IonFlow: a galaxy tool for the analysis of ionomics data sets.

INTRODUCTION: Inductively coupled plasma mass spectrometry (ICP-MS) experiments generate complex multi-dimensional data sets that require specialist data analysis tools. OBJECTIVE: Here we describe tools to facilitate analysis of the ionome composed of high-throughput elemental profiling data. METHODS: IonFlow is a Galaxy tool written in R for ionomics data analysis and is freely accessible at https://github.com/wanchanglin/ionflow . It is designed as a pipeline that can process raw data to enable exploration and interpretation using multivariate statistical techniques and network-based algorithms, including principal components analysis, hierarchical clustering, relevance network extraction and analysis, and gene set enrichment analysis. RESULTS AND CONCLUSION: The pipeline is described and tested on two benchmark data sets of the haploid S. Cerevisiae ionome and of the human HeLa cell ionome.

Impact of Azithromycin on the Quorum Sensing-Controlled Proteome of Pseudomonas aeruginosa.

The macrolide antibiotic, azithromycin (AZM), has been reported to improve the clinical outcome of cystic fibrosis patients, many of whom are chronically-infected with Pseudomonas aeruginosa. However, the highest clinically-achievable concentrations of this drug are well-below the minimum inhibitory concentration for P. aeruginosa, raising the question of why AZM exhibits therapeutic activity. One possibility that has been raised by earlier studies is that AZM inhibits quorum sensing (QS) by P. aeruginosa. To explicitly test this hypothesis the changes brought about by AZM treatment need to be compared with those associated with specific QS mutants grown alongside in the same growth medium, but this has not been done. In this work, we used quantitative 2D-difference gel electrophoresis and 1H-NMR spectroscopy footprint analysis to examine whether a range of clinically-relevant AZM concentrations elicited proteomic and metabolomic changes in wild-type cultures that were similar to those seen in cultures of defined QS mutants. Consistent with earlier reports, over half of the AZM-induced spot changes on the 2D gels were found to affect QS-regulated proteins. However, AZM modulated very few protein spots overall (compared with QS) and collectively, these modulated proteins comprised only a small fraction (12-13%) of the global QS regulon. We conclude that AZM perturbs a sub-regulon of the QS system but does not block QS per se. Reinforcing this notion, we further show that AZM is capable of attenuating virulence factor production in another Gram-negative species that secretes copious quantities of exoenzymes (Serratia marcescens), even in the absence of a functional QS system.

A metabolomic study of adipose tissue in mice with a disruption of the circadian system.

Adipose tissue functions in terms of energy homeostasis as a rheostat for blood triglyceride, regulating its concentration, in response to external stimuli. In addition it acts as a barometer to inform the central nervous system of energy levels which can vary dramatically between meals and according to energy demand. Here a metabolomic approach, combining both Mass Spectrometry and Nuclear Magnetic Resonance spectroscopy, was used to analyse both white and brown adipose tissue in mice with adipocyte-specific deletion of Arntl (also known as Bmal1), a gene encoding a core molecular clock component. The results are consistent with a peripheral circadian clock playing a central role in metabolic regulation of both brown and white adipose tissue in rodents and show that Arntl induced global changes in both tissues which were distinct for the two types. In particular, anterior subcutaneous white adipose tissue (ASWAT) tissue was effected by a reduction in the degree of unsaturation of fatty acids, while brown adipose tissue (BAT) changes were associated with a reduction in chain length. In addition the aqueous fraction of metabolites in BAT were profoundly affected by Arntl disruption, consistent with the dynamic role of this tissue in maintaining body temperature across the day-night cycle and an upregulation in fatty acid oxidation and citric acid cycle activity to generate heat during the day when rats are inactive (increases in 3-hydroxybutyrate and glutamate), and increased synthesis and storage of lipids during the night when rats feed more (increased concentrations of glycerol, choline and glycerophosphocholine).

Behavioural and molecular endophenotypes in psychotic disorders reveal heritable abnormalities in glutamatergic neurotransmission.

Psychotic disorders such as schizophrenia are biologically complex and carry huge population morbidity due to their prevalence, persistence and associated disability. Defined by features such as delusions and hallucinations, they involve cognitive dysfunction and neurotransmitter dysregulations that appear mostly to involve the dopaminergic and glutamatergic systems. A number of genetic and environmental factors are associated with these disorders but it has been difficult to identify the biological pathways underlying the principal symptoms. The endophenotype concept of stable, heritable traits that form a mechanistic link between genes and an overt expression of the disorder has potential to reduce the complexity of psychiatric phenotypes. In this study, we used a genetically sensitive design with individuals with a first episode of psychosis, their non-affected first-degree relatives and non-related healthy controls. Metabolomic analysis was combined with neurocognitive assessment to identify multilevel endophenotypic patterns: one concerned reaction times during the performance of cognitive and emotional tests that have previously been associated with the glutamate neurotransmission system, the other involved metabolites involved directly and indirectly in the co-activation of the N-methyl-D-aspartate receptor, a major receptor of the glutamate system. These cognitive and metabolic endophenotypes may comprise a single construct, such that genetically mediated dysfunction in the glutamate system may be responsible for delays in response to cognitive and emotional functions in psychotic disorders. This focus on glutamatergic neurotransmission should guide drug discovery and experimental medicine programmes in schizophrenia and related disorders.

Prostaglandin profiling reveals a role for haematopoietic prostaglandin D synthase in adipose tissue macrophage polarisation in mice and humans.

BACKGROUND/OBJECTIVES: Obesity has been associated with both changes in adipose tissue lipid metabolism and inflammation. A key class of lipid-derived signalling molecules involved in inflammation are the prostaglandins. In this study, we aimed to determine how obesity affects the levels of prostaglandins within white adipose tissue (WAT) and determine which cells within adipose tissue produce them. To avoid the effects of cellular stress on prostaglandin levels, we developed a multivariate statistical approach in which metabolite concentrations and transcriptomic data were integrated, allowing the assignment of metabolites to cell types. SUBJECTS/METHODS: Eicosanoids were measured by liquid chromatography-tandem mass spectrometry and mRNA levels using real-time PCR. Eicosanoid levels and transcriptomic data were combined using principal component analysis and hierarchical clustering in order to associate metabolites with cell types. Samples were obtained from C57Bl/6 mice aged 16 weeks. We studied the ob/ob genetically obese mouse model and diet-induced obesity model. We extended our results in mice to a cohort of morbidly obese humans undergoing bariatric surgery. RESULTS: Using our modelling approach, we determined that prostglandin D2 (PGD2) in adipose tissue was predominantly produced in macrophages by the haematopoietic isoform of prostaglandin D synthase (H-Pgds). Analysis of sub-fractionated WAT confirmed that H-Pgds was expressed in adipose tissue macrophages (ATMs). Furthermore, H-Pgds expression in ATMs isolated from lean and obese mice was consistent with it affecting macrophage polarisation. Functionally, we demonstrated that H-PGDS-produced PGD2 polarised macrophages toward an M2, anti-inflammatory state. In line with a potential anti-inflammatory role, we found that H-PGDS expression in ATMs was positively correlated with both peripheral insulin and adipose tissue insulin sensitivity in humans. CONCLUSIONS: In this study, we have developed a method to determine the cellular source of metabolites within an organ and used it to identify a new role for PGD2 in the control of ATM polarisation.

A nurse-run clinic for patients with incidentally discovered small abdominal aortic aneurysms is feasible and cost-effective.

INTRODUCTION: Patients with incidentally discovered small abdominal aortic aneurysms (AAA) require assessment by a vascular surgery department for possible enrollment in a surveillance programme. Our unit implemented a vascular nurse-run AAA clinic in October 2010. The aim of this study was to assess the feasibility of a specialist nurse-run small AAA clinic. METHODS: Demographic and clinical data were collected prospectively for all patients seen in the new vascular nurse clinic between October 2010 and November 2012. A validated AAA operative mortality score was used to aid decision making by the vascular nurse. RESULTS: Some 250 patients were seen in the clinic. 198 (79.2%) patients were enrolled in surveillance, 40 (16%) declined enrollment and 12 (4.8%) were referred to a consultant clinic for further assessment. The majority of patients were male and the mean age was 73.7 years. Co-morbidities included hypertension, a history of cardiovascular disease, and hyperlipidaemia. The majority of referrals were considered to be low operative risk. No aneurysms ruptured whilst under surveillance. CONCLUSIONS: A nurse-run clinic that assesses patients with incidentally discovered small AAAs for inclusion in AAA surveillance is a feasible alternative to assessment of these patients in a consultant-run clinic.

Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-ß-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.

Review metabolomics: a new approach to drug delivery in perinatology.

The purpose of this review is to describe the state of the art of the pharmacological applications in perinatal medicine and highlight how a new emerging discipline, metabolomics, may have a significant impact on understanding complex biological processes associated with the drugs actions. Currently, there is great demand for new information regarding the use of drugs, especially during the perinatal period in order to minimize the occurrence of adverse drug reactions and to maximize the desired therapeutic effect. Metabolomics is a functional genomic tool concerned with the high-throughput identification, quantification and characterization of small molecule metabolites. This new technique has been shown to have a great impact on classifying phenotypes, investigation of physiological status, diagnosing disease, measuring the response to treatment, discovering biomarkers, and identifying perturbed pathways due to disease or treatment. Metabolic profiles appear to be a key factor in predicting the outcome of a pathological condition and the individual's response to a pharmacological treatment. This new systems biology tool may have important potential implication for pharmacological science, in particular drug discovery, development, and prediction of the drug's effects on the body by explaining the mechanisms by which drug response causes adverse effects.

A metabolomics based test of independent action and concentration addition using the earthworm Lumbricus rubellus.

A major challenge in ecotoxicology is to understand the effects of multiple toxicants on organisms. Here we assess the effects on survival, weight change, cocoon production and metabolism caused by exposure to two similarly acting (imidacloprid/thiacloprid) and two dissimilarly acting (chlorpyrifos/Nickel) chemicals on the earthworm Lumbricus rubellus. We assessed the standard models of concentration addition (CA) and independent action (IA), in conjunction with a metabolomics based approach to elucidate mechanisms of effect. For imidacloprid and thiacloprid the reproductive effects indicated probable additivity. Although this suggests joint effects through a similar mechanism, metabolite changes for each pesticide actually indicated distinct effects. Further, earthworms exposed to a 0.5 toxic unit equitoxic mixture demonstrated metabolic effects intermediate between those for each pesticide, indicating a non-interactive, independent joint effect. For higher effect level mixtures (1 and 1.5 toxic units), metabolite changes associated with thiacloprid exposure began to dominate. The metabolomic effects of the two dissimilarly acting chemicals were distinct, confirming separate modes of action and both proved more toxic than anticipated from previous studies. In the mixtures, phenotypic effects were in accordance with IA estimates, while metabolite changes were dominated by Ni effects, even though chlorpyrifos contributed most to reproductive toxicity. This could be attributed to the greater systematic effect of Ni when compared to the more specifically acting chlorpyrifos.

Combined use of filtered and edited 1 H NMR spectroscopy to detect 13 C-enriched compounds in complex mixtures.

In conventional metabolism and pharmacokinetic studies, radioactive isotopes are used to identify and quantify the breakdown products of xenobiotics. However, the stable isotope (13) C provides a cheaper and less hazardous alternative. Metabolites of (13) C-enriched xenobiotics can be detected, quantified and identified by (13) C-filtered NMR spectroscopy. However, one obstacle to using (13) C is its 1.1% natural abundance that produces a background signal in (13) C-filtered NMR spectra of crude biological extracts. The signal makes it difficult to distinguish between (13) C-enriched xenobiotics resonances from endogenous metabolites unrelated to the xenobiotic. This study proposes that the (13) C background signal can be distinguished from resonances of (13) C-enriched xenobiotics by the absence of a (12) C component in the xenobiotic. This is detected by combined analysis of (13) C-filtered and -edited NMR spectra. The theory underlying the approach is described and the method is demonstrated by the detection of sub-microgram amounts of (13) C-enriched phenacetin in crude extracts of hepatocyte microsomes.

Evaluation of 2 sources of Angus cattle under South Florida subtropical conditions.

The objective of this study was to compare performance and aspects of adaptability attributes of cattle from a Florida Angus bloodline (local source from a mostly closed herd for over 50 yr) to cattle that are representative of modern Angus bloodlines (outside source) in US subtropical conditions. Embryos from both sources were transferred to Brahman-crossbred cows in South Florida, and calves (n=82) were born in 3 yr. Before weaning, summer tympanic temperatures were recorded hourly for 3 d in each year. Heifers were placed with fertile bulls until diagnosed pregnant. Traits relative to sexual maturation of bulls were recorded at 1- or 2-mo intervals until approximately 17 mo of age. Calves from outside sources had greater hip height at weaning than calves from the local source (P<0.001; 108.8 ± 0.62 and 104.7 ± 0.68 cm, respectively). Local-source calves (n=37) had greater (P=0.03) exit velocity (2.7 ± 0.3 m/s) than outside-source (n=45) calves (2.0 ± 0.29 m/s), which may be indicative of more nervous or temperamental disposition. However, no source differences were detected for other assessments of disposition (chute or pen score, P>0.8). Few source differences for minimum, maximum, or range of daily tympanic (inner ear) temperatures were detected. At 17 mo of age, outside-source heifers were heavier (P = 0.05) and had greater (P<0.001) hip height than Angus heifers from the local source. Heifers from the outside source were younger (P<0.001) at the time of their first conception (454 ± 17.5 d) than heifers from the local source (550 ± 16.9 d). Outside-source heifers also had greater (P<0.02) pregnancy and calving rates (0.7 ± 0.119 and 0.62 ± 0.125, respectively) from exposure to bulls within a year from weaning than the heifers from the local source (0.29 ± 0.089 and 0.19 ± 0.077, respectively). Bulls from the outside source were heavier (P=0.05) at 320 d of age than local-source bulls. From 14 through 17 mo of age, outside-source bulls had greater (P≤0.05) scrotal circumference and tended (P≤0.15) to be heavier than local-source bulls. There appeared to be no performance or adaptation advantages for the local-source Angus through 17 mo of age. The large source difference for age at first conception in heifers merits additional attention and comparison with cow lifetime production performance for the 2 sources.

¹H nuclear magnetic resonance spectroscopy characterisation of metabolic phenotypes in the medulloblastoma of the SMO transgenic mice.

BACKGROUND: Human medulloblastomas exhibit diverse molecular pathology. Aberrant hedgehog signalling is found in 20-30% of human medulloblastomas with largely unknown metabolic consequences. METHODS: Transgenic mice over-expressing smoothened (SMO) receptor in granule cell precursors with high incidence of exophytic medulloblastomas were sequentially followed up by magnetic resonance imaging (MRI) and characterised for metabolite phenotypes by ¹H MR spectroscopy (MRS) in vivo and ex vivo using high-resolution magic angle spinning (HR-MAS) ¹H MRS. RESULTS: Medulloblastomas in the SMO mice presented as T2 hyperintense tumours in MRI. These tumours showed low concentrations of N-acetyl aspartate and high concentrations of choline-containing metabolites (CCMs), glycine, and taurine relative to the cerebellar parenchyma in the wild-type (WT) C57BL/6 mice. In contrast, ¹H MRS metabolite concentrations in normal appearing cerebellum of the SMO mice were not different from those in the WT mice. Macromolecule and lipid ¹H MRS signals in SMO medulloblastomas were not different from those detected in the cerebellum of WT mice. The HR-MAS analysis of SMO medulloblastomas confirmed the in vivo ¹H MRS metabolite profiles, and additionally revealed that phosphocholine was strongly elevated in medulloblastomas accounting for the high in vivo CCM. CONCLUSIONS: These metabolite profiles closely mirror those reported from human medulloblastomas confirming that SMO mice provide a realistic model for investigating metabolic aspects of this disease. Taurine, glycine, and CCM are potential metabolite biomarkers for the SMO medulloblastomas. The MRS data from the medulloblastomas with defined molecular pathology is discussed in the light of metabolite profiles reported from human tumours.

Metabonomic analysis identifies molecular changes associated with the pathophysiology and drug treatment of bipolar disorder.

Bipolar affective disorder is a severe and debilitating psychiatric condition characterized by the alternating mood states of mania and depression. Both the molecular pathophysiology of the disorder and the mechanism of action of the mainstays of its treatment remain largely unknown. Here, (1)H NMR spectroscopy-based metabonomic analysis was performed to identify molecular changes in post-mortem brain tissue (dorsolateral prefrontal cortex) of patients with a history of bipolar disorder. The observed changes were then compared to metabolic alterations identified in rat brain following chronic oral treatment with either lithium or valproate. This is the first study to use (1)H NMR spectroscopy to study post-mortem bipolar human brain tissue, and it is the first to compare changes in disease brain with changes induced in rat brain following mood stabilizer treatment. Several metabolites were found to be concordantly altered in both the animal and human tissues. Glutamate levels were increased in post-mortem bipolar brain, while the glutamate/glutamine ratio was decreased following valproate treatment, and gamma-aminobutyric acid levels were increased after lithium treatment, suggesting that the balance of excitatory/inhibitory neurotransmission is central to the disorder. Both creatine and myo-inositol were increased in the post-mortem brain but depleted with the medications. Lastly, the level of N-acetyl aspartate, a clinically important metabolic marker of neuronal viability, was found to be unchanged following chronic mood stabilizer treatment. These findings promise to provide new insight into the pathophysiology of bipolar disorder and may be used to direct research into novel therapeutic strategies.

A metabolomic comparison of urinary changes in type 2 diabetes in mouse, rat, and human.

Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with multivariate statistics was applied to examine the urinary metabolic changes in two rodent models of type 2 diabetes mellitus as well as unmedicated human sufferers. The db/db mouse and obese Zucker (fa/fa) rat have autosomal recessive defects in the leptin receptor gene, causing type 2 diabetes. 1H-NMR spectra of urine were used in conjunction with uni- and multivariate statistics to identify disease-related metabolic changes in these two animal models and human sufferers. This study demonstrates metabolic similarities between the three species examined, including metabolic responses associated with general systemic stress, changes in the TCA cycle, and perturbations in nucleotide metabolism and in methylamine metabolism. All three species demonstrated profound changes in nucleotide metabolism, including that of N-methylnicotinamide and N-methyl-2-pyridone-5-carboxamide, which may provide unique biomarkers for following type 2 diabetes mellitus progression.

Characterization of the biochemical effects of 1-nitronaphthalene in rats using global metabolic profiling by NMR spectroscopy and pattern recognition.

Metabolic fingerprints, in the form of patterns of high-concentration endogenous metabolites, of 1-nitronaphthalene (NN)-induced lung toxicity have been elucidated in bronchoalveolar lavage fluid (BALF), urine, blood plasma, and intact lung and liver tissue using NMR spectroscopy-based metabolic profiling. A single dose of NN (75 mg kg(-1)) was administered orally to Sprague-Dawley rats. BALF and lung tissue were obtained 24 h after dosing from these animals and matched control rats post-mortem. High-resolution (1)H-NMR spectroscopy of BALF samples indicated that NN caused increases in concentrations of choline, amino acids (leucine, isoleucine and alanine) and lactate together with decreased concentrations of succinate, citrate, creatine, creatinine and glucose. In addition, the intact lung weights were higher in the NN-treated group (p<0.01), consistent with pulmonary oedema. The NMR-detected perturbations indicated that NN induces a perturbation in energy metabolism in both lung and liver tissue, as well as surfactant production and osmolyte levels in the lungs. As well as reporting the first NMR spectroscopic combined examination of BALF and intact lung, this study indicates that such holistic approaches to investigating mechanisms of lung toxicity may be of value in evaluating disease progression or the effects of therapeutic intervention in pulmonary conditions such as surfactant disorders or asthma.

Chemometric analysis of biofluids following toxicant induced hepatotoxicity: a metabonomic approach to distinguish the effects of 1-naphthylisothiocyanate from its products.

Metabonomics using high-resolution 1H-NMR spectroscopy of biofluids and pattern recognition is highly successful at distinguishing both organ- and sub-organ-specific toxicity. In the current study, this technique was investigated to distinguish the different biological effects caused by 1-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in the rat from that induced by exposure to 1-naphthylisocyanate (NI) and 1-naphthylamine (NA), two products of the metabolism of ANIT. While all three toxicants produced perturbations in similar urinary metabolites, principal components analysis of the temporal progression identified that the rapid initial glycosuria associated with ANIT toxicity was also present with NI but not NA dosing. However, longer-term perturbations in the urinary excretion of succinate, lactate and acetate were common to all three toxicants. The metabolic effects of the three compounds were also followed in blood plasma and liver tissue. Of the three toxicants, the most marked perturbations were induced by ANIT exposure, then NI, thereby indicating the effects of ANIT, NI and NA toxicity were distinct, with ANIT being the most, and NA the least, toxic of the three compounds. This indicates that metabonomics may be useful for following severity and mechanisms of toxicity in a series of related compounds during drug development.

MRS reveals additional hexose N-acetyl resonances in the brain of a mouse model for Sandhoff disease.

Sandhoff disease, one of several related lysosomal storage disorders, results from the build up of N-acetyl-containing glycosphingolipids in the brain and is caused by mutations in the genes encoding the hexosaminidase beta-subunit. Affected individuals undergo progressive neurodegeneration in response to the glycosphingolipid storage. (1)H magnetic resonance spectra of perchloric acid extracts of Sandhoff mouse brain exhibited several resonances ca 2.07 ppm that were not present in the corresponding spectra from extracts of wild-type mouse brain. High-performance liquid chromatography and mass spectrometry of the Sandhoff extracts post-MRS identified the presence of N-acetylhexosamine-containing oligosaccharides, which are the likely cause of the additional MRS resonances. MRS of intact brain tissue with magic angle spinning also showed additional resonances at ca 2.07 ppm in the Sandhoff case. These resonances appeared to increase with disease progression and probably arise, for the most part, from the stored glycosphingolipids, which are absent in the aqueous extracts. Hence in vivo MRS may be a useful tool for detecting early-stage Sandhoff disease and response to treatment.

High-resolution magic-angle spinning 13C NMR spectroscopy of cerebral tissue.

Monitoring the metabolism of (13)C-labelled substrates by biological tissues allows both the rate of metabolism and the relative importance of metabolic pathways to be determined. In this study high-resolution magic-angle spinning (HRMAS) (13)C NMR spectroscopy is assessed as a technique for determining the labelling of metabolites in brain slices. Freshly prepared rat brain slices were superfused in isotonic salt solution containing [1-(13)C] glucose. HRMAS (1)H and (13)C NMR spectra were acquired of the slices ( approximately 10 mg) at 3 degrees C. Using (1)H NMR spectroscopy it was demonstrated that the concentration of key metabolites indicative of metabolic degradation, including N-acetyl aspartate and lactate, did not change significantly across the approximately 11 h time period required for (13)C NMR spectra. The approach produced high-resolution spectra of intact tissue with the labelling patterns of tissues being indicative of both labelling via pyruvate dehydrogenase found in both neuronal and glial cells, and pyruvate carboxylase, found only within glial cells. This approach is a versatile tool for monitoring the compartmentation of metabolites directly, and will also allow the investigation of aqueous and lipid metabolites simultaneously.

Metabolic characterization of distinct neuroanatomical regions in rats by magic angle spinning 1H nuclear magnetic resonance spectroscopy.

High-resolution magic angle spinning (HRMAS) (1)H NMR spectroscopy has been applied to the biochemical characterization of specific brain regions in rats in order to establish baseline levels of tissue metabolite profiles with which to compare models of neuropathology or toxic lesion. Cores of tissue (20 mg) from the brain stem, cerebellum, frontal cortex, and hippocampus were obtained from histologically defined coronal slices of brain from 18 male Sprague-Dawley rats. HRMAS (1)H NMR spectra were acquired for each of the regions sampled and the degree of intersample variability, as assessed by principal components analysis and discriminant analysis by projection to latent structure was found to be low. Clear region-specific differences in the biochemical profiles were observed using both comparison of metabolite ratios and/or pattern recognition methods. Relatively low concentrations of GABA in the cerebellum, high concentrations of taurine and N-acetylaspartate in the cortex, and high levels of choline, glycerophosphocholine, and phosphocholine in the hippocampus predominantly influenced the classification of the different brain regions. Additionally, N-acetylaspartylglutamate was detected in the brain stem, but was largely absent from the other regions examined. Such analyses provide a baseline reference for further HRMAS NMR spectroscopic studies to monitor disease and pharmacological insults in specific regions of the brain.

Protoporphyrinogen oxidase inhibitor herbicide effects on pythium root rot of sugarcane, pythium species, and the soil microbial community.

ABSTRACT The effects of three protoporphyrinogen oxidase inhibitor herbicides, azafenidin, flumioxazin, and sulfentrazone, on Pythium root rot of sugarcane and the soil microbial community were evaluated in greenhouse experiments. Herbicides were applied as foliar and soil treatments. There were no consistent effects on plant growth or disease parameters. However, some herbicide treatments affected the relative frequency of isolation of Pythium spp. from roots and reduced colonization by the pathogenic species Pythium arrhenomanes. A comparison of sole carbon source utilization profiles indicated that soil-applied herbicides altered the functional diversity of the soil microbial community, with some variation depending on herbicide used. All three herbicides inhibited the in vitro mycelial growth of P. arrhenomanes, P. aphanidermatum, and P. ultimum. Active ingredients were less inhibitory than formulated product for azafenidin and flumioxazin but not for sulfentrazone.