Hydrophilic Metabolite Composition of Fruiting Bodies and Mycelia of Edible Mushroom Species (Agaricomycetes).

Mushrooms have two components, the fruiting body, which encompasses the stalk and the cap, and the mycelium, which supports the fruiting body underground. The part of the mushroom most commonly consumed is the fruiting body. Given that it is more time consuming to harvest the fruiting body versus simply the mycelia, we were interested in understanding the difference in metabolite content between the fruiting bodies and mycelia of four widely consumed mushrooms in Taiwan: Agrocybe cylindracea (AC), Coprinus comatus (CC), Hericium erinaceus (HE), and Hypsizygus marmoreus (HM). In total, we identified 54 polar metabolites using 1H NMR spectroscopy that included sugar alcohols, amino acids, organic acids, nucleosides and purine/pyrimidine derivatives, sugars, and others. Generally, the fruiting bodies of AC, CC, and HM contained higher amounts of essential amino acids than their corresponding mycelia. Among fruiting bodies, HE had the lowest essential amino acid content. Trehalose was the predominant carbohydrate in most samples except for the mycelia of AC, in which the major sugar was glucose. The amount of adenosine, uridine, and xanthine in the samples was similar, and was higher in fruiting bodies compared with mycelia, except for HM. The organic acid and sugar alcohol content between fruiting bodies and mycelia did not tend to be different. Although each mushroom had a unique metabolic profile, the metabolic profile of fruiting bodies and mycelia were most similar for CC and HE, suggesting that the mycelia of CC and HE may be good replacements for their corresponding fruiting bodies. Additionally, each mushroom species had a unique polar metabolite fingerprint, which could be utilized to identify adulteration.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analyses of Citrus sinensis (L.) Osbeck Graft-Inoculated with "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current CLas detection methods are unreliable during presymptomatic infection, and understanding CLas pathogenicity to help develop new detection techniques is challenging because CLas has yet to be isolated in pure culture. To understand how CLas affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange (Citrus sinensis (L.) Osbeck) plants were graft-inoculated with CLas and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during CLas infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Metabolome and Microbiome Signatures in the Roots of Citrus Affected by Huanglongbing.

Huanglongbing (HLB) is a severe, incurable citrus disease caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas). Although citrus leaves serve as the site of initial infection, CLas is known to migrate to and colonize the root system; however, little is known about the impact of CLas infection on root metabolism and resident microbial communities. Scions of 'Lisbon' lemon and 'Washington Navel' orange grafted onto 'Carrizo' rootstock were grafted with either CLas-infected citrus budwood or uninfected budwood. Roots were obtained from trees 46 weeks after grafting and analyzed via 1H nuclear magnetic resonance spectroscopy to identify water-soluble root metabolites and high-throughput sequencing of 16S rRNA and ITS gene amplicons to determine the relative abundance of bacterial and fungal taxa in the root rhizosphere and endosphere. In both citrus varieties, 27 metabolites were identified, of which several were significantly different between CLas(+) and control plants. CLas infection also appeared to alter the microbial community structure near and inside the roots of citrus plants. Nonmetric multidimensional scaling (NMDS) and a principal coordinate analysis (PCoA) revealed distinct metabolite and microbial profiles, demonstrating that CLas impacts the root metabolome and microbiome in a manner that is variety-specific.

Composition of Mycelia and Basidiomata of the Culinary-Medicinal Golden Oyster Mushroom, Pleurotus citrinopileatus (Agaricomycetes) by Nuclear Magnetic Resonance Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy has been used to obtain the profile of soluble metabolites on fungus. To enhance the ergothioneine content in Pleurotus citrinopileatus mycelium, amino acid precursors were used for submerged fermentation. The study aimed to analyze the metabolites of high-ergothioneine and regular P. citrinopileatus mycelia (HEPM and RPM) and P. citrinopileatus basidiomata (PCB) using NMR spectroscopy. Principal component analysis (PCA) was applied to differentiate and to describe the differences among three sets of metabolites. The results showed that 55 water-soluble metabolites of PCB, HEPM, and RPM were identified and classified into five groups including amino acids, carbohydrates, organic acids, purines and pyrimidines, and others. Contents of total soluble metabolites were in descending order as follows: PCB (135 mg/g) > HEPM (58 mg/g) > RPM (44 mg/g). The score plot and loading plot separated using PCA showed that the first two components were responsible for R2X = 97.1% and the variance [R2X(1) = 74% for principal component PC1 and R2X(2) = 23.1% for PC2]. A metabolic pathway map of P. citrinopileatus mycelium was established and the differences in the metabolites of RPM and HEPM with precursors added were illuminated.

NMR metabolomics of cerebrospinal fluid differentiates inflammatory diseases of the central nervous system.

BACKGROUND: Myriad infectious and noninfectious causes of encephalomyelitis (EM) have similar clinical manifestations, presenting serious challenges to diagnosis and treatment. Metabolomics of cerebrospinal fluid (CSF) was explored as a method of differentiating among neurological diseases causing EM using a single CSF sample. METHODOLOGY/PRINCIPAL FINDINGS: 1H NMR metabolomics was applied to CSF samples from 27 patients with a laboratory-confirmed disease, including Lyme disease or West Nile Virus meningoencephalitis, multiple sclerosis, rabies, or Histoplasma meningitis, and 25 controls. Cluster analyses distinguished samples by infection status and moderately by pathogen, with shared and differentiating metabolite patterns observed among diseases. CART analysis predicted infection status with 100% sensitivity and 93% specificity. CONCLUSIONS/SIGNIFICANCE: These preliminary results suggest the potential utility of CSF metabolomics as a rapid screening test to enhance diagnostic accuracies and improve patient outcomes.

Metabolomics reveals differences between three daidzein metabolizing phenotypes in adults with cardiometabolic risk factors.

SCOPE: The soy isoflavone, daidzein, is metabolized by gut microbiota to O-desmethylangolensin (ODMA) and/or equol. Producing equol is postulated as a contributing factor for the beneficial effects of soy. METHODS AND RESULTS: This randomized, controlled, cross-over design used an untargeted metabolomic approach to assess the metabolic profile of different daidzein metabolizers. Adults (n = 17) with cardiometabolic risk factors received soy nuts or control food for 4 weeks, separated by a 2-week washout. No significant differences were detected pre- and postintervention and between interventions. Examination of the ability to metabolize daidzein revealed three groups: ODMA only producers (n = 4), equol + ODMA producers (n = 8), and nonproducers (n = 5). Analysis of the serum metabolome revealed nonproducers could be distinguished from ODMA-only and equol + ODMA producers. Differences between these phenotypes were related to obesity and metabolic risk (methionine, asparagine, and trimethylamine) with equol + ODMA producers having lower concentrations, yet paradoxically higher pro-inflammatory cytokines. In urine, nonproducers clustered with ODMA producers and were distinct from equol + ODMA producers. Urinary metabolite profiles revealed significantly higher excretion of fumarate and 2-oxoglutarate, as well as pyroglutamate, alanine, and the gut microbial metabolite dimethylamine in equol + ODMA producers. CONCLUSION: These results emphasize that the serum and urine metabolomes are distinct based on the ability to metabolize isoflavones.

Quantification of Water-Soluble Metabolites in Medicinal Mushrooms Using Proton NMR Spectroscopy.

The water-soluble metabolites in 5 mushrooms were identified and quantified using proton nuclear magnetic resonance (NMR) spectroscopy and software for targeted metabolite detection and quantification. In total, 35 compounds were found in Agaricus brasiliensis, 25 in Taiwanofungus camphoratus, 23 in Ganoderma lucidum (Taiwan) and Lentinus edodes, and 16 in G. lucidum (China). Total amounts of all identified metabolites in A. brasiliensis, T. camphoratus, G. lucidum, G. lucidum (China), and L. edodes were 149,950.51, 12,834.18, 9,549.09, 2,788.41, and 111,726.51 mg/kg dry weight, respectively. These metabolites were categorized into 4 groups: free amino acids and derivatives, carbohydrates, carboxylic acids, and nucleosides. Carbohydrates were the most abundant metabolites among all 4 groups, with mannitol having the highest concentration among all analyzed metabolites (848-94,104 mg/kg dry weight). Principal components analysis (PCA) showed obvious distinction among the metabolites of the 5 different kinds of mushrooms analyzed in this study. Thus PCA could provide an optional analytical way of identifying and recognizing the compositions of flavor products. Furthermore, the results of this study demonstrate that NMRbased metabolomics is a powerful tool for differentiating between various medicinal mushrooms.

Consumption of vitamin D2 enhanced mushrooms is associated with improved bone health.

Mushrooms are the best nonanimal food source of vitamin D2. Pulsed irradiation can enhance vitamin D2 in mushrooms quickly. We investigated the effect of supplementing high vitamin D2Pleurotus ferulae mushrooms in a mouse model of osteoporosis. Thirty-two female C57BL/6JNarl mice were divided into four groups including sham, ovariectomized (OVX), OVX+nonpulsed mushroom (NPM) and OVX+pulsed mushroom (PM). After 23 weeks of treatment, serum samples were analyzed for osteoblast and osteoclast indicators, as well as metabolites using NMR spectroscopy. To examine bone density, femurs were analyzed using micro-computed tomography. The NPM and PM treatment mice showed increased bone density in comparison with OVX mice. In addition, the PM mice showed higher osteoblast and lower osteoclast indicators in comparison with OVX mice. Serum metabolomics analysis indicated several metabolites that were different in PM mice, some of which could be correlated with bone health. Taken together, these results suggest that pulsed irradiated mushrooms are able to increase bone density in osteoporotic mice possibly through enhanced bone metabolism. Further studies in humans are needed to show their efficacy in preventing osteoporosis.

An NMR metabolomic study on the effect of alendronate in ovariectomized mice.

Alendronate sodium (Fosamax) is most widely used for the prevention and treatment of osteoporosis. It is a type of anti-resorptive agent that reduces the risk of fractures by changing bone turnover and bone mineral density. We investigated the effect of Fosamax on a mouse model of osteoporosis. Twenty-seven female C57BL/6JNarl mice were divided into three groups: sham, ovariectomized (OVX) and OVX + Fosamax (Fosamax). After 23 weeks, bone density of femurs was analyzed using microcomputed tomography (micro-CT), and serum was analyzed for osteoblast and osteoclast activity, as well as metabolites using nuclear magnetic resonance (NMR) spectroscopy. Fosamax increased bone mineral density and cortical bone thickness, and decreased osteoblast activity slightly. Fosamax did not significantly change osteoclast activity. Serum metabolomics revealed that Fosamax had profound effects on overall metabolism, as significantly higher concentrations of metabolites associated with energy metabolism (including TCA-cycle intermediates and glucose), 3-hydroxybutyrate, taurine, allantoin, acetate, and ethanol, as well as lower concentrations of aspartate were observed in the Fosamax-treated mice compared with the OVX mice. These results suggest that alendronate may work by increasing bone density through altered metabolic activity.

Metabolite signature of Candidatus Liberibacter asiaticus infection in two citrus varieties.

Huanglongbing (HLB), also known as Citrus Greening Disease, is caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas) and is a serious threat to the citrus industry. To understand the effect of CLas infection on the citrus metabolome, juice from healthy (n = 18), HLB-asymptomatic (n = 18), and HLB-symptomatic Hamlin (n = 18), as well as from healthy (n = 18) and HLB-symptomatic (n = 18) Valencia sweet oranges (from southern and eastern Florida) were evaluated using (1)H NMR-based metabolomics. Differences in the concentration of several metabolites including phenylalanine, histidine, limonin, and synephrine between control or asymptomatic fruit and symptomatic fruit were observed regardless of the citrus variety or location. There were no clear differences between the metabolite profiles of Hamlin fruits classified by PCR as asymptomatic and control, suggesting that some of the control fruit may have been infected. Taken together, these data indicate that infection due to CLas presents a strong metabolic response that is observed across different cultivars and regions, suggesting the potential for generation of metabolite-based biomarkers of CLas infection.

Cross-talk between E. coli strains and a human colorectal adenocarcinoma-derived cell line.

Although there is great interest in the specific mechanisms of how gut microbiota modulate the biological processes of the human host, the extent of host-microbe interactions and the bacteria-specific metabolic activities for survival in the co-evolved gastrointestinal environment remain unclear. Here, we demonstrate a comprehensive comparison of the host epithelial response induced by either a pathogenic or commensal strain of Escherichia coli using a multi-omics approach. We show that Caco-2 cells incubated with E. coli display an activation of defense response genes associated with oxidative stress. Indeed, in the bacteria co-culture system, the host cells experience an altered environment compared with the germ-free system that includes reduced pH, depletion of major energy substrates, and accumulation of fermentation by-products. Measurement of intracellular Caco-2 cell metabolites revealed a significantly increased lactate concentration, as well as changes in TCA cycle intermediates. Our results will lead to a deeper understanding of acute microbial-host interactions.

Dietary pyrroloquinoline quinone (PQQ) alters indicators of inflammation and mitochondrial-related metabolism in human subjects.

Pyrroloquinoline quinone (PQQ) influences energy-related metabolism and neurologic functions in animals. The mechanism of action involves interactions with cell signaling pathways and mitochondrial function. However, little is known about the response to PQQ in humans. Using a crossover study design, 10 subjects (5 females, 5 males) ingested PQQ added to a fruit-flavored drink in two separate studies. In study 1, PQQ was given in a single dose (0.2 mg PQQ/kg). Multiple measurements of plasma and urine PQQ levels and changes in antioxidant potential [based on total peroxyl radical-trapping potential and thiobarbituric acid reactive product (TBAR) assays] were made throughout the period of 48 h. In study 2, PQQ was administered as a daily dose (0.3 mg PQQ/kg). After 76 h, measurements included indices of inflammation [plasma C-reactive protein, interleukin (IL)-6 levels], standard clinical indices (e.g., cholesterol, glucose, high-density lipoprotein, low-density lipoprotein, triglycerides, etc.) and (1)H-nuclear magnetic resonance estimates of urinary metabolites related in part to oxidative metabolism. The standard clinical indices were normal and not altered by PQQ supplementation. However, dietary PQQ exposure (Study 1) resulted in apparent changes in antioxidant potential based on malonaldehyde-related TBAR assessments. In Study 2, PQQ supplementation resulted in significant decreases in the levels of plasma C-reactive protein, IL-6 and urinary methylated amines such as trimethylamine N-oxide, and changes in urinary metabolites consistent with enhanced mitochondria-related functions. The data are among the first to link systemic effects of PQQ in animals to corresponding effects in humans.

Fertilisation and pesticides affect mandarin orange nutrient composition.

The effects of the application of foliar fertilisation and pesticide on nutritional quality of mandarin orange juices were evaluated using (1)H NMR metabolomics. Significant differences between the use of fertiliser and pesticides during fruit formation were observed, and included changes in sugar, amino acid and organic acid composition. To determine whether the difference in sugar concentration was enough for the consumer to detect, a sensory experiment was performed in which two orange juice samples were prepared to resemble the sweet/sour taste balance of juice from mandarin oranges in which foliar fertilisation was either applied or not. In a test using non-trained individuals, 68% could correctly identify which juice had a sourer, or less sweet, taste. The implications of this study could impact citrus growers, and ultimately aid in development of fruit with superior sensory quality.

Metabolomic analysis of citrus infection by 'Candidatus Liberibacter' reveals insight into pathogenicity.

Huanglongbing (HLB), considered the most serious citrus disease in the world, is associated with the nonculturable bacterium 'Candidatus Liberibacter asiaticus' (Las). Infection of citrus by this pathogen leads to reduced plant vigor and productivity, ultimately resulting in death of the infected tree. It can take up to two years following initial infection before outward symptoms become apparent, making detection difficult. The existing knowledge gap in our understanding of Las and its pathogenesis leading to HLB has stymied development of treatments and methods to mitigate the pathogen's influence. To evaluate the influence of Las on fruit quality in both symptomatic and asymptomatic fruit, and gain further insight into the pathogenesis of the disease, a 1H NMR metabolomics investigation, complemented with physicochemical and analyte-specific analyses, was undertaken. Comparison of the juice obtained from oranges gathered from Las+ (symptomatic and asymptomatic) and Las- (healthy) trees revealed significant differences in the concentrations of sugars, amino and organic acids, limonin glucoside, and limonin. This study demonstrates differing metabolic profiles in the juice of oranges from Las+ and Las- and proposes how Las may be able to evade citrus defense responses.

Dextran sulfate sodium inhibits alanine synthesis in Caco-2 cells.

To understand and characterize the pathogenic mechanisms of inflammatory bowel disease, dextran sulfate sodium (DSS) has been used to induce acute and chronic colitis in animal models by causing intestinal epithelium damage. The mechanism of action of DSS in producing this outcome is not well understood. In an effort to understand how DSS might impact epithelial cell metabolism, we studied the intestinal epithelial cell line Caco-2 incubated with 1% DSS over 56 hours using (1)H NMR spectroscopy. We observed no difference in cell viability as compared to control cultures, and an approximately 1.5-fold increase in IL-6 production upon incubation with 1% DSS. The effect on Caco-2 cell metabolism as measured through changes in the concentration of metabolites in the cell supernatant included a three-fold decrease in the concentration of alanine. Given that the concentrations of other amino acids in the cell culture supernatant were not different between treated and control cultures over 56 hours suggest that DSS inhibits alanine synthesis, specifically alanine aminotransferase, without affecting other key metabolic pathways. The importance of alanine aminotransferase in inflammatory bowel disease is discussed.

Elevation, rootstock, and soil depth affect the nutritional quality of mandarin oranges.

The effects of elevation, rootstock, and soil depth on the nutritional quality of mandarin oranges from 11 groves in California were investigated by nuclear magnetic resonance (NMR) spectroscopy by quantifying 29 compounds and applying multivariate statistical data analysis. A comparison of the juice from oranges in groves with deeper soil and trifoliate rootstock versus those with shallow soil and C-35 rootstock revealed differences in the concentrations of 4-aminobutyrate, ethanol, phenylalanine, succinate, and isoleucine. A comparison of fruit from trees grown at higher versus lower elevation revealed that those at higher elevation had higher concentrations of amino acids, succinate, and 4-aminobutyrate and lower concentrations of sugars and limonin glucoside. Such differences indicate that rootstock, soil depth, and differences in elevation influence the fruit nutrient composition. This study highlights how metabolomics coupled with multivariate statistical analysis can illuminate the metabolic characteristics of citrus, thereby aiding in the determination of the grove identity and fruit quality during orange production.