Metabolomics applied to urine samples in childhood asthma; differentiation between asthma phenotypes and identification of relevant metabolites

Asthma is a heterogeneous disorder and one of the most common chronic childhood diseases. An improved characterization of asthma phenotypes would be invaluable for the understanding of the pathogenic mechanisms and the correct treatment of this disease. The aim of this pilot study was to explore the potential of metabolomics applied to urine samples in characterizing asthma, and to identify the most representative metabolites. Urine samples of 41 atopic asthmatic children (further subdivided in sub-groups according to the symptoms) and 12 age-matched controls were analyzed. Untargeted metabolic profiles were collected by LC-MS, and studied by multivariate analysis. The group of the asthmatics was differentiated by a model that proved to be uncorrelated with the chronic assumption of controller drugs by part of the patients. The distinct sub-groups were also appropriately modeled. Further investigations revealed a reduced excretion of urocanic acid, methyl-imidazoleacetic acid and of a metabolite resembling the structure of an Ile-Pro fragment in the asthmatics. The meaning of these findings was discussed and mainly correlated with the modulation of immunity in asthma. Metabolic profiles from urines have revealed the potential to characterize asthma and enabled the identification of metabolites which may have a role in the underlying inflammation.JRC.I.6-Systems toxicolog

Different Real Time PCR Approaches for the Fine Quantification of SNP's Alleles in DNA Pools: Assays Development, Characterization and Pre-validation

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Comment on “Optimized Preprocessing of Ultra-Performance Liquid Chromatography/Mass Spectrometry Urinary Metabolic Profiles for Improved Information Recovery”

Metabolomics applied to urine samples has clearly demonstrated the potential to select possible biomarkers, despite the complexity of acquisition and elaboration of untargeted metabolic profiles. Many analytical aspects of this approach (chromatogram acquisition, “peak-picking” process and statistical analysis), have already been considered in scientific literature. In the paper of Veselkov and co-workers recently published some strategies to stabilize the technical variance within metabolic profiles acquired by UPLC-MS, and to normalize signal intensities were proposed (Anal. Chem. 2011, 83, 5864-5872). The described methods are interesting, but they also raise a number of questions that should be addressed. In particular we commented on the applicability and advantages of these methods in the context of a complete (e.g. from chromatogram acquisition to marker identification) metabolomics studies on human subjects.JRC.I.5-Systems Toxicolog

Critical aspects of urine profiling for the selection of potential biomarkers using UPLC-TOF-MS

Biomarker selection through the metabolomics approach involves the acquisition of non-targeted metabolic profiles. In this study, some critical factors that may affect this process were investigated using urine test samples and a UPLC-TOF system. Repeated injections of a single sample demonstrated that the percentage of undetected and poorly repeatable measurements (intensity RSD>15%) decreased from 22.5% to 5.8% and from 32.9% to 14.7%, respectively, as the scan time was increased up to 0.6 seconds (approximately 11 data points per peak). An additional critical factor was identified in the presence of broad concentration differences between the samples; the application of a dilution scheme that minimized these differences reduced the number of missing values in the final datasets by 36%. The impact of missing values was further investigated in the study of two groups of samples produced by using a spike as “artificial” marker. Missing values weakened the models used for the interpretation of the metabolic profiles, and greatly hindered the identification of possible markers. Finally, a simple strategy for an effective analysis of urine samples was outlined; it proved to limit the need for the post-acquisition elaboration of the data. The same strategy can easily be adapted to other matrices.JRC.I.5-Systems Toxicolog

Technical Issues Behind Molecular Monitoring in Chronic Myeloid Leukemia

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Development and Applications of Real Time PCR Standards for GMO Quantification Based on Tandem-Marker Plasmids

Abstract The need to comply with mandatory thresholds introduced for the labeling of GM food and feed by recent European Regulations is increasing the interest for reliable standards for GMO quantification. Certified reference materials (CRMs) for the different GMO lines are an essential element to calibrate measurement systems adopted to quantify GM ingredients. Unfortunately, conventional CRMs are affected not only by their high production costs but also by time consuming production and certification procedures. At the moment CRMs are available only for few of the GMOs authorized in Europe. In addition, latest developments in European legislation makes conceivable that the number of different authorized GM lines to be analysed will increase significantly, raising the need for additional CRMs. In the field of GMO detection and quantification, plasmids have been demonstrated to represent a cheap and reliable alternative to conventional CRMs. Here we describe the production of tandem-marker plasmids containing simultaneously specific target sequences for the GMO of interest and a species-specific reference marker sequence in a 1:1 ratio have been developed and used as real-time PCR standards. Strengths and weaknesses of these novel standards are discussed in detail.JRC.I.6-Biotechnology and GMO

Authentication of Trappist Beers by LC-MS Fingerprints and Multivariate Data Analysis

The aim of this study was to asses the applicability of LC-MS profiling to authenticate a selected Trappist beer as part of a program on traceability funded by the European Commission. A total of 232 beers were fingerprinted and classified through multivariate data analysis. The selected beer was clearly distinguished from beers of different brands, while only 3 samples (3.5% of the test set) were wrongly classified when compared with other types of beer of the same Trappist brewery. The fingerprints were further analyzed to extract the most discriminating variables, which proved to be sufficient for classification, even using a simplified unsupervised model. This reduced fingerprint allowed us to study the influence of batch to batch variability on the classification model. Our results can easily be applied to different matrices and they confirmed the effectiveness of LC-MS profiling in combination with multivariate data analysis for the characterization of food productsJRC.I.5-Systems Toxicolog

Different real time PCR approaches for the fine quantification of SNP's alleles in DNA pools: assays development, characterization and pre-validation.

7noneMATTARUCCHI E; MARSONI M; BINELLI G; PASSI A.G.; LO CURTO F; PASQUALI F; PORTA G.Mattarucchi, E; Marsoni, Milena; Binelli, GIORGIO PIETRO MARIO; Passi, Alberto; LO CURTO, Francesco; Pasquali, Francesco; Porta, Giovann

Molecular Monitoring of Residual Disease in Chronic Myeloid Leukemia by Genomic DNA Compared with Conventional mRNA Analysis

Translocation t(9;22), which produces the BCR-ABL gene, is pathognomonic of chronic myeloid leukemia. For clinical purposes, the amount of chimeric transcript is considered proportional to the leukemic clone; thus, mRNA is commonly used for molecular monitoring of patients. However, there is no consensus regarding the degree of increase in mRNA that should cause concern or whether the absence of transcript indicates a “cure.” In this study, we analyzed 57 samples from 10 chronic myeloid leukemia patients undergoing imatinib treatment. For each sample, we compared BCR-ABL mRNA levels with the actual proportion of leukemic cells, which were measured through a novel genomic approach based on the quantitative amplification of DNA breakpoints. The two approaches gave similar patterns of residual disease, and the majority of patients were still positive after an average treatment period of 2 years. Nevertheless, in one of two patients with confirmed undetectable levels of chimeric transcript, DNA still revealed the persistence of leukemic cells at 42 months. These findings appear to justify the clinical practice of maintaining imatinib treatment indefinitely. However, the absence of leukemic DNA (observed in 1 of 10 patients) could be used to identify possible candidates for drug discontinuation. In conclusion, DNA analysis proved to be a reliable index of residual disease with potential applications in the field of clinical diagnostics and research