The comet assay: past, present, and future

Langie, Sabine A. S.; Azqueta, Amaya; Collins, Andrew

doi:10.3389/fgene.2015.00266

Cited by 115 publications

(69 citation statements)

References 27 publications

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“…We utilized an alkaline comet assay, in The percentages of premature mitotic events (EdU and pH3 S10 positive) from the experiment shown in panel G were quantified as the percentage of cells positive for mitosis from the total cells in S phase. Error bars which DNA damage is visualized with single-cell resolution by a comet-like appearance of broken DNA migrating away from the genomic pellet in an electric field (24). As previously reported, BKPyV infection itself did not cause observable DNA damage since the comet tails were similar to those with mock infection ( Fig.…”

supporting

confidence: 72%

BK Polyomavirus Activates the DNA Damage Response To Prolong S Phase

Justice

Needham

Thompson

2019

J Virol

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BK polyomavirus (PyV) is a major source of kidney failure in transplant recipients. The standard treatment for patients with lytic BKPyV infection is to reduce immunosuppressive therapy, which increases the risk of graft rejection. PyVs are DNA viruses that rely upon host replication proteins for viral genome replication. A hallmark of PyV infection is activation of the DNA damage response (DDR) to prevent severe host and viral DNA damage that impairs viral production by an unknown mechanism. Therefore, we sought to better understand why BKPyV activates the DDR through the ATR and ATM pathways and how this prevents DNA damage and leads to increased viral production. When ATR was inhibited in BKPyV-infected primary kidney cells, severe DNA damage occurred due to premature Cdk1 activation, which resulted in mitosis of cells that were actively replicating host DNA in S phase. Conversely, ATM was required for efficient entry into S phase and to prevent normal mitotic entry after G 2 phase. The synergistic activation of these DDR kinases promoted and maintained BKPyV-mediated S phase to enhance viral production. In contrast to BKPyV infection, DDR inhibition did not disrupt cell cycle control in uninfected cells. This suggests that DDR inhibitors may be used to specifically target BKPyV-infected cells. IMPORTANCE BK polyomavirus (BKPyV) is an emerging pathogen that reactivates in immunosuppressed organ transplant patients. We wanted to understand why BKPyV-induced activation of the DNA damage response (DDR) enhances viral titers and prevents host DNA damage. Here, we show that the virus activates the DNA damage response in order to keep the infected cells in S phase to replicate the viral DNA. The source of DNA damage was due to actively replicating cells with uncondensed chromosomes entering directly into mitosis when the DDR was inhibited in BKPyV-infected cells. This study clarifies the previously enigmatic role of the DDR during BKPyV infection by demonstrating that the virus activates the DDR to maintain the cells in S phase in order to promote viral replication and that disruption of this cell cycle arrest can lead to catastrophic DNA damage for the host.

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supporting

confidence: 72%

BK Polyomavirus Activates the DNA Damage Response To Prolong S Phase

Justice

Needham

Thompson

2019

J Virol

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“…To detect the fragmentation of nuclear DNA, the use of the comet and TUNEL assays have widely increased in plant system as only a small number of cells are required to obtain the reliable results (Charzyńska et al, 2000; Langie et al, 2015). The comet assay is a rapid, visual, and sensitive technique used to measure DNA damage (DNA breakage or formation of alkali-labile sites) in individual cells (McKelvey-Martin et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum

et al. 2017

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A previously unknown disease which causes severe vein thickening and inward leaf curl was observed in a number of opium poppy (Papaver somniferum L.) plants. The sequence analysis of full-length viral genome and associated betasatellite reveals the occurrence of Ageratum enation virus (AEV) and Ageratum leaf curl betasatellite (ALCB), respectively. Co-infiltration of cloned agroinfectious DNAs of AEV and ALCB induces the leaf curl and vein thickening symptoms as were observed naturally. Infectivity assay confirmed this complex as the cause of disease and also satisfied the Koch’s postulates. Comprehensive microscopic analysis of infiltrated plants reveals severe structural anomalies in leaf and stem tissues represented by unorganized cell architecture and vascular bundles. Moreover, the characteristic blebs and membranous vesicles formed due to the virus-induced disintegration of the plasma membrane and intracellular organelles were also present. An accelerated nuclear DNA fragmentation was observed by Comet assay and confirmed by TUNEL and Hoechst dye staining assays suggesting virus-induced programmed cell death. Virus-infection altered the biosynthesis of several important metabolites. The biosynthesis potential of morphine, thebaine, codeine, and papaverine alkaloids reduced significantly in infected plants except for noscapine whose biosynthesis was comparatively enhanced. The expression analysis of corresponding alkaloid pathway genes by real time-PCR corroborated well with the results of HPLC analysis for alkaloid perturbations. The changes in the metabolite and alkaloid contents affect the commercial value of the poppy plants.

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“…The experimental protocol was standardized to obtain comets in positive controls, as recommended . Mice melanoma cells B16F10 (ATCC CRL‐6475) cultivated on endothelial cell basal medium were exposed to sphaeropsidine A at 5μM.…”

Section: Methodsmentioning

confidence: 99%

“…The experimental protocol was standardized to obtain comets in positive controls, as recommended. 15,16 Mice melanoma cells B16F10…”

Section: Positive Controlsmentioning

confidence: 99%