Crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> protein ybgI,a toroidal structure with a dinuclear metal site

Background

The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.

Results

The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.

Conclusion

The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

     

Metabolite and gene expression profiles suggest a putative mechanism through which high dietary carbohydrates reduce the content of hepatic betaine in <Emphasis Type="Italic">Megalobrama amblycephala</Emphasis>

Background

High-carbohydrate diets (HCD) are favoured by the aquaculture industry for economic reasons, but they can produce negative impacts on growth and induce hepatic steatosis. We hypothesised that the mechanism behind this is the reduction of hepatic betaine content.

Objective

We further explored this mechanism by supplementing betaine (1%) to the diet of a farmed fish Megalobrama amblycephala.

Methods

Four diet groups were designed: control (CD, 27.11% carbohydrates), high-carbohydrate (HCD, 36.75% carbohydrates), long-term betaine (LBD, 35.64% carbohydrates) and short-term betaine diet (SBD; 12 weeks HCD?+?4 weeks LBD). We analysed growth performance, body composition, liver condition, and expression of genes and profiles of metabolites associated with betaine metabolism.

Results

HCD resulted in poorer growth and liver health (compared to CD), whereas LBD improved these parameters (compared to HCD). HCD induced the expression of genes associated with glucose, serine and cystathionine metabolisms, and (non-significantly, p?=?.20) a betaine-catabolizing enzyme betaine-homocysteine-methyltransferase; and decreased the content of betaine, methionine, S-adenosylhomocysteine and carnitine. Betaine supplementation (LBD) reversed these patterns, and elevated betaine-homocysteine-methyltransferase, S-adenosylmethionine and S-adenosylhomocysteine (all p?≤?.05).

Conclusion

We hypothesise that HCD reduced the content of hepatic betaine by enhancing the activity of metabolic pathways from glucose to homocysteine, reflected in increased glycolysis, serine metabolism, cystathionine metabolism and homocysteine remethylation. Long-term dietary betaine supplementation improved the negative impacts of HCD, inculding growth parameters, body composition, liver condition, and betaine metabolism. However, betaine supplementation may have caused a temporary disruption in the metabolic homeostasis.

     

Comparative proteomic analysis between nitrogen supplemented and starved conditions in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Background

Fungi are constantly exposed to nitrogen limiting environments, and thus the efficient regulation of nitrogen metabolism is essential for their survival, growth, development and pathogenicity. To understand how the rice blast pathogen Magnaporthe oryzae copes with limited nitrogen availability, a global proteome analysis under nitrogen supplemented and nitrogen starved conditions was completed.

Methods

M. oryzae strain 70–15 was cultivated in liquid minimal media and transferred to media with nitrate or without a nitrogen source. Proteins were isolated and subjected to unfractionated gel-free based liquid chromatography-tandem mass spectrometry (LC-MS/MS). The subcellular localization and function of the identified proteins were predicted using bioinformatics tools.

Results

A total of 5498 M. oryzae proteins were identified. Comparative analysis of protein expression showed 363 proteins and 266 proteins significantly induced or uniquely expressed under nitrogen starved or nitrogen supplemented conditions, respectively. A functional analysis of differentially expressed proteins revealed that during nitrogen starvation nitrogen catabolite repression, melanin biosynthesis, protein degradation and protein translation pathways underwent extensive alterations. In addition, nitrogen starvation induced accumulation of various extracellular proteins including small extracellular proteins consistent with observations of a link between nitrogen starvation and the development of pathogenicity in M. oryzae.

Conclusion

The results from this study provide a comprehensive understanding of fungal responses to nitrogen availability.

     

Quantitative analysis of tetrahydrofolate metabolites from <Emphasis Type="Italic">clostridium autoethanogenum</Emphasis>

Introduction

Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.

Objective

To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.

Methods

Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.

Results

Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.

Conclusion

Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

     

Sex differences in hepatic one-carbon metabolism

Background

There are large differences between men and women of child-bearing age in the expression level of 5 key enzymes in one-carbon metabolism almost certainly caused by the sex hormones. These male-female differences in one-carbon metabolism are greatly accentuated during pregnancy. Thus, understanding the origin and consequences of sex differences in one-carbon metabolism is important for precision medicine.

Results

We have created a mathematical model of hepatic one-carbon metabolism based on the underlying physiology and biochemistry. We use the model to investigate the consequences of sex differences in gene expression. We give a mechanistic understanding of observed concentration differences in one-carbon metabolism and explain why women have lower S-andenosylmethionine, lower homocysteine, and higher choline and betaine. We give a new explanation of the well known phenomenon that folate supplementation lowers homocysteine and we show how to use the model to investigate the effects of vitamin deficiencies, gene polymorphisms, and nutrient input changes.

Conclusions

Our model of hepatic one-carbon metabolism is a useful platform for investigating the mechanistic reasons that underlie known associations between metabolites. In particular, we explain how gene expression differences lead to metabolic differences between males and females.

     

Extraction of natural products from bark of <Emphasis Type="Italic">Betula pendula</Emphasis> using ionic liquids

Introduction

Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.

Objectives

To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.

Methods

The extracted compounds were analyzed by LC/MS and GC/MS.

Results

The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.

Conclusion

From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

Chilling slows anaerobic metabolism to improve anoxia tolerance of insects

Background

Insects are renowned for their ability to survive anoxia. Anoxia tolerance may be enhanced during chilling through metabolic suppression.

Aims

Here, the metabolomic response of insects to anoxia, both with and without chilling, for different durations (12–36 h) was examined to assess the potential cross-tolerance mechanisms.

Results

Chilling during anoxia (cold anoxia) significantly improved survival relative to anoxia at warmer temperatures. Reduced intermediate metabolites and increased lactic acid, indicating a switch to anaerobic metabolism, were characteristic of larvae in anoxia.

Conclusions

Anoxia tolerance was correlated survival improvements after cold anoxia were correlated with a reduction in anaerobic metabolism.

     

Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

Mitochondrial aconitase is a key regulator of energy production for growth and protein expression in Chinese hamster ovary cells

Introduction

Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.

Objectives

The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.

Methods

To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.

Results

Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.

Conclusion

Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.

     

Squalene isolated from <Emphasis Type="Italic">Schizochytrium mangrovei</Emphasis> is a peroxisome proliferator-activated receptor-α agonist that regulates lipid metabolism in HepG2 cells

Objective

Peroxisome proliferator-activated receptor-alpha (PPARα) is a nuclear receptor that has critical roles in the treatment of atherosclerosis, hyperlipidemia and hepatic steatosis.

Results

Squalene is a novel nature PPARα agonist, identified from reporter gene assay and qPCR analysis. Cultured hepatocytes stimulated with squalene exhibited significantly decreased cellular triacylglycerols and cholesterol concentrations, while cellular uptake of fatty acids was increased. Quantitative PCR analysis revealed that expression of genes related to fatty acid uptake, fatty acid oxidation, ketogenesis and reverse cholesterol transport metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed in cell treated with squalene.

Conclusion

Squalene is hypolipidemic by activation of PPARα via a ligand-mediated mechanism that regulates the expression of lipid metabolism genes in hepatocytes.

     

<Emphasis Type="Italic">massPix</Emphasis>: an R package for annotation and interpretation of mass spectrometry imaging data for lipidomics

Introduction

Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools.

Objectives

We have developed massPix—an R package for analysing and interpreting data from MSI of lipids in tissue.

Methods

massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries.

Results

Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering.

Conclusion

massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.

     

Uncovering the metabolic response of abalone (<Emphasis Type="Italic">Haliotis midae)</Emphasis> to environmental hypoxia through metabolomics

Introduction

Oxygen is essential for metabolic processes and in the absence thereof alternative metabolic pathways are required for energy production, as seen in marine invertebrates like abalone. Even though hypoxia has been responsible for significant losses to the aquaculture industry, the overall metabolic adaptations of abalone in response to environmental hypoxia are as yet, not fully elucidated.

Objective

To use a multiplatform metabolomics approach to characterize the metabolic changes associated with energy production in abalone (Haliotis midae) when exposed to environmental hypoxia.

Methods

Metabolomics analysis of abalone adductor and foot muscle, left and right gill, hemolymph, and epipodial tissue samples were conducted using a multiplatform approach, which included untargeted NMR spectroscopy, untargeted and targeted LC–MS spectrometry, and untargeted and semi-targeted GC-MS spectrometric analyses.

Results

Increased levels of anaerobic end-products specific to marine animals were found which include alanopine, strombine, tauropine and octopine. These were accompanied by elevated lactate, succinate and arginine, of which the latter is a product of phosphoarginine breakdown in abalone. Primarily amino acid metabolism was affected, with carbohydrate and lipid metabolism assisting with anaerobic energy production to a lesser extent. Different tissues showed varied metabolic responses to hypoxia, with the largest metabolic changes in the adductor muscle.

Conclusions

From this investigation, it becomes evident that abalone have well-developed (yet understudied) metabolic mechanisms for surviving hypoxic periods. Furthermore, metabolomics serves as a powerful tool for investigating the altered metabolic processes in abalone.

     

Association of mitochondrial dysfunction and lipid metabolism with type 2 diabetes mellitus: A review of literature

Background

Diabetes mellitus (DM) is one of the most prevalent chronic diseases, and its prevalence continues to increase globally. The impact of mitochondrial dysfunction and lipid metabolism on diabetes mellitus and insulin resistance (IR) has been implicated in several previous reports; however, the results of studies are confusing despite four decades of study.

Methods/Results

This review has evaluated updated understanding of the role of mitochondrial dysfunction and lipid metabolism on type 2 diabetes, and found that mitochondrial dysfunction and lipid metabolism disorder induce the dysregulation of liver and pancreatic beta cells, insulin resistance, and type 2 diabetes.

Conclusion

Mitochondrial dysfunction and lipid metabolism induce metabolic dysregulation and finally increasing the possibility of diabetes.

     

Metabolic profiling identifies trehalose as an abundant and diurnally fluctuating metabolite in the microalga <Emphasis Type="Italic">Ostreococcus tauri</Emphasis>

Introduction

The picoeukaryotic alga Ostreococcus tauri (Chlorophyta) belongs to the widespread group of marine prasinophytes. Despite its ecological importance, little is known about the metabolism of this alga.

Objectives

In this work, changes in the metabolome were quantified when O. tauri was grown under alternating cycles of 12 h light and 12 h darkness.

Methods

Algal metabolism was analyzed by gas chromatography-mass spectrometry. Using fluorescence-activated cell sorting, the bacteria associated with O. tauri were depleted to below 0.1% of total cells at the time of metabolic profiling.

Results

Of 111 metabolites quantified over light–dark cycles, 20 (18%) showed clear diurnal variations. The strongest fluctuations were found for trehalose. With an intracellular concentration of 1.6 mM in the dark, this disaccharide was six times more abundant at night than during the day. This fluctuation pattern of trehalose may be a consequence of starch degradation or of the synchronized cell cycle. On the other hand, maltose (and also sucrose) was below the detection limit (~10 μM). Accumulation of glycine in the light is in agreement with the presence of a classical glycolate pathway of photorespiration. We also provide evidence for the presence of fatty acid methyl and ethyl esters in O. tauri.

Conclusions

This study shows how the metabolism of O. tauri adapts to day and night and gives new insights into the configuration of the carbon metabolism. In addition, several less common metabolites were identified.

     

The urinary metabolome in female mink (<Emphasis Type="Italic">Mustela neovison</Emphasis>) shows distinct changes in protein and lipid metabolism during the transition from diapause to implantation

Introduction

The mink exhibit an obligatory diapause. The metabolic changes during the transition from diapause to implantation and established pregnancy are currently unknown.

Objectives

The study aimed to characterize changes in the urinary metabolome in mink during the period from mating to early gestation and to identify the metabolites involved.

Methods

Urine samples were collected from 56 female mink on March 24, April 8, and April 15, covering the period from mating to early pregnancy. The urine samples were subjected to non-targeted LC-MS metabolomics. Processed data were evaluated by principal component analysis (PCA) and the peak area of identified metabolites were subjected to ANOVA.

Results

The samples showed clear clustering according to sampling date in a PCA scores plot, and 35 metabolites differing significantly between sampling days were identified. The excretion of dicarboxylic acids and acylcarnitines of dicarboxylic acids exhibited a decline on April 8, and the same trend was observed for four unidentified metabolites, two of which were putatively identified as acids of the furan fatty acid type. The decreased excretion of lipid components was suggested to be a result of increased oxidation of these compounds. In contrast, the excretion of amino acid-related metabolites showed an increase on April 8 which was attributed to increased metabolism of amino acids at this time point.

Conclusion

The urinary metabolic profile of mink showed distinct changes during the period studied. The major changes were observed at the time of implantation where increases in the lipid and protein metabolism were evident.

     

A metabolomics approach to uncover effects of different exercise modalities in type 1 diabetes

Introduction

Exercise-associated metabolism in type 1 diabetes (T1D) remains under-studied due to the complex interplay between exogenous insulin, counter-regulatory hormones and insulin-sensitivity.

Objective

To identify the metabolic differences induced by two exercise modalities in T1D using ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC–HRMS) based metabolomics.

Methods

Twelve T1D adults performed intermittent high-intensity (IHE) and continuous-moderate-intensity (CONT) exercise. Serum samples were analysed by UHPLC–HRMS.

Results

Metabolic profiling of IHE and CONT highlighted exercise-induced changes in purine and acylcarnitine metabolism.

Conclusion

IHE may increase beta-oxidation through higher ATP-turnover. UHPLC–HRMS based metabolomics as a data-driven approach without an a priori hypothesis may help uncover distinctive metabolic effects during exercise in T1D.Clinical trial registration number is www.clinicaltrials.gov: NCT02068638.