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  • In most cases patients will not present

    2020-01-29

    In most cases, patients will not present immediately after SM exposure because SM-induced clinical symptoms typically occur after a latency period of several hours [8], [9], [10]. When they do present, the detection of free, unbound alkylating agent is highly unlikely. For this reason, available rapid detections systems such as the Securetec Sulfur Mustard Detector©, which specifically detects free SM by antibody labeling, cannot be used in this context [11], [12]. The detection of SM DNA adducts extracted from exposed tissue (e.g. blister roofs) may be helpful in such a scenario. A slot blot-based method has been suggested for this purpose [13], [14].
    Materials and methods
    Results
    Discussion A significant decrease in fluorescence was observed after the treatment of pure DNA with alkylating compounds. This decrease was characterized by a dose-response relationship. Mass spectrometry and additional fluorescence measurements ruled out the covalent modification of EthBr by SM. The alkylation of DNA by SM results in around 14–20% of DNA crosslinks [3], [19], [20]; up to 50% of these crosslinks are assumed to represent interstrand crosslinks [21]. We thus assumed that DNA crosslinks may have caused DNA condensation and impaired the access of the fluorescent dyes to the DNA. Further experiments using bifunctional N-alkylating agents (i.e. HN-3) revealed an even more pronounced decrease in fluorescence, which underlined our hypothesis. However, some bifunctional and crosslinking agents (i.e. HN-1, HN-2) had only a minor effect on fluorescence. These agents were shown to exhibit slower kinetics with regard to DNA alkylation [14], [22], [23], [24]. This may also be a reason for the lower toxicity of these compounds [25]. We thus icilin synthesis assumed that, with regard to HN-1 and HN-2, lower reactivity was the reason for the only minor effects that we observed in our experiments. In order to counteract DNA condensation, we used restriction enzymes [26] to cleave alkylated DNA into small fragments. DNA fragments were thought to be more easily accessible for DNA dyes. Our results indeed demonstrated that the use of icilin synthesis enzymes restored the fluorescence signal of stained, alkylated DNA. The decrease in fluorescence intensity may, however, also be due to quenching effects. It is reported that especially chloride ions (Cl−) may impact fluorescence [27]. A single SM molecule is able to release two Cl− ions either due to SM hydrolysis or during DNA alkylation [2], [28]. In addition to the release of Cl− from the SM molecule, two protons per SM molecule are also produced, which results in the formation of hydrochloric acid [2], [29], [30], [31]. To mimic this fact, we performed additional experiments using NaCl or HCl instead of alkylating compounds. While NaCl had no effect on fluorescence, the use of HCl decreased fluorescence in a manner comparable to the alkylating compounds. Based on the results, we concluded that neither crosslinks nor Cl− but instead H+ were responsible for decreasing the fluorescence of SM-exposed DNA in aqueous, unbuffered solutions. This hypothesis was confirmed by repeating the experiments and using defined buffer systems (e.g. PBS, Tris-HCl) instead of water. In this case, the decrease in fluorescence was clearly prevented, especially when we used PBS or Tris-HCl. Other buffer systems that we tested were also suitable but were less effective.
    Within the last decade it has been established that extracellular DNA (eDNA) plays a pivotal role in bacterial biofilms. This ubiquitous matrix polymer contributes to the adhesion of single cells (, ), supports the structural integrity of young biofilms (, , , ), and is an abundant constituent of biofilm matrices in a myriad of bacterial species (, , ). For a comprehensive review see . Studies of eDNA often attempt to image eDNA of the biofilm matrix , using fluorescence microscopy. However, no comprehensive comparative studies have been done to determine which fluorescent stains are most sensitive and accurate for the visualization of eDNA. Due to the lack of standardization, eDNA images reported in the scientific literature are difficult to compare, and it is therefore equally difficult to evaluate the accuracy of the staining techniques used. To address this problem, we have systematically compared the most commonly used eDNA stains as well as several less common stains, and combined these with different counterstains for cell visualization. This has led us to recommend the combination of TOTO-1 with the cell permeable SYTO 60 as an optimal staining method for the visualization of DNA in dead cells and eDNA surrounding living and dead bacteria.