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Fitting the data of an electrochemical impedance spectroscopy (EIS) typically requires manual estimation of the initial values before regression algorithms such as complex nonlinear least squares (CNLS) can be applied. This makes the success rate of the fitting dependent on the user input. Furthermore, the Randles circuit consists of parameters with substantially differing magnitudes (e.g. capacitors and resistors), which can also strongly affect the success rate of the fitting due to numerical effects. The aim of this work is to investigate methods addressing the described limitations of fitting. Therefore, a Python implementation performing a fit for EIS is benchmarked with an equivalent open source library. The examined implementation optionally includes the normalization of the parameter values, the standardization of the impedances and a pre-fit. Applying the same equivalent circuit without additional signal processing steps and with fixed initial values defined by midpoints of the value ranges, both implementations were able to fit 46.50% of the simulated database with different spectra. Applying the normalization of the parameter values (76.25%) or the same method with additional pre-fit (97.75%) lead to a significant improvement of the success rate. The standardization of impedances did not affect the success rate.

Most food crops are susceptible to necrotrophic bacteria that cause rotting and wilting diseases in fleshy organs and foods. All varieties of cultivated potato ( Solanum tuberosum L.) are susceptible to diseases caused by Pectobacterium species, but resistance has been demonstrated in wild potato relatives including S. chacoense . Previous studies demonstrated that resistance is in part mediated by antivirulence activity of phytochemicals in stems and tubers. Little is known about the genetic basis of antivirulence traits, and the potential for inheritance and introgression into cultivated potato is unclear. Here, the metabolites and genetic loci associated with antivirulence traits in S. chacoense were elucidated by screening a sequenced S. tuberosum x S. chacoense recombinant inbred line (RIL) population for antivirulence traits of its metabolite extracts. Metabolite extracts from the RILs exhibited a quantitative distribution for two antivirulence traits that were positively correlated: quorum sensing inhibition and exo-protease inhibition, with some evidence of transgressive segregation, supporting the role of multiple loci and metabolites regulating these resistance-associated systems. Metabolomics was performed on the highly resistant and susceptible RILs that revealed 30 metabolites associated with resistance, including several alkaloids and terpenes. Specifically, several prenylated metabolites were more abundant in resistant RILs. We constructed a high-density linkage map with 795 SNPs mapped to 12 linkage groups, spanning a length of 1,507 cM and a density of 1 marker per 1.89 cM. Genetic mapping of the antivirulence and metabolite data identified five quantitative trait loci (QTLs) related to quorum sensing inhibition that explained 8-28% of the phenotypic variation and two QTLs for protease activity inhibition that explained 14-19% of the phenotypic variation. Several candidate genes including alkaloid, and secondary metabolite biosynthesis that are related to disease resistance were identified within these QTLs. Taken together, these data support that quorum sensing inhibition and exo-protease inhibition assays may serve as breeding targets to improve resistance to nectrotrophic bacterial pathogens in potato and other plants. The identified candidate genes and metabolites can be utilized in marker assisted selection and genomic selection to improve soft- rot and blackleg disease resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Joshi, Paudel, Eddy, Charkowski and Heuberger.)

The reuse of oilfield produced water (PW) for agricultural irrigation has received increased attention for utility in drought-stricken regions. It was recently demonstrated that PW irrigation can affect physiological processes in food crops. However, metabolomic evaluations are important to further discern specific mechanisms of how PW may contribute as a plant-environmental stressor. Herein, the primary metabolic responses of wheat irrigated with PW and matching salinity controls were investigated. Non-targeted gas chromatography mass spectrometry (GC-MS) metabolomics was combined with multivariate analysis and revealed that PW irrigation altered the primary metabolic profiles of both wheat leaf and grain. Over 600 compounds (183 annotated metabolites) were detected that varied between controls (salinity control and tap water) and PW irrigated plants. While some of these changed metabolites are related to salinity stress, over half were found to be unique to PW. The primary metabolites exhibiting changes in abundance in leaf and grain tissues were amines/amino acids, organic acids, and saccharides. Metabolite pathway analysis revealed that amino acid metabolism, sugar metabolism, and nitrogen remobilization are all impacted by PW irrigation, independent of regular plant responses to salinity stress. These data, when combined with prior physiological studies, support a multi-faceted, physio-metabolic response of wheat to the unique stressor imposed by irrigation with PW., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Potato is a major staple crop, and necrotrophic bacterial pathogens such as Pectobacterium spp. are a major threat to global food security. Most lines of cultivated potato ( Solanum tuberosum ) are susceptible to Pectobacterium spp., but some lines of wild potato are resistant, including Solanum chacoense M6. Despite the discovery of resistance in wild potatoes, specific resistance genes are yet to be discovered. Crude protein extract from M6 had a global effect on Pectobacterium brasiliense Pb1692 (Pb1692) virulence phenotypes. Specifically, M6 protein extracts resulted in reduced Pectobacterium exo-protease activity and motility, induced cell elongation, and affected bacterial virulence and metabolic gene expression. These effects were not observed from protein extracts of susceptible potato S. tuberosum DM1. A proteomics approach identified protease inhibitors (PIs) as candidates for S. chacoense resistance, and genomic analysis showed higher abundance and diversity of PIs in M6 than in DM1. We cloned five PIs that are unique or had high abundance in M6 compared with DM1 and purified the proteins (g18987, g28531, g39249, g40384, g6571). Four of the PIs significantly reduced bacterial protease activity, with strongest effects from g28531 and g6571. Three PIs (g18987, g28531, g6571) inhibited disease when co-inoculated with Pectobacterium pathogens into potato tubers. Two PIs (g28531, g6571) also significantly reduced Pb1692 motility and are promising as resistance genes. These results show that S. chacoense PIs contribute to bacterial disease resistance by inhibiting exo-proteases, motility, and tuber maceration and by modulating cell morphology and metabolism. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Potatoes are the most consumed vegetable worldwide and play an important role in the U.S. economy. Growers make critical decisions each year in choosing which cultivar to grow, based on factors such as yield, resilience to the growing environment, and utility in the food industry. Current research supports the finding that less-common specialty cultivars (SCs) have benefits for human health. However, growers have been slow to adopt SCs into mainstream operations. Here, we identify major factors in the decision-making process that determine whether a population of growers in the San Luis Valley, Colorado, a major potato-growing region, adopt SC potatoes. We used a combination of ethnographic techniques and quantitative methods to examine drivers of adoption. The data demonstrate grower perceptions within potato farming and the complexity of interacting factors in decision-making. An integration of the Theory of Planned Behavior, Rational Expectation Hypothesis, and Diffusion of Innovation models identifies economic and social factors that influence grower decision-making. Growers that were more aware of specialty cultivar innovation and associated consumer demand were more open to SCs adoption. Other influencing factors include a grower's experience selling a SC in the previous year and access to diverse markets. Based on these data, we developed a new model to explain grower decision-making processes in adopting SCs. The model demonstrates that one current barrier to adoption is access to buyers, including warehouses, retailers, and households. Taken together, this research demonstrates how rational expectations stem from economic outcomes, knowledge, and experience in the potato industry. These results are important in helping to consider opportunities for growers to access new, higher value markets, while also improving consumer access to nutritious cultivars., Competing Interests: The authors have declared that no competing interests exist.

Potato ( Solanum tuberosum L.) is the primary vegetable crop consumed worldwide and is largely affected by bacterial pathogens that can cause soft rot and blackleg disease. Recently, resistance to these diseases has been identified in the wild potato S. chacoense , and the mechanism of resistance is unknown. Here, it was hypothesized that S. chacoense stems or tubers have unique chemistry that confers resistance to the pathogen Pectobacterium brasiliense through bactericidal, bacteriostatic, or antivirulence activity. Stem and tuber metabolite extracts were collected from S. chacoense and tested for effects on Pectobacterium bacterial multiplication rates, and activity and expression of known exoenzymes and virulence genes using S. tuberosum extracts as a comparative control. Comparatively, the S. chacoense extracts did not affect bacterial multiplication rate; however, they did reduce pectinase, cellulase, and protease activities. The chemical extracts were profiled using a bioassay-guided fractionation, and a nontargeted metabolomics comparison of S. chacoense and S. tuberosum stems and tubers was performed. The data showed that selected alkaloids, phenolic amines, phenols, amines, and peptides are integrative chemical sources of resistance against the bacteria.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Background: Microgreens are the young leafy greens of many vegetables, herbs, grains, and flowers with potential to promote human health and sustainably diversify the global food system. For successful further integration into the global food system and evaluation of their health impacts, it is critical to elucidate and optimize their nutritional quality., Objectives: We aimed to comprehensively evaluate the metabolite and mineral contents of 6 microgreen species, and the influence of maturity on their contents., Methods: Plant species evaluated were from the Brassicaceae (arugula, broccoli, and red cabbage), Amaranthaceae (red beet and red amaranth), and Fabaceae (pea) plant families. Nontargeted metabolomics and ionomics analyses were performed to examine the metabolites and minerals, respectively, in each microgreen species and its mature counterpart., Results: Nontargeted metabolomics analysis detected 3321 compounds, 1263 of which were annotated and included nutrients and bioactive compounds. Ionomics analysis detected and quantified 26 minerals including macrominerals, trace minerals, ultratrace minerals, and other metals. Principal component analysis indicated that microgreens have distinct metabolite and mineral profiles compared with one another and with their mature counterparts. Several compounds were higher ( P   < 0.05; fold change ≥2) in microgreens compared with their mature counterparts, whereas some were not different or lower. In many cases, compounds that were higher in microgreens compared with the mature counterpart were also unique to that microgreen species., Conclusions: These data provide evidence for the nutritional quality of microgreens, and can inform future research and development aimed at characterizing and optimizing microgreen nutritional quality and health impacts., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Produced water (PW), a wastewater resulting from hydraulic fracturing and oil and gas production, has been utilized in arid regions for irrigation purposes and potentially presents a new water source for crop irrigation in areas of increasing water scarcity. However, there is a potential for both synthetic and geogenic contaminants in these waters to accumulate in irrigated food crops. This study assessed how water treatment technologies targeted at removal of salinity (i.e., total dissolved solids) and organic chemical content (i.e., dissolved organic carbon) from PW to achieve agricultural irrigation standards altered the impact of inorganic contaminants and nutrient uptake on two salt-tolerant food crops, sunflower (Helianthus annuus) and wheat (Triticum aestivum). The impacts of the treatment technologies on inorganic contaminant loadings in the irrigated soils were also assessed. Treatment technologies to improve PW quality decreased the adverse impacts on plant health; however, plant health was more affected by dilutions of PW than by the treatment technologies employed. Phenotypically, plants irrigated with 90% dilution (low) treatment groups, regardless of treatment technology, were comparable to controls; however, plants watered with high proportions (50%) of raw or treated PW displayed stunted growth, with reduced height and leaf area, and sunflower seed saw 100% yield loss. Although phenotypically similar, plants of the low treatment groups exhibited changes in the ionome, illustrating the influence of PW on plant uptake, translocation, and accumulation of metals, salts, and micronutrients. In addition, bioavailability of metals and nutrients was impacted by the unique and complex PW matrix: bioconcentration factors traditionally used to evaluate risk may therefore over or underestimate accumulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chocolate made from small-batch production is known for distinct sensory properties that differentiate its products from large-scale production. Specifically, small-batch processing includes a melanging step, a chocolate refining (a process involving time and temperature to refine texture and flavor) process that occurs in a stone wet-grinder. Chocolatiers understand that melanging is essential to flavor and overall quality, however the influence of melanging on the aroma chemistry of the finished chocolate is anecdotal and largely uncharacterized. Here, we evaluated the effects of time and temperature of melanging on the volatile chemistry of the finished chocolate. Specifically, chocolate aroma was profiled using HS/SPME-GC-MS for three different time and temperature combinations. A total of 88 compounds were annotated by mass spectrometry and included a diverse set of chemical classes such as pyrazines, aldehydes, terpenes, alcohols, esters, and ketones. Analysis of variance (ANOVA), principal component analysis (PCA), and partial least squares analysis (PLS) revealed that the overall aroma profile was influenced by the type of melanging, and time had a greater effect than temperature. Example compounds affected by time include 2-methylpropanal, dimethyl sulfide, and benzaldehyde. Particle size was also measured for each sample. Majority particle size was found to be below 25 microns generally at all time points beyond 8 h. Analysis showed significant p-values for the temperature variable for several compounds, but significant p-values for the time variable were apparent for a greater number of compounds. For compounds which showed dependency on both time and temperature, the p-value for the time variable was much smaller in most cases. Both PCA and OPLS analyses suggested the same trends. These data support that time is a critical factor in determining the aroma chemistry of chocolate and affects a diverse set of known flavor active compounds.

Barth syndrome is a mitochondrial myopathy resulting from mutations in the tafazzin ( TAZ ) gene encoding a phospholipid transacylase required for cardiolipin remodeling. Cardiolipin is a phospholipid of the inner mitochondrial membrane essential for the function of numerous mitochondrial proteins and processes. However, it is unclear how tafazzin deficiency impacts cardiac mitochondrial metabolism. To address this question while avoiding confounding effects of cardiomyopathy on mitochondrial phenotype, we utilized Taz -shRNA knockdown ( Taz KD ) mice, which exhibit defective cardiolipin remodeling and respiratory supercomplex instability characteristic of human Barth syndrome but normal cardiac function into adulthood. Consistent with previous reports from other models, mitochondrial H 2 O 2 emission and oxidative damage were greater in Taz KD than in wild-type (WT) hearts, but there were no differences in oxidative phosphorylation coupling efficiency or membrane potential. Fatty acid and pyruvate oxidation capacities were 40-60% lower in Taz KD mitochondria, but an up-regulation of glutamate oxidation supported respiration rates approximating those with pyruvate and palmitoylcarnitine in WT. Deficiencies in mitochondrial CoA and shifts in the cardiac acyl-CoA profile paralleled changes in fatty acid oxidation enzymes and acyl-CoA thioesterases, suggesting limitations of CoA availability or "trapping" in Taz KD mitochondrial metabolism. Incubation of Taz KD mitochondria with exogenous CoA partially rescued pyruvate and palmitoylcarnitine oxidation capacities, implicating dysregulation of CoA-dependent intermediary metabolism rather than respiratory chain defects in the bioenergetic impacts of tafazzin deficiency. These findings support links among cardiolipin abnormalities, respiratory supercomplex instability, and mitochondrial oxidant production and shed new light on the distinct metabolic consequences of tafazzin deficiency in the mammalian heart., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Le et al.)

Microgreens are an emerging functional food crop with promise for sustainably diversifying global food systems, facilitating adaptations to urbanization and global climate change, and promoting human health. Previous work suggests microgreens have high nutritional quality, low environmental impacts, and broad consumer acceptance. For better reception into the global food system and increased per capita consumption, research is needed to elucidate consumer acceptance of various microgreens species, including factors contributing to their acceptance or lack thereof. Using a consumer panel (n = 99), this study evaluated consumer sensory perception and acceptability of six microgreens species (arugula, broccoli, bull's blood beet, red cabbage, red garnet amaranth, and tendril pea), and potential drivers and barriers to consumer acceptance. All microgreens species received high mean liking scores for acceptability by consumers (means ranged from highly acceptable to slightly acceptable), with more distinct differences across microgreens species for flavor and overall acceptability, which appeared to be driven by specific sensory properties. Data from principal component analysis demonstrated that high acceptability scores were associated with higher intent to purchase microgreens and negatively associated with food neophobia. Participants indicated that factors such as knowledge and familiarity of microgreens, cost, access/availability, freshness/shelf life, among other factors, influence their intention to purchase microgreens. These findings suggest that further integration of microgreens into the global food system will be met with high consumer acceptability, but needs to be aligned with enhanced consumer education regarding microgreens, as well as considerations of cost, availability/access, and freshness/shelf life. PRACTICAL APPLICATION: Researchers investigated consumer sensory perception and acceptability of six microgreens species (arugula, broccoli, bull's blood beet, red cabbage, red garnet amaranth, and tendril pea), and potential drivers and barriers to consumer acceptance. All microgreens tested had high consumer acceptability, but certain factors such as sensory perception and food neophobia impacted their acceptability. Additionally, participants indicated that factors such as knowledge, access and availability, cost, freshness, and shelf life may impact the purchasing of microgreens and thus are important factors to consider for further integration of this emerging functional food crop into the global food system., (© 2020 Institute of Food Technologists®.)

Common wheat ( Triticum aestivum L.) is a global staple crop, and insect pests can impact grain yield. The wheat stem sawfly ( Cephus cinctus , WSS) is a major wheat pest, and while partial resistance has been deployed by breeding for a solid-stem trait, this trait is affected by environment. Here, a proteomics and metabolomics study was performed on four wheat cultivars to characterize a molecular response to WSS infestation. The cultivars Hatcher (hollow-stem partially tolerant), Conan (semisolid-stem-resistant), and Denali and Reeder (hollow-stem-susceptible) were infested with WSS, and changes in stem proteins and metabolites were characterized using liquid chromatography-mass spectrometry. The proteome was characterized as 1830 proteins that included five major biological processes, including metabolic processes and response to stimuli, and the metabolome (1823 metabolites) spanned eight chemical superclasses, including alkaloids, benzenoids, and lipids. All four varieties had a molecular response to WSS following infestation. Hatcher had the most distinct changes, whereby 62 proteins and 29 metabolites varied in metabolic pathways involving enzymatic detoxification, proteinase inhibition, and antiherbivory compound production via benzoxazinoids, neolignans, and phenolics. Taken together, these data demonstrate variation in the wheat stem molecular response to WSS infestation and support breeding for molecular resistance in hollow-stem cultivars.

The dietary fiber gap that is present in many countries co-exists with a low intake of grain legumes (pulses) that have 2-3 times more dietary fiber than cereal grains that are commonly recommended to increase fiber intake. Given the relationships among dietary fiber, gut health and chronic disease risk, a study was undertaken in a preclinical mouse model for obesity to examine how commonly consumed pulses, i.e., chickpea, common bean, dry pea and lentil, would impact gut microbes, intestinal function, and adiposity. Pulses were fed to C57BL/6 mice at similar levels of protein and fiber. Bacterial count in the cecum was elevated 3-fold by pulse consumption. At the phylum level, a 2.2- to 5-fold increase in Bacteriodetes relative to Firmicutes was observed. For Akkermansia muciniphila , a health-beneficial bacterium, differential effects were detected among pulses ranging from no effect to a 49-fold increase. Significant differences among pulses in biomarkers of intestinal function were not observed. Pulses reduced accumulation of lipid in adipose tissue with a greater reduction in the subcutaneous versus visceral depots. Metabolomics analysis indicated that 108 metabolites were highly different among pulse types, and several compounds are hypothesized to influence the microbiome. These results support recent recommendations to increase consumption of pulse-based foods for improved health, although all pulses were not equal in their effects., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Produced water (PW) from oil and gas operations is considered a potential resource for food crop irrigation because of increasing water scarcity in dryland agriculture. However, efforts to employ PW for agriculture have been met with limited success. A greenhouse study was performed to evaluate the effects of PW on physiological and morphological traits of spring wheat (Triticum aestivum). Plants were irrigated with water treatments containing 10 and 50% PW (PW10 and PW50, respectively) and compared to a matching 50% salinity (NaCl50) and 100% tap water controls. Compared to controls, plants watered with PW10 and PW50 exhibited developmental arrest and reductions in aboveground and belowground biomass, photosynthetic efficiency, and reproductive growth. Decreases in grain yield ranged from 70 to 100% in plants irrigated with PW compared to the tap water control. Importantly, the PW10 and NaCl50 treatments were comparable for morphophysiological effects, even though NaCl50 contained 5 times the total dissolved solids, suggesting that constituents other than NaCl in PW contributed to plant stress. These findings indicate that despite discharge and reuse requirements focused on total dissolved solids, salinity stress may not be the primary factor affecting crop health. The results of the present study are informative for developing guidelines for the use of PW in agriculture to ensure minimal effects on crop morphology and physiology. Environ Toxicol Chem 2019;38:1756-1769. © 2019 SETAC., (© 2019 SETAC.)

Ambient mass spectrometry is an analytical approach that enables ionization of molecules under open-air conditions with no sample preparation and very fast sampling times. Rapid evaporative ionization mass spectrometry (REIMS) is a relatively new type of ambient mass spectrometry that has demonstrated applications in both human health and food science. Here, we present an evaluation of REIMS as a tool to generate molecular scale information as an objective measure for the assessment of beef quality attributes. Eight different machine learning algorithms were compared to generate predictive models using REIMS data to classify beef quality attributes based on the United States Department of Agriculture (USDA) quality grade, production background, breed type and muscle tenderness. The results revealed that the optimal machine learning algorithm, as assessed by predictive accuracy, was different depending on the classification problem, suggesting that a "one size fits all" approach to developing predictive models from REIMS data is not appropriate. The highest performing models for each classification achieved prediction accuracies between 81.5-99%, indicating the potential of the approach to complement current methods for classifying quality attributes in beef.

Root exudation is an important plant process by which roots release small molecules into the rhizosphere that serve in overall plant functioning. Yet, there is a major gap in our knowledge in translating plant root exudation in artificial systems (i.e., hydroponics, sterile media) to crops, specifically for soils expected in field conditions. Sorghum ( Sorghum bicolor L. Moench) root exudation was determined using both ultra-performance liquid chromatography and gas chromatography mass spectrometry-based non-targeted metabolomics to evaluate variation in exudate composition of two sorghum genotypes among three substrates (sand, clay, and soil). Above and belowground plant traits were measured to determine the interaction between sorghum genotype and belowground substrate. Plant growth and quantitative exudate composition were found to vary largely by substrate. Two types of changes to rhizosphere metabolites were observed: rhizosphere-enhanced metabolites (REMs) and rhizosphere-abated metabolites (RAMs). More REMs and RAMs were detected in sand and clay substrates compared to the soil substrate. This study demonstrates that belowground substrate influences the root exudate profile in sorghum, and that two sorghum genotypes exuded metabolites at different magnitudes. However, metabolite identification remains a major bottleneck in non-targeted metabolite profiling of the rhizosphere., Competing Interests: The authors declare no conflict of interest.

The aim of this study was to compare fecal microbiome, plasma, fecal and urine metabolomes, and serum biochemistry of adult companion dogs according to body condition scores. Blood, serum/plasma, urine, and fecal samples were collected from 66 clinically healthy, adult companion dogs of either normal weight (NW), overweight (OW), or obese dogs (OB). analyses included fecal microbiome analyses via 16S ribosomal RNA gene amplicon; sequencing, nontargeted plasma, fecal, and urine metabolomics using liquid chromatography/gas chromatography-mass; spectrometry, and serum biochemistry for each dog. Few significant differences in serum biochemistry and fecal microbiome Operational Taxonomic Unit (OTU) were found between weight groups and there was high OTU variation between individual dogs. NW dogs had higher relative abundance of the genus Eubacterium (log-fold change 4.3, adjusted P value = .003) and lower relative abundance of the family Bifidobacteriaceae (log-fold change -3.6, adjusted P value = .02) compared to OB dogs. The microbiome of NW dogs had higher OTU richness compared with OB dogs. Metabolome analysis showed 185 plasma, 37 fecal, and 45 urine metabolites that significantly differed between NW and OW or OB dogs. There were notable significant differences in relative abundance of several plasma phospholipid moieties and fecal volatile fatty acids between weight phenotypes. The combinations of host and gut microbiota and metabolic shifts suggest a pattern that could help detection of early metabolic changes in overweight dogs before the development of obesity related disease. The results of this study support the need for continued investigation into sensitive measures of metabolic aberrancies in overweight dogs., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Beverage quality in the brewing industry is heavily influenced by ingredient properties. The contribution of raw ingredients such as yeast and hops to beer flavor is well understood. However, the influence of barley genotype and/or environment on flavor (the malt 'source') is largely unexplored. Here, a study was performed to determine (i) if there are metabolite differences among six commercial malt sources, (ii) if differences in malt chemistry are reflected in the chemistry of the beer, and (iii) if the differences in the beer chemistry impact sensory attributes of beer, through flavor and flavor stability. Six distinct sources of malts (six varieties from three maltsters) were brewed into six beers using a recipe designed to evaluate differences in flavor. Metabolomics and ionomics was used to characterize chemical variation among the six malts and beers using UHPLC- and HILIC-MS (non-volatile metabolites), HS-SPME/GC-MS (beer volatiles), and ICP-MS (malt metals). These analyses detected a total of 5042 compounds in malt, of which 217 were annotated and included amines, amino acids, fatty acids/lipids/fatty acyls, saccharides/glucosides/sugar acids/sugar alcohols, carboxylic acid derivatives, organic acids, phenolics/benzenoids, purines, pyrimidines/pyridines, terpenes, and organosulfurs. A total of 4568 compounds were detected in beer, of which 246 were annotated and included esters, aldehydes, and alcohols. Statistical analysis revealed chemical variation among the six malts (50/217 malt metabolites varied) and beers (150/246). The six beers were evaluated for flavor using a modified descriptive analysis for 45 sensory traits at 0, 4, and 8 weeks of storage at 4 °C. Principal component analysis of the sensory data revealed flavor differences among the six beers at 8 weeks, and the malt-type Full Pint was described as fruity and Meredith as corn chip. The metabolite and sensory data were integrated and revealed associations between flavor profiles in beer and the annotated malt and beer. The fruity or corn chip flavor profiles in beer were associated beer purines/pyrimidines, volatile ketones, amines, and phenolics, and malt lipids, saccharides, phenols, amines, and alkaloids. Taken together, these data support a role of malt source in beer flavor and flavor stability. As a raw ingredient, malting barley genotypes can be evaluated for a contribution to flavor, and this may be a future target for plant breeding, agronomy, and malting efforts to selectively improve flavor, flavor stability, and quality in beer., (Copyright © 2018. Published by Elsevier Ltd.)

Maternal body composition can be an important determinant for development of obesity and metabolic syndrome in adult offspring. Obesity-related outcomes in offspring may include epigenetic alterations; however, mechanisms of fetal programming remain to be fully elucidated. This study was conducted to determine the impact of maternal obesity in the absence of a high fat diet on equine endometrium and preimplantation embryos. Embryos were collected from normal and obese mares at 8 and 16 days and a uterine biopsy at 16 days (0 day = ovulation). With the exception of 8 day embryos, each sample was divided into two pieces. One piece was analyzed for gene expression markers related to carbohydrate metabolism, lipid homeostasis, inflammation, endoplasmic reticulum stress, oxidative stress, mitochondrial stress, and components of the insulin-like growth factor (IGF) system. The second piece was analyzed for lipid content using matrix-assisted laser desorption/ionization mass spectrometry. Obese mares had elevated concentrations of insulin, leptin, and total cholesterol, and they tended to have increased triglycerides and decreased insulin sensitivity. Embryos from obese mares had altered transcript abundance in genes for inflammation and lipid homeostasis, as well as endoplasmic reticulum, oxidative and mitochondrial stress and altered lipid fingerprints. Endometrium from obese mares had increased expression of inflammatory cytokines, lipid homeostasis regulation, mitochondrial stress, and the IGF2 system. This study demonstrates that increased adiposity in mares alters the uterine environment, transcript abundance of genes for cellular functions, and lipid profiles of embryos. These alterations could affect prenatal programming, with potential long-term effects in offspring.

Plant physiology and metabolism are important components of a plant response to microbial pathogens. Physiological resistance of common bean (Phaseolus vulgaris L.) to the fungal pathogen Sclerotinia sclerotiorum has been established, but the mechanisms of resistance are largely unknown. Here, the physiological and metabolic responses of bean varieties that differ in physiological resistance to S. sclerotiorum are investigated. Upon infection, the resistant bean variety A195 had a unique physiological response that included reduced photosynthesis and maintaining a higher leaf surface pH during infection. Leaf metabolomics was performed on healthy tissue adjacent to the necrotic lesion at 16, 24, and 48 hr post inoculation, and 144 metabolites were detected that varied between A195 and Sacramento following infection. The metabolites that varied in leaves included amines/amino acids, organic acids, phytoalexins, and ureides. The metabolic pathways associated with resistance included amine metabolism, uriede-based nitrogen remobilization, antioxidant production, and bean-specific phytoalexin production. A second experiment was conducted in stems of 13 bean genotypes with varying resistance. Stem resistance was associated with phytoalexin production, but unlike leaf metabolism, lipid changes were associated with susceptibility. Taken together, the data supports a multifaceted, physiometabolic response of common bean to S. sclerotiorum that mediates resistance., (© 2018 John Wiley & Sons Ltd.)

Potato ( Solanum tuberosum L.) is an important global food crop that contains phytochemicals with demonstrated effects on human health. Understanding sources of chemical variation of potato tuber can inform breeding for improved health attributes of the cooked food. Here, a comprehensive metabolomics (UPLC- and GC-MS) and ionomics (ICP-MS) analysis of raw and cooked potato tuber was performed on 60 unique potato genotypes that span 5 market classes including russet, red, yellow, chip, and specialty potatoes. The analyses detected 2,656 compounds that included known bioactives (43 compounds), nutrients (42), lipids (76), and 23 metals. Most nutrients and bioactives were partially degraded during cooking (44 out of 85; 52%), however genotypes with high quantities of bioactives remained highest in the cooked tuber. Chemical variation was influenced by genotype and market class. Specifically, ~53% of all detected compounds from cooked potato varied among market class and 40% varied by genotype. The most notable metabolite profiles were observed in yellow-flesh potato which had higher levels of carotenoids and specialty potatoes which had the higher levels of chlorogenic acid as compared to the other market classes. Variation in several molecules with known association to health was observed among market classes and included vitamins (e.g., pyridoxal, ~2-fold variation), bioactives (e.g., chlorogenic acid, ~40-fold variation), medicinals (e.g., kukoamines, ~6-fold variation), and minerals (e.g., calcium, iron, molybdenum, ~2-fold variation). Furthermore, more metabolite variation was observed within market class than among market class (e.g., α-tocopherol, ~1-fold variation among market class vs. ~3-fold variation within market class). Taken together, the analysis characterized significant metabolite and mineral variation in raw and cooked potato tuber, and support the potential to breed new cultivars for improved health traits.

Matrix-assisted desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) is a fast and effective tool for microbial species identification. However, current approaches are limited to species-level identification even when genetic differences are known. Here, we present a novel workflow that applies the statistical method of partial least squares discriminant analysis (PLS-DA) to MALDI-TOF-MS protein fingerprint data of Xanthomonas axonopodis, an important bacterial plant pathogen of fruit and vegetable crops. Mass spectra of 32 X. axonopodis strains were used to create a mass spectral library and PLS-DA was employed to model the closely related strains. A robust workflow was designed to optimize the PLS-DA model by assessing the model performance over a range of signal-to-noise ratios (s/n) and mass filter (MF) thresholds. The optimized parameters were observed to be s/n = 3 and MF = 0.7. The model correctly classified 83% of spectra withheld from the model as a test set. A new decision rule was developed, termed the rolled-up Maximum Decision Rule (ruMDR), and this method improved identification rates to 92%. These results demonstrate that MALDI-TOF-MS protein fingerprints of bacterial isolates can be utilized to enable identification at the strain level. Furthermore, the open-source framework of this workflow allows for broad implementation across various instrument platforms as well as integration with alternative modeling and classification algorithms. Graphical abstract ᅟ.

Wheat ( Triticum aestivum L.) is an important food crop, and biotic and abiotic stresses significantly impact grain yield. Wheat leaf and stem surface waxes are associated with traits of biological importance, including stress resistance. Past studies have characterized the composition of wheat cuticular waxes, however protocols can be relatively low-throughput and narrow in the range of metabolites detected. Here, gas chromatography-mass spectrometry (GC-MS) metabolomics methods were utilized to provide a comprehensive characterization of the chemical composition of cuticular waxes in wheat leaves and stems. Further, waxes from four wheat cultivars were assayed to evaluate the potential for GC-MS metabolomics to describe wax composition attributed to differences in wheat genotype. A total of 263 putative compounds were detected and included 58 wax compounds that can be classified (e.g., alkanes and fatty acids). Many of the detected wax metabolites have known associations to important biological functions. Principal component analysis and ANOVA were used to evaluate metabolite distribution, which was attributed to both tissue type (leaf, stem) and cultivar differences. Leaves contained more primary alcohols than stems such as 6-methylheptacosan-1-ol and octacosan-1-ol. The metabolite data were validated using scanning electron microscopy of epicuticular wax crystals which detected wax tubules and platelets. Conan was the only cultivar to display alcohol-associated platelet-shaped crystals on its abaxial leaf surface. Taken together, application of GC-MS metabolomics enabled the characterization of cuticular wax content in wheat tissues and provided relative quantitative comparisons among sample types, thus contributing to the understanding of wax composition associated with important phenotypic traits in a major crop., Competing Interests: The authors declare no conflict of interest.

A study was performed to identify metabolic processes associated with β-ODAP synthesis in grass pea using a metabolomics approach. GC-MS metabolomics was performed on seedlings at 2, 6, and 25 days after sowing. A total of 141 metabolites were detected among the three time points representing much of grass pea primary metabolism, including amino acids, carbohydrates, purines, and others. Principal component analysis revealed unique metabolite profiles of grass pea tissues among the three time points. Fold change, hierarchical clustering, and orthogonal projections to latent structures-discriminant analyses, and biochemical pathway ontologies were used to characterize covariance of metabolites with β-ODAP content. The data indicates that alanine and nitrogen metabolism, cysteine and sulfur metabolism, and purine, pyrimidine, and pyridine metabolism were associated with β-ODAP metabolism. Our results reveal the metabolite profiles in grass pea development and provide insights into mechanisms of β-ODAP accumulation and degradation.

Introduction: Catha edulis (qat, khat, mirra) is a woody plant species that is grown and consumed in East Africa and Yemen for its stimulant alkaloids cathinone, cathine and norephedrine. Two Celastraceae species, in addition to qat, have been noted for their stimulant properties in ethnobotanical literature. Recent phylogenetic reconstructions place four genera in a clade sister to Catha edulis, and these genera are primary candidates to search for cathine and related alkaloids., Objective: Determine if cathine or related alkaloids are present in species of Celastraceae other than Catha edulis., Methods: Leaf samples from 43 Celastraceae species were extracted in water followed by basification of the aqueous extract and partitioning with methyl-t-butyl ether to provide an alkaloid-enriched fraction. The extract was derivatised in a two-stage process and analysed using GC-MS for the presence of cathine. Related alkaloids and other metabolites in this alkaloid-enriched fraction were tentatively identified., Results: Cathinone, cathine and norephedrine were not detected in any of the 43 Celastraceae species assayed other than Catha edulis. However, the phenylalanine- or tyrosine-derived alkaloid phenylethylamine was identified in five species. Nine species were found to be enriched for numerous sterol- and terpene-like compounds., Conclusion: These results indicate that cathine is unique to Catha edulis, and not the compound responsible for the stimulant properties reported in related Celastraceae species. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Phloroglucinol (1,3,5-trihydroxybenzene; PG) and its derivatives are phenolic compounds that are used for various industrial applications. Current methods to synthesize PG are not sustainable due to the requirement for carbon-based precursors and co-production of toxic byproducts. Here, we describe a more sustainable production of PG using plants expressing a native bacterial or a codon-optimized synthetic PhlD targeted to either the cytosol or chloroplasts. Transgenic lines were analyzed for the production of PG using gas and liquid chromatography coupled to mass spectroscopy. Phloroglucinol was produced in all transgenic lines and the line with the highest PhlD transcript level showed the most accumulation of PG. Over 80% of the produced PG was glycosylated to phlorin. Arabidopsis leaves have the machinery to glycosylate PG to form phlorin, which can be hydrolyzed enzymatically to produce PG. Furthermore, the metabolic profile of plants with PhlD in either the cytosol or chloroplasts was altered. Our results provide evidence that plants can be engineered to produce PG using a bacterial gene. Phytoproduction of PG using a bacterial gene paves the way for further genetic manipulations to enhance the level of PG with implications for the commercial production of this important platform chemical in plants.

Metabolomics is an emerging method to improve our understanding of how genetic diversity affects phenotypic variation in plants. Recent studies have demonstrated that genotype has a major influence on biochemical variation in several types of plant tissues, however, the association between metabolic variation and variation in morphological and physiological traits is largely unknown. Sorghum bicolor (L.) is an important food and fuel crop with extensive genetic and phenotypic variation. Sorghum lines have been bred for differing phenotypes beneficial for production of grain (food), stem sugar (food, fuel), and cellulosic biomass (forage, fuel), and these varying phenotypes are the end products of innate metabolic programming which determines how carbon is allocated during plant growth and development. Further, sorghum has been adapted among highly diverse environments. Because of this geographic and phenotypic variation, the sorghum metabolome is expected to be highly divergent; however, metabolite variation in sorghum has not been characterized. Here, we utilize a phenotypically diverse panel of sorghum breeding lines to identify associations between leaf metabolites and morpho-physiological traits. The panel (11 lines) exhibited significant variation for 21 morpho-physiological traits, as well as broader trends in variation by sorghum type (grain vs. biomass types). Variation was also observed for cell wall constituents (glucan, xylan, lignin, ash). Non-targeted metabolomics analysis of leaf tissue showed that 956 of 1181 metabolites varied among the lines (81%, ANOVA, FDR adjusted p < 0.05). Both univariate and multivariate analyses determined relationships between metabolites and morpho-physiological traits, and 384 metabolites correlated with at least one trait (32%, p < 0.05), including many secondary metabolites such as glycosylated flavonoids and chlorogenic acids. The use of metabolomics to explain relationships between two or more morpho-physiological traits was explored and showed chlorogenic and shikimic acid to be associated with photosynthesis, early plant growth and final biomass measures in sorghum. Taken together, this study demonstrates the integration of metabolomics with morpho-physiological datasets to elucidate links between plant metabolism, growth, and architecture.

Flavour stability is vital to the brewing industry as beer is often stored for an extended time under variable conditions. Developing an accelerated model to evaluate brewing techniques that affect flavour stability is an important area of research. Here, we performed metabolomics on non-volatile compounds in beer stored at 37 °C between 1 and 14 days for two beer types: an amber ale and an India pale ale. The experiment determined high temperature to influence non-volatile metabolites, including the purine 5-methylthioadenosine (5-MTA). In a second experiment, three brewing techniques were evaluated for improved flavour stability: use of antioxidant crowns, chelation of pro-oxidants, and varying plant content in hops. Sensory analysis determined the hop method was associated with improved flavour stability, and this was consistent with reduced 5-MTA at both regular and high temperature storage. Future studies are warranted to understand the influence of 5-MTA on flavour and aging within different beer types., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Obesity is associated with disrupted reproductive cycles in mares, but the impact of obesity on follicles and oocytes has received minimal attention. We investigated the impact of obesity on 1) expression of selected genes in follicle cells for carbohydrate metabolism, inflammatory cytokines, lipid homeostasis, endoplasmic reticulum stress, and mitochondrial function; 2) follicular fluid content of metabolic hormones and metabolites; and 3) lipid fingerprint of oocytes. Mares (9-13 yr) were classified as control (n = 8, normal weight, body condition score [BCS] 5.1, 10.4% body fat) or obese (n = 9, BCS 7.9, 16.2% body fat). Gene expression from granulosa cells (GC) and cumulus cells (CC) was evaluated by RT-PCR. Serum and follicular fluid were evaluated for insulin, leptin, adiponectin, and metabolite profiling. Oocyte lipid fingerprints were acquired using matrix-assisted laser desorption/ionization mass spectrometry. Several genes for lipid homeostasis, endoplasmic reticulum stress, and mitochondrial function were different between groups in GC and CC. Obese had (P < 0.05) or tended to have (0.05 < P < 0.1) lower insulin sensitivity and higher insulin and leptin in serum and follicular fluid. Many metabolites differed between control and obese in serum and/or follicular fluid and correlated with BCS and/or insulin sensitivity. Oocytes from control had greater concentrations of lipids consistent with phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins, while lipids consistent with triglycerides tended to be higher in obese. These findings suggest that maternal obesity causes alterations in the follicle and oocyte; the extent to which these alterations impact the conceptus and offspring is still to be determined., (© 2016 by the Society for the Study of Reproduction, Inc.)

Dry edible bean ( Phaseolus vulgaris L.) is a globally relevant food crop. The bean genome was recently sequenced and annotated allowing for proteomics investigations aimed at characterization of leaf phenotypes important to agriculture. The objective of this study was to utilize a shotgun proteomics approach to characterize the leaf proteome and to identify protein abundance differences between two bean lines with known variation in their physiological resistance to biotic stresses. Overall, 640 proteins were confidently identified. Among these are proteins known to be involved in a variety of molecular functions including oxidoreductase activity, binding peroxidase activity, and hydrolase activity. Twenty nine proteins were found to significantly vary in abundance ( p -value < 0.05) between the two bean lines, including proteins associated with biotic stress. To our knowledge, this work represents the first large scale shotgun proteomic analysis of beans and our results lay the groundwork for future studies designed to investigate the molecular mechanisms involved in pathogen resistance.

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle., (© 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The advent of metabolomics technology and its application to small samples has allowed us to non-invasively monitor the metabolic activity of embryos in a complex culture environment. The aim of this study was to apply metabolomics technology to the analysis of individual embryos from several species during in vitro development to gain an insight into the metabolomics pathways used by embryos and their relationship with embryo quality. Alanine is produced by both in vivo- and in vitro-derived human, murine, bovine and porcine embryos. Glutamine is also produced by the embryos of these four species, but only those produced in vitro. Across species, blastocysts significantly consumed amino acids from the culture medium, whereas glucose was not significantly taken up. There are significant differences in the metabolic profile of in vivo- compared with in vitro-produced embryos at the blastocyst stage. For example, in vitro-produced murine embryos consume arginine, asparagine, glutamate and proline, whereas in vivo-produced embryos do not. Human embryos produce more alanine, glutamate and glutamine, and consume less pyruvate, at the blastocyst compared with cleavage stages. Glucose was consumed by human blastocysts, but not at a high enough level to reach significance. Consumption of tyrosine by cleavage stage human embryos is indicative of blastocyst development, although tyrosine consumption is not predictive of blastocyst quality. Similarly, although in vivo-produced murine blastocysts consumed less aspartate, lactate, taurine and tyrosine than those produced in vitro, consumption of these four amino acids by in vitro-derived embryos with high octamer-binding transcription factor 4 (Oct4) expression, indicative of high quality, did not differ from those with low Oct4 expression. Further application of metabolomic technologies to studies of the consumption and/or production of metabolites from individual embryos in a complete culture medium could transform our understanding of embryo physiology and improve our ability to produce developmentally competent embryos in vitro.

Heat-stabilized rice bran (SRB) has been shown to regulate blood lipids and glucose, modulate gut mucosal immunity and inhibit colorectal cancer in animal and human studies. However, SRB's effects on gut microbial composition and metabolism and the resulting implications for health remain largely unknown. A pilot, randomized-controlled trial was developed to investigate the effects of eating 30 g/day SRB on the stool microbiome and metabolome. Seven healthy participants consumed a study meal and snack daily for 28 days. The microbiome and metabolome were characterized using 454 pyrosequencing and gas chromatography-mass spectrometry (GC-MS) at baseline, two and four weeks post-intervention. Increases in eight operational taxonomic units (OTUs), including three from Bifidobacterium and Ruminococcus genera, were observed after two and four weeks of SRB consumption (p<0.01). Branched chain fatty acids, secondary bile acids and eleven other putative microbial metabolites were significantly elevated in the SRB group after four weeks. The largest metabolite change was a rice bran component, indole-2-carboxylic acid, which showed a mean 12% increase with SRB consumption. These data support the feasibility of dietary SRB intervention in adults and support that SRB consumption can affect gut microbial metabolism. These findings warrant future investigations of larger cohorts evaluating SRB's effects on intestinal health.

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes., (© 2014 American Society of Plant Biologists. All rights reserved.)

Metabolic processes in plants are key components of physiological and biochemical disease resistance. Metabolomics, the analysis of a broad range of small molecule compounds in a biological system, has been used to provide a systems-wide overview of plant metabolism associated with defense responses. Plant immunity has been examined using multiple metabolomics workflows that vary in methods of detection, annotation, and interpretation, and the choice of workflow can significantly impact the conclusions inferred from a metabolomics investigation. The broad range of metabolites involved in plant defense often requires multiple chemical detection platforms and implementation of a non-targeted approach. A review of the current literature reveals a wide range of workflows that are currently used in plant metabolomics, and new methods for analyzing and reporting mass spectrometry (MS) data can improve the ability to translate investigative findings among different plant-pathogen systems.

The process of breeding superior varieties for the agricultural industry is lengthy and expensive. Plant metabolites may act as markers of quality traits, potentially expediting the appraisal of experimental lines during breeding. Here, we evaluated the utility of metabolites as markers by assessing metabolic variation influenced by genetic and environmental factors in an advanced breeding setting and in relation to the phenotypic distribution of 20 quality traits. Nontargeted liquid chromatography-mass spectrometry metabolite profiling was performed on barley (Hordeum vulgare L.) grain and malt from 72 advanced malting barley lines grown at two distinct but climatically similar locations, with 2-row and 6-row barley as the main genetic factors. 27 420 molecular features were detected, and the metabolite and quality trait profiles were similarly influenced by genotype and environment; however, malt was more influenced by genotype compared with barley. An O2PLS model characterized molecular features and quality traits that covaried, and 1319 features associated with at least one of 20 quality traits. An indiscriminant MS/MS acquisition and novel data analysis method facilitated the identification of metabolites. The analysis described 216 primary and secondary metabolites that correlated with multiple quality traits and included amines, amino acids, alkaloids, polyphenolics and lipids. The mechanisms governing quality trait-metabolite associations were interpreted based on colocalization to genetic markers and their gene annotations. The results of this study support the hypothesis that metabolism and quality traits are co-influenced by relatively narrow genetic and environmental factors and illustrate the utility of grain metabolites as functional markers of quality traits., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Gas chromatography/mass spectrometry (GC/MS) is a primary tool used to identify compounds in complex samples. Both mass spectra and GC retention times are matched to those of standards; however, it is often impractical to have standards on hand for every compound of interest, so we must rely on shared databases of MS data and GC retention information. Unfortunately, retention databases (e.g., linear retention index libraries) are experimentally restrictive, notoriously unreliable, and strongly instrument dependent, relegating GC retention information to a minor, often negligible role in compound identification despite its potential power. A new methodology called "retention projection" has great potential to overcome the limitations of shared chromatographic databases. In this work, we tested the reliability of the methodology in five independent laboratories. We found that, even when each lab ran nominally the same method, the methodology was 3-fold more accurate than retention indexing because it properly accounted for unintentional differences between the GC/MS systems. When the laboratories used different methods of their own choosing, retention projections were 4- to 165-fold more accurate. More importantly, the distribution of error in the retention projections was predictable across different methods and laboratories, thus enabling automatic calculation of retention time tolerance windows. Tolerance windows at 99% confidence were generally narrower than those widely used even when physical standards are on hand to measure their retention. With its high accuracy and reliability, the new retention projection methodology makes GC retention a reliable, precise tool for compound identification, even when standards are not available to the user.

1,2,4-butanetriol (butanetriol) is a useful precursor for the synthesis of the energetic material butanetriol trinitrate and several pharmaceutical compounds. Bacterial synthesis of butanetriol from xylose or arabinose takes place in a pathway that requires four enzymes. To produce butanetriol in plants by expressing bacterial enzymes, we cloned native bacterial or codon optimized synthetic genes under different promoters into a binary vector and stably transformed Arabidopsis plants. Transgenic lines expressing introduced genes were analyzed for the production of butanetriol using gas chromatography coupled to mass spectrometry (GC-MS). Soil-grown transgenic plants expressing these genes produced up to 20 µg/g of butanetriol. To test if an exogenous supply of pentose sugar precursors would enhance the butanetriol level, transgenic plants were grown in a medium supplemented with either xylose or arabinose and the amount of butanetriol was quantified. Plants expressing synthetic genes in the arabinose pathway showed up to a forty-fold increase in butanetriol levels after arabinose was added to the medium. Transgenic plants expressing either bacterial or synthetic xylose pathways, or the arabinose pathway showed toxicity symptoms when xylose or arabinose was added to the medium, suggesting that a by-product in the pathway or butanetriol affected plant growth. Furthermore, the metabolite profile of plants expressing arabinose and xylose pathways was altered. Our results demonstrate that bacterial pathways that produce butanetriol can be engineered into plants to produce this chemical. This proof-of-concept study for phytoproduction of butanetriol paves the way to further manipulate metabolic pathways in plants to enhance the level of butanetriol production., (© 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study we used stool profiling to identify intestinal bacteria and metabolites that are differentially represented in humans with colorectal cancer (CRC) compared to healthy controls to identify how microbial functions may influence CRC development. Stool samples were collected from healthy adults (n = 10) and colorectal cancer patients (n = 11) prior to colon resection surgery at the University of Colorado Health-Poudre Valley Hospital in Fort Collins, CO. The V4 region of the 16s rRNA gene was pyrosequenced and both short chain fatty acids and global stool metabolites were extracted and analyzed utilizing Gas Chromatography-Mass Spectrometry (GC-MS). There were no significant differences in the overall microbial community structure associated with the disease state, but several bacterial genera, particularly butyrate-producing species, were under-represented in the CRC samples, while a mucin-degrading species, Akkermansia muciniphila, was about 4-fold higher in CRC (p<0.01). Proportionately higher amounts of butyrate were seen in stool of healthy individuals while relative concentrations of acetate were higher in stools of CRC patients. GC-MS profiling revealed higher concentrations of amino acids in stool samples from CRC patients and higher poly and monounsaturated fatty acids and ursodeoxycholic acid, a conjugated bile acid in stool samples from healthy adults (p<0.01). Correlative analysis between the combined datasets revealed some potential relationships between stool metabolites and certain bacterial species. These associations could provide insight into microbial functions occurring in a cancer environment and will help direct future mechanistic studies. Using integrated "omics" approaches may prove a useful tool in identifying functional groups of gastrointestinal bacteria and their associated metabolites as novel therapeutic and chemopreventive targets.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Metabolomics is maturing as an experimental approach in nutrition science, and it is a useful analysis for revealing systems biology outcomes associated with changes in diet. A major goal of this review is to present the rapidly evolving body of scientific literature that seeks to reveal connections between an individual's metabolic profile and experimentally manipulated or naturally varied dietary intakes. Metabolite profiles in tissue, serum, urine, or stool reflect changes in metabolic pathways that respond to dietary intervention which makes them accessible samples for revealing metabolic effects of diet. Three broadly defined areas of investigation related to dietary-metabolomic strategies include: (1) describing the metabolite variation within and between dietary exposures or interventions; (2) characterizing the metabolic response to dietary interventions with respect to time; and (3) assessing individual variation in baseline nutritional health and/or disease status. An overview of metabolites that were responsive to dietary interventions as reported from original research in human or animal studies is provided and illustrates the breadth of metabolites affected by dietary intervention. Advantages and drawbacks for assessing metabolic changes are discussed in relation to types of metabolite analysis platforms. A combination of targeted and non-targeted global profiling studies as a component of future dietary intervention trials will increase our understanding of nutrition in a systems context., Competing Interests: CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

The effect of temperature on non-volatile compounds in beer has not been well characterised during storage. Here, a metabolomics approach was applied to characterise the effect of storage temperature on non-volatile metabolite variation after 16weeks of storage, using fresh beer as a control. The metabolite profile of room temperature stored (RT) and cold temperature stored (CT) beer differed significantly from fresh, with the most substantial variation observed between RT and fresh beer. Metabolites that changed during storage included prenylated flavonoids, purines, and peptides, and all showed reduced quantitative variation under the CT storage conditions. Corresponding sensory panel observations indicated significant beer oxidation after 12 and 16weeks of storage, with higher values reported for RT samples. These data support that temperature affected beer oxidation during short-term storage, and reveal 5-methylthioadenosine (5-MTA) as a candidate non-volatile metabolite marker for beer oxidation and staling., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Obesity is closely associated with increased incidence of cardiovascular diseases, cancer, insulin resistance, and immune dysfunction, and thus obesity-mitigation strategies should take into account these secondary pathologies in addition to promoting weight loss. Recent studies indicate that black cumin (Nigella sativa) has cardio-protective, anti-cancer, anti-diabetic, antioxidant, and immune-modulatory properties. While black cumin and/or its major bioactive constituent, thymoquinone have demonstrated bioactivity in a variety of disease models, the mechanisms of action are largely unknown. Given the growing interest in and the use of functional foods and nutraceuticals, as well as the increase in obesity and chronic diseases worldwide, further research into the therapeutic/preventive effects of black cumin may be beneficial.

Emerging evidence supporting chronic disease fighting properties of rice bran has advanced the development of stabilized rice bran for human use as a functional food and dietary supplement. A global and targeted metabolomic investigation of stabilized rice bran fermented with Saccharomyces boulardii was performed in three rice varieties. Metabolites from S. boulardii-fermented rice bran were detected by gas chromatography-mass spectrometry (GC-MS) and assessed for bioactivity compared to nonfermented rice bran in normal and malignant lymphocytes. Global metabolite profiling revealed significant differences in the metabolome that led to discovery of candidate compounds modulated by S. boulardii fermentation. Fermented rice bran extracts from three rice varieties reduced growth of human B lymphomas compared to each variety's nonfermented control and revealed that fermentation differentially altered bioactive compounds. These data support that integration of global and targeted metabolite analysis can be utilized for assessing health properties of rice bran phytochemicals that are enhanced by yeast fermentation and that differ across rice varieties.

Emerging evidence supports that cooked rice (Oryza sativa L.) contains metabolites with biomedical activities, yet little is known about the genetic diversity that is responsible for metabolite variation and differences in health traits. Metabolites from ten diverse varieties of cooked rice were detected using ultra performance liquid chromatography coupled to mass spectrometry. A total of 3,097 compounds were detected, of which 25% differed among the ten varieties. Multivariate analyses of the metabolite profiles showed that the chemical diversity among the varieties cluster according to their defined subspecies classifications: indica, japonica, and aus. Metabolite-specific genetic diversity in rice was investigated by analyzing a collection of single nucleotide polymorphisms (SNPs) in genes from biochemical pathways of nutritional importance. Two classes of bioactive compounds, phenolics and vitamin E, contained nonsynonymous SNPs and SNPs in the 5' and 3' untranslated regions for genes in their biosynthesis pathways. Total phenolics and tocopherol concentrations were determined to examine the effect of the genetic diversity among the ten varieties. Per gram of cooked rice, total phenolics ranged from 113.7 to 392.6 µg (gallic acid equivalents), and total tocopherols ranged between 7.2 and 20.9 µg. The variation in the cooked rice metabolome and quantities of bioactive components supports that the SNP-based genetic diversity influenced nutritional components in rice, and that this approach may guide rice improvement strategies for plant and human health.

Background: Soybean cyst nematode (Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S. The Rhg1 locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance. The Glyma18g02680.1 gene at the Rhg1 locus that encodes an apparent leucine-rich repeat transmembrane receptor-kinase (LRR-kinase) has been proposed to be the SCN resistance gene, but its function has not been confirmed. Generation of fertile transgenic soybean lines is difficult but methods have been published that test SCN resistance in transgenic roots generated with Agrobacterium rhizogenes., Results: We report use of artificial microRNA (amiRNA) for gene silencing in soybean, refinements to transgenic root SCN resistance assays, and functional tests of the Rhg1 locus LRR-kinase gene. A nematode demographics assay monitored infecting nematode populations for their progress through developmental stages two weeks after inoculation, as a metric for SCN resistance. Significant differences were observed between resistant and susceptible control genotypes. Introduction of the Rhg1 locus LRR-kinase gene (genomic promoter/coding region/terminator; Peking/PI 437654-derived SCN-resistant source), into rhg1- SCN-susceptible plant lines carrying the resistant-source Rhg4+ locus, provided no significant increases in SCN resistance. Use of amiRNA to reduce expression of the LRR-kinase gene from the Rhg1 locus of Fayette (PI 88788 source of Rhg1) also did not detectably alter resistance to SCN. However, silencing of the LRR-kinase gene did have impacts on root development., Conclusion: The nematode demographics assay can expedite testing of transgenic roots for SCN resistance. amiRNAs and the pSM103 vector that drives interchangeable amiRNA constructs through a soybean polyubiqutin promoter (Gmubi), with an intron-GFP marker for detection of transgenic roots, may have widespread use in legume biology. Studies in which expression of the Rhg1 locus LRR-kinase gene from different resistance sources was either reduced or complemented did not reveal significant impacts on SCN resistance.