Metabolomics, the global study of small molecules in a particular system, has in the past few years risen to become a primary -omics platform for the study of metabolic processes. With the ...ever-increasing pool of quantitative data yielded from metabolomic research, specialized methods and tools with which to analyze and extract meaningful conclusions from these data are becoming more and more crucial. Furthermore, the depth of knowledge and expertise required to undertake a metabolomics oriented study is a daunting obstacle to investigators new to the field. As such, we have created a new statistical analysis workflow, MetaboLyzer, which aims to both simplify analysis for investigators new to metabolomics, as well as provide experienced investigators the flexibility to conduct sophisticated analysis. MetaboLyzer’s workflow is specifically tailored to the unique characteristics and idiosyncrasies of postprocessed liquid chromatography–mass spectrometry (LC–MS)-based metabolomic data sets. It utilizes a wide gamut of statistical tests, procedures, and methodologies that belong to classical biostatistics, as well as several novel statistical techniques that we have developed specifically for metabolomics data. Furthermore, MetaboLyzer conducts rapid putative ion identification and putative biologically relevant analysis via incorporation of four major small molecule databases: KEGG, HMDB, Lipid Maps, and BioCyc. MetaboLyzer incorporates these aspects into a comprehensive workflow that outputs easy to understand statistically significant and potentially biologically relevant information in the form of heatmaps, volcano plots, 3D visualization plots, correlation maps, and metabolic pathway hit histograms. For demonstration purposes, a urine metabolomics data set from a previously reported radiobiology study in which samples were collected from mice exposed to γ radiation was analyzed. MetaboLyzer was able to identify 243 statistically significant ions out of a total of 1942. Numerous putative metabolites and pathways were found to be biologically significant from the putative ion identification workflow.
After androgen deprivation, prostate cancer frequently becomes castration resistant (CRPC), with intratumoral androgen production from extragonadal precursors that activate the androgen receptor ...pathway. 3β-Hydroxysteroid dehydrogenase-1 (3βHSD1) is the rate-limiting enzyme for extragonadal androgen synthesis, which together lead to CRPC. Here, we show that cancer-associated fibroblasts (CAFs) increased epithelial 3βHSD1 expression, induced androgen synthesis, activated the androgen receptor, and induced CRPC. Unbiased metabolomics revealed that CAF-secreted glucosamine specifically induced 3βHSD1. CAFs induced higher GlcNAcylation in cancer cells and elevated expression of the transcription factor Elk1, which induced higher 3βHSD1 expression and activity. Elk1 genetic ablation in cancer epithelial cells suppressed CAF-induced androgen biosynthesis in vivo. In patient samples, multiplex fluorescent imaging showed that tumor cells expressed more 3βHSD1 and Elk1 in CAF-enriched areas compared with CAF-deficient areas. Our findings suggest that CAF-secreted glucosamine increases GlcNAcylation in prostate cancer cells, promoting Elk1-induced HSD3B1 transcription, which upregulates de novo intratumoral androgen synthesis to overcome castration.
Pancreatic Ductal Adenocarcinoma (PDAC) is highly resistant to chemotherapy. Effective alternative therapies have yet to emerge, as chemotherapy remains the best available systemic treatment. ...However, the discovery of safe and available adjuncts to enhance chemotherapeutic efficacy can still improve survival outcomes. We show that a hyperglycemic state substantially enhances the efficacy of conventional single- and multi-agent chemotherapy regimens against PDAC. Molecular analyses of tumors exposed to high glucose levels reveal that the expression of GCLC (glutamate-cysteine ligase catalytic subunit), a key component of glutathione biosynthesis, is diminished, which in turn augments oxidative anti-tumor damage by chemotherapy. Inhibition of GCLC phenocopies the suppressive effect of forced hyperglycemia in mouse models of PDAC, while rescuing this pathway mitigates anti-tumor effects observed with chemotherapy and high glucose.
Background & Aims Microbes may increase susceptibility to inflammatory bowel disease (IBD) by producing bioactive metabolites that affect immune activity and epithelial function. We undertook a ...family based study to identify microbial and metabolic features of IBD that may represent a predisease risk state when found in healthy first-degree relatives. Methods Twenty-one families with pediatric IBD were recruited, comprising 26 Crohn’s disease patients in clinical remission, 10 ulcerative colitis patients in clinical remission, and 54 healthy siblings/parents. Fecal samples were collected for 16S ribosomal RNA gene sequencing, untargeted liquid chromatography–mass spectrometry metabolomics, and calprotectin measurement. Individuals were grouped into microbial and metabolomics states using Dirichlet multinomial models. Multivariate models were used to identify microbes and metabolites associated with these states. Results Individuals were classified into 2 microbial community types. One was associated with IBD but irrespective of disease status, had lower microbial diversity, and characteristic shifts in microbial composition including increased Enterobacteriaceae, consistent with dysbiosis. This microbial community type was associated similarly with IBD and reduced microbial diversity in an independent pediatric cohort. Individuals also clustered bioinformatically into 2 subsets with shared fecal metabolomics signatures. One metabotype was associated with IBD and was characterized by increased bile acids, taurine, and tryptophan. The IBD-associated microbial and metabolomics states were highly correlated, suggesting that they represented an integrated ecosystem. Healthy relatives with the IBD-associated microbial community type had an increased incidence of elevated fecal calprotectin. Conclusions Healthy first-degree relatives can have dysbiosis associated with an altered intestinal metabolome that may signify a predisease microbial susceptibility state or subclinical inflammation. Longitudinal prospective studies are required to determine whether these individuals have a clinically significant increased risk for developing IBD.
Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here, we show that gut ...microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (
). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake but is instead associated with alterations in the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that gut microbial CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have potential as anti-obesity therapeutics.
Consistent compositional shifts in the gut microbiota are observed in IBD and other chronic intestinal disorders and may contribute to pathogenesis. The identities of microbial biomolecular ...mechanisms and metabolic products responsible for disease phenotypes remain to be determined, as do the means by which such microbial functions may be therapeutically modified.
The composition of the microbiota and metabolites in gut microbiome samples in 47 subjects were determined. Samples were obtained by endoscopic mucosal lavage from the cecum and sigmoid colon regions, and each sample was sequenced using the 16S rRNA gene V4 region (Illumina-HiSeq 2000 platform) and assessed by UPLC mass spectroscopy. Spearman correlations were used to identify widespread, statistically significant microbial-metabolite relationships. Metagenomes for identified microbial OTUs were imputed using PICRUSt, and KEGG metabolic pathway modules for imputed genes were assigned using HUMAnN. The resulting metabolic pathway abundances were mostly concordant with metabolite data. Analysis of the metabolome-driven distribution of OTU phylogeny and function revealed clusters of clades that were both metabolically and metagenomically similar.
The results suggest that microbes are syntropic with mucosal metabolome composition and therefore may be the source of and/or dependent upon gut epithelial metabolites. The consistent relationship between inferred metagenomic function and assayed metabolites suggests that metagenomic composition is predictive to a reasonable degree of microbial community metabolite pools. The finding that certain metabolites strongly correlate with microbial community structure raises the possibility of targeting metabolites for monitoring and/or therapeutically manipulating microbial community function in IBD and other chronic diseases.
The diagnosis of Behçet's disease (BD) remains challenging due to the lack of diagnostic biomarkers. This study aims to identify potential serum metabolites associated with BD and its disease ...activity.
Medical records and serum samples of 24 pretreated BD patients, 12 post-treated BD patients, and age-matched healthy controls (HC) were collected for metabolomics and lipidomics profiling using UPLC-QTOF-MS and UPLC-QTOF-MS
approaches. Additionally, serum samples from an independent cohort of BD patients, disease controls including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Takayasu's arteritis (TA), Crohn's disease (CD) patients, and HC were collected for further validation of two potential biomarkers using UPLC-QTOFMS analysis.
Unsupervised principal component analysis (PCA) showed a clear separation of metabolomics profiles of BD patients from HC. Statistical analysis of the data revealed differential metabolites between BD patients and HC. The serum levels of some phosphatidylcholines (PCs) were found to be significantly lower in BD patients, while the levels of several polyunsaturated fatty acids (PUFAs) were increased markedly in the BD group compared with HC. Furthermore, the serum level of two omega-6 PUFAs, linoleic acid (LA) and arachidonic acid (AA), were dramatically decreased in patients with remission. A validation cohort confirmed that the serum LA and AA levels in BD patients were significantly higher than those in HC and patients with RA, SLE, TA, and CD. In addition, receiver operating characteristic (ROC) analysis indicated good sensitivity and specificity.
The serum metabolomics profiles in BD patients are altered. Serum LA and AA are promising diagnostic biomarkers for BD.
Crohn's disease (CD) patients demonstrate distinct intestinal microbial compositions and metabolic characteristics compared to unaffected controls. However, the impact of inflammation and underlying ...genetic risk on these microbial profiles and their relationship to disease phenotype are unclear. We used lavage sampling to characterize the colonic mucosal-luminal interface (MLI) microbiome of CD patients in endoscopic remission and unaffected controls relative to obesity, disease genetics, and phenotype.
Cecum and sigmoid colon were sampled from 110 non-CD controls undergoing screening colonoscopy who were stratified by body mass index and 88 CD patients in endoscopic remission (396 total samples). CD polygenic risk score (GRS) was calculated using 186 known CD variants. MLI pellets were analyzed by 16S ribosomal RNA gene sequencing, and supernatants by untargeted liquid chromatography-mass spectrometry.
CD and obesity were each associated with decreased cecal and sigmoid MLI bacterial diversity and distinct bacterial composition compared to controls, including expansion of Escherichia/Shigella. Cecal and sigmoid dysbiosis indices for CD were significantly greater in obese controls than non-overweight controls. CD, but not obesity, was characterized by altered biogeographic relationship between the sigmoid and cecum. GRS was associated with select taxonomic shifts that overlapped with changes seen in CD compared to controls including Fusobacterium enrichment. Stricturing or penetrating Crohn's disease behavior was characterized by lower MLI bacterial diversity and altered composition, including reduced Faecalibacterium, compared to uncomplicated CD. Taxonomic profiles including reduced Parasutterella were associated with clinical disease progression over a mean follow-up of 3.7 years. Random forest classifiers using MLI bacterial abundances could distinguish disease state (area under the curve (AUC) 0.93), stricturing or penetrating Crohn's disease behavior (AUC 0.82), and future clinical disease progression (AUC 0.74). CD patients showed alterations in the MLI metabolome including increased cholate:deoxycholate ratio compared to controls.
Obesity, CD in endoscopic remission, and high CD genetic risk have overlapping colonic mucosal-luminal interface (MLI) microbiome features, suggesting a shared microbiome contribution to CD and obesity which may be influenced by genetic factors. Microbial profiling during endoscopic remission predicted Crohn's disease behavior and progression, supporting that MLI sampling could offer unique insight into CD pathogenesis and provide novel prognostic biomarkers.
ABSTRACT
The mechanism of inorganic carbon (Ci) uptake was examined in the marine green microalgae Stichococcus cylindricus and Stichococcus minor. External carbonic anhydrase (CA) activity was not ...detected in either species, by potentiometric assay or by mass spectrometry. Photosynthetic characteristics of Ci uptake indicate that both species have high apparent affinity for CO2 with a low K1/2(CO2) of about 10 µm. The O2 evolution rates in light exceeded the spontaneous CO2 formation rate by 2.5‐fold in both species, which thus have active bicarbonate uptake. Mass spectrometric monitoring of CO2 and O2 fluxes showed that rates of O2 evolution exceeded those of CO2 depletion by about three‐ and twofold in S. minor and S. cylindricus, respectively, and also showed, in cells photosynthesizing at pH 8.2, a rapid depletion of CO2 upon illumination to a CO2 compensation concentration of 15.42 and 12.03 µm in S. minor and S. cylindricus, respectively. Both species also exhibit active CO2 uptake: addition of bovine CA at CO2 compensation concentration caused a rapid rise in CO2 as the CO2–HCO3‐ equilibrium was restored. Accumulation of unfixed Ci by cells at pH 8.2 was calculated to be 84.33 mm in S. cylindricus, and 30.37 mm in S. minor to give internal accumulations of 23‐ and 8‐fold, respectively, compared to the external Ci concentration.
This paper is the first to describe the occurrence of both active CO2 and active bicarbonate uptake in marine green algae and corrects previous published studies on Stichococcus species, which contended that no active transport of inorganic carbon occurred in these algae at seawater pH. The data also demonstrates that the rate of active CO2 uptake greatly exceeds that of bicarbonate uptake in a marine alga and also demonstrates the effectiveness of external carbonic anhydrase in stimulating inorganic carbon uptake in a marine alga.
The Aster-C protein (encoded by the
gene) is an endoplasmic reticulum (ER) resident protein that has been reported to transport cholesterol from the plasma membrane to the ER. Although there is a ...clear role for the closely-related Aster-B protein in cholesterol transport and downstream esterification in the adrenal gland, the specific role for Aster-C in cholesterol homeostasis is not well understood. Here, we have examined whole body cholesterol balance in mice globally lacking Aster-C under low or high dietary cholesterol conditions.
Age-matched
and
mice were fed either low (0.02%, wt/wt) or high (0.2%, wt/wt) dietarycholesterol and levels of sterol-derived metabolites were assessed in the feces, liver, and plasma.
Compared to wild type controls (
) mice, mice lacking
(
) have no significant alterations in fecal, liver, or plasma cholesterol. Given the potential role for Aster C in modulating cholesterol metabolism in diverse tissues, we quantified levels of cholesterol metabolites such as bile acids, oxysterols, and steroid hormones. Compared to
controls,
mice had modestly reduced levels of select bile acid species and elevated cortisol levels, only under low dietary cholesterol conditions. However, the vast majority of bile acids, oxysterols, and steroid hormones were unaltered in
mice. Bulk RNA sequencing in the liver showed that
mice did not exhibit alterations in sterol-sensitive genes, but instead showed altered expression of genes in major urinary protein and cytochrome P450 (CYP) families only under low dietary cholesterol conditions.
Collectively, these data indicate nominal effects of Aster-C on whole body cholesterol transport and metabolism under divergent dietary cholesterol conditions. These results strongly suggest that Aster-C alone is not sufficient to control whole body cholesterol balance, but can modestly impact circulating cortisol and bile acid levels when dietary cholesterol is limited.
PDF
