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  1. Article ; Online: A contamination focused approach for optimizing the single-cell RNA-seq experiment

    Deronisha Arceneaux / Zhengyi Chen / Alan J. Simmons / Cody N. Heiser / Austin N. Southard-Smith / Michael J. Brenan / Yilin Yang / Bob Chen / Yanwen Xu / Eunyoung Choi / Joshua D. Campbell / Qi Liu / Ken S. Lau

    iScience, Vol 26, Iss 7, Pp 107242- (2023)

    2023  

    Abstract: Summary: Droplet-based single-cell RNA-seq (scRNA-seq) data are plagued by ambient contaminations caused by nucleic acid material released by dead and dying cells. This material is mixed into the buffer and is co-encapsulated with cells, leading to a ... ...

    Abstract Summary: Droplet-based single-cell RNA-seq (scRNA-seq) data are plagued by ambient contaminations caused by nucleic acid material released by dead and dying cells. This material is mixed into the buffer and is co-encapsulated with cells, leading to a lower signal-to-noise ratio. Although there exist computational methods to remove ambient contaminations post-hoc, the reliability of algorithms in generating high-quality data from low-quality sources remains uncertain. Here, we assess data quality before data filtering by a set of quantitative, contamination-based metrics that assess data quality more effectively than standard metrics. Through a series of controlled experiments, we report improvements that can minimize ambient contamination outside of tissue dissociation, via cell fixation, improved cell loading, microfluidic dilution, and nuclei versus cell preparation; many of these parameters are inaccessible on commercial platforms. We provide end-users with insights on factors that can guide their decision-making regarding optimizations that minimize ambient contamination, and metrics to assess data quality.
    Keywords Computational bioinformatics ; Transcriptomics ; Biology experimental methods ; Science ; Q
    Subject code 310
    Language English
    Publishing date 2023-07-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Clinically adaptable polymer enables simultaneous spatial analysis of colonic tissues and biofilms

    Mary C. Macedonia / Julia L. Drewes / Nicholas O. Markham / Alan J. Simmons / Joseph T. Roland / Paige N. Vega / Cherie’ R. Scurrah / Robert J. Coffey / Martha J. Shrubsole / Cynthia L. Sears / Ken S. Lau

    npj Biofilms and Microbiomes, Vol 6, Iss 1, Pp 1-

    2020  Volume 10

    Abstract: Abstract Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially ... ...

    Abstract Abstract Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in poloxamer-fixed samples. This in turn enabled us to integrate multimodal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and cross-link at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.
    Keywords Microbial ecology ; QR100-130
    Language English
    Publishing date 2020-09-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Quantitative assessment of cell population diversity in single-cell landscapes.

    Qi Liu / Charles A Herring / Quanhu Sheng / Jie Ping / Alan J Simmons / Bob Chen / Amrita Banerjee / Wei Li / Guoqiang Gu / Robert J Coffey / Yu Shyr / Ken S Lau

    PLoS Biology, Vol 16, Iss 10, p e

    2018  Volume 2006687

    Abstract: Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, ...

    Abstract Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes and regional specification, identification and definition of altered cell infiltrates in tumorigenesis, and benchmarking batch-correction tools. sc-UniFrac provides a framework for quantifying diversity or alterations in cell populations across conditions and has broad utility for gaining insight into tissue-level perturbations at the single-cell resolution.
    Keywords Biology (General) ; QH301-705.5
    Subject code 612
    Language English
    Publishing date 2018-10-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Heterogeneity and dynamics of active Kras-induced dysplastic lineages from mouse corpus stomach

    Jimin Min / Paige N. Vega / Amy C. Engevik / Janice A. Williams / Qing Yang / Loraine M. Patterson / Alan J. Simmons / R. Jarrett Bliton / Joshua W. Betts / Ken S. Lau / Scott T. Magness / James R. Goldenring / Eunyoung Choi

    Nature Communications, Vol 10, Iss 1, Pp 1-

    2019  Volume 16

    Abstract: How a precancerous form (dysplasia) becomes gastric cancer is unclear. Here, the authors assess the role of Kras activation in heterogenous dysplastic cells in murine stomach corpus organoids, identifying two dysplastic stem cell populations and show ... ...

    Abstract How a precancerous form (dysplasia) becomes gastric cancer is unclear. Here, the authors assess the role of Kras activation in heterogenous dysplastic cells in murine stomach corpus organoids, identifying two dysplastic stem cell populations and show that MEK inhibition causes alterations in cell behavior.
    Keywords Science ; Q
    Language English
    Publishing date 2019-12-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Heterogeneity and dynamics of active Kras-induced dysplastic lineages from mouse corpus stomach

    Jimin Min / Paige N. Vega / Amy C. Engevik / Janice A. Williams / Qing Yang / Loraine M. Patterson / Alan J. Simmons / R. Jarrett Bliton / Joshua W. Betts / Ken S. Lau / Scott T. Magness / James R. Goldenring / Eunyoung Choi

    Nature Communications, Vol 10, Iss 1, Pp 1-

    2019  Volume 16

    Abstract: How a precancerous form (dysplasia) becomes gastric cancer is unclear. Here, the authors assess the role of Kras activation in heterogenous dysplastic cells in murine stomach corpus organoids, identifying two dysplastic stem cell populations and show ... ...

    Abstract How a precancerous form (dysplasia) becomes gastric cancer is unclear. Here, the authors assess the role of Kras activation in heterogenous dysplastic cells in murine stomach corpus organoids, identifying two dysplastic stem cell populations and show that MEK inhibition causes alterations in cell behavior.
    Keywords Science ; Q
    Language English
    Publishing date 2019-12-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Cytometry‐based single‐cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF‐α‐induced apoptosis in vivo

    Alan J Simmons / Amrita Banerjee / Eliot T McKinley / Cherie’ R Scurrah / Charles A Herring / Leslie S Gewin / Ryota Masuzaki / Seth J Karp / Jeffrey L Franklin / Michael J Gerdes / Jonathan M Irish / Robert J Coffey / Ken S Lau

    Molecular Systems Biology, Vol 12, Iss 8, Pp n/a-n/a (2016)

    2016  

    Keywords Biology (General) ; QH301-705.5 ; Medicine (General) ; R5-920
    Language English
    Publishing date 2016-08-01T00:00:00Z
    Publisher Wiley
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Cytometry‐based single‐cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF‐α‐induced apoptosis in vivo

    Alan J Simmons / Amrita Banerjee / Eliot T McKinley / Cherie' R Scurrah / Charles A Herring / Leslie S Gewin / Ryota Masuzaki / Seth J Karp / Jeffrey L Franklin / Michael J Gerdes / Jonathan M Irish / Robert J Coffey / Ken S Lau

    Molecular Systems Biology, Vol 11, Iss 10, Pp n/a-n/a (2015)

    2015  

    Abstract: Abstract Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single‐cell resolution. However, probing signaling in epithelial tissues using cytometry‐based single‐cell analysis has been ... ...

    Abstract Abstract Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single‐cell resolution. However, probing signaling in epithelial tissues using cytometry‐based single‐cell analysis has been confounded by the necessity of single‐cell dissociation, where disrupting cell‐to‐cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue—DISSECT) that preserves native signaling for Cytometry Time‐of‐Flight (CyTOF) and fluorescent flow cytometry applications. A 21‐plex CyTOF analysis encompassing core signaling and cell‐identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor‐alpha (TNF‐α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF‐α‐induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p‐ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact‐dependent survival. Our novel single‐cell approach can broadly be applied, using both CyTOF and multi‐parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.
    Keywords apoptosis ; CyTOF ; epithelial signaling ; single‐cell biology ; TNF ; Biology (General) ; QH301-705.5 ; Medicine (General) ; R5-920
    Subject code 610
    Language English
    Publishing date 2015-10-01T00:00:00Z
    Publisher Wiley
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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