LC-MS System for Collecting Time-Resolved Metabolomics Data of Cultured Cells.

Temporal metabolic dynamics are difficult to capture but are critical to understanding biology. We developed an automated liquid chromatography-mass spectrometry system that collects time-resolved metabolomics data from cultured cells, enabling sub-minute sequential sampling, broad metabolite coverage, robust metabolite identification, and parallel monitoring of up to 72 experimental conditions. Using this system, we identified temporal metabolic phenotypes of and that could not be captured from single time points.

Metabolomics strategy for diagnosing urinary tract infections.

Metabolomics has emerged as a mainstream approach for investigating complex metabolic phenotypes but has yet to be integrated into routine clinical diagnostics. Metabolomics-based diagnosis of urinary tract infections (UTIs) is a logical application of this technology since microbial waste products are concentrated in the bladder and thus could be suitable markers of infection. We conducted an untargeted metabolomics screen of clinical specimens from patients with suspected UTIs and identified two metabolites, agmatine, and N6-methyladenine, that are predictive of culture-positive samples.

Metabolomics survey of uropathogenic bacteria in human urine.

Introduction: Urinary tract infections (UTIs) are one of the most prevalent infections in North America and are caused by a diverse range of bacterial species. Although uropathogenesis has been studied extensively in the context of macromolecular interactions, the degree to which metabolism may contribute to infection is unclear. Currently, most of what is known about the metabolic capacity of uropathogens has been derived from genomics, genetic knockout studies or transcriptomic analyses.

SCALiR: A Web Application for Automating Absolute Quantification of Mass Spectrometry-Based Metabolomics Data.

Quantitative liquid chromatography-mass spectrometry (LC-MS)-based metabolomics is becoming an important approach for studying complex biological systems but presents several technical challenges that limit its widespread use. Computing metabolite concentrations using standard curves generated from standard mixtures of known concentrations is a labor-intensive process that is often performed manually. Currently, there are few options for open-source software tools that can automatically calculate metabolite concentrations.

SCALiR: a web application for automating absolute quantification of mass spectrometry-based metabolomics data.

Metabolomics is an important approach for studying complex biological systems. Quantitative liquid chromatography-mass spectrometry (LC-MS)-based metabolomics is becoming a mainstream strategy but presents several technical challenges that limit its widespread use. Computing metabolite concentrations using standard curves generated from standard mixtures of known concentrations is a labor-intensive process which is often performed manually.

Rapid LC-MS assay for targeted metabolite quantification by serial injection into isocratic gradients.

Liquid chromatography mass spectrometry (LC-MS) has emerged as a mainstream strategy for metabolomics analyses. One advantage of LC-MS is that it can serve both as a biomarker discovery tool and as a platform for clinical diagnostics. Consequently, it offers an exciting opportunity to potentially transition research studies into real-world clinical tools.

Microbial containment device: A platform for comprehensive analysis of microbial metabolism without sample preparation.

Metabolomics is a mainstream strategy for investigating microbial metabolism. One emerging application of metabolomics is the systematic quantification of metabolic boundary fluxes - the rates at which metabolites flow into and out of cultured cells. Metabolic boundary fluxes can capture complex metabolic phenotypes in a rapid assay, allow computational models to be built that predict the behavior of cultured organisms, and are an emerging strategy for clinical diagnostics.

Biomarker enrichment medium: A defined medium for metabolomic analysis of microbial pathogens.

Microbes have diverse metabolic capabilities and differences in these phenotypes are critical for differentiating strains, species, and broader taxa of microorganisms. Recent advances in liquid chromatography-mass spectrometry (LC-MS) allow researchers to track the complex combinations of molecules that are taken up by each cell type and to quantify the rates that individual metabolites enter or exit the cells. This metabolomics-based approach allows complex metabolic phenotypes to be captured in a single assay, enables computational models of microbial metabolism to be constructed, and can serve as a diagnostic approach for clinical microbiology.

Methods for Quantifying the Metabolic Boundary Fluxes of Cell Cultures in Large Cohorts by High-Resolution Hydrophilic Liquid Chromatography Mass Spectrometry.

Metabolomics is a mainstream approach for investigating the metabolic underpinnings of complex biological phenomena and is increasingly being applied to large-scale studies involving hundreds or thousands of samples. Although metabolomics methods are robust in smaller-scale studies, they can be challenging to apply to larger cohorts due to the inherent variability of liquid chromatography mass spectrometry (LC-MS). Much of this difficulty results from the time-dependent changes in the LC-MS system, which affects both the qualitative and quantitative performances of the instrument.

Metabolic preference assay for rapid diagnosis of bloodstream infections.

Bloodstream infections (BSIs) cause >500,000 infections and >80,000 deaths per year in North America. The length of time between the onset of symptoms and administration of appropriate antimicrobials is directly linked to mortality rates. It currently takes 2-5 days to identify BSI pathogens and measure their susceptibility to antimicrobials - a timeline that directly contributes to preventable deaths.

Untargeted Metabolomics Investigation on Selenite Reduction to Elemental Selenium by SeITE01.

SeITE01 is an environmental isolate that transforms the oxyanion selenite ( ) into the less bioavailable elemental selenium (Se) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (NaSeO) by SeITE01 strain and the effect of exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with .

Bacterial cyclic diguanylate signaling networks sense temperature.

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change.

Staphylococcus aureus induces an itaconate-dominated immunometabolic response that drives biofilm formation.

Staphylococcus aureus is a prominent human pathogen that readily adapts to host immune defenses. Here, we show that, in contrast to Gram-negative pathogens, S. aureus induces a distinct airway immunometabolic response dominated by the release of the electrophilic metabolite, itaconate.

Thermogenic hydrocarbon biodegradation by diverse depth-stratified microbial populations at a Scotian Basin cold seep.

At marine cold seeps, gaseous and liquid hydrocarbons migrate from deep subsurface origins to the sediment-water interface. Cold seep sediments are known to host taxonomically diverse microorganisms, but little is known about their metabolic potential and depth distribution in relation to hydrocarbon and electron acceptor availability. Here we combined geophysical, geochemical, metagenomic and metabolomic measurements to profile microbial activities at a newly discovered cold seep in the deep sea.

Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells.

Due to their ability to standardize key physiological parameters, stirred suspension bioreactors can potentially scale the production of quality-controlled pluripotent stem cells (PSCs) for cell therapy application. Because of differences in bioreactor expansion efficiency between mouse (m) and human (h) PSCs, we investigated if conversion of hPSCs, from the conventional "primed" pluripotent state towards the "naïve" state prevalent in mPSCs, could be used to enhance hPSC production. Through transcriptomic enrichment of mechano-sensing signaling, the expression of epigenetic regulators, metabolomics, and cell-surface protein marker analyses, we show that the stirred suspension bioreactor environment helps maintain a naïve-like pluripotent state.

Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy.

Several species of intestinal bacteria have been associated with enhanced efficacy of checkpoint blockade immunotherapy, but the underlying mechanisms by which the microbiome enhances antitumor immunity are unclear. In this study, we isolated three bacterial species-, , and species-that significantly enhanced efficacy of immune checkpoint inhibitors in four mouse models of cancer. We found that intestinal modulated enhanced immunotherapy response through production of the metabolite inosine.

PelX is a UDP--acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation.

Pel is a GalNAc-rich bacterial polysaccharide that contributes to the structure and function of biofilms. The operon is highly conserved among diverse bacterial species, and Pel may therefore be a widespread biofilm determinant. Previous annotation of gene clusters has helped us identify an additional gene, , that is present adjacent to in >100 different bacterial species.

Metabolic potential of uncultured bacteria and archaea associated with petroleum seepage in deep-sea sediments.

The lack of microbial genomes and isolates from the deep seabed means that very little is known about the ecology of this vast habitat. Here, we investigate energy and carbon acquisition strategies of microbial communities from three deep seabed petroleum seeps (3 km water depth) in the Eastern Gulf of Mexico. Shotgun metagenomic analysis reveals that each sediment harbors diverse communities of chemoheterotrophs and chemolithotrophs.

An -methyltransferase from catalyzing the formation of ephedrine and pseudoephedrine enables microbial phenylalkylamine production.

Phenylalkylamines, such as the plant compounds ephedrine and pseudoephedrine and the animal neurotransmitters dopamine and adrenaline, compose a large class of natural and synthetic molecules with important physiological functions and pharmaceutically valuable bioactivities. The final steps of ephedrine and pseudoephedrine biosynthesis in members of the plant genus involve -methylation of norephedrine and norpseudoephedrine, respectively. Here, using a plant transcriptome screen, we report the isolation and characterization of an -methyltransferase (NMT) from able to catalyze the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines, including -methylcathinone and -methyl(pseudo)ephedrine.

Iron Sequestration in Microbiota Biofilms As A Novel Strategy for Treating Inflammatory Bowel Disease.

Significant alterations of intestinal microbiota and anemia are hallmarks of inflammatory bowel disease (IBD). It is widely accepted that iron is a key nutrient for pathogenic bacteria, but little is known about its impact on microbiota associated with IBD. We used a model device to grow human mucosa-associated microbiota in its physiological anaerobic biofilm phenotype.

Transcriptome profiling of khat (Catha edulis) and Ephedra sinica reveals gene candidates potentially involved in amphetamine-type alkaloid biosynthesis.

Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level.