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IgA Targets the Troublemakers
2014
Cell Host and Microbe
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Imaging Bacterial Colonization of the Zebrafish Gut with Selective Plane Illumination
2012
Biophysical Journal
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Zac Stephens, Joshua V. Troll, Karen Guillemin, Raghuveer Parthasarathy. University of Oregon, Eugene, OR, USA. ...
Current Opinion in Biotechnology 2007, 18, 287. 759-Pos Board B545 Imaging Bacterial Colonization of the Zebrafish Gut with Selective Plane Illumination Matthew Jemielita, Mike Taormina, W. ...
doi:10.1016/j.bpj.2011.11.833
fatcat:zk3tu7esaffjzn7agnjo3fnb2e
Study of Host–Microbe Interactions in Zebrafish
[chapter]
2011
Methods in Cell Biology
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All animals are ecosystems, home to diverse microbial populations. Animal-associated microbes play important roles in the normal development and physiology of their hosts, but can also be agents of infectious disease. Traditionally, mice have been used to study pathogenic and beneficial associations between microbes and vertebrate animals. The zebrafish is emerging as a valuable new model system for host-microbe interaction studies, affording researchers with the opportunity to survey large
doi:10.1016/b978-0-12-381320-6.00004-7
pmid:21951527
pmcid:PMC4700925
fatcat:khm77s66nnchxowrxdmio2xjie
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... lations of hosts and to visualize microbe-host associations at a cellular level in living animals. This chapter provides detailed protocols for the analysis of zebrafish-associated microbial communities, the derivation and husbandry of germ-free zebrafish, and the modeling of infectious disease in different stages of zebrafish development via different routes of inoculation. These protocols offer a starting point for researchers to address a multitude of questions about animals' coexistence with microorganisms.
Does MHC heterozygosity influence microbiota form and function?
2019
PLoS ONE
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MHC molecules are essential for the adaptive immune response, and they are the most polymorphic genetic loci in vertebrates. Extreme genetic variation at these loci is paradoxical given their central importance to host health. Classic models of MHC gene evolution center on antagonistic host-pathogen interactions to promote gene diversification and allelic diversity in host populations. However, all multicellular organisms are persistently colonized by their microbiota that perform essential
doi:10.1371/journal.pone.0215946
pmid:31095603
pmcid:PMC6522005
fatcat:l72mc5epgjd3lhstwcdyls33ae
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... bolic functions for their host and protect from infection. Here, we provide data to support the hypothesis that MHC heterozygote advantage (a main force of selection thought to drive MHC gene evolution), may operate by enhancing fitness advantages conferred by the host's microbiome. We utilized fecal 16S rRNA gene sequences and their predicted metagenome datasets collected from multiple MHC congenic homozygote and heterozygote mouse strains to describe the influence of MHC heterozygosity on microbiome form and function. We find that in contrast to homozygosity at MHC loci, MHC heterozygosity promotes functional diversification of the microbiome, enhances microbial network connectivity, and results in enrichment for a variety of microbial functions that are positively associated with host fitness. We demonstrate that taxonomic and functional diversity of the microbiome is positively correlated in MHC heterozygote but not homozygote animals, suggesting that heterozygote microbiomes are more functionally adaptive under similar environmental conditions than homozygote microbiomes. Our data complement previous observations on the role of MHC polymorphism in sculpting microbiota composition, but also provide functional insights into how MHC heterozygosity may enhance host health by modulating microbiome form and function. We also provide evidence to support that MHC heterozygosity limits functional redundancy among commensal microbes and may enhance the metabolic versatility of their microbiome. Results from our analyses yield multiple testable predictions regarding the role of MHC heterozygosity on the microbiome that will help guide future research in the area of MHC-microbiome interactions.
Investigating Bacterial-Animal Symbioses with Light Sheet Microscopy
2012
The Biological Bulletin
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Microbial colonization of the digestive tract is a crucial event in vertebrate development, required for maturation of host immunity and establishment of normal digestive physiology. Advances in genomic, proteomic, and metabolomic technologies are providing a more detailed picture of the constituents of the intestinal habitat, but these approaches lack the spatial and temporal resolution needed to characterize the assembly and dynamics of microbial communities in this complex environment. We
doi:10.1086/bblv223n1p7
pmid:22983029
pmcid:PMC3952068
fatcat:zxtn5lrgfffkbnnywfwxkmq46q
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... ort the use of light sheet microscopy to provide high resolution imaging of bacterial colonization of the zebrafish intestine. The methodology allows us to characterize bacterial population dynamics across the entire organ and the behaviors of individual bacterial and host cells throughout the colonization process. The large four-dimensional datasets generated by these imaging approaches require new strategies for image analysis. When integrated with other "omics" datasets, information about the spatial and temporal dynamics of microbial cells within the vertebrate intestine will provide new mechanistic insights into how microbial communities assemble and function within hosts.
Evidence for a core gut microbiota in the zebrafish
2011
The ISME Journal
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Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of
doi:10.1038/ismej.2011.38
pmid:21472014
pmcid:PMC3176511
fatcat:6nrpjltvwfbojebdfpnbmkw4ny
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... bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status.
Retinoic acid is required for endodermal pouch morphogenesis and not for pharyngeal endoderm specification
2006
Developmental Dynamics
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Because tissues from all three germ layers contribute to the pharyngeal arches, it is not surprising that all major signaling pathways are involved in their development. We focus on the role of retinoic acid (RA) signaling because it has been recognized for quite some time that alterations in this pathway lead to craniofacial malformations. Several studies exist that describe phenotypes observed upon RA perturbations in pharyngeal arch development; however, these studies did not address whether
doi:10.1002/dvdy.20905
pmid:16871626
fatcat:cjk2jalk2zb57bbutclqvbnkqy
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... RA plays multiple roles at distinct time points during development. Here, we report the resulting phenotypes in the hindbrain, the neural crest-derived tissues, and the pharyngeal endoderm when RA synthesis is disrupted during zebrafish gastrulation and pharyngeal arch morphogenesis. Our results demonstrate that RA is required for the postgastrulation morphogenesis and segmentation of endodermal pouches, and that loss of RA does not affect the length of the pharyngeal ectoderm or medial endoderm along the anterior-posterior axis. We also provide evidence that RA is not required for the specification of pharyngeal pouch endoderm and that the pharyngeal endoderm consists of at least two different cell populations, of which the pouch endoderm is sensitive to RA and the more medial pharyngeal endoderm is not. These results demonstrate that the developmental processes underlying pharyngeal arch defects differ depending on when RA signaling is disturbed during development.
Ontogenetic Differences in Dietary Fat Influence Microbiota Assembly in the Zebrafish Gut
2015
mBio
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Samples were named as follows: tank identifier (ID), followed by time point (5, 10, 35, or 70 dpf), followed by sample ID ("m" for water column, "s" for tank floor, "w" for tank wall, and the remaining ...
We therefore speculate that the observed differences between this study and that performed by Stephens et al. (6) , at comparable developmental stages, may be due in part to use of a constant diet and ...
doi:10.1128/mbio.00687-15
pmid:26419876
pmcid:PMC4611033
fatcat:nky3xpcoibdptgavnaolfsuyey
The composition of the zebrafish intestinal microbial community varies across development
2015
The ISME Journal
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Changes in the zebrafish microbiota across development W Zac Stephens et al Figure 1 1 Experimental design and zebrafish development. ...
Changes in the zebrafish microbiota across development
W Zac Stephens et al
Table 2 2 Results of multiple regressions comparing community dissimilarity with differences in host age, standard length ...
doi:10.1038/ismej.2015.140
pmid:26339860
pmcid:PMC4817687
fatcat:4ifwidhbcffojjj3nztxvvogj4
The enteric nervous system promotes intestinal health by constraining microbiota composition
2017
PLoS Biology
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Sustaining a balanced intestinal microbial community is critical for maintaining intestinal health and preventing chronic inflammation. The gut is a highly dynamic environment, subject to periodic waves of peristaltic activity. We hypothesized that this dynamic environment is a prerequisite for a balanced microbial community and that the enteric nervous system (ENS), a chief regulator of physiological processes within the gut, profoundly influences gut microbiota composition. We found that
doi:10.1371/journal.pbio.2000689
pmid:28207737
pmcid:PMC5331947
fatcat:xvhzjaknynhb7c6yb6zfs6gnn4
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... fish lacking an ENS due to a mutation in the Hirschsprung disease gene, sox10, develop microbiota-dependent inflammation that is transmissible between hosts. Profiling microbial communities across a spectrum of inflammatory phenotypes revealed that increased levels of inflammation were linked to an overabundance of pro-inflammatory bacterial lineages and a lack of anti-inflammatory bacterial lineages. Moreover, either administering a representative anti-inflammatory strain or restoring ENS function corrected the pathology. Thus, we demonstrate that the ENS modulates gut microbiota community membership to maintain intestinal health. Author summary Intestinal health depends on maintaining a balanced microbial community within the highly dynamic environment of the intestine. Every few minutes, this environment is rocked by peristaltic waves of muscular contraction and relaxation through a process regulated by the enteric nervous system (ENS). We hypothesized that normal, healthy intestinal microbial communities are adapted to this dynamic environment, and that their composition would become perturbed without a functional ENS. To test this idea, we used a model organism, the zebrafish, with a genetic mutation that prevents formation of PLOS Biology |
CIPR: a web-based R/shiny app and R package to annotate cell clusters in single cell RNA sequencing experiments
2020
BMC Bioinformatics
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Single cell RNA sequencing (scRNAseq) has provided invaluable insights into cellular heterogeneity and functional states in health and disease. During the analysis of scRNAseq data, annotating the biological identity of cell clusters is an important step before downstream analyses and it remains technically challenging. The current solutions for annotating single cell clusters generally lack a graphical user interface, can be computationally intensive or have a limited scope. On the other hand,
doi:10.1186/s12859-020-3538-2
pmid:32414321
fatcat:e4m4ennacbh4rgp2crbec2pb5q
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... manually annotating single cell clusters by examining the expression of marker genes can be subjective and labor-intensive. To improve the quality and efficiency of annotating cell clusters in scRNAseq data, we present a web-based R/Shiny app and R package, Cluster Identity PRedictor (CIPR), which provides a graphical user interface to quickly score gene expression profiles of unknown cell clusters against mouse or human references, or a custom dataset provided by the user. CIPR can be easily integrated into the current pipelines to facilitate scRNAseq data analysis. CIPR employs multiple approaches for calculating the identity score at the cluster level and can accept inputs generated by popular scRNAseq analysis software. CIPR provides 2 mouse and 5 human reference datasets, and its pipeline allows inter-species comparisons and the ability to upload a custom reference dataset for specialized studies. The option to filter out lowly variable genes and to exclude irrelevant reference cell subsets from the analysis can improve the discriminatory power of CIPR suggesting that it can be tailored to different experimental contexts. Benchmarking CIPR against existing functionally similar software revealed that our algorithm is less computationally demanding, it performs significantly faster and provides accurate predictions for multiple cell clusters in a scRNAseq experiment involving tumor-infiltrating immune cells. CIPR facilitates scRNAseq data analysis by annotating unknown cell clusters in an objective and efficient manner. Platform independence owing to Shiny framework and the requirement for a minimal programming experience allows this software to be used by researchers from different backgrounds. CIPR can accurately predict the identity of a variety of cell clusters and can be used in various experimental contexts across a broad spectrum of research areas.
Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth
2016
PLoS ONE
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Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human micro-RNAs that suppress or promote cell growth, revealing that
doi:10.1371/journal.pone.0153689
pmid:27081855
pmcid:PMC4833428
fatcat:bkvfpr73jbdkvn3ifgbkhccjj4
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... roRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how micro-RNAs contribute to human disease.
MyD88 Signaling in T Cells Directs IgA-Mediated Control of the Microbiota to Promote Health
2015
Cell Host and Microbe
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., 2011; Stephens and Round, 2014) . TiD often results in low-affinity, cross-reactive antibody. ...
doi:10.1016/j.chom.2014.12.009
pmid:25620548
pmcid:PMC4451207
fatcat:5ppbj7w6zzei7nwrymw7uak4ai
MicroRNA-155 coordinates the immunological landscape within murine melanoma and correlates with immunity in human cancers
2019
JCI Insight
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miR-155 has recently emerged as an important promoter of antitumor immunity through its functions in T lymphocytes. However, the impact of T cell-expressed miR-155 on immune cell dynamics in solid tumors remains unclear. In the present study, we used single-cell RNA sequencing to define the CD45+ immune cell populations at different time points within B16F10 murine melanoma tumors growing in either wild-type or miR-155 T cell conditional knockout (TCKO) mice. miR-155 was required for optimal T
doi:10.1172/jci.insight.126543
pmid:30721153
pmcid:PMC6482995
fatcat:6xlzrizmdbet5m4cws7tbbb2qy
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... ell activation and reinforced the T cell response at the expense of infiltrating myeloid cells. Further, myeloid cells from tumors growing in TCKO mice were defined by an increase in wound healing genes and a decreased IFN-γ-response gene signature. Finally, we found that miR-155 expression predicted a favorable outcome in human melanoma patients and was associated with a strong immune signature. Moreover, gene expression analysis of The Cancer Genome Atlas (TCGA) data revealed that miR-155 expression also correlates with an immune-enriched subtype in 29 other human solid tumors. Together, our study provides an unprecedented analysis of the cell types and gene expression signatures of immune cells within experimental melanoma tumors and elucidates the role of miR-155 in coordinating antitumor immune responses in mammalian tumors.
Identification of Population Bottlenecks and Colonization Factors during Assembly of Bacterial Communities within the Zebrafish Intestine
2015
mBio
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The zebrafish, Danio rerio , is a powerful model for studying bacterial colonization of the vertebrate intestine, but the genes required by commensal bacteria to colonize the zebrafish gut have not yet been interrogated on a genome-wide level. Here we apply a high-throughput transposon mutagenesis screen to Aeromonas veronii Hm21 and Vibrio sp. strain ZWU0020 during their colonization of the zebrafish intestine alone and in competition with each other, as well as in different colonization
doi:10.1128/mbio.01163-15
pmid:26507229
pmcid:PMC4626852
fatcat:fwvkjmlau5cvxeaz2hyoqeeypm
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... . We use these transposon-tagged libraries to track bacterial population sizes in different colonization regimes and to identify gene functions required during these processes. We show that intraspecific, but not interspecific, competition with a previously established bacterial population greatly reduces the ability of these two bacterial species to colonize. Further, using a simple binomial sampling model, we show that under conditions of interspecific competition, genes required for colonization cannot be identified because of the population bottleneck experienced by the second colonizer. When bacteria colonize the intestine alone or at the same time as the other species, we find shared suites of functional requirements for colonization by the two species, including a prominent role for chemotaxis and motility, regardless of the presence of another species. IMPORTANCE Zebrafish larvae, which are amenable to large-scale gnotobiotic studies, comprehensive sampling of their intestinal microbiota, and live imaging, are an excellent model for investigations of vertebrate intestinal colonization dynamics. We sought to develop a mutagenesis and tagging system in order to understand bacterial population dynamics and functional requirements during colonization of the larval zebrafish intestine. We explored changes in bacterial colonization dynamics and functional requirements when bacteria colonize a bacterium-free intestine, one previously colonized by their own species, or one colonized previously or simultaneously with a different species. This work provides a framework for rapid identification of colonization factors important under different colonization conditions. Furthermore, we demonstrate that when colonizing bacterial populations are very small, this approach is not accurate because random sampling of the input pool is sufficient to explain the distribution of inserts recovered from bacteria that colonized the intestines.
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