Metabolomics analysis reveals differences in evolution between maize and rice

Metabolites are the intermediate and final products of metabolism, which play essential roles in plant growth, evolution and adaptation to changing climates. However, it is unclear how evolution contributes to metabolic variation in plants. Here, we investigated the metabolomics data from leaf and seed tissues in maize and rice. Using principal components analysis based on leaf metabolites but not seed metabolites, metabolomics data could be clearly separated for rice Indica and Japonica accessions, while two maize subgroups, temperate and tropical, showed more visible admixture. Rice and maize seed exhibited significant interspecific differences in metabolic variation, while within rice, leaf and seed displayed similar metabolic variations. Among 10 metabolic categories, flavonoids had higher variation in maize than rice, indicating flavonoids are a key constituent of interspecific metabolic divergence. Interestingly, metabolic regulation was also found to be reshaped dramatically from positive to negative correlations, indicative of the differential evolutionary processes in maize and rice. Moreover, perhaps due to this divergence significantly more metabolic interactions were identified in rice than maize. Furthermore, in rice, the leaf was found to harbor much more intense metabolic interactions than the seed. Our result suggests that metabolomes are valuable for tracking evolutionary history, thereby complementing and extending genomic insights concerning which features are responsible for interspecific differentiation in maize and rice.     

A hypomorphic allele of dab1 reveals regional differences in reelin-Dab1 signaling during brain development

The disabled 1 (Dab1) p80 protein is essential for reelin signaling during brain development. p80 has an N-terminal domain for association with reelin receptors, followed by reelin-dependent tyrosine phosphorylation sites and about 310 C-terminal residues of unknown function. We have generated mutant mice that express only a natural splice form of Dab1, p45, that lacks the C-terminal region of p80. The normal development of these mice implies that the receptor-binding region and tyrosine phosphorylation sites of p80 are sufficient for reelin signaling. However, a single copy of the truncated gene does not support normal development of the neocortex and hippocampus. The CA1 region of the hippocampus is split into two well-organized layers, while the marginal zone of the neocortex is invaded by late-born cortical plate neurons. The haploinsufficiency of the p45 allele of Dab1 implies that the C terminus of p80 affects the strength of reelin-Dab1 signaling, yet there is no apparent change in reelin-dependent tyrosine phosphorylation of p45 relative to p80. Therefore, we suggest that the C-terminal region of Dab1 p80 is involved in signaling to downstream effector molecules. Furthermore, the presence of late-born cortical plate neurons in the marginal zone reveals a requirement for reelin-Dab1 signaling in late-born cortical plate neurons, and helps distinguish models for the cortical inversion in the reeler mutant mouse.     

Sex differences in regional brain response to aversive pelvic visceral stimuli

To explore sex differences in the response of seven brain regions to an aversive pelvic visceral stimulus, functional magnetic resonance images were acquired from 13 healthy adults (6 women) during 15 s of cued rectal distension at two pressures: 25 mmHg (uncomfortable), and 45 mmHg (mild pain), as well as during an expectation condition (no distension). Random-effects analyses combining subject data voxelwise found 45-mmHg pressure significantly activated the insular and anterior cingulate cortices in both sexes. In men only, the left thalamus and ventral striatum were also activated. Although all activations appeared more extensive in men, no sex difference attained significance. To explore the presence of deactivations, which are generally cancelled by more numerous activations when subjects are combined for each voxel, the number of activated voxels, number of deactivated voxels, and ratio of deactivated voxels to total voxels affected were assessed via random-effects, mixed-model analyses combining subject data at the region level. Greater insula activation in men compared with women was seen during the expectation condition and during the 25-mmHg distension. Greater deactivations in women were seen in the amygdala (25-mmHg distension) and midcingulate (45-mmHg distension). Women had a significantly higher proportion of deactivated voxels than men in all four subcortical structures during 25-mmHg distension. Greater familiarity of females with physiological pelvic visceral discomfort may have enhanced brain systems that dampen arousal networks during lower levels of discomfort.     

Metabolomics reveals abundant flavonoids in edible insect Antheraea pernyi

The natural flavonoids in foods of plant origin have been well-characterized due to their beneficial biological properties. However, the information regarding the flavonoid compounds in edible insects remains severely limited. In the present study, we used a metabolomics approach to identify the flavonoid compounds in the Chinese oak silkworm, Antheraea pernyi Guérin-Méneville (Lepidoptera: Saturniidae), an traditional edible insect. Our study identified over 200 flavonoid metabolites in the larval midgut of A. pernyi with LC-ESI-MS/MS system. These flavonoid metabolites come from eight subclasses, including flavones (1 0 3), flavonols (34), flavonoids (28), flavanones (20), polyphenols (19), isoflavones (9), anthocyanins (9), and proanthocyanidins (4). The relative content of the flavones is the most abundant, with a value of 36.74% of the total. The top five flavonoid components in A. pernyi are hyperoside, isoquercitroside, tricin 7-O-hexoside, hesperetin 5-O-glucoside and protocatechuic acid, accounting for 51.17% of the total flavonoids. Hyperoside is the most abundant flavonoid compound (18.07% of the total) in A. pernyi. Our findings indicated targeted metabolomics is a useful approach to identify flavonoids in edible insects which contain abundant flavonoids than we already knew.     

Metabolomics of prolonged fasting in humans reveals new catabolic markers

Fasting is one of the simplest metabolic challenges that can be performed in humans. We here report for the first time a comprehensive analysis of the human ??fasting metabolome?? obtained from analysis of plasma and urine samples in a small cohort of healthy volunteers, using nuclear magnetic resonance (NMR), gas chromatography- and liquid chromatography-mass spectrometry (GC-MS and LC-MS). Intra- and inter-individual variation of metabolites was on measurement of four overnight fasting samples collected from each volunteer over a four week period. One additional sample per volunteer was collected following a prolonged fasting period of 36?h. Amongst a total of 377 quantified entities in plasma around 44% were shown to change significantly in concentration when volunteers extended fasting from 12 to 36?h. In addition to known markers (plasma free fatty acids, glycerol, ketone bodies) that reflect changes in the body??s fuel management under fasting conditions a wide range of ??new?? entities such as ??-aminobutyrate as well as other amino and keto acids were identified as fasting markers. Based on multiple correlations amongst the metabolites and selected hormones in plasma such as leptin or insulin-like-growth-factor-1 (IGF-1), a robust metabolic network with coherent regulation of a wide range of metabolites could be identified. The metabolomics approach described here demonstrates the plasticity of human metabolism and identifies new and robust markers of the fasting state.     

Mass spectrometry-based phosphoproteomics reveals multisite phosphorylation on mammalian brain voltage-gated sodium and potassium channels

Voltage-gated sodium and potassium channels underlie electrical activity of neurons, and are dynamically regulated by diverse cell signaling pathways that ultimately exert their effects by altering the phosphorylation state of channel subunits. Recent mass spectrometric-based studies have led to a new appreciation of the extent and nature of phosphorylation of these ion channels in mammalian brain. This has allowed for new insights into how neurons dynamically regulate the localization, activity and expression through multisite ion channel phosphorylation.     

Metabolomics reveals organ-specific metabolic rearrangements during early tomato seedling development

Tomato seedlings (Solanum lycopersicum cv. MoneyMaker), grown under strictly controlled conditions, have been used to study alterations occurring in secondary metabolite biosynthetic pathways following developmental and environmental perturbations. Robustness and reproducibility of the system were confirmed using detailed statistical analyses of the metabolome. LCMS profiling was applied using whole germinated seeds as well as cotyledons, hypocotyls and roots from 3 to 9 days old seedlings to generate relative levels of 433 metabolites, of which 62 were annotated. Initial focus was given to the polyphenol pathway and several additional mass signals have been putatively annotated using high mass resolution fragmentation. Clear organ and developmental stage—specific differences were observed. Seeds accumulated saponin-like compounds; roots accumulated mainly alkaloids; cotyledons contained mainly glycosylated flavonols and; hypocotyls contained mainly anthocyanins. For each organ, the developmental changes in metabolite profiles were described by using linear mixed models. Across three independent experiments, 85 % of the metabolites showed similar developmental trends. This tomato seedling system has given us valuable additional insights into the complexity of seedling secondary metabolism. How metabolic profiles reflect an interplay between depletion of stored molecules and de novo synthesis and how the overall picture for this important crop plant contrasts to e.g. Arabidopsis are emphasised.     

Metabolomics characterization of energy metabolism reveals glycogen accumulation in gut-microbiota-lacking mice

Microbiota in the gut are considered an important environmental factor associated with host metabolism and physiology. Although gut microbiota are known to contribute to hepatic lipogenesis and fat storage, little is known about how the condition influences the deposition of glycogen in the liver. To better understand and characterize the host energy metabolism in guts lacking microbiota, we compared the liver metabolome of specific pathogen-free and germ-free mice by gas chromatography-mass spectrometry combined with partial least-squares discriminant analysis. We identified 30 of 52 highly reproducible peaks in chromatograms of liver tissue extracts from the two groups of mice. The two groups showed significant differences in metabolic profile. Changes in liver metabolism involved metabolites such as amino acids, fatty acids, organic acids and carbohydrates. The metabolic profile of germ-free mice suggests that they synthesize glycogen and accumulate it in the liver through gluconeogenesis and glycogenesis. Our findings shed light on a new perspective of the role of gut microbiota in energy metabolism and will be useful to help study probiotics, obesity and metabolic diseases.     

Geographical variation in genetic structure of an Atlantic Coastal Forest frog reveals regional differences in habitat stability

Climatic oscillations throughout the Pleistocene combined with geological and topographic complexity resulted in extreme habitat heterogeneity along the Atlantic coast of Brazil. Inferring how these historic landscape patterns have structured the current diversity of the region's biota is important both for our understanding of the factors promoting diversification, as well as the conservation of this biodiversity hotspot. Here we evaluate potential historical scenarios of diversification in the Atlantic Coastal Forest of Brazil by investigating the population genetic structure of a frog endemic to the region. Using mitochondrial and nuclear sequences, we generated a Bayesian population-level phylogeny of the Thoropa miliaris species complex. We found deep genetic divergences among five geographically distinct clades. Southern clades were monophyletic and nested within paraphyletic northern clades. Analyses of historical demographic patterns suggest an overall north to south population expansion, likely associated with regional differences in habitat stability during the Pliocene and early Pleistocene. However, genetic structure among southern populations is less pronounced and likely represents more recent vicariant events resulting from Holocenic sea-level oscillations. Our analyses corroborate that the Atlantic Coastal Forest has been a biogeographically dynamic landscape and suggest that the high diversity of its fauna and flora resulted from a combination of climatic and geologic events from the Pliocene to the present.     

A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes

Mammalian SWI/SNF complexes utilize either brahma (Brm) or brahma-related gene 1 (Brg1) catalytic subunits to remodel nucleosomes in an ATP-dependent manner. Brm was previously shown to be dispensable, suggesting that Brm and Brg1 are functionally redundant. To test this hypothesis, we have generated a Brg1 null mutation by gene targeting, and, surprisingly, homozygotes die during the periimplantation stage. Furthermore, blastocyst outgrowth studies indicate that neither the inner cell mass nor trophectoderm survives. However, experiments with other cell types demonstrate that Brg1 is not a general cell survival factor. In addition, Brg1 heterozygotes are predisposed to exencephaly and tumors. These results provide evidence that biochemically similar chromatin-remodeling complexes have dramatically different functions during mammalian development.     

Ecomorphology reveals Euler spiral of mammalian whiskers

Whiskers are present in many species of mammals. They are specialised vibrotactile sensors that sit within strongly innervated follicles. Whisker size and shape will affect the mechanical signals that reach the follicle, and hence the information that reaches the brain. However, whisker size and shape have not been quantified across mammals before. Using a novel method for describing whisker curvature, this study quantifies whisker size and shape across 19 mammalian species. We find that gross two-dimensional whisker shape is relatively conserved across mammals. Indeed, whiskers are all curved, tapered rods that can be summarised by Euler spiral models of curvature and linear models of taper, which has implications for whisker growth and function. We also observe that aquatic and semi-aquatic mammals have relatively thicker, stiffer, and more highly tapered whiskers than arboreal and terrestrial species. In addition, smaller mammals tend to have relatively long, slender, flexible whiskers compared to larger species. Therefore, we propose that whisker morphology varies between larger aquatic species, and smaller scansorial species. These two whisker morphotypes are likely to induce quite different mechanical signals in the follicle, which has implications for follicle anatomy as well as whisker function.     

Mechanism of the insect enzyme, tyramine beta-monooxygenase, reveals differences from the mammalian enzyme, dopamine beta-monooxygenase

Tyramine beta-monooxygenase (TbetaM) catalyzes the synthesis of the neurotransmitter, octopamine, in insects. Kinetic and isotope effect studies have been carried out to determine the kinetic mechanism of TbetaM for comparison with the homologous mammalian enzymes, dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase. A new and distinctive feature of TbetaM is very strong substrate inhibition that is dependent on the level of the co-substrate, O(2), and reductant as well as substrate deuteration. This has led to a model in which tyramine can bind to either the Cu(I) or Cu(II) forms of TbetaM, with substrate inhibition ameliorated at very high ascorbate levels. The rate of ascorbate reduction of the E-Cu(II) form of TbetaM is also reduced at high tyramine, leading us to propose the existence of a binding site for ascorbate to this class of enzymes. These findings may be relevant to the control of octopamine production in insect cells.     

Metabolomics reveals metabolite changes of patients with pulmonary arterial hypertension in China

The specific mechanism of pulmonary arterial hypertension (PAH) remains elusive. The present study aimed to explore the underlying mechanism of PAH through the identity of novel biomarkers for PAH using metabolomics approach. Serum samples from 40 patients with idiopathic PAH (IPAH), 20 patients with congenital heart disease‐associated PAH (CHD‐PAH) and 20 healthy controls were collected and analysed by ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry (UPLC‐HRMS). Orthogonal partial least square‐discriminate analysis (OPLS‐DA) was applied to screen potential biomarkers. These results were validated in monocrotaline (MCT)‐induced PAH rat model. The OPLS‐DA model was successful in screening distinct metabolite signatures which distinguished IPAH and CHD‐PAH patients from healthy controls, respectively (26 and 15 metabolites). Unbiased analysis from OPLS‐DA identified 31 metabolites from PAH patients which were differentially regulated compared to the healthy controls. Our analysis showed dysregulation of the different metabolic pathways, including lipid metabolism, glucose metabolism, amino acid metabolism and phospholipid metabolism pathways in PAH patients compared to their healthy counterpart. Among these metabolites from dysregulated metabolic pathways, a panel of metabolites from lipid metabolism and fatty acid oxidation (lysophosphatidylcholine, phosphatidylcholine, perillic acid, palmitoleic acid, N‐acetylcholine‐d ‐sphingomyelin, oleic acid, palmitic acid and 2‐Octenoylcarnitine metabolites) were found to have a close association with PAH. The results from the analysis of both real‐time quantitative PCR and Western blot showed that expression of LDHA, CD36, FASN, PDK1 GLUT1 and CPT‐1 in right heart/lung were significantly up‐regulated in MCT group than the control group.     

Metabolomics analysis of liver reveals profile disruption in bovines upon steroid treatment

Introduction

The surveillance of illegal anabolic practices in bovine meat production is necessary to guarantee consumers’ health. Screening strategies based on the recognition of indirect biological effects are considered by the community as promising tools to overcome some limitations of classical analytical methods and might therefore concur to ensure safer food for the consumer.

Objectives

The present work aims at characterizing the metabolic profile induced in liver by administration of anabolic steroids, and at identifying potential disturbances in the hepatic metabolism.

Methods

A total of 32 liver samples, 16 from untreated bulls and 16 from bulls treated with an ear implant (Revalor-XS^®) containing trenbolone acetate (200 mg) and β-estradiol (40 mg), were analyzed following a LC–MS-based metabolomic analysis combining RP and HILIC chromatographic separations. Different multivariate statistical tools were applied to the datasets to select common metabolites that may be considered as potential markers based on their significant changes in concentrations after administration of sexual steroids.

Results

Eight candidate markers were identified. Moreover, a subset of four markers was also validated by a different laboratory that performed the same analysis using an independent instrumental and elaboration platform, confirming the robustness of the results achieved.

Conclusion

This study was performed mimicking experimental conditions that may be used during a potential misuse practice. It is promising in the objective of setting up an analytical strategy to highlight sexual steroids abuse in livestock animals.

     

Metabolomics reveals determinants of weight loss during lifestyle intervention in obese children

The amount of weight loss in obese children during lifestyle intervention differs strongly between individuals. The metabolic processes underlying this variability are largely unknown. We hypothesize that metabolomics analyses of serum samples might help to identify metabolic predictors of weight loss. In this study, we investigated 80 obese children aged 6–15 years having completed the one-year lifestyle intervention program ‘Obeldicks’, 40 that achieved a substantial reduction of their body mass index standard deviation score (BMI-SDS) during this intervention (defined as BMI-SDS reduction ≥ 0.5), and 40 that did not improve their overweight status (BMI-SDS reduction < 0.1). Anthropometric and clinical parameters were measured and baseline fasting serum samples of all children were analyzed with a mass spectrometry-based metabolomics approach targeting 163 metabolites. Both univariate regression models and a multivariate least absolute shrinkage and selection operator (LASSO) approach identified lower serum concentrations of long-chain unsaturated phosphatidylcholines as well as smaller waist circumference as significant predictors of BMI-SDS reduction during intervention (p-values univariate models: 5.3E?03 to 1.0E?04). A permutation test showed that the LASSO model explained a significant part of BMI-SDS change (p = 4.6E?03). Our results suggest a role of phosphatidylcholine metabolism and abdominal obesity in body weight regulation. These findings might lead to a better understanding of the mechanisms behind the large inter-individual variation in response to lifestyle interventions, which is a prerequisite for the development of individualized intervention programs.     

Characterization of the enolase isozymes of rabbit brain: kinetic differences between mammalian and yeast enolases

Two isozymes of enolase, alpha alpha and gamma gamma, have been purified from rabbit brain and characterized. The kinetic properties of alpha alpha and gamma gamma (pH optimum, Km for phosphoglycerate and phosphoenolpyruvate, requirement for a divalent cation) are very similar to those of rabbit enolase, form beta beta, and to those of enolase isozymes from other species. However, several novel properties were observed. (i) All the enolases studied were inhibited by Na+ and Li+. (ii) The rabbit enolases, but not yeast enolase, were activated by K+, NH4+, Cs+, and Rb+. (iii) Rabbit enolase is more susceptible to inhibition by excess Mg2+ than is the yeast enolase; the increased inhibition by Mg2+ above pH 7.1 accounts, at least in part, for the observed differences between mammalian and yeast enolases in their pH optima for activity.     

Global and regional differences in brain anatomy of young children born small for gestational age

In children who are born small for gestational age (SGA), an adverse intrauterine environment has led to underdevelopment of both the body and the brain. The delay in body growth is (partially) restored during the first two years in a majority of these children. In addition to a negative influence on these physical parameters, decreased levels of intelligence and cognitive impairments have been described in children born SGA. In this study, we used magnetic resonance imaging to examine brain anatomy in 4- to 7-year-old SGA children with and without complete bodily catch-up growth and compared them to healthy children born appropriate for gestational age. Our findings demonstrate that these children strongly differ on brain organisation when compared with healthy controls relating to both global and regional anatomical differences. Children born SGA displayed reduced cerebral and cerebellar grey and white matter volumes, smaller volumes of subcortical structures and reduced cortical surface area. Regional differences in prefrontal cortical thickness suggest a different development of the cerebral cortex. SGA children with bodily catch-up growth constitute an intermediate between those children without catch-up growth and healthy controls. Therefore, bodily catch-up growth in children born SGA does not implicate full catch-up growth of the brain.     

Adaptation of the CHARM DNA methylation platform for the rat genome reveals novel brain region-specific differences

Comprehensive High-throughput Arrays for Relative Methylation (CHARM) was recently developed as an experimental platform and analytic approach to assess DNA methylation (DNAm) at a genome-wide level. Its initial implementation was for human and mouse. We adapted it for rat and sought to examine DNAm differences across tissues and brain regions in this model organism. We extracted DNA from liver, spleen, and three brain regions: cortex, hippocampus, and hypothalamus from adult Sprague Dawley rats. DNA was digested with McrBC, and the resulting methyl-depleted fraction was hybridized to the rat CHARM array along with a mock-treated fraction. Differentially methylated regions (DMRs) between tissue types were detected using normalized methylation log-ratios. In validating 24 of the most significant DMRs by bisulfite pyrosequencing, we detected large mean differences in DNAm, ranging from 33-59%, among the most significant DMRs in the across-tissue comparisons. The comparable figures for the hippocampus vs. hypothalamus DMRs were 14-40%, for the cortex vs. hippocampus DMRs, 12-29%, and for the cortex vs. hypothalamus DMRs, 5-35%, with a correlation of r² = 0.92 between the methylation differences in 24 DMRs predicted by CHARM and those validated by bisulfite pyrosequencing. Our adaptation of the CHARM array for the rat genome yielded highly robust results that demonstrate the value of this method in detecting substantial DNAm differences between tissues and across different brain regions. This platform should prove valuable in future studies aimed at examining DNAm differences in particular brain regions of rats exposed to environmental stimuli with potential epigenetic consequences.     

Adaptation of the CHARM DNA methylation platform for the rat genome reveals novel brain region-specific differences

Comprehensive High-throughput Arrays for Relative Methylation (CHARM) was recently developed as an experimental platform and analytic approach to assess DNA methylation (DNAm) at a genome-wide level. Its initial implementation was for human and mouse. We adapted it for rat and sought to examine DNAm differences across tissues and brain regions in this model organism. We extracted DNA from liver, spleen, and three brain regions: cortex, hippocampus, and hypothalamus from adult Sprague Dawley rats. DNA was digested with McrBC, and the resulting methyl-depleted fraction was hybridized to the rat CHARM array along with a mock-treated fraction. Differentially methylated regions (DMRs) between tissue types were detected using normalized methylation log-ratios. In validating 24 of the most significant DMRs by bisulfite pyrosequencing, we detected large mean differences in DNAm, ranging from 33-59%, among the most significant DMRs in the across-tissue comparisons. The comparable figures for the hippocampus vs. hypothalamus DMRs were 14-40%, for the cortex vs. hippocampus DMRs, 12-29%, and for the cortex vs. hypothalamus DMRs, 5-35%, with a correlation of r(2) = 0.92 between the methylation differences in 24 DMRs predicted by CHARM and those validated by bisulfite pyrosequencing. Our adaptation of the CHARM array for the rat genome yielded highly robust results that demonstrate the value of this method in detecting substantial DNAm differences between tissues and across different brain regions. This platform should prove valuable in future studies aimed at examining DNAm differences in particular brain regions of rats exposed to environmental stimuli with potential epigenetic consequences.