Quantitative 1-Step DNA Methylation Analysis with Native Genomic DNA as Template

DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis.

Sequence capture and next-generation resequencing of multiple tagged nucleic acid samples for mutation screening of urea cycle disorders

Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples.

Diagnostic accuracy of plasma glial fibrillary acidic protein for differentiating intracerebral hemorrhage and cerebral ischemia in patients with symptoms of acute stroke

Glial fibrillary acidic protein (GFAP) is a biomarker candidate indicative of intracerebral hemorrhage (ICH) in patients with symptoms of acute stroke. GFAP is released rapidly in the presence of expanding intracerebral bleeding, whereas a more gradual release occurs in ischemic stroke. In this study the diagnostic accuracy of plasma GFAP was determined in a prospective multicenter approach.

Detection of exon deletions within an entire gene (CFTR) by relative quantification on the LightCycler

BACKGROUND: Cystic fibrosis (CF) is associated with at least 1 pathogen point sequence variant on each CFTR allele. Some symptomatic patients, however, have only 1 detectable pathogen sequence variant and carry, on the other allele, a large deletion that is not detected by conventional screening methods. METHODS: For relative quantitative real-time PCR detection of large deletions in the CFTR gene, we designed DNA-specific primers for each exon of the gene and primers for a reference gene (beta2-microglobulin). For PCR we used a LightCycler system (Roche) and calculated the gene-dosage ratio of CFTR to beta2-microglobulin. We tested the method by screening all 27 exons in 3 healthy individuals and 2 patients with only 1 pathogen sequence variant. We then performed specific deletion screenings in 10 CF patients with known large deletions and a blinded analysis in which we screened 24 individuals for large deletions by testing 8 of 27 exons. RESULTS: None of the ratios for control samples were false positive (for deletions or duplications); moreover, for all samples from patients with known large deletions, the calculated ratios for deleted exons were close to 0.5. In addition, the results from the blinded analysis demonstrated that our method can also be used for the screening of single individuals. CONCLUSIONS: The LightCycler assay allows reliable and rapid screening for large deletions in the CFTR gene and detects the copy number of all 27 exons.

A meta-analysis of 16 randomized trials of sirolimus-eluting stents versus paclitaxel-eluting stents in patients with coronary artery disease

OBJECTIVES: Our purpose was to make a synthesis of the available evidence on the relative efficacy and safety of 2 drug-eluting stents (DES)--sirolimus-eluting stent (SES) and paclitaxel-eluting stent (PES)--in patients with coronary artery disease. BACKGROUND: It is not known whether there are differences in late outcomes between the 2 most commonly used DES: SES and PES. METHODS: Sixteen randomized trials of SES versus PES with a total number of 8,695 patients were included in this meta-analysis. A full set of individual outcome data from 5,562 patients was also available. Mean follow-up period ranged from 9 to 37 months. The primary efficacy end point was the need for reintervention (target lesion revascularization). The primary safety end point was stent thrombosis. Secondary end points were death and recurrent myocardial infarction (MI). RESULTS: No significant heterogeneity was found across trials. Compared with PES, SES significantly reduced the risk of reintervention (hazard ratio [HR] 0.74; 95% confidence interval [CI] 0.63 to 0.87, p < 0.001) and stent thrombosis (HR 0.66; 95% CI 0.46 to 0.94, p = 0.02) without significantly impacting on the risk of death (HR 0.92; 95% CI 0.74 to 1.13, p = 0.43) or MI (HR 0.84; 95% CI 0.69 to 1.03, p = 0.10). CONCLUSIONS: Sirolimus-eluting stents are superior to PES in terms of a significant reduction of the risk of reintervention and stent thrombosis. The risk of death was not significantly different between the 2 DES, but there was a trend toward a higher risk of MI with PES, especially after the first year from the procedure.

Monitoring thrombin generation by electrochemistry: development of an amperometric biosensor screening test for plasma and whole blood

BACKGROUND: Complete investigation of thrombophilic or hemorrhagic clinical presentations is a time-, apparatus-, and cost-intensive process. Sensitive screening tests for characterizing the overall function of the hemostatic system, or defined parts of it, would be very useful. For this purpose, we are developing an electrochemical biosensor system that allows measurement of thrombin generation in whole blood as well as in plasma. METHODS: The measuring system consists of a single-use electrochemical sensor in the shape of a strip and a measuring unit connected to a personal computer, recording the electrical signal. Blood is added to a specific reagent mixture immobilized in dry form on the strip, including a coagulation activator (e.g., tissue factor or silica) and an electrogenic substrate specific to thrombin. RESULTS: Increasing thrombin concentrations gave standard curves with progressively increasing maximal current and decreasing time to reach the peak. Because the measurement was unaffected by color or turbidity, any type of blood sample could be analyzed: platelet-poor plasma, platelet-rich plasma, and whole blood. The test strips with the predried reagents were stable when stored for several months before testing. Analysis of the combined results obtained with different activators allowed discrimination between defects of the extrinsic, intrinsic, and common coagulation pathways. Activated protein C (APC) predried on the strips allowed identification of APC-resistance in plasma and whole blood samples. CONCLUSIONS: The biosensor system provides a new method for assessing thrombin generation in plasma or whole blood samples as small as 10 microL. The assay is easy to use, thus allowing it to be performed in a point-of-care setting.

Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites

We investigated the protective potential of recombinant his-tagged antigens recNcMIC1, recNcMIC3 and recNcROP2, applied either as single vaccines or as vaccine combinations, in BALB/c mouse models for cerebral and fetal infection. Subsequently, mice were mated and challenged by i.p. inoculation of 2 x 10(6)Neospora caninum tachyzoites at day 7 of pregnancy. The mortality and morbidity of adult mice (non-pregnant and dams) and of the newborn pups was studied for a period of 40 days following birth. Vaccination of non-pregnant mice with recNcROP2 or combinations of recNcROP2 with recNcMIC antigens significantly reduced the numbers of mice suffering from clinical signs, and morbidity was completely prevented with the combination of all three antigens. Of the dams, the groups receiving either recNcROP2 alone or the combination of all three antigens did not exhibit any morbidity, the groups receiving ROP2 mixed with either MIC1 or MIC3 exhibited reduced numbers of deaths, and in the infection control group and the adjuvant group 50% and 43% of mice, respectively, succumbed to disease. For pups, the highest survival rates were noted for the groups receiving recNcROP2 (50%) and recNcROP2/NcMIC1/NcMIC3 (35%), while in the infection- and adjuvant- control groups all pups died, the latest at days 25 and 30, respectively. Quantification of parasite DNA by N. caninum-specific real-time PCR revealed consistently lower parasite burdens in brain tissue of pups from vaccinated groups compared with the controls. However, dense granule antigen 2 (GRA2) real-time reverse transcriptase-PCR on brain tissue of surviving pups (applied here to detect viable parasites) demonstrated that only the pups from the group vaccinated with all three antigens in combination appeared free of viable tachyzoites, while in all other groups viable parasites were still present. Serological analysis of humoral (total IgG, IgG1 and IgG2a) and serum cytokine (IL-4 and IFN-gamma) responses showed that this effect was associated with a Th-2-biased immune response, with a clearly elevated IL-4/IFN-gamma ratio in the mice receiving all three antigens in combination. In conclusion, a mixture of recombinant antigens representing important secretory micronemal and rhoptry proteins leads to a significant protection against vertical transmission of N. caninum in mice.

Evaluation of the diagnostic value of serologic microagglutination testing and a polymerase chain reaction assay for diagnosis of acute leptospirosis in dogs in a referral center.

OBJECTIVE To determine the diagnostic value of a serologic microagglutination test (MAT) and a PCR assay on urine and blood for the diagnosis of leptospirosis in dogs with acute kidney injury (AKI). DESIGN Cross-sectional study. Animals-76 dogs with AKI in a referral hospital (2008 to 2009). PROCEDURES Dogs' leptospirosis status was defined with a paired serologic MAT against a panel of 11 Leptospira serovars as leptospirosis-associated (n = 30) or nonleptospirosis-associated AKI (12). In 34 dogs, convalescent serologic testing was not possible, and leptospirosis status was classified as undetermined. The diagnostic value of the MAT single acute or convalescent blood sample was determined in dogs in which leptospirosis status could be classified. The diagnostic value of a commercially available genus-specific PCR assay was evaluated by use of 36 blood samples and 20 urine samples. RESULTS Serologic acute testing of an acute blood sample had a specificity of 100% (95% CI, 76% to 100%), a sensitivity of 50% (33% to 67%), and an accuracy of 64% (49% to 77%). Serologic testing of a convalescent blood sample had a specificity of 92% (65% to 99%), a sensitivity of 100% (87% to 100%), and an accuracy of 98% (88% to 100%). Results of the Leptospira PCR assay were negative for all samples from dogs for which leptospirosis status could be classified. CONCLUSIONS AND CLINICAL RELEVANCE Serologic MAT results were highly accurate for diagnosis of leptospirosis in dogs, despite a low sensitivity for early diagnosis. In this referral setting of dogs pretreated with antimicrobials, testing of blood and urine samples with a commercially available genus-specific PCR assay did not improve early diagnosis.

Luxembourgish farmers lack information about grain legume cultivation

Grain legume production in Europe has decreased in recent years, while legume demand has rapidly increased due to growth of meat production. Therefore, Europe imports grain legumes, principally soybeans, to meet feed protein requirements. Various investigations have identified problems and benefits of local grain legume cultivation. Nevertheless, grain legume cultivation has still not increased in the last years. Studies investigating why farmers do not cultivate grain legumes are missing. Here, we surveyed the knowledge of farmers about grain legume cultivation, problems and constraints of grain legume cultivation and the barriers faced by and incentives needed by farmers. We sent a questionnaire to 1373 farmers in Luxembourg, with a response rate of 29 %. Results show that only 17 % of all the responding farmers cultivated grain legumes; 88 % of the conventional farmers did not cultivate grain legumes, while 85 % of the organic farmers did. We observed that Luxembourgish farmers feel badly informed about grain legume cultivation; organic farmers generally feel better informed than their conventional colleagues. The main barrier, named by Luxemburgish farmers to not cultivate grain legumes, is not economic issues but a lack of knowledge and extension services for these crops. Main incentives needed to start grain legume cultivation in the future are economic issues. Even though grain legume producers mentioned several negative experiences with grain legume cultivation, they are not discouraged by the poor economic conditions and appreciate the benefits of grain legume cultivation. Overall, our findings show that research results on grain legume should be better disseminated to extension services and farmers.