Multi-Omics to Illuminate Signaling Molecules of the Gut Microbiota-Brain Axis

The human gastrointestinal (GI) tract harbors hundreds of trillions of microorganisms, collectively referred to as the gut microbiota. Complex, dynamic, and metabolically active by nature, these commensal microbes carry critical functional roles in mediating host physiology and health, with a perturbed or dysbiotic state linked to a long list of diseases. Recent studies support that gut microbiota, in close proximity to numerous local neurons and immune cells, also actively interacts with the brain, fueling enormous research interest as recently encapsulated as the “microbiota-gut-brain axis.” Although the neuronal gut-brain pathway (through the vagus nerve) is not disputable, the molecular underpinnings of a humoral route (through blood / lymphatic circulation) by which innumerable gut microbial molecular cues translocate from gut to circulating blood with potentials to cross the blood-brain barrier, remain largely unclear. From an environmental health perspective, although studies have been reported on xenobiotic-induced gut microbial perturbations and associated disease risks, whether and how a humoral gut microbiota-brain axis is involved in neurotoxicant exposure-induced effects remains unexplored. There is thus an urgent need to probe the neuroactive potential of microbiota for biomedical and public health causes. Recent upgrade of omics approaches, especially mass spectrometry-based metabolomics and associated cheminformatic algorithms for compound identification, shows promise for enabling high-coverage and untargeted metabolite profiling with much improved accuracy. This dissertation thus aims to integrate these cutting-edge tools and rules for metabolome-wide analysis to probe the metabolome-wide patterns while identifying high-impact molecular cues of the humoral microbiota-brain axis. In Chapter 1, we first conducted a comprehensive review of existing studies to evaluate the functional roles of reported gut microbial molecules (including small molecules and peptides) in lieu of the gut-brain axis and associated neurologic / mental disorders. We call for massive multi-omics mining efforts in resolving the complexity of the gut microbiota-metabolome network while highlighting related chances and challenges for biomedical, nutritional, and environmental causes. In Chapter 2, for a preliminary check, we developed a targeted metabolomics assay to quantitatively determine literature-reported neurotransmitters, tryptophan metabolites, and indole derivatives (n=50) in specific pathogen-free C57BL/6 mice. We validated the assay, evaluated distribution in vivo for these molecules, and collected information as needed for method development of global metabolomics analysis applied to later chapters. To probe the microbiota-brain axis, for a proof-of-concept, we combine high-resolution mass spectrometry profiling, targeted and untargeted compound annotation procedures, and 16S rRNA gene sequencing (as needed) to comparatively profile feces, blood sera, and cerebral cortical brain tissues of C57BL/6 mice under steady state (germ-free vs. conventionally raised; Chapter 3), under neurotoxicant exposure (perfluorooctanoic acid-treated vs. control; Chapter 4), and under dietary treatment of reportedly neuroprotective components (black raspberry-based diet vs. standard AIN-76A diet; Chapter 5). In all three scenarios, we identified distinct metabolome-wide (and microbial) patterns owing to the presence of or perturbed gut microbiota, with biochemical details in support of an active role of the humoral microbiota-brain axis. Together, the massive, novel, and highly informative datasets generated in this dissertation may provide fundamental insights into how resident microbiota interacts with host bodies via the gut-brain axis while laying the foundation for incorporating the role of microbiota into neurotoxicity assessment and for the development of biomarkers and countermeasures against risks induced by the ever-prevailing environmental chemical exposures.Doctor of Philosoph

Exposomics by Liquid Chromatography-ESI-Drift-Tube Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry: A Proof-of-Principle Study

Exposomics is challenging due to the low abundance, vast dynamic range, and structural diversity of exposome molecules, and is further complicated by matrix effects. The recent coupling of ion mobility spectrometry to mass spectrometry (IMS-MS) has been proposed as a promising solution for exposomics because of its increase in coverage, peak capacity, dynamic range, without compromising sensitivity and throughput compared to mass spectrometry alone. Here, we propose the first proof-of-principle study testing an actual IMS-MS instrument for exposomic analysis of complex biological matrices. In this study, a novel drift-tube ion mobility-quadrupole time-of-flight mass spectrometer (DTIMS-QTOF MS) was employed, coupled with cutting-edge phospholipid-removing solid phase extraction (SPE) and hydrophilic interaction chromatography (HILIC). An in-depth evaluation of this SPE-LC-DTIMS-QTOF workflow was conducted via a spiking trial, for which common exposome compounds were queried, collected, and spiked into human plasma at health-relevant levels. Results of this study provide strong evidence endorsing the incorporation of DTIMS into LC-QTOF for exposomics: (i) improved peak capacity without substantial compromise in duty cycle; (ii) EIC noise level isobar ions were revealed as separated by IMS drift spectrum; (iii) a linear dynamic range comparable to LC-QTOF, with some ions observed at much lower concentration levels; (iv) collision cross sections (CCS) (Single-Field calibration) was highly reproducible (0.2% RSD) and accurate (0.9% RSD), and correlated well with mass (R2 > 0.91). This work may be useful for future exposomics practitioners or IMS-MS users in biomedical and environmental arenas

Effects of the Artificial Sweetener Neotame on the Gut Microbiome and Fecal Metabolites in Mice

Although artificial sweeteners are widely used in food industry, their effects on human health remain a controversy. It is known that the gut microbiota plays a key role in human metabolism and recent studies indicated that some artificial sweeteners such as saccharin could perturb gut microbiome and further affect host health, such as inducing glucose intolerance. Neotame is a relatively new low-caloric and high-intensity artificial sweetener, approved by FDA in 2002. However, the specific effects of neotame on gut bacteria are still unknown. In this study, we combined high-throughput sequencing and gas chromatography–mass spectrometry (GC-MS) metabolomics to investigate the effects of neotame on the gut microbiome and fecal metabolite profiles of CD-1 mice. We found that a four-week neotame consumption reduced the alpha-diversity and altered the beta-diversity of the gut microbiome. Firmicutes was largely decreased while Bacteroidetes was significantly increased. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis also indicated that the control mice and neotame-treated mice have different metabolic patterns and some key genes such as butyrate synthetic genes were decreased. Moreover, neotame consumption also changed the fecal metabolite profiles. Dramatically, the concentrations of multiple fatty acids, lipids as well as cholesterol in the feces of neotame-treated mice were consistently higher than controls. Other metabolites, such as malic acid and glyceric acid, however, were largely decreased. In conclusion, our study first explored the specific effects of neotame on mouse gut microbiota and the results may improve our understanding of the interaction between gut microbiome and neotame and how this interaction could influence the normal metabolism of host bodies

Coupling coal pyrolysis with char gasification in a multi-stage fluidized bed to co-produce high-quality tar and syngas

A multi-stage fluidized bed (MSFB) by configuring the distributor with an overflow standpipe between its neighboring stages was developed to couple the powder coal pyrolysis with its resultant char gasification for co-production of tar and syngas. This work succeeded in the smooth operation of MSFB for coal staged conversion. The three modes of coupling pyrolysis and gasification in terms of the one-stage, two-stage and three-stage bed characterized by temperature drop from the bottom up were investigated to evaluate the quality of the liquid and gas products. Coupling low- and mid-temperature tandem coal pyrolysis with high-temperature char gasification in the MSFB improved the quality of tar and syngas. The obtained tar yield was over 80% of the Gray King assay tar yield and its light tar fraction (boiling point &lt; 360 degrees C) was as high as 70-80% in the MSFB. Syngas with CH4 content of 5.2 vol.% was produced that was suitable for SNG production. Inside the reactor, the flow direction of pyrolysis volatiles toward the temperature drop avoided the deep secondary reaction of tar. Syngas and steam from the bottom gasification section could contribute to the formation of light tar and CH4 by affecting the top coal pyrolysis. A comparison with the typical pyrolysis processes suggested that the MSFB process had its own advantages in treating powder coal to produce the high-quality tar and syngas.</p

Conditioning micro fluidized bed for maximal approach of gas plug flow

We have for the first time investigated the gas-solid fluidization behavior in size-reduced beds called micro fluidized bed (Liu. X et al. Chem. Eng. J., 2008, 137: 302-307), but up to now there is not yet any clear definition for the micro fluidized beds (MFBs). This study intends to characterize MFBs in terms of gas back-mixing. The residence time distribution (RTD) and extent of back-mixing of gas were investigated in beds with inner diameters of up to 21 mm for particles of FCC, glass beads and silica sand. The RTD curves of gas, shown as E(t) versus time t, in such beds were determined on the basis of axial dispersion model to obtain the mean residence time (t) over bar, variance sigma(2)(t) and peak height of a given E(t) curve. In terms of these parameters the degree of gas backmixing and its variation were evaluated with respect to particles, bed diameter (D), superficial gas velocity (U-g) and static particle bed height (H-g). It was found that the RTD of gas is subject to a unique correlation between the height of E(t) peak and the variance sigma(2)(t), which makes it clear that the gas flow in an MFB has limited gas dispersion and is highly approaching to a plug flow if sigma(2)(t) is below 0.25 or the height of E(t) peak is larger than 1.0. This refers to the feature that is most desired for an MFB and can thus be the criterion for defining MFB.</p

Serum Metabolomics Identifies Altered Bioenergetics, Signaling Cascades in Parallel with Exposome Markers in Crohn’s Disease

Inflammatory bowel disease (IBD) has stimulated much interest due to its surging incidences and health impacts in the U.S. and worldwide. However, the exact cause of IBD remains incompletely understood, and biomarker is lacking towards early diagnostics and effective therapy assessment. To tackle these, the emerging high-resolution mass spectrometry (HRMS)-based metabolomics shows promise. Here, we conducted a pilot untargeted LC/MS metabolomic profiling in Crohn&#8217;s disease, for which serum samples of both active and inactive cases were collected, extracted, and profiled by a state-of-the-art compound identification workflow. Results show a distinct metabolic profile of Crohn&#8217;s from control, with most metabolites downregulated. The identified compounds are structurally diverse, pointing to important pathway perturbations ranging from energy metabolism (e.g., &#946;-oxidation of fatty acids) to signaling cascades of lipids (e.g., DHA) and amino acid (e.g., L-tryptophan). Importantly, an integral role of gut microbiota in the pathogenesis of Crohn&#8217;s disease is highlighted. Xenobiotics and their biotransformants were widely detected, calling for massive exposomic profiling for future cohort studies as such. This study endorses the analytical capacity of untargeted metabolomics for biomarker development, cohort stratification, and mechanistic interpretation; the findings might be valuable for advancing biomarker research and etiologic inquiry in IBD

An Actionable Annotation Scoring Framework for Gas Chromatography - High Resolution Mass Spectrometry (GCHRMS)

Omics-based technologies have enabled comprehensive characterization of our exposure to environmental chemicals (exposome) as well as assessment of the corresponding biological responses at the molecular level (e.g., metabolome, lipidome, proteome, and genome). By systematically measuring personal exposures and linking these stimuli to biological perturbations, researchers can determine specific chemical exposures of concern, identify mechanisms and biomarkers of toxicity, and design interventions to reduce exposures. However, further advancement of metabolomics and exposomics approaches is limited by a lack of standardization and approaches for assigning confidence to chemical annotations. While a wealth of chemical data is generated by gas chromatography high-resolution mass spectrometry (GC-HRMS), incorporating GC-HRMS data into an annotation framework, and communicating confidence in these assignments is challenging. It is essential to be able to compare chemical data for exposomics studies across platforms to build upon prior knowledge and advance the technology. Here we discuss the major pieces of evidence provided by common GC-HRMS workflows, including retention time and retention index, electron ionization, positive chemical ionization, electron capture negative ionization, and atmospheric pressure chemical ionization spectral matching, molecular ion, accurate mass, isotopic patterns, database occurrence, and occurrence in blanks. We then provide a qualitative framework for incorporating these various lines of evidence for communicating confidence in GC-HRMS data by adapting the Schymanski scoring schema developed for reporting confidence levels by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Validation of our framework is presented using standards spiked in plasma, and confident annotations in outdoor and indoor air samples, showing a false positive rate of 12% for suspect screening for chemical identifications assigned as Level 2 (when structurally similar isomers are not considered false positives). This framework is easily adaptable to various workflows and provides a concise means to communicate confidence in annotations. Further validation, refinements, and adoption of this framework will ideally lead to harmonization across the field, helping to improve the quality and interpretability of compound annotations obtained in GC-HRMS

Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in a murine model of PSC

Objective Conflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. Goal: define the function of complex resident microbes and their association relevant to PSC patients by studying germ-free (GF) and antibiotic-treated specific pathogen-free (SPF) multidrug-resistant 2 deficient ( mdr2 −/− ) mice and microbial profiles in PSC patient cohorts. Design We measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, faecal 16S rRNA gene profiling and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2 −/− mice and targeted metagenomic analysis in PSC patients. Results GF mdr2 −/− mice had 100% mortality by 8 weeks with increasing hepatic bile acid (BA) accumulation and cholestasis. Early SPF autologous stool transplantation rescued liver-related mortality. Inhibition of ileal BA transport attenuated antibiotic-accelerated liver disease and decreased total serum and hepatic BAs. Depletion of vancomycin-sensitive microbiota exaggerated hepatobiliary disease. Vancomycin selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased Enterococcus faecalis and Escherichia coli liver translocation. Colonisation of GF mdr2 −/− mice with translocated E. faecalis and E. coli strains accelerated hepatobiliary inflammation and mortality. Lachnospiraceae colonisation of antibiotic pretreated mdr2 −/− mice reduced liver fibrosis, inflammation and translocation of pathobionts, and SCFA-producing Lachnospiraceae and purified SCFA decreased fibrosis. Faecal Lachnospiraceae negatively associated, and E. faecalis/ Enterobacteriaceae positively associated, with PSC patients’ clinical severity by Mayo risk scores. Conclusions We identified novel functionally protective and detrimental resident bacterial species in mdr2 −/− mice and PSC patients with associated clinical risk score. These insights may guide personalised targeted therapeutic interventions in PSC patients