Potential Role of Inflammation-Promoting Biliary Microbiome in Primary Sclerosing Cholangitis and Cholangiocarcinoma.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a major risk factor for cholangiocarcinoma (CCA). We investigated biliary and fecal microbiota to determine whether specific microbes in the bile or stool are associated with PSC or CCA. METHODS: Bile was obtained from 32 patients with PSC, 23 with CCA with PSC, 26 with CCA without PSC, and 17 controls. Over 90% of bile samples were from patients with perihilar CCA. Stool was obtained from 31 patients with PSC (11 were matched to bile), 16 with CCA with PSC (10 matched to bile), and 11 with CCA without PSC (6 matched to bile). Microbiota composition was assessed using 16SrRNA-marker-based sequencing and was compared between groups. RESULTS: Bile has a unique microbiota distinguished from negative DNA controls and stool. Increased species richness and abundance of Fusobacteria correlated with duration of PSC and characterized the biliary microbiota in CCA. Stool microbiota composition showed no significant differences between groups. CONCLUSIONS: We identified a unique microbial signature in the bile of patients with increased duration of PSC or with CCA, suggesting a role for microbiota-driven inflammation in the pathogenesis and or progression to perihilar CCA. Further studies are needed to test this hypothesis.

Comparison of Three Commercial Tools for Metagenomic Shotgun Sequencing Analysis.

Metagenomic shotgun sequencing for the identification of pathogens is being increasingly utilized as a diagnostic method. Interpretation of large and complicated data sets is a significant challenge, for which multiple commercial tools have been developed. Three commercial metagenomic shotgun sequencing tools, CosmosID, One Codex, and IDbyDNA, were compared to determine whether they result in similar interpretations of the same sequencing data. We selected 24 diverse samples from a previously characterized data set derived from DNA extracted from biofilms dislodged from the surfaces of resected arthroplasties (sonicate fluid). Sequencing data sets were analyzed using the three commercial tools and compared to culture results and prior metagenomic analysis interpretation. Identical interpretations from all three tools occurred for 6 samples. The total number of species identified included 28 by CosmosID, 59 by One Codex, and 41 by IDbyDNA. All of the tools performed similarly in detecting those microorganisms identified by culture, including polymicrobial mixes. These data show that while all of the tools performed well overall, there were some differences, particularly in their predilection for identifying low-abundance or contaminant organisms as present.

Whole genome amplification of single epithelial cells dissociated from snap-frozen tissue samples in microfluidic platform.

Single cell sequencing is a technology capable of analyzing the genome of a single cell within a population. This technology is mostly integrated with microfluidics for precise cell manipulation and fluid handling. So far, most of the microfluidic-based single cell genomic studies have been focused on lab-cultured species or cell lines that are relatively easy to handle following standard microfluidic-based protocols without additional adjustments. The major challenges for performing single cell sequencing on clinical samples is the complex nature of the samples which requires additional sample processing steps to obtain intact single cells of interest without using amplification-inhibitive agents. Fluorescent-activated cell sorting is a common option to obtain single cells from clinical samples for single cell applications but requires >100 000 viable cells in suspension and the need for specialized laboratory and personnel. In this work, we present a protocol that can be used to obtain intact epithelial cells from snap-frozen postsurgical human endometrial tissues for single cell whole genome amplification. Our protocol includes sample thawing, cell dissociation, and labeling for genome amplification of targeted cells. Between 80% and 100% of single cell replicates lead to >25 ng of DNA after amplification with no measurable contamination, sufficient for downstream sequencing.

Distinct microbes, metabolites, and ecologies define the microbiome in deficient and proficient mismatch repair colorectal cancers.

BACKGROUND: Links between colorectal cancer (CRC) and the gut microbiome have been established, but the specific microbial species and their role in carcinogenesis remain an active area of inquiry. Our understanding would be enhanced by better accounting for tumor subtype, microbial community interactions, metabolism, and ecology. METHODS: We collected paired colon tumor and normal-adjacent tissue and mucosa samples from 83 individuals who underwent partial or total colectomies for CRC. Mismatch repair (MMR) status was determined in each tumor sample and classified as either deficient MMR (dMMR) or proficient MMR (pMMR) tumor subtypes. Samples underwent 16S rRNA gene sequencing and a subset of samples from 50 individuals were submitted for targeted metabolomic analysis to quantify amino acids and short-chain fatty acids. A PERMANOVA was used to identify the biological variables that explained variance within the microbial communities. dMMR and pMMR microbial communities were then analyzed separately using a generalized linear mixed effects model that accounted for MMR status, sample location, intra-subject variability, and read depth. Genome-scale metabolic models were then used to generate microbial interaction networks for dMMR and pMMR microbial communities. We assessed global network properties as well as the metabolic influence of each microbe within the dMMR and pMMR networks. RESULTS: We demonstrate distinct roles for microbes in dMMR and pMMR CRC. Bacteroides fragilis and sulfidogenic Fusobacterium nucleatum were significantly enriched in dMMR CRC, but not pMMR CRC. These findings were further supported by metabolic modeling and metabolomics indicating suppression of B. fragilis in pMMR CRC and increased production of amino acid proxies for hydrogen sulfide in dMMR CRC. CONCLUSIONS: Integrating tumor biology and microbial ecology highlighted distinct microbial, metabolic, and ecological properties unique to dMMR and pMMR CRC. This approach could critically improve our ability to define, predict, prevent, and treat colorectal cancers.

Direct Detection and Identification of Prosthetic Joint Infection Pathogens in Synovial Fluid by Metagenomic Shotgun Sequencing.

Metagenomic shotgun sequencing has the potential to transform how serious infections are diagnosed by offering universal, culture-free pathogen detection. This may be especially advantageous for microbial diagnosis of prosthetic joint infection (PJI) by synovial fluid analysis since synovial fluid cultures are not universally positive and since synovial fluid is easily obtained preoperatively. We applied a metagenomics-based approach to synovial fluid in an attempt to detect microorganisms in 168 failed total knee arthroplasties. Genus- and species-level analyses of metagenomic sequencing yielded the known pathogen in 74 (90%) and 68 (83%) of the 82 culture-positive PJIs analyzed, respectively, with testing of two (2%) and three (4%) samples, respectively, yielding additional pathogens not detected by culture. For the 25 culture-negative PJIs tested, genus- and species-level analyses yielded 19 (76%) and 21 (84%) samples with insignificant findings, respectively, and 6 (24%) and 4 (16%) with potential pathogens detected, respectively. Genus- and species-level analyses of the 60 culture-negative aseptic failure cases yielded 53 (88%) and 56 (93%) cases with insignificant findings and 7 (12%) and 4 (7%) with potential clinically significant organisms detected, respectively. There was one case of aseptic failure with synovial fluid culture growth; metagenomic analysis showed insignificant findings, suggesting possible synovial fluid culture contamination. Metagenomic shotgun sequencing can detect pathogens involved in PJI when applied to synovial fluid and may be particularly useful for culture-negative cases.

Synthesis of multi-omic data and community metabolic models reveals insights into the role of hydrogen sulfide in colon cancer.

Multi-omic data and genome-scale microbial metabolic models have allowed us to examine microbial communities, community function, and interactions in ways that were not available to us historically. Now, one of our biggest challenges is determining how to integrate data and maximize data potential. Our study demonstrates one way in which to test a hypothesis by combining multi-omic data and community metabolic models. Specifically, we assess hydrogen sulfide production in colorectal cancer based on stool, mucosa, and tissue samples collected on and off the tumor site within the same individuals. 16S rRNA microbial community and abundance data were used to select and inform the metabolic models. We then used MICOM, an open source platform, to track the metabolic flux of hydrogen sulfide through a defined microbial community that either represented on-tumor or off-tumor sample communities. We also performed targeted and untargeted metabolomics, and used the former to quantitatively evaluate our model predictions. A deeper look at the models identified several unexpected but feasible reactions, microbes, and microbial interactions involved in hydrogen sulfide production for which our 16S and metabolomic data could not account. These results will guide future in vitro, in vivo, and in silico tests to establish why hydrogen sulfide production is increased in tumor tissue.

Identification of Prosthetic Joint Infection Pathogens Using a Shotgun Metagenomics Approach.

Background: Metagenomic shotgun sequencing has the potential to change how many infections, particularly those caused by difficult-to-culture organisms, are diagnosed. Metagenomics was used to investigate prosthetic joint infections (PJIs), where pathogen detection can be challenging. Methods: Four hundred eight sonicate fluid samples generated from resected hip and knee arthroplasties were tested, including 213 from subjects with infections and 195 from subjects without infection. Samples were enriched for microbial DNA using the MolYsis basic kit, whole-genome amplified, and sequenced using Illumina HiSeq 2500 instruments. A pipeline was designed to screen out human reads and analyze remaining sequences for microbial content using the Livermore Metagenomics Analysis Toolkit and MetaPhlAn2 tools. Results: When compared to sonicate fluid culture, metagenomics was able to identify known pathogens in 94.8% (109/115) of culture-positive PJIs, with additional potential pathogens detected in 9.6% (11/115). New potential pathogens were detected in 43.9% (43/98) of culture-negative PJIs, 21 of which had no other positive culture sources from which these microorganisms had been detected. Detection of microorganisms in samples from uninfected aseptic failure cases was conversely rare (7/195 [3.6%] cases). The presence of human and contaminant microbial DNA from reagents was a challenge, as previously reported. Conclusions: Metagenomic shotgun sequencing is a powerful tool to identify a wide range of PJI pathogens, including difficult-to-detect pathogens in culture-negative infections.

A comprehensive analysis of breast cancer microbiota and host gene expression.

The inflammatory tumoral-immune response alters the physiology of the tumor microenvironment, which may attenuate genomic instability. In addition to inducing inflammatory immune responses, several pathogenic bacteria produce genotoxins. However the extent of microbial contribution to the tumor microenvironment biology remains unknown. We utilized The Cancer Genome Atlas, (TCGA) breast cancer data to perform a novel experiment utilizing unmapped and mapped RNA sequencing read evidence to minimize laboratory costs and effort. Our objective was to characterize the microbiota and associate the microbiota with the tumor expression profiles, for 668 breast tumor tissues and 72 non-cancerous adjacent tissues. The prominent presence of Proteobacteria was increased in the tumor tissues and conversely Actinobacteria abundance increase in non-cancerous adjacent tissues. Further, geneset enrichment suggests Listeria spp to be associated with the expression profiles of genes involved with epithelial to mesenchymal transitions. Moreover, evidence suggests H. influenza may reside in the surrounding stromal material and was significantly associated with the proliferative pathways: G2M checkpoint, E2F transcription factors, and mitotic spindle assembly. In summary, further unraveling this complicated interplay should enable us to better diagnose and treat breast cancer patients.

Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis.

Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.

Photophysics of EGFP (E222H) Mutant, with Comparisons to Model Chromophores: Excited State pK's, Progressions, Quenching and Exciton Interaction.

A novel version of the well-known and commercially successful Green Fluorescent Protein (GFP) variant known as EGFP, with an introduced E222H mutation, was produced in this laboratory. Given the current state of hypotheses about the role of glutamate 222, and the observed dominance of the phenolate absorption with an E222H variant observed from earlier study, the new mutant was considered a natural choice to investigate more fully the acid-base behavior of the chromophore in absorption and fluorescence. The bulk of this investigation concerns fitting the excitation, emission and absorption spectra to vibrational progressions of a novel 'q-deformed' type at various values of pH, and protein concentration. From these data, and from temperature-dependent fluorescence lifetime data and other experiments (with lanthanide doped gels into which H/EGFP is embedded), we construct a picture of excited inter- state conversion mechanisms, and quenching mechanisms, that attempts to explain many features of the GFP system. Graphical Abstract Hypothetical proton current loop (orange) upon excitation; electron motion in purple H/EGFP. Solid boxes about waters project toward viewer, dashed boxes project away.

The Microbiome of Aseptically Collected Human Breast Tissue in Benign and Malignant Disease.

Globally breast cancer is the leading cause of cancer death among women. The breast consists of epithelium, stroma and a mucosal immune system that make up a complex microenvironment. Growing awareness of the role of microbes in the microenvironment recently has led to a series of findings important for human health. The microbiome has been implicated in cancer development and progression at a variety of body sites including stomach, colon, liver, lung, and skin. In this study, we assessed breast tissue microbial signatures in intraoperatively obtained samples using 16S rDNA hypervariable tag sequencing. Our results indicate a distinct breast tissue microbiome that is different from the microbiota of breast skin tissue, breast skin swabs, and buccal swabs. Furthermore, we identify distinct microbial communities in breast tissues from women with cancer as compared to women with benign breast disease. Malignancy correlated with enrichment in taxa of lower abundance including the genera Fusobacterium, Atopobium, Gluconacetobacter, Hydrogenophaga and Lactobacillus. This work confirms the existence of a distinct breast microbiome and differences between the breast tissue microbiome in benign and malignant disease. These data provide a foundation for future investigation on the role of the breast microbiome in breast carcinogenesis and breast cancer prevention.

Multiple sclerosis patients have a distinct gut microbiota compared to healthy controls.

Multiple sclerosis (MS) is an immune-mediated disease, the etiology of which involves both genetic and environmental factors. The exact nature of the environmental factors responsible for predisposition to MS remains elusive; however, it's hypothesized that gastrointestinal microbiota might play an important role in pathogenesis of MS. Therefore, this study was designed to investigate whether gut microbiota are altered in MS by comparing the fecal microbiota in relapsing remitting MS (RRMS) (n = 31) patients to that of age- and gender-matched healthy controls (n = 36). Phylotype profiles of the gut microbial populations were generated using hypervariable tag sequencing of the V3-V5 region of the 16S ribosomal RNA gene. Detailed fecal microbiome analyses revealed that MS patients had distinct microbial community profile compared to healthy controls. We observed an increased abundance of Psuedomonas, Mycoplana, Haemophilus, Blautia, and Dorea genera in MS patients, whereas control group showed increased abundance of Parabacteroides, Adlercreutzia and Prevotella genera. Thus our study is consistent with the hypothesis that MS patients have gut microbial dysbiosis and further study is needed to better understand their role in the etiopathogenesis of MS.

Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.

Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing.

Nitric oxide promotes endothelial cell survival signaling through S-nitrosylation and activation of dynamin-2.

Endothelial cell-based angiogenesis requires activation of survival signals that generate resistance to external apoptotic stimuli, such as tumor necrosis factor-alpha (TNF-alpha), during pathobiologic settings. Mechanisms by which this is achieved are not fully defined. Here, we use a model in which the multifunctional cytokine nitric oxide counterbalances TNF-alpha-induced apoptosis, to define a role for membrane trafficking in the process of endothelial cell survival signaling. By perturbing dynamin GTPase function, we identify a key role of dynamin for ensuing downstream endothelial cell survival signals and vascular tube formation. Furthermore, nitric oxide is directly demonstrated to promote dynamin function through specific cysteine residue nitrosylation, which promotes endocytosis and endothelial cell survival signaling. Thus, these studies identify a novel role for dynamin as a survival factor in endothelial cells, through a mechanism by which dynamin S-nitrosylation regulates the counterbalances of TNF-alpha-induced apoptosis and nitric oxide-dependent survival signals, with implications highly relevant to angiogenesis.

Disruption of an SP2/KLF6 repression complex by SHP is required for farnesoid X receptor-induced endothelial cell migration.

The farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain- and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility.

Mechanisms of nitric oxide interplay with Rho GTPase family members in modulation of actin membrane dynamics in pericytes and fibroblasts.

Migration of pericytes such as hepatic stellate cells is fundamentally important for diverse biological and pathological processes including tumor invasion and fibrosis. In prototypical migratory cells such as fibroblasts, the small GTPases Rac1 and RhoA govern the assembly of lamellipodia and stress fibers, respectively, cytoskeletal structures that are integral to the cell migration process. The gaseous signaling molecule nitric oxide (NO) influences growth factor chemotactic responses, although this occurs primarily in cell-type-specific ways and through cell biological effects that are poorly characterized. In this study, we use complementary molecular and cell biological approaches to delineate important roles for Rac1, RhoA, and NO in migration of the human hepatic stellate cell line LX2 and primary rat hepatic stellate cells. Both platelet-derived growth factor (PDGF) and Rac1 overexpression drove migration through formation of actin-positive filopodia spikes in LX2 as compared to the formation of lamellipodia in fibroblasts. NO inhibited PDGF- and Rac1-driven migration in LX2 by abrogating filopodia formation and inhibited migration of fibroblasts by attenuating lamellipodial protrusions. Additionally, RhoA conferred resistance to NO inhibition of migration and restored chemotactic responses to PDGF in the absence of functional Rac1 in LX2. In conclusion, these studies identify novel crosstalk between small GTPases, cytoskeletal structures, and NO in pericyte-specific pathways, providing counterbalances in the chemotactic responses to growth factors.

Microtubule destabilization and nuclear entry are sequential steps leading to toxicity in Huntington's disease.

There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntington's disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is "downstream" of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival.

The proline-rich domain of dynamin-2 is responsible for dynamin-dependent in vitro potentiation of endothelial nitric-oxide synthase activity via selective effects on reductase domain function.

The GTPase dynamin-2 (dyn-2) binds and positively regulates the nitric oxide-generating enzyme, endothelial nitric-oxide synthase (eNOS) (Cao, S., Yao, Y., McCabe, T., Yao, Q., Katusic, Z., Sessa, W., and Shah, V. (2001) J. Biol. Chem. 276, 14249-14256). Here we demonstrate, using purified proteins, that this occurs through a selective influence of the dyn-2 proline-rich domain (dyn-2 PRD) on the eNOS reductase domain. In vitro studies demonstrate that dyn-2 PRD fused with glutathione S-transferase (GST) binds recombinant eNOS protein specifically and with binding kinetics comparable with that observed between dyn-2 full-length and eNOS. Additionally, GST-dyn-2 PRD binds the in vitro transcribed (35)S-eNOS reductase domain but not the (35)S-eNOS oxygenase domain. Furthermore GST-dyn-2 PRD binds a (35)S-labeled eNOS reductase domain fragment (amino acids 645-850) that partially overlaps with the FAD binding domain of eNOS. A recombinant form of the SH3-containing protein Fyn competes the binding of recombinant eNOS protein with dyn-2 PRD, thereby implicating the SH3-like region contained within this reductase domain fragment as the dyn-2 binding region. Mammalian two-hybrid screen corroborates these interactions in cells as well. Functional studies demonstrate that dyn-2 PRD selectively potentiates eNOS activity in a concentration-dependent manner in an order of magnitude similar to that observed with dyn-2 full-length and in a manner that requires calmodulin. Although dyn-2 PRD does not influence eNOS oxygenase domain function or ferricyanide reduction, it does potentiate the ability of recombinant eNOS to reduce cytochrome c, supporting an influence of dyn-2 PRD on electron transfer between FAD and FMN. (These data indicate that the binding domains of dyn-2 and eNOS reside within the dyn-2 PRD domain and the FAD binding region of the eNOS reductase domains, respectively, and that dyn-2 PRD is sufficient to mediate dyn-2-dependent potentiation of eNOS activity, at least in part, by potentiating electron transfer.)