucleolytic processing by Rnt1 and Rat1, respectively, or remain unchanged. In the latter case, the m7G cap becomes hypermethylated by Tgs1. cRT-PCR and northern blot analyses suggest that processing of snoRNA 3' and 5' termini is tightly coupled. Inhibition of 5’ end maturation, in rnt1Δ strain, cause the accumulation of snoRNA precursors that don't possess mature 3’ ends but carry polyadenylated extensions. This defect is further increased when components of the cap-binding complex (CBC) or methyltransferase Tgs1 are missing. Although we observe an accumulation of pre-snoRNAs in rnt1Δ strain, there are also mature snoRNAs present. These results suggest the existence of an alternative pathway of 5' end processing, that most likely involves pre-snoRNA cap removal by Dcp1/Dcp2 complex. Interestingly most pre-snoRNAs that accumulates in dcp2Δ strain, are not cleaved by Rnt1, which was confirmed by 5´ RACE analysis. Obtained data strongly suggest the existence of a quality control mechanism that coordinates 5’ and 3’ end processing. Results were obtained thanks to the financial support given by “Generacja Przyszłości” Ministry of Science and Higher Education Funding Programme.

Introduction
Pseudorabies virus (PRV) belongs to the Alphaherpesvirinae subfamily. PRV is a commonly used transsynaptic neural tracer and it is also a used as a model for the study of transcriptional gene regulation. The viral DNA genome is double-stranded, encodes 70 protein coding genes. Two latency associated long non-coding transcripts (LAT and AST) were previously described and characterized. In this project we have identified and characterized two, formerly unknown, long non coding RNAs of PRV.
Materials and Methods
Immortalized porcine kidney 15 (PK-15) cells were infected with Kaplan (Ka), ep0 knock-out (KO) mutant, or VHS KO-mutant strain of PRV. Total RNA were purified and cDNAs were synthesized from the samples. For the analysis of the abundance of the novel transcripts, Illumina sequencing and Real-Time RT PCR techniques were used. The kinetics of these transcripts was characterized by PacBio sequencing and Real-Time RT PCR. The effects of the KO mutations on the expression of the novel transcripts were tested by Real-Time RT PCR.
Results
Two novel, polyadenylated, 3’-coterminal non protein coding RNAs were identified using Illumina and PacBio sequencers as well as Northern-blot analysis. These transcripts (termed CTOs) located between the ul21 and ul22 genes, close to the replication origin of the viral genome. The length of the short intergenic lncRNA (CTO-S) is 286 base pairs. The longer (CTO-L) transcript overlaps OriL, and it is 2615 bp. CTO-L originates from the promoter of the ul21 gene. The expression levels of the two CTOs were higher than that of the wt PRV in the vhs KO virus. Our data show that the early protein 0 (encoded by the ep0 gene) exerts a down-regulatory effect on the transcription of CTOs throughout the whole life cycle of PRV.

Anjali Pai is an Engineer in Biotechnology, India and has completed her Masters in Cancer Biology from University College London. She is currently pursuing her PhD in Cancer Biology under the prestigious PhD Scholars Program in Cancer Biology , UCC, Ireland under the supervision of Dr. Pavel Baranov.

Translation initiation is considered to be the rate limiting step in mRNA translation. Translation initiation begins with the assembly of the preitiation complex of 40S ribosomal subunit and initiation factors at the mRNA 5' cap. The efficiency of ribosome loading may depend on the sequence specificity of the extreme 5' end of the mRNA. A well studied example of such dependency is the sensitivity of mRNAs containing 5'Terminal OligoPyrimidine tracts (5'TOP) to mTOR inhibition. To systematically explore how sequences at mRNA 5' end might affect translation efficiency we designed a library of mRNA molecules containing all possible decamers at mRNA 5' ends. Enrichment of particular decamers found in polysome fractions can be used as an indicator of efficient translation. Such an assay could be used to rank mRNA 5'ends in terms of their affect on translation efficiency under various cellular conditions including stress and cancer. Here we will present our progress in achieving this goal.

Sushma Venkat did her bachelor degree(2007) from Genetics as combination subjects, followed by Masters in Genetics (2009) and joined Ph.D in 2011 in Department of Genetics under the supervision of Prof. K. Rudrama Devi. She have a heartful interest in research and especially to work on woman related cancers. Thus, she choose a topic of Breast Cancer. She has collaborated with Indo American Cancer Hospitals for specimen collection and collected about 150 paired tissue specimens for the research work. Therefore she is into her submission of thesis and planning for Post Doctorate studies soon. Two of her manuscripts have been accepted in international journals. Have been working on 4 different gene polymorphisms and doing the correlation study with each impact on Breast Cancer.

Weak immunity surveillance has been the main reason for progression and rapid development of various cancers reported. Though the association between cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene 49 G/A polymorphism and breast cancer has been widely assessed, a definitive conclusion remains elusive and still focused by scientific community. The present study aims to evaluate the potential influences of CTLA-4 gene polymorphisms on breast cancer risk in invasive ductal carcinoma of tumor tissue and healthy tissues. Tumor tissue of forty confirmed breast cancer patients were included as cases. The same forty patients’ adjacent non-tumor tissue samples were considered as controls. The prevalence of AA, GG and AG genotypes was 42.1. %, 15.8.2% and 42.8% in the tumor tissue and non-tumor tissues respectively. Individuals containing tumor and non-tumor tissue samples showed significance in breast cancer, in which homozygous A/A and heterozygote G/A found to be more significant than G/G genotype. The study confirmed that the presence of at least one ‘A’ allele may increase the risk of breast cancer when compared to the presence of ‘G’ allele in same patient.

Sujitha Mariappan has completed B.Tech Biotechnology from Kalasalingam University 2011 and currently pursuing final year M.Tech Biotechnology from Karunya University, India. She has communicated one paper in Thomson Reuter’s journal on title “Association of the SHANK3 gene mutations coupled with 22q13.3 deletion syndrome”. She has also participated in “Stimulating Bio-Entrepreneurs Talk” conducted by ABLE Biotech. Attended national level workshop on “Recent Techniques in Cell Culture Cytotoxity Assay” at Karunya University, Coimbatore.

Phelan-McDermid syndrome (PMS) is a postnatal neurodegenerative disorder which is caused by the mutations in the gene called SHANK3 which is a member of the highly conserved SHANK family of synaptic scaffolding proteins. The function of SHANK3 is to connect ion channels and receptors in the post-synaptic membrane to the cytoskeleton and to signal transduction pathways. The disruption of SHANK3(Src homology 3 (SH3) and multiple ankyrin repeat domains 3) / ProSAP2(Proline-rich synapse-associated protein 2) gene by balanced rearrangement and the alteration of SHANK3 by a frame shift mutation lead to the language deficits and autistic characters of PMS and also SHANK3 has increased actin polymerization and promoted spine formation. Haploinsufficiency of SHANK3 gene is the cause of the major neurological features associated with 22q13 deletion. The PMS is a microdeletion syndrome resulting from loss of 22q13 by simple deletion, unbalanced translocation, ring chromosome formation, or other irregular structural change. PMS is characterized by developmental delay, delayed or absent speech, autistic-like behavior, normal to accelerated growth, and minor dysmorphic features including dolichocephaly, full brow and large or prominent ears. The genetic alterations that lead to the syndrome will mainly affect the gene SHANK3 which codes for a scaffold protein localized in the postsynaptic densities of excitatory synapses, connecting membrane receptors to cytoskeleton. The present aim is to explain in depth of the clinical and genetic facets of the PMS, with more concentration on the domain architecture of SHANK3 protein, by shedding light on the mutations involved in the protein families.

Hyalomma dromedarii is a common parasite of the camel, exophilic preferring a warm and wet climate. It is probably the most completely desert- adapted of all hard ticks. In Tunisia, H. dromedarii is found in the southern region, mainly in Gafsa and Tataouine Kebelli. Similarly to other hematophagous, the salivary glands of the slower feeding hard ticks such as H. dromedarii might be a rich source of many compounds with biological activities that interact directly with host systems, such as blood clotting, platelet aggregation, cell death, among others. Knowledge of the tick salivary protein repertoire may lead to vaccine targets to disrupt feeding and/or parasite transmission as well as to the discovery of novel pharmacological agents. Male saliva may also assist reproduction because males use their mouthparts to lubricate and introduce their spermatophores into the females' genital pore. The analyses of the sialomes of male and female ticks independently allow us to understand the strategy used by each gender to feed successfully. We sequenced cDNA libraries from pools of salivary glands from adult male and female Hyalomma dromedarii Salivary gland extract, using the Illumina HiSeq protocol.We have also generated the proteome, from the salivary gland extracts of male and female H. dromedarii,resulting a total of 1740 and 923 proteins respectively which were identified by one or more peptides with at least 95% confidence. Our results showed a complex composition of H. dromedarii SGE, in a good agreement with the complexity of the ixodid sialomes. The most representative proteins found were housekeeping/intracellular, hemelipoproteins and other transporter proteins; conserved proteins, lipocalins, peptidase inhibitors, glycine-rich proteins, and host proteins… This study represents the first sialotranscriptomic/ proteomic study on H. dromedarii salivary glands. Our results assist the understanding of the tick-host molecules interaction during the blood feeding and discovery of new pharmacologically active proteins of the ticks of Hyalomma genera.

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 (PBMs) represent a major systemic cell type for bone metabolism by serving as progenitors of osteoclasts and producing cytokines important for osteoclastogenesis. Protein coding genes for osteoporosis have been widely studied by mRNA analyses of monocytes in high vs. low hip BMD (bone mineral density) subjects. However, long non-coding RNAs (lncRNAs), which account for a large proportion of human transcriptome, are few reported. In our study, microarray analyses of monocytes were performed using Affymetrix 1.0 ST arrays in 73 Caucasian females (age: 47-56) with extremely high (mean ZBMD =1.38, n=42, 16 pre- and 26 postmenopausal subjects) or low hip BMD (mean ZBMD=-1.05, n=31, 15 pre- and 16 postmenopausal subjects). LncRNA profile was analyzed by re-annotating exon array for lncRNAs detection via noncoder software. 575 lncRNAs were differentially expressed between the two groups. In high BMD subject, 309 lncRNAs were upregulated and 266 were downregulated. To investigate the relationship between protein coding genes and lncRNAs, we identified the genes transcribed within 10kb nearby lncRNAs via UCSC Genome Browser. Four genes and lncRNAs pairs were found to be significantly correlated and cooperatively differentially expressed in high vs. low BMD subjects. In these four pairs, the protein coding genes, NR4A1 and ABCG1, have been reported to be important for monocytes differentiation and survival in tissues. Overall, our findings for the first time reported the lncRNAs profiles for osteoporosis and may suggest the regulatory mechanism between lncRNAs and protein coding genes in bone metabolism.

Since the introduction of microarray technology, a variety of statistical methods have been developed in an endeavor to reconstruct the underlying dynamics of transcription regulated gene networks. Pairwise correlation coefficients have been calculated to create gene co-expression networks. Co-expression networks have been used to predict gene function through association with functionally related clusters. PPI networks have served very similar predictive purposes. Like PPI networks, co-expression networks are static networks and therefore lack the temporal features that yield the hierarchical structure necessary for representing causal dynamics. In the present work, we report on the use of GeneNet to infer Arabidopsis gene regulatory networks (GRNs) from microarray data. GeneNet computes partial correlation coefficients to generate edge associations (topology) and partial variances are computed to infer edge direction (causality). GRNs were inferred from the AtGenExpress microarray data sets cataloged at GEO. We compared the edge sets of the GRNs to edge sets of Arabidopsis co-expression networks generated from the AtGenExpress microarray data and to the edge set belonging to the Bio-Analytical Resource Arabidopsis PPI. Furthermore, we evaluated the topology of all three types of networks for graph theoretical coefficients unique to topological features related to the propagation of information throughout the underlying biological organization. We report that the use of graph theoretical analyses demonstrates that the structure of gene regulatory networks differs from the structure of both co-expression and PPI networks.