Anti-Inflammatory Diet Prevents Subclinical Colonic Inflammation and Alters Metabolomic Profile of Ulcerative Colitis Patients in Clinical Remission.

A relationship between ulcerative colitis (UC) and diet has been shown in epidemiological and experimental studies. In a 6-month, open-label, randomized, placebo-controlled trial, adult UC patients in clinical remission were randomized to either an "Anti-inflammatory Diet (AID)" or "Canada's Food Guide (CFG)". Menu plans in the AID were designed to increase the dietary intake of dietary fiber, probiotics, antioxidants, and omega-3 fatty acids and to decrease the intake of red meat, processed meat, and added sugar. Stool was collected for fecal calprotectin (FCP) and microbial analysis. Metabolomic analysis was performed on urine, serum, and stool samples at the baseline and study endpoint. In this study, 53 patients were randomized. Five (19.2%) patients in the AID and 8 (29.6%) patients in the CFG experienced a clinical relapse. The subclinical response to the intervention (defined as FCP < 150 µg/g at the endpoint) was significantly higher in the AID group (69.2 vs. 37.0%, p = 0.02). The patients in the AID group had an increased intake of zinc, phosphorus, selenium, yogurt, and seafood versus the control group. Adherence to the AID was associated with significant changes in the metabolome, with decreased fecal acetone and xanthine levels along with increased fecal taurine and urinary carnosine and p-hydroxybenzoic acid levels. The AID subjects also had increases in fecal Bifidobacteriaceae, Lachnospiraceae, and Ruminococcaceae. In this study, we found thatdietary modifications involving the increased intake of anti-inflammatory foods combined with a decreased intake of pro-inflammatory foods were associated with metabolic and microbial changes in UC patients in clinical remission and were effective in preventing subclinical inflammation.

Identification of Primary Natural Killer Cell Modulators by Chemical Library Screening with a Luciferase-Based Functional Assay.

Natural killer (NK) cells are essential players of the innate immune response that secrete cytolytic factors and cytokines such as IFN-γ when contacting virus-infected or tumor cells. They represent prime targets in immunotherapy as defects in NK cell functions are hallmarks of many pathological conditions, such as cancer and chronic infections. The functional screening of chemical libraries or biologics would greatly help identify new modulators of NK cell activity, but commonly used methods such as flow cytometry are not easily scalable to high-throughput settings. Here we describe an efficient assay to measure the natural cytotoxicity of primary NK cells where the bioluminescent enzyme NanoLuc is constitutively expressed in the cytoplasm of target cells and is released in co-culture supernatants when lysis occurs. We fully characterized this assay using either purified NK cells or total peripheral blood mononuclear cells (PBMCs), including some patient samples, as effector cells. A pilot screen was also performed on a library of 782 metabolites, xenobiotics, and common drugs, which identified dextrometorphan and diphenhydramine as novel NK cell inhibitors. Finally, this assay was further improved by developing a dual-reporter cell line to simultaneously measure NK cell cytotoxicity and IFN-γ secretion in a single well, extending the potential of this system.

Microbiome and metabolome modifying effects of several cardiovascular disease interventions in apo-E-/- mice.

BACKGROUND: There is strong evidence indicating that gut microbiota have the potential to modify, or be modified by the drugs and nutritional interventions that we rely upon. This study aims to characterize the compositional and functional effects of several nutritional, neutraceutical, and pharmaceutical cardiovascular disease interventions on the gut microbiome, through metagenomic and metabolomic approaches. Apolipoprotein-E-deficient mice were fed for 24 weeks either high-fat/cholesterol diet alone (control, HFC) or high-fat/cholesterol in conjunction with one of three dietary interventions, as follows: plant sterol ester (PSE), oat ß-glucan (OBG) and bile salt hydrolase-active Lactobacillus reuteri APC 2587 (BSH), or the drug atorvastatin (STAT). The gut microbiome composition was then investigated, in addition to the host fecal and serum metabolome. RESULTS: We observed major shifts in the composition of the gut microbiome of PSE mice, while OBG and BSH mice displayed more modest fluctuations, and STAT showed relatively few alterations. Interestingly, these compositional effects imparted by PSE were coupled with an increase in acetate and reduction in isovalerate (p < 0.05), while OBG promoted n-butyrate synthesis (p < 0.01). In addition, PSE significantly dampened the microbial production of the proatherogenic precursor compound, trimethylamine (p < 0.05), attenuated cholesterol accumulation, and nearly abolished atherogenesis in the model (p < 0.05). However, PSE supplementation produced the heaviest mice with the greatest degree of adiposity (p < 0.05). Finally, PSE, OBG, and STAT all appeared to have considerable impact on the host serum metabolome, including alterations in several acylcarnitines previously associated with a state of metabolic dysfunction (p < 0.05). CONCLUSIONS: We observed functional alterations in microbial and host-derived metabolites, which may have important implications for systemic metabolic health, suggesting that cardiovascular disease interventions may have a significant impact on the microbiome composition and functionality. This study indicates that the gut microbiome-modifying effects of novel therapeutics should be considered, in addition to the direct host effects.

Metabolome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants.

BACKGROUND: Recent progress toward the elucidation of benzylisoquinoline alkaloid (BIA) metabolism has focused on a small number of model plant species. Current understanding of BIA metabolism in plants such as opium poppy, which accumulates important pharmacological agents such as codeine and morphine, has relied on a combination of genomics and metabolomics to facilitate gene discovery. Metabolomics studies provide important insight into the primary biochemical networks underpinning specialized metabolism, and serve as a key resource for metabolic engineering, gene discovery, and elucidation of governing regulatory mechanisms. Beyond model plants, few broad-scope metabolomics reports are available for the vast number of plant species known to produce an estimated 2500 structurally diverse BIAs, many of which exhibit promising medicinal properties. RESULTS: We applied a multi-platform approach incorporating four different analytical methods to examine 20 non-model, BIA-accumulating plant species. Plants representing four families in the Ranunculales were chosen based on reported BIA content, taxonomic distribution and importance in modern/traditional medicine. One-dimensional (1)H NMR-based profiling quantified 91 metabolites and revealed significant species- and tissue-specific variation in sugar, amino acid and organic acid content. Mono- and disaccharide sugars were generally lower in roots and rhizomes compared with stems, and a variety of metabolites distinguished callus tissue from intact plant organs. Direct flow infusion tandem mass spectrometry provided a broad survey of 110 lipid derivatives including phosphatidylcholines and acylcarnitines, and high-performance liquid chromatography coupled with UV detection quantified 15 phenolic compounds including flavonoids, benzoic acid derivatives and hydroxycinnamic acids. Ultra-performance liquid chromatography coupled with high-resolution Fourier transform mass spectrometry generated extensive mass lists for all species, which were mined for metabolites putatively corresponding to BIAs. Different alkaloids profiles, including both ubiquitous and potentially rare compounds, were observed. CONCLUSIONS: Extensive metabolite profiling combining multiple analytical platforms enabled a more complete picture of overall metabolism occurring in selected plant species. This study represents the first time a metabolomics approach has been applied to most of these species, despite their importance in modern and traditional medicine. Coupled with genomics data, these metabolomics resources serve as a key resource for the investigation of BIA biosynthesis in non-model plant species.

Detection of fortification of ginkgo products using nanoelectrospray ionization mass spectrometry.

We report here a negative ionization nanoelectrospray ionization mass spectrometry (nanoESI-MS) technique that simultaneously detects active components, terpenes and intact flavonol glycosides, and toxic ginkgolic acids in ginkgo products. Unlike the conventional methods that hydrolyze flavonol glycosides to flavonoids for analysis, this technique directly detects intact flavonol glycosides, enabling differentiation of these natural glycosides from the synthetic flavonoids. Thus, it allows the detection of fortification of ginkgo products, alleviating a common problem encountered by the conventional methods. Analysis of 14 commercial ginkgo products using this technique demonstrates large variations and deviation from the well-accepted standardized ginkgo extract. Four products showed evidence of fortification with synthetic surrogates. Two products were found to have toxic ginkgolic acids that exceed the 5 microg g(-1) limit by as much as 60000 fold. These results emphasize the importance of appropriate monitoring of ginkgo product quality.