Metabotypes of breast cancer cell lines revealed by non-targeted metabolomics

We present an analysis of intracellular metabolism by non-targeted, high-throughput metabolomics profiling of 18 breast cell lines. We profiled >900 putatively annotated metabolite ions for >100 samples collected under both normoxic and hypoxic conditions and revealed extensive heterogeneity across all metabolic pathways and cell lines. Cell line–specific metabolome profiles dominated over patterns associated with malignancy or with the clinical nomenclature of breast cancer cells. Such characteristic metabolome profiles were reproducible across different laboratories and experiments and exhibited mild to robust changes with change in experimental conditions. To extract a functional overview of cell line heterogeneity, we devised an unsupervised metabotyping procedure that for each pathway automatically recognized metabolic types from metabolome data and assigned cell lines. Our procedure provided a condensed yet global representation of cell line metabolism, revealing the fine structure of metabolic heterogeneity across all tested pathways and cell lines. In follow-up experiments on selected pathways, we confirmed that different metabolic types correlated to differences in the underlying fluxes and difference sensitivity to gene knockdown or pharmacological inhibition. Thus, the identified metabotypes recapitulated functional differences at the pathway level. Metabotyping provides a powerful compression of multi-dimensional data that preserves functional information and serves as a resource for reconciling or understanding heterogeneous metabolic phenotypes or response to inhibition of metabolic pathways.     

State-of-the-art non-targeted metabolomics in the study of chronic kidney disease

Here we report a metabolomics discovery study conducted on blood serum samples of patients in different stages of chronic kidney disease (CKD). Metabolites were monitored on a quality controlled holistic platform combining reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry in both negative and positive ionization mode and gas chromatography coupled to quadrupole mass spectrometry. A substantial portion of the serum metabolome was thereby covered. Eighty-five metabolites were shown to evolve with CKD progression of which 43 metabolites were a confirmation of earlier reported uremic retention solutes and/or uremic toxins. Thirty-one unique metabolites were revealed which were increasing significantly throughout CKD progression, by a factor surpassing the level observed for creatinine, the currently used biomarker for kidney function. Additionally, 11 unique metabolites showed a decreasing trend.     

High‐altitude adaptation in vertebrates as revealed by mitochondrial genome analyses

The high‐altitude environment may drive vertebrate evolution in a certain way, and vertebrates living in different altitude environments might have different energy requirements. We hypothesized that the high‐altitude environment might impose different influences on vertebrate mitochondrial genomes (mtDNA). We used selection pressure analyses and PIC (phylogenetic independent contrasts) analysis to detect the evolutionary rate of vertebrate mtDNA protein‐coding genes (PCGs) from different altitudes. The results showed that the ratio of nonsynonymous/synonymous substitutions (dN/dS) in the mtDNA PCGs was significantly higher in high‐altitude vertebrates than in low‐altitude vertebrates. The seven rapidly evolving genes were shared by the high‐altitude vertebrates, and only one positive selection gene (ND5 gene) was detected in the high‐altitude vertebrates. Our results suggest the mtDNA evolutionary rate in high‐altitude vertebrates was higher than in low‐altitude vertebrates as their evolution requires more energy in a high‐altitude environment. Our study demonstrates the high‐altitude environment (low atmospheric O₂ levels) drives vertebrate evolution in mtDNA PCGs.     

Metabolic differentiations of dwarf elder by NMR-based metabolomics

Sambucus plants have prominent place in folk medicine of the people from Europe and the Middle East. Sambucus ebulus (both above and under ground parts) preparations showed anti-neoplastic, antimicrobial (incl. antiviral) and anti-inflammatory properties. Elderberries accumulate sugars and fibers, vitamins and minerals, besides abundant secondary metabolites, as flavonoids, anthocyanins, phytosterols, triterpenes and iridoid glycosides, among others. Sambucus plants, however, also accumulate cyanogenic glycosides, whose presence is undesirable. Despite many applications so far the knowledge of the metabolites, accumulated in Sambucus species, is still limited and based mainly on determination of the major compounds. Here we report the application of ¹H NMR metabolic fingerprinting in combination with principal component and hierarchical clustering analyses to reveal the metabolic differences of Sambucus mature and immature fruits, and plant leaves. Moreover, we show that immature fruits and leaves of S. ebulus have similar metabolome, which apparently undergoes significant changes during the fruit ripening stage. Sambunigrin was not detectable in any sample. To the best of our knowledge this is the first report on the systematic analysis of S. ebulus metabolome.     

Insulin sensitivity is reflected by characteristic metabolic fingerprints--a Fourier transform mass spectrometric non-targeted metabolomics approach

Background

A decline in body insulin sensitivity in apparently healthy individuals indicates a high risk to develop type 2 diabetes. Investigating the metabolic fingerprints of individuals with different whole body insulin sensitivity according to the formula of Matsuda, et al. (ISI_Matsuda) by a non-targeted metabolomics approach we aimed a) to figure out an unsuspicious and altered metabolic pattern, b) to estimate a threshold related to these changes based on the ISI, and c) to identify the metabolic pathways responsible for the discrimination of the two patterns.

Methodology and Principal Findings

By applying infusion ion cyclotron resonance Fourier transform mass spectrometry, we analyzed plasma of 46 non-diabetic subjects exhibiting high to low insulin sensitivities. The orthogonal partial least square model revealed a cluster of 28 individuals with alterations in their metabolic fingerprints associated with a decline in insulin sensitivity. This group could be separated from 18 subjects with an unsuspicious metabolite pattern. The orthogonal signal correction score scatter plot suggests a threshold of an ISI_Matsuda of 15 for the discrimination of these two groups. Of note, a potential subgroup represented by eight individuals (ISI_Matsuda value between 8.5 and 15) was identified in different models. This subgroup may indicate a metabolic transition state, since it is already located within the cluster of individuals with declined insulin sensitivity but the metabolic fingerprints still show some similarities with unaffected individuals (ISI >15). Moreover, the highest number of metabolite intensity differences between unsuspicious and altered metabolic fingerprints was detected in lipid metabolic pathways (arachidonic acid metabolism, metabolism of essential fatty acids and biosynthesis of unsaturated fatty acids), steroid hormone biosyntheses and bile acid metabolism, based on data evaluation using the metabolic annotation interface MassTRIX.

Conclusions

Our results suggest that altered metabolite patterns that reflect changes in insulin sensitivity respectively the ISI_Matsuda are dominated by lipid-related pathways. Furthermore, a metabolic transition state reflected by heterogeneous metabolite fingerprints may precede severe alterations of metabolism. Our findings offer future prospects for novel insights in the pathogenesis of the pre-diabetic phase.     

High altitude adaptation of the schizothoracine fishes (Cyprinidae) revealed by the mitochondrial genome analyses

The schizothoracine fishes, also known as “mountain carps” are widely distributed in the Qinghai-Tibetan Plateau and its peripheral regions. Although they provide a prime example of high altitude adaptation, the phylogenetic relationships and the divergence times among these carp lineages are still controversial. Moreover, the genetic basis for high altitude adaptation is also poorly understood. In this study, we determined the mitochondrial genomes from two species of the schizothoracine fishes, representing a “morphologically primitive” clade and “morphologically specialized” clade, respectively. The phylogenetic tree and the divergence times were estimated within the evolutionary framework of the entire order Cypriniformes. Our results indicate a polyphylyetic relationship of the schizothoracine fishes and suggest two independent migration events into the Qinghai-Tibetan Plateau: one by the “morphologically primitive” clade in the Late Miocene and another by the “morphologically specialized” clade in the Eocene. Rapid speciation events of each clade from the Late Miocene to the Pliocene correspond to the timing of the geologic acceleration of the Qinghai-Tibetan Plateau. Interestingly, we found evidence for positive selection acting on the protein coding genes in the mitochondrial genomes of the “morphologically specialized” clade, implying a possible genetic basis for high altitude adaptation in this derived lineage of cypriniform fishes.     

Metabolic differentiations and classification of Verbascum species by NMR-based metabolomics

The genus Verbascum L. (mulleins) comprises of about 360 species of flowering plants in the Scrophulariaceae family. Mulleins have been used in the traditional folk medicine for centuries, for treatment of a wide range of human ailments, inter alia bronchitis, tuberculosis, asthma, and different inflammations. Despite all applications the knowledge of the metabolites, accumulated in different mullein species, is still limited and based mainly on determination of the major compounds. Here we report the application of ¹H NMR metabolic fingerprinting in combination with principal component analyses (PCA) in five different Verbascum species. Based on the obtained results mulleins were divided in two groups: group A (Verbascum phlomoides and Verbascum densiflorum) and group B (Verbascum xanthophoeniceum, Verbascum nigrum and Verbascum phoeniceum). Further it was found that the plants in group B accumulate higher amounts of bioactive iridoid and phenylethanoid glycosides. V. xanthophoeniceum and V. nigrum accumulate higher amounts of the pharmaceutically-important harpagoside (∼0.5% on dry weight basis) and verbascoside, forsythoside B and leucosceptoside B (in total 5.6–5.8% on dry weight basis), which underlines the possibility for their application in pharmaceutical industry. To the best of our knowledge this is the first report on the analyses of Verbascum sp. leaf metabolome.     

Metabolic aspects of temperature adaptation by fish

During 8-hour acclimation to changes in the water temperature by 10 degrees within the range of 20-30 degrees C the metabolic compensation of the temperature effects in the carp liver mitochondria is manifested at the level of thermogenesis, activity of succinate dehydrogenase and rate of oxidative phosphorylation. No temperature compensation is found for the activity of cytochromoxidase, ATPase, rate of nonphosphorylating oxidation, content of ATP, phosphorus and calcium in the mentioned organelles.     

Spatiotopic visual maps revealed by saccadic adaptation in humans

Saccadic adaptation [1] is a powerful experimental paradigm to probe the mechanisms of eye movement control and spatial vision, in which saccadic amplitudes change in response to false visual feedback. The adaptation occurs primarily in the motor system [2, 3], but there is also evidence for visual adaptation, depending on the size and the permanence of the postsaccadic error [4-7]. Here we confirm that adaptation has a strong visual component and show that the visual component of the adaptation is spatially selective in external, not retinal coordinates. Subjects performed?a memory-guided, double-saccade, outward-adaptation task designed to maximize visual adaptation and to dissociate the visual and motor corrections. When the memorized saccadic target was in the same position (in external space) as that used in the adaptation training, saccade targeting was strongly influenced by adaptation (even if not matched in retinal or cranial position), but when in the same retinal or cranial but different external spatial position, targeting was unaffected by adaptation, demonstrating unequivocal spatiotopic selectivity. These results point to the existence of a spatiotopic neural representation for eye movement control that adapts in response to saccade error signals.     

1,5-Anhydroglucitol predicts CKD progression in macroalbuminuric diabetic kidney disease: results from non-targeted metabolomics

Introduction

Metabolomics allows exploration of novel biomarkers and provides insights on metabolic pathways associated with disease. To date, metabolomics studies on CKD have been largely limited to Caucasian populations and have mostly examined surrogate end points.

Objective

In this study, we evaluated the role of metabolites in predicting a primary outcome defined as dialysis need, doubling of serum creatinine or death in Brazilian macroalbuminuric DKD patients.

Methods

Non-targeted metabolomics was performed on plasma from 56 DKD patients. Technical triplicates were done. Metabolites were identified using Agilent Fiehn GC/MS Metabolomics and NIST libraries (Agilent MassHunter Work-station Quantitative Analysis, version B.06.00). After data cleaning, 186 metabolites were left for analyses.

Results

During a median follow-up time of 2.5 years, the PO occurred in 17 patients (30.3%). In non-parametric testing, 13 metabolites were associated with the PO. In univariate Cox regression, only 1,5-anhydroglucitol (HR 0.10; 95% CI 0.01–0.63, p?=?.01), norvaline and l-aspartic acid were associated with the PO. After adjustment for baseline renal function, 1,5-anhydroglucitol (HR 0.10; 95% CI 0.02–0.63, p?=?.01), norvaline (HR 0.01; 95% CI 0.001–0.4, p?=?.01) and aspartic acid (HR 0.12; 95% CI 0.02–0.64, p?=?.01) remained significantly and inversely associated with the PO.

Conclusion

Our results show that lower levels of 1,5-anhydroglucitol, norvaline and l-aspartic acid are associated with progression of macroalbuminuric DKD. While norvaline and l-aspartic acid point to interesting metabolic pathways, 1,5-anhydroglucitol is of particular interest since it has been previously shown to be associated with incident CKD. This inverse biomarker of hyperglycemia should be further explored as a new tool in DKD.

     

A non-targeted metabolomics approach to quantifying differences in root storage between fast- and slow-growing plants

Life history theory posits that slower-growing species should invest proportionally more resources to storage, structural (e.g. stems) or defence traits than fast-growing species. Previously, we showed that the slower-growing monocarpic plants had lower mortality rates and higher bolting probabilities after two defoliation events. Here, we consider a mechanistic explanation, that the slower-growing species invested relatively more resources to storage. We compared the relative levels of root storage compounds between eight monocarpic species using metabolomic profiling, and characterized plant growth using a size-corrected estimate of relative growth rate (RGR). Growth rate was negatively correlated with the proportional allocation of root metabolites identified as sucrose, raffinose and stachyose and with amino acids known for their roles in nitrogen storage, particularly proline and arginine. The total amount and concentration of energy-corrected carbohydrates were also negatively correlated with RGR. Our results show for the first time that slower-growing species invest proportionally more of their total root metabolites in carbon- and nitrogen-storage compounds. We conclude that the increased investment in these reserves is an important resource allocation strategy underlying the growth-survival trade-off in plants.     

Microbial metabolism of catechin stereoisomers by human faecal microbiota: Comparison of targeted analysis and a non-targeted metabolomics method

Microbial metabolism of the stereoisomers (+)-catechin and (−)-epicatechin was compared by two analytical techniques, GC/MS for quantitative targeted analysis and GC×GC-TOF for global characterization of the metabolome, using human faecal microbiota as an inoculum of converting microbiota. The ring-fission site changed when the inocula originated from two different groups of donors, but dehydroxylation progressed similarly regardless of the inoculum. Whereas GC/MS proved to be an appropriate tool for the study of specific expected metabolites of catechin stereoisomers, GC×GC-TOF-based metabolomics analysis also revealed new metabolites not included in the targeted analyses. Quantitation and verification of identification can also be performed in a metabolomics platform, if authentic standards are available.     

Visual neuroscience: face-encoding mechanisms revealed by adaptation

Faces convey a great variety of information, for example about the species, gender, age, identity and even mood or intentions. A recent study sheds light on the neural mechanisms for encoding a face's gaze direction.     

Phylogenetic clusters of rhizobia revealed by genome structures

Rhizobia, bacteria that fix atmospheric nitrogen, are important agricultural resources. In order to establish the evolutionary relationships among rhizobia isolated from different geographic regions and different plant hosts for systematic studies, we evaluated the use of physical structure of the rhizobial genomes as a phylogenetic marker to categorize these bacteria. In this work, we analyzed the features of genome structures of 64 rhizobial strains. These rhizobial strains were divided into 21 phylogenetic clusters according to the features of genome structures evaluated by the endonuclease I-Ceul. These clusters were supported by 16S rRNA comparisons and genomic sequences of four rhizobial strains, but they are largely different from those based on the current taxonomic scheme (except 16S rRNA).     

Phylogenetic clusters of rhizobia revealed by genome structures

Rhizobiaareagriculturallyandenvironmentallyimportantbacteria.Theirsymbiosiswithleguminousplantsisresponsibleformostoftheatmosphericni-trogenfixedonland.Classificationofthesebacteriabasedontheirnaturalrelationshipswillpromotetheirapplication.Thisresearchemploysanewphylogeneticmethod,i.e.,revelationandcomparisonofgenomestructure,tocategorizerhizobia.Phylogenyisthestudyoftheevolutionaryrelationshipsamongorgan-isms[1].Currentlyphylogeneticrelationshipsamongrhizobiaaremostlyinferredfromcomparisons…     

Bacterial phylogenetic clusters revealed by genome structure.

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.