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Journal articleSarafian MH, Lewis MR, Pechlivanis A, et al., 2015,
Bile Acid Profiling and Quantification in Biofluids Using Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry
, Analytical Chemistry, Vol: 87, Pages: 9662-9670, ISSN: 1520-6882Bile acids are important end products of cholesterol metabolism. While they have been identified as key factors in lipid emulsification and absorption due to their detergent properties, bile acids have also been shown to act as signaling molecules and intermediates between the host and the gut microbiota. To further the investigation of bile acid functions in humans, an advanced platform for high throughput analysis is essential. Herein, we describe the development and application of a 15 min UPLC procedure for the separation of bile acid species from human biofluid samples requiring minimal sample preparation. High resolution time-of-flight mass spectrometry was applied for profiling applications, elucidating rich bile acid profiles in both normal and disease state plasma. In parallel, a second mode of detection was developed utilizing tandem mass spectrometry for sensitive and quantitative targeted analysis of 145 bile acid (BA) species including primary, secondary, and tertiary bile acids. The latter system was validated by testing the linearity (lower limit of quantification, LLOQ, 0.25–10 nM and upper limit of quantification, ULOQ, 2.5–5 μM), precision (≈6.5%), and accuracy (81.2–118.9%) on inter- and intraday analysis achieving good recovery of bile acids (serum/plasma 88% and urine 93%). The ultra performance liquid chromatography–mass spectrometry (UPLC-MS)/MS targeted method was successfully applied to plasma, serum, and urine samples in order to compare the bile acid pool compositional difference between preprandial and postprandial states, demonstrating the utility of such analysis on human biofluids.
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Journal articleCox MJ, Moffatt MF, Cookson WOC, 2015,
Outside In: Sequencing the Lung Microbiome
, American Journal of Respiratory and Critical Care Medicine, Vol: 192, Pages: 403-404, ISSN: 1535-4970 -
Journal articleShoaie S, Ghaffari P, Kovatcheva-Datchary P, et al., 2015,
Quantifying Diet-Induced Metabolic Changes of the Human Gut Microbiome
, Cell Metabolism, Vol: 22, Pages: 320-331, ISSN: 1932-7420The human gut microbiome is known to be associated with various human disorders, but a major challenge is to go beyond association studies and elucidate causalities. Mathematical modeling of the human gut microbiome at a genome scale is a useful tool to decipher microbe-microbe, diet-microbe and microbe-host interactions. Here, we describe the CASINO (Community And Systems-level INteractive Optimization) toolbox, a comprehensive computational platform for analysis of microbial communities through metabolic modeling. We first validated the toolbox by simulating and testing the performance of single bacteria and whole communities in vitro. Focusing on metabolic interactions between the diet, gut microbiota, and host metabolism, we demonstrated the predictive power of the toolbox in a diet-intervention study of 45 obese and overweight individuals and validated our predictions by fecal and blood metabolomics data. Thus, modeling could quantitatively describe altered fecal and serum amino acid levels in response to diet intervention.
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Journal articleBrill S, Law M, El-Emir E, et al., 2015,
Effects of different antibiotic classes on airwaybacteria in stable COPD using culture and molecularQ1 techniques: a randomised controlled trial
, Thorax, Vol: 70, Pages: 930-938, ISSN: 1468-3296BackgroundLong term antibiotic therapy is used to prevent exacerbations of chronic obstructive pulmonary disease (COPD) but there is uncertainty over whether this reduces airway bacteria. The optimum antibiotic choice remains unknown. We conducted an exploratory trial in stable patients with COPD comparing three antibiotic regimens against placebo. MethodsThis was a single-centre, single-blind, randomised placebo-controlled trial (clinicaltrials.gov number NCT01398072). Patients ≥45 years with COPD, FEV1<80% predicted and chronic productive cough were randomised to receive either moxifloxacin 400mg daily for 5 days/4 weeks, doxycycline 100mg/day, azithromycin 250mg 3x/week or one placebo tablet daily for 13 weeks. The primary outcome was the change in total cultured bacterial load in sputum from baseline; secondary outcomes included bacterial load by 16S qPCR, sputum inflammation and antibiotic resistance. Results99 patients were randomised; 86 completed follow-up, were able to expectorate sputum and were analysed. After adjustment, there was a mean reduction in bacterial load of 0.42 log10 cfu/ml (95% CI -0.08, 0.91, p=0.10) with moxifloxacin, 0.11 (-0.33, 0.55, p=0.62) with doxycycline, and 0.08 (-0.38, 0.54, p=0.73) with azithromycin from placebo, respectively. There were also no significant changes in bacterial load measured by 16S qPCR or in airway inflammation. More treatment-related adverse events occurred with moxifloxacin. Of note, mean inhibitory concentrations of cultured isolates increased by at least 3 times over placebo in all treatment arms.ConclusionsTotal airway bacterial load did not decrease significantly after three months of antibiotic therapy. Large increases in antibiotic resistance were seen in all treatment groups and this has important implications for future studies.
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Journal articleShaw AG, Sim K, Randell P, et al., 2015,
Late-onset bloodstream infection and perturbed maturation of the gastrointestinal microbiota in premature infants
, PLOS One, Vol: 10, ISSN: 1932-6203 -
Journal articleSim K, Shaw AG, Randell P, et al., 2015,
Dysbiosis anticipating necrotizing enterocolitis in very premature infants
, Clinical Infectious Diseases, Vol: 60, Pages: 389-397, ISSN: 1537-6591Background. Necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease of premature infants speculatively associated with infection. Suspected NEC can be indistinguishable from sepsis, and in established cases an infant may die within hours of diagnosis. Present treatment is supportive. A means of presymptomatic diagnosis is urgently needed. We aimed to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants.Methods. Fecal samples and clinical data were collected from a 2-year cohort of 369 premature neonates. Next-generation sequencing of 16S ribosomal RNA gene regions was used to characterize the microbiota of prediagnosis fecal samples from 12 neonates with NEC, 8 with suspected NEC, and 44 controls. Logistic regression was used to determine clinical characteristics and operational taxonomic units (OTUs) discriminating cases from controls. Samples were cultured and isolates identified using matrix-assisted laser desorption/ionization–time of flight. Clostridial isolates were typed and toxin genes detected.Results. A clostridial OTU was overabundant in prediagnosis samples from infants with established NEC (P = .006). Culture confirmed the presence of Clostridium perfringens type A. Fluorescent amplified fragment-length polymorphism typing established that no isolates were identical. Prediagnosis samples from NEC infants not carrying profuse C. perfringens revealed an overabundance of a Klebsiella OTU (P = .049). Prolonged continuous positive airway pressure (CPAP) therapy with supplemental oxygen was also associated with increased NEC risk.Conclusions. Two fecal microbiota signatures (Clostridium and Klebsiella OTUs) and need for prolonged CPAP oxygen signal increased risk of NEC in presymptomatic infants. These biomarkers will assist development of a screening tool to allow very early diagnosis of NEC.Clinical Trials Registration. NCT01102738.
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Journal articleSalter SJ, Cox MJ, Turek EM, et al., 2014,
Reagent and laboratory contamination can critically impact sequence-based microbiome analyses
, BMC Biology, Vol: 12, ISSN: 1741-7007BackgroundThe study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents.ResultsIn this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination.ConclusionsThese results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.
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Journal articleMolyneaux PL, Cox MJ, Willis-Owen SAG, et al., 2014,
The role of bacteria in the pathogenesis and progression of idiopathic pulmonary fibrosis
, American Journal of Respiratory and Critical Care Medicine, Vol: 190, Pages: 906-913, ISSN: 1535-4970Rationale:Idiopathic pulmonaryfibrosis (IPF)isa progressivelung diseaseof unknown cause that leads to respiratory failure and death within 5 yearsof diagnosis. Overt respiratory infection and immunosuppression carrya high morbidity and mortality, and polymorphisms in genes related toepithelial integrity and host defense predispose to IPF.Objectives: To investigate the role of bacteria in the pathogenesisand progression of IPF.Methods: We prospectively enrolled patients diagnosed with IPFaccording to international criteria together with healthy smokers,nonsmokers, and subjectswithmoderate chronic obstructive pulmonarydisease as control subjects. Subjects underwent bronchoalveolarlavage (BAL), from which genomic DNA was isolated. The V3–V5region of the bacterial 16S rRNA gene was amplified, allowingquantification of bacterial load and identification of communities by 16SrRNA quantitative polymerase chain reaction and pyrosequencing. Measurements and Main Results: Sixty-five patients with IPFhad double the burden of bacteria in BAL fluid compared with 44 controlsubjects. Baseline bacterial burden predicted the rate of decline in lungvolume and risk of death and associated independently with thers35705950 polymorphism of the MUC5B mucin gene, a proven hostsusceptibilityfactorfor IPF. Sequencing yielded912,883 high-quality readsfrom all subjects.WeidentifiedHaemophilus, Streptococcus,Neisseria, andVeillonella spp. to be more abundant in cases than control subjects.Regression analyses indicated that these specific operational taxonomicunits as well as bacterial burden associated independently with IPF.Conclusions: IPF is characterized by an increased bacterial burdenin BAL that predicts decline in lung function and death. Trials ofantimicrobial therapy are needed to determine if microbial burdenis pathogenic in the disease.
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Journal articleVenkatesh M, Mukherjee S, Wang H, et al., 2014,
Symbiotic bacterial metabolites regulate gastrointestinal barrier function via the Xenobiotic sensor PXR and toll-like receptor 4
, Immunity, Vol: 41, Pages: 296-310, ISSN: 1074-7613Intestinal microbial metabolites are conjectured to affect mucosal integrity through an incompletely characterized mechanism. Here we showed that microbial-specific indoles regulated intestinal barrier function through the xenobiotic sensor, pregnane X receptor (PXR). Indole 3-propionic acid (IPA), in the context of indole, is a ligand for PXR in vivo, and IPA downregulated enterocyte TNF-α while it upregulated junctional protein-coding mRNAs. PXR-deficient (Nr1i2−/−) mice showed a distinctly “leaky” gut physiology coupled with upregulation of the Toll-like receptor (TLR) signaling pathway. These defects in the epithelial barrier were corrected in Nr1i2−/−Tlr4−/− mice. Our results demonstrate that a direct chemical communication between the intestinal symbionts and PXR regulates mucosal integrity through a pathway that involves luminal sensing and signaling by TLR4.
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PatentMartin FP, Boulange CL, Montoliu Roura I, et al., 2014,
Trimethylamine-N-oxide as biomarker for the predisposition for weight gain and obesity
, WO2014086604The present invention relates generally to the field of nutrition and health. In particular, the present invention relates to a new biomarker, its use and a nnethod that allows it to diagnose the likelihood to resist diet induced weight gain, and/or to be susceptible to a diet induced weight gain. For example, the biomarker may be trimethylamine-N-oxide.
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