WO2016125243A1 - Procédé de dosage d'exosomes et procédé d'extraction d'exosomes - Google Patents

Procédé de dosage d'exosomes et procédé d'extraction d'exosomes Download PDF

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Publication number
WO2016125243A1
WO2016125243A1 PCT/JP2015/052909 JP2015052909W WO2016125243A1 WO 2016125243 A1 WO2016125243 A1 WO 2016125243A1 JP 2015052909 W JP2015052909 W JP 2015052909W WO 2016125243 A1 WO2016125243 A1 WO 2016125243A1
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exosome
exosomes
gold
solution
fine particles
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PCT/JP2015/052909
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English (en)
Japanese (ja)
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美南 庄子
武田　健一
崇秀 横井
裕一 内保
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株式会社日立製作所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration

Definitions

  • the present invention relates to a method for measuring the amount of exosomes contained in a sample and a method for extracting exosomes from a sample.
  • Exosome is a kind of extracellular vesicle (or extracellular granule).
  • a vesicle is a bag-like structure wrapped in a membrane inside a cell, and is used not only to store the substance in the cell but also to transport the substance into and out of the cell.
  • Vesicles are naturally formed structures due to the chemical properties of lipid membranes. Most vesicles have some specialized function, and the function varies depending on the substance contained in the vesicle. Among them, exosome is a general term for vesicles of about 40 nm to 200 nm that are secreted by various cells.
  • An exosome is a vesicle covered with a lipid bilayer, and retains therein a microRNA that plays an important role in suppressing gene expression in vivo.
  • the outer membrane of exosome contains characteristic lipid components such as ceramide, unlike ordinary cell membranes.
  • Exosomes contain nucleic acid substances such as 100 kinds or more of micro RNA and 1000 kinds or more of messenger RNA.
  • the microRNA contained in the exosome is a small RNA of 18 to 25 bases, and is present not only in cells but also in body fluids such as blood. Since the expression pattern of microRNA is tissue-specific, the expression pattern differs between cancer tissue and non-cancer tissue, and further, the expression pattern differs depending on the cancer type. Thus, microRNA in blood is attracting attention as it can serve as a biomarker for identifying the primary tumor focus and identifying the cancer type.
  • microRNA contained in exosomes is highly tissue-specific, and other microRNAs and dead cells that exist in the blood can be removed in the process of separating exosomes. It is expected as a biomarker that can be identified.
  • cancer diagnosis based on the number of exosomes is expected rather than microRNA analysis.
  • An ELISA Enzyme-Linked ImmunoSorbent Assay
  • ELISA is a method used when detecting and quantifying the concentration of an antibody or antigen contained in a sample, and is used for quantitative evaluation of proteins and the like. Quantitative evaluation is possible by detecting changes in absorbance at a specific wavelength. This method has been reported to be applicable to exosomes, and it uses a highly specific antigen-antibody reaction for proteins expressed on the exosome surface to detect color development and luminescence based on enzymatic reactions. Shows that the amount of exosomes in a sample can be measured (Patent Document 1).
  • Patent Document 2 As another known exosome extraction method, there is a method in which a specimen is processed by an ultracentrifuge and extracted (Patent Document 2).
  • Patent Document 1 The technique using ELISA and the antigen-antibody reaction of Patent Document 1 can only measure exosomes having matching antigens, and cannot perform quantitative evaluation reflecting all exosomes contained in the sample.
  • the exosome measuring method includes a step of mixing a metal ion solution with a specimen containing exosome to form a mixed solution, a step of measuring an index representing the amount of metal fine particles aggregated in the mixed solution, and a measured index. And evaluating the exosome concentration based on.
  • the index absorbance of the mixed solution, scattered light generated when the mixed solution is irradiated with light, and the like can be used.
  • the specimen can be serum and plasma obtained by centrifuging a blood sample.
  • the metal ion can be a gold ion.
  • the exosome extraction method includes a step of mixing a gold ion solution into a sample containing exosome to form a mixed solution, a step of centrifuging and precipitating gold fine particles aggregated in the mixed solution, and removing the supernatant. It includes a step of dropping sodium thiosulfate, a step of centrifuging again to precipitate gold fine particles, and a step of recovering the supernatant and extracting exosomes.
  • the present invention makes it possible to measure the total amount of exosomes in a specimen. In addition, it is possible to extract all exosomes suspended in the specimen in a short time without using an antigen-antibody reaction or ultracentrifugation.
  • the schematic block diagram of an exosome measuring apparatus The flowchart of exosome amount evaluation in a sample and exosome extraction.
  • exosomes having a lipid bilayer membrane and metal ions are adsorbed, and the metal ions aggregate to form metal fine particles.
  • the amount of exosome can be quantified by measuring the metal fine particles through a change in absorbance by localized surface plasmon resonance. Specifically, this localized surface plasmon resonance changes the wavelength and absorbance at which a peak occurs depending on the size, shape, and amount of the metal fine particles.
  • the amount of the metal fine particles is measured by measuring the absorbance of the exosomes on which the metal fine particles have been adsorbed with a spectroscope after a certain period of time, and the amount of exosomes in the specimen is quantified.
  • exosomes having a lipid bilayer and gold ions are adsorbed, and gold ions aggregate to form gold fine particles.
  • Exosomes with gold fine particles with a size of 100 nm or more attached can be settled by centrifuging for about 5 minutes with a normal centrifuge ( ⁇ 10,000 rpm). After removing the supernatant, an aqueous solution of sodium thiosulfate is added dropwise. Since the binding force of gold is smaller than that of sodium thiosulfate and gold, exosomes and gold are separated. The solution in which exosome and gold-sodium thiosulfate are mixed is centrifuged again, and the exosome is extracted by collecting the supernatant.
  • FIG. 1 is a schematic configuration diagram of the exosome measuring apparatus according to the present embodiment.
  • the exosome measuring apparatus according to the present embodiment measures the absorbance of a sample as an index representing the amount of gold fine particles aggregated by exosome, and quantifies the amount of exosome based on the change in absorbance.
  • the absorbance represents the intensity ratio between incident light and transmitted light.
  • the exosome measuring apparatus of the present embodiment has a light source 1 that irradiates irradiation light 4 to a cell 2 that contains a specimen, a detector 3 that detects light that has passed through the cell 2, and a processing unit 5, and has a predetermined absorbance. Acquired every time, and the processing unit 5 stores and analyzes the data.
  • the processing unit 5 may be configured as a control module or a computer that executes a processing program.
  • FIG. 2 is a diagram showing a flow of quantitatively evaluating the amount of exosomes in a specimen in which exosomes are suspended, and then extracting exosomes in the specimen.
  • a sample containing exosomes is pretreated to remove substances having a membrane structure other than exosomes, and a specimen containing only exosomes as a substance having a membrane structure is prepared (S11).
  • a metal ion solution is mixed into the cell 2 in which the specimen prepared in this way is placed to obtain a mixed solution (S12).
  • the cell 2 is irradiated with light from the light source 1, the transmitted light spectrum of the mixed solution is detected by the detector 3, and the absorbance is measured (S 13).
  • the measurement result of the absorbance is stored in the processing unit 5, and the absorbance peak described later is calculated (S14).
  • the steps of absorbance measurement and absorbance peak calculation are repeated until the measurement end condition is satisfied in step S15.
  • the concentration of exosome is evaluated based on the result of calculating the absorbance peak (S16).
  • gold ions are used as the metal ions in step S12, and the sample prepared from the sample is irradiated with light having a wavelength of 400 nm to 1100 nm, and each wavelength by localized surface plasmon resonance generated by the substance in the sample is detected. The absorbance at was measured. In localized surface plasmon resonance, the absorption wavelength and absorption intensity are determined by the size, shape, molecule, etc. of the substance contained in the sample to be measured. In this example, the absorbance of the gold microparticles by the localized surface plasmon resonance is measured. It was measured.
  • the absorbance in the wavelength band from 400 nm to 900 nm was measured. As shown in FIG. 4, a point having the largest absorbance in the wavelength range from 400 nm to 900 nm is “maximum absorbance”, and a point having the smallest absorbance on the shorter wavelength side than the maximum absorbance in the wavelength range from 400 nm to 900 nm is called “minimum absorbance”. Defined. The difference between the maximum absorbance and the minimum absorbance was defined as “absorbance peak (h)”.
  • the absorbance of the specimen is acquired at an arbitrary sampling interval and stored in the processing unit 5, and the measurement is repeated for a predetermined time, for example, 20 to 30 minutes.
  • the absorbance measurement result is stored in the processing unit 5 every sampling, and the process of calculating the absorbance peak in the processing unit 5 is repeated until the condition for termination of measurement is satisfied.
  • the maximum absorbance peak (h max ) is identified from the acquired absorbance peaks, and the amount of exosome of the sample is calculated by comparing with the calibration data stored in the processing unit 5.
  • the sample for which the amount of exosome was measured is centrifuged at 10,000 rpm for 5 minutes by using a centrifuge, gold and gold-attached exosomes are allowed to settle, and the supernatant is removed with a pipette (S17). Next, sodium thiosulfate is added and mixed (S18). Next, the specimen is again centrifuged and centrifuged at 10,000 rpm for 5 minutes, and then the exosome is extracted by collecting the supernatant (S19).
  • step S11 If the following processing is performed in step S11 before step S12 on the sample for measuring the exosome concentration, the measurement accuracy is improved. Since gold ions in the gold ion solution have a property of non-specific adsorption to a structure having a membrane structure, it is preferable that the sample mixed with the gold ion solution is in a state where only exosomes are present. If the sample is human blood, a specimen with a membrane structure that contains only exosomes can be separated by centrifuging around 900G to 1000G with an ordinary centrifuge without using an ultracentrifuge. It is possible to prepare. As another method for separating membrane structural substances other than exosomes from the sample, only the exosomes may be extracted by passing the collected blood through a filtration filter of 500 ⁇ m or less.
  • FIG. 3 is a diagram showing a flow for measuring the total amount of exosomes in blood and extracting and analyzing only exosomes. Steps S21 and S22 shown in FIG. 3 are preprocessing steps corresponding to step S11 of the flow shown in FIG.
  • a blood sample collected from a human is centrifuged (S21), and serum and plasma are extracted (S22).
  • the collected blood sample contains cells such as red blood cells and white blood cells in addition to exosomes. In the case of blood collected from humans, these cells are small and about 1 ⁇ m, which can be separated by centrifuging for about several minutes with a normal centrifuge.
  • exosomes When serum and plasma are extracted by centrifugation, exosomes, water, proteins (albumin, fibrinogen, immunoglobulin), lipids, saccharides (glucose), and inorganic salts exist in the serum and plasma.
  • the exosome is the only substance having a membrane structure and a structure of about several tens of nanometers. Therefore, the concentration of exosome can be evaluated by the absorbance peak due to the size of the gold fine particles. Therefore, by performing the same processes as steps S12 to S19 shown in FIG. 2 on the specimen obtained in step S22, it is possible to measure the concentration of exosomes in blood and extract exosomes.
  • HBS-N Absorbance is measured as an index indicating the amount of gold microparticles aggregated by exosomes in solutions with different exosome amounts in buffer solutions 0.01M HEPES, 0.15M NaCl (hereinafter referred to as HBS-N).
  • a halogen lamp was used as the light source 1
  • an absorbance measurement device USB2000 (Ocean Optics Co., Ltd.) equipped with a spectroscope capable of splitting the visible light wavelength was used as the detector 3.
  • the cell 2 was made of acrylic resin.
  • the specimen used was an exosome solution in which exosomes were suspended in buffer HBS-N (pH 7.4).
  • An exosome solution having a concentration of 100% and an exosome solution diluted with HBS-N so that the concentrations of the exosome solution were 10% and 30% were prepared.
  • the gold ion solution to be mixed for each concentration sample was 1/10 of the total amount of the specimen.
  • the gold ion solution to be mixed with the exosome-suspended solution (specimen) used GoldEnhance (TM) of Nanoprobes, Sakai Inc.
  • FIG. 4 shows the results of measuring samples with different exosome solution concentrations. From this, it was found that the absorbance varies depending on the exosome solution concentration.
  • FIG. 5 shows the relationship between the exosome solution concentration and the maximum absorbance peak.
  • the measurement end condition was set as the measurement time, and the gold ion solution was mixed with the specimen, and the measurement was completed after 20 minutes. After completion of the measurement, an absorbance peak was calculated for each sampling from the obtained absorbance, and the maximum absorbance peak was taken as the maximum absorbance peak h max .
  • FIG. 6 shows an SEM observation image of the HBS-N solution in which exosomes are suspended. Since the structure of 40 nm or more in this solution is only an exosome, the structure having a diameter of about 90 nm in FIG. 6 is considered an exosome.
  • FIG. 7 is an SEM observation image of the specimen 25 minutes after mixing the gold ion solution with the HBS-N solution in which exosomes are suspended. From the image of FIG. 7, it can be determined that a bright structure having a dimension of about 300 nm is a gold fine particle, and a dark structure having a diameter of about 90 nm is an exosome. Moreover, as a result of measuring exosomes and gold fine particles existing in the visual field range other than those shown in FIG.
  • FIG. 7 shows an SEM observation image of a specimen 25 minutes after the gold ion solution was mixed with the HBS-N solution. Spherical gold fine particles with a diameter of about 50 nm at the maximum could be confirmed on the substrate. Further, gold fine particles having a size of about 300 nm to 500 nm similar to those in FIG. 7 could not be confirmed. From this, it was found from FIG. 7 and FIG. 8 that there is a difference of about 10 times in the size of the gold microparticles aggregated in the presence or absence of exosomes regardless of the treatment time of the gold ion solution.
  • the amount of exosomes can be quantified by measuring the number of gold fine particles with a size of 50 nm or more.
  • gold fine particles having a size of 50 nm or more have an absorption wavelength in a wavelength region of about 530 nm or more, the absorbance in the wavelength region of 530 nm or more is considered to represent the absorbance of the gold fine particles generated in response to exosomes.
  • the gold fine particles are grown to 100 nm or more with respect to the exosome surface, it can be sedimented by a centrifuge. Therefore, after precipitating gold and gold adsorbed exosomes with a centrifuge, the solution was exchanged with an aqueous sodium thiosulfate solution and centrifuged again. As a result, a solution in which only exosomes were suspended was extracted from the supernatant. did it. This is because the gold-exosome adsorption force is very small compared to the binding force of gold-sodium thiosulfate, so that the exosome is separated from the gold microparticles, and only the gold microparticles are settled by centrifuging again. It is thought that it was present in the supernatant without settling because of its small diameter. By extracting only exosomes, PCR and RNA analysis can be performed and used for analysis of gene information and the like possessed by exosomes.
  • the solution used for the measurement was centrifuged for 5 minutes, the supernatant was removed, and an aqueous sodium thiosulfate solution was added dropwise. Then, since exosomes and gold microparticles were separated by gold-thiol bonds, this solution was centrifuged again, and the supernatant was extracted, so that exosomes were suspended and exosomes could be extracted from the solution.
  • the process from quantitative evaluation to exosome extraction was performed as a series of operations, but the quantitative analysis method and the exosome extraction method may be performed independently.
  • the process up to the step of mixing the gold ion solution with the specimen is common.
  • quantitatively analyzing exosomes it may be performed up to a quantitative evaluation step.
  • extracting an exosome it is good to start from the centrifugation process of step S17, after arbitrary time passes, after mixing a gold ion solution.
  • FIG. 9 is a diagram showing an optical system for measuring the exosome concentration of a solution in which exosomes are suspended by scattered light. Since the Rayleigh scattering of the irradiation light 4 is generated by the gold fine particles adsorbed on the exosome and the amount of scattered light is proportional to the amount of exosomes present in the specimen, the scattered light 6 is measured as an index representing the amount of exosomes, that is, the amount of gold fine particles. In particular, it is possible to measure the exosome concentration. As shown in FIG.
  • the position of the detector 3 is preferably between 1 ° and 180 ° with respect to the optical axis of the irradiation light 4 from the light source 1 starting from the cell 2.
  • the detection accuracy is best when it is installed between 1 ° and 90 °.
  • the amount of exosomes may be calculated by irradiating a solution in which exosomes are suspended with laser light and measuring the amount of gold fine particles by detecting the backscattered light generated by the gold fine particles.
  • the amount of gold fine particles may be measured by measuring the Brownian motion of the gold fine particles with a dynamic light scattering device, and the amount of exosomes may be calculated. That is, as an index representing the amount of gold fine particles aggregated by exosomes, backscattered light or dynamic light scattering based on Brownian motion can be used in addition to absorbance.
  • a gold ion solution was used for metal modification to exosomes, but a silver ion solution may be used. This is because local fine surface plasmon resonance occurs in silver fine particles.
  • the wavelength to be measured is 200 to 1000 nm.
  • the metal ion solution can be palladium or platinum in addition to silver.
  • the wavelength range to be measured is 100 nm to 1000 nm, and the lamp used as the light source may be a mercury lamp or the like that can generate ultraviolet light.
  • Nanoprobes, Inc. GoldEnhance (TM) was used as a gold ion solution for gold modification to exosomes, but other gold ion solutions may be used. However, it is better to use a neutral gold ion solution so as not to destroy the exosome morphology.
  • a halogen lamp is used as the light source, but any light source that emits light in the vicinity of the plasmon resonance wavelength of the metal ion solution to be used may be used.
  • white light sources include halogen lamps, tungsten lamps, and xenon lamps.
  • a single wavelength light source LED or laser may be used.
  • a gold ion solution one that emits light within a range of 500 to 650 nm is preferable.
  • a centrifuge was used for the separation process of exosomes adsorbed with gold fine particles, but the following method may be used. Separation of exosomes and gold fine particles is achieved by separating exosomes and gold fine particles with sodium thiosulfate and dropping them onto a substrate with thiol termination, so that only the gold fine particles are fixed by thiol bonds, so that only exosomes can be extracted. It becomes.
  • separation using a filter is possible when the particle size of the gold fine particle exceeds 400 nm, it is separated by mixing sodium thiosulfate with a solution in which exosomes are adsorbed to the gold fine particle and passing through the filter. Also good.
  • the measurement end condition is the time after mixing the gold ion solution.
  • a predetermined threshold value may be set for the obtained absorbance, and the measurement may be terminated when the predetermined threshold is exceeded. It is also possible to set a condition that the change amount of the sex is calculated, and the measurement is terminated when the change amount is greater than or less than the predetermined change amount.
  • the maximum absorbance peak h max among the obtained absorbance peaks was used for exosome quantitative evaluation data, but the absorbance peak h after a predetermined time (for example, 10 minutes later) was used for exosome quantitative evaluation. It may be used for the data. It may also be used measuring elapsed time with the maximum absorbance peak h max data for exosomes quantitatively evaluated.
  • the wavelength-dependent slope of absorbance may be used as data for quantitative evaluation of exosomes.
  • the time change of absorbance is detected by irradiating with light of a certain wavelength, and the data obtained there, for example, stable absorbance after elapse of a predetermined time after mixing the gold ion solution, is used for quantitative evaluation of exosomes. May be used for data.
  • this invention is not limited to the above-mentioned Example, Various modifications are included.
  • the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described.
  • a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment.

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Abstract

L'invention concerne un échantillon contenant des exosomes qui est mélangé avec une solution d'ions métalliques telle qu'une solution d'ions or pour obtenir une solution mixte. La solution mixte est examinée relativement à la quantité de fines particules métalliques qui ont été agrégées en son sein, au moyen de l'absorbance, etc. La concentration d'exosomes est évaluée à partir de la quantité des fines particules métalliques agrégées. Pendant ce temps, des ions or sont utilisés comme ions métalliques, et de fines particules d'or qui se sont agrégées dans la solution mixte sont sédimentées par centrifugation. Le surnageant est retiré de celle-ci, et le thiosulfate de sodium est ensuite ajouté goutte à goutte au résidu. Le mélange est à nouveau centrifugé pour sédimenter les fines particules d'or. Le surnageant est récupéré à partir de celui-ci pour extraire les exosomes.
PCT/JP2015/052909 2015-02-03 2015-02-03 Procédé de dosage d'exosomes et procédé d'extraction d'exosomes WO2016125243A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017141947A1 (fr) * 2016-02-15 2017-08-24 凸版印刷株式会社 Procédé de formation de complexe d'exosomes
CN111148828A (zh) * 2017-07-26 2020-05-12 罗塞塔外排体株式会社 利用阳离子分离细胞外囊泡的方法
JP2021506324A (ja) * 2017-12-19 2021-02-22 ウニヴェルシタ デリ ストゥディ ディ トレント 生体材料から細胞外小胞を単離する方法および固定相

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JP2012507300A (ja) * 2008-10-30 2012-03-29 カリス ライフ サイエンシズ ルクセンブルク ホールディングス Ｒｎａパターンを評価する方法
WO2014030590A1 (fr) * 2012-08-24 2014-02-27 国立大学法人東京大学 Méthode d'analyse d'un exosome, appareil d'analyse d'un exosome, complexe anticorps-exosome et puce pour l'électrophorèse d'un exosome
JP2014519331A (ja) * 2011-06-10 2014-08-14 日立化成株式会社 小胞捕捉デバイスおよびそれを用いるための方法

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Publication number Priority date Publication date Assignee Title
JP2012507300A (ja) * 2008-10-30 2012-03-29 カリス ライフ サイエンシズ ルクセンブルク ホールディングス Ｒｎａパターンを評価する方法
JP2014519331A (ja) * 2011-06-10 2014-08-14 日立化成株式会社 小胞捕捉デバイスおよびそれを用いるための方法
WO2014030590A1 (fr) * 2012-08-24 2014-02-27 国立大学法人東京大学 Méthode d'analyse d'un exosome, appareil d'analyse d'un exosome, complexe anticorps-exosome et puce pour l'électrophorèse d'un exosome

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017141947A1 (fr) * 2016-02-15 2017-08-24 凸版印刷株式会社 Procédé de formation de complexe d'exosomes
CN111148828A (zh) * 2017-07-26 2020-05-12 罗塞塔外排体株式会社 利用阳离子分离细胞外囊泡的方法
JP2020528766A (ja) * 2017-07-26 2020-10-01 ロゼッタ エクソソーム 陽イオンを利用した細胞外小胞体の分離方法
EP3660142A4 (fr) * 2017-07-26 2021-05-05 Rosetta Exosome Procédé d'isolement de vésicules extracellulaires à l'aide de cations
US11904259B2 (en) 2017-07-26 2024-02-20 Rosetta Exosome Method for isolating extracellular vesicles using cations
JP2021506324A (ja) * 2017-12-19 2021-02-22 ウニヴェルシタ デリ ストゥディ ディ トレント 生体材料から細胞外小胞を単離する方法および固定相

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