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

Dual Quaternion Synthesis of Constrained Robots

This paper presents a synthesis methodology for robots that have less than six degrees of freedom, termed constrained robots. The goal is to determine the physical parameters of the chain that fit its workspace to a given set of spatial positions. Our formulation uses the dual quaternion form of the kinematics equations of the constrained robot. Here we develop the theory and formulate the synthesis equations for the spatial RPR robot. Their solution ensures that the three dimensional workspace of this robot contains a given set of four spatial positions.Peer ReviewedPostprint (author's final draft

A systematic review of CD14 and toll-like receptors in relation to asthma in Caucasian children

The aetiology of childhood asthma is complex. An early dysfunction in immunological development of the innate immune system in combination environmental factors possibly triggers asthma. CD14 and toll-like important components of the innate immune system. The aim of this review was to obtain a better insight into the relation between CD14 and toll-like receptors and childhood asthma in Caucasians. We searched EMBASE for relevant articles. In total, 44 articles were included. The the selected studies was independently assessed by the first two authors the Newcastle-Ottawa quality assessment scale. Toll-like receptor 2, receptor 6, toll-like receptor 9, and toll-like receptor 10 appear to association with childhood asthma in Caucasians. The evidence for a CD14 with childhood asthma is limited. In conclusion, there is no evidence yet for a role of CD14 and toll-like receptors in relation to asthma. Future studies should include haplotype analysis and take factors into account to further clarify the role of CD14 and toll-like on childhood asthma