Uncovering information on expression of natural antisense transcripts in Affymetrix MOE430 datasets

<p>Abstract</p> <p>Background</p> <p>The function and significance of the widespread expression of natural antisense transcripts (NATs) is largely unknown. The ability to quantitatively assess changes in NAT expression for many different transcripts in multiple samples would facilitate our understanding of this relatively new class of RNA molecules.</p> <p>Results</p> <p>Here, we demonstrate that standard expression analysis Affymetrix MOE430 and HG-U133 GeneChips contain hundreds of probe sets that detect NATs. Probe sets carrying a "Negative Strand Matching Probes" annotation in NetAffx were validated using Ensembl by manual and automated approaches. More than 50 % of the 1,113 probe sets with "Negative Strand Matching Probes" on the MOE430 2.0 GeneChip were confirmed as detecting NATs. Expression of selected antisense transcripts as indicated by Affymetrix data was confirmed using strand-specific RT-PCR. Thus, Affymetrix datasets can be mined to reveal information about the regulated expression of a considerable number of NATs. In a correlation analysis of 179 sense-antisense (SAS) probe set pairs using publicly available data from 1637 MOE430 2.0 GeneChips a significant number of SAS transcript pairs were found to be positively correlated.</p> <p>Conclusion</p> <p>Standard expression analysis Affymetrix GeneChips can be used to measure many different NATs. The large amount of samples deposited in microarray databases represents a valuable resource for a quantitative analysis of NAT expression and regulation in different cells, tissues and biological conditions.</p

Optimierte Strahlformungs- und Fokussieroptik zur optischen Mikromanipulation

Optische Pinzetten werden oft mit konventionellen Mikroskopobjektiven mit hoher numerischer Apertur aufgebaut. Diese sind meist auf die gängige Deckglasdicke von 170µm angepasst und dadurch für die Mikromanipulation auf diesen Arbeitsabstand beschränkt. Dies stellt ein erhebliches Problem für die Manipulation z.B. in Fluidikkanälen dar, die aus Gründen der mechanischen Stabilität zumeist mit Wandstärken hergestellt werden, die 0,5 mm oder mehr betragen. Um in diesen Kanalsystemen dennoch mit optischen Pinzetten arbeiten zu können, kann mit relativ aufwändigen Counterpropagating Pinzetten oder mit integrierten Faserpinzetten gearbeitet werden. Ziel unserer Arbeiten ist der Entwurf und die Demonstration einer optimierten Spezialoptik für diese Anwendung. Unterstützt durch numerische Simulation der optischen Kraftwirkung in elektromagnetischen Feldern wir ein optimiertes Design für optische Pinzetten abgeleitet. Daran anschließend wird ein integriertes monolithisches und Fokussierungsobjektiv entworfen. Die Herstellung des Systems erfolgt durch mechanische Ultrapräzisionsbearbeitung der optischen Flächen. Erste Ergebnisse und eine Charakterisierung des Konzepts werden vorgestellt

Metabolic Profiling as Well as Stable Isotope Assisted Metabolic and Proteomic Analysis of RAW 264.7 Macrophages Exposed to Ship Engine Aerosol Emissions: Different Effects of Heavy Fuel Oil and Refined Diesel Fuel

Exposure to air pollution resulting from fossil fuel combustion has been linked to multiple short-term and long term health effects. In a previous study, exposure of lung epithelial cells to engine exhaust from heavy fuel oil (HFO) and diesel fuel (DF), two of the main fuels used in marine engines, led to an increased regulation of several pathways associated with adverse cellular effects, including pro-inflammatory pathways. In addition, DF exhaust exposure was shown to have a wider response on multiple cellular regulatory levels compared to HFO emissions, suggesting a potentially higher toxicity of DF emissions over HFO. In order to further understand these effects, as well as to validate these findings in another cell line, we investigated macrophages under the same conditions as a more inflammationrelevant model. An air-liquid interface aerosol exposure system was used to provide a more biologically relevant exposure system compared to submerged experiments, with cells exposed to either the complete aerosol (particle and gas phase), or the gas phase only (with particles filtered out). Data from cytotoxicity assays were integrated with metabolomics and proteomics analyses, including stable isotope-assisted metabolomics, in order to uncover pathways affected by combustion aerosol exposure in macrophages. Through this approach, we determined differing phenotypic effects associated with the different components of aerosol. The particle phase of diluted combustion aerosols was found to induce increased cell death in macrophages, while the gas phase was found more to affect the metabolic profile. In particular, a higher cytotoxicity of DF aerosol emission was observed in relation to the HFO aerosol. Furthermore, macrophage exposure to the gas phase of HFO leads to an induction of a pro-inflammatory metabolic and proteomic phenotype. These results validate the effects found in lung epithelial cells, confirming the role of inflammation and cellular stress in the response to combustion aerosols

CIP2A Promotes T-Cell Activation and Immune Response to Listeria monocytogenes Infection

The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A ( CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2A(HOZ)) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2A(HOZ) mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4(+) T-cells and CD8(+) effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2A(HOZ) as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects.Peer reviewe

Emissions from a modern log wood masonry heater and wood pellet boiler : Composition and biological impact on air-liquid interface exposed human lung cancer cells

The consumption of wood fuel is markedly increasing in developing and industrialized countries. Known side effects of wood smoke inhalation manifest in proinflammatory signaling, oxidative stress, DNA damage and hence increased cancer risk. In this study, the composition and acute biological impact of emissions of state-of-the-art wood combustion compliances: masonry heater (MH) and pellet boiler (PB) were investigated. Therefore A549 cells were exposed to emission aerosols in an automated air-liquid interface exposure station followed by cytotoxicity, transcriptome and proteome analyses. In parallel, aerosols were subjected to a chemical and physical haracterization. Compared to PB, the MH combustion at the same dilution ratio resulted in a 3-fold higher particle mass concentration (PM2.5) and deposited dose (PB: 27 $\pm$ 2 ng/cm2, MH; 73 $\pm$ 12 ng/cm2). Additionally, the MH aerosol displayed a substantially larger concentration of aldehydes, polycyclic aromatic hydrocarbons (PAH) or oxidized PAH. Gene ontology analysis of transcriptome of A549 cells exposed to MH emissions revealed the activation of proinflammatory response and key signaling cascades MAP kinase and JAK-STAT. Furthermore, CYP1A1, an essential enzyme in PAH metabolism, was induced. PB combustion aerosol activated the proinflammatory marker IL6 and different transport processes. The proteomics data uncovered induction of DNA damage-associated proteins in response to PB and DNA doublestrand break processing proteins in response to MH emissions. Taking together, the MH produces emissions with a higher particle dose and more toxic compounds while causing only mild biological responses. This finding points to a significant mitigating effect of antioxidative compounds in MH wood smoke

Uncovering information on expression of natural antisense transcripts in Affymetrix MOE430 datasets-4

<p><b>Copyright information:</b></p><p>Taken from "Uncovering information on expression of natural antisense transcripts in Affymetrix MOE430 datasets"</p><p>http://www.biomedcentral.com/1471-2164/8/200</p><p>BMC Genomics 2007;8():200-200.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1929078.</p><p></p>ed to the displayed categories based on the orientation of the probe set sequence ("sense", "antisense", "overlap") found. Also depicted is the frequency of cross-hybridizing probe sets and those without match to an annotated transcript

Uncovering information on expression of natural antisense transcripts in Affymetrix MOE430 datasets-1

<p><b>Copyright information:</b></p><p>Taken from "Uncovering information on expression of natural antisense transcripts in Affymetrix MOE430 datasets"</p><p>http://www.biomedcentral.com/1471-2164/8/200</p><p>BMC Genomics 2007;8():200-200.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1929078.</p><p></p>transcripts of the category "overlap"; right panel: transcripts of the category "antisense". B) RT-PCR products of transcripts of the category "overlap" with indicated product sizes. C) RT-PCR products of transcripts of the category "antisense". D) Strand-specific RT-PCR for three transcripts with no prior evidence of SAS pairing. : RT reaction with 3' reverse primer; : RT reaction with 5' forward primer; no primer during RT reaction; no template control

Uncovering information on expression of natural antisense transcripts in Affymetrix MOE430 datasets-3

<p><b>Copyright information:</b></p><p>Taken from "Uncovering information on expression of natural antisense transcripts in Affymetrix MOE430 datasets"</p><p>http://www.biomedcentral.com/1471-2164/8/200</p><p>BMC Genomics 2007;8():200-200.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1929078.</p><p></p>found probe set pairs with probe sets in sense and antisense orientation, respectively. B) Exemplary scatterplots of the expression values for two SAS probe set pairs. Left: no correlation between probesets 1437874_s_at and 1426591_at; right: positive correlation for the sense (1418840_at) and antisense (1456393_at) probe set pair shown in A). Data represents vsn-normalized and scaled expression values (unit:natural logarithm) from 1637 mouse MOE420 2.0 microarrays downloaded from the Gene Expression Omnibus database. Correlation was calculated using Pearson Correlation. C) Distribution of Pearson correlation coefficients using the data matrix described in B) for 179 SAS probe set pairs (gray bars) and 150 randomly picked control probeset pairs (hatched bars). same as left, but with Pearson Correlation performed for each individual dataset

Uncovering information on expression of natural antisense transcripts in Affymetrix MOE430 datasets-0

<p><b>Copyright information:</b></p><p>Taken from "Uncovering information on expression of natural antisense transcripts in Affymetrix MOE430 datasets"</p><p>http://www.biomedcentral.com/1471-2164/8/200</p><p>BMC Genomics 2007;8():200-200.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1929078.</p><p></p>ed to the displayed categories based on the orientation of the probe set sequence ("sense", "antisense", "overlap") found. Also depicted is the frequency of cross-hybridizing probe sets and those without match to an annotated transcript