Plasma and Serum Metabolite Association Networks: Comparability within and between Studies Using NMR and MS Profiling.

Blood is one of the most used biofluids in metabolomics studies, and the serum and plasma fractions are routinely used as a proxy for blood itself. Here we investigated the association networks of an array of 29 metabolites identified and quantified via NMR in the plasma and serum samples of two cohorts of ∼1000 healthy blood donors each. A second study of 377 individuals was used to extract plasma and serum samples from the same individual on which a set of 122 metabolites were detected and quantified using FIA-MS/MS. Four different inference algorithms (ARANCE, CLR, CORR, and PCLRC) were used to obtain consensus networks. The plasma and serum networks obtained from different studies showed different topological properties with the serum network being more connected than the plasma network. On a global level, metabolite association networks from plasma and serum fractions obtained from the same blood sample of healthy people show similar topologies, and at a local level, some differences arise like in the case of amino acids.

Probabilistic networks of blood metabolites in healthy subjects as indicators of latent cardiovascular risk.

The complex nature of the mechanisms behind cardiovascular diseases prevents the detection of latent early risk conditions. Network representations are ideally suited to investigate the complex interconnections between the individual components of a biological system that underlies complex diseases. Here, we investigate the patterns of correlations of an array of 29 metabolites identified and quantified in the plasma of 864 healthy blood donors and use a systems biology approach to define metabolite probabilistic networks specific for low and high latent cardiovascular risk. We adapted methods based on the likelihood of correlation and methods from information theory and combined them with resampling techniques. Our results show that plasma metabolite networks can be defined that associate with latent cardiovascular disease risk. The analysis of the networks supports our previous finding of a possible association between cardiovascular risk and impaired mitochondrial activity and highlights post-translational modifications (glycosilation and oxidation) of lipoproteins as a possible target-mechanism for early detection of latent cardiovascular risk.

NMR metabolomic fingerprinting distinguishes milk from different farms.

A fast and reproducible protocol for milk Nuclear Magnetic Resonance (NMR) metabolomic fingerprinting was developed, allowing for an accurate discrimination among milk samples from large-scale distribution, as well as among milk sample from different farms located in the same restricted geographical area. Seasonal variations in milk composition and correlations with cows' nutritional patterns are also assessed, underlining relationships between feeding and metabolites. The most important difference was related to the use of silage feeding. This finding is relevant to assess the suitability of milk for different dairy products. A prominent example is parmesan cheese, the preparation protocol of which excludes milk from silage-fed cows.

Extreme Hypoxic Conditions Induce Selective Molecular Responses and Metabolic Reset in Detached Apple Fruit.

The ripening physiology of detached fruit is altered by low oxygen conditions with profound effects on quality parameters. To study hypoxia-related processes and regulatory mechanisms, apple (Malus domestica, cv Granny Smith) fruit, harvested at commercial ripening, were kept at 1°C under normoxic (control) and hypoxic (0.4 and 0.8 kPa oxygen) conditions for up to 60 days. NMR analyses of cortex tissue identified eight metabolites showing significantly different accumulations between samples, with ethanol and alanine displaying the most pronounced difference between hypoxic and normoxic treatments. A rapid up-regulation of alcohol dehydrogenase and pyruvate-related metabolism (lactate dehydrogenase, pyruvate decarboxylase, alanine aminotransferase) gene expression was detected under both hypoxic conditions with a more pronounced effect induced by the lowest (0.4 kPa) oxygen concentration. Both hypoxic conditions negatively affected ACC synthase and ACC oxidase transcript accumulation. Analysis of RNA-seq data of samples collected after 24 days of hypoxic treatment identified more than 1000 genes differentially expressed when comparing 0.4 vs. 0.8 kPa oxygen concentration samples. Genes involved in cell-wall, minor and major CHO, amino acid and secondary metabolisms, fermentation and glycolysis as well as genes involved in transport, defense responses, and oxidation-reduction appeared to be selectively affected by treatments. The lowest oxygen concentration induced a higher expression of transcription factors belonging to AUX/IAA, WRKY, HB, Zinc-finger families, while MADS box family genes were more expressed when apples were kept under 0.8 kPa oxygen. Out of the eight group VII ERF members present in apple genome, two genes showed a rapid up-regulation under hypoxia, and western blot analysis showed that apple MdRAP2.12 proteins were differentially accumulated in normoxic and hypoxic samples, with the highest level reached under 0.4 kPa oxygen. These data suggest that ripe apple tissues finely and specifically modulate sensing and regulatory mechanisms in response to different hypoxic stress conditions.

NMR fingerprinting as a tool to evaluate post-harvest time-related changes of peaches, tomatoes and plums.

The time-related changes of three agricultural products, coming from two distribution routes, have been followed using NMR fingerprinting to monitor metabolic variations occurring during several days of cold storage. An NMR profiling approach was employed to evaluate the variations in metabolic profile and metabolite content in three different agricultural products highly consumed in Italy (peaches, tomatoes and plums) coming from Tuscanian farms and how they change with time after collection. For each product, we followed the time-related changes during cold storage along three different collection periods. We monitored the variations in metabolic fingerprint and the trend of a set of metabolites, focusing our attention on nutritive and health-promoting metabolites (mainly, essential amino acids and antioxidants) as well as metabolites that contribute to the taste. Concurrently, for comparison, the time-dependent changes of the same kind of products coming from large-scale distribution have been also analyzed under the same conditions. In this second category, only slight variations in the metabolic fingerprint and metabolite levels were seen during cold storage. Unsupervised and supervised multivariate statistics was also employed to enlighten the differences between the three collections. In particular it seems that the metabolic fingerprint of large-scale distribution products is quite similar in the early, middle and late collection, while peaches and plums locally collected are markedly different among the three periods. The metabolic profiles of the agricultural products belonging to these two different distribution routes are intrinsically different, and they show different changes during the time of cold storage.

BRAFV600E detection in melanoma is highly improved by COLD-PCR.

BACKGROUND: The BRAF gene has been identified as an oncogene in human cancer and the V600E mutation has been shown to be associated with clinico pathological features of primary invasive melanomas. As BRAF may be an attractive therapeutic target, it is crucial to have a sensitive method for detecting mutated DNA in biological samples. Our aim was to investigate COLD-PCR (co-amplification at lower denaturation temperature-PCR) as a new approach for the pre-analytical enrichment of the BRAFV600E variant in formalin fixed paraffin embedded (FFPE) melanoma tissues. METHODS: COLD-PCR was used to selectively amplify BRAFV600E minority alleles from mixtures of wild-type and mutated sequences, and from biological samples. The method shows higher specificity than other conventional PCR-based methods in detecting somatic mutations. RESULTS: We used COLD-PCR to increase the theoretical sensitivity of three different post-PCR methods: sequencing, pyrosequencing and HRMA. The gain in sensitivity seems to be more evident for HRMA, which allows the detection of 3.1% mutated alleles. More than 20% of patients initially classified negative for BRAFV600E were found positive after COLD-PCR. CONCLUSIONS: COLD-PCR was confirmed as a suitable method for the enrichment of mutated alleles, particularly for samples in which the percentage of tumor cells is very low.

The use of COLD-PCR and high-resolution melting analysis improves the limit of detection of KRAS and BRAF mutations in colorectal cancer.

Fast and reliable tests to detect mutations in human cancers are required to better define clinical samples and orient targeted therapies. KRAS mutations occur in 30-50% of colorectal cancers (CRCs) and represent a marker of clinical resistance to cetuximab therapy. In addition, the BRAF V600E is mutated in about 10% of CRCs, and the development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAF (V600E) detection. Traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer, resulting in false negative diagnoses. In this study, we designed a protocol to detect mutations of KRAS and BRAF(V600E) in 117 sporadic CRCs based on coamplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM). Using traditional PCR and direct sequencing, we found KRAS mutations in 47 (40%) patients and BRAF(V600E) in 10 (8.5%). The use of COLD-PCR in apparently wild-type samples allowed us to identify 15 newly mutated CRCs (10 for KRAS and 5 for BRAF (V600E)), raising the percentage of mutated CRCs to 48.7% for KRAS and to 12.8% for BRAF (V600E). Therefore, COLD-PCR combined with HRM permits the correct identification of less represented mutations in CRC and better selection of patients eligible for targeted therapies, without requiring expensive and time-consuming procedures.