Microbiology

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    Protocols in Current Issue
    A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA
    Authors:  Benno Zehnder, Stephan Urban and Thomas Tu, date: 04/20/2021, view: 56, Q&A: 0

    Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral

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    Measuring Cytosolic Translocation of Mycobacterium marinum in RAW264.7 Macrophages with a CCF4-AM FRET Assay
    Authors:  Javier Aguilera and Jianjun Sun, date: 04/20/2021, view: 32, Q&A: 0
    The CCF4-AM Förster resonance energy transfer (FRET) assay is a sensitive approach to measure bacterial cytosolic translocation in live cells. The FRET pair hydroxycoumarin (donor) and fluorescein (acceptor) are linked by a CCF4-AM β-lactam ring, ...
    Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry
    Authors:  Gareth Howell and Robert-Jan Bleichrodt, date: 04/20/2021, view: 30, Q&A: 0

    Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but

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    Screening for Lysogen Activity in Therapeutically Relevant Bacteriophages
    Authors:  Fernando L. Gordillo Altamirano and Jeremy J. Barr, date: 04/20/2021, view: 142, Q&A: 0

    Lysogenic phages can integrate into their bacterial host’s genome, potentially transferring any genetic information they possess including virulence or resistance genes, and are therefore routinely excluded from therapeutic applications.

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    A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA
    Authors:  Benno Zehnder, Stephan Urban and Thomas Tu, date: 04/20/2021, view: 56, Q&A: 0
    [Abstract]

    Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain

    ...
    Measuring Cytosolic Translocation of Mycobacterium marinum in RAW264.7 Macrophages with a CCF4-AM FRET Assay
    Authors:  Javier Aguilera and Jianjun Sun, date: 04/20/2021, view: 32, Q&A: 0
    [Abstract] The CCF4-AM Förster resonance energy transfer (FRET) assay is a sensitive approach to measure bacterial cytosolic translocation in live cells. The FRET pair hydroxycoumarin (donor) and fluorescein (acceptor) are linked by a CCF4-AM β-lactam ring, the substrate of β-lactamase. The exogenously added, neutral charged-FRET reagent can diffuse across ...
    Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry
    Authors:  Gareth Howell and Robert-Jan Bleichrodt, date: 04/20/2021, view: 30, Q&A: 0
    [Abstract]

    Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but it has low throughput and cannot be used to select individual cells for downstream experiments. Here

    ...
    Screening for Lysogen Activity in Therapeutically Relevant Bacteriophages
    Authors:  Fernando L. Gordillo Altamirano and Jeremy J. Barr, date: 04/20/2021, view: 142, Q&A: 0
    [Abstract]

    Lysogenic phages can integrate into their bacterial host’s genome, potentially transferring any genetic information they possess including virulence or resistance genes, and are therefore routinely excluded from therapeutic applications. Lysogenic behavior is typically seen in phages that create turbid plaques or possess subpar bactericidal

    ...
    A Potent Vaccine Delivery System
    Authors:  Guangzu Zhao, Armira Azuar, Istvan Toth and Mariusz Skwarczynski, date: 04/05/2021, view: 202, Q&A: 0
    [Abstract]

    Most vaccines require co-delivery of an adjuvant in order to generate the desired immune responses. However, many currently available adjuvants are non-biodegradable, have limited efficacy, and/or poor safety profile. Thus, new adjuvants, or self-adjuvanting vaccine delivery systems, are required. Here, we proposed a self-adjuvanting delivery

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    Measurements of Root Colonized Bacteria Species
    Authors:  Vílchez Juan Ignacio, Yu Yang, Dian Yi and Huiming Zhang, date: 04/05/2021, view: 292, Q&A: 0
    [Abstract]

    Root-associated bacteria are able to influence plant fitness and vigor. A key step in understanding the belowground plant-bacteria interactions is to quantify root colonization by the bacteria of interest. Probably, genetic engineering with fluorescence markers is the most powerful way to monitor bacterial strains in plant. However, this could

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    Brain-localized and Intravenous Microinjections in the Larval Zebrafish to Assess Innate Immune Response
    Authors:  Alison M. Rojas and Celia E. Shiau, date: 04/05/2021, view: 360, Q&A: 0
    [Abstract]

    Creating a robust and controlled infection model is imperative for studying the innate immune response. Leveraging the particular strengths of the zebrafish model system, such as optical transparency, ex utero development, and large clutch size, allows for the development of methods that yield consistent and reproducible results. We created a

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    A Spectrofluorophotometrical Method Based on Fura-2-AM Probe to Determine Cytosolic Ca2+ Level in Pseudomonas syringae Complex Bacterial Cells
    [Abstract]

    Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca2+]i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an

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    Detection and Quantification of African Swine Fever Virus in MA-104 Cells
    [Abstract]

    Detection of live African swine fever virus (ASFV) has historically relied on the use of primary swine macrophages (PSM). PSM do not replicate and have to be isolated fresh from donor swine. We previously identified that a MA-104 cells (ATCC #CRL-2378.1), a commercially available cell line isolated from African green monkey (Cercopithecus

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    Colorimetric RT-LAMP and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples
    [Abstract]

    During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric

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