D-statistic versus FDR relationship at putative TREs, across the time-series (IGB view)

<p><b>Copyright information:</b></p><p>Taken from "Differential analysis for high density tiling microarray data"</p><p>http://www.biomedcentral.com/1471-2105/8/359</p><p>BMC Bioinformatics 2007;8():359-359.</p><p>Published online 24 Sep 2007</p><p>PMCID:PMC2231405.</p><p></p> Examples of enrichment fragments are observed within and upstream of the second intron of the HIC gene (pink). The upstream fragment is possibly un-annotated (UA), in so far as no RefSeq annotation is available. The top four tracks represent the HisH4 p-value graphs at 0 (red), 2 (light-blue), 8 (dark-blue) and 32 (green) hours, scaled appropriately for comparison; the subsequent tracks represent the d-statistic (top) and FDR (bottom) pair for the 0–2 (red), 2–8 (cyan) and 8–32 (blue) hour time intervals. The horizontal lines associated with the FDR data refer to the 5 percent threshold in each case

A representative density profile of the d-statistic for change in H3K27T histone modification between 0 and 2 hours of retinoic acid treatment for the ENCODE region on chromosome 1

<p><b>Copyright information:</b></p><p>Taken from "Differential analysis for high density tiling microarray data"</p><p>http://www.biomedcentral.com/1471-2105/8/359</p><p>BMC Bioinformatics 2007;8():359-359.</p><p>Published online 24 Sep 2007</p><p>PMCID:PMC2231405.</p><p></p> The curves of different colors illustrate differential change for the H3K27T modification in exonic (green), intronic (black) and intergenic (blue) regions. The shift into the negative territory for the d-statistic for all classes of regions suggest is a consistent downward trend for this modification between 0 and 2 hours

The histogram summarizes the differential expression profiles in each ENCODE region on each chromosome

<p><b>Copyright information:</b></p><p>Taken from "Differential analysis for high density tiling microarray data"</p><p>http://www.biomedcentral.com/1471-2105/8/359</p><p>BMC Bioinformatics 2007;8():359-359.</p><p>Published online 24 Sep 2007</p><p>PMCID:PMC2231405.</p><p></p> Chromosome region specific differential expression is observed across the time-points – 30 percent change on chromosome 8 to no detectable change on chromosome 10. Globally, the highest fraction of differential expression when summarized across all transfrag is observed between 8–32 hours (53.8 percent),. The most statistically significant (FDR ≤12 percent) changes are also observed between 8–32 hours

Proteomics Analysis of Co-Purifying Cellular Proteins Associated with rAAV Vectors

<div><p>Recombinant adeno-associated vectors (rAAV) are commonly purified by either chromatography or equilibrium CsCl gradient. Nevertheless, even after purification various cellular proteins often associate with rAAV vector capsids. Such co-purifying cellular proteins may raise concern about safety of gene therapy. Here we report identification and characterization of the co-purifying cellular protein in the vector preparations by using a combination of two proteomics approaches, GeLC-MS (gel electrophoresis liquid chromatography-mass spectrometry) and 2DE (two-dimensional gel electrophoresis). Most prominent bands revealed by Coomassie Blue staining were mostly similar to the AAV capsid proteins. Posttranslational modifications of capsid proteins were detected by the proteomics analysis. A total of 13 cellular proteins were identified in the rAAV vectors purified by two rounds of cesium chloride gradient centrifugation, including 9 by the GeLC-MS analysis and 4 by the 2DE analysis. Selected cellular proteins were verified by western blot. Furthermore, the cellular proteins could be consistently found associated with different AAV serotypes and carrying different transgenes. Yet, the proteins were not integral components of the viral capsis since a stringent washing procedure by column purification could remove them. These co-purified proteins in AAV vector preparations may have a role in various stages of the AAV life cycle.</p></div

Detection of protein SET in different fractions of the AAV2-dsEGFP preparation.

<p>The vector was produced by triple plasmid transfection and purified by two rounds of cesium chloride ultra-centrifugation. The fractions with different densities were collected after the second round of ultra-centrifugation. Protein form 1×10¹⁰ viral particles was resolved on 10% SDS/PAGE. The full AAV vector particles have buoyant densities in CsCl from 1.41 to 1.45 g/cm³ while empty particles have the density of 1.32 g/cm³. A, silver staining; and B, western blot.</p

List of cellular proteins identified using GeLC-MS analysis of adeno-associated virus vectors.

<p>List of cellular proteins identified using GeLC-MS analysis of adeno-associated virus vectors.</p

2DE map of recombinant AAV vector.

<p>The top 2DE map was obtained using a wide pH range of IPG strip pH–10, and the bottom one using a narrow pH range of IPG strip pH 4–7. The circled and numbered spots were identified as human proteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086453#pone-0086453-t002" target="_blank">Table 2</a>), the spots only circled were identified as AAV capsid proteins.</p

List of human cellular proteins identified from 2DE analysis via nano-LC IT MS.

<p>List of human cellular proteins identified from 2DE analysis via nano-LC IT MS.</p

Identification of celluar proteins co-purifed with recombinant AAV vector.

<p>A, 1DE-SDS PAGE of recombinant AAV vector. The seven marked bands were excised and processed for MALDI-TOF analysis. B, All seven bands were identified as capsid-related proteins by the sequence homology. The Mowse score and the number of peptides matched were from the matching results against capsid protein VP1.</p

The effect of AAV serotypes, transgenes and purification methods on the presence of SET proteins in the rAAV vector preparations.

<p>Protein from 1×1010 viral particles was resolved on 10% SDS/PAGE. AAV2-EGFP/C and AAV2-FIX/C were purified by ion exchange chromatography. A, silver staining; and B, western blot.</p