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Papers by David Watson

Computational and Structural Biotechnology Journal, 2016

Metabolomic profiling was carried out on 53 post-mortem brain samples from subjects diagnosed wit... more Metabolomic profiling was carried out on 53 post-mortem brain samples from subjects diagnosed with schizophrenia, depression, bipolar disorder (SDB), diabetes, and controls. Chromatography on a ZICpHILIC column was used with detection by Orbitrap mass spectrometry. Data extraction was carried out with m/z Mine 2.14 with metabolite searching against an in-house database. There was no clear discrimination between the controls and the SDB samples on the basis of a principal components analysis (PCA) model of 755 identified or putatively identified metabolites. Orthogonal partial least square discriminant analysis (OPLSDA) produced clear separation between 17 of the controls and 19 of the SDB samples (R2CUM 0.976, Q2 0.671, p-value of the cross-validated ANOVA score 0.0024). The most important metabolites producing discrimination were the lipophilic amino acids leucine/isoleucine, proline, methionine, phenylalanine, and tyrosine; the neurotransmitters GABA and NAAG and sugar metabolites sorbitol, gluconic acid, xylitol, ribitol, arabinotol, and erythritol. Eight samples from diabetic brains were analysed, six of which grouped with the SDB samples without compromising the model (R2 CUM 0.850, Q2 CUM 0.534, p-value for cross-validated ANOVA score 0.00087). There appears on the basis of this small sample set to be some commonality between metabolic perturbations resulting from diabetes and from SDB.

Phytochemical analysis : PCA, Jan 11, 2015

A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria ... more A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria (RSN), with regards to chemical composition and activity against Trypanosoma brucei in comparison with other African propolis. To carry out fractionation and biological testing of Nigerian propolis in order to isolate compounds with anti-trypanosomal activity. To compare the composition of the RSN propolis with the composition of Brazilian red propolis. Profiling was carried out using HPLC-UV-ELSD and HPLC-Orbitrap-FTMS on extracts of two samples collected from RSN with data extraction using MZmine software. Isolation was carried out by normal phase and reversed phase MPLC. Elucidation of the compounds with a purity > 95% was performed by 1D/2D NMR HRMS and HRLC-MS(n) . Ten phenolic compounds were isolated or in the case of liquiritigenin partially purified. Data for nine of these correlated with literature reports of known compounds i.e. one isoflavanone, calycosin (1); two flavanone...

In order to assess the effect of silica gel structure on retention in hydrophilic interaction chr... more In order to assess the effect of silica gel structure on retention in hydrophilic interaction chromatography, a test system was developed which used quaternary ammonium ions as probes with tetramethylammonium acetate (TMAA) as the counter-ion competing against the interaction of the test probes with ionised silanols in the stationary phase. Four silica gel columns and a silica hydride column were examined. Retention times were obtained for the test probes at 20, 40, 60, 80 and 90% acetonitrile (ACN) with all the mobile phase mixtures containing 10 mM TMAA buffer at pH 6.0. All phases gave "U" shaped plots for log k against percentage of acetonitrile with the steepest rise in retention occurring between 80 and 90% acetonitrile.

Bears produce the most altricial neonates of any placental mammal. We hypothesized that the trans... more Bears produce the most altricial neonates of any placental mammal. We hypothesized that the transition from colostrum to mature milk in bears reflects a temporal and biochemical adaptation for altricial development and immune protection. Comparison of bear milks with milks of other eutherians yielded distinctive protein profiles. Proteomic and metabolomic analysis of serial milk samples collected from six giant pandas showed a prolonged transition from colostrum to main-phase lactation over approximately 30 days. Particularly striking are the persistence or sequential appearance of adaptive and innate immune factors. The endurance of immunoglobulin G suggests an unusual duration of trans-intestinal absorption of maternal antibodies, and is potentially relevant to the underdeveloped lymphoid system of giant panda neonates. Levels of certain milk oligosaccharides known to exert anti-microbial activities and/or that are conducive to the development of neonatal gut microbiomes underwent an almost complete changeover around days 20–30 postpartum, coincident with the maturation of the protein profile. A potential metabolic marker of starvation was detected, the prominence of which may reflect the natural postpartum period of anorexia in giant panda mothers. Early lactation in giant pandas, and possibly in other ursids, appears to be adapted for the unique requirements of unusually altricial eutherian neonates.

Comparative genomic analyses of Leishmania species have revealed relatively minor het-erogeneity ... more Comparative genomic analyses of Leishmania species have revealed relatively minor het-erogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falcipa... more Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10 Dpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10 Dpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13 C-U-D-glucose and 13 C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10 Dpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13 C, 15 N-U-glutamine was similar in both parasite lines, although the flux was lower in D10 Dpepc ; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO 2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery.

Physical activity and exercise are potent lifestyle interventions for the reduction of several ps... more Physical activity and exercise are potent lifestyle interventions for the reduction of several psy-cho-somatic risks, including cardiovascular disease, diabetes and mild-to-moderate depression. The current review describes the studies which have utilised a metabolomics/ metabolite profiling approach in combination with exercise in the period between 1999-2014 in predominantly healthy subjects (N:64) and in a smaller subset of patient cohorts (N:10) between 2011-2014. A broad range of intervention types were used in both healthy and patient cohorts with varying exercise regimes with or without supplement use. Effects on purine metabolism , which have been known for over 40 years, were by far the most well-cited and persistent finding. Other reported effects included observations of: glucocorticoid and androgen metabolism, glycolysis and Krebs cycle, gluconeogenesis, fatty acid metabolism, fatty acid oxidation and pantothenic acid, amino acid oxidation, tryptophan metabolism and effects on microbiome metabolites. The discussion section draws observations from the very disparate studies together and links them to the general aspects of human metabolism.

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metaboli... more Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypano-soma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabo-lism, its transamination product, alanine, is also produced in significant quantities. The oxi-dative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via

Nine samples of propolis from different parts of the UK were extracted and profiled by high resol... more Nine samples of propolis from different parts of the UK were extracted and profiled by high resolution LC-MS. The data were aligned and features were extracted into 0.02 amu windows. The profiles contained thousands of features. In order to establish a platform for comparison of the samples the top 125 features by average peak intensity across the samples, after excluding abundant dimer peaks, were selected for further characterisation by MS 2. Of the top 125 features around 90% of the peaks could be assigned an identity with some degree of confidence. Only ca 50% of these puta-tively identified compounds had been reported in propolis before. The compounds fell into a few major categories: fla-vonoid esters and possibly some flavonoid ethers, phenyl propanoid esters, glycerol esters, flavonoid glycosides and hy-droxylated fatty acids. Pinobanksin was the most abundant compound by average response across nine samples. The fla-vonoids pinocembrin, pinobanksin, galangin and chrysin showed a relatively low degree of variation across the samples whereas some compounds such as flavonoid esters and glycerol esters were much more variable in their abundance. The role of propolis in preventing infections in the bee hive has yet to be established but the approach taken in this paper provides a potential method for trying to correlate hive health with the composition of the propolis gathered by the hive if suitable metadata were collected.

Yellow (y) encodes a protein which is closely similar to major royal jelly proteins produced by b... more Yellow (y) encodes a protein which is closely similar to major royal jelly proteins produced by bees. However, the function of y remains largely unknown. Metabolomic profiling was carried out on third instar Oregon R (OR) and yellow (y) Drosophila melanogaster larvae. Phenylalanine, tyrosine and DOPA were all elevated in y as might be expected since the mutation blocks melanin biosynthe-sis. The most consistent effects were related to lysine metabolism, with the lysine metabolite sac-charopine being much higher in y. In addition, lysine acetate was elevated, and the levels of methyl lysines were lower, in y than in OR.

In metabolomics studies, optimal liquid chromatography separations prior to mass spectrometric an... more In metabolomics studies, optimal liquid chromatography separations prior to mass spectrometric analysis are important with regard to identification of metabolites as distinct from their isomers, and for differentiation of genuine me-tabolites from fragments and adducts of other molecules and from environmental and system peaks. The role of liquid chromatography in mass spectrometry based metabolomics is reviewed. The basic principles behind liquid chroma-tographic separations are discussed with regard to the types of interaction which can occur with stationary phases. Examples are given in order to illustrate the importance of liquid chromatography in verifying metabolite identity and also to illustrate the different types of separation produced by the various chromatographic phases available. Applications of reversed phase chromatography in metabolomics studies are reviewed for the last three years. Some examples of derivatisa-tion, prior to liquid chromatography, which can be used in order to enhance mass spectrometric detection are covered. The review of other chromatographic methods which are less commonly used than reversed phase chromatography covers a period of six years since these methods present a wider range of stationary phase chemistries. There is a short review of lipidomics methods using liquid chromatography mass spectrometry drawn from the last two years.

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. I... more Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.

Human exhibit wide variations in their metabolic profiles because of differences in genetic facto... more Human exhibit wide variations in their metabolic profiles because of differences in genetic factors, diet and lifestyle. Therefore in order to detect metabolic differences between individuals robust analytical methods are required. A protocol was produced based on the use of Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) in combination with orthogonal Hydrophilic Interaction (HILIC) and Reversed Phase (RP) liquid chromatography methods for the analysis of the urinary metabolome, which was then evaluated as a diagnostic tool for prostate cancer (a common but highly heterogeneous condition). The LC-HRMS method was found to be robust and exhibited excellent repeatability for retention times (,61%), and mass accuracy (,61 ppm). Based on normalised data (against creatinine levels, osmolality or MS total useful signals/MSTUS) coupled with supervised multivariate analysis using Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA), we were able to discriminate urine samples from men with or without prostate cancer with R2Y(cum) .0.9. In addition, using the receiver operator characteristics (ROC) test, the area under curve (AUC) for the combination of the four best characterised biomarker compounds was 0.896. The four biomarker compounds were also found to differ significantly (P,0.05) between an independent patient cohort and controls. This is the first time such a rigorous test has been applied to this type of model. If validated, the established protocol provides a robust approach with a potentially wide application to metabolite profiling of human biofluids in health and disease.

Metabolomics can provide exciting insights into organismal function, but most work on simple mode... more Metabolomics can provide exciting insights into organismal function, but most work on simple models has focussed on the whole organism metabolome, so missing the contributions of individual tissues. Comprehensive metabolite profiles for ten tissues from adult Drosophila melanogaster were obtained here by two chromatographic methods, a hydrophilic interaction (HILIC) method for polar metabolites and a lipid profiling method also based on HILIC, in combination with an Orbitrap Exactive instrument. Two hundred and forty two polar metabolites were putatively identified in the various tissues, and 251 lipids were observed in positive ion mode and 61 in negative ion mode. Although many metabolites were detected in all tissues, every tissue showed characteristically abundant metabolites which could be rationalised against specific tissue functions. For example, the cuticle contained high levels of glutathione, reflecting a role in oxidative defence; the alimentary canal (like vertebrate gut) had high levels of acylcarnitines for fatty acid metabolism, and the head contained high levels of ether lipids. The male accessory gland uniquely contained decarboxylated S-adenosylmethionine. These data thus both provide valuable insights into tissue function, and a reference baseline, compatible with the FlyAtlas.org transcriptomic resource, for further metabolomic analysis of this important model organism, for example in the modelling of human inborn errors of metabolism, aging or metabolic imbalances such as diabetes.

Metabolite profiles of ten batches of male and female Drosophila melanogaster were compared using... more Metabolite profiles of ten batches of male and female Drosophila melanogaster were compared using three different chromatographic methods interfaced to an Orbitrap Exactive mass spectrometer. Several thousand features were observed and were reduced after data extraction and careful checking of around 390 metabolites excluding lipids. Chromatographic traces for these metabolites are presented as supplementary information. There were many significant differences between male and female flies. Female flies contained much higher levels of methylated lysines and methylated arginine suggesting differences in histone metabolism. In addition, there were differences in the methylation of nucleosides, and S-adenosylmethionine metabolism. Differences in the methylome may relate to the requirement of compensation for the single X chromosome present in males since methylated histones inhibit gene transcription. Nucleoside phosphate levels were elevated in female flies which may relate to increased requirement for DNA biosynthesis for egg production. A series of acylated amino acids previously observed in Drosophila was further characterised and these metabolites were present to a much greater extent in female flies and may be associated with the microbiome.

Aerobic exercise, in spite of its multi-organ benefit and potent effect on the metabolome, has ye... more Aerobic exercise, in spite of its multi-organ benefit and potent effect on the metabolome, has yet to be investigated comprehensively via an untargeted metabolomics technology. We conducted an exploratory untargeted liquid chromatography mass spectrometry study to investigate the effects of a one-h aerobic exercise session in the urine of three physically active males. Individual urine samples were collected over a 37-h protocol (two pre-exercise and eight post-exercise). Raw data were subjected to a variety of normalization techniques, with the most effective measure dividing each metabolite by the sum response of that metabolite for each individual across the 37-h protocol expressed as a percentage. This allowed the metabolite responses to be plotted on a normalised scale. Our results highlight significant metabolites located in the following systems: purine pathway, tryptophan metabolism, carnitine metabolism, cortisol metabolism, androgen metabolism, amino acid oxidation, as well as metabolites from the gastrointestinal microbiome. Many of the significant changes observed in our pilot investigation mirror previous research studies, of various methodological designs, published within the last 15 years, although they have never been reported at the same time in a single study.

Ten physically active subjects underwent two cycling exercise trials. In the first, aerobic capac... more Ten physically active subjects underwent two cycling exercise trials. In the first, aerobic capacity (VO 2max) was determined and the second was a 45 min submaximal exercise test. Urine samples were collected separately the day before (day 1) , the day of (day 2), and the day after (day 3) the submaximal exercise test (12 samples per subject). Metabolomic profiling of the samples was carried out using hydrophilic interaction chromatography (HILIC) coupled to an Orbitrap Exactive mass spectrometer. Data were extracted, database searched and then subjected to principle components (PCA) and orthogonal partial least squares (OPLSDA) modelling. The best results were obtained from pre-treating the data by normalising the metabolites to their mean output on days 1 and 2 of the trial. This allowed PCA to separate the day 2 first void samples (D2S1) from the day 2 post-exercise samples (D2S3) PCA also separated the equivalent samples obtained on day 1 (D1S1 and D1S3). OPLSDA modelling separated both the D2S1 and D2S3 samples and D1S1 and D1S3 samples. The metabolites affected by the exercise samples included a range of purine metabolites and several acyl carnitines. Some metabolites were subject to diurnal variation these included bile acids and several amino acids, the variation of these metabolites was similar on day 1 and day 2 despite the exercise intervention on day 2. Using OPLS modelling it proved possible to identify a single abundant urinary metabolite provisionally identified as oxo-aminohexanoic acid (OHA) as being strongly correlated with VO 2max when the levels in the D2S3 samples were considered.

Ursids (bears) in general, and giant pandas in particular, are highly altricial at birth. The com... more Ursids (bears) in general, and giant pandas in particular, are highly altricial at birth. The components of bear milks and their changes with time may be uniquely adapted to nourish relatively immature neonates, protect them from pathogens, and support the maturation of neonatal digestive physiology. Serial milk samples collected from three giant pandas in early lactation were subjected to untargeted metabolite profiling and multivariate analysis. Changes in milk metabolites with time after birth were analysed by Principal Component Analysis, Hierarchical Cluster Analysis and further supported by Orthogonal Partial Least Square-Discriminant Analysis, revealing three phases of milk maturation: days 1–6 (Phase 1), days 7–20 (Phase 2), and beyond day 20 (Phase 3). While the compositions of Phase 1 milks were essentially indistinguishable among individuals, divergences emerged during the second week of lactation. OPLS regression analysis positioned against the growth rate of one cub tentatively inferred a correlation with changes in the abundance of a trisaccha-ride, isoglobotriose, previously observed to be a major oligosaccharide in ursid milks. Three artificial milk formulae used to feed giant panda cubs were also analysed, and were found to differ markedly in component content from natural panda milk. These findings have implications for the dependence of the ontogeny of all species of bears, and potentially other members of the Carnivora and beyond, on the complexity and sequential changes in maternal provision of micrometabolites in the immediate period after birth.

Computational and Structural Biotechnology Journal, 2016

Metabolomic profiling was carried out on 53 post-mortem brain samples from subjects diagnosed wit... more Metabolomic profiling was carried out on 53 post-mortem brain samples from subjects diagnosed with schizophrenia, depression, bipolar disorder (SDB), diabetes, and controls. Chromatography on a ZICpHILIC column was used with detection by Orbitrap mass spectrometry. Data extraction was carried out with m/z Mine 2.14 with metabolite searching against an in-house database. There was no clear discrimination between the controls and the SDB samples on the basis of a principal components analysis (PCA) model of 755 identified or putatively identified metabolites. Orthogonal partial least square discriminant analysis (OPLSDA) produced clear separation between 17 of the controls and 19 of the SDB samples (R2CUM 0.976, Q2 0.671, p-value of the cross-validated ANOVA score 0.0024). The most important metabolites producing discrimination were the lipophilic amino acids leucine/isoleucine, proline, methionine, phenylalanine, and tyrosine; the neurotransmitters GABA and NAAG and sugar metabolites sorbitol, gluconic acid, xylitol, ribitol, arabinotol, and erythritol. Eight samples from diabetic brains were analysed, six of which grouped with the SDB samples without compromising the model (R2 CUM 0.850, Q2 CUM 0.534, p-value for cross-validated ANOVA score 0.00087). There appears on the basis of this small sample set to be some commonality between metabolic perturbations resulting from diabetes and from SDB.

Phytochemical analysis : PCA, Jan 11, 2015

A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria ... more A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria (RSN), with regards to chemical composition and activity against Trypanosoma brucei in comparison with other African propolis. To carry out fractionation and biological testing of Nigerian propolis in order to isolate compounds with anti-trypanosomal activity. To compare the composition of the RSN propolis with the composition of Brazilian red propolis. Profiling was carried out using HPLC-UV-ELSD and HPLC-Orbitrap-FTMS on extracts of two samples collected from RSN with data extraction using MZmine software. Isolation was carried out by normal phase and reversed phase MPLC. Elucidation of the compounds with a purity > 95% was performed by 1D/2D NMR HRMS and HRLC-MS(n) . Ten phenolic compounds were isolated or in the case of liquiritigenin partially purified. Data for nine of these correlated with literature reports of known compounds i.e. one isoflavanone, calycosin (1); two flavanone...

In order to assess the effect of silica gel structure on retention in hydrophilic interaction chr... more In order to assess the effect of silica gel structure on retention in hydrophilic interaction chromatography, a test system was developed which used quaternary ammonium ions as probes with tetramethylammonium acetate (TMAA) as the counter-ion competing against the interaction of the test probes with ionised silanols in the stationary phase. Four silica gel columns and a silica hydride column were examined. Retention times were obtained for the test probes at 20, 40, 60, 80 and 90% acetonitrile (ACN) with all the mobile phase mixtures containing 10 mM TMAA buffer at pH 6.0. All phases gave "U" shaped plots for log k against percentage of acetonitrile with the steepest rise in retention occurring between 80 and 90% acetonitrile.

Bears produce the most altricial neonates of any placental mammal. We hypothesized that the trans... more Bears produce the most altricial neonates of any placental mammal. We hypothesized that the transition from colostrum to mature milk in bears reflects a temporal and biochemical adaptation for altricial development and immune protection. Comparison of bear milks with milks of other eutherians yielded distinctive protein profiles. Proteomic and metabolomic analysis of serial milk samples collected from six giant pandas showed a prolonged transition from colostrum to main-phase lactation over approximately 30 days. Particularly striking are the persistence or sequential appearance of adaptive and innate immune factors. The endurance of immunoglobulin G suggests an unusual duration of trans-intestinal absorption of maternal antibodies, and is potentially relevant to the underdeveloped lymphoid system of giant panda neonates. Levels of certain milk oligosaccharides known to exert anti-microbial activities and/or that are conducive to the development of neonatal gut microbiomes underwent an almost complete changeover around days 20–30 postpartum, coincident with the maturation of the protein profile. A potential metabolic marker of starvation was detected, the prominence of which may reflect the natural postpartum period of anorexia in giant panda mothers. Early lactation in giant pandas, and possibly in other ursids, appears to be adapted for the unique requirements of unusually altricial eutherian neonates.

Comparative genomic analyses of Leishmania species have revealed relatively minor het-erogeneity ... more Comparative genomic analyses of Leishmania species have revealed relatively minor het-erogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falcipa... more Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10 Dpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10 Dpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13 C-U-D-glucose and 13 C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10 Dpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13 C, 15 N-U-glutamine was similar in both parasite lines, although the flux was lower in D10 Dpepc ; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO 2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery.

Physical activity and exercise are potent lifestyle interventions for the reduction of several ps... more Physical activity and exercise are potent lifestyle interventions for the reduction of several psy-cho-somatic risks, including cardiovascular disease, diabetes and mild-to-moderate depression. The current review describes the studies which have utilised a metabolomics/ metabolite profiling approach in combination with exercise in the period between 1999-2014 in predominantly healthy subjects (N:64) and in a smaller subset of patient cohorts (N:10) between 2011-2014. A broad range of intervention types were used in both healthy and patient cohorts with varying exercise regimes with or without supplement use. Effects on purine metabolism , which have been known for over 40 years, were by far the most well-cited and persistent finding. Other reported effects included observations of: glucocorticoid and androgen metabolism, glycolysis and Krebs cycle, gluconeogenesis, fatty acid metabolism, fatty acid oxidation and pantothenic acid, amino acid oxidation, tryptophan metabolism and effects on microbiome metabolites. The discussion section draws observations from the very disparate studies together and links them to the general aspects of human metabolism.

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metaboli... more Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypano-soma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabo-lism, its transamination product, alanine, is also produced in significant quantities. The oxi-dative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via

Nine samples of propolis from different parts of the UK were extracted and profiled by high resol... more Nine samples of propolis from different parts of the UK were extracted and profiled by high resolution LC-MS. The data were aligned and features were extracted into 0.02 amu windows. The profiles contained thousands of features. In order to establish a platform for comparison of the samples the top 125 features by average peak intensity across the samples, after excluding abundant dimer peaks, were selected for further characterisation by MS 2. Of the top 125 features around 90% of the peaks could be assigned an identity with some degree of confidence. Only ca 50% of these puta-tively identified compounds had been reported in propolis before. The compounds fell into a few major categories: fla-vonoid esters and possibly some flavonoid ethers, phenyl propanoid esters, glycerol esters, flavonoid glycosides and hy-droxylated fatty acids. Pinobanksin was the most abundant compound by average response across nine samples. The fla-vonoids pinocembrin, pinobanksin, galangin and chrysin showed a relatively low degree of variation across the samples whereas some compounds such as flavonoid esters and glycerol esters were much more variable in their abundance. The role of propolis in preventing infections in the bee hive has yet to be established but the approach taken in this paper provides a potential method for trying to correlate hive health with the composition of the propolis gathered by the hive if suitable metadata were collected.

Yellow (y) encodes a protein which is closely similar to major royal jelly proteins produced by b... more Yellow (y) encodes a protein which is closely similar to major royal jelly proteins produced by bees. However, the function of y remains largely unknown. Metabolomic profiling was carried out on third instar Oregon R (OR) and yellow (y) Drosophila melanogaster larvae. Phenylalanine, tyrosine and DOPA were all elevated in y as might be expected since the mutation blocks melanin biosynthe-sis. The most consistent effects were related to lysine metabolism, with the lysine metabolite sac-charopine being much higher in y. In addition, lysine acetate was elevated, and the levels of methyl lysines were lower, in y than in OR.

In metabolomics studies, optimal liquid chromatography separations prior to mass spectrometric an... more In metabolomics studies, optimal liquid chromatography separations prior to mass spectrometric analysis are important with regard to identification of metabolites as distinct from their isomers, and for differentiation of genuine me-tabolites from fragments and adducts of other molecules and from environmental and system peaks. The role of liquid chromatography in mass spectrometry based metabolomics is reviewed. The basic principles behind liquid chroma-tographic separations are discussed with regard to the types of interaction which can occur with stationary phases. Examples are given in order to illustrate the importance of liquid chromatography in verifying metabolite identity and also to illustrate the different types of separation produced by the various chromatographic phases available. Applications of reversed phase chromatography in metabolomics studies are reviewed for the last three years. Some examples of derivatisa-tion, prior to liquid chromatography, which can be used in order to enhance mass spectrometric detection are covered. The review of other chromatographic methods which are less commonly used than reversed phase chromatography covers a period of six years since these methods present a wider range of stationary phase chemistries. There is a short review of lipidomics methods using liquid chromatography mass spectrometry drawn from the last two years.

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. I... more Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.

Human exhibit wide variations in their metabolic profiles because of differences in genetic facto... more Human exhibit wide variations in their metabolic profiles because of differences in genetic factors, diet and lifestyle. Therefore in order to detect metabolic differences between individuals robust analytical methods are required. A protocol was produced based on the use of Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) in combination with orthogonal Hydrophilic Interaction (HILIC) and Reversed Phase (RP) liquid chromatography methods for the analysis of the urinary metabolome, which was then evaluated as a diagnostic tool for prostate cancer (a common but highly heterogeneous condition). The LC-HRMS method was found to be robust and exhibited excellent repeatability for retention times (,61%), and mass accuracy (,61 ppm). Based on normalised data (against creatinine levels, osmolality or MS total useful signals/MSTUS) coupled with supervised multivariate analysis using Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA), we were able to discriminate urine samples from men with or without prostate cancer with R2Y(cum) .0.9. In addition, using the receiver operator characteristics (ROC) test, the area under curve (AUC) for the combination of the four best characterised biomarker compounds was 0.896. The four biomarker compounds were also found to differ significantly (P,0.05) between an independent patient cohort and controls. This is the first time such a rigorous test has been applied to this type of model. If validated, the established protocol provides a robust approach with a potentially wide application to metabolite profiling of human biofluids in health and disease.

Metabolomics can provide exciting insights into organismal function, but most work on simple mode... more Metabolomics can provide exciting insights into organismal function, but most work on simple models has focussed on the whole organism metabolome, so missing the contributions of individual tissues. Comprehensive metabolite profiles for ten tissues from adult Drosophila melanogaster were obtained here by two chromatographic methods, a hydrophilic interaction (HILIC) method for polar metabolites and a lipid profiling method also based on HILIC, in combination with an Orbitrap Exactive instrument. Two hundred and forty two polar metabolites were putatively identified in the various tissues, and 251 lipids were observed in positive ion mode and 61 in negative ion mode. Although many metabolites were detected in all tissues, every tissue showed characteristically abundant metabolites which could be rationalised against specific tissue functions. For example, the cuticle contained high levels of glutathione, reflecting a role in oxidative defence; the alimentary canal (like vertebrate gut) had high levels of acylcarnitines for fatty acid metabolism, and the head contained high levels of ether lipids. The male accessory gland uniquely contained decarboxylated S-adenosylmethionine. These data thus both provide valuable insights into tissue function, and a reference baseline, compatible with the FlyAtlas.org transcriptomic resource, for further metabolomic analysis of this important model organism, for example in the modelling of human inborn errors of metabolism, aging or metabolic imbalances such as diabetes.

Metabolite profiles of ten batches of male and female Drosophila melanogaster were compared using... more Metabolite profiles of ten batches of male and female Drosophila melanogaster were compared using three different chromatographic methods interfaced to an Orbitrap Exactive mass spectrometer. Several thousand features were observed and were reduced after data extraction and careful checking of around 390 metabolites excluding lipids. Chromatographic traces for these metabolites are presented as supplementary information. There were many significant differences between male and female flies. Female flies contained much higher levels of methylated lysines and methylated arginine suggesting differences in histone metabolism. In addition, there were differences in the methylation of nucleosides, and S-adenosylmethionine metabolism. Differences in the methylome may relate to the requirement of compensation for the single X chromosome present in males since methylated histones inhibit gene transcription. Nucleoside phosphate levels were elevated in female flies which may relate to increased requirement for DNA biosynthesis for egg production. A series of acylated amino acids previously observed in Drosophila was further characterised and these metabolites were present to a much greater extent in female flies and may be associated with the microbiome.

Aerobic exercise, in spite of its multi-organ benefit and potent effect on the metabolome, has ye... more Aerobic exercise, in spite of its multi-organ benefit and potent effect on the metabolome, has yet to be investigated comprehensively via an untargeted metabolomics technology. We conducted an exploratory untargeted liquid chromatography mass spectrometry study to investigate the effects of a one-h aerobic exercise session in the urine of three physically active males. Individual urine samples were collected over a 37-h protocol (two pre-exercise and eight post-exercise). Raw data were subjected to a variety of normalization techniques, with the most effective measure dividing each metabolite by the sum response of that metabolite for each individual across the 37-h protocol expressed as a percentage. This allowed the metabolite responses to be plotted on a normalised scale. Our results highlight significant metabolites located in the following systems: purine pathway, tryptophan metabolism, carnitine metabolism, cortisol metabolism, androgen metabolism, amino acid oxidation, as well as metabolites from the gastrointestinal microbiome. Many of the significant changes observed in our pilot investigation mirror previous research studies, of various methodological designs, published within the last 15 years, although they have never been reported at the same time in a single study.

Ten physically active subjects underwent two cycling exercise trials. In the first, aerobic capac... more Ten physically active subjects underwent two cycling exercise trials. In the first, aerobic capacity (VO 2max) was determined and the second was a 45 min submaximal exercise test. Urine samples were collected separately the day before (day 1) , the day of (day 2), and the day after (day 3) the submaximal exercise test (12 samples per subject). Metabolomic profiling of the samples was carried out using hydrophilic interaction chromatography (HILIC) coupled to an Orbitrap Exactive mass spectrometer. Data were extracted, database searched and then subjected to principle components (PCA) and orthogonal partial least squares (OPLSDA) modelling. The best results were obtained from pre-treating the data by normalising the metabolites to their mean output on days 1 and 2 of the trial. This allowed PCA to separate the day 2 first void samples (D2S1) from the day 2 post-exercise samples (D2S3) PCA also separated the equivalent samples obtained on day 1 (D1S1 and D1S3). OPLSDA modelling separated both the D2S1 and D2S3 samples and D1S1 and D1S3 samples. The metabolites affected by the exercise samples included a range of purine metabolites and several acyl carnitines. Some metabolites were subject to diurnal variation these included bile acids and several amino acids, the variation of these metabolites was similar on day 1 and day 2 despite the exercise intervention on day 2. Using OPLS modelling it proved possible to identify a single abundant urinary metabolite provisionally identified as oxo-aminohexanoic acid (OHA) as being strongly correlated with VO 2max when the levels in the D2S3 samples were considered.

Ursids (bears) in general, and giant pandas in particular, are highly altricial at birth. The com... more Ursids (bears) in general, and giant pandas in particular, are highly altricial at birth. The components of bear milks and their changes with time may be uniquely adapted to nourish relatively immature neonates, protect them from pathogens, and support the maturation of neonatal digestive physiology. Serial milk samples collected from three giant pandas in early lactation were subjected to untargeted metabolite profiling and multivariate analysis. Changes in milk metabolites with time after birth were analysed by Principal Component Analysis, Hierarchical Cluster Analysis and further supported by Orthogonal Partial Least Square-Discriminant Analysis, revealing three phases of milk maturation: days 1–6 (Phase 1), days 7–20 (Phase 2), and beyond day 20 (Phase 3). While the compositions of Phase 1 milks were essentially indistinguishable among individuals, divergences emerged during the second week of lactation. OPLS regression analysis positioned against the growth rate of one cub tentatively inferred a correlation with changes in the abundance of a trisaccha-ride, isoglobotriose, previously observed to be a major oligosaccharide in ursid milks. Three artificial milk formulae used to feed giant panda cubs were also analysed, and were found to differ markedly in component content from natural panda milk. These findings have implications for the dependence of the ontogeny of all species of bears, and potentially other members of the Carnivora and beyond, on the complexity and sequential changes in maternal provision of micrometabolites in the immediate period after birth.