Direct quantification of mitochondrial DNA and its 4.9-kb common deletion without DNA purification.

Quantitative analysis of mitochondrial DNA (mtDNA) and its common deletion (CD) are sensitive and early markers for mitochondrial mutations and suffering. However, the use of purified DNA can lead to quantification errors because of variable DNA extraction yields due to the significant differences in size and structure between genomic DNA (gDNA) and mtDNA. We report a real-time qPCR-based protocol directly on tissue lysate, without DNA extraction. This method, which allows both absolute and relative measure, increases the measuring accuracy of the mtDNA/gDNA ratio and leads to reliable and more reproducible results when measuring the deleted/total mtDNA ratio.

Quality of care of patients with type 1 diabetes: population-based results in a French region.

AIM: Although the incidence of type 1 diabetes (T1D) has been increasing, little is known of its quality of care. Thus, our survey was designed to retrospectively evaluate this issue in French patients. METHODS: Patients with T1D living in northeastern France were identified thanks to the healthcare system (CPAM) database, and the resulting list reviewed by local diabetes specialists. All of the listed patients and their primary physicians were asked to fill in a questionnaire including clinical data, laboratory results and follow-up habits. The 'optimized results' included CPAM-based results plus any specialized care provided during hospitalizations in diabetes and non-diabetes units, according to questionnaire data. RESULTS: A total of 227 individuals, for whom CPAM data were available, were identified as having T1D. From these patients, 174 questionnaires were answered, and optimized results (having both CPAM data and a completely filled-in questionnaire) were available for 149 patients. Of the 169 patients who responded, 71.3% reported at least a yearly visit with a diabetologist. This number reached 77.9% when optimized results were considered. Patients who received specialized care were younger, underwent HbA(1c) tests more often and were more frequently on optimal treatment; however, there was no difference in HbA(1c) values or in the prevalence of complications. Eye examinations and kidney tests had been performed at least once over the 2-year period in more than 87% of the patients, whereas around 30%, 21% and 23% had an eye exam, creatinine test and urinary albumin excretion measurement, respectively, only once over the same time period. CONCLUSION: This is the first large-scale study of the quality of care in patients with T1DM in France, and it could serve as a preliminary survey for a national study. Although the follow-up was better than previously reported, there is still considerable room for improvement.

Anti-inflammatory effect of vagus nerve stimulation in a rat model of inflammatory bowel disease.

Vagus nerve stimulation of afferents is used as an adjunctive treatment for drug-resistant epilepsy and depression. In addition, anti-inflammatory properties of vagus nerve stimulation have been reported in various experimental models of inflammation but not in colitis. These effects are thought to be mediated via peripheral release of acetylcholine from the vagus and subsequent activation of macrophages. Our aim was to evaluate in rats the anti-inflammatory effects of chronic vagus nerve stimulation on colonic inflammation. Colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid. Vagus nerve stimulation (left cervical) was performed in freely moving animals 3 h per day for five consecutive days. Assessment of colonic inflammation was obtained using physiological (e.g. body weight, temperature and locomotor activity) parameters, macroscopical (area of lesions), histological, and biological parameters (e.g. myeloperoxidase activity, cytokine and cytokine-related mRNAs), both at the level of the damaged colon and the colon immediately above. A global multivariate index of colitis was then generated for a better characterization of colonic inflammation. Vagus nerve stimulation reduced the degree of body weight loss and inflammatory markers as observed above the lesion by histological score and myeloperoxidase quantification. This anti-inflammatory effect was also demonstrated by the improvement of the multivariate index of colitis. These data argue for an anti-inflammatory role of vagus nerve stimulation chronically performed in freely moving rats with colitis and provide potential therapeutic applications for patients with inflammatory bowel diseases.

Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers.

Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased.