References of "O'Grady, Tina"
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See detailTumor microenvironment affects the composition of endothelial-derived exosomes: impact in tumor progression
Njock, Makon-Sébastien ULiege; O'Grady, Tina ULiege; Nivelles, Olivier ULiege et al

Conference (2018, September 13)

The tumor microenvironment plays a crucial role in the progression of tumor growth and metastasis by deregulating various physiological processes including angiogenesis and inflammation. Several studies ... [more ▼]

The tumor microenvironment plays a crucial role in the progression of tumor growth and metastasis by deregulating various physiological processes including angiogenesis and inflammation. Several studies have previously demonstrated that tumor-derived exosomes are actively involved in the mediation of tumorigenesis by “reprogramming” target cells (e.g. endothelial cells (ECs)) through transfer of pro-angiogenic microRNAs. But the function of exosomes released by target cells is poorly studied. Consequently, we sought to determine the composition of exosomes released by ECs under tumor microenvironment, and to assess whether these vesicles present different functional properties. Using RNA-seq approaches, we demonstrated that exosomes released by ECs in tumor microenvironment context present a specific repertory of microRNAs associated to tumor angiogenesis and inflammatory pathways. Furthermore, we showed that these vesicles were able to optimize inflammatory response of immune cells by transferring several dysregulated microRNAs. Currently, we are identifying the molecular pathways modulated by endothelial-derived exosomes in tumor microenvironment. [less ▲]

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See detailIntegrating long and short sequencing data for a global overview of the endothelial extracellular vesicle RNA landscape
O'Grady, Tina ULiege; Njock, Makon-Sébastien ULiege; Lion, Michelle ULiege et al

Poster (2018, May)

Background: Although extracellular vesicles (EVs) are well known to be enriched in miRNAs and other short RNA species, long RNA transcripts such as mRNA and lncRNA have been reported by several groups. We ... [more ▼]

Background: Although extracellular vesicles (EVs) are well known to be enriched in miRNAs and other short RNA species, long RNA transcripts such as mRNA and lncRNA have been reported by several groups. We use RNA-Seq analysis in conjunction with wet bench techniques to provide a global analysis of the RNA content of endothelial cell EVs. Methods: EVs from primary human umbilical vein endothelial cell (HUVEC) cultures were isolated by sequential ultracentrifugation and immunocapture. RNA was extracted from cells and EVs, then short and long RNA libraries were prepared and sequenced. Reads were mapped to the human genome and transcriptome and mapped reads were analysed to determine transcript type and differential abundance between cells and EVs. RT-PCR was used to investigate integrity of long RNA transcripts. Gene ontology analyses were performed to determine enrichment of functional terms. Results: RNA in primary endothelial EVs is highly diverse in terms of length, type and abundance. As expected, endothelial EV RNA content is dominated by short RNA molecules, in particular snRNA and piRNA. Additionally, long rRNA, mRNA and lncRNA transcripts are present. Many of these transcripts are intact, putatively functional transcripts and are detectable at robust levels. Analysis of differential abundance between EVs and cells reveals significant differences in miRNA, snRNA, piRNA, mRNA and lncRNA profiles. LncRNAs in particular show a striking distribution, with about 13 times more lncRNA transcripts being enriched in EVs than in cells. Few of these lncRNAs have been fully functionally characterized, but gene ontology analysis of EV-enriched mRNA transcripts reveals an overabundance of genes coding for ribosomal proteins, elongation factors and other translation-related proteins. Summary/conclusion: Endothelial EVs are enriched for short and long regulatory RNA with the potential to control gene expression at both transcriptional and post-transcriptional levels. [less ▲]

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See detailAnalysis of EBV Transcription Using High-Throughput RNA Sequencing.
O'Grady, Tina ULiege; Baddoo, Melody; Flemington, Erik K.

in Methods in Molecular Biology (2017), 1532

High-throughput sequencing of RNA is used to analyze the transcriptomes of viruses and cells, providing information about transcript structure and abundance. A wide array of programs and pipelines has ... [more ▼]

High-throughput sequencing of RNA is used to analyze the transcriptomes of viruses and cells, providing information about transcript structure and abundance. A wide array of programs and pipelines has been created to manage and interpret the abundance of data generated from high-throughput RNA sequencing experiments. This protocol details the use of free and open-source programs to align RNA-Seq reads to a reference genome, visualize read coverage and splice junctions, estimate transcript abundance, and evaluate differential expression of transcripts in different conditions. Particular concerns related to EBV and viral transcriptomics are addressed and access to EBV reference files is provided. [less ▲]

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See detailTRIMD: Transcriptome Resolution by Integration of Multi-platform Data
O'Grady, Tina ULiege

Software (2016)

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See detailGlobal transcript structure resolution of high gene density genomes through multi-platform data integration.
O'Grady, Tina ULiege; Wang, Xia; Honer Zu Bentrup, Kerstin et al

in Nucleic Acids Research (2016), 44(18), 145

Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second ... [more ▼]

Annotation of herpesvirus genomes has traditionally been undertaken through the detection of open reading frames and other genomic motifs, supplemented with sequencing of individual cDNAs. Second generation sequencing and high-density microarray studies have revealed vastly greater herpesvirus transcriptome complexity than is captured by existing annotation. The pervasive nature of overlapping transcription throughout herpesvirus genomes, however, poses substantial problems in resolving transcript structures using these methods alone. We present an approach that combines the unique attributes of Pacific Biosciences Iso-Seq long-read, Illumina short-read and deepCAGE (Cap Analysis of Gene Expression) sequencing to globally resolve polyadenylated isoform structures in replicating Epstein-Barr virus (EBV). Our method, Transcriptome Resolution through Integration of Multi-platform Data (TRIMD), identifies nearly 300 novel EBV transcripts, quadrupling the size of the annotated viral transcriptome. These findings illustrate an array of mechanisms through which EBV achieves functional diversity in its relatively small, compact genome including programmed alternative splicing (e.g. across the IR1 repeats), alternative promoter usage by LMP2 and other latency-associated transcripts, intergenic splicing at the BZLF2 locus, and antisense transcription and pervasive readthrough transcription throughout the genome. [less ▲]

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See detailExamining lncRNAs in highly transcribed, gene-dense genomes
O'Grady, Tina ULiege; Wang, Xia; Cao, Subing et al

Poster (2015, October)

Recent studies using high-throughput methods have revealed an extraordinarily complex transcriptional landscape across the genomes of multiple herpesviruses, with noncoding RNA transcripts overlapping and ... [more ▼]

Recent studies using high-throughput methods have revealed an extraordinarily complex transcriptional landscape across the genomes of multiple herpesviruses, with noncoding RNA transcripts overlapping and antisense to protein-coding genes as well as arising from presumed intergenic regions. This pervasive transcription across dense viral genomes presents challenges in correctly delineating individual transcripts and their functions. We have used Pacific Biosciences SMRT sequencing and Illumina RNA-seq to generate detailed transcriptome annotation for the ubiquitous human gammaherpesvirus Epstein-Barr virus (EBV) during lytic reactivation. We report the structures of over 100 novel polyadenylated transcripts, more than doubling the number of known EBV lytic genes. More detailed investigations into several specific transcripts reveal viral “late” gene kinetics, suggesting roles in processes such as viral genome packaging or virion assembly, though functional assays indicate that some of these transcripts influence viral and cellular mRNA levels. These multifold novel transcripts likely possess as diverse an array of functions as their protein-coding counterparts and could represent the key to elucidating some of the enduring mysteries of the herpesvirus lytic cycle, such as control of late gene expression and virion envelopment and egress. [less ▲]

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See detailNovel IL20 isoforms in EBV-infected B cells
O'Grady, Tina ULiege; Flemington, Erik K.

Poster (2015, March)

Members of the IL20 subfamily of cytokines, including IL19, IL20, IL22, IL24 and IL26, have been implicated as messengers in the communication between immune system cells and epithelial cells. Upon ... [more ▼]

Members of the IL20 subfamily of cytokines, including IL19, IL20, IL22, IL24 and IL26, have been implicated as messengers in the communication between immune system cells and epithelial cells. Upon stimulation with microbial products or other cytokines, myeloid and lymphoid cells can produce IL20 cytokines. These cytokines stimulate epithelial cells to proliferate and differentiate, produce antimicrobial peptides and secrete pro-inflammatory cytokines and chemokines1. In this project, second- and third-generation RNA sequencing has revealed alternative isoforms of IL19 and IL20 initiating from a novel promoter that is antisense to the more distantly related IL10. Expression of these isoforms is induced during B-cell receptor signaling and viral replication in the Epstein Barr Virus (EBV)-infected B-cell line Akata. These novel isoforms may represent additional insights into the transcriptional and translational control of the IL10 family and its IL20 subfamily of cytokines. [less ▲]

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See detailNew Noncoding Lytic Transcripts Derived from the Epstein-Barr Virus Latency Origin of Replication, oriP, Are Hyperedited, Bind the Paraspeckle Protein, NONO/p54nrb, and Support Viral Lytic Transcription.
Cao, Subing; Moss, Walter; O'Grady, Tina ULiege et al

in Journal of Virology (2015), 89(14), 7120-32

UNLABELLED: We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV ... [more ▼]

UNLABELLED: We have previously shown that the Epstein-Barr virus (EBV) likely encodes hundreds of viral long noncoding RNAs (vlncRNAs) that are expressed during reactivation. Here we show that the EBV latency origin of replication (oriP) is transcribed bi-directionally during reactivation and that both leftward (oriPtLs) and rightward (oriPtRs) transcripts are largely localized in the nucleus. While the oriPtLs are most likely noncoding, at least some of the oriPtRs contain the BCRF1/vIL10 open reading frame. Nonetheless, oriPtR transcripts with long 5' untranslated regions may partially serve noncoding functions. Both oriPtL and oriPtR transcripts are expressed with late kinetics, and their expression is inhibited by phosphonoacetic acid. RNA sequencing (RNA-seq) analysis showed that oriPtLs and oriPtRs exhibited extensive "hyperediting" at their Family of Repeat (FR) regions. RNA secondary structure prediction revealed that the FR region of both oriPtLs and oriPtRs may form large evolutionarily conserved and thermodynamically stable hairpins. The double-stranded RNA-binding protein and RNA-editing enzyme ADAR was found to bind to oriPtLs, likely facilitating editing of the FR hairpin. Further, the multifunctional paraspeckle protein, NONO, was found to bind to oriPt transcripts, suggesting that oriPts interact with the paraspeckle-based innate antiviral immune pathway. Knockdown and ectopic expression of oriPtLs showed that it contributes to global viral lytic gene expression and viral DNA replication. Together, these results show that these new vlncRNAs interact with cellular innate immune pathways and that they help facilitate progression of the viral lytic cascade. IMPORTANCE: Recent studies have revealed that the complexity of lytic herpesviral transcriptomes is significantly greater than previously appreciated with hundreds of viral long noncoding RNAs (vlncRNAs) being recently discovered. Work on cellular lncRNAs over the past several years has just begun to give us an initial appreciation for the array of functions they play in complex formation and regulatory processes in the cell. The newly identified herpesvirus lncRNAs are similarly likely to play a variety of different functions, although these functions are likely tailored to specific needs of the viral infection cycles. Here we describe novel transcripts derived from the EBV latency origin of replication. We show that they are hyperedited, that they interact with a relatively newly appreciated antiviral pathway, and that they play a role in facilitating viral lytic gene expression. These investigations are a starting point to unraveling the complex arena of vlncRNA function in herpesvirus lytic replication. [less ▲]

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See detailHigh-throughput RNA sequencing-based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project.
Cao, Subing; Strong, Michael J.; Wang, Xia et al

in Journal of Virology (2015), 89(1), 713-29

UNLABELLED: Using high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the ... [more ▼]

UNLABELLED: Using high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus type 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of murine leukemia virus (MuLV) transcripts was observed in DEL cells, and we identified four transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes, including interleukin-15 (IL-15), IL-6ST, STAT5B, HIVEP1, and IL-9R. Although KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL-10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL-6, K4/vIL-8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles. IMPORTANCE: Viruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus type 1 are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes, including cytokine regulators, that are disrupted by virus integration, and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL-10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas. [less ▲]

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See detailGlobal bidirectional transcription of the Epstein-Barr virus genome during reactivation.
O'Grady, Tina ULiege; Cao, Subing; Strong, Michael J. et al

in Journal of Virology (2014), 88(3), 1604-16

Epstein-Barr virus (EBV) reactivation involves the ordered induction of approximately 90 viral genes that participate in the generation of infectious virions. Using strand-specific RNA-seq to assess the ... [more ▼]

Epstein-Barr virus (EBV) reactivation involves the ordered induction of approximately 90 viral genes that participate in the generation of infectious virions. Using strand-specific RNA-seq to assess the EBV transcriptome during reactivation, we found extensive bidirectional transcription extending across nearly the entire genome. In contrast, only 4% of the EBV genome is currently bidirectionally annotated. Most of the newly identified transcribed regions show little evidence of coding potential, supporting noncoding roles for most of these RNAs. Based on previous cellular long noncoding RNA size calculations, we estimate that there are likely hundreds more EBV genes expressed during reactivation than was previously known. Limited 5' and 3' rapid amplification of cDNA ends (RACE) experiments and findings of novel splicing events by RNA-seq suggest that the complexity of the viral genome during reactivation may be even greater. Further analysis of antisense transcripts at some of the EBV latency gene loci showed that they are "late" genes, they are nuclear, and they tend to localize in areas of the nucleus where others find newly synthesized viral genomes. This raises the possibility that these transcripts perform functions such as new genome processing, stabilization, organization, etc. The finding of a significantly more complex EBV transcriptome during reactivation changes our view of the viral production process from one that is facilitated and regulated almost entirely by previously identified viral proteins to a process that also involves the contribution of a wide array of virus encoded noncoding RNAs. Epstein-Barr virus (EBV) is a herpesvirus that infects the majority of the world's population, in rare cases causing serious disease such as lymphoma and gastric carcinoma. Using strand-specific RNA-seq, we have studied viral gene expression during EBV reactivation and have discovered hundreds more viral transcripts than were previously known. The finding of alternative splicing and the prevalence of overlapping transcripts indicate additional complexity. Most newly identified transcribed regions do not encode proteins but instead likely function as noncoding RNA molecules which could participate in regulating gene expression, gene splicing or even activities such as viral genome processing. These findings broaden the scope of what we need to consider to understand the viral manufacturing process. As more detailed studies are undertaken they will likely change the way we view this process as a whole. [less ▲]

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See detailEpstein-Barr virus and human herpesvirus 6 detection in a non-Hodgkin's diffuse large B-cell lymphoma cohort by using RNA sequencing.
Strong, Michael J.; O'Grady, Tina ULiege; Lin, Zhen et al

in Journal of Virology (2013), 87(23), 13059-62

Comprehensive virome analysis of RNA sequence (RNA-seq) data sets from 118 non-Hodgkin's B-cell lymphomas revealed a small subset that is positive for Epstein-Barr virus (EBV) or human herpesvirus 6B (HHV ... [more ▼]

Comprehensive virome analysis of RNA sequence (RNA-seq) data sets from 118 non-Hodgkin's B-cell lymphomas revealed a small subset that is positive for Epstein-Barr virus (EBV) or human herpesvirus 6B (HHV-6B), with one coinfection. EBV transcriptome analysis revealed expression of the latency genes RPMS1, LMP1, and LMP2, with one sample additionally showing a high level of early lytic expression and another sample showing a high level of EBNA2 expression. HHV-6B transcriptome analysis revealed that the majority of genes were transcribed. [less ▲]

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See detailLong noncoding RNA expression in lymphoma
O'Grady, Tina ULiege; Flemington, Erik K.

Poster (2013)

Diffuse large B cell lymphoma (DLBCL), the most common non-Hodgkin’s lymphoma, is an aggressive lymphoma that affects patients worldwide. Although it is curable in some cases, many patients remain ... [more ▼]

Diffuse large B cell lymphoma (DLBCL), the most common non-Hodgkin’s lymphoma, is an aggressive lymphoma that affects patients worldwide. Although it is curable in some cases, many patients remain refractory to treatment and a better understanding of disease mechanisms could lead to improved therapies. Using expression data from protein-coding genes DLBCL can be classified into the activated B cell (ABC) and germinal centre B cell (GCB) subtypes (1), which are predictive of different outcomes (2). However, many tumours remain unclassifiable with this data. Furthermore, DLBCL can be difficult to distinguish from the more easily treatable Burkitt’s Lymphoma (BL) using histology and even protein-coding gene expression (3). The proliferation of publicly available datasets and lncRNA annotation offers the opportunity to re-examine this clinically important problem. We are using RNA-Seq datasets from DLBCL and BL available from the NCBI’s Sequence Read Archive (4, 5) in conjunction with the transcript assembler Cufflinks and noncoding RNA catalogs from the ENCODE project and John Rinn’s laboratory (6) to detect and quantify novel and known long noncoding transcripts in these tumours. Using hierarchical clustering and differential expression analysis we are characterizing lncRNA expression profiles in both established and lncRNA-defined subtypes. In addition, both DLBCL and BL are known in some cases to be driven by Epstein-Barr Virus (EBV), a factor that is potentially confounding to attempts to classify tumours and characterize pathways. Using the RNA Comprehensive Multi-Processor Analysis System for Sequencing (RNA CoMPASS) developed in our laboratory (7), we are able to sensitively detect and quantify EBV RNA expression in RNA-Seq datasets. LncRNA expression in different tumours can then be analyzed in this context, and [less ▲]

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See detailAnnTools: a comprehensive and versatile annotation toolkit for genomic variants.
Makarov, Vladimir; O'Grady, Tina ULiege; Cai, Guiqing et al

in Bioinformatics (2012), 28(5), 724-5

UNLABELLED: AnnTools is a versatile bioinformatics application designed for comprehensive annotation of a full spectrum of human genome variation: novel and known single-nucleotide substitutions (SNP/SNV ... [more ▼]

UNLABELLED: AnnTools is a versatile bioinformatics application designed for comprehensive annotation of a full spectrum of human genome variation: novel and known single-nucleotide substitutions (SNP/SNV), short insertions/deletions (INDEL) and structural variants/copy number variation (SV/CNV). The variants are interpreted by interrogating data compiled from 15 constantly updated sources. In addition to detailed functional characterization of the coding variants, AnnTools searches for overlaps with regulatory elements, disease/trait associated loci, known segmental duplications and artifact prone regions, thereby offering an integrated and comprehensive analysis of genomic data. The tool conveniently accepts user-provided tracks for custom annotation and offers flexibility in input data formats. The output is generated in the universal Variant Call Format. High annotation speed makes AnnTools suitable for high-throughput sequencing facilities, while a low-memory footprint and modest CPU requirements allow it to operate on a personal computer. The application is freely available for public use; the package includes installation scripts and a set of helper tools. AVAILABILITY: http://anntools.sourceforge.net/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. [less ▲]

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See detailDissecting p63 function in renal development: a ChIP-Seq approach
O'Grady, Tina ULiege; Liu, J; Saifudeen, Z et al

Poster (2012)

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See detailSurvival and success beyond grad school: improving library services to postdoctoral researchers
McCrillis, A; Vieira, D; Beam, P et al

Conference (2012)

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See detailPostdoctoral scholars: a forgotten library constituency?
O'Grady, Tina ULiege; Beam, P. S.

in Science & Technology Libraries (2011), 30(1), 76-79

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See detailFraming the clinical question: a collaboration to enhance data warehouse user education
Anderson, E; O'Grady, Tina ULiege

Poster (2011)

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