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  • 1
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    Heme activation by DNA: isoguanine pentaplexes, but not quadruplexes, bind heme and enhance its oxidative activity (2015)
    Oxford University Press
    Details
    Publication Date: 2015-05-03
    Description: Guanine-rich, single-stranded, DNAs and RNAs are able to fold to form G-quadruplexes that are held together by guanine base quartets. G-quadruplexes are known to bind ferric heme [Fe(III)-protoporphyrin IX] and to strongly activate such bound hemes toward peroxidase (1-electron oxidation) as well as oxygenase/peroxygenase (2-electron oxidation) activities. However, much remains unknown about how such activation is effected. Herein, we investigated whether G-quadruplexes were strictly required for heme activation or whether related multi-stranded DNA/RNA structures such as isoguanine (iG) quadruplexes and pentaplexes could also bind and activate heme. We found that iG-pentaplexes did indeed bind and activate heme comparably to G-quadruplexes; however, iG-quadruplexes did neither. Earlier structural and computational studies had suggested that while the geometry of backbone-unconstrained iG-quintets templated by cations such as Na + or NH 4 + was planar, that of iG-quartets deviated from planarity. We hypothesize that the binding as well as activation of heme by DNA or RNA is strongly supported by the planarity of the nucleobase quartet or quintet that interacts directly with the heme.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines (2015)
    Oxford University Press
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    Publication Date: 2015-02-18
    Description: The CRISPR/Cas has been recently shown to be a powerful genome-editing tool in a variety of organisms. However, these studies are mainly focused on protein-coding genes. The present study aims to determine whether this technology can be applied to non-coding genes. One of the challenges for knockout of non-coding genes is that a small deletion or insertion generated by the standard CRISPR/Cas system may not necessarily lead to functional loss of a given non-coding gene because of lacking an open reading frame, especially in polyploidy human cell lines. To overcome this challenge, we adopt a selection system that allows for marker genes to integrate into the genome through homologous recombination (HR). Moreover, we construct a dual guide RNA vector that can make two cuts simultaneously at designated sites such that a large fragment can be deleted. With these approaches, we are able to successfully generate knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 in various human cell lines. Finally, we show that the HR-mediated targeting efficiency can be further improved by suppression of the non-homologous end joining pathway. Together, these results demonstrate the feasibility of knockout for non-coding genes by the CRISPR/Cas system in human cell lines.
    Keywords: Targeted inhibition of gene function, Targeted gene modification
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    Loss of nNOS inhibits compensatory muscle hypertrophy and exacerbates inflammation and eccentric contraction-induced damage in mdx mice (2014)
    Oxford University Press
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    Publication Date: 2014-12-24
    Description: Approaches targeting nitric oxide (NO) signaling show promise as therapies for Duchenne and Becker muscular dystrophies. However, the mechanisms by which NO benefits dystrophin-deficient muscle remain unclear, but may involve nNOSβ, a newly discovered enzymatic source of NO in skeletal muscle. Here we investigate the impact of dystrophin deficiency on nNOSβ and use mdx mice engineered to lack nNOSμ and nNOSβ to discern how the loss of nNOS impacts dystrophic skeletal muscle pathology. In mdx muscle, nNOSβ was mislocalized and its association with the Golgi complex was reduced. nNOS depletion from mdx mice prevented compensatory skeletal muscle cell hypertrophy, decreased myofiber central nucleation and increased focal macrophage cell infiltration, indicating exacerbated dystrophic muscle damage. Reductions in muscle integrity in nNOS-null mdx mice were accompanied by decreases in specific force and increased susceptibility to eccentric contraction-induced muscle damage compared with mdx controls. Unexpectedly, muscle fatigue was unaffected by nNOS depletion, revealing a novel latent compensatory mechanism for the loss of nNOS in mdx mice. Together with previous studies, these data suggest that localization of both nNOSμ and nNOSβ is disrupted by dystrophin deficiency. They also indicate that nNOS has a more complex role as a modifier of dystrophic pathology and broader therapeutic potential than previously recognized. Importantly, these findings also suggest nNOSβ as a new drug target and provide a new conceptual framework for understanding nNOS signaling and the benefits of NO therapies in dystrophinopathies.
    Print ISSN: 0964-6906
    Electronic ISSN: 1460-2083
    Topics: Biology , Medicine
    Published by Oxford University Press
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  • 4
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    ExoMol molecular line lists - XIV. The rotation-vibration spectrum of hot SO2 (2016)
    Oxford University Press
    Details
    Publication Date: 2016-05-22
    Description: Sulphur dioxide is well-known in the atmospheres of planets and satellites, where its presence is often associated with volcanism, and in circumstellar envelopes of young and evolved stars as well as the interstellar medium. This work presents a line list of 1.3 billion 32 S 16 O 2 vibration–rotation transitions computed using an empirically adjusted potential energy surface and an ab initio dipole moment surface. The list gives complete coverage up to 8000 cm –1 (wavelengths longer than 1.25 μm) for temperatures below 2000 K. Infrared absorption cross-sections are recorded at 300 and 500 C are used to validated the resulting ExoAmes line list. The line list is made available in electronic form as supplementary data to this article and at www.exomol.com .
    Print ISSN: 0035-8711
    Electronic ISSN: 1365-2966
    Topics: Physics
    Published by Oxford University Press
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  • 5
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    SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity (2014)
    Oxford University Press
    Details
    Publication Date: 2014-07-04
    Description: Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high- SPIB and low- BATF or the reciprocal low- SPIB and high- BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIB high /BATF low -ABC-DLBCL is enriched for mutation of MYD88, and SPIB high /BATF low -ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 6
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    Disruption of myocardial Gata4 and Tbx5 results in defects in cardiomyocyte proliferation and atrioventricular septation (2014)
    Oxford University Press
    Details
    Publication Date: 2014-09-10
    Description: Mutations in GATA4 and TBX5 are associated with congenital heart defects in humans. Interaction between GATA4 and TBX5 is important for normal cardiac septation, but the underlying molecular mechanisms are not well understood. Here, we show that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle, but after E15.5, ventricular expression of Tbx5 decreases. Co-localization and co-immunoprecipitation studies demonstrate an interaction of Gata4 and Tbx5 in the developing atria and ventricles, but the ventricular interaction declines after E14.5. Gata4 +/– ;Tbx5 +/– mouse embryos display decreased atrial and ventricular myocardial thickness at E11.5, prior to cardiac septation. To determine the cell lineage in which the interaction was functionally significant in vivo , mice heterozygous for Gata4 in the myocardium or endocardium and heterozygous for Tbx5 ( Gata4 MyoDel/wt ;Tbx5 +/– and Gata4 EndoDel/wt ;Tbx5 +/– , respectively) were generated. Gata4 MyoDel/wt ;Tbx5 +/– mice displayed embryonic lethality, thin myocardium with reduced cell proliferation, and atrioventricular septation defects similar to Gata4;Tbx5 compound heterozygotes while Gata4 EndoDel/wt ;Tbx5 +/– embryos were normal. Cdk4 and Cdk2 , cyclin-dependent kinases required for myocardial development and septation were reduced in Gata4 +/– ;Tbx5 +/– hearts. Cdk4 is a known direct target of Gata4 and the regulation of Cdk2 in the developing heart has not been studied. Chromatin immunoprecipitation and transactivation studies demonstrate that Gata4 and Tbx5 directly regulate Cdk4 while only Tbx5 activates Cdk2 expression. These findings highlight the mechanisms by which disruption of the Gata4 and Tbx5 interaction in the myocardium contributes to cardiac septation defects in humans.
    Print ISSN: 0964-6906
    Electronic ISSN: 1460-2083
    Topics: Biology , Medicine
    Published by Oxford University Press
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  • 7
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    ImmuCo: a database of gene co-expression in immune cells (2015)
    Oxford University Press
    Details
    Publication Date: 2015-01-16
    Description: Current gene co-expression databases and correlation networks do not support cell-specific analysis. Gene co-expression and expression correlation are subtly different phenomena, although both are likely to be functionally significant. Here, we report a new database, ImmuCo ( http://immuco.bjmu.edu.cn ), which is a cell-specific database that contains information about gene co-expression in immune cells, identifying co-expression and correlation between any two genes. The strength of co-expression of queried genes is indicated by signal values and detection calls, whereas expression correlation and strength are reflected by Pearson correlation coefficients. A scatter plot of the signal values is provided to directly illustrate the extent of co-expression and correlation. In addition, the database allows the analysis of cell-specific gene expression profile across multiple experimental conditions and can generate a list of genes that are highly correlated with the queried genes. Currently, the database covers 18 human cell groups and 10 mouse cell groups, including 20 283 human genes and 20 963 mouse genes. More than 8.6 x 10 8 and 7.4 x 10 8 probe set combinations are provided for querying each human and mouse cell group, respectively. Sample applications support the distinctive advantages of the database.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 8
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    Lessons from a decade of integrating cancer copy number alterations with gene expression profiles (2012)
    Oxford University Press
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    Publication Date: 2012-05-23
    Description: Over the last decade, multiple functional genomic datasets studying chromosomal aberrations and their downstream effects on gene expression have accumulated for several cancer types. A vast majority of them are in the form of paired gene expression profiles and somatic copy number alterations (CNA) information on the same patients identified using microarray platforms. In response, many algorithms and software packages are available for integrating these paired data. Surprisingly, there has been no serious attempt to review the currently available methodologies or the novel insights brought using them. In this work, we discuss the quantitative relationships observed between CNA and gene expression in multiple cancer types and biological milestones achieved using the available methodologies. We discuss the conceptual evolution of both, the step-wise and the joint data integration methodologies over the last decade. We conclude by providing suggestions for building efficient data integration methodologies and asking further biological questions.
    Print ISSN: 1467-5463
    Electronic ISSN: 1477-4054
    Topics: Biology , Computer Science
    Published by Oxford University Press
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  • 9
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    Integrative analysis of gene and miRNA expression profiles with transcription factor-miRNA feed-forward loops identifies regulators in human cancers (2012)
    Oxford University Press
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    Publication Date: 2012-09-27
    Description: We describe here a novel method for integrating gene and miRNA expression profiles in cancer using feed-forward loops (FFLs) consisting of transcription factors (TFs), miRNAs and their common target genes. The dChip-GemiNI (Gene and miRNA Network-based Integration) method statistically ranks computationally predicted FFLs by their explanatory power to account for differential gene and miRNA expression between two biological conditions such as normal and cancer. GemiNI integrates not only gene and miRNA expression data but also computationally derived information about TF–target gene and miRNA–mRNA interactions. Literature validation shows that the integrated modeling of expression data and FFLs better identifies cancer-related TFs and miRNAs compared to existing approaches. We have utilized GemiNI for analyzing six data sets of solid cancers (liver, kidney, prostate, lung and germ cell) and found that top-ranked FFLs account for ~20% of transcriptome changes between normal and cancer. We have identified common FFL regulators across multiple cancer types, such as known FFLs consisting of MYC and miR-15/miR-17 families, and novel FFLs consisting of ARNT, CREB1 and their miRNA partners. The results and analysis web server are available at http://www.canevolve.org/dChip-GemiNi .
    Keywords: Computational Methods, Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 10
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    Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method (2013)
    Oxford University Press
    Details
    Publication Date: 2013-04-14
    Description: The genome-wide distribution patterns of the ‘6th base’ 5-hydroxymethylcytosine (5hmC) in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, it has been challenging to directly compare different data sets and samples using data generated by this method. Here, we report a new comparative hMeDIP-seq method, which involves barcoding different input DNA samples at the start and then performing hMeDIP-seq for multiple samples in one hMeDIP reaction. This approach extends the barcode technology from simply multiplexing the DNA deep sequencing outcome and provides significant advantages for quantitative control of all experimental steps, from unbiased hMeDIP to deep sequencing data analysis. Using this improved method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESC-derived neural progenitor cells (NPCs). We identified differentially hydroxymethylated regions (DHMRs) between ESCs and NPCs and uncovered an intricate relationship between the alteration of DNA hydroxymethylation and changes in gene expression during neural lineage commitment of ESCs. Presumably, the DHMRs between ESCs and NPCs uncovered by this approach may provide new insight into the function of 5hmC in gene regulation and neural differentiation. Thus, this newly developed comparative hMeDIP-seq method provides a cost-effective and user-friendly strategy for direct genome-wide comparison of DNA hydroxymethylation across multiple samples, lending significant biological, physiological and clinical implications.
    Keywords: Massively Parallel (Deep) Sequencing, Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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