Live Cell MicroRNA Imaging Using Exonuclease III-Aided Recycling

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Live Cell MicroRNA Imaging Using Exonuclease IIIAided Recycling Amplification Based on AIEgens Xuehong Min, Mengshi Zhang, Fujian Huang, Xiaoding Lou, and Fan Xia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01581 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 25, 2016

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ACS Applied Materials & Interfaces

Live Cell MicroRNA Imaging Using Exonuclease III-Aided Recycling Amplification Based on AIEgens Xuehong Min‡, Mengshi Zhang‡, Fujian Huang, Xiaoding Lou* and Fan Xia

Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China. KEYWORDS: AIEgens, live cell, microRNA, urine specimens, recycling

ABSTRACT: Enzyme-assisted detection strategies of microRNAs (miRNAs) in vitro have accomplished both great sensitivity and specificity. However, low expression of miRNAs and complex environment in cells induces big challenges for the monitoring and tracking of miRNAs in vivo. The work reports the attempt to carry miRNA imaging inside live cells, by enzyme-aided recycling amplification. We utilize a facile probe based yellow emission AIEgens with super photo-stable property but without quencher, which are applied to monitoring miRNAs not only from urine sample extracts (in vitro) but also in live cells (in vivo). The assay could distinguish the cancer patients’ urine samples and the normal persons’ due to the good specificity. Moreover, the probe showed much higher fluorescence intensity in breast cancer cells (MCF-7) (miR-21 in high expression) than that in cervical cancer cells (HeLa) and human lung fibroblast cells (HLF)

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(miR-21 in low expression) in more than 60 minutes, which showed the good performance and super photo-stability for the probe in vivo. As controls, another two probes with FAM/Cy3 and corresponding quenchers respectively, could perform miRNAs detections in vitro, and parts of in vivo tests but not suitable for the long-term cell tracking due to the photo-bleach phenomena, which also demonstrates that the probe with AIEgens is the potential candidate for the accurate identification of cancer biomarkers.

INTRODUCTION

MiRNAs, a series of small and noncoding RNAs with the 18-25 nt, act as the key controller of gene expressions and are expressed in the vast majority of eukaryotes, including human beings, which, therefore, are potential biomarkers and targets for diagnostic and therapeutic applications respectively.1-4 But monitoring and tracking of miRNAs in situ in live cells induces big challenges due to the low expression level of miRNA in cells and complex environment in vivo. Varieties of enzyme assisted amplification assays, for examples, duplex-specific nuclease (DSN) amplification,5-7 loop-mediated isothermal amplification (LAMP),8-14 Exonuclease III based amplification,15-17 rolling circle amplification (RCA),18-22 and hairpin-mediated quadratic enzyme amplification (HDEA),23,24 were, therefore, developed and achieved multiple signal outputs (the ratio of targets to signal probes is 1 : N or even 1 : N2). The above assays supply extreme amplified output signals for trace amounts of the miRNAs in vitro, extracted from cells, urine samples and even tissue samples. Very recently, Li’s group designed a toehold-initiated rolling circle amplification (TIRCA) monitoring individual miRNAs in situ in fixed cells.25 Tan’s group engineered an enzyme-free hairpin DNA cascade amplifier (HDCA) for specific mRNA targets in living cells.26 However, the complex and elaborate probe designing processes, for examples,


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the length of toe-hold or two hairpin-shaped metastable DNA substrates, block the universal utility in live cells.27,28 Moreover, most of these strategies still needed both the fluorophore and quencher group for the monitoring process.29 However, the organic synthesis of both fluorophore and quencher group in the nucleic acid probe systems,30,31 not only induced the burden synthesis works, but also brought the complexity for the planning of the precisely positions of fluorophore and quencher in the nucleic acid sequences. Further inconvenient thing to be designed is that the transformation of relative distance between fluorophore and quencher before and after the miRNAs/probe binding needed to be accurately considered, which further increased the difficulties for the probe system designing, and also limited the applications of in vivo tracking for miRNAs. Another point needed to be concerned is that the fluorophores used as signal-output in the above methods showed photo-bleach phenomena especially in confocal microscopy time course observation processes, which also impede the long-time monitoring miRNAs in live cells. So, the assignments turn into developing the easy designed probes with photo-stable signal output for miRNA imaging in situ in live cells. Previously,

we

reported

not

only

the

hairpin-mediated

quadratic

enzyme-based

amplification,17,24 but also a fluorescence probe with blue Aggregation-induced emission luminogens, AIEgens, with no quencher to detect microRNA from clinical urine samples.32 The assay based on AIEgens showed different calibration curves by controlling the experimental temperature, due to various enzyme activity in different temperature.17,24,32 However, the probe was not able to be applied to the monitoring of miRNAs in live cells, because the short wavelength emissions is easy to be interference by the organisms. For monitoring the analysts in cells, the probes with longer-wavelength emission luminescent materials, which hardly suffer


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interferences from optical self-absorption and auto fluorescence from the background,

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therefore, are highly needed.

Figure 1. (Left) TPEPy-LDNA (5’-(TPEPy)–CAGTCTGATAAGCTA-3’) contains a hydrophilic linear DNA part, a linker and a hydrophobic fluorophore but without quencher. LDNA part and AIEgens part (TPEPy) are chemically conjugated by the click reaction (see the Supporting Information). (Middle) FAM-LDNA contains a hydrophilic linear DNA part, FAM and quencher, DABCYL, (5’-(FAM)–CAGTCTGAT-(DABCYL) AAGCTA-3’). (Right) Cy3MBDNA contains a hydrophilic molecular beacon DNA part, Cy3 and quencher, BHQ2, (5’(Cy3)–TCAGACCGTCCCAGTCTGAT-(BHQ2) AAGCTA-3’) (Table S1). Herein, we developed a facile probe with yellow photo stable AIEgens, which was applied to monitor miRNAs not only from urine sample extracts (in vitro) but also in live cells (in vivo). The assay could distinguish the cancer patients’ urine samples and the normal person’s due to the


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good specificity. Moreover, the probe showed much higher fluorescence intensity in breast cancer cells (MCF-7) (miR-21 in high expression) than that in cervical cancer cells (HeLa) and human lung fibroblast cells (HLF) (miR-21 in low expression) in 60 minutes in vivo, which showed the photo stable of the yellow fluorophore. As controls, another two probes with FAM/Cy3 and corresponding quenchers respectively, could perform miRNAs detections in vitro, and parts of in vivo tests but not suitable for the long-term cell tracking due to the photo-bleach phenomena, which also demonstrates that the probe with AIEgens is the potential candidate for the accurate identification of cancer biomarkers. We synthesize a facile fluorescent nucleic acid probe without quencher, which contains a hydrophobic yellow fluorophore (4-{2-[4-(1,2,2triphenylvinly)phenyl]vinyl}-1-methylpyridinium hexafluorophosphate (TPEPy) unit, a linker and a hydrophilic linear DNA sequence (5’-CAGTCTGATAAGCTA-3’) (Left in Figure 1). TPEPy was synthesized via a click reaction according to the synthetic routes shown in Figure S1. There was a strong peak at 5268.2 in the mass spectra (Figure S2 and S3) for the probe with TPEPy and linear DNA (TPEPy-LDNA), which was consistent with the calculated molecular weight 533.3 (TPEPy) + (4736.1 (linear DNA) = 5269.4). It demonstrated that we successfully synthesized the probes with fluorophore and nucleic acid.


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Figure 2. (a) The calibration curve for the probe, based on TPEPy-LDNA, showed that the fluorescence intensity increased with the miR-21 concentration from 1 pM to 1000 pM. Inset: Linear relationships between fluorescence enhancement and the logarithm of concentration of miR-21. (b) The calibration curve for the probe, based on Cy3-MBDNA, showed the fluorescence intensity increased with the miR-21 concentration from 1 pM to 1000 pM. Inset: Linear relationships between fluorescence enhancement and the logarithm of concentration of miR-21 (c) The calibration curve for the probe, based on FAM-LDNA, showed the fluorescence intensity increased with the miR-21 concentration from 10 pM to 1000 pM. Inset: Linear relationships between fluorescence enhancement and the logarithm of concentration of miR-21. Control tests in (d) PBS, BSA, thrombin, Bst DNA polymerase, Trypsin, and T4 DNA Ligase were used to instead the Exo III under the same experimental conditions, among which PBS is used as the control experimental condition. Specificity tests (e) demonstrate that the assay, based on TPEPy-LDNA, could distinguish the perfect match (PM) targets with control (miR-155, miR210, and miR-221) and single/triple mismatched targets. There are 25 clinical urine samples used


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in this work, 20 of them from bladder cancer patients, and 5 of them from healthy persons. The box charts in Figure 2f represent miR-21 of clinical samples extracted from 20 bladder patients (red) and 5 healthy persons (green), which demonstrated that the assay based on TPEPy-LDNA could discriminates cancer patients’ sample from the healthy ones. The error bars in the figures reveals the standard deviation of different triplicate tests. All of the experiments were carried out at 37 oC. EXPERIMENTAL SECTION Materials and Reagents. The following items, such as copper (I) bromide, sodium ascorbate and acetonitrile, were purchased from Aladdin and used as received without further purification. Other chemicals were purchased from Sigma Aldrich and used as received without further purification. FAM-LDNA and Cy3-MBDNA were obtained from the DNA synthesis company, Takara in China. Organic synthesis of TPEPy-DNA. We synthesized the TPEPy according to the reported reference.35 All the DNA sequences were obtained from the company, Takara in China. We synthesized the TPEPy-DNA by using the process, which was used in our previous work.32 Figure S1 showed the synthesis process for TPEPy-DNA. FAM-LDNA and Cy3-MBDNA were synthesized by the company (Takara), and mass spectrum of them was given in Figure S4. Meanwhile dynamic light scattering measurements of the liberated TPEPy after inducing different concentrations (10 pM, 100 pM, and 1nM) miR-21 under the enzyme Exo III from TPEPy-LDNA (10 µM) was provided in Figure S5.

Cell culture for MCF-7, Hela and HLF. MCF-7 cells and HeLa cells were obtained from Xiangya Central Experiment Laboratory (Hunan Province, China). Human lung fibroblast (HLF)


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cells were obtained from China Center (Hubei Province, China) for Type Culture Collection. HeLa and MCF-7 cells were maintained under the standard culture conditions (atmosphere of 5% CO2 and 95% air at 37 °C) in RPMI 1640 medium, supplemented with 10% FBS (fetal calf serum) and 100 IU/mL penicillin–streptomycin. HLF cells were cultured in Minimum Essential Medium, MEM, plus 10% FBS and 100 IU/mL penicillin–streptomycin in 5% CO2 and 95% air at 37 °C. HeLa, MCF-7 and HLF cells in the exponential phase were plated on 35-mm glassbottom culture dishes (Φ 20 mm) for 2 days to reach around 80% confluency respectively. Confocal microscopy imaging of Cells with probes. Cells were starved with 1 mL serumfree medium Opti-MEM for 2 hours before they were transfected with the detection probe (TPEPy-LDNA, FAM-LDNA and Cy3-MBDNA) and Exonuclease III by liposome transfection. The transfection liquid solution was a mixture of two different solutions. The first one contained 10 μL Lipofectamine® 2000 Reagent and 140 μL Opti-MEM, standing 5 minutes after fully mixing. The second one was added with the appropriate concentrations of enzyme and detection probes to make the total volume of 150 μL Opti-MEM. The transfection liquid solution (300 μL total volume) was finally formed when the two solutions were kept in static for 15 minutes after fully mixing. Then the cells were incubated with the prepared transfection liquid solution which contained TPEPy-LDNA (6.6 μM) and Exonuclease III (1.33 U/μL) in an atmosphere of 5% CO2 and 95% air for 60 minutes at 37 °C. The other two transfection liquid solution contained Cy3-MBDNA (500 nM) and Exonuclease III (1.33 U/μL) or FAM-LDNA (500 nM) and Exonuclease III (1.33 U/μL), respectively. And the transfection process was the same to that of TPEPy-LDNA. Cells were washed by three times with 1 mL PBS at room temperature and then they were added with 1 mL PBS culture medium to observe under a confocal microscopy (Olympus FV1200)


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with a 60×oil-immersion objective. To monitor dynamic progress of the intracellular miR-21 in the corresponding cells, scanning was stopped after the completion of the first scanning picture. The light irradiation was kept for 10 minutes in situ before the following scanning. The scanning time interval was 10 minutes, and the total light irradiation time is 60 minutes. For Cy3MBDNA, excitation: 543 nm, emission collected: 560-620 nm. For FAM-LDNA, excitation: 488 nm, emission collected: 510-540 nm. For TPEPy-LDNA, excitation: 405 nm, emission collected: 560-620 nm. MicroRNAs extraction from urine samples. MicroRNA were obtained from urine samples by using Norgen’s Urine microRNA Purification Kit according to our previous work.29 RESULTS AND DISCUSSION Detection of miR-21 in vitro. We still chose Exonuclease III (Exo III), which could only catalyzes the stepwise removal of mononucleotides from 3’ termini of double strand DNA, 41 in this work for the in vivo monitoring as our previous work for the in vitro detection.32 Just as previous works, here we still chose miR-21 as the targets that is highly expressions in liver,42 brain,43 prostate,44 pancreas45 and bladder.46 Our probe, TPEPy-LDNA, contains two parts: the recognize component (DNA, which hybridizes with miR-21) and the fluorescence signal component (TPEPy molecules show the yellow emission when aggregated). The very hydrophilic recognize component makes the whole TPEPy-LDNA hydrophilic,

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which

induced the nanoaggregate states of TPEPy, showing weak fluorescence signal. At the beginning, the miR-21 hybridized with DNA part in the probe, which induced the conformation change from random coil single-DNA (the DNA part in TPEPy-LDNA) to duplex DNA-RNA (the DNA part in TPEPy-LDNA and miR-21) with a blunt 3’ terminus. The enzyme, Exo III, then cuts off the recognize parts from the whole probe, release both of miR-21 and AIEgens. The


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released miR-21 could bind with another probe, beginning the second period. One miR-21 could trigger many periods, and release a number of hydrophobic AIEgens, which could be aggregated together, initiating the RIM process (Restriction of Intramolecular Motions (RIM) is the main cause for the AIE effects)

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to activate the yellow fluorescence (Left in Figure 1). The

calibration curve (Figure 2a) shows the detect limit for the TPEPy-LDNA based assay is 1 pM in 37 oC. Corresponding three-dimensional map (Figure S6) of MCF-7 cells was listed to make sure that the dye indeed enter the cell rather than attach on the cell surface. The catalytic amplification of our TPEPy-LDNA probe was compared with two nucleic acid probes with different dyes: FAM-LDNA and Cy3-MBDNA (Middle and Right in Figure 1) both in vitro and in vivo. The detection limit for FAM-LDNA and Cy3-MBDNA is 10 pM (Figure 2b) and 1 pM (Figure 2c) in 37 oC, respectively, which are similar to the results for TPEPy-LDNA. The above shows all of the three probes with different dyes (AIEgens, FAM and Cy3) and different DNA conformations (linear and molecular beacon) works well in vitro. Figure 2d demonstrates that Exo III is the key factor for the detection assay. The specificity tests in Figure 2e reveals the strategy, based on TPEPy-LDNA, could distinguish the perfect match (PM) targets with control (miR-155) and single/triple mismatched targets. Moreover, our assay based on TPEPy-LDNA is challenged by miR-21 of clinical samples from 20 bladder patients and 5 healthy persons (Figure 2f, Figure S7 and Table S2). All the testing results are fully consistent with the clinical results, which prove the good specificity of our strategy. All the above testing results showed that the three probes (TPEPy-LDNA, FAM-LDNA and Cy3-MBDNA) could accomplish the miR-21 detection in vitro just like our previously introduced probe (TPE-LDNA), 32 which could not achieve in vivo detection mission due to its blue emissions easy to be interference by the cells. 3034


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Figure 3. Live-cell fluorescence analysis. (a, d) MCF-7 live cells in the presence of TPEPyLDNA. (b, e) HeLa live cells in the presence of TPEPy-LDNA. (c, f) HLF live cells in the presence of TPEPy-LDNA. The fluorescence intensity in MCF-7 is distinctively higher than that in Hela and HLF. The results consistently suggest the high expression miR-21 in MCF-7 and low expression miR-21 in both HeLa and HLF. The reaction time is 20 minutes. Detection of miR-21 in vivo. Having verified the amplification of the three probes in vitro, we next did the research about their imaging capability in vivo (in live cells). Three kinds of cells were chosen in the following tests: MCF-7 (miR-21 high expression), HeLa (miR-21 low expression) and HLF (miR-21 low expression). After transfected into the above three different cells via lipofectamine 2000, the TPEPy-LDNA, FAM-LDNA and Cy3-MBDNA were incubated with cells at 37o C for 1 h, respectively. All the three probes revealed high intensity of fluorescence signals in MCF-7, but relatively low intensity in HeLa and HLF (Figure 3, Figure 4 and Figure 5). The negative control experiment, which shows very weak fluorescence intensity


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in the absence of EXO III (Figure S8), illustrates the enzyme is indispensable. The above results consistently suggest that all the three different catalytic amplification probes could differentiate the high miR-21 expression cells and low miR-21 expression cells.

Figure 4. Live-cell fluorescence analysis. (a, d) MCF-7 live cells in the presence of FAMLDNA. (b, e) HeLa live cells in the presence of FAM-LDNA. (c, f) HLF live cells in the presence of FAM-LDNA. The fluorescence intensity in MCF-7 is distinctively higher than that in Hela and HLF. The results consistently suggest the high expression miR-21 in MCF-7 and low expression miR-21 in both HeLa and HLF. The reaction time is 20 minutes.


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Figure 5. Live-cell fluorescence analysis. (a, d) MCF-7 live cells in the presence of Cy3MBDNA. (b, e) HeLa live cells in the presence of Cy3-MBDNA. (c, f) HLF live cells in the presence of Cy3-MBDNA. The fluorescence intensity in MCF-7 is distinctively higher than that in Hela and HLF. The results consistently suggest the high expression miR-21 in MCF-7 and low expression miR-21 in both HeLa and HLF. The reaction time is 20 minutes. The probes in live-cells life time fluorescence signals. To further test whether TPEPy-LDNA could be applied to dynamic monitoring the intracellular miR-21. After incubation of MCF-7 cells, the probes and Exo III were, respectively, added to each dish, and the mixtures were incubated for 0.5 h to perform the confocal imaging. With the time, the fluorescence intensity of MCF-7 cells, with TPEPy-LDNA, become stronger (Figure 6a, 6d), indicating the timedependent of the intracellular detection process, which was consistent with in vitro results. While the time-course monitoring with FAM-LDNA, the fluorescence intensity decreased from 10 min to 60 min, and finally became negligible (Figure 6b, 6e). Comparing with the above two probes,


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Cy3-MBDNA, however, showed different results. The fluorescence signals increased from 10 min to 50 min, but began to fade at 60 min (Figure 6c, 6f). Additional life-time studies (Figure S9, Figure S10 and Figure S11) were performed to support the results shown in Figure 6. The phenomena for these probes may be attributed to the photo-bleach properties for the ACQ dyes. FAM-LDNA and Cy3-MBDNA worked well in vitro (Figure 2), but showed the photo-bleach in vivo (Figure 6b and 6c), probably due to the much higher intensity of excited lights in confocal microscopy than that in fluorescence spectroscopy. The above reveals that the nanoaggregates of TPEPy from the probe own better photostability than the single fluorescent molecule in dilute solutions.50

Figure 6. Time-dependent detection process of miR-21 in MCF-7 with TPEPy-LDNA (6.6 μM) (a), FAM-LDNA (500 nM) (b) and Cy3-MBDNA (500 nM) (c), respectively, which showed


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totally different behaviors. (d) The signal increased gradually within 60min for TPEPy-LDNA in vivo. (e) The signal of FAM-LDNA decreased from 10 min to 60 min in MCF. (f) The signal of Cy3-MBDNA increased from 10 min to 50 min, but began to fade at 60 min finally. The scale bar in the figure is 20 μm. CONCLUSION In conclusion, comparing our previous probe, TPE-LDNA with blue emission, TPEPy-LDNA (yellow emission) and other two probes, FAM-LDNA (green emission) and Cy3-MBDNA (red emission), could accomplish the miR-21 detection in vitro with 1 pM, 10 pM and 1 pM detection limit respectively. TPEPy-LDNA could also be applied to clinical urine sample detections, differentiating 20 cancer patients’ samples from 5 healthy ones. While only TPEPy-LDNA works well for the long-term in vivo test due to AIEgens photo-stable properties. The practicality of the proposed probe has been demonstrated by dynamic monitoring of the time-dependent detection process in vivo. We anticipate that the probe will accelerate the uncovering of the basic role of miRNA in various biological events and contribute to the clinical diagnosis and therapeutic monitoring cancer.

ASSOCIATED CONTENT Supporting Information. Detailed description of the experimental procedures, DNA sequence, and additional figures. The supporting information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author


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* Phone: +86-27-87559484. E-mail: [email protected] Author Contributions ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by National Basic Research Program of China (973 program, 2015CB932600, 2013CB933000), the National Natural Science Foundation of China (21525523, 21375042, 21574048, 21405054) and 1000 Young Talent (to Fan Xia). REFERENCES (1) He, L.; He, X.; Lim, L. P.; de Stanchina, E.; Xuan, Z.; Liang, Y.; Xue, W.; Zender, L.; Magnus, J.; Ridzon, D.; Jackson, A. L.; Linsley, P. S.; Chen, C.; Lowe, S. W.; Cleary, M. A.; Hannon, G. J. A MicroRNA Component of the p53 Tumour Suppressor Network. Nature 2007, 447, 1130-1134. (2) Arenz, C. MicroRNAs-Future Drug Targets? Angew. Chem. Int. Ed. 2006, 45, 5048-5050. (3) Croce, C. M. Causes and Consequences of MicroRNA Dysregulation in Cancer. Nat. Rev. Genet. 2009, 10, 704-714. (4) Zhang, H.; Wang, Y.; Zhao, D.; Zeng, D.; Xia, J.; Aldalbahi, A.; Wang, C,; San, L.; Fan, C.; Zuo, X.; Mi, X. Universal Fluorescence Biosensor Platform Based on Graphene Quantum Dots


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Live Cell MicroRNA Imaging Using Exonuclease III-Aided Recycling

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