|Ahead of print publication
Regulation of XPA could play a role in inhibition of radiation-induced bystander effects in QU-DB cells at high doses
Mohammad Taghi Bahreyni Toossi1, Hosein Azimian1, Shokouhozaman Soleymanifard2, Habibeh Vosoughi3, Elham Dolat3, Abdul Rahim Rezaei4, Sara Khademi5
1 Medical Physics Research Center; Department of Medical Physics, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
2 Medical Physics Research Center; Department of Medical Physics, School of Medicine, Mashhad University of Medical Sciences; Department of Medical Physics, Omid Hospital, Mashhad, Iran
3 Department of Medical Physics, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
4 Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
5 Department of Radiology Technology, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran
Medical Physics Research Center, Mashhad University of Medical Sciences, Mashhad
Department of Radiology Technology, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad
Source of Support: None, Conflict of Interest: None
Introduction: Radiation-induced bystander effects (RIBE) is the radiobiological effects detected in nonirradiated cells that have received signals from neighboring irradiated cells. In some studies, there are observations that RIBE unexpectedly reduces at high doses. In this study, the expression of two selected apoptotic and repair genes and their possible role in the formation of this unexpected reduction is examined.
Materials and Methods: The QU-DB cells were irradiated with gamma rays of a60 Co teletherapy unit at doses of 2, 4, 6, and 8 Gy. One hour following irradiation, their culture media were transferred to bystander cells to induced RIBE. After 24 h incubation, the RNA of cells was isolated and cDNA synthesized. Expression levels of BAX, XPA, and XPA/BAX ratio were examined by relative quantitative reverse transcription-polymerase chain reaction.
Results: In target cells, up-regulation of both genes was observed at all doses. In bystander cells, at the low dose (2 Gy), the expression of BAX was more than XPA; at 4 Gy, the ratio was balanced. A significant correlation was found between the XPA/BAX ratio and the dose, at high doses pattern of gene expression dominated by DNA repair gene.
Conclusion: Gene expression profile was distinctive in bystander cells compared to target cells. The observed linear increasing of the ratio of XPA/BAX could support the hypothesis that the DNA repair system is stimulated and causes a reduction in RIBE at high doses.
Keywords: Bystander effects, DNA repair, gene expression, ionizing radiation, lung carcinoma
|How to cite this URL:|
Toossi MT, Azimian H, Soleymanifard S, Vosoughi H, Dolat E, Rezaei AR, Khademi S. Regulation of XPA could play a role in inhibition of radiation-induced bystander effects in QU-DB cells at high doses. J Can Res Ther [Epub ahead of print] [cited 2020 Feb 28]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=269912
| > Introduction|| |
Radiation-induced bystander effects (RIBEs) are the radiobiological effects detected in nonirradiated cells which have gotten signals from neighboring or distant irradiated cells., The nature of RIBE and the way that it affects nonirradiated cells remain to be unknown.,,, Radiation bystander effect has important role during radiotherapy. In some radiotherapy techniques such as brachytherapy, stereotactic radiosurgery, intraoperative radiotherapy and intensity modulated radiotherapy, high doses per fraction are applied so, it is necessary to study RIBE at high doses., In a study carried out by Soleymanifard et al., RIBE increased in QU-DB cells as the dose increased from 0.5 to 4 Gy; however, it decreased when dose rose to the higher amount. This observation should be paid attention carefully. If RIBE reduction at high doses is confirmed, the effectiveness of grid therapy and tolerability of high doses used in hypofractionated protocols, mentioned above, may be explained. There are three hypotheses regarding the reduction of RIBE. According to the hypothesis proposed by Gow et al., the increase of dose raises the number of signals produced by the target cells. It creates a negative feedback in the bystander cells, which causes a reduction in RIBE response. The correctness of this hypothesis was shown in our previous study., It was observed that at high dose, RIBE abolished. Therefore, the medium extracted from target cells was diluted to decrease the number of bystander signals and then it was transferred to the bystander cells. Expectedly, it revived the abolished RIBE observed at high doses. It confirmed the negative feedback hypothesis. A decrease of bystander signals due to dilution of the culture medium caused a revived and increased RIBE. In contrast with the first hypothesis, the second refers that the death of target cells at high dose causes a reduction in the number of bystander signals, and consequently reduces the RIBE level. Our observation (RIBE revival as a result of medium dilution) could not be explained by this hypothesis. In fact, despite the decrease of bystander signals, diluted medium caused an increase in RIBE response. According to another hypothesis proposed by Mackonis et al., high amounts of bystander signals produced by target cells at high dose activate repair system in bystander cells, which decrease RIBE. To assess the correctness of this hypothesis, the present study was designed. In the first step, gene expression levels of the selected repair gene (XPA) was measured in directly irradiated and bystander QU-DB cells. XPA gene is associated with the nucleotide excision repair (NER) pathway to remove DNA lesions caused by carcinogen agents. DNA double strand break (DSB) is one of the most serious kinds of damage induced by ionizing radiation (IR). However, recent studies have reported growing evidence that various DNA repair mechanisms are not separated, but well-interlinked. In other words, DSB formation can be consequence of the production of NER breaks on opposite DNA strands. It has been proposed that NER are highly involved in DNA DSB repairs. Zhang et al. revealed that non-DSB repair genes expression, such as XPA, influenced the IR induced cytogenetic aberrations. These evidence suggest that this gene is extremely involved in DSB repair.
In addition, expression levels of selected apoptotic gene (BAX) was measured to evaluate cell death by apoptotic pathway activation. BAX as a pro-apoptotic member of Bcl-2–family proteins control the cell reaction to radiation and control apoptosis. It was speculated if the repair mechanism hypothesis is true, the higher level of repair and lower level of apoptosis would be observed in bystander cells at high doses.
| > Materials and Methods|| |
Cell culture, irradiation, and the medium transfer
Human lung carcinoma (QU-DB) cell line was obtained from the Pasteur Institute, Tehran, Iran. It was treated as target and bystander cells. The cells were grown in RPMI-1640 media (Biosera, England) supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 U/ml penicillin. The cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. Two days before to irradiation, QU-DB cells were trypsinized and cultured in (2.5 × 105) 12 cm2 flasks. Two main groups were defined: Target and bystander groups. Two hours before irradiation, the culture media of target flasks were replaced with fresh medium. Irradiation was performed with a60 Co teletherapy unit (Theratron, phoenix model, average dose-rate of 60/79 cGy/min) at doses of 2, 4, 6, and 8 Gy. The radiation field size was 15 cm × 15 cm and source to medium distance was 80 cm. Following irradiation, target flasks were returned to the incubator. After 1 h, irradiated culture medium extracted from irradiated flasks was filtered through 0.22 μm acetate cellulose filter (Orange Scientific, Belgium) to remove any dead cells. Then, they were transferred to specified bystander flasks. Following to medium transfer, all groups including irradiated, sham-irradiated and bystander flasks were incubated for 24 h.
Expression of apoptosis and repair genes in direct and bystander irradiation samples using real-time polymerase chain reaction and SYBR green method
Following incubation, culture media of the flasks were removed, the cells in the flasks were washed with phosphate-buffered saline and then 1.2 ml TriPure isolation reagent (Roche Applied Science, Germany) was added directly to the cells. Pipetting was done for several times. Each flask was incubated for 5 min at room temperature. Lysate cells were transferred to a polypropylene centrifuge tube. Phase separation was added to them (150 μl Chloroform, Merck, Darmstadt, Germany) and after manual shaking for 15 s, all samples were incubated at room temperature for 10 min. To separate the solution into three phases, the mixture was centrifuged at 12,000 g for 15 min at 4°C. The colorless upper fluid phase as transferred to a unused centrifuge tube and 0.5 ml Isopropanol precipitation (Merck, Darmstadt, Germany) was included to them. At that point, the samples were incubated at room temperature for 10 min to permit the RNA precipitate to make. The solution was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was diposed of, and the RNA pellets were washed with 1 ml of 75% Ethanol (Merck, Darmstadt, Germany), air-dried and that point resuspended in 20 μL diethylpyrocarbonate-treated RNase-free water. The solution was pipped by pipette several times, then incubated for 10–15 min at 55°C to 60°C. Next, it was stored at-80 for subsequent studies.
All reactions were carried out in add up to volume of 20 μL. Each reaction mix contained 200 U of M-MuLV Reverse Transcriptase, 4 μl of 5 × Reaction Buffer, 20 U of Ribolock™ RNase inhibitor, 2 μl of Deoxyribonucleotide triphosphate (final 1 mM), 1 μl of oligo (dt) 18 primer (0.5 μg) and 1 μg of total RNA. cDNA was synthesized according to the manufacturer recommendations (RevertAid™First Strand cDNA Synthesis Kit, Fermentas). Each cDNA was confirmed by control polymerase chain reaction (PCR) reaction using primers for the glyceraldehyde 3-phosphate dehydrogenase according to the manufacturer protocol (Prime Taq DNA polymerase, Genet Bio, South Korea). The PCR product (5 μl) was loaded on a 1% agarose gel, stained with ethidium bromide, and observed with an ultraviolet transilluminator.
Gene expression analyzed by real-time polymerase chain reaction
Reverse transcription (RT)-PCR is a sensible methodology for the detection of mRNA expression levels. Evaluation of the expressions level of BAX and XPA were performed using the AccuPower 2X GreenStar qPCR Master Mix (Bioneer, Daejeon, Korea) in an Applied Biosystems 48-well StepOne™ Real Time PCR System. The following primers were used for evaluating BAX and XPA expressions: BAX forward 5'-GCT TCA GGG TTT CAT CCA G-3', reverse 5'-GGC GGC AAT CAT CCT CTG-3'. XPA forward 5'-CTGGAGGCATGGCTAATG-3', reverse 5'-CAAATTCCATAACAGGTCCTG-3', Beta-2 Microglobulin (β2M) forward 5'-GTA TGC CTG CCG TGT GAA C-3', reverse 5'-AAC CTC CAT GAT GCT TAC-3'. Primers were purchased from Metabion (Martinsried, Germany). RT-PCR experiments were carried out using the MicroAmp™ Fast Optical 48-Well reaction plate. The wells for each sample were duplicate so that the total volume of them was 15 μl, containing 1.5 μl of cDNA, 0.3 μl of forward and reverse primers (300 nM), 7.5 μl of 2X SYBR and 0.3 μl of 50X ROX Dye (Bioneer, Daejeon, Korea) and 5.1 μl of dH2O. The PCR program consisted of the following steps; PCR primary activation stage for 60 s at 95°C, denaturation for 10 s at 95°C, annealing for 30 s at 60°C.
Analysis of gene expression data
Relative quantification analysis directly from the Step One software v. 2.1 (Applied Biosystems) from the RT-PCR experiments using SYBR green fluorescence was used to determine the apoptosis and repair genes expression levels in target and reference genes. The reference (housekeeping or 'stably expressed') gene (β2M) normalized sample to sample differences and was imperative to determine the changes in expression of different genes, according to dose. To measure the final relative quantity; the normalized quantity of the treated groups was compared with the normalized quantity of the control group.
The aim was to study the effect of both direct and indirect irradiation on the expression of the selected target genes. This method enables the measurement of gene expression changes of target genes normalized to β2M (housekeeping gene) monitored at 2, 4, 6 and 8 Gy at 24 h and exposures and relative to the expression at 0 Gy (control). The quantity of the mean-fold change at 0 Gy (control) was calibrated at 1. The mean fold changes in gene expression of each target gene were plotted by Excel software, and the graph presents either up-regulation or down-regulation of samples (target genes) which indicates whether their result is above or below the control sample amount of 1.
The real-time RT-PCR data were assessed using Kolmogorov–Smirnov analysis for all groups. One-way analysis of variance and Tukey's tests were performed to determine the significance of the experimental data. The correlation between the variables was estimated by the Pearson correlation coefficient. Statistical calculations were performed with the GraphPad Prism, (version 7.01.; GraphPad Software, La Jolla, CA, USA). P < 0.05 was considered statistically significant.
| > Results|| |
Genes expression in target and bystander cells
The results indicate BAX proapoptotic gene is upregulated at the all doses in target cell although the difference is not statistically significant. Interestingly, significant down-regulation of BAX gene expression induced by 6 and 8 Gy in bystander cells [Figure 1]. In the same circumstance, expression of XPA repair gene was up-regulated at 2, 4, 6, and 8 Gy in target cell. However, at 8 Gy, it was significantly down-regulated in bystander cells [Figure 1].
|Figure 1: Mean values of BAX and XPA genes expression in QU-DB target and bystander cells after 24 h. Each value is the mean of three replications, and error bars indicate ± standard deviation. Gene expression data are given regarding the base 2 logarithms of the ratio; positive and negative values represent increased and decreased expression level, respectively. *Represent P = 0.05|
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To determine the relationship between repair and apoptotic pathways, the in-vitro variation of XPA/BAX ratio upon irradiation with 0–8 Gy was determined in both target and bystander cells. The results have been shown in [Table 1].
|Table 1: The ratio of XPA gene expression to BAX gene expression at different radiation doses in bystander cells|
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In bystander cells, at the low dose (2 Gy), the expression of BAX proapoptotic gene was more than XPA, at 4 Gy the ratio was balanced. However; at doses of 6 and 8 Gy, the ratio changed and pattern of gene expression dominated by XPA NER gene. The correlation between XPA/BAX ratio (as an indicator of the dominant mechanism) and dose in bystander cells examined by linear regression analysis. Interestingly, a significant correlation was found between the ratio and the dose [Table 1] and [Figure 2]. On the other hand, for directly irradiated cells, BAX gene dominates the repair XPA gene at all doses [Table 1].
|Figure 2: There is a linear relationship (R2 =0.97 and P = 0.01) between XPA/BAX ratio and the dose|
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| > Discussion|| |
The aim of this study was to assess the unexpected responses in indirectly irradiated cells (at high doses) and possible association with apoptosis and repair genes expression. The results show the apoptosis gene expression in QU-DB bystander cells increased compared to the control group but, it decreased when dose rose to more than 4 Gy. In the previous study, it was found that in the dose range of 0.5–4 Gy, the number of micronucleus (MN) in bystander cells increased and their survival fraction decreased. When dose exceeded to more than 4 Gy, survival fraction of bystander cells increased and MN value decreased. In other words, RIBE increased in the dose range of 0–4 Gy, and decreased at the higher dose (6 and 8 Gy). In this study, we found pro-apoptotic gene expression likewise other endpoints decreases at high dose. The decrease of RIBE level at high dose has also been observed by other researchers.,,, In conformity with the present results, Gow et al., demonstrated that bystander cell survival decreased at low doses (0–5 Gy) while at high doses, it increased, although this phenomenon may vary in different types of cells. Alterations in gene expression and protein levels induced by IR were reported more than 30 years ago. Nevertheless, there is exceptionally little information accessible regarding the alterations in gene expression of bystander cells that may support signaling pathways involved in sustaining damage to these cells and it give information concerning the signaling factors and variable involved in this process., Previous studies had observed apoptosis in nonirradiated cells when some target cells were irradiated by microbeam.,, The culture medium of irradiated cells induces early events in apoptotic cascades such as loss of mitochondrial membrane potential, an increase in reactive oxygen species, and mobilization of intracellular calcium, in bystander cells. All of these responses are mediated via genes that control complex regulatory pathways. In this study, the ratios of XPA expression to BAX expression (XPA/BAX) were determined at different doses for both bystander and target cells. Interestingly, the results revealed that the XPA/BAX ratio in bystander cells has a linear relationship with dose. Some studies have demonstrated that the XPA gene is involved in DNA repair and that it thereby influences apoptosis induced by DNA damage. Increased expression of XPA versus BAX at 6 and 8 Gy may explain the cause of RIBE reduction in QU-DB bystander cells at these doses, which was observed in our previous Studies., This corroborates the ideas of Makonis et al., who suggested that extracellular signaling pathways in the bystander effect may modulate death programs and cellular repair. The signal may contain growth-promoting action and could be related to an increase in DNA repair and cell cycle/cell growth regulation. The result shown in [Table 1] indicates that radiation-induced gene expression profile in directly irradiated cells is different from bystander QU-DB cells. In contrary to bystander cells, XPA/BAX ratio at all doses was less than one in target cells. Therefore, it may be suggested the pathways leading to biological effects in the bystander cells are distinctive from directly irradiated cells. It was observed for target cells, BAX proapoptotic gene expression is always more than XPA, while there was not the constantly upward trend for BAX expression. Mechanisms of NER and apoptosis through XPA and BAX genes respectively, at high doses (6 and 8 Gy) for target and bystander QU-DB cells are shown in [Figure 3]. Contrary to the present results, there are some reports that indicated cell survival of target cells declined with increase of radiation dose. To explain this discrepancy, it may be suggested that other kinds of cell death or other pathways of apoptosis dominate apoptosis via BAX gene expression in target cells at high dose. The role of the DNA repair process in bystander response was previously reported through the regulation of Rad51 which assists in DNA DSB repair.
|Figure 3: Mechanisms of nucleotide excision repair and apoptosis through XPA and BAX genes respectively, at high doses (6 and 8 Gy) are shown for target and bystander QU-DB cells|
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Present results showed, for the first time, that NER process could play a role in RIBE inhibition of QU-DB cells at high doses. The mechanism may operate, at least partly, through the regulation of XPA in the ability to DNA repair.
| > Conclusion|| |
RIBE at different doses may be different through the regulation of some genes. Accordingly, in this study, it was observed that the gene expression profile was different in bystander cells compared to target cells. The observed linear increasing of the XPA/BAX ratio could support the hypothesis that the DNA repair mechanism is stimulated and causes a reduction in RIBE at high doses. Nevertheless, the phenomenological picture of RIBE depends on the culture media, experimental design, and cell type, so more research on this topic is recommended to investigate these processes with other new biomarkers.
We would like to give our sincere appreciation to the reviewers for their helpful comments on this article. The authors are also grateful to Omid hospital for use of the60 Co tele-therapy unit. This article is based on the results extracted from an M. Sc. thesis (code no: 911173) presented to the Medical Physics Department of Mashhad University of Medical Sciences (MUMS).
Financial support and sponsorship
The authors would like to thank the office of the Vice-President for Research affairs of MUMS for funding this work.
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Soleymanifard S, Toossi MT, Samani RK, Mohebbi S. Investigation of the bystander effect in MRC5 cells after acute and fractionated irradiation in vitro
. J Med Phys 2014;39:93-7.
] [Full text]
Kong EY, Cheng SH, Yu KN. Induction of autophagy and interleukin 6 secretion in bystander cells: Metabolic cooperation for radiation-induced rescue effect? J Radiat Res 2018;59:129-40.
Wang R, Coderre JA. A bystander effect in alpha-particle irradiations of human prostate tumor cells. Radiat Res 2005;164:711-22.
Matsumoto H, Hamada N, Takahashi A, Kobayashi Y, Ohnishi T. Vanguards of paradigm shift in radiation biology: Radiation-induced adaptive and bystander responses. J Radiat Res 2007;48:97-106.
Hamada N, Matsumoto H, Hara T, Kobayashi Y. Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects. J Radiat Res 2007;48:87-95.
Rzeszowska-Wolny J, Przybyszewski WM, Widel M. Ionizing radiation-induced bystander effects, potential targets for modulation of radiotherapy. Eur J Pharmacol 2009;625:156-64.
Widel M. Intercellular Communication in Response to Radiation Induced Stress: Bystander Effects in Vitro
and in Vivo
and Their Possible Clinical Implications, in Radioisotopes-Applications in Physical Sciences, Singh N, Editor. InTech. 2011.
Hei TK, Zhou H, Chai Y, Ponnaiya B, Ivanov VN. Radiation induced non-targeted response: Mechanism and potential clinical implications. Curr Mol Pharmacol 2011;4:96-105.
Gow MD, Seymour CB, Byun SH, Mothersill CE. Effect of dose rate on the radiation-induced bystander response. Phys Med Biol 2008;53:119-32.
Bahreyni Toossi MT, Khademi S, Azimian H, Mohebbi S, Soleymanifard S. Assessment of the dose-response relationship of radiation-induced bystander effect in two cell lines exposed to high doses of ionizing radiation (6 and 8 gy). Cell J 2017;19:434-42.
Mackonis EC, Suchowerska N, Zhang M, Ebert M, McKenzie DR, Jackson M, et al.
Cellular response to modulated radiation fields. Phys Med Biol 2007;52:5469-82.
Bahreyni-Toossi MT, Vosoughi H, Azimian H, Rezaei AR, Momennezhad M.In vivo
exposure effects of 99mTc-methoxyisobutylisonitrile on the FDXR and XPA genes expression in human peripheral blood lymphocytes. Asia Ocean J Nucl Med Biol 2018;6:32-40.
Zhang Y, Rohde LH, Wu H. Involvement of nucleotide excision and mismatch repair mechanisms in double strand break repair. Curr Genomics 2009;10:250-8.
Zhang Y, Rohde LH, Emami K, Hammond D, Casey R, Mehta SK, et al.
Suppressed expression of non-DSB repair genes inhibits gamma-radiation-induced cytogenetic repair and cell cycle arrest. DNA Repair (Amst) 2008;7:1835-45.
Azimian H, Dayyani M, Toossi MT, Mahmoudi M. Bax/Bcl-2 expression ratio in prediction of response to breast cancer radiotherapy. Iran J Basic Med Sci 2018;21:325-32.
Shao C, Aoki M, Furusawa Y. Bystander effect in lymphoma cells vicinal to irradiated neoplastic epithelial cells: Nitric oxide is involved. J Radiat Res 2004;45:97-103.
Maguire P, Mothersill C, Seymour C, Lyng FM. Medium from irradiated cells induces dose-dependent mitochondrial changes and BCL2 responses in unirradiated human keratinocytes. Radiat Res 2005;163:384-90.
Soleymanifard S, Bahreyni MT. Comparing the level of bystander effect in a couple of tumor and normal cell lines. J Med Phys 2012;37:102-6. [Full text]
Mitchell SA, Randers-Pehrson G, Brenner DJ, Hall EJ. The bystander response in C3H 10T1/2 cells: The influence of cell-to-cell contact. Radiat Res 2004;161:397-401.
Ariyoshi K, Miura T, Kasai K, Akifumi N, Fujishima Y, Yoshida MA, et al.
Radiation-induced bystander effect in large Japanese field mouse (Apodemus speciosus) embryonic cells. Radiat Environ Biophys 2018;57:223-31.
Mothersill C, Seymour C. Radiation-induced bystander effects: Are they good, bad or both? Med Confl Surviv 2005;21:101-10.
Furlong H, Mothersill C, Lyng FM, Howe O. Apoptosis is signalled early by low doses of ionising radiation in a radiation-induced bystander effect. Mutat Res 2013;741-742:35-43.
Ghandhi SA, Yaghoubian B, Amundson SA. Global gene expression analyses of bystander and alpha particle irradiated normal human lung fibroblasts: Synchronous and differential responses. BMC Med Genomics 2008;1:63.
Lyng FM, Seymour CB, Mothersill C. Initiation of apoptosis in cells exposed to medium from the progeny of irradiated cells: A possible mechanism for bystander-induced genomic instability? Radiat Res 2002;157:365-70.
Enokido Y, Inamura N, Araki T, Satoh T, Nakane H, Yoshino M, et al.
Loss of the xeroderma pigmentosum group A gene (XPA) enhances apoptosis of cultured cerebellar neurons induced by UV but not by low-K+medium. J Neurochem 1997;69:246-51.
Wang XC, Zhang TJ, Guo ZJ, Xiao CY, Ding XW, Fang F, et al.
Overexpression of SKP2 inhibits the radiation-induced bystander effects of esophageal carcinoma. Int J Environ Res Public Health 2017;14. pii: E155.
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