Development and validation of 'AutoRIF': software for the automated analysis of radiation-induced foci

Abstract

BackgroundThe quantification of radiation-induced foci (RIF) to investigate the induction and subsequent repair of DNA double strands breaks is now commonplace. Over the last decade systems specific for the automatic quantification of RIF have been developed for this purpose, however to ask more mechanistic questions on the spatio-temporal aspects of RIF, an automated RIF analysis platform that also quantifies RIF size/volume and relative three-dimensional (3D) distribution of RIF within individual nuclei, is required.ResultsA java-based image analysis system has been developed (AutoRIF) that quantifies the number, size/volume and relative nuclear locations of RIF within 3D nuclear volumes. Our approach identifies nuclei using the dynamic Otsu threshold and RIF by enhanced Laplacian filtering and maximum entropy thresholding steps and, has an application 'batch optimisation' process to ensure reproducible quantification of RIF. AutoRIF was validated by comparing output against manual quantification of the same 2D and 3D image stacks with results showing excellent concordance over a whole range of sample time points (and therefore range of total RIF/nucleus) after low-LET radiation exposure.ConclusionsThis high-throughput automated RIF analysis system generates data with greater depth of information and reproducibility than that which can be achieved manually and may contribute toward the standardisation of RIF analysis. In particular, AutoRIF is a powerful tool for studying spatio-temporal relationships of RIF using a range of DNA damage response markers and can be run independently of other software, enabling most personal computers to perform image analysis. Future considerations for AutoRIF will likely include more complex algorithms that enable multiplex analysis for increasing combinations of cellular markers.

Polymorphisms in XPD and ERCC1 Associated with Colorectal Cancer Outcome

“…Mention should also be made of the particular importance of decreased expression of ERCC1 in progression to colon cancer, shown in recent studies. A 2012 report indicates that epigenetic repression of ERCC1 is found in 40% of about 1 million crypts in large field defects surrounding colon cancers (in which progression to cancer occurred) and in 100% of colon cancers themselves [26]. Local and systemic treatment modalities, like preoperative chemoradiotherapy, should be planned for patients carrying these risk factors.…”

Molecular toxicity mechanism of nanosilver

“…Nanosilvers not only bind to cytosolic proteins causing a corona, but also express genes involved in DNA damage. Increased ataxia telangiectasia mutated (ATM) and ATM-related levels in fibroblast cells indicate double DNA strand breakage (129). Piao et al (85, 117) conducted a comparative study with nanosilver and AgNO 3 in human Chang liver cells.…”

Live Dynamics of 53BP1 Foci Following Simultaneous Induction of Clustered and Dispersed DNA Damage in U2OS Cells

“…X-rays and gamma radiations ionise sparsely, have a low LET, and induce predominantly simple DSBs which are randomly distributed throughout cell nuclei [ 17 ]. When nuclear foci are analysed in cells exposed to high and low LET radiation, quantitative and qualitative differences are seen: per unit dose, high LET radiation is less effective in inducing microscopically visible DSB focus numbers than low LET radiation, which reflects the fewer cell hits which suffice to reach a given dose [ 20 , 21 , 22 ]. While the majority of high LET radiation-induced foci are large, most foci induced by low LET radiation are small [ 20 , 23 ].…”

“…While the majority of high LET radiation-induced foci are large, most foci induced by low LET radiation are small [ 20 , 23 ]. Moreover, the kinetics of focus formation is different: large high LET-induced foci form and disappear slowly, while small foci appear and disappear fast [ 21 , 22 , 24 ]. These results fit well with the notion that high LET radiation-induced complex DSBs and non-DSB clustered lesions pose a more serious problem for the DNA repair machinery as compared to simple DSBs [ 25 , 26 , 27 , 28 ].…”