In This Issue
In this issue
The “Trojan Horse” within DNA template
The “click chemistry” approach utilizing 5-ethynyl-2′-deoxyuridine (EdU) as a DNA precursor, recently introduced to detect DNA replication, offers several advantages over the classical BrdU methodology. However, in this issue Zhao and coworkers present evidence that EdU when incorporated into DNA of A549, TK6 or WTK1 cells during short pulse leads to severe problems, particularly exacerbated during the second round of replication. This is discernible by DNA damage signaling reported by phosphorylation of ATM on Ser1981, histone H2AX on Ser139, p53 on Ser15 and Chk2 on Thr68. The data indicate that DNA replication using a template containing incorporated EdU is protracted and results in DNA damage, including formation of DNA double strand breaks. Progression of EdU labeled cells through the cell cycle, mainly through G2, is perturbed likely in response to activation of Chk2. Subsequently the cells undergo apoptosis. Severity of the effects varies in cells having different status (wt versus mutated) of p53.
In this issue: page 979
Fenton chemistry for BrdU determination
The use of copper ions and ascorbic acid has been previously described for the detection of cells that incorporate EdU through a “click chemistry” approach (copper-catalyzed cycloaddition). Cappella, Giansanti, Pulici, and Gasparri demostrate that, alongside “click” cycloaddiction, Fenton reactions may also occur under particular experimental conditions, catalyzing the formation of reactive hydroxyl radicals which introduce DNA double-strand breaks and allow immunodetection of incorporated halogen deoxyuridine in viable cells. Fenton and “click” reactions can be combined for BrdU/EdU pulse-and-case experiments and can be employed for multiplexed analysis of DNA replication and immunofluorescence of labile epitopes.
In this issue: page 989
Enabling tools for cell reaction monitoring
Immunofluorescence microscopy is a well-established technique in bioimaging. Combined with super resolution microscopy techniques, it is able to provide single-molecule imaging and has become a powerful tool for understanding cellular pathways. However, these high-resolution techniques present some disadvantages with respect to conventional microscopy: they are costly and slow and that prevents the processing or a multitude of biological samples. Fortunately, we can use signal processing to leverage data extraction from conventional microscope images. Based on a case study, Ghaye and coworkers monitor toll-like receptor 2 proteins expressed in Caco-2 cell cultures. The authors propose a comparative study of various image segmentation algorithms for extracting regions of interest within fluorescent images. The performance of T-point, Otsu and Sauvola's segmentation algorithms are studied along with a novel approach. These algorithms are de facto tools for a deeper, cost-effective automated analysis of cells. In particular, guidelines are provided on algorithm selection for segmenting regions populated with receptors.
In this issue: page 1001
DNA-damage analysis by automated indirect immunofluorescence
There is a rising demand for efficient DNA-damage analysis techniques in basic research and clinical diagnostics. Detection of phosphorylated histone protein H2AX (γH2AX) foci has been reported as the most sensitive method for the analysis of DNA double-strand breaks so far. Efficient analysis and interpretation of γH2AX foci requires automation and standardization of respective indirect immunofluorescence patterns which were addressed by Willitzki and coworkers. By demonstrating the usefulness of pattern recognition algorithms of the Aklides™ platform for detecting new features of γH2AX foci apart from classical foci counting, this new approach has the potential to become a routine assay. Thus, automated analysis of γH2AX is an alternative to the classical time-consuming manual foci counting and an intriguing new approach for biomarker-based personalized medicine particularly in diagnosis and monitoring of cancer. Furthermore, it will facilitate the identification of drugs inducing or modulating DNA damage as shown by the authors.
In this issue: page 1017