scholarly journals Evaluation of machine learning models for automatic detection of DNA double strand breaks after irradiation using a γH2AX foci assay

PLoS ONE ◽  
2020 ◽  
Vol 15 (2) ◽  
pp. e0229620
Author(s):  
Tim Hohmann ◽  
Jacqueline Kessler ◽  
Dirk Vordermark ◽  
Faramarz Dehghani
2018 ◽  
Vol 63 (1) ◽  
pp. 28-34 ◽  
Author(s):  
Н. Воробьева ◽  
N. Vorob'eva ◽  
В. Уйба ◽  
V. Uyba ◽  
О. Кочетков ◽  
...  

Purpose: To estimate the impact of 3H-thymidine on DNA double strand breaks (DSBs) induction in cultured human mesenchymal stem cells (MSC). Material and methods: Isolation and cultivation of human bone marrow MSC was carried out according to a standard procedure. A sterile solution of 3H-thymidine with different specific radioactivity was added to the cell culture and incubated under the conditions of the CO2 incubator for 24 hours. The specific radioactivity of 3H-thymidine in the incubation medium was 50–1600 kBq/ml. To evaluate quantitatively the DSBs, an immunocytochemical analysis of the DSB marker – γH2AX foci histone was used. Additionally, the proportion of dividing cells was estimated using an immunocytochemical analysis of the cell proliferation marker, the Ki67 protein. Results: It was shown that 24 h incubation of human MSC in a culture medium results in a dose-dependent increase in γH2AX foci. There is a linear increase in the foci γH2AX in the range of 50–400 kBq/ml, after which the relative quantitative yield of foci per unit of specific radioactivity begins to decrease. In general, the dose-effect relationship is approximated by the quadratic function y = 3.13 + 50.80x – 12.38x2 (R2 = 0.99), where y is the number of foci γH2AX in the cell nucleus, and x is the specific radioactivity in 1000 kBq/ml. It was found that incubation of human MSC in a culture medium containing 800 and 1600 kBq/ml of 3H-thymidine resulted in a statistically significant decrease in the cells proliferative activity compared to the control of ~1.25 and 1.41 respectively. The peculiar biological limitation of tritium accumulation in the cell nucleus explains well the nonlinear character of the dependence of the formation of DSBs on the specific radioactivity of 3H-thymidine in the culture medium observed in our study. Conclusion: Quantitative analysis of γH2AX foci has proved to be a highly reproducible and highly sensitive method for evaluating the induction of DSBs in living cells under the action of 3H-thymidine. An analysis of the foci of γH2AX will be useful for accurate estimating the quantitative yield of DBS in living cells per dose of 3H-thymidine β-radiation. To do this, it is necessary to make a correct calculation of the doses received by the cells taking into account the microdistribution of 3H-thymidine in the cell volume and its accumulation in the DNA of living cells.


2020 ◽  
Vol 196 (12) ◽  
pp. 1116-1127
Author(s):  
Sebastian Zahnreich ◽  
Hans-Peter Rösler ◽  
Carina Schwanbeck ◽  
Heiko Karle ◽  
Heinz Schmidberger

Abstract Purpose Biodosimetric assessment and comparison of radiation-induced deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by γH2AX immunostaining in peripheral leukocytes of patients with painful heel spur after radiation therapy (RT) with orthovoltage X‑rays or a 6-MV linear accelerator (linac). The treatment response for each RT technique was monitored as a secondary endpoint. Patients and methods 22 patients were treated either with 140-kV orthovoltage X‑rays (n = 11) or a 6-MV linac (n = 11) with two weekly fractions of 0.5 Gy for 3 weeks. In both scenarios, the dose was prescribed to the International Commission on Radiation Units and Measurements (ICRU) dose reference point. Blood samples were obtained before and 30 min after the first RT session. γH2AX foci were quantified by immunofluorescence microscopy to assess the yield of DSBs at the basal level and after radiation exposure ex vivo or in vivo. The treatment response was assessed before and 3 months after RT using a five-level functional calcaneodynia score. Results RT for painful heel spurs induced a very mild but significant increase of γH2AX foci in patients’ leukocytes. No difference between the RT techniques was observed. High and comparable therapeutic responses were documented for both treatment modalities. This trial was terminated preliminarily after an interim analysis (22 patients randomized). Conclusion Low-dose RT for painful heel spurs with orthovoltage X‑rays or a 6-MV linac is an effective treatment option associated with a very low and comparable radiation burden to the patient, as confirmed by biodosimetric measurements.


2016 ◽  
Vol 88 (1) ◽  
pp. 127-136 ◽  
Author(s):  
TANJA BULAT ◽  
OTILIJA KETA ◽  
LELA KORIĆANAC ◽  
JELENA ŽAKULA ◽  
IVAN PETROVIĆ ◽  
...  

ABSTRACT Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.


2017 ◽  
Vol 268 ◽  
pp. 100-108 ◽  
Author(s):  
Isaac Fernández-Varela ◽  
Elena Hernández-Pereira ◽  
Diego Álvarez-Estévez ◽  
Vicente Moret-Bonillo

2019 ◽  
Vol 58 (05) ◽  
pp. 387-394
Author(s):  
Roswitha Runge ◽  
Liane Oehme ◽  
Sabine Grosche-Schlee ◽  
Anja Braune ◽  
Robert Freudenberg ◽  
...  

Abstract Aim The aim of this study is to assess if the number of radiation-induced double strand breaks (DSB) in lymphocytes of prostate cancer patients is affected after repeated Ra-223 therapies. In addition, we investigated the repair of ex vivo induced DSB to investigate the repair proficiency in patient’s lymphocytes over the therapy course. Methods Before each of six therapy cycles, blood samples were obtained from seventeen patients. After separation of lymphocytes, the cells were subjected to immunofluorescence staining for detection of DSB-marking γH2AX foci. The number of foci per cell per patient sample was determined for each cycle (X1-X6, baseline foci per cell). Additionally, appropriate samples were exposed ex vivo to an X-ray dose of 1 Gy. The number of γH2AX foci per cell were analyzed after 0.5 h, 2 h and 24 h of recovery. Results Patient-specific linear regression of the baseline foci per cell over the therapy cycles revealed no significant slopes in the regression lines. Likewise, the mean baseline foci per cell of all patients for cycles X2-X6 was not significantly elevated in comparison to the pre-therapeutic value (X1). The differences between the percentages of residual DSB and cycles were not significant, both at 2 h and 24 h repair time. Consideration of the X6/X1 ratios of both the number of lymphocytes and the amount of residual damage at 24 h indicated a significant correlation. Conclusion Our findings indicate that the number of γH2AX foci per cell was not changed in dependence on the Ra-223 therapy cycles. The ability of patient’s lymphocytes to repair ex vivo induced DSB remained unaffected throughout the entire therapy course.


2018 ◽  
Author(s):  
Dasheng Bi

AbstractSleep spindles are characteristic events in EEG signals during non-REM sleep, and are known to be important biological markers. Manually labeling spindles by visual inspection, however, has proved to be a tedious task. Automatic detection algorithms generalize weakly for versatile spindle forms, and machine-learning methods require large datasets to train, which are unfeasible to acquire particularly for experimental animal groups. Here, a novel, integrated system based on a process of iterative “Selection-Revision” (iSR) is introduced to aid in the efficient detection of spindles. By coupling low-threshold automatic detection of spindle events based on selected parameters with manual “Revision,” the human task is effectively simplified from searching across signal traces to binary verification. Convergence was observed between resulting spindle sets through iSR, largely independent of their initial labeling, demonstrating the robustness of the method. Although possible breakdown of the revised spindle sets could be seen after multiple rounds of Revision, due to overfitting of the revised set to the initial human labeling, this could be compensated for by a Selection scheme tolerant to higher False-Negative rates of the machine labeling relative to the standard set. It was also found that iSR is generalizable to different datasets, and that initial human labeling could be substituted by low-threshold machine detection. Overall, this human-machine coupled approach allows for fast labeling to obtain consistent spindle sets, which can also be used to train machine-learning models in the future. The principle of iSR may also be applied for many different data types to assist with other pattern detection tasks.Significance StatementElectroencephalography (EEG) recordings are widely adopted in brain research. Abnormalities in the occurrence of particular EEG waveforms, such as sleep spindles, can be used to diagnose psychiatric diseases. Traditionally, human experts have labeled EEG traces for sleep spindles, a time consuming process; automated detection algorithms, however, often yield inaccurate results. This study introduces a new method for efficient sleep spindle detection with a human-machine coupled system that can iteratively revise labeled datasets, enabling convergence towards a robust, accurate spindle labeling. This system eases large-scale sleep spindle detection, which can yield datasets for both biological analyses and for training machine-learning models. Furthermore, the underlying method of iterative revision can be used to analyze other types of patterns efficiently.


2021 ◽  
Vol 22 (9) ◽  
pp. 4622
Author(s):  
Honoka Obata ◽  
Atsushi B. Tsuji ◽  
Hitomi Sudo ◽  
Aya Sugyo ◽  
Katsuyuki Minegishi ◽  
...  

Due to their short-range (2–500 nm), Auger electrons (Auger e−) have the potential to induce nano-scale physiochemical damage to biomolecules. Although DNA is the primary target of Auger e−, it remains challenging to maximize the interaction between Auger e− and DNA. To assess the DNA-damaging effect of Auger e− released as close as possible to DNA without chemical damage, we radio-synthesized no-carrier-added (n.c.a.) [189, 191Pt]cisplatin and evaluated both its in vitro properties and DNA-damaging effect. Cellular uptake, intracellular distribution, and DNA binding were investigated, and DNA double-strand breaks (DSBs) were evaluated by immunofluorescence staining of γH2AX and gel electrophoresis of plasmid DNA. Approximately 20% of intracellular radio-Pt was in a nucleus, and about 2% of intra-nucleus radio-Pt bound to DNA, although uptake of n.c.a. radio-cisplatin was low (0.6% incubated dose after 25-h incubation), resulting in the frequency of cells with γH2AX foci was low (1%). Nevertheless, some cells treated with radio-cisplatin had γH2AX aggregates unlike non-radioactive cisplatin. These findings suggest n.c.a. radio-cisplatin binding to DNA causes severe DSBs by the release of Auger e− very close to DNA without chemical damage by carriers. Efficient radio-drug delivery to DNA is necessary for successful clinical application of Auger e−.


2020 ◽  
Vol 21 (24) ◽  
pp. 9488
Author(s):  
Nelly Babayan ◽  
Natalia Vorobyeva ◽  
Bagrat Grigoryan ◽  
Anna Grekhova ◽  
Margarita Pustovalova ◽  
...  

Laser-driven accelerators allow to generate ultrashort (from femto- to picoseconds) high peak dose-rate (up to tens of GGy/s) accelerated particle beams. However, the radiobiological effects of ultrashort pulsed irradiation are still poorly studied. The aim of this work was to compare the formation and elimination of γH2AX and 53BP1 foci (well known markers for DNA double-strand breaks (DSBs)) in Hela cells exposed to ultrashort pulsed electron beams generated by Advanced Research Electron Accelerator Laboratory (AREAL) accelerator (electron energy 3.6 MeV, pulse duration 450 fs, pulse repetition rates 2 or 20 Hz) and quasi-continuous radiation generated by Varian accelerator (electron energy 4 MeV) at doses of 250–1000 mGy. Additionally, a study on the dose–response relationships of changes in the number of residual γH2AX foci in HeLa and A549 cells 24 h after irradiation at doses of 500–10,000 mGy were performed. We found no statistically significant differences in γH2AX and 53BP1 foci yields at 1 h after exposure to 2 Hz ultrashort pulse vs. quasi-continuous radiations. In contrast, 20 Hz ultrashort pulse irradiation resulted in 1.27-fold higher foci yields as compared to the quasi-continuous one. After 24 h of pulse irradiation at doses of 500–10,000 mGy the number of residual γH2AX foci in Hela and A549 cells was 1.7–2.9 times higher compared to that of quasi-continuous irradiation. Overall, the obtained results suggest the slower repair rate for DSBs induced by ultrashort pulse irradiation in comparison to DSBs induced by quasi-continuous irradiation.


Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3833
Author(s):  
Anne-Sophie Wozny ◽  
Arnaud Gauthier ◽  
Gersende Alphonse ◽  
Céline Malésys ◽  
Virginie Varoclier ◽  
...  

Hypoxia-Inducible Factor 1α (HIF-1α), which promotes cancer cell survival, is the main regulator of oxygen homeostasis. Hypoxia combined with photon and carbon ion irradiation (C-ions) stabilizes HIF-1α. Silencing HIF-1α under hypoxia leads to substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and C-ions. Thus, this study aimed to clarify a potential involvement of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). After confirming the nucleoshuttling of HIF-1α in response to both exposure under hypoxia, we showed that silencing HIF-1α in non-CSCs and CSCs decreased the initiation of the DSB detection (P-ATM), and increased the residual phosphorylated H2AX (γH2AX) foci. While HIF-1α silencing did not modulate 53BP1 expression, P-DNA-PKcs (NHEJ-c) and RAD51 (HR) signals decreased. Altogether, our experiments demonstrate the involvement of HIF-1α in the detection and signaling of DSBs, but also in the main repair pathways (NHEJ-c and HR), without favoring one of them. Combining HIF-1α silencing with both types of radiation could therefore present a potential therapeutic benefit of targeting CSCs mostly present in tumor hypoxic niches.


Sign in / Sign up

Export Citation Format

Share Document