NMR-based metabolomics at work in phytochemistry

Metabolomics is defined as both the qualitative and quantitative analysis of all metabolites in an organism unraveling correlation with other OMICs data. Many of the technologies used in metabolomics have method-specific advantages and drawbacks in terms of diversity of metabolites detected, sensitivity, or resolution. In this paper, the potential of NMR spectrometry applied to metabolomics is reviewed using examples of Nicotiana tabacum and Catharanthus roseus.     

Plant metabolomics: large-scale phytochemistry in the functional genomics era

Metabolomics or the large-scale phytochemical analysis of plants is reviewed in relation to functional genomics and systems biology. A historical account of the introduction and evolution of metabolite profiling into today's modern comprehensive metabolomics approach is provided. Many of the technologies used in metabolomics, including optical spectroscopy, nuclear magnetic resonance, and mass spectrometry are surveyed. The critical role of bioinformatics and various methods of data visualization are summarized and the future role of metabolomics in plant science assessed.     

Taxus sp. cell, tissue and organ cultures as alternative sources for taxoids production: a literature survey

The literature concerning the tissue culture of Taxus sp. as an alternative source for taxoid production is reviewed. The aim of this review is to summarize and discuss the progress achieved with the approaches and methods used for the establishment of various Taxus culture systems, the methods used for the evaluation of taxoid production, the multiple factors which control taxoid production and the feasibility of the in vitro production of taxoids on a commercial scale.     

Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?

Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and rigorous sample collection and sample handling protocols. The sensitivity of the MS analysis relies on the quality of the sample; consequently, the blood sample preparation step is crucial to obtain pure and concentrated samples and enrichment of the proteins and peptides of interest. This review focuses on the serum sample preparation step prior to protein profiling by MALDI MS analysis, with particular focus on various SPE methods. The application of SPE techniques with different chromatographic properties such as RP, ion exchange, or affinity binding to isolate specific subsets of molecules (subproteomes) is advantageous for increasing resolution and sensitivity in the subsequent MS analysis. In addition, several of the SPE sample preparation methods are simple and scalable and have proven easy to automate for higher reproducibility and throughput, which is important in a clinical proteomics setting.     

Metabolomics and its role in understanding cellular responses in plants

A natural shift is taking place in the approaches being adopted by plant scientists in response to the accessibility of systems-based technology platforms. Metabolomics is one such field, which involves a comprehensive non-biased analysis of metabolites in a given cell at a specific time. This review briefly introduces the emerging field and a range of analytical techniques that are most useful in metabolomics when combined with computational approaches in data analyses. Using cases from Arabidopsis and other selected plant systems, this review highlights how information can be integrated from metabolomics and other functional genomics platforms to obtain a global picture of plant cellular responses. We discuss how metabolomics is enabling large-scale and parallel interrogation of cell states under different stages of development and defined environmental conditions to uncover novel interactions among various pathways. Finally, we discuss selected applications of metabolomics. This special review article is dedicated to the commemoration of the retirement of Dr. Oluf L. Gamborg after 25 years of service as Founding Managing Editor of Plant Cell Reports. RB and KN have contributed equally to this review.     

ReviewMethods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from l-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects consitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

1H NMR and GC/MS metabolomics of earthworm responses to sub-lethal DDT and endosulfan exposure

The metabolic response of the earthworm Eisenia fetida to two pesticides, dichlorodiphenyltrichloroethane (DDT) and endosulfan, was characterized in contact tests using proton nuclear magnetic resonance (¹H NMR) and principal component analysis (PCA). PCA loading plots suggested that maltose, leucine and alanine were important metabolites contributing to the differences in dosed and control earthworms for both compounds at doses of 0.5, 1.0 and 2.0 μg/cm². Gas chromatography/mass spectrometry (GC/MS) was used to quantify the metabolites identified in E. fetida and determine if the changes in maltose, leucine and alanine following exposure to DDT and endosulfan (at 0.5 and 1.0 μg/cm²) were reproducible and greater than the natural variability. Quantification by GC/MS suggested that maltose was not a reliable biomarker since it both increased and decreased in earthworms exposed to DDT and increased by just 3% with exposure to endosulfan. Leucine was not stable with the GC/MS derivitization method used in this study and could not be confirmed as a reliable biomarker. However, alanine consistently increased for both DDT and endosulfan exposed E. fetida. Alanine showed considerable variability in control earthworms (±41.6%), yet the variability in alanine to glycine ratios was just ±10.5%. Increases in the alanine to glycine ratio were statistically significant at the P = 0.05 level for the 1.0 μg/cm² DDT dose and both the 0.5 and 1.0 μg/cm² endosulfan doses, suggesting that deviations from the normal homeostatic ratio of 1.5 for alanine to glycine is a potential biomarker of DDT and endosulfan exposure warranting further study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Environmental Metabolomics Special Issue of Metabolomics.     

Inducible gene expression systems inLactococcus lactis

Lactococcus lactis is industrially important microorganism used in many dairy fermentations. Numerous genes and gene expression signals from this organism have now been identified and characterized. Recently, several naturally occurring, inducible gene-expression systems have also been described inL. lactis. The main features of these systems can be exploited to design genetically engineered expression cassettes for controlled production of various proteins and enzymes. Novel gene-expression systems inLactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.     

Applications of mass spectrometry in metabolomic studies of animal model and invertebrate systems

Metabolomics provides rich datasets for systems biology. Massspectrometric (MS) techniques are rapidly gaining in importancefor untargeted metabolic profiling. In this review, we surveythe various techniques for sample preparation and analysis relatingto the various MS techniques and illustrate the potential ofthese techniques for both observing complete metabolomes anddetecting changes in the metabolism resulting from genetic mutationof other perturbations. The use of some of these techniquesin the study of model organisms including rodent and variousinvertebrate models is described. The invertebrate systems areof particular interest since such organisms have valuable mutantresources, such as RNAi panels directed against nearly all thegenes in the genome. The demonstration that they are readilycompatible with metabolomic approaches is particularly importantfor systems approaches to metabolic pathways.      

The effect of water temperature,current velocity and suspended solids on the distribution,growth and seasonality of Lemanea fluviatilis (C. Ag.), Rhodophyta,in the river Usk and other South Wales rivers

Two surveys of Lamanea in South Wales rivers have given details of its distribution, seasonality and range of tolerance to various environmental parameters, with particular reference to temperature, current velocity and suspended solids.Temperature, current velocity and suspended solids had most effect. Such findings were confirmed by principal component and correlation coefficient analysis.These investigations also suggest that abundant growths of Lemanea may be used in a limited manner as indicative of oligotrophic and metal free waters especially during Spring.In addition, transplants of Lemanea from the upper to the lower reaches of the River Usk and vice versa were studied to clarify the effect of suspended solids in nature.Using the ¹⁴C technique for primary production proved to be a successful method with Lamanea for comparison with chlorophyll a extraction and phycoerythrin. The importance of the latter in energy transfer for photosynthesis was revealed for this genus. A combination of various simple and cheap methods for the extraction of the pigment phycoerythrin was used successfully for the first time in red algal studies.Procedures for estimating percentage cover, standing crop and the presence/absence of the plant are given and the statistical reliability of the methods is evaluated.     

A comparison of two techniques for measuring and crossdating tree rings

Crossdating is the core principle of dendrochronology. Our study compared two techniques for measuring and crossdating tree rings using Juniperus virginiana L. (eastern redcedar) as a case study. We used a pseudo 2 × 2 study design comparing the traditional skeleton plot/sliding measuring stage technique to a semi-automatic image analysis program across two technicians. Crossdating was evaluated in COFECHA. Raw measurements of total, earlywood, and latewood widths from the two methods were analyzed using the Verify for Windows program, ANOVA, and correlation matrices. Total ring width and earlywood width were well correlated between techniques and technicians but questionable ring boundaries from image analysis program should be checked under a stereoscope. Juniperus virginiana latewood widths were significantly different between techniques and technicians; therefore, we do not recommend combining latewood measurements from species with limited latewood variability for dendrochronological analysis. A standard definition of the earlywood-latewood boundary that can be replicated across technicians is needed to combine latewood measurements from the sliding measuring stage and image analysis systems.     

Floral bract function, flowering process and breeding systems ofSarcandra andChloranthus (Chloranthaceae)

Structure and function of floral bracts, anthesis and breeding systems were investigated in greenhouse plants ofSarcandra chloranthoides, Sarcandra glabra, andChloranthus spicatus (Chloranthaceae). In early developmental stages the floral bract replaces the lacking perianth as a protective structure by enclosing the floral organs completely in a pocket-like structure, which is formed by the u-shaped attachment zone of the floral bract to the spike axis. The floral bract has a tip with an epithem tissue, from which traces of secretion seem to be released by the stomata. All species investigated have protogynous and longlasting flowers. The female phase begins five to seven days earlier than the male phase. It continues during the male phase and for some time thereafter. The male phase lasts between one day (Chloranthus spicatus) and seven days (Sarcandra chloranthoides). This is the first study on breeding systems of Chloranthaceae. They are of special interest in such a basal family of the angiosperms. Although the investigatedSarcandra andChloranthus species have similar flowers with entomophilous features, surprisingly their breeding systems are diverse:Sarcandra glabra is self-compatible,S. chloranthoides is agamospermous andChloranthus spicatus is self-incompatible.     

The ABRF Metabolomics Research Group 2013 Study: Investigation of Spiked Compound Differences in a Human Plasma Matrix

Metabolomics is an emerging field that involves qualitative and quantitative measurements of small molecule metabolites in a biological system. These measurements can be useful for developing biomarkers for diagnosis, prognosis, or predicting response to therapy. Currently, a wide variety of metabolomics approaches, including nontargeted and targeted profiling, are used across laboratories on a routine basis. A diverse set of analytical platforms, such as NMR, gas chromatography-mass spectrometry, Orbitrap mass spectrometry, and time-of-flight-mass spectrometry, which use various chromatographic and ionization techniques, are used for resolution, detection, identification, and quantitation of metabolites from various biological matrices. However, few attempts have been made to standardize experimental methodologies or comparative analyses across different laboratories. The Metabolomics Research Group of the Association of Biomolecular Resource Facilities organized a “round-robin” experiment type of interlaboratory study, wherein human plasma samples were spiked with different amounts of metabolite standards in 2 groups of biologic samples (A and B). The goal was a study that resembles a typical metabolomics analysis. Here, we report our efforts and discuss challenges that create bottlenecks for the field. Finally, we discuss benchmarks that could be used by laboratories to compare their methodologies.     

Metabolomics and its application to studying metal toxicity

Here we explain the omics approach of metabolomics and how it can be applied to study a physiological response to toxic metal exposure. This review aims to educate the metallomics field to the tool of metabolomics. Metabolomics is becoming an increasingly used tool to compare natural and challenged states of various organisms, from disease states in humans to toxin exposure to environmental systems. This approach is key to understanding and identifying the cellular or biochemical targets of metals and the underlying physiological response. Metabolomics steps are described and overviews of its application to metal toxicity to organisms are given. As this approach is very new there are yet only a small number of total studies and therefore only a brief overview of some metal metabolomics studies is described. A frank critical evaluation of the approach is given to provide newcomers to the method a clear idea of the challenges and the rewards of applying metabolomics to their research.     

Suspended cultivation of Gracilaria in the sea

Suspended rope cultivation systems for Gracilaria or Gracilariopsis can be seeded from spores but vegetative propagation is used for those which are currently commercially successful. Such systems and methods have been recently developed for the shallow lagoon areas around Lüderitz, Namibia and deeper open water sites off the Araya Peninsula, Venezuela. Various sites, substrata, depths and seeding methods have been assessed and have resulted in average commercial growth rates of about 3.5% per day. Netlon ‘Superope’ has been demonstrated to be more suitable than laid rope as a substratum for outplanted vegetative Gracilaria gracilis in Namibia as it does not compress the thalli as they grow. An efficient method, using hooks, has been developed to ‘seed’ the Superope. There is a very low incidence of algal epiphytism: mainly Ceramium and Ulva sp. In Venezuela, pilot rope cultivation systems for Gracilariopsis tenuifrons proved successful and have good commercial prospects.     

Monitoring novel metabolic pathways using metabolomics and machine learning: induction of the phosphoketolase pathway in Aspergillus nidulans cultivations

The physiological phenotype of Aspergillus nidulans was determined under different environmental conditions through the quantification of the intracellular and extracellular metabolite pools and clear evidence of the presence of a novel fungal metabolic pathway, the phosphoketolase pathway, was obtained. Induction of the phosphoketolase pathway resulted after blocking the EMP pathway through the deactivation of glyceraldehyde-3-P dehydrogenase (G3PD). Deactivation of G3PD in cultivations of A. nidulans on glucose and xylose led to a 10-fold decrease in the specific growth rate; however, growth could still be sustained solely through the phosphoketolase pathway. Metabolomics and machine learning tools were successfully used to monitor the alteration caused by the inhibition of G3PD in the metabolism of A. nidulans grown on glucose, xylose, acetate as well as mixtures of glucose or xylose with acetate. This is the first study that demonstrates in vivo that the fungal central carbon metabolism includes an active phosphoketolase pathway.     

Ammonium and methylammonium uptake in a fertilizer-degrading strain of Ochrobactrum anthropi

The transport of ammonium and methylammonium was studied in a strain of Ochrobactrum anthropi, a microorganism isolated from garden soil and able to degrade methyleneureas which are used as slow-release nitrogen fertilizer. The activity of both transport systems was determined using [¹⁴C]methylammonium. Differences between the two transport systems were observed with regard to their pH- and temperature dependence as well as their kinetic parameters and regulation during growth with various nitrogen sources. Ammonium transport was subject to repression by ammonium and to derepression in its absence, while the methylammonium carrier was induced in the presence of methylamine. The ammonium but not the methylammonium transport system was severely inhibited by ammonium, and metabolic poisons inhibited both uptake systems. The analysis of intracellular metabolites using thin-layer chromatography and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry indicated that methylammonium was rapidly metabolized to N-methylglutamate via -N-methylglutamine.     

Analysis of rhamnolipid biosurfactants by methylene blue complexation

Rhamnolipids, produced by Pseudomonas aeruginosa, represent an important group of biosurfactants having various industrial, environmental, and medical applications. Current methods for rhamnolipid quantification involve the use of strong hazardous acids/chemicals, indirect measurement of the concentration of sugar moiety, or require the availability of expensive equipment (HPLC-MS). A safer, easier method that measures the whole rhamnolipid molecules would significantly enhance strain selection, metabolic engineering, and process development for economical rhamnolipid production. A semi-quantitative method was reported earlier to differentiate between the rhamnolipid-producing and non-producing strains using agar plates containing methylene blue and cetyl trimethylammonium bromide (CTAB). In this study, a rapid and simple method for rhamnolipid analysis was developed by systematically investigating the complexation of rhamnolipids and methylene blue, with and without the presence of CTAB. The method relies on measuring the absorbance (at 638 nm) of the rhamnolipid−methylene blue complex that partitions into the chloroform phase. With P. aeruginosa fermentation samples, the applicability of this method was verified by comparison of the analysis results with those obtained from the commonly used anthrone reaction technique.