Promoter methylation analysis of O6-methylguanine-DNA methyltransferase in glioblastoma: detection by locked nucleic acid based quantitative PCR using an imprinted gene (SNURF) as a reference

<p>Abstract</p> <p>Background</p> <p>Epigenetic silencing of the <it>MGMT </it>gene by promoter methylation is associated with loss of <it>MGMT </it>expression, diminished DNA-repair activity and longer overall survival in patients with glioblastoma who, in addition to radiotherapy, received alkylating chemotherapy with carmustine or temozolomide. We describe and validate a rapid methylation sensitive quantitative PCR assay (MS-qLNAPCR) using Locked Nucleic Acid (LNA) modified primers and an imprinted gene as a reference.</p> <p>Methods</p> <p>An analysis was made of a database of 159 GBM patients followed between April 2004 and October 2008. After bisulfite treatment, methylated and unmethylated CpGs were recognized by LNA primers and molecular beacon probes. The <it>SNURF </it>promoter of an imprinted gene mapped on 15q12, was used as a reference. This approach was used because imprinted genes have a balanced copy number of methylated and unmethylated alleles, and this feature allows an easy and a precise normalization.</p> <p>Results</p> <p>Concordance between already described nested MS-PCR and MS-qLNAPCR was found in 158 of 159 samples (99.4%). The MS-qLNAPCR assay showed a PCR efficiency of 102% and a sensitivity of 0.01% for LNA modified primers, while unmodified primers revealed lower efficiency (69%) and lower sensitivity (0.1%). <it>MGMT </it>promoter was found to be methylated using MS-qLNAPCR in 70 patients (44.02%), and completely unmethylated in 89 samples (55.97%). Median overall survival was of 24 months, being 20 months and 36 months, in patients with <it>MGMT </it>unmethylated and methylated, respectively. Considering <it>MGMT </it>methylation data provided by MS-qLNAPCR as a binary variable, overall survival was different between patients with GBM samples harboring <it>MGMT </it>promoter unmethylated and other patients with any percentage of <it>MGMT </it>methylation (p = 0.003). This difference was retained using other cut off values for <it>MGMT </it>methylation rate (i.e. 10% and 20% of methylated allele), while the difference was lost when 50% of <it>MGMT </it>methylated allele was used as cut-off.</p> <p>Conclusions</p> <p>We report and clinically validate an accurate, robust, and cost effective MS-qLNAPCR protocol for the detection and quantification of methylated <it>MGMT </it>alleles in GBM samples. Using MS-qLNAPCR we demonstrate that even low levels of <it>MGMT </it>promoter methylation have to be taken into account to predict response to temozolomide-chemotherapy.</p

A Physical Impact of Organic Fouling Layers on Bacterial Adhesion During Nanofiltration

Organic conditioning films have been shown to alter properties of surfaces, such as hydrophobicity and surface free energy. Furthermore, initial bacterial adhesion has been shown to depend on the conditioning film surface properties as opposed to the properties of the virgin surface. For the particular case of nanofiltration membranes under permeate flux conditions, however, the conditioning film thickens to form a thin fouling layer. This study hence sought to determine if a thin fouling layer deposited on a nanofiltration membrane under permeate flux conditions governed bacterial adhesion in the same manner as a conditioning film on a surface. Thin fouling layers (less than 50 μm thick) of humic acid or alginic acid were formed on Dow Filmtec NF90 membranes and analysed using Atomic Force Microscopy (AFM), confocal microscopy and surface energy techniques. Fluorescent microscopy was then used to quantify adhesion of Pseudomonas fluorescens bacterial cells onto virgin or fouled membranes under filtration conditions.It was found that instead of adhering on or into the organic fouling layer, the bacterial cells penetrated the thin fouling layer and adhered directly to the membrane surface underneath. Contrary to what surface energy measurements of the fouling layer would indicate, bacteria adhered to a greater extent onto clean membranes (24 ± 3% surface coverage) than onto those fouled with humic acid (9.8 ± 4%) or alginic acid (7.5 ± 4%). These results were confirmed by AFM measurements which indicated that a considerable amount of energy (10−7 J/μm) was dissipated when attempting to penetrate the fouling layers compared to adhering onto clean NF90 membranes (10−15 J/μm). The added resistance of this fouling layer was thusly seen to reduce the number of bacterial cells which could reach the membrane surface under permeate conditions. This research has highlighted an important difference between fouling layers for the particular case of nanofiltration membranes under permeate flux conditions and surface conditioning films which should be considered when conducting adhesion experiments under filtration conditions. It has also shown AFM to be an integral tool for such experiments.Science Foundation IrelandEuropean Research Counci

“Working the System”—British American Tobacco's Influence on the European Union Treaty and Its Implications for Policy: An Analysis of Internal Tobacco Industry Documents

Katherine Smith and colleagues investigate the ways in which British American Tobacco influenced the European Union Treaty so that new EU policies advance the interests of major corporations, including those that produce products damaging to health

Contribution of oxic methane production to surface methane emission in lakes and its global importance

Recent discovery of oxic methane production in sea and lake waters, as well as wetlands demands re-thinking of the global methane cycle and re-assessment of the contribution of oxic waters to atmospheric methane emission. Here we analysed system-wide sources and sinks of surface-water methane in a temperate lake. Using a mass balance analysis, we show that internal methane production in well-oxygenated surface water is an important source for surface-water methane during the stratified period. Combining our results and literature reports, oxic methane contribution to emission follows a predictive function of littoral sediment area and surface mixed layer volume. The contribution of oxic methane source(s) is predicted to increase with lake size, accounting for the majority (>50 %) of surface methane emission for lakes with surface areas >1 km2