Although it has long been recognized that the enteric community of bacteria that inhabit the human distal intestinal track broadly impacts human health, the biochemical details that underlie these effects remain largely undefined. Here, we report a broad MS-based metabolomics study that demonstrates a surprisingly large effect of the gut ''microbiome'' on mammalian blood metabolites. Plasma extracts from germ-free mice were compared with samples from conventional (conv) animals by using various MS-based methods. Hundreds of features were detected in only 1 sample set, with the majority of these being unique to the conv animals, whereas Ϸ10% of all features observed in both sample sets showed significant changes in their relative signal intensity. Amino acid metabolites were particularly affected. For example, the bacterial-mediated production of bioactive indole-containing metabolites derived from tryptophan such as indoxyl sulfate and the antioxidant indole-3-propionic acid (IPA) was impacted. Production of IPA was shown to be completely dependent on the presence of gut microflora and could be established by colonization with the bacterium Clostridium sporogenes. Multiple organic acids containing phenyl groups were also greatly increased in the presence of gut microbes. A broad, drug-like phase II metabolic response of the host to metabolites generated by the microbiome was observed, suggesting that the gut microflora has a direct impact on the drug metabolism capacity of the host. Together, these results suggest a significant interplay between bacterial and mammalian metabolism.

Translocation of bacteria and their products across the intestinal barrier is common in patients with liver disease, and there is evidence that experimental liver fibrosis depends on bacterial translocation. The purpose of our study was to investigate liver fibrosis in conventional and germ‐free (GF) C57BL/6 mice. Chronic liver injury was induced by administration of thioacetamide (TAA) in the drinking water for 21 wk or by repeated intraperitoneal injections of carbon tetrachloride (CCl4). Increased liver fibrosis was observed in GF mice compared with conventional mice. Hepatocytes showed more toxin‐induced oxidative stress and cell death. This was accompanied by increased activation of hepatic stellate cells, but hepatic mediators of inflammation were not significantly different. Similarly, a genetic model using Myd88/Trif‐deficient mice, which lack downstream innate immunity signaling, had more severe fibrosis than wild‐type mice. Isolated Myd88/Trif‐deficient hepatocytes were more susceptible to toxin‐induced cell death in culture. In conclusion, the commensal microbiota prevents fibrosis upon chronic liver injury in mice. This is the first study describing a beneficial role of the commensal microbiota in maintaining liver homeostasis and preventing liver fibrosis.—Mazagova, M., Wang, L., Anfora, A. T., Wissmueller, M., Lesley, S. A., Miyamoto, Y., Eckmann, L., Dhungana, S., Pathmasiri, W., Sumner, S., Westwater, C., Brenner, D. A., Schnabl, B., Commensal microbiota is hepatoprotective and prevents liver fibrosis in mice. FASEB J. 29, 1043–1055 (2015). http://www.fasebj.org

In this study we show that deletion of the genes encoding L-serine deaminases SdaA and SdaB resulted in a mutant that accumulates higher intracellular levels of L-serine than CFT073. CFT073 sdaA sdaB has a mild competitive colonization defect whereas a CFT073 dsdA sdaA sdaB triple mutant shows a greater loss in competitive colonization ability. Thus, the inability to generate serine-specific catabolic products does not result in hypercolonization and the ability to catabolize serine represents a positive physiological trait during murine UTI. CFT073 dsdC and CFT073 dsdC dsdA mutants continue to outcompete the wild type in the UTI model. These results confirm that loss of DsdA activity results in the hypercolonization phenotype and that DsdC does not play a direct role in the elevated-colonization phenotype. Interestingly, a CFT073 dsdA mutant with deletions of D-serine transporter genes dsdX and cycA shows wild-type colonization levels of the bladder but is attenuated for kidney colonization. Thus, D-serine acts as a signal for hypercolonization and virulence gene expression by CFT073 dsdA, whereas overall catabolism of serine represents a positive Escherichia coli fitness trait during UTI.Urinary tract infections (UTIs) in adult women impose an estimated cost of $2.4 billion per year in the United States (12). Most women will experience at least one UTI in their lifetime, resulting in an estimated 6.8 million physician visits, 1.2 million emergency room visits, and nearly a quarter million hospitalizations each year. Escherichia coli remains by far the primary causative agent of community-acquired UTIs (11).The urinary tract is a normally sterile environment, and it poses daunting challenges to colonization by E. coli and other microorganisms. In addition to the cleansing flow of urine, numerous innate and acquired immune factors challenge the growth of uropathogenic Escherichia coli (UPEC) in the urinary tract. The host defense involves phagocytic attack, antimicrobial peptides, complement lytic and opsonizing factors, and reactive oxygen and nitrogen species. In addition, the urinary tract offers high-salt and high-osmolarity conditions while limiting E. coli nutrients common to the intestinal tract, especially neutral sugars and iron (4). Thus, we hypothesize that the ability of UPEC to import and metabolize the available carbon and nitrogen sources present in the urinary tract plays a special role in its ability to colonize and cause disease at that site.From a bacterial nutritional standpoint, urine is a dilute mixture of amino acids and small peptides, quite similar to tryptone broth, with the notable exception of the abundance of urea in urine (4). The growth of E. coli in tryptone broth is well characterized, where growing cells preferentially and sequentially utilize serine and then aspartate while secreting acetate. Once these amino acids are depleted, cells then import and use tryptophan and acetate, followed by alanine, glutamine, and threonine (31). This order of nutrient preference holds true for ...

D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl.

D-Serine is an amino acid present in mammalian urine that is inhibitory toEscherichia coli is a normal resident of the vertebrate large intestine, and certain pathogenic strains are capable of infecting sites outside of the intestine. The sequencing of multiple E. coli genomes has allowed for a better insight into what genes may permit success in niches outside the intestine. Comparison of an E. coli K-12 isolate (MG1655) to an O157:H7 enterohemorrhagic E. coli isolate (EDL933) and a uropathogenic E. coli (UPEC) isolate (CFT073) showed numerous genetic differences between the strains. Less than 50% of the apparent genes were common to all three strains; these common genes represent what can be thought of as the core chromosome of E. coli. The variable regions specific to each isolate include genes potentially important to disease or growth in different environments (38). One such variable site is a genetic island starting 3Ј to the argW tRNA gene and ending with the dsdCXA locus. The dsdCXA locus is intact in MG1655 and CFT073 but is truncated in EDL933 (28). In fact, the dsdCXA locus is intact in most extraintestinal pathogenic E. coli (ExPEC) strains but is truncated in the vast majority of diarrheagenic E. coli strains (R. L. Moritz and R. A. Welch, submitted for publication). This region of the E. coli chromosome frequently replaces dsdC and dsdX with csrRAKB genes responsible for sucrose catabolism (12,28).The E. coli dsdCXA locus permits growth on D-serine as a sole carbon and nitrogen source. The E. coli K-12 dsdCXA locus was extensively studied by McFall and coworkers (23). The DNA sequence of the locus was originally described by this group, but apparent sequence assembly problems and the inability at the time to create targeted, site-specific mutations prevented proper identification and functional analysis of dsdX. The dsdC gene encodes a Lys-R-type transcriptional regulator that induces transcription of dsdX and dsdA in the presence of D-serine and inhibits its own transcription in the absence of D-serine (23). DsdX has been hypothesized to act as a D-serine transporter (23). The dsdA gene encodes a pyridoxal phosphate-dependent D-serine deaminase (DsdA) that degrades D-serine to ammonia and pyruvate (18). D-Serine catabolism is biologically important because D-serine is available in some environments as a readily utilizable nutrient source, but it can also have also inhibitory effects on growth. D-Serine is bacteriostatic to cells lacking DsdA grown in minimal medium (16). D-Serine toxicity on minimal medium can be reversed with expression of functional DsdA or by the addition of pantothenate or ␤-alanine to the medium; this suggests that the inhibitory effect of D-serine is associated with pantothenate biosynthesis due to the structural similarity between D-serine and ␤-alanine (2, 4, 7, 15). A D-serine deaminase gene can serve as a selective marker on par with antibiotic resistance genes for bacteria (16), yeast (35), or plant transformations (8) due to the toxicity of D-serine. Despite the fact t...

In vivo accumulation of D-serine by Escherichia coli CFT073 leads to elevated expression of PAP fimbriae and hemolysin by an unknown mechanism. Loss of D-serine catabolism by CFT073 leads to a competitive advantage during murine urinary tract infection (UTI), but loss of both D-and L-serine catabolism results in attenuation. Serine is the first amino acid to be consumed in closed tryptone broth cultures and precedes the production of acetyl phosphate, a high-energy molecule involved in intracellular signaling, and the eventual secretion of acetate. We propose that the colonization defect associated with the loss of serine catabolism is due to perturbations of acetate metabolism. CFT073 grows more rapidly on acetogenic substrates than does E. coli K-12 isolate MG1655. As shown by transcription microarray results, D-serine is catabolized into acetate via the phosphotransacetylase (pta) and acetate kinase (ackA) genes while downregulating expression of acetyl coenzyme A synthase (acs). CFT073 acs, which is unable to reclaim secreted acetate, colonized mouse bladders and kidneys in the murine model of UTI indistinguishably from the wild type. Both pta and ackA are involved in the maintenance of intracellular acetyl phosphate. CFT073 pta and ackA mutants were screened to investigate the role of acetyl phosphate in UTI pathogenesis. Both single mutants are at a competitive disadvantage relative to the wild type in the kidneys but normally colonize the bladder. CFT073 ackA pta was attenuated in both the bladder and the kidneys. Thus, we demonstrate that CFT073 is adapted to acetate metabolism as a result of requiring a proper cycling of the acetyl phosphate pathway for colonization of the upper urinary tract.Urinary tract infections (UTIs) place a significant burden on the United States healthcare system, costing upwards of $2.4 billion per year (28). The majority of women will experience a UTI in their lifetime, and every year there are approximately 6.8 million physician visits, 1.2 million emergency room visits, a quarter million hospitalizations, and thousands of deaths due to complications of UTIs, most often sepsis (10,18,28,47). Escherichia coli is the most commonly isolated causative agent of community-acquired UTIs (10).The transition from residence in the gastrointestinal tract, where uropathogenic Escherichia coli (UPEC) transiently resides, to the urinary tract represents a significant change in environment. While the gastrointestinal tract is densely populated with many different species of bacteria, the bladder is normally a sterile environment yet one that presents significant challenges to bacterial growth. In addition to the cleansing flow of urine, numerous innate and acquired immune factors challenge the growth of UPEC in the urinary tract. The host defense involves phagocytic attack, antimicrobial peptides, complement lytic and opsonizing factors, and reactive oxygen and nitrogen species. In addition, the urinary tract as reflected in urine is limited in nutrients common to the intestinal tract, esp...

Introduction: Gene fusions, usually the result of chromosomal rearrangements, are frequently associated with many cancer types, and hence clinically actionable, making fusion detection an important part of cancer disease management. We developed a new, optimized version of the Seraseq® Fusion RNA Reference Material, and demonstrate its consistent performance across different NGS enrichment assays, sequencing coverage depths, bioinformatics pipelines, and RNA mass inputs from a multi-lab investigation (Sites A, B, C, D & E). Methods: Biosynthetic DNA was used for transcription of 18 RNA fusions, including more common fusions of ALK, RET, and ROS1, as well as rare fusion events such as PAX-PPARG and ETV6-NTRK3. The in vitro transcribed RNAs were mixed with total RNA extracted from GM24385 reference cell line (The 1000 Genomes Project, Coriell). Digital PCR with TaqMan® chemistry was used to determine the target fusion RNA concentration and serve as the “truth” data set for comparison to NGS, which can be variable depending on input, assay, and bioinformatics. The fusion-total RNA mix was analyzed by five external laboratories; it was tested using the ArcherDx FusionPlex™ Solid Tumor Panel (Site A, Site B, Site C), the ArcherDx FusionPlex™ CTL Panel (Site C), the ArcherDx FusionPlex™ Lung Panel (Site B), a custom ArcherDx FusionPlex™ Panel (Site D), and the TruSight Tumor 170 Panel (Site E). Results: All eighteen (18) fusions in the new Seraseq Fusion RNA Mix v4 reference standard were detected as expected on each NGS platform with an average of greater than 85% of on-target reads across all assays. Even at inputs as low as 20 ng, all 18 fusions were typically detected above fusion-calling thresholds. In general, the results for individual fusions among the different NGS panels and among replicates were concordant, with observed variance in reads across some fusion junctions between assays and replicates. Within FusionPlex assay results, the average percent of reads supporting the fusion call across all fusions was about 63%, regardless of input (a range between 20 to 250 ng). Collectively, the multi-lab results confirm that the Seraseq® Fusion RNA Mix v4 reference standard is compatible with both amplicon and hybridization-capture based NGS assays. Conclusions: Seraseq RNA Fusion Mix v4 has broad NGS assay compatibility and allows for reliable and simultaneous detection of 18 clinically relevant RNA fusions even at low input amounts. The data from a multi-lab study support the use of this reference standard for targeted NGS assay development, assay validation, bioinformatics pipeline optimization, and as positive controls in clinical NGS RNA fusion assays. The biosynthetic manufacturing approach produces reference materials that provide consistent results for a wide variety of common and rare gene fusions. Citation Format: Dana J. Ruminski Lowe, Deepika Philkana, Catherine Huang, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella. Consistent performance of highly multiplexed RNA fusion reference materials across different NGS assays in a multi-lab study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1709.

In this study we demonstrate precision quantification of Seraseq ctDNA EGFR T790M mutation mix at AF0.1% (mutant/wild-type ratio) using a novel one-step digital PCR (dPCR) platform. This novel yet simple workflow has the potential to make cancer liquid biopsy a clinical application. EGFR is an important drug target for the treatment of non-small cell lung carcinoma (NSCLC). During the treatment of NSCLC with tyrosine kinase inhibitors (TKIs), there is typically a significant response initially, followed by a secondary mutation as the carcinoma develops resistance. Early detection of cancer can better inform patient treatment and guide drug selection. One key EGFR mutation that leads to TKI resistance is the T790M mutation. Only a few clinical assays have been approved as companion diagnostics in patient biopsies (FFPE or plasma), while a larger number of laboratory developed assays (LDTs) under CLIA/CAP guidance are finding routine use in cancer disease diagnosis or treatment monitoring. Herein we describe the validation of a novel, fully integrated dPCR platform for the detection and absolute quantification of EGFR T790M. The integrated dPCR platform consists of a patented micro-molded plastic consumable and a fully-integrated instrument combining consumable sample loading, thermal cycling and 5-color fluorescence detection. The platform was designed to have a simplified, single-step workflow and provide results in less than one hour. In addition, developments in the micro-molded plastic consumable allow for near-zero dead volume. The novel integrated instrument is 100% dry and contamination-free, making it attractive for the transition to clinical applications. Seraseq ctDNA EGFR T790M mutation mix AF1% and AF0.1% reference standards in combination with a commercially available EGFR T790M dPCR assay were used to validate the novel integrated platform. In conclusion, we highlight the capability to precisely quantify samples as low as 0.1% T790M EGFR in a background of wild-type EGFR with high reproducibility and high accuracy. Citation Format: Megan E. Dueck, Robert Lin, Andrew Anfora, Andrew Zayac, Steve Gallagher, Omo Clement, Dana Ruminsky-Lowe, Paul Hung. Validation of a novel one-step digital PCR platform with precision circulating cell-free DNA standards [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2296.