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DDC: 610 × Subject: DNA ×

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Now showing 1 - 3 of 3
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    In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1
    (San Francisco, California, US : PLOS, 2023) Blanch-Asensio, Marc; Dey, Sourik; Sankaran, Shrikrishnan
    Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 μg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.
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    H2A.X Phosphorylation in Oxidative Stress and Risk Assessment in Plasma Medicine
    (Austin, Tex. : Landes Bioscience, 2021) Schütz, Clarissa S.; Stope, Matthias B.; Bekeschus, Sander
    At serine139-phosphorylated gamma histone H2A.X (γH2A.X) has been established over the decades as sensitive evidence of radiation-induced DNA damage, especially DNA double-strand breaks (DSBs) in radiation biology. Therefore, γH2A.X has been considered a suitable marker for biomedical applications and a general indicator of direct DNA damage with other therapeutic agents, such as cold physical plasma. Medical plasma technology generates a partially ionized gas releasing a plethora of reactive oxygen and nitrogen species (ROS) simultaneously that have been used for therapeutic purposes such as wound healing and cancer treatment. The quantification of γH2A.X as a surrogate parameter of direct DNA damage has often been used to assess genotoxicity in plasma-treated cells, whereas no sustainable mutagenic potential of the medical plasma treatment could be identified despite H2A.X phosphorylation. However, phosphorylated H2A.X occurs during apoptosis, which is associated with exposure to cold plasma and ROS. This review summarizes the current understanding of γH2A.X induction and function in oxidative stress in general and plasma medicine in particular. Due to the progress towards understanding the mechanisms of H2A.X phosphorylation in the absence of DSB and ROS, observations of γH2A.X in medical fields should be carefully interpreted.
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    Elevated H2AX Phosphorylation Observed with kINPen Plasma Treatment Is Not Caused by ROS-Mediated DNA Damage but Is the Consequence of Apoptosis
    (London: Hindawi, 2019) Bekeschus, Sander; Schütz, Clarissa S.; Nießner, Felix; Wende, Kristian; Weltmann, Klaus-Dieter; Gelbrich, Nadine; von Woedtke, Thomas; Schmidt, Anke; Stope, Matthias B.
    Phosphorylated histone 2AX (γH2AX) is a long-standing marker for DNA double-strand breaks (DSBs) from ionizing radiation in the field of radiobiology. This led to the perception of γH2AX being a general marker of direct DNA damage with the treatment of other agents such as low-dose exogenous ROS that unlikely act on cellular DNA directly. Cold physical plasma confers biomedical effects majorly via release of reactive oxygen and nitrogen species (ROS). In vitro, increase of γH2AX has often been observed with plasma treatment, leading to the conclusion that DNA damage is a direct consequence of plasma exposure. However, increase in γH2AX also occurs during apoptosis, which is often observed with plasma treatment as well. Moreover, it must be questioned if plasma-derived ROS can reach into the nucleus and still be reactive enough to damage DNA directly. We investigated γH2AX induction in a lymphocyte cell line upon ROS exposure (plasma, hydrogen peroxide, or hypochlorous acid) or UV-B light. Cytotoxicity and γH2AX induction was abrogated by the use of antioxidants with all types of ROS treatment but not UV radiation. H2AX phosphorylation levels were overall independent of analyzing either all nucleated cells or segmenting γH2AX phosphorylation for each cell cycle phase. SB202190 (p38-MAPK inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited γH2AX induction upon ROS but not UV treatment. Finally, and despite γH2AX induction, UV but not plasma treatment led to significantly increased micronucleus formation, which is a functional read-out of genotoxic DNA DSBs. We conclude that plasma-mediated and low-ROS γH2AX induction depends on caspase activation and hence is not the cause but consequence of apoptosis induction. Moreover, we could not identify lasting mutagenic effects with plasma treatment despite phosphorylation of H2AX.