Publications by authors named "Paul Ruegger"

25 Publications

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Particle Radiation Side-Effects: Intestinal Microbiota Composition Shapes Interferon-γ-Induced Osteo-Immunogenicity.

Radiat Res 2022 02;197(2):184-192

Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, California.

Microbiota can both negatively and positively impact radiation-induced bone loss. Our prior research showed that compared to mice with conventional gut microbiota (CM), mice with restricted gut microbiota (RM) reduced inflammatory tumor necrosis factor (TNF) in bone marrow, interleukin (IL)-17 in blood, and chemokine (C-C motif) ligand 20 (CCL20) in bone marrow under anti-IL-17 treatment. We showed that Muribaculum intestinale was more abundant in intestinal epithelial cells (IECs) from the small intestine of female RM mice and positively associated with augmented skeletal bone structure. Female C57BL/6J pun RM mice, which were injected with anti-IL-17 antibody one day before exposure to 1.5 Gy 28Si ions of 850 MeV/u, showed high trabecular numbers in tibiae at 6 weeks postirradiation. Irradiated CM mice were investigated for lower interferon-γ and IL-17 levels in the small intestine than RM mice. IL-17 blockage resulted in bacterial indicator phylotypes being different between both microbiota groups before and after irradiation. Analysis of the fecal bacteria were performed in relation to bone quality and body weight, showing reduced tibia cortical thickness in irradiated CM mice (-15%) vs. irradiated RM mice (-9.2%). Correlation analyses identified relationships among trabecular bone parameters (TRI-BV/TV, Tb.N, Tb.Th, Tb.Sp) and Bacteroides massiliensis, Muribaculum sp. and Prevotella denticola. Turicibacter sp. was found directly correlated with trabecular separation in anti-IL-17 treated mice, whereas an unidentified Bacteroidetes correlated with trabecular thickness in anti-IL-17 neutralized and radiation-exposed mice. We demonstrated radiation-induced osteolytic damage to correlate with bacterial indicator phylotypes of the intestinal microbiota composition, and these relationships were determined from the previously discovered dose-dependent particle radiation effects on cell proliferation in bone tissue. New translational approaches were designed to investigate dynamic changes of gut microbiota in correlation with conditions of treatment and disease as well as mechanisms of systemic side-effects in radiotherapy.
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http://dx.doi.org/10.1667/RADE-21-00065.1DOI Listing
February 2022

Early-life effects of juvenile Western diet and exercise on adult gut microbiome composition in mice.

J Exp Biol 2021 02 25;224(Pt 4). Epub 2021 Feb 25.

Department of Evolution, Ecology, and Organismal Biology, University of California, Riverside, Riverside, CA 91521, USA

Alterations to the gut microbiome caused by changes in diet, consumption of antibiotics, etc., can affect host function. Moreover, perturbation of the microbiome during critical developmental periods potentially has long-lasting impacts on hosts. Using four selectively bred high runner and four non-selected control lines of mice, we examined the effects of early-life diet and exercise manipulations on the adult microbiome by sequencing the hypervariable internal transcribed spacer region of the bacterial gut community. Mice from high runner lines run ∼3-fold more on wheels than do controls, and have several other phenotypic differences (e.g. higher food consumption and body temperature) that could alter the microbiome, either acutely or in terms of coevolution. Males from generation 76 were given wheels and/or a Western diet from weaning until sexual maturity at 6 weeks of age, then housed individually without wheels on standard diet until 14 weeks of age, when fecal samples were taken. Juvenile Western diet reduced bacterial richness and diversity after the 8-week washout period (equivalent to ∼6 human years). We also found interactive effects of genetic line type, juvenile diet and/or juvenile exercise on microbiome composition and diversity. Microbial community structure clustered significantly in relation to both line type and diet. Western diet also reduced the relative abundance of These results constitute one of the first reports of juvenile diet having long-lasting effects on the adult microbiome after a substantial washout period. Moreover, we found interactive effects of diet with early-life exercise exposure, and a dependence of these effects on genetic background.
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http://dx.doi.org/10.1242/jeb.239699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929929PMC
February 2021

Herpes simplex virus infection, Acyclovir and IVIG treatment all independently cause gut dysbiosis.

PLoS One 2020 6;15(8):e0237189. Epub 2020 Aug 6.

Department of Molecular Immunology, Beckman Research Institute of City of Hope, Duarte, California, United States of America.

Herpes simplex virus 1 (HSV) is a ubiquitous human virus resident in a majority of the global population as a latent infection. Acyclovir (ACV), is the standard of care drug used to treat primary and recurrent infections, supplemented in some patients with intravenous immunoglobulin (IVIG) treatment to suppress infection and deleterious inflammatory responses. As many diverse medications have recently been shown to change composition of the gut microbiome, we used Illumina 16S rRNA gene sequencing to determine the effects of ACV and IVIG on the gut bacterial community. We found that HSV, ACV and IVIG can all independently disrupt the gut bacterial community in a sex biased manner when given to uninfected C57BL/6 mice. Treatment of HSV infected mice with ACV or IVIG alone or together revealed complex interactions between these drugs and infection that caused pronounced sex biased dysbiosis. ACV reduced Bacteroidetes levels in male but not female mice, while levels of the Anti-inflammatory Clostridia (AIC) were reduced in female but not male mice, which is significant as these taxa are associated with protection against the development of graft versus host disease (GVHD) in hematopoietic stem cell transplant (HSCT) patients. Gut barrier dysfunction is associated with GVHD in HSCT patients and ACV also decreased Akkermansia muciniphila, which is important for maintaining gut barrier functionality. Cumulatively, our data suggest that long-term prophylactic ACV treatment of HSCT patients may contribute to GVHD and also potentially impact immune reconstitution. These data have important implications for other clinical settings, including HSV eye disease and genital infections, where ACV is given long-term.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237189PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410316PMC
October 2020

The autoimmune susceptibility gene, , restricts expansion of a novel mouse adherent-invasive .

Gut Microbes 2020 11 26;11(6):1547-1566. Epub 2020 Jun 26.

Division of Biomedical Sciences, University of California Riverside , Riverside, California, USA.

Inflammatory bowel disease (IBD) pathogenesis involves significant contributions from genetic and environmental factors. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 () gene increase IBD risk and are associated with altered microbiome population dynamics in IBD. Expansion of intestinal pathobionts, such as adherent-invasive (AIEC), is strongly implicated in IBD pathogenesis as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation - suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. We aimed to determine if deficiency alters intestinal microbiome composition to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking , we identified increased abundance of a novel mouse AIEC (AIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages, to the IBD-associated AIEC, LF82. Furthermore, AIEC caused disease when administered to mice lacking segmented-filamentous bacteria (SFB), and in germ-free mice but only when reconstituted with a microbiome, thus supporting its classification as a pathobiont, not a pathogen. Moreover, AIEC infection increased the severity of, and prevented recovery from, induced colitis. Although AIEC genome sequence analysis showed >90% similarity to LF82, AIEC contained putative virulence genes with >50% difference in gene/protein identities from LF82 indicating potentially distinct genetic features of AIEC. We show for the first time that an IBD susceptibility gene, , modulates the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD and identifies a new model to study AIEC-host interactions.
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http://dx.doi.org/10.1080/19490976.2020.1775538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524159PMC
November 2020

High Levels of Oxidative Stress Create a Microenvironment That Significantly Decreases the Diversity of the Microbiota in Diabetic Chronic Wounds and Promotes Biofilm Formation.

Front Cell Infect Microbiol 2020 3;10:259. Epub 2020 Jun 3.

Molecular, Cell and Systems Biology Department, University of California, Riverside, Riverside, CA, United States.

Diabetics chronic wounds are characterized by high levels of oxidative stress (OS) and are often colonized by biofilm-forming bacteria that severely compromise healing and can result in amputation. However, little is known about the role of skin microbiota in wound healing and chronic wound development. that high OS levels lead to chronic wound development by promoting the colonization of biofilm-forming bacteria over commensal/beneficial bacteria. To test this hypothesis, we used our mouse model for chronic wounds where pathogenic biofilms develop naturally after induction of high OS immediately after wounding. We sequenced the bacterial rRNA internal transcribed spacer (ITS) gene of the wound microbiota from wound initiation to fully developed chronic wounds. Indicator species analysis, which considers a species' fidelity and specificity, was used to determine which bacterial species were strongly associated with healing wounds or chronic wounds. We found that healing wounds were colonized by a diverse and dynamic bacterial microbiome that never developed biofilms even though biofilm-forming bacteria were present. Several clinically relevant species that are present in human chronic wounds, such as sp., sp., and , were highly associated with healing wounds. These bacteria may serve as bioindicators of healing and may actively participate in the processes of wound healing and preventing pathogenic bacteria from colonizing the wound. In contrast, chronic wounds, which had high levels of OS, had low bacterial diversity and were colonized by several clinically relevant, biofilm-forming bacteria such as , and sp. We observed unique population trends: for example, associated with aggressive biofilm development, whereas was only present early after injury. These findings show that high levels of OS in the wound significantly altered the bacterial wound microbiome, decreasing diversity and promoting the colonization of bacteria from the skin microbiota to form biofilm. , bacteria associated with non-chronic or chronic wounds could function as bioindicators of healing or non-healing (chronicity), respectively. Moreover, a better understanding of bacterial interactions between pathogenic and beneficial bacteria within an evolving chronic wound microbiota may lead to .
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http://dx.doi.org/10.3389/fcimb.2020.00259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283391PMC
June 2021

An Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-" Liberibacter asiaticus" Properties.

Appl Environ Microbiol 2020 04 1;86(8). Epub 2020 Apr 1.

Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, California, USA

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against , a culturable surrogate for the nonculturable " Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against that include the fungi and and bacterial species of , , and Purified bioactive natural products with anti-"" activity were identified from the fungus Bioassay-guided fractionation of an organic extract of yielded the natural products cladosporols A, C, and D as the active agents against This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-" Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem. Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using , a culturable surrogate for the unculturable HLB-associated bacterium " Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-" Liberibacter asiaticus" natural products with potential anti-" Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to .
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http://dx.doi.org/10.1128/AEM.02883-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117939PMC
April 2020

Dominant Role of the Gut Microbiota in Chemotherapy Induced Neuropathic Pain.

Sci Rep 2019 12 30;9(1):20324. Epub 2019 Dec 30.

Department of Molecular Immunology, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA.

Chemotherapy induced peripheral neuropathy (CIPN), a toxic side effect of some cancer treatments, negatively impacts patient outcomes and drastically reduces survivor's quality of life (QOL). Uncovering the mechanisms driving chemotherapy-induced CIPN is urgently needed to facilitate the development of effective treatments, as currently there are none. Observing that C57BL/6 (B6) and 129SvEv (129) mice are respectively sensitive and resistant to Paclitaxel-induced pain, we investigated the involvement of the gut microbiota in this extreme phenotypic response. Reciprocal gut microbiota transfers between B6 and 129 mice as well as antibiotic depletion causally linked gut microbes to Paclitaxel-induced pain sensitivity and resistance. Microglia proliferated in the spinal cords of Paclitaxel treated mice harboring the pain-sensitive B6 microbiota but not the pain-resistant 129 microbiota, which exhibited a notable absence of infiltrating immune cells. Paclitaxel decreased the abundance of Akkermansia muciniphila, which could compromise barrier integrity resulting in systemic exposure to bacterial metabolites and products - that acting via the gut-immune-brain axis - could result in altered brain function. Other bacterial taxa that consistently associated with both bacteria and pain as well as microglia and pain were identified, lending support to our hypothesis that microglia are causally involved in CIPN, and that gut bacteria are drivers of this phenotype.
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http://dx.doi.org/10.1038/s41598-019-56832-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937259PMC
December 2019

High Levels of Oxidative Stress and Skin Microbiome are Critical for Initiation and Development of Chronic Wounds in Diabetic Mice.

Sci Rep 2019 12 17;9(1):19318. Epub 2019 Dec 17.

Department of Molecular, Cell and Systems Biology, University of California, Riverside, USA.

A balanced redox state is critical for proper healing. Although human chronic wounds are characterized by high levels of oxidative stress (OS), whether OS levels are critical for chronic wound development is not known. For these studies, we used our chronic wound model in diabetic mice that has similar characteristics as human chronic wounds, including naturally developed biofilm. We hypothesize that OS levels in wound tissues are critical for chronic wound initiation and development. We show that increased OS levels in the wound correlate with increased chronicity. Moreover, without increased OS levels, biofilm taken from chronic wounds and placed in new excision wounds do not create chronic wounds. Similarly, high OS levels in the wound tissue in the absence of the skin microbiome do not lead to chronic wounds. These findings show that both high OS levels and bacteria are needed for chronic wound initiation and development. In conclusion, OS levels in the wound at time of injury are critical for biofilm formation and chronic wound development and may be a good predictor of the degree of wound chronicity. Treating such wounds might be accomplished by managing OS levels with antioxidants combined with manipulation of the skin microbiome after debridement.
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http://dx.doi.org/10.1038/s41598-019-55644-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917777PMC
December 2019

Intestinal bacterial indicator phylotypes associate with impaired DNA double-stranded break sensors but augmented skeletal bone micro-structure.

Carcinogenesis 2020 06;41(4):483-489

Department of Environmental Health Sciences, Fielding School of Public Health, University of California, Los Angeles 650 Charles E. Young Dr. South, Los Angeles, CA, USA.

Intestinal microbiota are considered a sensor for molecular pathways, which orchestrate energy balance, immune responses, and cell regeneration. We previously reported that microbiota restriction promoted higher levels of systemic radiation-induced genotoxicity, proliferative lymphocyte activation, and apoptotic polarization of metabolic pathways. Restricted intestinal microbiota (RM) that harbors increased abundance of Lactobacillus johnsonii (LBJ) has been investigated for bacterial communities that correlated radiation-induced genotoxicity. Indicator phylotypes were more abundant in RM mice and increased in prevalence after whole body irradiation in conventional microbiota (CM) mice, while none of the same ten most abundant phylotypes were different in abundance between CM mice before and after heavy ion irradiation. Muribaculum intestinale was detected highest in female small intestines in RM mice, which were lacking Ureaplasma felinum compared with males, and thus these bacteria could be contributing to the differential amounts of radiation-induced systemic genotoxicity between the CM and RM groups. Helicobacter rodentium and M.intestinale were found in colons in the radiation-resistant CM phenotype. While the expression of interferon-γ was elevated in the small intestine, and lower in blood in CM mice, high-linear energy transfer radiation reduced transforming growth factor-β with peripheral interleukin (IL)-17 in RM mice, particularly in females. We found that female RM mice showed improved micro-architectural bone structure and anti-inflammatory radiation response compared with CM mice at a delayed phase 6 weeks postexposure to particle radiation. However, microbiota restriction reduced inflammatory markers of tumor necrosis factor in marrow, when IL-17 was reduced by intraperitoneal injection of IL-17 neutralizing antibody.
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http://dx.doi.org/10.1093/carcin/bgz204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456343PMC
June 2020

Bacterial Microbiome Dynamics in Post Pull-Through Hirschsprung-Associated Enterocolitis (HAEC): An Experimental Study Employing the Endothelin Receptor B-Null Mouse Model.

Front Surg 2018 6;5:30. Epub 2018 Apr 6.

Division of Pediatric Surgery and Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, United States.

Purpose: Hirschsprung-associated enterocolitis (HAEC) is the most frequent potentially life-threatening complication in children with Hirschsprung disease (HSCR) even after definitive corrective surgery. Mounting evidence suggests that intestinal microbiota likely contribute to the etiology of enterocolitis, so the aim of this study was to use a mouse model of post pull-through HAEC to compare the fecal bacterial communities of animals which developed HAEC to those free of enterocolitis.

Methods: Ten and 8wild type mice underwent the microsurgical pull-through surgery, and stool was collected at the time of surgery, and then either at 2 and 4 weeks after the operation, or when the mice developed enterocolitis. The mid-colon of all animals was collected, prepared and histologically graded for enterocolitis. Fecal DNA was isolated and bacterial 16S rRNA genes analyzed using Illumina sequencing.

Results: Six mice developed HAEC with a mean enterocolitis score of 5.7, while the remaining 4 mutant and 8 WT mice remained free of enterocolitis by 4 weeks. The HAEC group had lower alpha diversity by Chao1 analysis compared with WT group, while the mice demonstrated distinct bacterial communities from WT mice on beta diversity analysis. The most striking finding was increased proportion of and reduced Bacteroidetes compared with the NO HAEC and WT groups, suggesting may contribute to development of enterocolitis while Bacteroidetes may be protective. Less abundant genera that were reduced in HAEC were and XIVa which may play a protective role.

Conclusions: This is the first study to identify as potentially playing a role in HAEC, either as a pathobiont taxa contributing to pathogenesis of enterocolitis, or possibly a protective commensal taxa expanded in response to inflammation. These findings characterized the dynamic shifts in the gut microbial communities through the onset of post pull-through HAEC, and suggests that there may be identifiable bacterial community differences in HSCR patients that are high risk for developing HAEC.
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http://dx.doi.org/10.3389/fsurg.2018.00030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897423PMC
April 2018

Concurrent gut transcriptome and microbiota profiling following chronic ethanol consumption in nonhuman primates.

Gut Microbes 2018 07 27;9(4):338-356. Epub 2018 Apr 27.

b Department of Molecular Biology and Biochemistry , University of California-Irvine , Irvine , CA , USA.

Alcohol use disorder (AUD) results in increased intestinal permeability, nutrient malabsorption, and increased risk of colorectal cancer (CRC). Our understanding of the mechanisms underlying these morbidities remains limited because studies to date have relied almost exclusively on short-term heavy/binge drinking rodent models and colonic biopsies/fecal samples collected from AUD subjects with alcoholic liver disease (ALD). Consequently, the dose- and site-dependent impact of chronic alcohol consumption in the absence of overt liver disease remains poorly understood. In this study, we addressed this knowledge gap using a nonhuman primate model of voluntary ethanol self-administration where rhesus macaques consume varying amounts of 4% ethanol in water for 12 months. Specifically, we performed RNA-Seq and 16S rRNA gene sequencing on duodenum, jejunum, ileum, and colon biopsies collected from 4 controls and 8 ethanol-consuming male macaques. Our analysis revealed that chronic ethanol consumption leads to changes in the expression of genes involved in protein trafficking, metabolism, inflammation, and CRC development. Additionally, we observed differences in the relative abundance of putatively beneficial bacteria as well as those associated with inflammation and CRC. Given that the animals studied in this manuscript did not exhibit signs of ALD or CRC, our data suggest that alterations in gene expression and bacterial communities precede clinical disease and could serve as biomarkers as well as facilitate future studies aimed at developing interventions to restore gut homeostasis.
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http://dx.doi.org/10.1080/19490976.2018.1441663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219653PMC
July 2018

A Disease-Associated Microbial and Metabolomics State in Relatives of Pediatric Inflammatory Bowel Disease Patients.

Cell Mol Gastroenterol Hepatol 2016 Nov 2;2(6):750-766. Epub 2016 Jul 2.

Susan and Leonard Feinstein Inflammatory Bowel Disease Center, Department of Pediatrics, Icahn School of Medicine, Mount Sinai, New York.

Background & Aims: Microbes may increase susceptibility to inflammatory bowel disease (IBD) by producing bioactive metabolites that affect immune activity and epithelial function. We undertook a family based study to identify microbial and metabolic features of IBD that may represent a predisease risk state when found in healthy first-degree relatives.

Methods: Twenty-one families with pediatric IBD were recruited, comprising 26 Crohn's disease patients in clinical remission, 10 ulcerative colitis patients in clinical remission, and 54 healthy siblings/parents. Fecal samples were collected for 16S ribosomal RNA gene sequencing, untargeted liquid chromatography-mass spectrometry metabolomics, and calprotectin measurement. Individuals were grouped into microbial and metabolomics states using Dirichlet multinomial models. Multivariate models were used to identify microbes and metabolites associated with these states.

Results: Individuals were classified into 2 microbial community types. One was associated with IBD but irrespective of disease status, had lower microbial diversity, and characteristic shifts in microbial composition including increased Enterobacteriaceae, consistent with dysbiosis. This microbial community type was associated similarly with IBD and reduced microbial diversity in an independent pediatric cohort. Individuals also clustered bioinformatically into 2 subsets with shared fecal metabolomics signatures. One metabotype was associated with IBD and was characterized by increased bile acids, taurine, and tryptophan. The IBD-associated microbial and metabolomics states were highly correlated, suggesting that they represented an integrated ecosystem. Healthy relatives with the IBD-associated microbial community type had an increased incidence of elevated fecal calprotectin.

Conclusions: Healthy first-degree relatives can have dysbiosis associated with an altered intestinal metabolome that may signify a predisease microbial susceptibility state or subclinical inflammation. Longitudinal prospective studies are required to determine whether these individuals have a clinically significant increased risk for developing IBD.
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http://dx.doi.org/10.1016/j.jcmgh.2016.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247316PMC
November 2016

Microbial, metabolomic, and immunologic dynamics in a relapsing genetic mouse model of colitis induced by T-synthase deficiency.

Gut Microbes 2017 01 22;8(1):1-16. Epub 2016 Nov 22.

b Department of Pathology , University of California Los Angeles , Los Angeles , California , USA.

Intestinal dysbiosis is thought to confer susceptibility to inflammatory bowel disease (IBD), but it is unknown whether dynamic changes in the microbiome contribute to fluctuations in disease activity. We explored this question using mice with intestine-specific deletion of C1galt1 (also known as T-synthase) (Tsyn mice). These mice develop spontaneous microbiota-dependent colitis with a remitting/relapsing course due to loss of mucin core-1 derived O-glycans. 16S rRNA sequencing and untargeted metabolomics demonstrated age-specific perturbations in the intestinal microbiome and metabolome of Tsyn mice compare with littermate controls at weeks 3 (disease onset), 5 (during remission), and 9 (after relapse). Colitis remission corresponded to increased levels of FoxP3+RORγt+CD4+ T cells in the colonic lamina propria that were positively correlated with operational taxonomic units (OTUs) in the S24-7 family and negatively correlated with OTUs in the Clostridiales order. Relapse was characterized by marked expansion of FoxP3-RORγt+CD4+ T cells expressing IFNγ and IL17A, which were associated with Clostridiales OTUs distinct from those negatively correlated with FoxP3+RORγt+CD4+ T cells. Our findings suggest that colitis remission and relapse in the Tsyn model may reflect alterations in the microbiome due to reduced core-1 O-glycosylation that shift the balance of regulatory and pro-inflammatory T cell subsets. We investigated whether genetic variation in C1galt1 correlated with the microbiome in a cohort of 78 Crohn's disease patients and 101 healthy controls. Polymorphisms near C1galt1 (rs10486157) and its molecular chaperone, Cosmc (rs4825729), were associated with altered composition of the colonic mucosal microbiota, supporting the relevance of core-1 O-glycosylation to host regulation of the microbiome.
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http://dx.doi.org/10.1080/19490976.2016.1257469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341916PMC
January 2017

Chemopreventive Metabolites Are Correlated with a Change in Intestinal Microbiota Measured in A-T Mice and Decreased Carcinogenesis.

PLoS One 2016 13;11(4):e0151190. Epub 2016 Apr 13.

Department of Environmental Health Sciences, Fielding School of Public Health, University of California Los Angeles, Los Angeles, California, United States of America.

Intestinal microbiota play a significant role in nutrient metabolism, modulation of the immune system, obesity, and possibly in carcinogenesis, although the underlying mechanisms resulting in disease or impacts on longevity caused by different intestinal microbiota are mostly unknown. Herein we use isogenic Atm-deficient and wild type mice as models to interrogate changes in the metabolic profiles of urine and feces of these mice, which are differing in their intestinal microbiota. Using high resolution mass spectrometry approach we show that the composition of intestinal microbiota modulates specific metabolic perturbations resulting in a possible alleviation of a glycolytic phenotype. Metabolites including 3-methylbutyrolactone, kyneurenic acid and 3-methyladenine known to be onco-protective are elevated in Atm-deficient and wild type mice with restricted intestinal microbiota. Thus our approach has broad applicability to study the direct influence of gut microbiome on host metabolism and resultant phenotype. These results for the first time suggest a possible correlation of metabolic alterations and carcinogenesis, modulated by intestinal microbiota in A-T mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151190PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830457PMC
August 2016

Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer.

J Microbiol Methods 2014 Oct 15;105:82-7. Epub 2014 Jul 15.

Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA. Electronic address:

Current high throughput sequencing (HTS) methods are limited in their ability to resolve bacteria at or below the genus level. While the impact of this limitation may be relatively minor in whole-community analyses, it constrains the use of HTS as a tool for identifying and examining individual bacteria of interest. The limited resolution is a consequence of both short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA (SSU) gene. The goal of this work was to improve the resolving power of bacterial HTS. We developed an assay targeting the hypervariable rRNA internal transcribed spacer (ITS) region residing between the SSU and large-subunit (LSU) rRNA genes. Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. This report presents a new HTS method for analyzing bacterial composition with improved capabilities. The greater resolving power enabled by the ITS region arises from its high sequence variation across a wide range of bacterial taxa and an associated decrease in taxonomic heterogeneity within its OTUs. Although the method should be adaptable to any HTS platform, this report presents PCR primers, amplification parameters, and protocols for Illumina-based analyses.
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http://dx.doi.org/10.1016/j.mimet.2014.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160368PMC
October 2014

Reprograming of gut microbiome energy metabolism by the FUT2 Crohn's disease risk polymorphism.

ISME J 2014 Nov 29;8(11):2193-206. Epub 2014 Apr 29.

1] Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA [2] Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.

Fucosyltransferase 2 (FUT2) is an enzyme that is responsible for the synthesis of the H antigen in body fluids and on the intestinal mucosa. The H antigen is an oligosaccharide moiety that acts as both an attachment site and carbon source for intestinal bacteria. Non-secretors, who are homozygous for the loss-of-function alleles of FUT2 gene (sese), have increased susceptibility to Crohn's disease (CD). To characterize the effect of FUT2 polymorphism on the mucosal ecosystem, we profiled the microbiome, meta-proteome and meta-metabolome of 75 endoscopic lavage samples from the cecum and sigmoid of 39 healthy subjects (12 SeSe, 18 Sese and 9 sese). Imputed metagenomic analysis revealed perturbations of energy metabolism in the microbiome of non-secretor and heterozygote individuals, notably the enrichment of carbohydrate and lipid metabolism, cofactor and vitamin metabolism and glycan biosynthesis and metabolism-related pathways, and the depletion of amino-acid biosynthesis and metabolism. Similar changes were observed in mice bearing the FUT2(-/-) genotype. Metabolomic analysis of human specimens revealed concordant as well as novel changes in the levels of several metabolites. Human metaproteomic analysis indicated that these functional changes were accompanied by sub-clinical levels of inflammation in the local intestinal mucosa. Therefore, the colonic microbiota of non-secretors is altered at both the compositional and functional levels, affecting the host mucosal state and potentially explaining the association of FUT2 genotype and CD susceptibility.
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http://dx.doi.org/10.1038/ismej.2014.64DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992076PMC
November 2014

HIV Infection is associated with compositional and functional shifts in the rectal mucosal microbiota.

Microbiome 2013 Oct 12;1(1):26. Epub 2013 Oct 12.

Pathology and Laboratory Medicine, UCLA, 10833 Le Conte Ave 13-188 CHS, Los Angeles, CA 90095, USA.

Background: Regardless of infection route, the intestine is the primary site for HIV-1 infection establishment and results in significant mucosal CD4+ T lymphocyte depletion, induces an inflammatory state that propagates viral dissemination, facilitates microbial translocation, and fosters establishment of one of the largest HIV reservoirs. Here we test the prediction that HIV infection modifies the composition and function of the mucosal commensal microbiota.

Results: Rectal mucosal microbiota were collected from human subjects using a sponge-based sampling methodology. Samples were collected from 20 HIV-positive men not receiving combination anti-retroviral therapy (cART), 20 HIV-positive men on cART and 20 healthy, HIV-negative men. Microbial composition of samples was analyzed using barcoded 16S Illumina deep sequencing (85,900 reads per sample after processing). Microbial metagenomic information for the samples was imputed using the bioinformatic tools PICRUST and HUMAnN. Microbial composition and imputed function in HIV-positive individuals not receiving cART was significantly different from HIV-negative individuals. Genera including Roseburia, Coprococcus, Ruminococcus, Eubacterium, Alistipes and Lachnospira were depleted in HIV-infected subjects not receiving cART, while Fusobacteria, Anaerococcus, Peptostreptococcus and Porphyromonas were significantly enriched. HIV-positive subjects receiving cART exhibited similar depletion and enrichment for these genera, but were of intermediate magnitude and did not achieve statistical significance. Imputed metagenomic functions, including amino acid metabolism, vitamin biosynthesis, and siderophore biosynthesis differed significantly between healthy controls and HIV-infected subjects not receiving cART.

Conclusions: HIV infection was associated with rectal mucosal changes in microbiota composition and imputed function that cART failed to completely reverse. HIV infection was associated with depletion of some commensal species and enrichment of a few opportunistic pathogens. Many imputed metagenomic functions differed between samples from HIV-negative and HIV-positive subjects not receiving cART, possibly reflecting mucosal metabolic changes associated with HIV infection. Such functional pathways may represent novel interventional targets for HIV therapy if normalizing the microbial composition or functional activity of the microbiota proves therapeutically useful.
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http://dx.doi.org/10.1186/2049-2618-1-26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971626PMC
October 2013

Integrative analysis of the microbiome and metabolome of the human intestinal mucosal surface reveals exquisite inter-relationships.

Microbiome 2013 Jun 5;1(1):17. Epub 2013 Jun 5.

Pathology and Laboratory Medicine UCLA, Los Angeles, CA, USA.

Background: Consistent compositional shifts in the gut microbiota are observed in IBD and other chronic intestinal disorders and may contribute to pathogenesis. The identities of microbial biomolecular mechanisms and metabolic products responsible for disease phenotypes remain to be determined, as do the means by which such microbial functions may be therapeutically modified.

Results: The composition of the microbiota and metabolites in gut microbiome samples in 47 subjects were determined. Samples were obtained by endoscopic mucosal lavage from the cecum and sigmoid colon regions, and each sample was sequenced using the 16S rRNA gene V4 region (Illumina-HiSeq 2000 platform) and assessed by UPLC mass spectroscopy. Spearman correlations were used to identify widespread, statistically significant microbial-metabolite relationships. Metagenomes for identified microbial OTUs were imputed using PICRUSt, and KEGG metabolic pathway modules for imputed genes were assigned using HUMAnN. The resulting metabolic pathway abundances were mostly concordant with metabolite data. Analysis of the metabolome-driven distribution of OTU phylogeny and function revealed clusters of clades that were both metabolically and metagenomically similar.

Conclusions: The results suggest that microbes are syntropic with mucosal metabolome composition and therefore may be the source of and/or dependent upon gut epithelial metabolites. The consistent relationship between inferred metagenomic function and assayed metabolites suggests that metagenomic composition is predictive to a reasonable degree of microbial community metabolite pools. The finding that certain metabolites strongly correlate with microbial community structure raises the possibility of targeting metabolites for monitoring and/or therapeutically manipulating microbial community function in IBD and other chronic diseases.
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http://dx.doi.org/10.1186/2049-2618-1-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3971612PMC
June 2013

Intestinal bacteria modify lymphoma incidence and latency by affecting systemic inflammatory state, oxidative stress, and leukocyte genotoxicity.

Cancer Res 2013 Jul;73(14):4222-32

Department of Pathology and Lab Medicine, David Geffen School of Medicine, University of California, California, USA.

Ataxia-telangiectasia is a genetic disorder associated with high incidence of B-cell lymphoma. Using an ataxia-telangiectasia mouse model, we compared lymphoma incidence in several isogenic mouse colonies harboring different bacterial communities, finding that intestinal microbiota are a major contributor to disease penetrance and latency, lifespan, molecular oxidative stress, and systemic leukocyte genotoxicity. High-throughput sequence analysis of rRNA genes identified mucosa-associated bacterial phylotypes that were colony-specific. Lactobacillus johnsonii, which was deficient in the more cancer-prone mouse colony, was causally tested for its capacity to confer reduced genotoxicity when restored by short-term oral transfer. This intervention decreased systemic genotoxicity, a response associated with reduced basal leukocytes and the cytokine-mediated inflammatory state, and mechanistically linked to the host cell biology of systemic genotoxicity. Our results suggest that intestinal microbiota are a potentially modifiable trait for translational intervention in individuals at risk for B-cell lymphoma, or for other diseases that are driven by genotoxicity or the molecular response to oxidative stress.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-0022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718495PMC
July 2013

Correlations between root-associated microorganisms and peach replant disease symptoms in a California soil.

PLoS One 2012 5;7(10):e46420. Epub 2012 Oct 5.

Department of Nematology, University of California Riverside, Riverside, California, United States of America.

Background: Replant disease often occurs when certain crops are "replanted" in a soil that had previously supported the same or similar plant species. This disease typically leads to reductions in plant growth, crop yields, and production duration, and its etiology remains ill-defined. The objective of this study was to identify microorganisms associated with peach replant disease symptoms at a field location in California, USA. Soil samples were subjected to treatments to create various levels of replant disease symptoms. Clonal peach seedlings were grown in the treated soils in greenhouse trials. After 6 weeks, plant growth parameters were measured, and both culture and culture-independent analyses were performed to identify root-associated bacteria, fungi and stramenopiles.

Results: A total of 295,785 bacterial operational taxonomic units (OTU) were identified by an Illumina-based, high throughput sequence analysis of rRNA genes. Among the 60 most abundant OTUs, 27 showed significant (P<0.05) negative correlation with peach shoot weights while 10 were positively correlated. Most of these OTUs belonged to the bacterial phylum Proteobacteria (96%), including the classes Gammaproteobacteria (44.4%), Betaproteobacteria (33.3%) and Alphaproteobacteria (22.2%), and the orders Pseudomonadales, Burkholderiales, Chromatiales, Rhodocyclales, and Sphingomonadales. The most abundant fungi were Trichoderma asperellum, Trichoderma virens, Fusarium oxysporum, Ceratocystis fimbriata and Fusarium solani. The most abundant stramenopiles were Pythium vexans, Pythium violae and an unidentified Aplanochytrium species. Validation experiments using sequence-selective quantitative PCR analyses identified negative and positive associations between P. vexans and Trichoderma spp. and peach shoot weights, respectively.

Conclusions: This study identified numerous microorganisms associated with peach replant symptoms, some of which have been previously identified while others represent new candidates. Subsequent Koch's postulates investigations will assess their possible roles in this replant disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046420PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465339PMC
April 2013

Improving oligonucleotide fingerprinting of rRNA genes by implementation of polony microarray technology.

J Microbiol Methods 2012 Sep 25;90(3):235-40. Epub 2012 May 25.

Department of Plant Pathology and Microbiology, University of California, Riverside, CA, USA.

Improvements to oligonucleotide fingerprinting of rRNA genes (OFRG) were obtained by implementing polony microarray technology. OFRG is an array-based method for analyzing microbial community composition. Polonies are discrete clusters of DNA, produced by solid-phase PCR in hydrogels, and derived from individual, spatially isolated DNA molecules. The advantages of a polony-based OFRG method include higher throughput and reductions in the PCR-induced errors and compositional skew inherent in all other PCR-based community composition methods, including high-throughput sequencing of rRNA genes. Given the similarities between polony microarrays and certain aspects of sequencing methods such as the Illumina platform, we suggest that if concepts presented in this study were implemented in high-throughput sequencing protocols, a reduction of PCR-induced errors and compositional skew may be realized.
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http://dx.doi.org/10.1016/j.mimet.2012.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404216PMC
September 2012

Improving probe set selection for microbial community analysis by leveraging taxonomic information of training sequences.

BMC Bioinformatics 2011 Oct 10;12:394. Epub 2011 Oct 10.

Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA.

Background: Population levels of microbial phylotypes can be examined using a hybridization-based method that utilizes a small set of computationally-designed DNA probes targeted to a gene common to all. Our previous algorithm attempts to select a set of probes such that each training sequence manifests a unique theoretical hybridization pattern (a binary fingerprint) to a probe set. It does so without taking into account similarity between training gene sequences or their putative taxonomic classifications, however. We present an improved algorithm for probe set selection that utilizes the available taxonomic information of training gene sequences and attempts to choose probes such that the resultant binary fingerprints cluster into real taxonomic groups.

Results: Gene sequences manifesting identical fingerprints with probes chosen by the new algorithm are more likely to be from the same taxonomic group than probes chosen by the previous algorithm. In cases where they are from different taxonomic groups, underlying DNA sequences of identical fingerprints are more similar to each other in probe sets made with the new versus the previous algorithm. Complete removal of large taxonomic groups from training data does not greatly decrease the ability of probe sets to distinguish those groups.

Conclusions: Probe sets made from the new algorithm create fingerprints that more reliably cluster into biologically meaningful groups. The method can readily distinguish microbial phylotypes that were excluded from the training sequences, suggesting novel microbes can also be detected.
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http://dx.doi.org/10.1186/1471-2105-12-394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224148PMC
October 2011

Neutral Zone Classifiers Using a Decision-Theoretic Approach With Application to DNA Array Analyses.

J Agric Biol Environ Stat 2010 Dec;15(4):474-490

Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA.

Two-class neutral zone classifiers were recently proposed for use in microbial community profiling applications. These classifiers allow a region of neutrality for cases where probe hybridization outcomes are too ambiguous to have adequate confidence in assigning a "binding" or "no binding" result. In this paper, we generalize the idea of neutral zone classifiers to an arbitrary number of classes and apply it to improve the process of microbial community profiling by considering a third class for the outcome of probe hybridization experiments, "partial binding." We introduce a family of class distributions that uses a mixture of Gaussian distributions as a model for a Box-Cox power transformation of the raw intensity measurements. Stratified cross-validation analyses are used to assess the efficacy of the proposed three-class neutral zone classifier. This article has supplementary material online.
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http://dx.doi.org/10.1007/s13253-010-0034-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3137885PMC
December 2010

Host-microbe relationships in inflammatory bowel disease detected by bacterial and metaproteomic analysis of the mucosal-luminal interface.

Inflamm Bowel Dis 2012 Mar 22;18(3):409-17. Epub 2011 Jun 22.

Department of Plant Pathology and Microbiology, University of California, Riverside, CA 90095, USA.

Background: Host-microbe interactions at the intestinal mucosal-luminal interface (MLI) are critical factors in the biology of inflammatory bowel disease (IBD).

Methods: To address this issue, we performed a series of investigations integrating analysis of the bacteria and metaproteome at the MLI of Crohn's disease, ulcerative colitis, and healthy human subjects. After quantifying these variables in mucosal specimens from a first sample set, we searched for bacteria exhibiting strong correlations with host proteins. This assessment identified a small subset of bacterial phylotypes possessing this host interaction property. Using a second and independent sample set, we tested the association of disease state with levels of these 14 "host interaction" bacterial phylotypes.

Results: A high frequency of these bacteria (35%) significantly differentiated human subjects by disease type. Analysis of the MLI metaproteomes also yielded disease classification with exceptional confidence levels. Examination of the relationships between the bacteria and proteins, using regularized canonical correlation analysis (RCCA), sorted most subjects by disease type, supporting the concept that host-microbe interactions are involved in the biology underlying IBD. Moreover, this correlation analysis identified bacteria and proteins that were undetected by standard means-based methods such as analysis of variance, and identified associations of specific bacterial phylotypes with particular protein features of the innate immune response, some of which have been documented in model systems.

Conclusions: These findings suggest that computational mining of mucosa-associated bacteria for host interaction provides an unsupervised strategy to uncover networks of bacterial taxa and host processes relevant to normal and disease states. (Inflamm Bowel Dis 2012;).
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http://dx.doi.org/10.1002/ibd.21793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3179764PMC
March 2012

PRISE (PRImer SElector): software for designing sequence-selective PCR primers.

J Microbiol Methods 2008 Mar 23;72(3):263-7. Epub 2007 Dec 23.

Department of Plant Pathology, University of California, Riverside, CA 92521, USA.

This report presents PRImer Selector (PRISE), a new software package that implements several features that improve and streamline the design of sequence-selective PCR primers. The PRISE design process involves two main steps. In the first step, target and non-target DNA sequences are identified. In the second step, primers are designed to amplify target (but not non-target) sequences. One important feature of PRISE is that it automates the task of placing primer-template mismatches at the 3' end of the primers - a property that is crucial for sequence selectivity. Once a list of candidate primers has been produced, sorting tools in PRISE speed up the selection process by allowing a user to sort the primers by properties such as amplicon length, GC content and sequence selectivity. PRISE can be used to design primers with a range of specificities, targeting individual sequences as well as diverse assemblages of genes. PRISE also allows user-defined primers to be analyzed, enabling their properties to be examined in relation to target and non-target sequences. The utility of PRISE was demonstrated by using it to design sequence-selective PCR primers for an rRNA gene from the fungus Pochonia chlamydosporia.
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http://dx.doi.org/10.1016/j.mimet.2007.12.004DOI Listing
March 2008
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