Next-Generation Probiotics Targeting \u3ci\u3eClostridium difficile\u3c/i\u3e through Precursor- Directed Antimicrobial Biosynthesis

Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of C. difficile, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials

iCircos: Visual Analytics for Translational Bioinformatics

ABSTRACT Translational bioinformatics increasingly involves the discovery of associations between molecular and phenotype information, with the goal of transforming those discoveries into novel methods for diagnosis and treatment. To enable such complex analyses, researchers need approaches that provide the simultaneous representation and interactive analysis of patients, and their molecular and phenotype information. Because few existing visual analytical systems provide appropriate capabilities, we developed a prototypical visual analytical system called iCircos, which enables the simultaneous and interactive exploration of molecular and phenotype information. We discuss our overall method for developing the prototype by integrating user needs and design heuristics from visual analytics, with agile programming in HTML5 and SVG. A demonstration of the prototype to explore molecular and phenotype associations in two disease datasets suggests that iCircos has the potential to accelerate translational discoveries in complex disease datasets. We conclude by discussing insights about designing visual analytical systems for translational bioinformatics, and present our future plans for user testing and adding advanced interactivity to the prototype

Ligand Diversity of Human and Chimpanzee CYP3A4: Activation of Human CYP3A4 by Lithocholic Acid Results from Positive SelectionS⃞

For currently unknown reasons, the evolution of CYP3A4 underwent acceleration in the human lineage after the split from chimpanzee. We investigated the significance of this event by comparing Escherichia coli-expressed CYP3A4 from humans, chimpanzee, and their most recent common ancestor. The expression level of chimpanzee CYP3A4 was ∼50% of the human CYP3A4, whereas ancestral CYP3A4 did not express in E. coli. Steady-state kinetic analysis with 7-benzyloxyquinoline, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone showed no significant differences between human and chimpanzee CYP3A4. Upon addition of α-naphthoflavone (25 μM), human CYP3A4 showed a slightly decreased substrate concentration at which 50% of the maximal rate Vmax is reached for 7-BFC, whereas chimpanzee CYP3A4 showed a >2-fold increase. No significant differences in inhibition/activation were found for a panel of 43 drugs and endogenous compounds, suggesting that the wide substrate spectrum of human CYP3A4 precedes the human-chimpanzee split. A striking exception was the hepatotoxic secondary bile acid lithocholic acid, which at saturation caused a 5-fold increase in 7-BFC debenzylation by human CYP3A4 but not by chimpanzee CYP3A4. Mutagenesis of human CYP3A4 revealed that at least four of the six amino acids positively selected in the human lineage contribute to the activating effect of lithocholic acid. In summary, the wide functional conservation between chimpanzee and human CYP3A4 raises the prospect that phylogenetically more distant primate species such as rhesus and squirrel monkey represent suitable models of the human counterpart. Positive selection on the human CYP3A4 may have been triggered by an increased load of dietary steroids, which led to a novel defense mechanism against cholestasis