CN115851745B - Application of substance for regulating Esrrb activity in preparation of product for intervening retinal cleavage disease - Google Patents
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Abstract
The invention provides application of a substance for regulating and controlling Esrrb activity in preparation of a product for intervening in retinal splitting, and relates to the technical field of biology. The retinal cleavage disease mediated by the substance for regulating the Esrrb activity is mainly retinal cleavage disease caused by the mutation or deletion of the RS1 gene; esrrb can also be used as a target point for regulating and controlling the expression of the vision function protein for non-diagnosis and treatment purposes, and the technical problem of poor treatment effect on the retinal rival disease product caused by the mutation or deletion of the RS1 gene in the prior art is solved.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of a substance for regulating and controlling Esrrb activity in preparation of a product for intervening in retinal splitting.
Background
X chromosome linked congenital retinal cleavage disease (X-Linked Retinoschisis, XLRS) is a relatively common early-onset central retinal degenerative disease, the worldwide incidence rate is 1/5000-1/25000, and is one of the most main causes of male adolescent macular degeneration. XLRS primarily involves both eyes and clinical features include progressive central vision loss, foveal cleavage induced radial streaks, peripheral retinal interlayer cleavage, retinal detachment and vitreous hemorrhage. At present, no effective treatment method for XLRS exists, and clinical observation and treatment of complications are still the main treatment method. The gene mutation is the main cause of XLRS, and the pathogenic gene is RS1.RS1 encodes retinal cleavage protein 1 (retinoscisin 1, RS 1) which plays an important role in retinal development and maintenance structure. The mutation of RS1 leads to abnormal functions of RS1 protein, such as synthesis and secretion of RS1, and further leads to abnormal functions of Muller cell fibers, thereby leading to retinal cleavage. Gene therapy is currently the primary treatment for XLRS, but the clinical trial efficacy is not ideal. However, there is currently no other method of alleviation and treatment internationally other than gene therapy. Therefore, how to improve the therapeutic products of XLRS is a current problem to be solved.
In view of this, the present invention has been made.
Disclosure of Invention
The invention is based on the use of the parthenogenic estrogen receptor Esrrb in the preparation of the medicineRS1The invention aims to provide an application of a substance for regulating and controlling Esrrb activity in preparing a product for interfering with retinal split diseases and a method for regulating and controlling expression of opsin for non-diagnostic and therapeutic purposes, which relieves the existing treatment in the prior artRS1The technical problem of poor treatment effect of retinal rival disease products caused by gene mutation or deletion.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the invention there is provided the use of a substance which modulates Esrrb activity in the manufacture of a product for use in the intervention of retinal rivalry; the retinal split diseaseIs thatRS1Retinal cleavage due to gene mutation or deletion.
Preferably, the retinal riving disease is X chromosome linked congenital retinal riving disease;
or, the retinal cleavage disease is a knockout mammalRS1Retinal cleavage after the gene.
Preferably, the mammal comprises a mouse, rat, rabbit, pig, cow, dog, monkey, or human;
preferably, the mammal comprises a mouse.
Preferably, the product for intervening in retinal cleavage is a product for preventing, treating or alleviating retinal cleavage, and the substance that modulates the activity of Esrrb is an Esrrb expression vector or an Esrrb agonist;
preferably, the Esrrb agonist comprises DY131.
Preferably, the Esrrb inhibitor comprises siRNA, shRNA or miRNA;
preferably, the Esrrb inhibitor is an siRNA;
preferably, the nucleotide sequence of the siRNA is as shown in seq_1 or seq_7.
According to another aspect of the invention, there is provided a method of modulating expression of a opsin protein for non-diagnostic and therapeutic purposes, comprising modulating Esrrb activity to effect modulation of the expression level of the opsin protein;
preferably, the opsin protein comprises one or more of Rho, rgs9 and Kcnb 1.
Preferably, the modulating the expression of the opsin protein is up-regulating the expression of the opsin protein, the modulating the Esrrb activity comprises over-expressing the Esrrb protein, or using an Esrrb agonist to increase the Esrrb activity;
preferably, the up-regulating the expression of the opsin protein is up-regulating the expression of the Kcnb1 protein;
preferably, the Esrrb agonist comprises DY131;
preferably, the up-regulating functionally protein is expressed as up-regulatedRS1Knocking out the expression of the visual function protein of the mammal;
preferably, the mammal comprises a mouse, rat, rabbit, pig, cow, dog, monkey, or human;
preferably, the saidRS1The mammal is gene knockout mammalRS1A knockout mouse.
Preferably, said modulating expression of a opsin protein is down-regulating expression of a opsin protein, said modulating Esrrb activity comprising down-regulating expression of Esrrb using an Esrrb inhibitor;
preferably, the downregulating expression of the opsin protein comprises downregulating expression of one or more of Rho, rgs9 and Kcnb 1;
preferably, the Esrrb inhibitor comprises siRNA, shRNA or miRNA;
preferably, the Esrrb inhibitor is an siRNA;
preferably, the nucleotide sequence of the siRNA is as shown in seq_1 or seq_7.
According to another aspect of the invention, the invention also provides an siRNA for silencing Esrrb, the nucleotide sequence of which is shown as seq_1.
According to another aspect of the present invention, there is also provided a product for down-regulating expression of a opsin protein, the product comprising the above siRNA.
Compared with the prior art, the invention has the following beneficial effects:
the parthenogenic estrogen receptor Esrrb (orphan nuclear hormone receptor estrogen-linked receptor beta), also known as Nr3b2, is one of the important members of the estrogen receptor family. Current Esrrb targeted drugs are mainly hormone analogues, used for climacteric and postmenopausal disorders. The parthenogenetic estrogen receptor Esrrb is never applied to an action target point of a therapeutic drug for eye diseases.
The invention finds that Esrrb is related to the expression of opsin proteins. Esrrb in wild-type mouse MEF cells were knocked down, down regulating opsin expression. At the position ofRS1The expression of opsin was also reduced in knockout mice. Overexpression ofRS1The expression of the Esrrb protein and the visual function protein of the gene knockout mice is recovered. To the direction ofRS1Esrrb agonist was administered to the knockout mice and cleavage cavity visualization of the mice was observed by OCTThe results show that the Esrrb activity can be obviously improved by reducing the amountRS1Retinal morphology in knockout mice.
Based on the findings, the application of the substance for regulating and controlling the Esrrb activity in the preparation of the product for intervening in the retinal rivalry disease can be used for preparing the interveningRS1Retinal cleavage caused by gene mutation or deletion provides a new drug target. Meanwhile, the provided method for regulating and controlling the expression of the vision function protein for non-diagnosis and treatment purposes provides an important research means for researching the vision function protein and related mechanisms or pathological conditions thereof.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the immunofluorescence of wild-type mouse MEF cells in example 2;
FIG. 2 is a schematic diagram of example 2Rs1Knocking out the MEF cell immunofluorescence result of the mice;
FIG. 3 is a graph showing the effect of knock-down Esrrb on expression of a functional protein in murine MEF cells in example 2;
FIG. 4 is a graph showing the effect of Esrrb on expression of a functionally active protein in overexpressing mouse MEF cells in example 2;
FIG. 5 is a schematic diagram of example 1Rs1Knocking out the mouse DNA identification result;
FIG. 6 is a diagram of wild type mice and in example 1Rs1Knocking out the expression condition of the Rs1 protein in the retina of the mouse;
FIG. 7 is a schematic diagram of example 3Rs1The retinal morphology of the mice after intraperitoneal injection of the Esrrb agonist was knocked out.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references, as well as laboratory procedures and techniques thereof, are those well known and commonly used in the art.
It should be noted that:
in the present invention, all embodiments and preferred methods of implementation mentioned herein may be combined with each other to form new technical solutions, if not specifically stated; all technical features and preferred features mentioned herein may be combined with each other to form new solutions; the components involved or their preferred components can be combined with one another to form new technical solutions.
The parthenogenic estrogen receptor Esrrb (orphan nuclear hormone receptor estrogen-linked receptor beta), also known as Nr3b2, is one of the important members of the estrogen receptor family. Esrrb is significantly expressed in retinal photoreceptor cells. Overexpression of Esrrb induces NrL lacking rod photoreceptors −/− Both mice and wild-type mice express rod-like specific genes such as rhodopsin Rho, slc24a1, etc. At present, the medicaments taking Esrrb as targets are mainly non-steroidal female hormones, such as diethylstilbestrol, estradiol, tamoxifen and the like. These drugs are mainly used as hormone analogues for climacteric and postmenopausal disorders, and the parthenogenetic estrogen receptor Esrrb has never been applied to the treatment of ocular diseases.
The invention has been found through experiments that,RS1gene knockout mouseRs1Expression of the visual function proteins Rho, rgs9 and Kcnb1 in-KO) MEF cells was reduced, while siRNA was used against wild typeEsrrb in mouse MEF cells was knocked down, and expression of the visual function proteins Rho, rgs9 and Kcnb1 was also reduced, indicating that Esrrb was associated with expression of visual function proteins.
Further, the inventors have achieved by overexpression ofRs1Esrrb protein in KO mouse MEF cells, and it was found that overexpression of Esrrb up-regulates expression of the opsin protein Kcnb1, demonstrated inRS1Overexpression of Esrrb protein in knock-out cell lines, which can restore the effects of the geneRS1Reduced expression of opsin due to deletion of gene.
Further, the inventors are directed toRs1Mice with KO were administered the Esrrb agonist DY131, observed by OCT (ptical coherence tomography optical coherence tomography)Rs1Morphology of KO mice, finding administration of Esrrb agonist DY131Rs1The cleavage cavity of the KO mouse is obviously reduced compared with that of a blank control, and the result proves that the Esrrb activity can be obviously improvedRs1Retinal morphology in KO mice.
Based on the above findings, in some alternatives, there is provided the use of a substance that modulates Esrrb activity in the manufacture of a product for use in the intervention of retinal rivalry disease.
Wherein "an agent that modulates Esrrb activity" refers to an agent that increases or decreases Esrrb activity. Increasing Esrrb activity is optionally achieved by increasing the expression level of endogenous Esrrb or exogenous Esrrb, or alternatively by increasing Esrrb activity of the cell or body itself; the reduction of Esrrb activity is optionally a down-regulation of Esrrb expression in the cell or body, or alternatively a reduction of Esrrb activity of the cell or body itself. Wherein increasing or decreasing Esrrb activity is compared to the non-interfered wild-type Esrrb activity.
"intervening in retinal detachment" includes preventing, treating, or alleviating retinal detachment.
The retinal cleavage disease regulated in the above embodiment isRS1Retinal cleavage due to gene mutation or deletion.RS1Gene mutations include, but are not limited to, point mutations, or deletion, insertion, and cleavage site mutations.RS1The retinal cleavage induced by the gene mutation is optionally X-chromosome Linked congenital retinal cleavage (X-Linked retinin)oschisis, XLRS), or artificially mutatedRS1Gene-induced retinal detachment, such as those obtained by animals by constructing animal models of retinal detachment. Also optionally a knockout mammalRS1Retinal cleavage after the gene. Wherein the mammal comprises a mouse, rat, rabbit, pig, cow, dog, monkey, or human. Experiments prove that the improvement is achievedRS1Gene knockout mouse Esrrb activity is relievedRS1Retinal cleavage in knockout mice, therefore, in at least one embodiment, agents that modulate Esrrb activity can be used to prepare interventionsRS1Products of retinal cleavage in knockout mice.
In some alternative embodiments, the product for intervention in retinal detachment is a product for preventing, treating, or alleviating retinal detachment, and the substance that modulates Esrrb activity is an Esrrb expression vector or an Esrrb agonist. The Esrrb expression vector is transformed into a host, and can express exogenous Esrrb protein in the host, so that the expression of Esrrb is up-regulated, the amount of Esrrb is increased, and the aim of improving the activity of Esrrb in cells or organisms is fulfilled. The Esrrb agonist can enhance the Esrrb activity and promote downstream reaction, thereby achieving the purpose of enhancing the Esrrb activity in cells or organisms. An example of an Esrrb agonist is DY131 (CAS: 95167-41-2), DY131 (GSK 9089) is an ERRβ and ERRγ selective agonist with very low activity towards ERRα, ERα and ERβ.
The expression quantity of the vision function protein is also found to be changed while the intervention of the retina split disease is realized by regulating and controlling the Esrrb activity. Thus in other alternatives, a method of modulating the expression of a opsin protein for non-diagnostic and therapeutic purposes is provided, wherein the opsin protein includes, but is not limited to, one or more of Rho, rgs9 and Kcnb 1. The test shows that the Esrrb activity in the cell or host is changed, and the expression level of the opsin protein is also changed.
In some alternative embodiments, modulating expression of the opsin protein as up-regulating expression of the opsin protein, modulating Esrrb activity comprises over-expressing the Esrrb protein, or using an Esrrb agonist to increase Esrrb activity. The up-regulating expression of the functional protein is preferably up-regulating Kcnb1 proteinExpression, preferably up-regulation of Kcnb1 protein expression by overexpression of Esrrb; if an Esrrb agonist is used to up-regulate expression of the opsin protein, an alternative example of an Esrrb agonist includes DY131. Up-regulating expression of functionally protein is optionally up-regulatingRS1The expression of the opsin protein in a mammal is knocked out, wherein the mammal includes, but is not limited to, mice, rats, rabbits, pigs, cattle, dogs, monkeys or humans.RS1The knockout mammal is preferablyRS1A knockout mouse.
In other alternative embodiments, modulating expression of a opsin protein is down-regulating expression of the opsin protein, and modulating Esrrb activity comprises down-regulating expression of Esrrb using an Esrrb inhibitor, the down-regulating expression of the opsin protein including, but not limited to, down-regulating expression of one or more of Rho, rgs9, and Kcnb 1. Wherein the Esrrb inhibitor includes, but is not limited to, an siRNA, shRNA or miRNA, for example an siRNA with a nucleotide sequence as shown in seq_1 or seq_7. The SiRNA can also optionally be modified in a manner known in the art, for example by adding two thymines at 3' to increase the stability of the siRNA, the sequence of which is shown in seq_7.
The invention also provides the siRNA for silencing Esrrb, and experiments prove that the expression quantity of the functional proteins Rho, rgs9 and Kcnb1 is reduced by using the siRNA for silencing Esrrb. Correspondingly, the invention also provides a product for down-regulating expression of the vision functional protein, which comprises the siRNA.
The technical solution and technical effects of the present invention are further described below in connection with preferred embodiments.
Example 1
ConstructionRs1Knockout mouse
(1) Preparation of sgrnas and Cas9-mRNA:
rs1-gRNA1 sequence GTTAACTGTAAGGCGAGTAAT (5 'to3', seq_2),
rs1-gRNA2 sequence GAGACTCGTGGGCCATACGCC (5 'to3', seq_3).
The DNA fragment of Rs1-sgRNA was amplified by PCR using sgRNA-Vector as a template, and then recovered as a template for in vitro transcription of the sgRNA. And then the sgRNA is subjected to in-vitro transcription, purification and recovery, and split charging and storage are carried out in a refrigerator at-80 ℃ for standby. Linearizing the Cas9 transcription vector, and then carrying out in vitro transcription, purification, recovery, split charging and storage in a refrigerator at-80 ℃ for later use.
(2) Microinjection: cas9-mRNA and Rs1-sgRNA were co-microinjected into C57 mouse embryos, which were transplanted into the oviduct of surrogate recipient mice after injection.
(3) Embryo transfer and F0 mouse identification and propagation: the fertilized eggs after microinjection are returned to the oviduct of the surrogate mother, and the mice are born about 21 days after embryo transplantation, and genotyping is completed about 2 weeks after birth.
(4)Rs1-KO mouse phenotype identification:
Rs1the DNA extracted from the tail of the knockout mouse was cut and purified by PCR to identify homozygotes, the sequence of the PCR primer is shown in Table 1, and the sequence comparison proves that the XLRS model mouse is successfully constructed, as shown in FIG. 5, the Homo (++) representsRs1KO mouse homozygote, heter (+ -) stands forRs1KO mouse heterozygotes, WT representing wild-type mice, primers and fragments of interest used in each group are shown in Table 2. The Rs1 protein (green fluorescence) visible on the retina of a wild mouse is found through Foundation murine sequence alignment and immunofluorescence research, andRs1KO mice retina showed no Rs1 protein expression, as shown in FIG. 6, demonstrating that XLRS model mice were successfully constructed.
TABLE 1
TABLE 2
Example 2
Effect of Esrrb on XLRS cell model
(1) Construction of XLRS disease modelRs1MEF cell line of KO mice
1. Will become pregnant for 13.5 daysRs1Knockout mice (and control mice) were sacrificed by carbon dioxide inhalation and immersed in 75% alcohol. The abdomen was then rubbed with 75% ethanol, the abdomen was cut with a sterilized tool, the uterus was removed and placed in a 10cm petri dish and rinsed with PBS. With scissorsThe embryo membrane was cut, the embryos removed, transferred to a new 10cm dish, and placenta and other maternal tissues removed. The head (eyes and above) was cut off and all viscera were removed, leaving the limbs of the mice. Washed with sterile PBS. Embryoid bodies were placed in separate 10cm plates, embryos were minced, after which 10ml trypsin was added for digestion (trypsin was allowed to cover cells). After more cells are digested and overflowed, the cells are incubated in a cell culture incubator at 37 ℃ for 30-45 minutes, and then an equivalent amount of MEF culture solution (90% DMEM+10% FBS+1% P/S) is added to terminate the digestion. After the supernatant was sucked through a 200 mesh cell sieve by gentle pipetting, cells were harvested by centrifugation at 1000 rpm for 5 minutes. Then, the cells were resuspended, the cell suspension was transferred to a flask, and 10mL MEF medium was added to allow the cells to grow to confluence (3-4 days).
2. Identification of MEF cell lines
MEF cells were seeded in 24-well plates, after cell attachment, the medium was aspirated and washed with PBS, 0.1% triton x-100 was added and permeabilized for 30min at room temperature, and PBS was washed three times. Blocking with 5% BSA blocking solution was performed for 2 hours at room temperature, and the cells were washed with PBS. Vimentin antibody was added and incubated overnight at 4 ℃. The next day fluorescent secondary antibody was added and incubated for 2 hours at room temperature in the dark. Washing with PBS, adding DAPI dye solution, and incubating for 5min in dark place. After washing, the images were observed and photographed by a fluorescence microscope. Wild-type mouse MEF cellsRs1The results of the KO murine MEF cell immunofluorescence are shown in FIGS. 1 and 2.
(2) Esrrb down-regulation may cause reduced expression of opsin proteins
Wild-type mouse MEF (WT-MEF) cells were plated in 6-well plates, 2.5X10 5 Well cells, protein was extracted by transfecting siRNA, 3 μl (10 μm working concentration) of siRNA per well (GGAUGGAGAUUCUCAUCUUtt, seq_7) and 9 μl lipranaimax transfection reagent, and harvesting the cells after 48 h. WB experiments demonstrated that knockdown Esrrb down regulated expression of opsin proteins. Compared to wild-type mouse MEF (WT-MEF) cells,Rs1knockout of MEF cells in miceRs1-KO-MEF) expression of the opsin proteins Rho, rgs9, kcnb1 is reduced. Expression of the opsin Rho, rgs9, kcnb1 was down-regulated by siRNA knockdown of Esrrb in wild-type mouse MEF (WT-MEF) cells, and the results are shown in fig. 3. From FIG. 3, it can be seen thatIt appears that the knockdown Esrrb down regulates the expression of the functionally active protein. Compared to wild-type mouse MEF (WT-MEF) cells,Rs1expression of the opsin Rho, rgs9, kcnb1 proteins in the (Rs 1-KO-MEF) cells of the knockout mice was reduced. Expression of the visual function proteins Rho, rgs9 and Kcnb1 can be down-regulated after the Esrrb is knocked down by siRNA in wild-type mouse MEF (WT-MEF) cells, which indicates that the Esrrb is related to the expression of the visual function proteins, and the down-regulation of the expression of the Esrrb leads to the reduction of the expression quantity of the visual function proteins.
(3) Esrrb up-regulation can save the vision function protein damage caused by RS1 deletion
Will beRs1Knockout of mouse MEFRs1-KO-MEF) cells were plated in 6cm dishes, 8X 10 5 Cells per dish, protein was extracted by transfecting pcDNA3.1-Esrrb plasmid, 6. Mu.g DNA and 20. Mu.l transfection reagent per well, and harvesting cells after 48 h. The results are shown in FIG. 4, and can be seen from FIG. 4 inRs1Knockout of MEF cells in miceRs1-KO-MEF) overexpression of Esrrb up-regulates expression of the opsin protein Kcnb1, WB experiments confirm that overexpression of Esrrb up-regulates expression of the opsin protein Kcnb 1.
Example 3
Esrrb agonist DY131 versus XLRS mouse modelRs1Therapeutic action of KO mice
(1) Treatment of XLRS mice
Will beRs1KO mice are divided into two groups of 10-15 mice each. At the position ofRs1KO mice were given separate injections of PBS and Esrrb agonist DY131 (CAS: 95167-41-2) 1 mg/kg intraperitoneally 7 days after birth, every other day, for two months. Low dose stimulationRs1KO mice activate the Esrrb receptor.
(2) DY131 can improve visual function of XLRS mice
The morphology of Rs1-KO mice was observed by OCT in 15D, 1M, 1.5M, and 2M, respectively, of the Rs1-KO mice injected with Esrrb agonist DY131 intraperitoneally. As shown in FIG. 7, the cleavage cavity of Rs1-KO mice, to which Esrrb agonist DY131 was administered intraperitoneally, was significantly reduced compared to the blank (PBS) for two months, and as a result, it was confirmed that DY 131-treated groups significantly improved the retinal morphology of Rs1-KO mice.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (7)
1. Use of a substance that modulates Esrrb activity in the preparation of a product for treating or alleviating retinal detachment; the retinal cleavage disease is thatRS1Retinal cleavage due to gene deletion;
the substance for regulating the activity of the Esrrb is an expression vector for over-expressing the Esrrb or an Esrrb agonist DY131.
2. The use according to claim 1, wherein the retinal cleavage is X-chromosome linked congenital retinal cleavage.
3. The use according to claim 1, wherein the retinal split is a knockout mouseRS1Retinal cleavage after the gene.
4. The use according to claim 3, wherein the substance that modulates Esrrb activity is Esrrb agonist DY131.
5. A method of modulating expression of an opsin protein for non-diagnostic and therapeutic purposes, wherein said modulating is down-regulating expression of one or more of Rho, rgs9 and Kcnb1 by over-expressing an Esrrb protein by an expression vector to up-regulate expression of an opsin protein Kcnb1 or by down-regulating expression of Esrrb by an siRNA shown in SEQ ID No. 7.
6. The method of claim 5, wherein up-regulating the expression of the functional protein Kcnb1 is up-regulatedRS1The visual function protein Kcnb1 of the gene knockout mouse is expressed.
7. siRNA for silencing Esrrb, wherein the nucleotide sequence of the siRNA is shown in SEQ ID NO. 7.
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CN114848850A (en) * | 2022-04-28 | 2022-08-05 | 武汉大学 | Application of RS1 gene in preparation of XLRS therapeutic agent and therapeutic agent |
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