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Sökning: L773:1754 2189 OR L773:1750 2799

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  • Andersson, Christopher, et al. (författare)
  • Using the chicken embryo to assess virulence of Listeria monocytogenes and to model other microbial infections
  • 2015
  • Ingår i: Nature Protocols. - : Nature Publishing Group. - 1754-2189 .- 1750-2799. ; 10:8, s. 1155-1164
  • Tidskriftsartikel (refereegranskat)abstract
    • Microbial infections are a global health problem, particularly as microbes are continually developing resistance to antimicrobial treatments. An effective and reliable method for testing the virulence of different microbial pathogens is therefore a useful research tool. This protocol describes how the chicken embryo can be used as a trustworthy, inexpensive, ethically desirable and quickly accessible model to assess the virulence of the human bacterial pathogen Listeria monocytogenes, which can also be extended to other microbial pathogens. We provide a step-by-step protocol and figures and videos detailing the method, including egg handling, infection strategies, pathogenicity screening and isolation of infected organs. From the start of incubation of the fertilized eggs, the protocol takes <4 weeks to complete, with the infection part taking only 3 d. We discuss the appropriate controls to use and potential adjustments needed for adapting the protocol for other microbial pathogens.
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  • Bárcenas-Walls, José Ramón, et al. (författare)
  • Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution
  • 2024
  • Ingår i: Nature Protocols. - 1754-2189 .- 1750-2799.
  • Tidskriftsartikel (refereegranskat)abstract
    • The ability to comprehensively analyze the chromatin state with single-cell resolution is crucial for understanding gene regulatory principles in heterogenous tissues or during development. Recently, we developed a nanobody-based single-cell CUT&Tag (nano-CT) protocol to simultaneously profile three epigenetic modalities-two histone marks and open chromatin state-from the same single cell. Nano-CT implements a new set of secondary nanobody-Tn5 fusion proteins to direct barcoded tagmentation by Tn5 transposase to genomic targets labeled by primary antibodies raised in different species. Such nanobody-Tn5 fusion proteins are currently not commercially available, and their in-house production and purification can be completed in 3-4 d by following our detailed protocol. The single-cell indexing in nano-CT is performed on a commercially available platform, making it widely accessible to the community. In comparison to other multimodal methods, nano-CT stands out in data complexity, low sample requirements and the flexibility to choose two of the three modalities. In addition, nano-CT works efficiently with fresh brain samples, generating multimodal epigenomic profiles for thousands of brain cells at single-cell resolution. The nano-CT protocol can be completed in just 3 d by users with basic skills in standard molecular biology and bioinformatics, although previous experience with single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is beneficial for more in-depth data analysis. As a multimodal assay, nano-CT holds immense potential to reveal interactions of various chromatin modalities, to explore epigenetic heterogeneity and to increase our understanding of the role and interplay that chromatin dynamics has in cellular development. This protocol involves profiling gene regulatory dynamics in complex tissues at the single-cell level. This is achieved by generating two nanobody-Tn5 fusion proteins to recognize primary antibodies against widespread histone modifications.The nanobody-Tn5 fusions are combined with ATAC-seq to simultaneously profile three epigenetic modalities from the same single cell, thousands of cells at the same time. A bioinformatic pipeline, Nanoscope, for seamless analysis of nano-CT datasets is also described. Mapping of chromatin states at single-cell resolution is still challenging. This protocol describes nano-CT, a novel method to simultaneously characterize up to three epigenetic modalities at single-cell resolution.
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  • Beloqui, Ana, et al. (författare)
  • A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation
  • 2017
  • Ingår i: Nature Protocols. - : NATURE PUBLISHING GROUP. - 1754-2189 .- 1750-2799. ; 12:7, s. 1387-1399
  • Tidskriftsartikel (refereegranskat)abstract
    • The specialized microfold cells (M cells) in the follicle-associated epithelium (FAE) of intestinal Peyer's patches serve as antigen-sampling cells of the intestinal innate immune system. Unlike 'classical' enterocytes, they are able to translocate diverse particulates without digesting them. They act as pathways for microorganism invasion and mediate food tolerance by transcellular transport of intestinal microbiota and antigens. Their ability to transcytose intact particles can be used to develop oral drug delivery and oral immunization strategies. This protocol describes a reproducible and versatile human M-cell-like in vitro model. This model can be exploited to evaluate M-cell transport of microparticles and nanoparticles for protein, drug or vaccine delivery and to study bacterial adherence and translocation across M cells. The inverted in vitro M-cell model consists of three main steps. First, Caco-2 cells are seeded at the apical side of the inserts. Second, the inserts are inverted and B lymphocytes are seeded at the basolateral side of the inserts. Third, the conversion to M cells is assessed. Although various M-cell culture systems exist, this model provides several advantages over the rest: (i) it is based on coculture with well-established differentiated human cell lines; (ii) it is reproducible under the conditions described herein; (iii) it can be easily mastered; and (iv) it does not require the isolation of primary cells or the use of animals. The protocol requires skills in cell culture and microscopy analysis. The model is obtained after 3 weeks, and transport experiments across the differentiated model can be carried out over periods of up to 10 h.
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  • Bender, Brian J., et al. (författare)
  • A practical guide to large-scale docking
  • 2021
  • Ingår i: Nature Protocols. - : Springer Nature. - 1754-2189 .- 1750-2799. ; 16:10, s. 4799-4832
  • Tidskriftsartikel (refereegranskat)abstract
    • Structure-based docking screens of large compound libraries have become common in early drug and probe discovery. As computer efficiency has improved and compound libraries have grown, the ability to screen hundreds of millions, and even billions, of compounds has become feasible for modest-sized computer clusters. This allows the rapid and cost-effective exploration and categorization of vast chemical space into a subset enriched with potential hits for a given target. To accomplish this goal at speed, approximations are used that result in undersampling of possible configurations and inaccurate predictions of absolute binding energies. Accordingly, it is important to establish controls, as are common in other fields, to enhance the likelihood of success in spite of these challenges. Here we outline best practices and control docking calculations that help evaluate docking parameters for a given target prior to undertaking a large-scale prospective screen, with exemplification in one particular target, the melatonin receptor, where following this procedure led to direct docking hits with activities in the subnanomolar range. Additional controls are suggested to ensure specific activity for experimentally validated hit compounds. These guidelines should be useful regardless of the docking software used. Docking software described in the outlined protocol (DOCK3.7) is made freely available for academic research to explore new hits for a range of targets. Structure-based docking screens of compound libraries are common in early drug and probe discovery. This protocol outlines best practices and control calculations to evaluate docking parameters prior to undertaking a large-scale prospective screen.
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  • Berg, Ina, 1982-, et al. (författare)
  • Efficient imaging of amyloid deposits in Drosophila models of human amyloidoses
  • 2010
  • Ingår i: Nature Protocols. - : Macmillan Publishers Ltd.. - 1754-2189 .- 1750-2799. ; 5:5, s. 935-944
  • Tidskriftsartikel (refereegranskat)abstract
    • Drosophila melanogaster is emerging as an important model system for neurodegenerative disease research. In this protocol, we describe an efficient method for imaging amyloid deposits in the Drosophila brain, by the use of a luminescent-conjugated oligothiophene (lco), p-Ftaa polymer probe. We also demonstrate the feasibility of co-staining with antibodies and compare the lco staining with standard amyloid-specific probes. the lco protocol enables high-resolution imaging of several different protein aggregates, such as aβ1-42, aβ1-42e22G, transthyretin V30M and human tau, in the Drosophila brain. aβ and tau aggregates could also be distinguished from each other because of distinct lco emission spectra. Furthermore, this protocol enables threedimensional brain mapping of amyloid distribution in whole-mount Drosophila brains. the use of p-Ftaa combined with other probes, antibodies and/or dyes will aid the rapid characterization of various amyloid deposits in the rapidly growing number of Drosophila models of neurodegenerative diseases.
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  • Blaise, Benjamin J., et al. (författare)
  • Statistical analysis in metabolic phenotyping
  • 2021
  • Ingår i: Nature Protocols. - : Nature Publishing Group. - 1754-2189 .- 1750-2799. ; 16:9, s. 4299-4326
  • Forskningsöversikt (refereegranskat)abstract
    • Metabolic phenotyping is an important tool in translational biomedical research. The advanced analytical technologies commonly used for phenotyping, including mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, generate complex data requiring tailored statistical analysis methods. Detailed protocols have been published for data acquisition by liquid NMR, solid-state NMR, ultra-performance liquid chromatography (LC-)MS and gas chromatography (GC-)MS on biofluids or tissues and their preprocessing. Here we propose an efficient protocol (guidelines and software) for statistical analysis of metabolic data generated by these methods. Code for all steps is provided, and no prior coding skill is necessary. We offer efficient solutions for the different steps required within the complete phenotyping data analytics workflow: scaling, normalization, outlier detection, multivariate analysis to explore and model study-related effects, selection of candidate biomarkers, validation, multiple testing correction and performance evaluation of statistical models. We also provide a statistical power calculation algorithm and safeguards to ensure robust and meaningful experimental designs that deliver reliable results. We exemplify the protocol with a two-group classification study and data from an epidemiological cohort; however, the protocol can be easily modified to cover a wider range of experimental designs or incorporate different modeling approaches. This protocol describes a minimal set of analyses needed to rigorously investigate typical datasets encountered in metabolic phenotyping.
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  • Bludau, Isabell, et al. (författare)
  • Complex-centric proteome profiling by SEC-SWATH-MS for the parallel detection of hundreds of protein complexes
  • 2020
  • Ingår i: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 15:8, s. 2341-2386
  • Tidskriftsartikel (refereegranskat)abstract
    • Most catalytic, structural and regulatory functions of the cell are carried out by functional modules, typically complexes containing or consisting of proteins. The composition and abundance of these complexes and the quantitative distribution of specific proteins across different modules are therefore of major significance in basic and translational biology. However, detection and quantification of protein complexes on a proteome-wide scale is technically challenging. We have recently extended the targeted proteomics rationale to the level of native protein complex analysis (complex-centric proteome profiling). The complex-centric workflow described herein consists of size exclusion chromatography (SEC) to fractionate native protein complexes, data-independent acquisition mass spectrometry to precisely quantify the proteins in each SEC fraction based on a set of proteotypic peptides and targeted, complex-centric analysis where prior information from generic protein interaction maps is used to detect and quantify protein complexes with high selectivity and statistical error control via the computational framework CCprofiler (https://github.com/CCprofiler/CCprofiler). Complex-centric proteome profiling captures most proteins in complex-assembled state and reveals their organization into hundreds of complexes and complex variants observable in a given cellular state. The protocol is applicable to cultured cells and can potentially also be adapted to primary tissue and does not require any genetic engineering of the respective sample sources. At present, it requires ~8 d of wet-laboratory work, 15 d of mass spectrometry measurement time and 7 d of computational analysis.
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