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Identification of transcriptome signature for myocardial reductive stress

Justin M. Quiles, Madhusudhanan Narasimhan, Timothy Mosbruger, Gobinath Shanmugam, David Crossman, Namakkal S. Rajasekaran
2017 Redox Biology  
A B S T R A C T The nuclear factor erythroid 2 like 2 (Nfe2l2/Nrf2) is a master regulator of antioxidant gene transcription. We recently identified that constitutive activation of Nrf2 (CaNrf2) caused reductive stress (RS) in the myocardium. Here we investigate how chronic Nrf2 activation alters myocardial mRNA transcriptome in the hearts of CaNrf2 transgenic (TG-low and TG-high) mice using an unbiased integrated systems approach and next generation RNA sequencing followed by qRT-PCR methods. A
more » ... total of 246 and 1031 differentially expressed genes (DEGs) were identified in the heart of TGL and TGH in relation to NTG littermates at~6 months of age. Notably, the expression and validation of the transcripts were gene-dosage dependent and statistically significant. Ingenuity Pathway Analysis identified enriched biological processes and canonical pathways associated with myocardial RS in the CaNrf2-TG mice. In addition, an overrepresentation of xenobiotic metabolic signaling, glutathionemediated detoxification, unfolded protein response, and protein ubiquitination was observed. Other, non-canonical signaling pathways identified include: eNOS, integrin-linked kinase, glucocorticoid receptor, PI3/AKT, actin cytoskeleton, cardiac hypertrophy, and the endoplasmic reticulum stress response. In conclusion, this mRNA profiling identified a "biosignature" for pro-reductive (TGL) and reductive stress (TGH) that can predict the onset, rate of progression, and clinical outcome of Nrf2-dependent myocardial complications. We anticipate that this global sequencing analysis will illuminate the undesirable effect of chronic Nrf2 signaling leading to RSmediated pathogenesis besides providing important guidance for the application of Nrf2 activation-based cytoprotective strategies. http://dx.
doi:10.1016/j.redox.2017.07.013 pmid:28768233 pmcid:PMC5536881 fatcat:zjcgtot6vjecfkptvzm2ieqxi4

Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth

Jared Wallace, Ruozhen Hu, Timothy L. Mosbruger, Timothy J. Dahlem, W. Zac Stephens, Dinesh S. Rao, June L. Round, Ryan M. O'Connell, Daniel T Starczynowski
2016 PLoS ONE  
Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human micro-RNAs that suppress or promote cell growth, revealing that
more » ... roRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how micro-RNAs contribute to human disease.
doi:10.1371/journal.pone.0153689 pmid:27081855 pmcid:PMC4833428 fatcat:bkvfpr73jbdkvn3ifgbkhccjj4

AnthOligo: Automating the design of oligonucleotides for capture/enrichment technologies [article]

Pushkala Jayaraman, Timothy Mosbruger, Taishan Hu, Nikolaos G Tairis, Chao Wu, Peter M Clark, Monica D'Arcy, Deborah Ferriola, Katarzyna Mackiewicz, Xiaowu Gai, Dimitrios Monos, Mahdi Sarmady
2019 bioRxiv   pre-print
AbstractSummaryA number of methods have been devised to address the need for targeted genomic resequencing. One of these methods, Region-specific extraction (RSE) of DNA is characterized by the capture of long DNA fragments (15-20 kb) by magnetic beads, after enzymatic extension of oligonucleotides hybridized to selected genomic regions. Facilitating the selection of the most optimal capture oligos targeting a region of interest, satisfying the properties of temperature (Tm) and entropy (ΔG),
more » ... ile minimizing the formation of primer dimers in a pooled experiment is therefore necessary. Manual design and selection of oligos becomes an extremely arduous task complicated by factors such as length of the target region and number of targeted regions. Here we describe, AnthOligo, a web-based application developed to optimally automate the process of generation of oligo sequences to be used for the targeting and capturing the continuum of large and complex genomic regions. Apart from generating oligos for RSE, this program may have wider applications in the design of customizable internal oligos to be used as baits for gene panel analysis or even probes for large-scale comparative genomic hybridization (CGH) array processes.Implementation and AvailabilityThe application written in Java8 and run on Tomcat9 is a lightweight Java Spring MVC framework that provides the user with a simple interface to upload an input file in BED format and customize parameters for each task. A Redis-like MapReduce framework is implemented to run sub-tasks in parallel to optimize time and system resources alongside a 'task-queuing' system that runs submitted jobs as a server-side background daemon. The task of probe design in AnthOligo commences when a user uploads an input file and concludes with the generation of a result-set containing an optimal set of region-specific oligos.AnthOligo is currently available as a public web application with URL: http://antholigo.chop.edu.
doi:10.1101/2019.12.12.873497 fatcat:nczdd5mq55bxrofvig6rj27kgq

Nkx2.5-dependent alterations of the embryonic heart DNA methylome identify novel cis-regulatory elements in cardiac development [article]

Bushra Gorsi, Timothy Mosbruger, Megan Smith, Jonathon Hill, H. Joseph Yost
2017 bioRxiv   pre-print
Transcription factor Nkx2.5 is frequently mutated in congenital heart disease, but the mechanisms by which Nkx2.5 regulates heart development are poorly understood. By generating comprehensive DNA methylome maps from zebrafish embryonic hearts in nxk2.5 mutants and siblings, we discovered that Nkx2.5 regulates DNA methylation patterns during cardiac morphogenesis. We identified hundreds of Nkx-dependent heart specific Differentially Methylated Regions (nhDMRs). A majority of the nhDMRs were
more » ... methylated in nkx2.5-/- hearts, correlating with changes in the mutant transcriptome, suggesting Nkx2.5 functions largely as a repressor. Distinct Nkx DNA-binding motifs were significantly enriched in subclasses of nhDMRs. Furthermore, nhDMRs were significantly associated with histone H3K4me1 and H3K27ac posttranslational modifications, suggesting Nkx2.5 regulates gene expression by differential methylation of cis-regulatory elements. Using transgenics, we validated several nhDMRs with enhancer activities in the heart. We propose a novel role of Nkx2.5 mediated DNA methylation is integral in activating and repressing Nkx2.5 target genes during heart development.
doi:10.1101/186395 fatcat:vr3ohs74frbzhefqnfofbjtwci

Nucleotide resolution analysis ofTMPRSS2andERGrearrangements in prostate cancer

Christopher Weier, Michael C Haffner, Timothy Mosbruger, David M Esopi, Jessica Hicks, Qizhi Zheng, Helen Fedor, William B Isaacs, Angelo M. De Marzo, William G Nelson, Srinivasan Yegnasubramanian
2013 Journal of Pathology  
TMPRSS2-ERG rearrangements occur in approximately 50% of prostate cancers and therefore represent one of the most frequently observed structural rearrangements in all cancers. However, little is known about the genomic architecture of such rearrangements. We therefore designed and optimized a pipeline involving target-capture of TMPRSS2 and ERG genomic sequences coupled with paired-end next generation sequencing to resolve genomic rearrangement breakpoints in TMPRSS2 and ERG at nucleotide
more » ... tion in a large series of primary prostate cancer specimens (n = 83). This strategy showed >90% sensitivity and specificity in identifying TMPRSS2-ERG rearrangements, and allowed identification of intra-and inter-chromosomal rearrangements involving TMPRSS2 and ERG with known and novel fusion partners. Our results indicate that rearrangement breakpoints show strong clustering in specific intronic regions of TMPRSS2 and ERG. The observed TMPRSS2-ERG rearrangements often exhibited complex chromosomal architecture associated with several intra-and inter-chromosomal rearrangements. Nucleotide resolution analysis of breakpoint junctions revealed that the majority of TMPRSS2 and ERG rearrangements (~88%) occurred at or near regions of microhomology or involved insertions of one or more base pairs. This architecture implicates nonhomologous end joining (NHEJ) and microhomology mediated end joining (MMEJ) pathways in the generation of such rearrangements. These analyses have provided important insights into the molecular mechanisms involved in generating prostate cancer-specific recurrent rearrangements.
doi:10.1002/path.4186 pmid:23447416 pmcid:PMC3668093 fatcat:vwnogyhsgfhipk2dgo46ialqne

Comprehensive analyses of CD8+ T cell responses during longitudinal study of acute human hepatitis C

Andrea L. Cox, Timothy Mosbruger, Georg M. Lauer, Drew Pardoll, David L. Thomas, Stuart C. Ray
2005 Hepatology  
We comprehensively studied the cellular immune response during acute human hepatitis C virus (HCV) infection by monthly prospective sampling of persons with high risk of infection. In 19 of 23 subjects, interferon-gamma secreting T cells specific for one or more peptides spanning the entire HCV polyprotein were detected 1-3 months after infection. The median time to development of interferon gamma responses to HCV peptides was 33 days (range 29 to 50 days), and these responses peaked between
more » ... and 360 days. Nineteen subjects had sufficient follow-up to determine outcome, with 15 (79%) developing persistent viremia and 4 (21%) clearing viremia spontaneously. Of those with progression to chronic infection and detectable T cell responses, all lost recognition of one or more antigens recognized during acute infection and the median reduction in the magnitude of responses was 85%. Most significantly, despite ongoing viremia those who had persistent infection did not develop new epitope specificities after the first six months of infection. In conclusion, these results suggest that in the majority of individuals, the CD8+ T cell responses generated early in HCV infection decline in peripheral blood and are not replaced with new responses.
doi:10.1002/hep.20749 pmid:15962289 pmcid:PMC2759395 fatcat:wr6h4zjivzawfbyjyv2rx367ti

Exosome-delivered microRNAs modulate the inflammatory response to endotoxin

Margaret Alexander, Ruozhen Hu, Marah C. Runtsch, Dominique A. Kagele, Timothy L. Mosbruger, Tanya Tolmachova, Miguel C. Seabra, June L. Round, Diane M. Ward, Ryan M. O'Connell
2015 Nature Communications  
doi:10.1038/ncomms8321 pmid:26084661 pmcid:PMC4557301 fatcat:rd2d2xgewre3pic575ajuspyei

Spatial Constrains and Information Content of Sub-Genomic regions of the Human Genome

Leonidas P. Karakatsanis, Evgenios G. Pavlos, George Tsoulouhas, Georgios L. Stamokostas, Timothy Mosbruger, Jamie L. Duke, George P. Pavlos, Dimitri S. Monos
2021 iScience  
Complexity metrics and machine learning (ML) models have been utilized to analyze the lengths of segmental genomic entities of DNA sequences (exonic, intronic, intergenic, repeat, unique) with the purpose to ask questions regarding the segmental organization of the human genome within the size distribution of these sequences. For this we developed an integrated methodology that is based upon the reconstructed phase space theorem, the non-extensive statistical theory of Tsallis, ML techniques,
more » ... d a technical index, integrating the generated information, which we introduce and named complexity factor (COFA). Our analysis revealed that the size distribution of the genomic regions within chromosomes are not random but follow patterns with characteristic features that have been seen through its complexity character, and it is part of the dynamics of the whole genome. Finally, this picture of dynamics in DNA is recognized using ML tools for clustering, classification, and prediction with high accuracy.
doi:10.1016/j.isci.2021.102048 pmid:33554061 pmcid:PMC7843455 fatcat:3o55hkifwzcrrm3crv7cllot24

Cellular immune selection with hepatitis C virus persistence in humans

Andrea L. Cox, Timothy Mosbruger, Qing Mao, Zhi Liu, Xiao-Hong Wang, Hung-Chih Yang, John Sidney, Alessandro Sette, Drew Pardoll, David L. Thomas, Stuart C. Ray
2005 Journal of Experimental Medicine  
Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific cellular immune responses. To determine if immunologically driven sequence variation occurs with HCV persistence, we coordinately analyzed sequence evolution and CD8 ϩ T cell responses to epitopes covering the entire HCV polyprotein in subjects who were followed prospectively from before infection to beyond the first year. There were no substitutions in T cell epitopes for a year after infection in a
more » ... who cleared viremia. In contrast, in subjects with persistent viremia and detectable T cell responses, we observed substitutions in 69% of T cell epitopes, and every subject had a substitution in at least one epitope. In addition, amino acid substitutions occurred 13-fold more often within than outside T cell epitopes (P Ͻ 0.001, range 5-38). T lymphocyte recognition of 8 of 10 mutant peptides was markedly reduced compared with the initial sequence, indicating viral escape. Of 16 nonenvelope substitutions that occurred outside of known T cell epitopes, 8 represented conversion to consensus (P ϭ 0.015). These findings reveal two distinct mechanisms of sequence evolution involved in HCV persistence: viral escape from CD8 ϩ T cell responses and optimization of replicative capacity.
doi:10.1084/jem.20050121 pmid:15939790 pmcid:PMC2213263 fatcat:5cm6mmepsnf7fgjqzlj6tp6544

Characterization of 108 Genomic DNA Reference Materials for 11 Human Leukocyte Antigen Loci

Maria P. Bettinotti, Deborah Ferriola, Jamie L. Duke, Timothy L. Mosbruger, Nikolaos Tairis, Lawrence Jennings, Lisa V. Kalman, Dimitri Monos
2018 Journal of Molecular Diagnostics  
The highly polymorphic human leukocyte antigen (HLA) genes, located in the human major histocompatibility complex, encode the class I and II antigen-presenting molecules, which are centrally involved in the immune response. HLA typing is used for several clinical applications, such as transplantation, pharmacogenetics, and diagnosis of autoimmune disease. HLA typing is highly complex because of the homology of HLA genes and pseudogenes and the extensive polymorphism in the population. The
more » ... s for Disease Control and Prevention established the Genetic Testing Reference Materials Coordination Program (GeT-RM) in partnership with the genetics community to improve the availability of genomic DNA reference materials necessary for quality assurance of genetic laboratory testing. The GeT-RM together with three clinical laboratories and the Coriell Cell Repositories have characterized genomic DNA obtained from a panel of 108 cell lines for all HLA classic polymorphic loci: HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, and DPB1. The goal was to develop a publicly available and renewable source of well-characterized genomic DNA reference materials to support molecular HLA typing assay development, validation, and verification, quality control, and proficiency testing. These genomic DNA samples are publicly available from the National Institutes of General Medical Science Repository at the Coriell Cell Repositories.
doi:10.1016/j.jmoldx.2018.05.009 pmid:29959025 pmcid:PMC6939753 fatcat:eobbkokrivbyvfdgd5uboau6w4

MEK Inhibitors Reverse Growth of Embryonal Brain Tumors Derived from Oligoneural Precursor Cells

Katarzyna Modzelewska, Elena F. Boer, Timothy L. Mosbruger, Daniel Picard, Daniela Anderson, Rodney R. Miles, Mitchell Kroll, William Oslund, Theodore J. Pysher, Joshua D. Schiffman, Randy Jensen, Cicely A. Jette (+2 others)
2016 Cell Reports  
Graphical Abstract Highlights d Subsets of CNS-PNETs express oligodendrocyte precursor cell genes SOX10 and OLIG2 d Activating NRAS/MAPK signaling in OPCs generates CNS-PNET-like tumors in zebrafish d Cancer genomes of zebrafish and human NB-FOXR2 CNS-PNETs are highly conserved d MEK inhibitors are identified as a potential treatment for oligoneural/NB-FOXR2 CNS-PNETs SUMMARY Malignant brain tumors are the leading cause of cancer-related deaths in children. Primitive neuroectodermal tumors of
more » ... e CNS (CNS-PNETs) are particularly aggressive embryonal tumors of unknown cellular origin. Recent genomic studies have classified CNS-PNETs into molecularly distinct subgroups that promise to improve diagnosis and treatment; however, the lack of cell-or animal-based models for these subgroups prevents testing of rationally designed therapies. Here, we show that a subset of CNS-PNETs co-express oligoneural precursor cell (OPC) markers OLIG2 and SOX10 with coincident activation of the RAS/MAPK (mitogen-activated protein kinase) pathway. Modeling NRAS activation in embryonic OPCs generated malignant brain tumors in zebrafish that closely mimic the human oligoneural/NB-FOXR2 CNS-PNET subgroup by histology and comparative oncogenomics. The zebrafish CNS-PNET model was used to show that MEK inhibitors selectively eliminate Olig2 + /Sox10 + CNS-PNET tumors in vivo without impacting normal brain development. Thus, MEK inhibitors represent a promising rationally designed therapy for children afflicted with oligoneural/NB-FOXR2 CNS-PNETs.
doi:10.1016/j.celrep.2016.09.081 pmid:27783941 fatcat:neywpd7kbjcf5jmwzpc6eesu24

Reversible LSD1 inhibition with HCI-2509 induces the p53 gene expression signature and disrupts the MYCN signature in high-risk neuroblastoma cells

Sumati Gupta, Kelly Doyle, Timothy L. Mosbruger, Andrew Butterfield, Alexis Weston, Allison Ast, Mohan Kaadige, Anupam Verma, Sunil Sharma
2018 OncoTarget  
Lysine-Specific Demethylase 1 (LSD1) over-expression correlates with poorly differentiated neuroblastoma and predicts poor outcome despite multimodal therapy. We have studied the efficacy of reversible and specific LSD1 inhibition with HCI-2509 in neuroblastoma cell lines and particularly the effect of HCI-2509 on the transcriptomic profile in MYCN amplified NGP cells. Cell survival assays show that HCI-2509 is cytotoxic to poorly differentiated neuroblastoma cell lines in low micromole or
more » ... doses. Transcriptional profiling of NGP cells treated with HCI-2509 shows a significant effect on p53, cell cycle, MYCN and hypoxia pathway gene sets. HCI-2509 results in increased histone methyl marks and p53 levels along with cell cycle arrest in the G2/M phase and inhibition of colony formation of NGP cells. Our findings indicate that LSD1 inhibition with HCI-2509 has a multi-target effect in neuroblastoma cell lines, mediated in part via p53. MYCN-amplified neuroblastoma cells have a targeted benefit as HCI-2509 downregulates the MYCN upregulated gene set.
doi:10.18632/oncotarget.24035 pmid:29515779 pmcid:PMC5839410 fatcat:3fj7b6dbjfbepfd2lphnlrli4m

Large‐Scale Candidate Gene Analysis of Spontaneous Clearance of Hepatitis C Virus

Timothy L. Mosbruger, Priya Duggal, James J. Goedert, Gregory D. Kirk, W. Keith Hoots, Leslie H. Tobler, Michael Busch, Marion G. Peters, Hugo R. Rosen, David L. Thomas, Chloe L. Thio
2010 Journal of Infectious Diseases  
et al • JID 2010:201 (1 May) • Mosbruger et al  ...  71408363 rs2298456 .75 1 71411859 rs2298457 .75 1 Downloaded from https://academic.oup.com/jid/article-abstract/201/9/1371/877063 by guest on 28 July 2018 • JID 2010:201 (1 May) • Mosbruger  ... 
doi:10.1086/651606 pmid:20331378 pmcid:PMC2853721 fatcat:pefa6fxmwvhwzgn4g3coi5dj2a

Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution

Vahid Khoddami, Archana Yerra, Timothy L. Mosbruger, Aaron M. Fleming, Cynthia J. Burrows, Bradley R. Cairns
2019 Proceedings of the National Academy of Sciences of the United States of America  
The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously,
more » ... ide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1–2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.
doi:10.1073/pnas.1817334116 pmid:30872485 pmcid:PMC6452723 fatcat:6n5yddkwdfhmvocg2w5inzwala

Tracking the clonal origin of lethal prostate cancer

Michael C. Haffner, Timothy Mosbruger, David M. Esopi, Helen Fedor, Christopher M. Heaphy, David A. Walker, Nkosi Adejola, Meltem Gürel, Jessica Hicks, Alan K. Meeker, Marc K. Halushka, Jonathan W. Simons (+4 others)
2013 Journal of Clinical Investigation  
Recent controversies surrounding prostate cancer overtreatment emphasize the critical need to delineate the molecular features associated with progression to lethal metastatic disease. Here, we have used whole-genome sequencing and molecular pathological analyses to characterize the lethal cell clone in a patient who died of prostate cancer. We tracked the evolution of the lethal cell clone from the primary cancer to metastases through samples collected during disease progression and at the
more » ... of death. Surprisingly, these analyses revealed that the lethal clone arose from a small, relatively low-grade cancer focus in the primary tumor, and not from the bulk, higher-grade primary cancer or from a lymph node metastasis resected at prostatectomy. Despite being limited to one case, these findings highlight the potential importance of developing and implementing molecular prognostic and predictive markers, such as alterations of tumor suppressor proteins PTEN or p53, to augment current pathological evaluation and delineate clonal heterogeneity. Furthermore, this case illustrates the potential need in precision medicine to longitudinally sample metastatic lesions to capture the evolving constellation of alterations during progression. Similar comprehensive studies of additional prostate cancer cases are warranted to understand the extent to which these issues may challenge prostate cancer clinical management.
doi:10.1172/jci70354 pmid:24135135 pmcid:PMC3809798 fatcat:frs6lo5qznhkfbn2evc677bmcy
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