JP7602932B2 - Agent for maintaining stem cell status of sweat gland myoepithelial cells, method for maintaining stem cell status of sweat gland myoepithelial cells, and topical skin preparation - Google Patents

Description

The present invention relates to an agent for maintaining the stem cell status of sweat gland myoepithelial cells, a method for maintaining the stem cell status of sweat gland myoepithelial cells, and an external preparation for the skin.

Sweat glands, which are an accessory organ of the skin, are important organs that regulate body temperature. Absence or dysfunction of sweat glands can induce heat stroke or cholinergic urticaria, which is associated with constant dry skin. To treat absence or dysfunction of sweat glands, it is important to elucidate the mechanisms of sweat gland formation and homeostasis.

Stem cells play an important role in maintaining homeostasis. Sweat gland myoepithelial cells exist as stem cells in sweat glands. However, the detailed mechanism by which sweat gland myoepithelial cells maintain their stemness remains unclear.

Patent Document 1 describes a medium for in vitro culture of colon epithelial stem cells and/or colon epithelial cells, which contains serum albumin, Wnt3a, and R-spondin-1. Patent Documents 2 to 5 also disclose agents for maintaining stem cell properties of various types of cells.

Re-table No. 2013-061608 JP 2019-026617 A JP 2015-044788 A JP 2013-189389 A JP 2013-209302 A

In elucidating the mechanism of sweat glands, a method of culturing sweat gland myoepithelial cells in a form similar to a sweat gland-like structure by three-dimensional culture (spheroid culture) can be a useful technique. However, the conventional techniques described in the above-mentioned Patent Documents 1 to 5 are not targeted at sweat gland myoepithelial cells.

The inventors' research has revealed that when sweat gland myoepithelial cells are cultured in 3D, they lose their stemness in about one month and differentiate into luminal cells, etc., creating a new problem in that they cannot be cultured for long periods in a state close to a sweat gland-like structure. Therefore, in order to elucidate the mechanism of sweat glands, it is necessary to establish a culture method that maintains the stemness of sweat gland myoepithelial cells in 3D culture.

The objective of the present invention is to realize a technology that can maintain the stemness of sweat gland myoepithelial cells under three-dimensional culture.

The present inventors have conducted extensive research to solve the above problems, and have found that the stemness of sweat gland myoepithelial cells can be maintained in three-dimensional culture by using a substance that enhances LGR4 signaling, thereby completing the present invention. That is, the present invention includes the following features.
<1> An agent for maintaining the stemness of sweat gland myoepithelial cells, which contains a substance that enhances LGR4 signal transduction.
<2> The agent for maintaining stem cell properties of sweat gland myoepithelial cells according to <1>, wherein the substance is at least one selected from the group consisting of Wnt3a and R-spondin1.
<3> A method for maintaining the stemness of sweat gland myoepithelial cells, comprising a step of contacting the agent for maintaining stemness of sweat gland myoepithelial cells according to <1> or <2> with sweat gland myoepithelial cells in vitro.
<4> An external skin preparation comprising the agent for maintaining the stem cell properties of sweat gland myoepithelial cells according to <1> or <2>.

The present invention provides a technology that can maintain the stemness of sweat gland myoepithelial cells under three-dimensional culture.

FIG. 1 shows the expression level (relative value) of the LGR4 gene in representative cells contained in sweat glands. FIG. 13 shows the expression levels (relative values) of aSMA gene and KRT18 gene in control sweat gland spheroids or sweat gland spheroids cultured with Wnt3a or R-spondin1.

The present invention is described below, but is not limited thereto. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims. In addition, embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic and patent documents described in this specification are incorporated herein by reference.

Unless otherwise specified in this specification, "X to Y" indicating a numerical range means "X or more and Y or less."

1. Overview of the Invention
The present inventors attempted to identify factors capable of maintaining the stemness of sweat gland myoepithelial cells as follows. Hereinafter, sweat gland myoepithelial cells are also simply referred to as "myoepithelial cells."

In general, factors released by cells surrounding stem cells are important for maintaining stem cells in the epidermis of the skin or hair follicles. Therefore, the inventors used single-cell RNA analysis, which allows for the analysis of individual cells, to comprehensively analyze cells found around sweat glands. As a result, it was found that the gene for LGR4, a receptor involved in the wnt signaling pathway, is specifically expressed in myoepithelial cells, which are sweat gland stem cells. It was suggested that LGR4 is a receptor for factors that can maintain stemness.

From the above results, the inventors speculated that substances that enhance LGR4 signal transduction could be factors that can maintain stemness. Therefore, the inventors selected R-spondin1, which binds to LGR4, and Wnt3a, which binds to R-spondin1, as candidates for factors that can maintain stemness, and evaluated the effects of these on the culture of sweat gland spheroids. As a result, it was confirmed that in sweat gland spheroids cultured in a medium containing R-spondin1 or Wnt3a, the expression of myoepithelial cell markers was maintained and the expression of luminal cell markers was reduced compared to sweat gland spheroids cultured in a control medium. This suggests that substances that enhance LGR4 signal transduction, including R-spondin1 and Wnt3a, may contribute to the maintenance of stemness of sweat gland myoepithelial cells. The present invention was completed based on these new findings.

[2. Agent for maintaining stem cell status of sweat gland myoepithelial cells]
The agent for maintaining stemness of sweat gland myoepithelial cells according to the present invention contains a substance that enhances signal transduction of LGR4. This allows the substance that enhances signal transduction of LGR4 to act on sweat gland myoepithelial cells, thereby maintaining the stemness of sweat gland myoepithelial cells under three-dimensional culture. Hereinafter, the "agent for maintaining stemness of sweat gland myoepithelial cells" is also simply referred to as "agent for maintaining stemness".

Sweat gland myoepithelial cells are one of the cells that make up sweat glands, and are involved in the movement of sweat glands when sweat is secreted. Sweat gland myoepithelial cells are also the stem cells of sweat glands. Therefore, sweat gland myoepithelial cells can be used to evaluate sweat gland function in order to develop means to improve sweat gland dysfunction and abnormally increased function.

In this specification, "maintaining the stemness of sweat gland myoepithelial cells" means maintaining a state in which sweat gland myoepithelial cells have pluripotency. It can also be said that the stemness maintaining agent reduces the differentiation ability of sweat gland myoepithelial cells into luminal cells, etc. "Maintaining the stemness of sweat gland myoepithelial cells" also includes proliferating sweat gland myoepithelial cells to an extent that they do not become cancerous. In addition, "under three-dimensional culture" refers to the culture of spheroids containing sweat gland myoepithelial cells.

In this specification, "sweat gland cells" refers to the general term for cells that make up sweat glands, including sweat gland myoepithelial cells and luminal cells. "Sweat gland spheroids" are three-dimensional aggregates of cells that make up sweat glands. Sweat gland spheroids may be cell aggregates consisting of only sweat gland myoepithelial cells, or cell aggregates that contain a mixture of sweat gland myoepithelial cells and other sweat gland cells.

The method for determining whether stemness is maintained under three-dimensional culture is not particularly limited, and a known method may be used. For example, it may be confirmed that the expression level of myoepithelial cell markers is maintained and/or increased in sweat gland spheroids contacted with a stemness maintenance agent compared to sweat gland spheroids not contacted with the stemness maintenance agent. It may also be confirmed that the expression level of markers contained in differentiated cells, such as luminal cells, is decreased in sweat gland spheroids contacted with a stemness maintenance agent compared to sweat gland spheroids not contacted with the stemness maintenance agent. Examples of myoepithelial cell markers include aSMA. Examples of luminal cell markers include KRT18 (Keratin18).

LGR4 is a type of G protein-coupled receptor that contains leucine-rich repeats. Examples of substances that enhance the signal transduction of LGR4 include ligands that bind to LGR4 and substances that bind to the ligands. Substances that enhance the signal transduction of LGR4 can also be said to be substances that activate a signal transduction pathway in which LGR4 is involved. Hereinafter, substances that enhance the signal transduction of LGR4 are also referred to as active ingredients. Examples of such active ingredients include R-spondin1 (accession number: NP_001033722), Wnt3a (accession number: BAB61052), R-spondin2 (accession number: NP_848660), and R-spondin3 (accession number: NP_116173). Among these, the active ingredient is preferably at least one selected from the group consisting of Wnt3a and R-spondin1.

The stem cell maintenance agent may contain pharma- ceutically acceptable ingredients in addition to the above-mentioned active ingredients. Examples of such ingredients include buffers, pH adjusters, isotonicity agents, preservatives, antioxidants, high molecular weight polymers, excipients, and solvents.

Examples of the buffering agent include phosphoric acid or a phosphate salt, boric acid or a borate salt, citric acid or a citrate salt, acetic acid or an acetate salt, carbonic acid or a carbonate salt, tartaric acid or a tartrate salt, ε-aminocaproic acid, trometamol, etc. Examples of the phosphate salt include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, etc. Examples of the borate salt include borax, sodium borate, potassium borate, etc. Examples of the citrate salt include sodium citrate, disodium citrate, trisodium citrate, etc. Examples of the acetate salt include sodium acetate, potassium acetate, etc. Examples of the carbonate salt include sodium carbonate, sodium bicarbonate, etc. Examples of the tartrate salt include sodium tartrate, potassium tartrate, etc.

Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, etc.

Examples of the above isotonicity agents include ionic isotonicity agents (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc.) and non-ionic isotonicity agents (glycerin, propylene glycol, sorbitol, mannitol, etc.).

Examples of the above preservatives include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

Examples of the antioxidants include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

Examples of the high molecular weight polymers include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

Examples of the above excipients include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

Examples of the above solvents include water, physiological saline, alcohol, etc.

The amount of the active ingredient contained in the stem cell property maintenance agent is not particularly limited. For example, the amount of the active ingredient may be 0.001% to 100% by mass, 0.01% to 100% by mass, 0.1% to 100% by mass, 0.1% to 95% by mass, 0.1% to 90% by mass, 0.1% to 80% by mass, 0.1% to 70% by mass, 0.1% to 60% by mass, 0.1% to 50% by mass, 0.1% to 40% by mass, 0.1% to 30% by mass, 0.1% to 20% by mass, or 0.1% to 10% by mass, relative to the total mass of the stem cell property maintenance agent.

The amount of the components other than the active ingredient contained in the stem cell property maintenance agent is not particularly limited. The amount of the components other than the active ingredient may be, for example, 0% by mass to 99.999% by mass, 0% by mass to 99.99% by mass, 0% by mass to 99.9% by mass, 5% by mass to 99.9% by mass, 10% by mass to 99.9% by mass, 20% by mass to 99.9% by mass, 30% by mass to 99.9% by mass, 40% by mass to 99.9% by mass, 50% by mass to 99.9% by mass, 60% by mass to 99.9% by mass, 70% by mass to 99.9% by mass, 80% by mass to 99.9% by mass, or 90% by mass to 99.9% by mass.

3. Method for maintaining stemness of sweat gland myoepithelial cells
The method for maintaining the stemness of sweat gland myoepithelial cells according to the present invention includes a step of contacting the above-mentioned agent for maintaining stemness of sweat gland myoepithelial cells with sweat gland myoepithelial cells in vitro. This step can also be said to be a step of culturing sweat gland myoepithelial cells in the presence of the agent for maintaining stemness. Examples of the method for contacting the agent for maintaining stemness with sweat gland myoepithelial cells in vitro include a method of adding the agent for maintaining stemness to a basal medium in which sweat gland myoepithelial cells are cultured, and a method of seeding sweat gland myoepithelial cells in a basal medium to which the agent for maintaining stemness has been added.

The sweat gland myoepithelial cells may be cells isolated from, for example, skin tissue of a human or non-human mammal. The sweat gland myoepithelial cells may be in a mixed state with other sweat gland cells. The sweat gland myoepithelial cells may form the sweat gland spheroids described above. In addition, sweat gland spheroids may be formed after contact of the sweat gland myoepithelial cells with a stem cell maintenance agent.

The basal medium is preferably a medium containing nutritional components that allow sweat gland myoepithelial cells to survive. The basal medium may be, for example, a serum medium, a low-serum medium, or a serum-free medium supplemented with nutritional components, or may be a medium that is readily commercially available. Examples of nutritional components include, but are not limited to, amino acids, vitamins, inorganic salts, sugars, and cell growth promoting factors (e.g., epidermal growth factor, basic fibroblast growth factor, hydrocortisone-21-hemisuccinate, etc.).

The concentration of the nutritional components in the basal medium is not particularly limited, and can be set appropriately depending on the type of skin tissue source from which the sweat gland myoepithelial cells are derived, the type of serum medium, low-serum medium or serum-free medium, the type of nutritional components, etc. These nutritional components may be used alone or in combination of two or more types.

When the source of skin tissue is human, the concentration of epidermal growth factor is usually preferably 0.01 ng/mL or more, more preferably 1 ng/mL or more, from the viewpoint of moderate cell proliferation, and preferably 1 μg/mL or less, more preferably 100 ng/mL or less, from the viewpoint of suppressing excessive cell proliferation. The same applies to the concentration of basic fibroblast growth factor.

The concentration of the stem cell property maintenance agent added to the basal medium is not particularly limited and can be appropriately determined depending on the type of skin tissue source from which the sweat gland myoepithelial cells are derived, the shape of the cells, the cell density in the basal medium, the components of the basal medium, the culture method, the type of active ingredient, etc. When the source of skin tissue is human, the concentration of the stem cell property maintenance agent in the basal medium is preferably 0.5 to 1000 ng/mL, more preferably 10 to 500 ng/mL.

The culture may be performed by adhesion culture or suspension culture. The culture conditions (e.g., culture temperature, culture time, pH of the medium, carbon dioxide concentration in the culture atmosphere, etc.) are not particularly limited and can be appropriately determined depending on the type of skin tissue source from which the sweat gland myoepithelial cells are derived.

The following are examples of preferred culture conditions from the viewpoint of better maintaining the functions and properties of sweat gland myoepithelial cells when the source of skin tissue is human. The culture temperature is preferably 35 to 38°C, more preferably 36.5 to 37.5°C. The culture time is preferably 60 to 672 hours, more preferably 144 to 168 hours. The pH of the basal medium is preferably 6.8 to 7.6, more preferably 7.0 to 7.4. The carbon dioxide concentration in the culture atmosphere is preferably 4 to 10% by volume, more preferably 5 to 7% by volume.

In the basal medium, only sweat gland myoepithelial cells may be present, or other sweat gland cells may be present. The density of cells in the basal medium is not particularly limited, and can be appropriately set according to the shape of the cells. When the cells are in a state of being dissociated into individual cells, the density of cells in the medium at the time of starting contact between the sweat gland myoepithelial cells and the stem cell property maintenance agent is preferably 10 cells/ ^cm2 or more, more preferably 100 cells/ ^cm2 or more, from the viewpoint of cell proliferation, and is preferably 1,000,000 cells/ ^cm2 or less, more preferably 100,000 cells/cm2 or ^less , from the viewpoint of cell survival.

4. Skin preparations for external use
The skin topical preparation according to the present invention includes the above-mentioned agent for maintaining stem cell properties of sweat gland myoepithelial cells. The skin topical preparation may contain, in addition to the agent for maintaining stem cell properties, ingredients that can be generally contained in skin topical preparations. Examples of such ingredients include oils, surfactants, moisturizing agents, powders, UV absorbers, fragrances, colorants, chelating agents, cooling agents, thickeners, vitamins, neutralizing agents, amino acids, whitening agents, anti-inflammatory agents, bactericides, deodorants, animal and plant extracts, and metal sequestering agents. The dosage form of the skin topical preparation may be liquid, gel, milky, creamy, or the like.

The present invention also includes a method for treating, improving, or preventing a disease associated with a defect or dysfunction of sweat glands, comprising the step of administering to a subject an agent for maintaining stemness of sweat gland myoepithelial cells or an external skin preparation. The subject may be a subject in need of treatment, improvement, or prevention of a disease associated with a defect or dysfunction of sweat glands. For example, the subject may be a subject suffering from the disease. The subject may be a human or a non-human mammal. Examples of diseases associated with a defect or dysfunction of sweat glands include hyperhidrosis, anhidrosis, heat stroke, and cholinergic urticaria. The present invention also includes a method for promoting sweating, comprising the step of administering to a subject an agent for maintaining stemness of sweat gland myoepithelial cells or an external skin preparation. The stemness maintaining agent or external skin preparation may be applied to the skin of the subject.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments.

One embodiment of the present invention is described below.

[Single cell RNA analysis]
Using single-cell RNA analysis, we identified a receptor that is specifically expressed in myoepithelial cells, which are stem cells in sweat glands. The specific procedure is as follows:

First, sweat glands were isolated from human skin tissue. Then, the sweat glands were individually digested using 0.5% Trypsin (Thermo Fisher Scientific, Cat. No. 15240062) and 5 mg/mL Dispase (Thermo Fisher Scientific, Cat. No. 17105041). This resulted in a sample containing multiple types of cells contained in the sweat glands. Using Chromium Single Cell 3' Reagent Kits (10x Genomics, Cat. No. 1000075), Chromium Single Cell 3' Feature Barcode Library Kit (10x Genomics, Cat. No. 1000079), and Chromium Single Cell B Chip Kit (10x Genomics, Cat. No. 1000154), a cDNA library was prepared from the RNA of individual cells contained in the sample according to the procedure described in the user guide. The library was sequenced using NovaSeq 6000 (Illumina). Using the obtained sequence data, analyses including normalization, feature selection, scaling, principal component analysis, and clustering were performed. The analyses were performed using a commonly standardized pipeline in Cell Ranger-v3.1.0 (manufactured by 10x Genomics). Based on the results of this analysis, receptors specifically expressed in myoepithelial cells were selected. Specifically, first, multiple genes that satisfied p<0.05 and log2 fold change>0.5 were found in the cell population contained in the sweat gland. Then, from these multiple genes, a gene for a receptor specifically expressed in myoepithelial cells was selected. Note that the above "p<0.05" means that the gene expression level is significantly higher than other genes at a risk of p<0.05. In addition, the above "log2 fold change" means the relative value of the gene expression level (log2-transformed fold change) with all cells other than the selected cell population as the control.

As a result of the selection, the corresponding gene was determined to be the LGR4 gene. Figure 1 shows the relative expression level (log2 fold change) of the LGR4 gene in representative cells contained in sweat glands. This figure also makes it clear that the LGR4 gene is a receptor gene that is specifically expressed in myoepithelial cells.

Based on these results, the inventors speculated that substances involved in LGR4 signal transduction may contribute to the maintenance of stemness in myoepithelial cells. Below, we investigated whether R-spondin1, which binds to LGR4, and Wnt3a, which binds to R-spondin1, may contribute to the maintenance of stemness in myoepithelial cells.

[Sweat gland spheroid culture]
A sample containing multiple types of cells contained in sweat glands was prepared from human skin tissue in the same manner as described above. A basal medium was prepared by adding the following components to MammoCult Human Medium (manufactured by STEMCELL Technologies). The numerical values written next to the names of each component indicate the concentration of each component in the completed basal medium.
Epidermal growth factor (EGF) (manufactured by PeproTech)...10ng/mL
Basic Fibroblast growth factor (bFGF) (manufactured by STEMCELL Technologies)...10 ng/mL
Heparin (STEMCELL Technologies)...4 μg/mL
Hydrocortisone 21-hemisuccinate (Sigma-Adrich, Cat. No. H2882-1) ... 0.5 μg/mL
BD Matrigel Matrix Growth Factor Reduced (BD Biosciences)...2% by volume
MammoCult proliferation supplements (manufactured by STEMCELL Technologies)...10% by volume
Antibiotic-Antimycotic...1% by volume
The above samples were seeded in the basal medium at a density of 7.5×10 ³ to 1.5×10 ⁴ cells/cm ^2. The basal medium was placed in an incubator set to an environment of 37°C and a carbon dioxide concentration of 5% by volume, and the cells contained in the above samples were cultured. The basal medium was replaced every two days, and sweat gland spheroids were collected on the 10th day of culture.

The basal medium used was a basal medium supplemented with the human recombinant protein Wnt3a, a basal medium supplemented with the human recombinant protein R-spondin1, and a control basal medium supplemented with no human recombinant protein. Here, the Wnt3a and R-spondin1 shown in Table 1 were used. The concentrations in Table 1 indicate the concentrations in the basal medium.

[Real-time PCR]
Total RNA was extracted from the collected sweat gland spheroids using TRI Reagent LS (Molecular Research Center). Chloroform was added to the obtained RNA extract, and the aqueous layer containing RNA was collected. Isopropanol was added to the collected aqueous layer to precipitate RNA. Next, the RNA was washed with 70% ethanol, and then the RNA was dissolved in RNase-free water to obtain an RNA sample. cDNA was synthesized from this RNA sample using reverse transcriptase QuantiTect Reverse Transcription Kit (QIAGEN). A PCR reaction solution was prepared using THUNDERBIRD SYBER qPCR Mix (Toyobo Co., Ltd., Cat. No. QPS-201). Analysis of gene expression levels was performed by quantitative real-time PCR. Quantitative real-time PCR was performed using a real-time PCR device (ViiA ^™ 7 Real Time PCR System) with 40 cycles of 95°C for 15 seconds, 55°C for 10 seconds, and 72°C for 30 seconds. In addition, the relative expression levels of aSMA, a myoepithelial cell marker, and KRT18, a luminal cell marker, were calculated. β-actin (ACTB) was used as an endogenous control to calculate the relative expression levels. The primers used for gene analysis are shown in Table 2.

Figure 2 shows the expression levels (relative values) of the aSMA gene and the KRT18 gene in control sweat gland spheroids or sweat gland spheroids cultured with Wnt3a or R-spondin1. As described above, aSMA is a myoepithelial cell marker, and KRT18 is a luminal cell marker. As shown in Figure 2, it was found that the expression level of the aSMA gene was maintained and the expression level of the KRT18 gene was decreased in sweat gland spheroids cultured with Wnt3a or R-spondin1. This shows that the stemness of myoepithelial cells can be maintained in 3D culture by using Wnt3a and R-spondin1, which are substances that enhance the signal transduction of LGR4, which is specifically expressed in myoepithelial cells.

This invention can be widely used in the fields of cosmetics and medicine.

Claims (5)

A stem cell maintenance agent for sweat gland myoepithelial cells that contains a substance that enhances LGR4 signaling. The agent for maintaining stemness of sweat gland myoepithelial cells according to claim 1, wherein the substance is at least one selected from the group consisting of Wnt3a and R-spondin1. A method for maintaining the stemness of sweat gland myoepithelial cells, comprising a step of contacting the agent for maintaining stemness of sweat gland myoepithelial cells according to claim 1 or 2 with sweat gland myoepithelial cells in vitro. An external skin preparation for treating, ameliorating or preventing a disease associated with a defect or dysfunction of sweat glands or for promoting sweating, comprising the agent for maintaining stemness of sweat gland myoepithelial cells according to claim 1 or 2. An external skin preparation for maintaining the stemness of sweat gland myoepithelial cells, comprising the agent for maintaining the stemness of sweat gland myoepithelial cells according to claim 1 or 2.

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