-
[show abstract]
[hide abstract]
ABSTRACT: A new version of the Gavin software (V. Behrends, G. D. Tredwell, J. G. Bundy, Anal Biochem 415, 206–208 (2011)), implementing a new GUI.
-
[show abstract]
[hide abstract]
ABSTRACT: Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used nuclear magnetic resonance (NMR) spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles, e.g. for two-component system mutants, the cognate response regulator and sensor kinase genes clustered tightly together. Some strains had metabolic phenotypes (metabotypes) that could be related to the known gene function; e.g. pyruvate dehydrogenase mutants accumulated large amounts of pyruvate in the medium. In other cases, the metabolic phenotypes were not easily interpretable. The rpoN mutant, which lacks the alternative sigma factor RpoN (σ54), accumulated high levels of gluconate in the medium. In addition, endometabolome profiling of intracellular metabolites identified a number of systemic metabolic changes. We linked this to indirect regulation of the catabolite repression protein Crc via the noncoding RNA crcZ, and found that a crcZ (but not crc) mutant also shared the high-gluconate phenotype. We profiled an additional set of relevant metabolic enzymes and transporters, including Crc targets, and showed that the Crc-regulated edd mutant (gluconate-6-phosphate dehydratase) had similar gluconate levels to the rpoN mutant. Finally, a set of clinical isolates showed patient- and RAPD-type-specific differences in gluconate production, which were significantly associated with resistance across four antibiotics (tobramycin, ciprofloxacin, aztreonam, imipenem), indicating that this has potential clinical relevance.
Journal of Biological Chemistry 04/2013; · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We grew Pseudomonas aeruginosa in LB and artificial sputum medium (filtered and unfiltered), and quantified metabolite utilization and excretion by NMR spectroscopy (metabolic footprinting, or extracellular metabolomics). Utilization was similar between media, but there were differences in excretion - e.g. acetate was produced only in unfiltered ASM.
Applied and environmental microbiology 01/2013; · 3.69 Impact Factor
-
Peter Kille,
Jane Andre,
Craig Anderson,
Hui Na Ang,
Michael W. Bruford, Jacob G. Bundy,
Robert Donnelly,
Mark E. Hodson,
Gabriela Juma,
Elma Lahive,
A. John Morgan,
Stephen R. Stürzenbaum,
David J. Spurgeon
[show abstract]
[hide abstract]
ABSTRACT: Evidence is emerging that earthworms can evolve tolerance to trace element enriched soils. However, few studies have sought to establish whether such tolerance is determined through adaption or plasticity. Here we report results from a combined analysis of mitochondrial (cytochrome oxidase II, COII), nuclear (amplified fragment length polymorphism, AFLP) variation and DNA methylation in populations of the earthworm Lumbricus rubellus from sites across an abandoned arsenic and copper mine. Earthworms from the mine site population demonstrated clear arsenic tolerance in comparison to a naïve strain. COII and AFLP results suggest that L. rubellus from the unexposed and the adapted populations comprises two cryptic lineages (Linages A and B) each of which was present across all of the sites. AFLP analysis by lineage highlighted variations associated with soil metal/metalloid concentrations (most clearly for Lineage A) suggesting a genetic component to the observed tolerance. The methylation sensitive AFLP (Me-AFLP) identified a high genome methylation content (average 13.5%) in both lineages. For Lineage A, Me-AFLP analysis did not identify a strong association with soil arsenic levels. For Lineage B, however, a clear association of methylation patterns with soil arsenic concentrations was found. This suggests that Lineage B earthworms utilise epigenetic mechanisms to adapt to the presence of contamination. These fundamentally different genetic adjustments in the two clades indicate that the two lineages employ distinct adaptive strategies (genetic or epigenetic) in response to arsenic exposure. Mechanisms driving this variation may be founded within the colonisation histories of the lineages.
Soil Biology and Biochemsitry. 01/2013; 57:524-532.
-
[show abstract]
[hide abstract]
ABSTRACT: Aging is a complex process, which involves changes in different cellular functions that all can be integrated on the metabolite level. This means that different gene regulation pathways that affect aging might lead to similar changes in metabolism and result in a metabolic signature of senescence. In this chapter, we describe how to establish a metabolic signature of senescence by analyzing the metabolome of various longevity mutants of the model organism Caenorhabditis elegans using gas chromatography-mass spectrometry (GC-MS). Since longevity-associated genes exist for other model organisms and humans, this analysis could be universally applied to body fluids or whole tissue samples for studing the relationship between senescence and metabolism.
Methods in molecular biology (Clifton, N.J.) 01/2013; 965:355-71.
-
[show abstract]
[hide abstract]
ABSTRACT: The ability to accumulate osmoprotectant compounds, such as betaines, is an important evolutionary feature in many organisms. This is particularly the case for organisms that live in variable environments, which may have fluctuations in moisture and salinity levels. There is, surprisingly, very little known about betaines in soil invertebrates in general, and there is almost no information about earthworms - a group that are important 'ecosystem engineers' and key indicators of soil health. Here, we describe a fast and reliable (1)H-(13)C heteronuclear single quantum coherence (HSQC) 2D NMR approach for the metabolic profiling of a series of betaines and related metabolites in tissue extracts, and list (1)H and (13)C chemical shifts for the trimethylammonium signal for 23 such compounds. The analysis of ten different species from three different families (Lumbricidae, Megascolecidae and Glossoscolecidae) showed an unexpected diversity of betaines present in earthworms. In total ten betaines were identified, including hydroxyproline-betaine, proline-betaine, taurine-betaine, GABA-betaine and histidine-betaine, and a further eleven as-yet unassigned putative betaine metabolites detected. The findings clearly indicate a hitherto-unappreciated important role for betaine metabolism in earthworms.
Biochemical and Biophysical Research Communications 12/2012; · 2.48 Impact Factor
-
James K Ellis,
Toby J Athersuch,
Laura D K Thomas,
Friederike Teichert,
Miriam Pérez-Trujillo,
Claus Svendsen,
David J Spurgeon,
Rajinder Singh,
Lars Järup, Jacob G Bundy,
Hector C Keun
[show abstract]
[hide abstract]
ABSTRACT: The 'exposome' represents the accumulation of all environmental exposures across a lifetime. Top-down strategies are required to assess something this comprehensive, and could transform our understanding of how environmental factors affect human health. Metabolic profiling (metabonomics/metabolomics) defines an individual's metabolic phenotype, which is influenced by genotype, diet, lifestyle, health and xenobiotic exposure, and could also reveal intermediate biomarkers for disease risk that reflect adaptive response to exposure. We investigated changes in metabolism in volunteers living near a point source of environmental pollution: a closed zinc smelter with associated elevated levels of environmental cadmium.
High-resolution ¹H NMR spectroscopy (metabonomics) was used to acquire urinary metabolic profiles from 178 human volunteers. The spectral data were subjected to multivariate and univariate analysis to identify metabolites that were correlated with lifestyle or biological factors. Urinary levels of 8-oxo-deoxyguanosine were also measured, using mass spectrometry, as a marker of systemic oxidative stress.
Six urinary metabolites, either associated with mitochondrial metabolism (citrate, 3-hydroxyisovalerate, 4-deoxy-erythronic acid) or one-carbon metabolism (dimethylglycine, creatinine, creatine), were associated with cadmium exposure. In particular, citrate levels retained a significant correlation to urinary cadmium and smoking status after controlling for age and sex. Oxidative stress (as determined by urinary 8-oxo-deoxyguanosine levels) was elevated in individuals with high cadmium exposure, supporting the hypothesis that heavy metal accumulation was causing mitochondrial dysfunction.
This study shows evidence that an NMR-based metabolic profiling study in an uncontrolled human population is capable of identifying intermediate biomarkers of response to toxicants at true environmental concentrations, paving the way for exposome research.
BMC Medicine 06/2012; 10:61. · 6.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Metabolic profiling of tissues needs special attention, because the compartmentalization of cellular constituents will be abolished by sample homogenization. This loss of partitioning leads to protein and metabolite instability in extracts, and therefore metabolite extraction protocols need to ensure very rapid inactivation of mac- romolecules as well as solubilization of metabolites. There are many published methods for tissue metabolome anal- ysis, but no universally accepted standard, and a lack of measurable quality benchmarks. We developed a protocol for efficient tissue disruption and metabolite extraction of the earthworm Lumbricus rubellus guided by prior biological knowledge as well as metrics based on the data. In particular, we identified an unusual degree of instability of L. rubellus tissue extracts, and evaluated different approaches such as heating and filtration to counteract this. Finally, we evaluated four different solvent systems for comprehensive metabolite extraction using three analytical platforms (1H NMR spectroscopy, GC–MS, and direct- infusion FT-ICR-MS), and also compared bead-beating and cryogenic milling for tissue disruption. Initially we ranked methods by common analytical criteria (e.g. num- bers and total intensity of detected peaks) in order to compare protocols. These approaches to assess protocol suitability proved to be inadequate to judge earthworm tissue extraction methods because of sample instability. Existing tissue extraction protocols should not be assumed to be automatically applicable to novel species.
Metabolomics 01/2012; · 4.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Fluorodeoxyuridine (FUdR) is often used to help maintain synchronous populations of Caenorhabditis elegans adults, for instance as would typically be the case in studying age-related effects. However, given that FUdR inhibits DNA synthesis and therefore reproduction, it will clearly have significant wide-ranging biological effects. It is often assumed that these can be compensated for using appropriate controls. We show here that this is not the case for a metabolomic analysis of a long-lived daf-2 mutant strain: not only were the effects of FUdR much greater than the effects of the mutation, there were clear interactions between FUdR and genotype, such that identification of daf-2-dependent metabolites would have been compromised on FUdR plates. This indicates that FUdR should only be used with caution for C. elegans ageing experiments, and should not be assumed to be independent of other factors being studied.
Mechanisms of ageing and development 11/2011; 133(1):46-9. · 4.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nuclear magnetic resonance (NMR) spectroscopy is widely used as an analytical platform for metabolomics. Many studies make use of 1D spectra, which have the advantages of relative simplicity and rapid acquisition times. The spectral data can then be analyzed either with a chemometric workflow or by an initial deconvolution or fitting step to generate a list of identified metabolites and associated sample concentrations. Various software tools exist to simplify the fitting process, but at least for 1D spectra, this still requires a degree of skilled operator input. It is of critical importance that we know how much person-to-person variability affects the results, in order to be able to judge between different studies. Here we tested a commercially available software package (Chenomx' NMR Suite) for fitting metabolites to a set of NMR spectra of yeast extracts and compared the output of five different people for both metabolite identification and quantitation. An initial comparison showed good agreement for a restricted set of common metabolites with characteristic well-resolved resonances but wide divergence in the overall identities and number of compounds fitted; refitting according to an agreed set of metabolites and spectral processing approach increased the total number of metabolites fitted but did not dramatically increase the quality of the metabolites that could be fitted without prior knowledge about peak identity. Hence, robust peak assignments are required in advance of manual deconvolution, when the widest range of metabolites is desired. However, very low concentration metabolites still had high coefficients of variation even with shared information on peak assignment. Overall, the effect of the person was less than the experimental group (in this case, sampling method) for almost all of the metabolites.
Analytical Chemistry 11/2011; 83(22):8683-7. · 5.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The relationship of measured or modelled Cd concentrations in soil, house dust and available to plants with human urinary Cd concentrations were assessed in a population living around a Cd/Pb/Zn smelter in the UK. Modelled air concentrations explained 35% of soil Cd variation indicating the smelter contributed to soil Cd loads. Multi-variate analysis confirmed a significant role of biological and life-style factors in determining urinary Cd levels. Significant correlations of urinary Cd with soil, house dust and modelled plant available Cd concentrations were not, however, found. Potential reasons for the absence of clear relationships include limited environmental contact in urban populations; the role of undefined factors in determining exposure; and the limited spatial scope of the survey which did not sample from the full pollution gradient. Further, the absence of any significant relationship indicates that environmental measures provide limited advantage over atmospheric model outputs for first stage human exposure assessment.
Environmental pollution (Barking, Essex: 1987) 09/2011; 159(12):3425-32. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The software package AMDIS performs gas chromatography-mass spectrometry (GC-MS) peak deconvolution but tends to produce false positives and leaves missing values where peaks are found in only a proportion of a set of chromatograms. We have developed a software complement to AMDIS that (i) allows rapid manual inspection of chromatographic peaks across all samples to confirm data quality and (ii) for a given sample set, integrates peak areas across all samples even where AMDIS deconvolution would leave missing values. The freely available package runs within the commercial Matlab environment and is useful where GC-MS is used to profile complex mixtures.
Analytical Biochemistry 08/2011; 415(2):206-8. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The genome of the nematode Caenorhabditis elegans encodes for two multifunctional metal binding metallothioneins (MTs), CeMT-1 and CeMT-2. Here we applied qPCR to identify a transcriptional up-regulation following the exposure to free radical generators (ROS) paraquat or hydrogen peroxide, a trend that was confirmed with Pmtl::GFP expressing alleles. The deletion of the MT loci resulted in an elevation of in vivo levels of hydrogen peroxide and exposure to ROS caused a reduction in total egg production, growth and life span in wild type nematodes, effects that were particularly pronounced in the CeMT-2 and double knockout. Moreover, in vitro incubation of recombinant MTs with hydrogen peroxide demonstrated the presence of direct oxidation of the CeMTs, with zinc released from both isoforms and concomitant formation of intra-molecular disulfides. Finally, metabolic profiling (metabolomic) analysis of wild type and MT knockouts in the presence/absence of oxidative stressors, confirmed the overall trend described by the other experiments, and identified 2-aminoadipate as a potential novel small-molecule marker of oxidative stress. In summary, this study highlights that C. elegans metallothioneins scavenge and protect against reactive oxygen species and potentially against oxidative stress, with CeMT-2 being more effective than CeMT-1.
Molecular BioSystems 06/2011; 7(8):2397-406. · 3.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The nematode Caenorhabditis elegans is widely used as a model organism in many areas of the life sciences. Metabolite profiling (metabolomics/metabonomics) is a powerful means of assigning phenotypes to experimentally perturbed C. elegans samples (e.g., mutants, RNAi, or chemical treatments). Tissue extraction is a key step, and high-quality and reproducible extractions are essential to the success of metabolomics studies. We have performed an extensive comparison of different tissue extraction techniques with C. elegans, comparing two different solvent systems (chloroform/methanol and aqueous methanol) and six different tissue disruption techniques (including manual grinding in a cooled mortar, homogenization, and various grinding media in both reciprocating and orbital tissue mills). All twelve combinations were then compared by GC/MS, (1)H NMR spectroscopy, and UPLC-MS, and the results were evaluated by both overall multivariate clustering approaches as well as distributions over individual metabolites/metabolite features of coefficient of variation and yield. The choice of solvent had more influence than the disruption method used, although the homogenizer results were clearly outliers. Overall, we concluded that bead-beating with 80% methanol solution was a good trade-off, although it is important to note that the definition of the apparent "best" method depended on which analytical platform was used to evaluate the results.
Analytical Chemistry 05/2011; 83(10):3730-6. · 5.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate mechanisms of osmotic tolerance in Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc) of closely related strains, which is of clinical as well as environmental importance.
We employed NMR-based metabolic profiling (metabolomics) to elucidate the metabolic consequences of high osmotic stress for five isolates of B. cenocepacia. The strains differed significantly in their levels of osmotic stress tolerance, and we identified three different sets of metabolic responses with the strains least impacted by osmotic stress exhibiting higher levels of the osmo-protective metabolites glycine-betaine and/or trehalose. Strains either increased concentrations or had constitutively high levels of these metabolites.
Even within the small set of B. cenocepacia isolates, there was a surprising degree of variability in the metabolic responses to osmotic stress.
The metabolic responses, and hence osmotic stress tolerance, vary between different B. cenocepacia isolates. This study provides a first look into the potentially highly diverse physiology of closely related strains of one species of the Bcc and illustrates that physiological or clinically relevant phenotypes are unlikely to be inferable from genetic relatedness within this species group.
Letters in Applied Microbiology 03/2011; 52(6):619-25. · 1.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris.
PLoS ONE 01/2011; 6(1):e16286. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Metabolic footprinting has shown enormous potential as a phenotyping tool and we are interested in applying it to understand the physiology of the opportunistic pathogen Pseudomonas aeruginosa during its chronic infection of the lungs of cystic fibrosis patients. The selection pressures of surviving in the CF lung environment lead to genetic adaptations of the bacterium. A common adaptation is mutation of the mucA gene, resulting in a loss-of-function mutation to the anti-sigma factor MucA, which leads to a mucoid phenotype as a consequence of the overproduction of the extracellular polysaccharide alginate. However, apart from the mucoid phenotype little is known about the overall metabolic and physiological changes caused by mucA mutation. We investigated the pleiotropic metabolic effects of this mutation using time-resolved metabolic footprinting (extracellular metabolomics), and found changes in the levels of various metabolites associated with osmotic tolerance, including glycine-betaine, trehalose and glutamate. Physiological experiments confirmed that the isogenic mucA22 mutant is less resistant to osmotic stress than the parental PA01 wild-type strain, but only in the stationary phase of growth. Quantitative comparison of the endometabolome of the cells showed differences in the accumulation of osmoprotective metabolites by the wild-type and mucA22 mutant strains, suggesting a switch in osmo-protectant preference from glycine-betaine to trehalose.
Molecular BioSystems 03/2010; 6(3):562-9. · 3.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Many Caenorhabditis elegans mutations increase longevity and much evidence suggests that they do so at least partly via changes in metabolism. However, up until now there has been no systematic investigation of how the metabolic networks of long-lived mutants differ from those of normal worms. Metabolomic technologies, that permit the analysis of many untargeted metabolites in parallel, now make this possible. Here we use one of these, 1H nuclear magnetic resonance spectroscopy, to investigate what makes long-lived worms metabolically distinctive.
We examined three classes of long-lived worms: dauer larvae, adult Insulin/IGF-1 signalling (IIS)-defective mutants, and a translation-defective mutant. Surprisingly, these ostensibly different long-lived worms share a common metabolic signature, dominated by shifts in carbohydrate and amino acid metabolism. In addition the dauer larvae, uniquely, had elevated levels of modified amino acids (hydroxyproline and phosphoserine). We interrogated existing gene expression data in order to integrate functional (metabolite-level) changes with transcriptional changes at a pathway level.
The observed metabolic responses could be explained to a large degree by upregulation of gluconeogenesis and the glyoxylate shunt as well as changes in amino acid catabolism. These responses point to new possible mechanisms of longevity assurance in worms. The metabolic changes observed in dauer larvae can be explained by the existence of high levels of autophagy leading to recycling of cellular components.See associated minireview: http://jbiol.com/content/9/1/7.
BMC Biology 02/2010; 8:14. · 5.75 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cystic fibrosis (CF) is caused by a defect in the CF transmembrane regulator that leads to depletion and dehydration of the airway surface liquid (ASL) of the lung epithelium, providing an environment that can be infected by bacteria leading to increased morbidity and mortality. Pseudomonas aeruginosa chronically infects more than 80% of CF patients and one hallmark of infection is the emergence of a mucoid phenotype associated with a worsening prognosis and more rapid decline in lung function. Hypertonic saline (HS) is a clinically proven treatment that improves mucociliary clearance through partial rehydration of the ASL of the lung. Strikingly, while HS therapy does not alter the prevalence of P. aeruginosa in the CF lung it does decrease the frequency of episodes of acute, severe illness known as infective exacerbations among CF patients. In this article, we propose a hypothesis whereby the positive clinical effects of HS treatment are explained by the osmotic sensitivity of the mucoid sub-population of P. aeruginosa in the CF lung leading to selection against this group in favor of the osmotically resistant non-mucoid variants.
Frontiers in microbiology. 01/2010; 1:120.
-
Jacob G. Bundy,
Eva M. Lenz,
Nigel J. Bailey,
Claire L. Gavaghan,
Claus Svendsen,
David Spurgeon,
Peter K. Hankard,
Daniel Osborn,
Jason M. Weeks,
Sunia A. Trauger,
Paul Speir,
Ian Sanders,
John C Lindon,
Jeremy K. Nicholson,
Huiru Tang
[show abstract]
[hide abstract]
ABSTRACT: High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy can be used to produce a biochemical fingerprint of low-molecular-weight metabolites from complex biological mixtures such as tissue extracts and biofluids. Changes in such fingerprint profiles can be used to characterize the effects of toxic insult in in vivo systems. The technique is nonselective and requires little sample preparation or derivatization. In the present study, earthworms (Eisenia veneta) were exposed to three different model xenobiotics by a standard filter paper contact test, and toxicant-induced biochemical changes were then investigated by characterizing the changes in endogenous metabolites visible in 600-MHz 1H NMR spectra of tissue extracts. The NMR spectral intensities were converted to discrete numerical values and tabulated in order to provide data matrices suitable for multivariate analysis. Principal component analysis showed that changes had occurred in the biochemical profiles relative to the undosed controls. The 2-fluoro-4-methylaniline—treated worms showed a decrease in a resonance from a compound identified as 2-hexyl-5-ethyl-3-furansulfonate using a combination of high-performance liquid chromatography (HPLC)—Fourier transform mass spectrometry (IonSpec, Lake Forest, CA, USA) and 1H and 13C NMR spectroscopy. An increase in inosine monophosphate was also observed. The 4-fluoroaniline—treated worms showed a decrease in maltose concentrations, and 3,5-difluoroaniline exerted the same effect as 2-fluoro-4-methylaniline but to a lesser extent. These changes could potentially be used as novel biomarkers of xenobiotic toxicity and could be used to determine the mechanism of action of other toxic chemicals.
Environmental Toxicology and Chemistry 11/2009; 21(9):1966 - 1972. · 2.81 Impact Factor
