Identification and Microbial Production of the Raspberry Phenol Salidroside that Is Active against Huntington's Disease.

Edible berries are considered to be among nature's treasure chests as they contain a large number of (poly)phenols with potentially health-promoting properties. However, as berries contain complex (poly)phenol mixtures, it is challenging to associate any interesting pharmacological activity with a single compound. Thus, identification of pharmacologically interesting phenols requires systematic analyses of berry extracts. Here, raspberry (Rubus idaeus, var Prestige) extracts were systematically analyzed to identify bioactive compounds against pathological processes of neurodegenerative diseases. Berry extracts were tested on different Saccharomyces cerevisiae strains expressing disease proteins associated with Alzheimer's, Parkinson's, or Huntington's disease, or amyotrophic lateral sclerosis. After identifying bioactivity against Huntington's disease, the extract was fractionated and the obtained fractions were tested in the yeast model, which revealed that salidroside, a glycosylated phenol, displayed significant bioactivity. Subsequently, a metabolic route to salidroside was reconstructed in S cerevisiae and Corynebacterium glutamicum The best-performing S cerevisiae strain was capable of producing 2.1 mm (640 mg L-1) salidroside from Glc in shake flasks, whereas an engineered C glutamicum strain could efficiently convert the precursor tyrosol to salidroside, accumulating up to 32 mm (9,700 mg L-1) salidroside in bioreactor cultivations (yield: 0.81 mol mol-1). Targeted yeast assays verified that salidroside produced by both organisms has the same positive effects as salidroside of natural origin.

Extraction, Enrichment, and LC-MSn-Based Characterization of Phlorotannins and Related Phenolics from the Brown Seaweed, Ascophyllum nodosum.

Phenolic components from the edible brown seaweed, Ascophyllum nodosum, have been associated with considerable antioxidant activity but also bioactivities related to human health. This study aims to select and identify the main phlorotannin components from this seaweed which have been previously associated with potential health benefits. Methods to enrich phenolic components then further select phlorotannin components from ethanolic extracts of Ascophyllum nodosum were applied. The composition and phenolic diversity of these extracts were defined using data dependent liquid chromatography mass spectroscopic (LC-MSn) techniques. A series of phlorotannin oligomers with apparent degree of polymerization (DP) from 10 to 31 were enriched by solid phase extraction and could be selected by fractionation on Sephadex LH-20. Evidence was also obtained for the presence of dibenzodioxin linked phlorotannins as well as sulphated phlorotannins and phenolic acids. As well as diversity in molecular size, there was evidence for potential isomers at each DP. MS2 fragmentation analyses strongly suggested that the phlorotannins contained ether linked phloroglucinol units and were most likely fucophlorethols and MS3 data suggested that the isomers may result from branching within the chain. Therefore, application of these LC-MSn techniques provided further information on the structural diversity of the phlorotannins from Ascophyllum, which could be correlated against their reported bioactivities and could be further applied to phlorotannins from different seaweed species.

Engineering Lactococcus lactis for the production of unusual anthocyanins using tea as substrate.

Plant material rich in anthocyanins has been historically used in traditional medicines, but only recently have the specific pharmacological properties of these compounds been the target of extensive studies. In addition to their potential to modulate the development of various diseases, coloured anthocyanins are valuable natural alternatives commonly used to replace synthetic colourants in food industry. Exploitation of microbial hosts as cell factories is an attractive alternative to extraction of anthocyanins and other flavonoids from plant sources or chemical synthesis. In this study, we present the lactic acid bacterium Lactococcus lactis as an ideal host for the production of high-value plant-derived bioactive anthocyanins using green tea as substrate. Besides the anticipated red-purple compounds cyanidin and delphinidin, orange and yellow pyranoanthocyanidins with unexpected methylation patterns were produced from green tea by engineered L. lactis strains. The pyranoanthocyanins are currently attracting significant interest as one of the most important classes of anthocyanin derivatives and are mainly formed during the aging of wine, contributing to both colour and sensory experience.

Rapid UHPLC-MS metabolite profiling and phenotypic assays reveal genotypic impacts of nitrogen supplementation in oats.

INTRODUCTION: Oats (Avena sativa L.) are a whole grain cereal recognised for their health benefits and which are cultivated largely in temperate regions providing both a source of food for humans and animals, as well as being used in cosmetics and as a potential treatment for a number of diseases. Oats are known as being a cereal source high in dietary fibre (e.g. ß-glucans), as well as being high in antioxidants, minerals and vitamins. Recently, oats have been gaining increased global attention due to their large number of beneficial health effects. Consumption of oats has been proven to lower blood LDL cholesterol levels and blood pressure, thus reducing the risk of heart disease, as well as reducing blood-sugar and insulin levels. OBJECTIVES: Oats are seen as a low input cereal. Current agricultural guidelines on nitrogen application are believed to be suboptimal and only consider the effect of nitrogen on grain yield. It is important to understand the role of both variety and of crop management in determining nutritional quality of oats. In this study the response of yield, grain quality and grain metabolites to increasing nitrogen application to levels greater than current guidelines were investigated. METHODS: Four winter oat varieties (Mascani, Tardis, Balado and Gerald) were grown in a replicated nitrogen response trial consisting of a no added nitrogen control and four added nitrogen treatments between 50 and 200 kg N ha-1 in a randomised split-plot design. Grain yield, milling quality traits, ß-glucan, total protein and oil content were assessed. The de-hulled oats (groats) were also subjected to a rapid Ultra High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) metabolomic screening approach. RESULTS: Application of nitrogen had a significant effect on grain yield but there was no significant difference between the response of the four varieties. Grain quality traits however displayed significant differences both between varieties and nitrogen application level. ß-glucan content significantly increased with nitrogen application. The UHPLC-MS approach has provided a rapid, sub 15 min per sample, metabolite profiling method that is repeatable and appropriate for the screening of large numbers of cereal samples. The method captured a wide range of compounds, inclusive of primary metabolites such as the amino acids, organic acids, vitamins and lipids, as well as a number of key secondary metabolites, including the avenanthramides, caffeic acid, and sinapic acid and its derivatives and was able to identify distinct metabolic phenotypes for the varieties studied. Amino acid metabolism was massively upregulated by nitrogen supplementation as were total protein levels, whilst the levels of organic acids were decreased, likely due to them acting as a carbon skeleton source. Several TCA cycle intermediates were also impacted, potentially indicating increased TCA cycle turn over, thus providing the plant with a source of energy and reductant power to aid elevated nitrogen assimilation. Elevated nitrogen availability was also directed towards the increased production of nitrogen containing phospholipids. A number of both positive and negative impacts on the metabolism of phenolic compounds that have influence upon the health beneficial value of oats and their products were also observed. CONCLUSIONS: Although the developed method has broad applicability as a rapid screening method or a rapid metabolite profiling method and in this study has provided valuable metabolic insights, it still must be considered that much greater confidence in metabolite identification, as well as quantitative precision, will be gained by the application of higher resolution chromatography methods, although at a large expense to sample throughput. Follow up studies will apply higher resolution GC (gas chromatography) and LC (reversed phase and HILIC) approaches, oats will be also analysed from across multiple growth locations and growth seasons, effectively providing a cross validation for the results obtained within this preliminary study. It will also be fascinating to perform more controlled experiments with sampling of green tissues, as well as oat grains, throughout the plants and grains development, to reveal greater insight of carbon and nitrogen metabolism balance, as well as resource partitioning into lipid and secondary metabolism.

Application of HPLC-PDA-MS metabolite profiling to investigate the effect of growth temperature and day length on blackcurrant fruit.

INTRODUCTION: Blackcurrant (Ribes nigrum L.) is an excellent example of a "super fruit" with potential health benefits. Both genotype and cultivation environment are known to affect the chemical composition of blackcurrant, especially ascorbic acid and various phenolic compounds. Environmental conditions, like temperature, solar radiation and precipitation can also have significant impact on fruit chemical composition. The relevance of the study is further accentuated by the predicted and ongoing changes in global climate. OBJECTIVES: The aim of the present study was to provide new knowledge and a deeper understanding of the effects of post flowering environmental conditions, namely temperature and day length, on fruit quality and chemical composition of blackcurrant using an untargeted high performance liquid chromatography-photo diode array-mass spectrometry (HPLC-PDA-MS) metabolomics approach. METHODS: A phytotron experiment with cultivation of single-stemmed potted plants of blackcurrant cv. Narve Viking was conducted using constant temperatures of 12, 18 or 24 °C and three different photoperiods (short day, short day with night interruption, and natural summer daylight conditions). Plants were also grown under ambient outdoor conditions. Ripe berries were analysed using an untargeted HPLC-PDA-MS metabolomics approach to detect the presence and concentration of molecules as affected by controlled climatic factors. RESULTS: The untargeted metabolomics dataset contained a total of 7274 deconvolved retention time-m/z pairs across both electrospray ionisation (ESI) positive and negative polarities, from which 549 metabolites were identified or minimally annotated based upon accurate mass MS. Conventional principal component analysis (PCA) in combination with the Friedman significance test were applied to first identify which metabolites responded to temperature in a linear fashion. Multi-block hierarchical PCA in combination with the Friedman significance test was secondly applied to identify metabolites that were responsive to different day length conditions. Temperature had significant effect on a total of 365 metabolites representing a diverse range of chemical classes. It was observed that ripening of the blackcurrant berries under ambient conditions, compared to controlled conditions, resulted in an increased accumulation of 34 annotated metabolites, mainly anthocyanins and flavonoids. 18 metabolites were found to be regulated differentially under the different daylength conditions. Moreover, based upon the most abundant anthocyanins, a comparison between targeted and untargeted analyses, revealed a close convergence of the two analytical methods. Therefore, the study not just illustrates the value of non-targeted metabolomics approaches with respect to the huge diversity and numbers of significantly changed metabolites detected (and which would be missed by conventional targeted analyses), but also shows the validity of the non-targeted approach with respect to its precision compared to targeted analyses. CONCLUSIONS: Blackcurrant maturation under controlled ambient conditions revealed a number of insightful relationships between environment and chemical composition of the fruit. A prominent reduction of the most abundant anthocyanins under the highest temperature treatments indicated that blackcurrant berries in general may accumulate lower total anthocyanins in years with extreme hot summer conditions. HPLC-PDA-MS metabolomics is an excellent method for broad analysis of chemical composition of berries rich in phenolic compounds. Moreover, the experiment in controlled phytotron conditions provided additional knowledge concerning plant interactions with the environment.

Cinnamon Shows Antidiabetic Properties that Are Species-Specific: Effects on Enzyme Activity Inhibition and Starch Digestion.

Despite considerable research the evidence around the antidiabetic properties of cinnamon remains equivocal, and this may be due to varietal differences which is an aspect that is understudied. This study systematically compared the anti-hyperglycaemic properties of the four major commercial cinnamon types used around the world (Chinese; Cinnamomum cassia [CC], Indonesian; C. burmanii [IC], Vietnamese; C. loureirii [VC], and Ceylon; C. zeylanicum [SC]). LC-MS analysis showed distinct diffrences in the phytochemical profiles of cinnamon with SC showing the lowest coumarin concentration. CC and IC had the highest polyphenol levels and antioxidant potential, and all four types differed significantly in their content (P < 0.001). All cinnamon types showed potent species-specific effects on starch digestion enzyme activity inhibition (P < 0.001), CC was most effective against α-amylase and all four strongly inhibited α-glucosidase compared to acarbose. Cinnamon significantly reduced starch breakdown during oral (P = 0.006) and gastric (P = 0.029) phases of gastro-intestinal digestion with IC and SC showing consistent effects. No effects of cinnamon were seen in the intestinal phase. IC, VC and SC showed the greatest potential to inhibit formation of advanced glycation endproducts (AGEs) during digestion. In conclusion, cinnamon demonstrates anti-hyperglycaemic properties, however effects are species-specific with best overall properties seen for Ceylon cinnamon.

Regulation of hepatic glucose production and AMPK by AICAR but not by metformin depends on drug uptake through the equilibrative nucleoside transporter 1 (ENT1).

AIM: Recently we have observed differences in the ability of metformin and AICAR to repress glucose production from hepatocytes using 8CPT-cAMP. Previous results indicate that, in addition to activating protein kinase A, 8CPT-modified cAMP analogues suppress the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter ENT1. We aimed to exploit 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR, which is highly selective for a high-affinity binding-site on ENT1, to investigate the role of ENT1 in the liver-specific glucose-lowering properties of AICAR and metformin. METHODS: Primary mouse hepatocytes were incubated with AICAR and metformin in combination with cAMP analogues, glucagon, forskolin and NBMPR. Hepatocyte glucose production (HGP) and AMPK signalling were measured, and a uridine uptake assay with supporting LC-MS was used to investigate nucleoside depletion from medium by cells. RESULTS: AICAR and metformin increased AMPK pathway phosphorylation and decreased HGP induced by dibutyryl cAMP and glucagon. HGP was also induced by 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR; however, in each case this was resistant to suppression by AICAR but not by metformin. Cross-validation of tracer and mass spectrometry studies indicates that 8CPT-cAMP, 8CPT-2-Methyl-O-cAMP and NBMPR inhibited the effects of AICAR, at least in part, by impeding its uptake into hepatocytes. CONCLUSIONS: We report for the first time that suppression of ENT1 induces HGP. ENT1 inhibition also impedes uptake and the effects of AICAR, but not metformin, on HGP. Further investigation of nucleoside transport may illuminate a better understanding of how metformin and AICAR each regulate HGP.

Intermittent energy restriction induces changes in breast gene expression and systemic metabolism.

BACKGROUND: Observational studies suggest weight loss and energy restriction reduce breast cancer risk. Intermittent energy restriction (IER) reduces weight to the same extent as, or more than equivalent continuous energy restriction (CER) but the effects of IER on normal breast tissue and systemic metabolism as indicators of breast cancer risk are unknown. METHODS: We assessed the effect of IER (two days of 65 % energy restriction per week) for one menstrual cycle on breast tissue gene expression using Affymetrix GeneChips, adipocyte size by morphometry, and systemic metabolism (insulin resistance, lipids, serum and urine metabolites, lymphocyte gene expression) in 23 overweight premenopausal women at high risk of breast cancer. Unsupervised and supervised analyses of matched pre and post IER biopsies in 20 subjects were performed, whilst liquid and gas chromatography mass spectrometry assessed corresponding changes in serum and urine metabolites in all subjects after the two restricted and five unrestricted days of the IER. RESULTS: Women lost 4.8 % (±2.0 %) of body weight and 8.0 % (±5.0 %) of total body fat. Insulin resistance (homeostatic model assessment (HOMA)) reduced by 29.8 % (±17.8 %) on the restricted days and by 11 % (±34 %) on the unrestricted days of the IER. Five hundred and twenty-seven metabolites significantly increased or decreased during the two restricted days of IER. Ninety-one percent of these returned to baseline after 5 days of normal eating. Eleven subjects (55 %) displayed reductions in energy restriction-associated metabolic gene pathways including lipid synthesis, gluconeogenesis and glycogen synthesis. Some of these women also had increases in genes associated with breast epithelial cell differentiation (secretoglobulins, milk proteins and mucins) and decreased collagen synthesis (TNMD, PCOLCE2, TIMP4). There was no appreciable effect of IER on breast gene expression in the other nine subjects. These groups did not differ in the degree of changes in weight, total body fat, fat cell size or serum or urine metabolomic markers. Corresponding gene changes were not seen in peripheral blood lymphocytes. CONCLUSION: The transcriptional response to IER is variable in breast tissue, which was not reflected in the systemic response, which occurred in all subjects. The mechanisms of breast responsiveness/non-responsiveness require further investigation. TRIAL REGISTRATION: ISRCTN77916487 31/07/2012.

MUSCLE: automated multi-objective evolutionary optimization of targeted LC-MS/MS analysis.

Developing liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses of (bio)chemicals is both time consuming and challenging, largely because of the large number of LC and MS instrument parameters that need to be optimized. This bottleneck significantly impedes our ability to establish new (bio)analytical methods in fields such as pharmacology, metabolomics and pesticide research. We report the development of a multi-platform, user-friendly software tool MUSCLE (multi-platform unbiased optimization of spectrometry via closed-loop experimentation) for the robust and fully automated multi-objective optimization of targeted LC-MS/MS analysis. MUSCLE shortened the analysis times and increased the analytical sensitivities of targeted metabolite analysis, which was demonstrated on two different manufacturer's LC-MS/MS instruments.

Nontargeted LC-MSn Profiling of Compounds in Ileal Fluids That Decrease after Raspberry Intake Identifies Consistent Alterations in Bile Acid Composition.

Ileostomy studies provide a unique insight into the digestion of foods, allowing identification of physiologically relevant dietary phytochemicals and their metabolites that are important to gut health. We previously reported an increase of components, including novel triterpenoids, in ileal fluids of 11 ileostomates following consumption of raspberries using nontargeted LC-MSn techniques in combination with data deconvolution software. The current study focused on components that consistently decreased postsupplementation. After data deconvolution, 32 components were identified that met exclusion parameters of m/z signals and which decreased significantly in ileal fluids from eight of 11 participants post-raspberry supplementation. Two-thirds of these components were identified putatively from their MS properties. Consistent decreases were observed in components that possibly reflected "washing out" of presupplementation intake of common foods/drinks including (poly)phenol metabolites. Metabolites associated with fat metabolism such as hydroxylated fatty acids and cholate-type bile acids were specifically reduced. However, more directed re-examination of the data revealed that although some cholates were consistently reduced, the more polar glyco- and tauro-linked bile acid derivatives increased consistently, by as much as 100-fold over presupplementation levels. The possible reasons for these substantial alterations in bile acid composition in ileal fluids in response to raspberry intake are discussed.

HAMMER: automated operation of mass frontier to construct in silico mass spectral fragmentation libraries.

SUMMARY: Experimental MS(n) mass spectral libraries currently do not adequately cover chemical space. This limits the robust annotation of metabolites in metabolomics studies of complex biological samples. In silico fragmentation libraries would improve the identification of compounds from experimental multistage fragmentation data when experimental reference data are unavailable. Here, we present a freely available software package to automatically control Mass Frontier software to construct in silico mass spectral libraries and to perform spectral matching. Based on two case studies, we have demonstrated that high-throughput automation of Mass Frontier allows researchers to generate in silico mass spectral libraries in an automated and high-throughput fashion with little or no human intervention required. AVAILABILITY AND IMPLEMENTATION: Documentation, examples, results and source code are available at http://www.biosciences-labs.bham.ac.uk/viant/hammer/.

Metabolic Profiling of Geobacter sulfurreducens during Industrial Bioprocess Scale-Up.

During the industrial scale-up of bioprocesses it is important to establish that the biological system has not changed significantly when moving from small laboratory-scale shake flasks or culturing bottles to an industrially relevant production level. Therefore, during upscaling of biomass production for a range of metal transformations, including the production of biogenic magnetite nanoparticles by Geobacter sulfurreducens, from 100-ml bench-scale to 5-liter fermentors, we applied Fourier transform infrared (FTIR) spectroscopy as a metabolic fingerprinting approach followed by the analysis of bacterial cell extracts by gas chromatography-mass spectrometry (GC-MS) for metabolic profiling. FTIR results clearly differentiated between the phenotypic changes associated with different growth phases as well as the two culturing conditions. Furthermore, the clustering patterns displayed by multivariate analysis were in agreement with the turbidimetric measurements, which displayed an extended lag phase for cells grown in a 5-liter bioreactor (24 h) compared to those grown in 100-ml serum bottles (6 h). GC-MS analysis of the cell extracts demonstrated an overall accumulation of fumarate during the lag phase under both culturing conditions, coinciding with the detected concentrations of oxaloacetate, pyruvate, nicotinamide, and glycerol-3-phosphate being at their lowest levels compared to other growth phases. These metabolites were overlaid onto a metabolic network of G. sulfurreducens, and taking into account the levels of these metabolites throughout the fermentation process, the limited availability of oxaloacetate and nicotinamide would seem to be the main metabolic bottleneck resulting from this scale-up process. Additional metabolite-feeding experiments were carried out to validate the above hypothesis. Nicotinamide supplementation (1 mM) did not display any significant effects on the lag phase of G. sulfurreducens cells grown in the 100-ml serum bottles. However, it significantly improved the growth behavior of cells grown in the 5-liter bioreactor by reducing the lag phase from 24 h to 6 h, while providing higher yield than in the 100-ml serum bottles.

Acclimation of metabolism to light in Arabidopsis thaliana: the glucose 6-phosphate/phosphate translocator GPT2 directs metabolic acclimation.

Mature leaves of plants transferred from low to high light typically increase their photosynthetic capacity. In Arabidopsis thaliana, this dynamic acclimation requires expression of GPT2, a glucose 6-phosphate/phosphate translocator. Here, we examine the impact of GPT2 on leaf metabolism and photosynthesis. Plants of wild type and of a GPT2 knockout (gpt2.2) grown under low light achieved the same photosynthetic rate despite having different metabolic and transcriptomic strategies. Immediately upon transfer to high light, gpt2.2 plants showed a higher rate of photosynthesis than wild-type plants (35%); however, over subsequent days, wild-type plants acclimated photosynthetic capacity, increasing the photosynthesis rate by 100% after 7 d. Wild-type plants accumulated more starch than gpt2.2 plants throughout acclimation. We suggest that GPT2 activity results in the net import of glucose 6-phosphate from cytosol to chloroplast, increasing starch synthesis. There was clear acclimation of metabolism, with short-term changes typically being reversed as plants acclimated. Distinct responses to light were observed in wild-type and gpt2.2 leaves. Significantly higher levels of sugar phosphates were observed in gpt2.2. We suggest that GPT2 alters the distribution of metabolites between compartments and that this plays an essential role in allowing the cell to interpret environmental signals.

Selective induction and subcellular distribution of ACONITASE 3 reveal the importance of cytosolic citrate metabolism during lipid mobilization in Arabidopsis.

Arabidopsis thaliana has three genes that encode distinct aconitases (ACO), but little is known about the function of each isoenzyme during plant development. In newly emerged seedlings of Arabidopsis, transcript and protein levels for ACO3 were selectively induced to yield more than 80% of total aconitase activity. Characterization of knockout mutants for each of the three ACOs suggests a major role for only ACO3 in citrate metabolism. The aco3 mutant showed delayed early seedling growth, altered assimilation of [14C]acetate feeding and elevated citrate levels, which were nearly 4-fold greater than in wild-type, aco1 or aco2. However, both ACO1 and ACO2 are active in seedlings as shown by inhibition of aco3 growth by the toxin monofluoroacetate, and altered [14C]acetate assimilation and metabolite levels in aco1 and aco2. Relative levels of fumarate and malate differed between aco2 and aco3, indicating metabolically isolated pools of these metabolites in seedlings. Our inability to enrich ACO protein through mitochondria isolation, and the reduced cytosolic ACO activity of the iron-sulfur centre assembly mutant atm3-1, indicated a cytosolic localization of ACO3 in 3-day-old seedlings. Subsequently, we determined that more than 90% of ACO3 was cytosolic. We conclude that ACO3 is cytosolic in young seedlings and functions in citrate catabolism consistent with the operation of the classic glyoxylate and not direct catabolism of citrate within mitochondria.

Multiple metabolomics of uropathogenic E. coli reveal different information content in terms of metabolic potential compared to virulence factors.

No single analytical method can cover the whole metabolome and the choice of which platform to use may inadvertently introduce chemical selectivity. In order to investigate this we analysed a collection of uropathogenic Escherichia coli. The selected strains had previously undergone extensive characterisation using classical microbiological methods for a variety of metabolic tests and virulence factors. These bacteria were analysed using Fourier transform infrared (FT-IR) spectroscopy; gas chromatography mass spectrometry (GC-MS) after derivatisation of polar non-volatile analytes; as well as reversed-phase liquid chromatography mass spectrometry in both positive (LC-MS(+ve)) and negative (LC-MS(-ve)) electrospray ionisation modes. A comparison of the discriminatory ability of these four methods with the metabolic test and virulence factors was made using Procrustes transformations to ascertain which methods produce congruent results. We found that FT-IR and LC-MS(-ve), but not LC-MS(+ve), were comparable with each other and gave highly similar clustering compared with the virulence factors tests. By contrast, FT-IR and LC-MS(-ve) were not comparable to the metabolic tests, and we found that the GC-MS profiles were significantly more congruent with the metabolic tests than the virulence determinants. We conclude that metabolomics investigations may be biased to the analytical platform that is used and reflects the chemistry employed by the methods. We therefore consider that multiple platforms should be employed where possible and that the analyst should consider that there is a danger of false correlations between the analytical data and the biological characteristics of interest if the full metabolome has not been measured.

Optimization of parameters for the quantitative surface-enhanced Raman scattering detection of mephedrone using a fractional factorial design and a portable Raman spectrometer.

A new optimization strategy for the SERS detection of mephedrone using a portable Raman system has been developed. A fractional factorial design was employed, and the number of statistically significant experiments (288) was greatly reduced from the actual total number of experiments (1722), which minimized the workload while maintaining the statistical integrity of the results. A number of conditions were explored in relation to mephedrone SERS signal optimization including the type of nanoparticle, pH, and aggregating agents (salts). Through exercising this design, it was possible to derive the significance of each of the individual variables, and we discovered four optimized SERS protocols for which the reproducibility of the SERS signal and the limit of detection (LOD) of mephedrone were established. Using traditional nanoparticles with a combination of salts and pHs, it was shown that the relative standard deviations of mephedrone-specific Raman peaks were as low as 0.51%, and the LOD was estimated to be around 1.6 µg/mL (9.06 × 10(-6) M), a detection limit well beyond the scope of conventional Raman and extremely low for an analytical method optimized for quick and uncomplicated in-field use.

Fingerprinting food: current technologies for the detection of food adulteration and contamination.

Major food adulteration and contamination events seem to occur with some regularity, such as the widely publicised adulteration of milk products with melamine and the recent microbial contamination of vegetables across Europe for example. With globalisation and rapid distribution systems, these can have international impacts with far-reaching and sometimes lethal consequences. These events, though potentially global in the modern era, are in fact far from contemporary, and deliberate adulteration of food products is probably as old as the food processing and production systems themselves. This review first introduces some background into these practices, both historically and contemporary, before introducing a range of the technologies currently available for the detection of food adulteration and contamination. These methods include the vibrational spectroscopies: near-infrared, mid-infrared, Raman; NMR spectroscopy, as well as a range of mass spectrometry (MS) techniques, amongst others. This subject area is particularly relevant at this time, as it not only concerns the continuous engagement with food adulterers, but also more recent issues such as food security, bioterrorism and climate change. It is hoped that this introductory overview acts as a springboard for researchers in science, technology, engineering, and industry, in this era of systems-level thinking and interdisciplinary approaches to new and contemporary problems.

Qualitative phytochemical analysis and in vitro investigation of the immunomodulatory properties of honeys produced in Kazakhstan.

Honey is known to have antimicrobial, immunomodulatory and wound healing properties. The biological properties of honey have been attributed to phytochemicals derived from their source plants and research has focused on identifying the bioactive phytochemicals with therapeutic potential. In this study, we determined the ability of 5 honeys from Kazakhstan and manuka honey to stimulate TNF-α and TGF-ß production by human keratinocytes. TNF-α and TGF-ß levels increased over time in honey treated and untreated keratinocytes, whereas cells treated with sugar solutions that matched those of the honeys had reduced levels of both cytokines. This suggests that the non-sugar phytochemical components of the honeys may have prevented this decrease. Analysis by LC-MS confirmed that the honeys contained a diverse range of phytochemicals. Some phytochemicals e.g. pinobanksin and vanillin were present at different levels across the honey types, whereas other components, e.g. dicarboxylic acids and their glycosides, were abundant in all honeys.

Dissecting the impact of environment, season and genotype on blackcurrant fruit quality traits.

This work aims to determine the effect of genotype x environment (GxE) interaction that influence blackcurrant (Ribes nigrum) fruit quality. We applied metabolomics-driven analysis on fruits from four cultivars grown in contrasting European-locations over two seasons. By integrating metabolomics and sensory analysis, we also defined specific metabolic signatures associated with consumer acceptance. Our results showed that rainfall is a crucial factor associated with accumulation of delphinidin- and cyanidin-3-O-glucoside, the two mayor blackcurrant pigments meanwhile temperature affects the main organic acid levels which can be decisive for fruit taste. Sensorial analysis showed that increases in terpenoid and acetate ester volatiles were strongly associated with higher appreciation score, while proacacipetalin, a cyanogenic-glycoside, was positively associated to bitter taste. Our results pave the way for the selection of high-quality cultivars and suitable production sites for blackcurrant cultivation.

Liquid chromatography-mass spectrometry calibration transfer and metabolomics data fusion.

Metabolic profiling is routinely performed on multiple analytical platforms to increase the coverage of detected metabolites, and it is often necessary to distribute biological and clinical samples from a study between instruments of the same type to share the workload between different laboratories. The ability to combine metabolomics data arising from different sources is therefore of great interest, particularly for large-scale or long-term studies, where samples must be analyzed in separate blocks. This is not a trivial task, however, due to differing data structures, temporal variability, and instrumental drift. In this study, we employed blood serum and plasma samples collected from 29 subjects diagnosed with small cell lung cancer and analyzed each sample on two liquid chromatography-mass spectrometry (LC-MS) platforms. We describe a method for mapping retention times and matching metabolite features between platforms and approaches for fusing data acquired from both instruments. Calibration transfer models were developed and shown to be successful at mapping the response of one LC-MS instrument to another (Procrustes dissimilarity = 0.04; Mantel correlation = 0.95), allowing us to merge the data from different samples analyzed on different instruments. Data fusion was assessed in a clinical context by comparing the correlation of each metabolite with subject survival time in both the original and fused data sets: a simple autoscaling procedure (Pearson's R = 0.99) was found to improve upon a calibration transfer method based on partial least-squares regression (R = 0.94).