IntroductionMetabolic profiling of intact tumor tissue by high-resolution magic angle spinning (HR MAS) MR spectroscopy (MRS) provides important biological information possibly useful for clinical diagnosis and development of novel treatment strategies. However, generation of high-quality data requires that sample handling from surgical resection until analysis is performed using systematically validated procedures. In this study, we investigated the effect of postsurgical freezing delay time on global metabolic profiles and stability of individual metabolites in intact tumor tissue.Materials and methodsTumor tissue samples collected from two patient-derived breast cancer xenograft models (n = 3 for each model) were divided into pieces that were snap-frozen in liquid nitrogen at 0, 15, 30, 60, 90, and 120 min after surgical removal. In addition, one sample was analyzed immediately, representing the metabolic profile of fresh tissue exposed neither to liquid nitrogen nor to room temperature. We also evaluated the metabolic effect of prolonged spinning during the HR MAS experiments in biopsies from breast cancer patients (n = 14). All samples were analyzed by proton HR MAS MRS on a Bruker Avance DRX600 spectrometer, and changes in metabolic profiles were evaluated using multivariate analysis and linear mixed modeling.ResultsMultivariate analysis showed that the metabolic differences between the two breast cancer models were more prominent than variation caused by freezing delay time. No significant changes in levels of individual metabolites were observed in samples frozen within 30 min of resection. After this time point, levels of choline increased, whereas ascorbate, creatine, and glutathione (GS) levels decreased. Freezing had a significant effect on several metabolites but is an essential procedure for research and biobank purposes. Furthermore, four metabolites (glucose, glycine, glycerophosphocholine, and choline) were affected by prolonged HR MAS experiment time possibly caused by physical release of metabolites caused by spinning or due to structural degradation processes.ConclusionThe MR metabolic profiles of tumor samples are reproducible and robust to variation in postsurgical freezing delay up to 30 min.
Background Cryptosporidium infection is a worldwide cause of diarrheal disease. To gain insight into the epidemiology of the infection in a certain geographic area, molecular methods are needed to determine the species/genotypes and subtypes.Methodology/Principal FindingsFrom 2004 to 2009, 161 cryptosporidiosis cases were detected in two hospitals in Barcelona. Diagnosis was performed by microscopic observation of oocysts in stool specimens following modified Ziehl-Neelsen staining. Most cases (82%) occurred in children. The number of cases increased in summer and autumn. Molecular characterization of Cryptosporidium was performed in 69 specimens, and C. hominis and C. parvum were identified in 88.4% and 10.1% of the cases, respectively. C. meleagridis was detected in one specimen. Subtyping based on the gp60 polymorphism showed six subtypes, four C. hominis and two C. parvum. Subtype IbA10G2 was the most prevalent subtype corresponding to 90% of all C. hominis isolates. This is the first report on the distribution of specific Cryptosporidium subtypes from humans in Spain.Conclusions/SignificanceIn our geographic area, the anthroponotic behavior of C. hominis, the lower infective dose, and the higher virulence of certain subtypes may contribute to the high incidence of human cryptosporidiosis caused by the IbA10G2 subtype. Further studies should include populations with asymptomatic shedding of the parasite.
To identify factors related with the risk to develop microalbuminuria using combined genomic and metabolomic values from a general population study. One thousand five hundred and two subjects, Caucasian, more than 18 years, representative of the general population, were included. Blood pressure measurement and albumin/creatinine ratio were measured in a urine sample. Using SNPlex, 1251 SNPs potentially associated to urinary albumin excretion (UAE) were analyzed. Serum metabolomic profile was assessed by 1H NMR spectra using a Brucker Advance DRX 600 spectrometer. From the total population, 1217 (mean age 54±19, 50.6% men, ACR>30 mg/g in 81 subjects) with high genotyping call rate were analysed. A characteristic metabolomic profile, which included products from mitochondrial and extra mitochondrial metabolism as well as branched amino acids and their derivative signals, were observed in microalbuminuric as compare to normoalbuminuric subjects. The comparison of the metabolomic profile between subjects with different UAE status for each of the genotypes associated to microalbuminuria revealed two SNPs, the rs10492025_TT of RPH3A gene and the rs4359_CC of ACE gene, with minimal or no statistically significant differences. Subjects with and without microalbuminuria, who shared the same genotype and metabolomic profile, differed in age. Microalbuminurics with the CC genotype of the rs4359 polymorphism and with the TT genotype of the rs10492025 polymorphism were seven years older and seventeen years younger, respectively as compared to the whole microalbuminuric subjects. With the same metabolomic environment, characteristic of subjects with microalbuminuria, the TT genotype of the rs10492025 polymorphism seems to increase and the CC genotype of the rs4359 polymorphism seems to reduce risk to develop microalbuminuria.
Delayed enhancement cardiovascular magnetic resonance (MR) is the gold-standard for non-invasive assessment after myocardial infarction (MI). MR microscopy (MRM) provides a level of detail comparable to the macro objective of light microscopy. We used MRM and correlative histopathology to identify infarct and remote tissue in contrast agent-free multi-sequence MRM in swine MI hearts. One control group (n = 3 swine) and two experimental MI groups were formed: 90 min of ischemia followed by 1 week (acute MI = 6 swine) or 1 month (chronic MI = 5 swine) reperfusion. Representative samples of each heart were analysed by contrast agent-free multi-sequence (T1-weighting, T2-weighting, T2*-weighting, T2-mapping, and T2*-mapping). MRM was performed in a 14-Tesla vertical axis imager (Bruker-AVANCE 600 system). Images from MRM and the corresponding histopathological stained samples revealed differences in signal intensities between infarct and remote areas in both MI groups (p-value < 0.001). The multivariable models allowed us to precisely classify regions of interest (acute MI: specificity 92% and sensitivity 80%; chronic MI: specificity 100% and sensitivity 98%). Probabilistic maps based on MRM images clearly delineated the infarcted regions. As a proof of concept, these results illustrate the potential of MRM with correlative histopathology as a platform for exploring novel contrast agent-free MR biomarkers after MI.
The results reported here provide a basis for a new hypotheses connecting EGFR gene amplification in GB cells with ARF1 gene promoter methylation, vesicle trafficking, membrane turnover and tumor metabolism. The mechanism(s) underlying these connections and their functional consequences remain to be established.
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