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1.  Microbiota-Dependent Hepatic Lipogenesis Mediated by Stearoyl CoA Desaturase 1 (SCD1) Promotes Metabolic Syndrome in TLR5-Deficient Mice 
Cell metabolism  2015;22(6):983-996.
SUMMARY
The gut microbiota plays a key role in host metabolism. Toll-Like Receptor 5 (TLR5), a flagellin receptor, is required for gut microbiota homeostasis. Accordingly, TLR5 deficient (T5KO) mice are prone to develop microbiota-dependent metabolic syndrome. Here we observed that T5KO mice display elevated neutral lipids with a compositional increase of oleate [C18:1 (n9)] relative to wild-type littermates. Increased oleate contribution to hepatic lipids and liver SCD1 expression were both microbiota-dependent. Analysis of short chain fatty acids (SCFA) and 13C-acetate label incorporation revealed elevated SCFA in ceca and hepatic portal blood and, increased liver de novo lipogenesis in T5KO mice. Dietary SCFA further aggravated metabolic syndrome in T5KO mice. Deletion of hepatic SCD1 not only prevented hepatic neutral lipid oleate enrichment but also ameliorated metabolic syndrome in T5KO mice. Collectively, these results underscore the key role of the gut microbiota-liver axis in the pathogenesis of metabolic diseases.
Graphical Abstract
doi:10.1016/j.cmet.2015.09.028
PMCID: PMC4670569  PMID: 26525535
Toll-like receptor 5; Gut bacteria; Short chain fatty acids; Hepatic neutral lipids; Monounsaturated fatty acids; Low-grade inflammation; Metabolic diseases
2.  Farnesoid X Receptor Signaling Shapes the Gut Microbiota and Controls Hepatic Lipid Metabolism 
mSystems  2016;1(5):e00070-16.
The farnesoid X receptor (FXR) plays an important role in mediating the dialog between the host and gut microbiota, particularly through modulation of enterohepatic circulation of bile acids. Mounting evidence suggests that genetic ablation of Fxr in the gut or gut-restricted chemical antagonism of the FXR promotes beneficial health effects, including the prevention of nonalcoholic fatty liver disease in rodent models. However, questions remain unanswered, including whether modulation of FXR activity plays a role in shaping the gut microbiota community structure and function and what metabolic pathways of the gut microbiota contribute in an FXR-dependent manner to the host phenotype. In this report, new insights are gained into the metabolic contribution of the gut microbiota to the metabolic phenotypes, including establishing a link between FXR antagonism, bacterial bile salt hydrolase activity, and fermentation. Multiple approaches, including unique mouse models as well as metabolomics and genome-scale metabolic models, were employed to confirm these results.
ABSTRACT
The gut microbiota modulates obesity and associated metabolic phenotypes in part through intestinal farnesoid X receptor (FXR) signaling. Glycine-β-muricholic acid (Gly-MCA), an intestinal FXR antagonist, has been reported to prevent or reverse high-fat diet (HFD)-induced and genetic obesity, insulin resistance, and fatty liver; however, the mechanism by which these phenotypes are improved is not fully understood. The current study investigated the influence of FXR activity on the gut microbiota community structure and function and its impact on hepatic lipid metabolism. Predictions about the metabolic contribution of the gut microbiota to the host were made using 16S rRNA-based PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), then validated using 1H nuclear magnetic resonance-based metabolomics, and results were summarized by using genome-scale metabolic models. Oral Gly-MCA administration altered the gut microbial community structure, notably reducing the ratio of Firmicutes to Bacteroidetes and its PICRUSt-predicted metabolic function, including reduced production of short-chain fatty acids (substrates for hepatic gluconeogenesis and de novo lipogenesis) in the ceca of HFD-fed mice. Metabolic improvement was intestinal FXR dependent, as revealed by the lack of changes in HFD-fed intestine-specific Fxr-null (FxrΔIE) mice treated with Gly-MCA. Integrative analyses based on genome-scale metabolic models demonstrated an important link between Lactobacillus and Clostridia bile salt hydrolase activity and bacterial fermentation. Hepatic metabolite levels after Gly-MCA treatment correlated with altered levels of gut bacterial species. In conclusion, modulation of the gut microbiota by inhibition of intestinal FXR signaling alters host liver lipid metabolism and improves obesity-related metabolic dysfunction.
IMPORTANCE The farnesoid X receptor (FXR) plays an important role in mediating the dialog between the host and gut microbiota, particularly through modulation of enterohepatic circulation of bile acids. Mounting evidence suggests that genetic ablation of Fxr in the gut or gut-restricted chemical antagonism of the FXR promotes beneficial health effects, including the prevention of nonalcoholic fatty liver disease in rodent models. However, questions remain unanswered, including whether modulation of FXR activity plays a role in shaping the gut microbiota community structure and function and what metabolic pathways of the gut microbiota contribute in an FXR-dependent manner to the host phenotype. In this report, new insights are gained into the metabolic contribution of the gut microbiota to the metabolic phenotypes, including establishing a link between FXR antagonism, bacterial bile salt hydrolase activity, and fermentation. Multiple approaches, including unique mouse models as well as metabolomics and genome-scale metabolic models, were employed to confirm these results.
doi:10.1128/mSystems.00070-16
PMCID: PMC5080402  PMID: 27822554
bile acid; farnesoid X receptor; genome-scale metabolic models; gut microbiota; metabolomics; nonalcoholic fatty liver disease
3.  Expression of the aryl hydrocarbon receptor contributes to the establishment of intestinal microbial community structure in mice 
Scientific Reports  2016;6:33969.
Environmental and genetic factors represent key components in the establishment/maintenance of the intestinal microbiota. The aryl hydrocarbon receptor (AHR) is emerging as a pleiotropic factor, modulating pathways beyond its established role as a xenobiotic sensor. The AHR is known to regulate immune surveillance within the intestine through retention of intraepithelial lymphocytes, functional redistribution of Th17/Treg balance. Consequently, environmental/genetic manipulation of AHR activity likely influences host-microbe homeostasis. Utilizing C57BL6/J Ahr−/+ and Ahr−/− co-housed littermates followed by 18 days of genotypic segregation, we examined the influence of AHR expression upon intestinal microbe composition/functionality and host physiology. 16S sequencing/quantitative PCR (qPCR) revealed significant changes in phyla abundance, particularly Verrucomicrobia together with segmented filamentous bacteria, and an increase in species diversity in Ahr−/− mice following genotypic segregation. Metagenomics/metabolomics indicate microbial composition is associated with functional shifts in bacterial metabolism. Analysis identified Ahr−/−-dependent increases in ileal gene expression, indicating increased inflammatory tone. Transfer of Ahr−/− microbiota to wild-type germ-free mice recapitulated the increase Verrucomicrobia and inflammatory tone, indicating Ahr−/−-microbial dependence. These data suggest a role for the AHR in influencing the community structure of the intestinal microbiota.
doi:10.1038/srep33969
PMCID: PMC5034278  PMID: 27659481
4.  THE RELIABILITY OF MAGNETIC RESONANCE ELASTOGRAPHY USING MULTISLICE 2D SPIN-ECHO ECHO-PLANAR IMAGING (SE-EPI) AND 3D INVERSION RECONSTRUCTION FOR ASSESSING RENAL STIFFNESS 
PURPOSE
To evaluate the reliability of MRE using a spin-echo echo-planar imaging (SE-EPI) renal MRE technique in healthy volunteers
MATERIALS AND METHODS
Institutional review board approved prospective study in which all participants provided written informed consent. Sixteen healthy volunteers comprising seven males and nine females with a median age of 35 years (age range: 23 to 59 years) were included. Coronal 90-Hz and 60-Hz MRE acquisitions were performed twice within a 30-minute interval between examinations. Renal MRE reliability was assessed by i) test-retest repeatability, and ii) inter-rater agreement between two independent readers. The MRE-measured averaged renal stiffness values were evaluated using: intraclass correlation coefficient (ICC), Bland-Altman and the within-subject coefficient of variation (COV).
RESULTS
For test-retest repeatability, Bland-Altman showed a mean stiffness difference between examinations of 0.07 kPa (95% limits of agreement: −1.41, 1.54) at 90-Hz and 0.01 kPa (95% limits of agreement: −0.51, 0.53) at 60-Hz. Coefficient of repeatability was 1.47 kPa and 0.52 kPa at 90-Hz and 60-Hz, respectively. The within-subject COV was 13.6% and 7.7% at 90-Hz and 60-Hz, respectively. ICC values were 0.922 and 0.907 for test-retest repeatability and 0.998 and 0.989 for inter-rater agreement, respectively (p < 0.001).
CONCLUSION
SE-EPI renal MRE is a reliable technique
doi:10.1002/jmri.24826
PMCID: PMC4560097  PMID: 25537823
Magnetic resonance elastography; Kidneys; Diagnostic reliability
5.  Metabolomics Reveals that Aryl Hydrocarbon Receptor Activation by Environmental Chemicals Induces Systemic Metabolic Dysfunction in Mice 
Environmental science & technology  2015;49(13):8067-8077.
Environmental exposure to dioxins and dioxin-like compounds poses a significant health risk for human health. Developing a better understanding of the mechanisms of toxicity through activation of the aryl hydrocarbon receptor (AHR) is likely to improve the reliability of risk assessment. In this study, the AHR-dependent metabolic response of mice exposed to 2,3,7,8-tetrachlorodibenzofuran (TCDF) were assessed using global 1H nuclear magnetic resonance (NMR)-based metabolomics and targeted metabolic profiling of extracts obtained from serum and liver. 1H NMR analyses revealed that TCDF exposure suppressed gluconeogenesis and glycogenolysis, stimulated lipogenesis, and triggered inflammatory gene expression in an Ahr-dependent manner. Targeted analyses using gas chromatography mass spectrometry showed TCDF treatment altered the ratio of unsaturated/saturated fatty acids. Consistent with this observation, an increase in hepatic expression of stearoyl coenzyme A desaturase 1 was also observed. In addition, TCDF exposure resulted in inhibition of de novo fatty acid biosynthesis manifested by down-regulation of acetyl-CoA, malonyl-CoA and palmitoyl-CoA metabolites and related mRNA levels. In contrast, no significant changes in the levels of glucose and lipid were observed in serum and liver obtained from Ahr-null mice following TCDF treatment, thus strongly supporting the important role of the AHR in mediating the metabolic effects seen following TCDF exposure.
doi:10.1021/acs.est.5b01389
PMCID: PMC4890155  PMID: 26023891
Metabolomics; TCDF; Nuclear magnetic resonance (NMR); AHR; Gas and liquid chromatography-mass spectrometry (GC/LC-MS)
6.  Role of fibroblast growth factor 21 in the early stage of NASH induced by methionine- and choline-deficient diet 
Biochimica et biophysica acta  2015;1852(7):1242-1252.
Fibroblast growth factor 21 (FGF21) is a modulator of energy homeostasis and is increased in human nonalcoholic liver disease (NAFLD) and after feeding of methionine- and choline-deficient diet (MCD), a conventional inducer of murine nonalcoholic steatohepatitis (NASH). However, the significance of FGF21 induction in the occurrence of MCD-induced NASH remains undetermined. C57BL/6J Fgf21-null and wild-type mice were treated with MCD for 1 week. Hepatic Fgf21 mRNA was increased early after commencing MCD treatment independent of peroxisome proliferator-activated receptor (PPAR) α and farnesoid X receptor. While no significant differences in white adipose lipolysis were seen in both genotypes, hepatic triglyceride (TG) contents were increased in Fgf21-null mice, likely due to the up-regulation of genes encoding CD36 and phosphatidic acid phosphatase 2a/2c, involved in fatty acid (FA) uptake and diacylglycerol synthesis, respectively, and suppression of increased mRNAs encoding carnitine palmitoyl-CoA transferase 1α, PPARγ coactivator 1α, and adipose TG lipase, which are associated with lipid clearance in the liver. The MCD-treated Fgf21-null mice showed increased hepatic endoplasmic reticulum (ER) stress. Exposure of primary hepatocytes to palmitic acid elevated the mRNA levels encoding DNA damage-inducible transcript 3, an indicator of ER stress, and FGF21 in a PPARα-independent manner, suggesting that lipid-induced ER stress can enhance hepatic FGF21 expression. Collectively, FGF21 is induced in the early stage of MCD-induced NASH likely to minimize hepatic lipid accumulation and ensuing ER stress. These results provide a possible mechanism on how FGF21 is increased in NAFLD/NASH.
doi:10.1016/j.bbadis.2015.02.012
PMCID: PMC4433820  PMID: 25736301
ER stress; lipotoxicity; palmitic acid; ATGL; PGC1α; PPARα
7.  Reversing methanogenesis to capture methane for liquid biofuel precursors 
Background
Energy from remote methane reserves is transformative; however, unintended release of this potent greenhouse gas makes it imperative to convert methane efficiently into more readily transported biofuels. No pure microbial culture that grows on methane anaerobically has been isolated, despite that methane capture through anaerobic processes is more efficient than aerobic ones.
Results
Here we engineered the archaeal methanogen Methanosarcina acetivorans to grow anaerobically on methane as a pure culture and to convert methane into the biofuel precursor acetate. To capture methane, we cloned the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable organism, anaerobic methanotrophic archaeal population 1 (ANME-1) from a Black Sea mat, into M. acetivorans to effectively run methanogenesis in reverse. Starting with low-density inocula, M. acetivorans cells producing ANME-1 Mcr consumed up to 9 ± 1 % of methane (corresponding to 109 ± 12 µmol of methane) after 6 weeks of anaerobic growth on methane and utilized 10 mM FeCl3 as an electron acceptor. Accordingly, increases in cell density and total protein were observed as cells grew on methane in a biofilm on solid FeCl3. When incubated on methane for 5 days, high-densities of ANME-1 Mcr-producing M. acetivorans cells consumed 15 ± 2 % methane (corresponding to 143 ± 16 µmol of methane), and produced 10.3 ± 0.8 mM acetate (corresponding to 52 ± 4 µmol of acetate). We further confirmed the growth on methane and acetate production using 13C isotopic labeling of methane and bicarbonate coupled with nuclear magnetic resonance and gas chromatography/mass spectroscopy, as well as RNA sequencing.
Conclusions
We anticipate that our metabolically-engineered strain will provide insights into how methane is cycled in the environment by Archaea as well as will possibly be utilized to convert remote sources of methane into more easily transported biofuels via acetate.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-015-0397-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12934-015-0397-z
PMCID: PMC4714516  PMID: 26767617
Reverse methanogenesis; Anaerobic oxidation of methane; Methyl-coenzyme M reductase
8.  Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction 
Nature Communications  2015;6:10166.
The farnesoid X receptor (FXR) regulates bile acid, lipid and glucose metabolism. Here we show that treatment of mice with glycine-β-muricholic acid (Gly-MCA) inhibits FXR signalling exclusively in intestine, and improves metabolic parameters in mouse models of obesity. Gly-MCA is a selective high-affinity FXR inhibitor that can be administered orally and prevents, or reverses, high-fat diet-induced and genetic obesity, insulin resistance and hepatic steatosis in mice. The high-affinity FXR agonist GW4064 blocks Gly-MCA action in the gut, and intestine-specific Fxr-null mice are unresponsive to the beneficial effects of Gly-MCA. Mechanistically, the metabolic improvements with Gly-MCA depend on reduced biosynthesis of intestinal-derived ceramides, which directly compromise beige fat thermogenic function. Consequently, ceramide treatment reverses the action of Gly-MCA in high-fat diet-induced obese mice. We further show that FXR signalling in ileum biopsies of humans positively correlates with body mass index. These data suggest that Gly-MCA may be a candidate for the treatment of metabolic disorders.
The nuclear farnesoid X receptor (FXR) is activated by bile acids and influences energy metabolism. Here, the authors report a small molecule inhibitor of FXR, glycine-ß-muricholic acid, which inhibits FXR in the intestine and improves metabolic homeostasis by repressing intestinal ceramide synthesis.
doi:10.1038/ncomms10166
PMCID: PMC4682112  PMID: 26670557
9.  Microbial determinants of biochemical individuality and their impact on toxicology and pharmacology 
Cell metabolism  2014;20(5):761-768.
SUMMARY
Humans exhibit remarkable inter-individual variations in the concentration of small molecules found throughout the body, due in part to concurrent variations in each person’s associated microbial communities. Recent studies have begun to uncover how microbes interface with their host during exposure to drugs, dietary compounds, and environmental toxicants, with broader implications regarding the causes and consequences of biochemical individuality. Progress in this area will likely be an essential component of personalized medicine and might be accelerated through the implementation of experimental designs and theoretical principles honed through decades of work in the fields of toxicology and pharmacology.
doi:10.1016/j.cmet.2014.07.002
PMCID: PMC4252706  PMID: 25156450
human microbiome; pharmacology; toxicology; xenobiotic metabolism
10.  Crucial role of macrophage selenoproteins in experimental colitis 
Inflammation is a hallmark of inflammatory bowel disease (IBD) that involves macrophages. Given the inverse link between selenium (Se) status and IBD-induced inflammation, our objective was to demonstrate that selenoproteins in macrophages were essential to suppress pro-inflammatory mediators, in part, by the modulation of arachidonic acid metabolism. Acute colitis was induced using 4% DSS in wild type mice maintained on Se-deficient (<0.01 ppm Se), Se-adequate (0.1 ppm; sodium selenite), and two supraphysiological levels in the form of Se-supplemented (0.4 ppm; sodium selenite) and high Se (1.0 ppm; sodium selenite) diets. Transfer RNASec (tRNA[sec]) knockout mice (Trspfl/flLysMCre) were used to examine the role of selenoproteins in macrophages on disease progression and severity using histopathological evaluation, expression of pro-inflammatory and anti-inflammatory genes, and modulation of prostaglandin (PG) metabolites in urine and plasma. While Se-deficient and Se-adequate mice showed increased colitis and exhibited poor survival, Se supplementation at 0.4 and 1.0 ppm increased survival of mice and decreased colitis-associated inflammation with an up-regulation of expression of pro-inflammatory and anti-inflammatory genes. Metabolomic profiling of urine suggested increased oxidation of PGE2 at supraphysiological levels of Se that also correlated well with Se-dependent upregulation of 15-hydroxy-PG dehydrogenase (15-PGDH) in macrophages. Pharmacological inhibition of 15-PGDH, lack of selenoprotein expression in macrophages, and depletion of infiltrating macrophages indicated that macrophage-specific selenoproteins and upregulation of 15-PGDH expression were key for Se-dependent anti-inflammatory and pro-resolving effects. Selenoproteins in macrophages protect mice from DSS-colitis by enhancing 15-PGDH-dependent oxidation of PGE2 to alleviate inflammation, suggesting a therapeutic role for Se in IBD.
doi:10.4049/jimmunol.1400347
PMCID: PMC4170023  PMID: 25187657
Selenium; macrophage activation; PGE2; IBD; 15-PGDH
11.  Targeted transperineal biopsy of the prostate has limited additional benefit over background cores for larger MRI-identified tumors 
World Journal of Urology  2015;34:501-508.
Purpose
To compare histological outcomes in patients undergoing MRI–transrectal ultrasound fusion transperineal (MTTP) prostate biopsy and determine the incremental benefit of targeted cores.
Methods
Seventy-six consecutive patients with 89 MRI-identified targets underwent MTTP biopsy. Separate targeted biopsies and background cores were obtained according to a standardized protocol. Target biopsies were considered of added diagnostic value if these cores showed a higher Gleason grade than non-targeted cores taken from the same sector (Group 1, n = 41). Conversely, where background cores demonstrated an equal or higher Gleason grade, target cores were considered to be non-beneficial (Group 2, n = 48).
Results
There was no significant difference in age, PSA, prostate volume, time-to-biopsy, and number of cores obtained between the groups. A greater proportion of target cores were positive for cancer (158/228; 69.3 %) compared to background (344/1881; 18.38 %). The median target volume was 0.54 cm3 for Group 1 (range 0.09–2.79 cm3) and 1.65 cm3 for Group 2 (0.3–9.07 cm3), p < 0.001. The targets in Group 1 had statistically lower diameters for short and long axes, even after correction for gland size. The highest area under the receiver operating characteristic curve was demonstrated when a lesion cutoff value of 1.0 cm in short axis was applied, resulting in a sensitivity of 83.3 % and a specificity of 82.9 %.
Conclusions
When a combined systematic and targeted transperineal prostate biopsy is performed, there is limited benefit in acquiring additional cores from larger-volume targets with a short axis diameter >1.0 cm.
doi:10.1007/s00345-015-1650-0
PMCID: PMC4799791  PMID: 26238348
Image-guided biopsy; Magnetic resonance imaging; Ultrasound; Transperineal; Prostate cancer
12.  Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles 
Scientific Reports  2015;5:12689.
Ligand activation of the aryl hydrocarbon (AHR) has profound effects upon the immunological status of the gastrointestinal tract, establishing and maintaining signaling networks, which facilitate host-microbe homeostasis at the mucosal interface. However, the identity of the ligand(s) responsible for such AHR-mediated activation within the gut remains to be firmly established. Here, we combine in vitro ligand binding, quantitative gene expression, protein-DNA interaction and ligand structure activity analyses together with in silico modeling of the AHR ligand binding domain to identify indole, a microbial tryptophan metabolite, as a human-AHR selective agonist. Human AHR, acting as a host indole receptor may exhibit a unique bimolecular (2:1) binding stoichiometry not observed with typical AHR ligands. Such bimolecular indole-mediated activation of the human AHR within the gastrointestinal tract may provide a foundation for inter-kingdom signaling between the enteric microflora and the immune system to promote commensalism within the gut.
doi:10.1038/srep12689
PMCID: PMC4522678  PMID: 26235394
13.  Lack of soluble fiber drives diet-induced adiposity in mice 
Diet-induced obesity is often modeled by comparing mice fed high-fat diet (HFD), which is made from purified ingredients, vs. normal chow diet (NCD), which is a low-fat assemblage of relatively unrefined plant and animal products. The mechanism by which HFD promotes adiposity is complex but thought to involve low-grade inflammation and altered gut microbiota. The goal of this study was to investigate the extent to which HFD-induced adiposity is driven by fat content vs. other factors that differentiate HFD vs. NCD. Mice were fed NCD, HFD, or other compositionally defined diets (CDD), designed to mimic NCD and/or explore the role of HFD components. A range of metabolic parameters reflecting low-grade inflammation and adiposity were assayed. Relative to NCD, HFD, and to a lesser, but, nonetheless, significant extent, CDD induced increased adiposity, indicating both lipid content and other aspects of HFD are obesogenic. Moreover, HFD and CDD induced a rapid and marked loss of cecal and colonic mass. Such CDD-induced effects were not affected by adjusting dietary protein levels/types but could be largely eliminated by exchanging insoluble fiber (cellulose) for soluble fiber (inulin). Replacing cellulose with inulin in HFD also protected mice against decreased intestinal mass, hyperphagia, and increased adiposity. Such beneficial effects of inulin were microbiota dependent, correlated with elevated fecal short-chain fatty acid levels analyzed via 1H-NMR-based metabolomics and were partially recapitulated by administration of short-chain fatty acid. HFD-induced obesity is strongly promoted by its lack of soluble fiber, which supports microbiota-mediated intestinal tissue homeostasis that prevents inflammation driving obesity and metabolic syndrome.
doi:10.1152/ajpgi.00172.2015
PMCID: PMC4593822  PMID: 26185332
microbiota; short-chain fatty acids; low-grade inflammation; metabolic syndrome
14.  Persistent Organic Pollutants Modify Gut Microbiota–Host Metabolic Homeostasis in Mice Through Aryl Hydrocarbon Receptor Activation 
Environmental Health Perspectives  2015;123(7):679-688.
Background
Alteration of the gut microbiota through diet and environmental contaminants may disturb physiological homeostasis, leading to various diseases including obesity and type 2 diabetes. Because most exposure to environmentally persistent organic pollutants (POPs) occurs through the diet, the host gastrointestinal tract and commensal gut microbiota are likely to be exposed to POPs.
Objectives
We examined the effect of 2,3,7,8-tetrachlorodibenzofuran (TCDF), a persistent environmental contaminant, on gut microbiota and host metabolism, and we examined correlations between gut microbiota composition and signaling pathways.
Methods
Six-week-old male wild-type and Ahr–/– mice on the C57BL/6J background were treated with 24 μg/kg TCDF in the diet for 5 days. We used 16S rRNA gene sequencing, 1H nuclear magnetic resonance (NMR) metabolomics, targeted ultra-performance liquid chromatography coupled with triplequadrupole mass spectrometry, and biochemical assays to determine the microbiota compositions and the physiological and metabolic effects of TCDF.
Results
Dietary TCDF altered the gut microbiota by shifting the ratio of Firmicutes to Bacteroidetes. TCDF-treated mouse cecal contents were enriched with Butyrivibrio spp. but depleted in Oscillobacter spp. compared with vehicle-treated mice. These changes in the gut microbiota were associated with altered bile acid metabolism. Further, dietary TCDF inhibited the farnesoid X receptor (FXR) signaling pathway, triggered significant inflammation and host metabolic disorders as a result of activation of bacterial fermentation, and altered hepatic lipogenesis, gluconeogenesis, and glycogenolysis in an AHR-dependent manner.
Conclusion
These findings provide new insights into the biochemical consequences of TCDF exposure involving the alteration of the gut microbiota, modulation of nuclear receptor signaling, and disruption of host metabolism.
Citation
Zhang L, Nichols RG, Correll J, Murray IA, Tanaka N, Smith PB, Hubbard TD, Sebastian A, Albert I, Hatzakis E, Gonzalez FJ, Perdew GH, Patterson AD. 2015. Persistent organic pollutants modify gut microbiota–host metabolic homeostasis in mice through aryl hydrocarbon receptor activation. Environ Health Perspect 123:679–688; http://dx.doi.org/10.1289/ehp.1409055
doi:10.1289/ehp.1409055
PMCID: PMC4492271  PMID: 25768209
15.  Non-invasive urinary metabolomic profiling identifies diagnostic and prognostic markers in lung cancer 
Cancer research  2014;74(12):3259-3270.
Lung cancer remains the most common cause of cancer deaths worldwide, yet there is currently a lack of diagnostic noninvasive biomarkers that could guide treatment decisions. Small molecules (<1500 Da) were measured in urine collected from 469 lung cancer patients and 536 population controls using unbiased liquid chromatography-mass spectrometry. Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points and further validated in an independent sample set, which comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared to the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA), were each significantly (P <0.00001) elevated in non–small cell lung cancer (NSCLC) and associated with worse prognosis (hazard ratio (HR) =1.81 [P =0.0002], and 1.54 [P =0.025], respectively). Creatine riboside was the strongest classifier of lung cancer status in all and stage I–II cases, important for early detection, and also associated with worse prognosis in stage I–II lung cancer (HR =1.71, P =0.048). All measurements were highly reproducible with intraclass correlation coefficients ranging from 0.82 – 0.99. Both metabolites were significantly (P <0.03) enriched in tumor tissue compared to adjacent non-tumor tissue (N =48), thus revealing their direct association with tumor metabolism. Creatine riboside and NANA may be robust urinary clinical metabolomic markers that are elevated in tumor tissue and associated with early lung cancer diagnosis and worse prognosis.
doi:10.1158/0008-5472.CAN-14-0109
PMCID: PMC4100625  PMID: 24736543
lung cancer; metabolomics; urine; diagnosis; prognosis
16.  Non-Directed Allylic C–H Acetoxylation in the Presence of Lewis Basic Heterocycles 
We outline a strategy to enable non-directed Pd(II)-catalyzed C–H functionalization in the presence of Lewis basic heterocycles. In a high-throughput screen of two Pd-catalyzed C–H acetoxylation reactions, addition of a variety of N-containing heterocycles is found to cause low product conversion. A pyridine-containing test substrate is selected as representative of heterocyclic scaffolds that are hypothesized to cause catalyst arrest. We pursue two approaches in parallel that allow product conversion in this representative system: Lewis acids are found to be effective in situ blocking groups for the Lewis basic site, and a pre-formed pyridine N-oxide is shown to enable high yield of allylic C–H acetoxylation. Computational studies with density functional theory (M06) of binding affinities of selected heterocycles to Pd(OAc)2 provide an inverse correlation of the computed heterocycle–Pd(OAc)2 binding affinities with the experimental conversions to products. Additionally, 1H NMR binding studies provide experimental support for theoretical calculations.
doi:10.1039/C3SC53414F
PMCID: PMC4323382  PMID: 25685311
18.  Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease 
Nonalcoholic fatty liver disease (NAFLD) is a major worldwide health problem. Recent studies suggest that the gut microbiota influences NAFLD pathogenesis. Here, a murine model of high-fat diet–induced (HFD-induced) NAFLD was used, and the effects of alterations in the gut microbiota on NAFLD were determined. Mice treated with antibiotics or tempol exhibited altered bile acid composition, with a notable increase in conjugated bile acid metabolites that inhibited intestinal farnesoid X receptor (FXR) signaling. Compared with control mice, animals with intestine-specific Fxr disruption had reduced hepatic triglyceride accumulation in response to a HFD. The decrease in hepatic triglyceride accumulation was mainly due to fewer circulating ceramides, which was in part the result of lower expression of ceramide synthesis genes. The reduction of ceramide levels in the ileum and serum in tempol- or antibiotic-treated mice fed a HFD resulted in downregulation of hepatic SREBP1C and decreased de novo lipogenesis. Administration of C16:0 ceramide to antibiotic-treated mice fed a HFD reversed hepatic steatosis. These studies demonstrate that inhibition of an intestinal FXR/ceramide axis mediates gut microbiota–associated NAFLD development, linking the microbiome, nuclear receptor signaling, and NAFLD. This work suggests that inhibition of intestinal FXR is a potential therapeutic target for NAFLD treatment.
doi:10.1172/JCI76738
PMCID: PMC4382255  PMID: 25500885
19.  Kernel approaches for differential expression analysis of mass spectrometry-based metabolomics data 
BMC Bioinformatics  2015;16(1):77.
Background
Data generated from metabolomics experiments are different from other types of “-omics” data. For example, a common phenomenon in mass spectrometry (MS)-based metabolomics data is that the data matrix frequently contains missing values, which complicates some quantitative analyses. One way to tackle this problem is to treat them as absent. Hence there are two types of information that are available in metabolomics data: presence/absence of a metabolite and a quantitative value of the abundance level of a metabolite if it is present. Combining these two layers of information poses challenges to the application of traditional statistical approaches in differential expression analysis.
Results
In this article, we propose a novel kernel-based score test for the metabolomics differential expression analysis. In order to simultaneously capture both the continuous pattern and discrete pattern in metabolomics data, two new kinds of kernels are designed. One is the distance-based kernel and the other is the stratified kernel. While we initially describe the procedures in the case of single-metabolite analysis, we extend the methods to handle metabolite sets as well.
Conclusions
Evaluation based on both simulated data and real data from a liver cancer metabolomics study indicates that our kernel method has a better performance than some existing alternatives. An implementation of the proposed kernel method in the R statistical computing environment is available at http://works.bepress.com/debashis_ghosh/60/.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-015-0506-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12859-015-0506-3
PMCID: PMC4359587  PMID: 25887233
Differential expression analysis; Distance-based kernel; Metabolite; Stratified kernel
20.  The use of error-category mapping in pharmacokinetic model analysis of dynamic contrast-enhanced MRI data 
Magnetic Resonance Imaging  2015;33(2):246-251.
This study introduces the use of ‘error-category mapping’ in the interpretation of pharmacokinetic (PK) model parameter results derived from dynamic contrast-enhanced (DCE-) MRI data.
Eleven patients with metastatic renal cell carcinoma were enrolled in a multiparametric study of the treatment effects of bevacizumab. For the purposes of the present analysis, DCE-MRI data from two identical pre-treatment examinations were analysed by application of the extended Tofts model (eTM), using in turn a model arterial input function (AIF), an individually-measured AIF and a sample-average AIF. PK model parameter maps were calculated. Errors in the signal-to-gadolinium concentration ([Gd]) conversion process and the model-fitting process itself were assigned to category codes on a voxel-by-voxel basis, thereby forming a colour-coded ‘error-category map’ for each imaged slice.
These maps were found to be repeatable between patient visits and showed that the eTM converged adequately in the majority of voxels in all the tumours studied. However, the maps also clearly indicated sub-regions of low Gd uptake and of non-convergence of the model in nearly all tumours. The non-physical condition ve ≥ 1 was the most frequently indicated error category and appeared sensitive to the form of AIF used.
This simple method for visualisation of errors in DCE-MRI could be used as a routine quality-control technique and also has the potential to reveal otherwise hidden patterns of failure in PK model applications.
doi:10.1016/j.mri.2014.10.010
PMCID: PMC4728188  PMID: 25460333
PK, pharmacokinetic; DCE-MRI, dynamic contrast-enhanced magnetic resonance imaging; eTM, extended Tofts model; AIF, arterial input function; AIC, Akaike information criterion; mRCC, metastatic renal cell carcinoma; MFA, multiple flip angles; ROI, region of interest; DCE-MRI; pharamacokinetic modeling; error analysis; metastatic renal cell carcinoma; repeatability
21.  Glucocorticoid Modulates Angiotensin II Receptor Expression Patterns and Protects the Heart from Ischemia and Reperfusion Injury 
PLoS ONE  2014;9(9):e106827.
Glucocorticoid regulates angiotensin II receptor (ATR) expression via activating glucocorticoid receptors and binding to glucocorticoid response elements. The regulation of ATR by glucocorticoids in the context of myocardial injury from ischemia/reperfusion (I/R) is yet to be elucidated. The present study determined the role of ATR in glucocorticoid-induced cardiac protection. Adult male rats were administered once a day i.p. 1 mg/kg/day dexamethasone or dexamethasone plus 10 mg/kg/day RU486 for 5 days. Hearts were then isolated and subjected to I/R injury in a Langendorff preparation. Dexamethasone treatment significantly decreased I/R injury and improved post-ischemic recovery of cardiac function. Dexamethasone increased glucocorticoid receptor binding to glucocorticoid response elements at AT1aR and AT2R promoters, resulting in a significant increase in expression of AT1R protein but a decrease in AT2R expression in the heart. In addition, dexamethasone treatment significantly increased PKCε expression and p-PKCε protein abundance. These dexamethasone-mediated effects were blocked by RU486. More importantly, blockade of AT1R and AT2R with losartan and PD123319 abrogated dexamethasone-induced protection of the heart from I/R injury. The results indicate that glucocorticoid promotes a cardioprotective phenotype associated with the upregulation of AT1R and PKCε and downregulation of AT2R in the heart.
doi:10.1371/journal.pone.0106827
PMCID: PMC4180065  PMID: 25265380
22.  Species-specific ant brain manipulation by a specialized fungal parasite 
Background
A compelling demonstration of adaptation by natural selection is the ability of parasites to manipulate host behavior. One dramatic example involves fungal species from the genus Ophiocordyceps that control their ant hosts by inducing a biting behavior. Intensive sampling across the globe of ants that died after being manipulated by Ophiocordyceps suggests that this phenomenon is highly species-specific. We advance our understanding of this system by reconstructing host manipulation by Ophiocordyceps parasites under controlled laboratory conditions and combining this with field observations of infection rates and a metabolomics survey.
Results
We report on a newly discovered species of Ophiocordyceps unilateralis sensu lato from North America that we use to address the species-specificity of Ophiocordyceps-induced manipulation of ant behavior. We show that the fungus can kill all ant species tested, but only manipulates the behavior of those it infects in nature. To investigate if this could be explained at the molecular level, we used ex vivo culturing assays to measure the metabolites that are secreted by the fungus to mediate fungus-ant tissue interactions. We show the fungus reacts heterogeneously to brains of different ant species by secreting a different array of metabolites. By determining which ion peaks are significantly enriched when the fungus is grown alongside brains of its naturally occurring host, we discovered candidate compounds that could be involved in behavioral manipulation by O. unilateralis s.l.. Two of these candidates are known to be involved in neurological diseases and cancer.
Conclusions
The integrative work presented here shows that ant brain manipulation by O. unilateralis s.l. is species-specific seemingly because the fungus produces a specific array of compounds as a reaction to the presence of the host brain it has evolved to manipulate. These studies have resulted in the discovery of candidate compounds involved in establishing behavioral manipulation by this specialized fungus and therefore represent a major advancement towards an understanding of the molecular mechanisms underlying this phenomenon.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-014-0166-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12862-014-0166-3
PMCID: PMC4174324  PMID: 25085339
Behavioral manipulation; Host specificity; Secretome; Metabolomics; Ophiocordyceps unilateralis
23.  Optimization of Improved Motion-sensitized Driven-equilibrium (iMSDE) blood suppression for carotid artery wall imaging 
Background
Improved motion-sensitized driven-equilibrium (iMSDE) preparations have been successfully used in carotid artery wall imaging to achieve blood suppression, but it causes notable signal loss, mostly due to inherent T2 decay, eddy current effects and B1+ inhomogeneity. In this study, we investigate the signal to noise ratio (SNR) and blood suppression performance of iMSDE using composite RF pulses and sinusoidal gradients. Optimized first moment (m1) values for iMSDE prepared T1- and T2- weighted (T1- and T2-w) imaging are presented.
Methods
Twelve healthy volunteers and six patients with carotid artery disease underwent iMSDE and double inversion recovery (DIR) prepared T1- and T2-w fast spin echo (FSE) MRI of the carotid arteries. Modified iMSDE module using composite RF pulses and sinusoidal gradients were evaluated with a range of m1. SNR of adjacent muscle, vessel wall and the lumen were reported. The optimized iMSDE module was also tested in a 3D variable flip angle FSE (CUBE) acquisition.
Results
The SNR of muscle was highest using sinusoidal gradients, and the relative improvement over the trapezoidal gradient increased with higher m1 (p<0.001). Optimal SNR was observed using an iMSDE preparation scheme containing two 180° composite pulses and standard 90° and -90° pulses (p=0.151). iMSDE produced better blood suppression relative to DIR preparations even with a small m1 of 487 mT*ms2/m (p<0.001). In T1-w iMSDE, there was a SNR decrease and an increased T2 weighting with increasing m1. In T2-w iMSDE, by matching the effective echo time (TE), the SNR was equivalent when m1 was <= 1518 mT*ms2/m, however, higher m1 values (2278 – 3108 mT*ms2/m) reduced the SNR. In the patient study, iMSDE improved blood suppression but reduced vessel wall CNR efficiency in both T1-w and T2-w imaging. iMSDE also effectively suppressed residual flow artifacts in the CUBE acquisition.
Conclusions
iMSDE preparation achieved better blood suppression than DIR preparation with reduced vessel wall CNR efficiency in T1-w and T2-w images. The optimized m1s are 487 mT*ms2/m for T1-w imaging and 1518 mT*ms2/m for T2-w imaging. Composite 180° refocusing pulses and sinusoidal gradients improve SNR performance. iMSDE further improves the inherent blood suppression of CUBE.
doi:10.1186/s12968-014-0061-5
PMCID: PMC4145260  PMID: 25160911
Carotid atheroma; Blood suppression optimization; Fast spin echo; Motion-sensitized driven-equilibrium
24.  Stable Isotope- and Mass Spectrometry-based Metabolomics as Tools in Drug Metabolism: A Study Expanding Tempol Pharmacology 
Journal of proteome research  2013;12(3):1369-1376.
The application of mass spectrometry-based metabolomics in the field of drug metabolism has yielded important insights not only into the metabolic routes of drugs but has provided unbiased, global perspectives of the endogenous metabolome that can be useful for identifying biomarkers associated with mechanism of action, efficacy, and toxicity. In this report, a stable isotope- and mass spectrometry-based metabolomics approach that captures both drug metabolism and changes in the endogenous metabolome in a single experiment is described. Here the antioxidant drug tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) was chosen because its mechanism of action is not completely understood and its metabolic fate has not been studied extensively. Furthermore, its small size (MW = 172.2) and chemical composition (C9H18NO2) makes it challenging to distinguish from endogenous metabolites. In this study, mice were dosed with tempol or deuterated tempol (C9D17HNO2) and their urine profiled using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Principal component analysis of the urinary metabolomics data generated a Y-shaped scatter plot containing drug metabolites (protonated and deuterated) that were clearly distinct from the endogenous metabolites. Ten tempol drug metabolites, including eight novel metabolites, were identified. Phase II metabolism was the major metabolic pathway of tempol in vivo, including glucuronidation and glucosidation. Urinary endogenous metabolites significantly elevated by tempol treatment included 2,8-dihydroxyquinoline (8.0-fold, P<0.05) and 2,8-dihydroxyquinoline-β-D-glucuronide (6.8-fold, P<0.05). Urinary endogenous metabolites significantly attenuated by tempol treatment including pantothenic acid (1.3-fold, P<0.05) and isobutrylcarnitine (5.3-fold, P<0.01). This study underscores the power of a stable isotope- and mass spectrometry-based metabolomics in expanding the view of drug pharmacology.
doi:10.1021/pr301023x
PMCID: PMC3594779  PMID: 23301521
Tempol; Stable Isotope; Metabolomics; Mass spectrometry; Drug Metabolism
25.  Metabolomics: An Essential Tool to Understand the Function of Peroxisome Proliferator–Activated Receptor Alpha 
Toxicologic pathology  2012;41(2):410-418.
The peroxisome proliferator–activated receptor (PPAR) family of nuclear hormone transcription factors (PPARα, PPARβ/δ, and PPARγ) is regulated by a wide array of ligands including natural and synthetic chemicals. PPARs have important roles in control of energy metabolism and are known to influence inflammation, differentiation, carcinogenesis, and chemical toxicity. As such, PPARs have been targeted as therapy for common disorders such as cancer, metabolic syndrome, obesity, and diabetes. The recent application of metabolomics, or the global, unbiased measurement of small molecules found in biofluids, or extracts from cells, tissues, or organisms, has advanced our understanding of the varied and important roles that the PPARs have in normal physiology as well as in pathophysiological processes. Continued development and refinement of analytical platforms, and the application of new bioinformatics strategies, have accelerated the widespread use of metabolomics and have allowed further integration of small molecules into systems biology. Recent studies using metabolomics to understand PPARα function, as well as to identify PPARα biomarkers associated with drug efficacy/toxicity and drug-induced liver injury, will be discussed.
doi:10.1177/0192623312466960
PMCID: PMC3690496  PMID: 23197196
metabolomics; liver; PPARα; chromatography; mass spectrometry

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