Cornulin expression promoter

Abstract

To elucidate the molecular mechanisms involved in photoaging and to provide a novel method for the prevention or mitigation of photoaging.SOLUTION: As a result of intensive studies, the inventors found that cornulin is a molecule involved in photoaging and that the amount of cornulin decreases with photoaging. Having obtained knowledge that cornulin has an effect of counteracting photoaging, they conducted extensive research on components capable of promoting the expression of cornulin. Surprisingly, it was found that the combination of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) and retinol (RO) promotes the expression of cornulin in the epidermis. Accordingly, the present disclosure provides: a cornulin expression promoter containing 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof or a salt of them, and retinol or a retinol derivative; a photoaging inhibitor, a skin barrier function improver and a rough-skin suppressor each containing the cornulin expression promoter; and a method for screening a photoaging inhibitor.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to the technical field of promoting cornulin expression. By promoting cornulin expression, effects such as suppressing photoaging, improving skin barrier function, and suppressing rough skin can be achieved. [Background technology]

[0002] Photoaging generally refers to changes in the appearance and function of skin that are observed as a result of repeated exposure to sunlight. Clinically, photoaging can be identified as rough skin, wrinkle formation, mottled pigmentation, sallowness, age spots, increased vulnerability to blemishes, atrophy, and other conditions. Photoaging typically occurs in skin habitually exposed to sunlight, such as the face, ears, head, neck, and hands, and significantly impairs the aesthetic appearance of the skin. Therefore, various research efforts have been conducted to inhibit photoaging. For example, it has been shown that inhibiting the production of matrix metalloproteinase 1 in the skin (Patent No. 7377005) is effective in preventing wrinkle formation, a type of photoaging. Further research into the inhibition of photoaging is needed, including the search for other substances associated with photoaging.

[0003] On the other hand, cornulin, which is also known as a differentiation marker for epidermal cells, is expressed during the process in which epidermal cells that divide at the basement membrane differentiate towards the stratum corneum, and is known to be strongly expressed in cells from the spinous layer to the granular layer. Cornulin is also known as a late differentiation marker factor for the epidermis. Cornulin is a member of the same S100 fused-type protein family as filaggrin, and is a Ca 2+ binds to Ca and contributes to differentiation 2+ is thought to be involved in the control of [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7377005 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to elucidate the molecular mechanism involved in photoaging and to provide a new method for preventing or ameliorating photoaging. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors discovered that cornulin is a molecule involved in photoaging and that the amount of cornulin decreases with photoaging. Having learned that cornulin has the effect of combating photoaging, the inventors conducted extensive research into components that can promote cornulin expression and surprisingly found that a combination of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) and retinol (RO) promotes cornulin expression in the epidermis. Therefore, the present invention relates to the following: [1] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, comprising the following: The following formula: [ka] (In the formula, n is an integer from 1 to 3, R 1 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH2- or -N(R 2 )-, R 2 is a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group. A cornulin expression promoter comprising a compound represented by the formula (I) and retinol or a retinol derivative. [2] The cornulin expression promoter according to item 1, wherein the retinol derivative is selected from the group consisting of retinol palmitate, retinol acetate, retinal, tretinoin, isotretinoin, retinoic acid, retinol propionate, retinol linoleate, and hydrogenated retinol. [3] A photoaging inhibitor comprising the cornulin expression promoter according to item 1 or 2. [4] A skin barrier function improving agent comprising the cornulin expression promoter according to item 1 or 2. [5] A rough skin inhibitor containing the cornulin expression promoter described in item 1 or 2. [6] A transdermal composition containing the cornulin expression promoter described in item 1 or 2. [7] The composition according to item 6, which is a cosmetic composition. [8] culturing a culture comprising keratinocytes in a medium containing a candidate drug; After culturing in a medium containing a candidate drug, measuring the expression of cornulin in the culture; and A step of comparing the measured expression level with a control to determine the photoaging inhibitory effect of the candidate drug. A method for screening a photoaging inhibitor, comprising: [9] The method according to Item 8, wherein the control is the expression level of cornulin in a culture containing keratinocytes cultured in a medium containing no candidate drug, or a threshold determined from the expression level.

[10] In a subject for whom suppression of photoaging is desired, 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, which is one of the following: formula: [ka] (In the formula, n is an integer from 1 to 3, R 1 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH2- or -N(R 2 )-, R 2 is a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group. A method for promoting cornulin expression, comprising applying a compound represented by the formula (I) and retinol or a retinol derivative.

[11] The cosmetic method according to Item 10, wherein the subject for whom inhibition of photoaging is desired is a subject selected from the group consisting of a subject with photoaging, a subject with reduced skin barrier function, and a subject with rough skin.

[12] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, for use in preventing or improving photoaging through the promotion of cornulin expression, comprising: The following formula: [ka] (In the formula, n is an integer from 1 to 3, R 1 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH2- or -N(R 2 )-, R 2 is a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group. and retinol or a retinol derivative.

[13] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, for use in improving skin barrier function through promotion of cornulin expression, the derivative comprising: The following formula: [ka] (In the formula, n is an integer from 1 to 3, R 1 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH2- or -N(R 2 )-, R 2 is a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group. and retinol or a retinol derivative.

[14] 1. A method for treating rough skin by promoting cornulin expression, comprising: The following formula: [ka] (In the formula, n is an integer from 1 to 3, R 1 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH2- or -N(R 2 )-, R 2 is a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group. and retinol or a retinol derivative.

[15] In the production of a cornulin expression promoter, 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, comprising the following: The following formula: [ka] (In the formula, n is an integer from 1 to 3, R 1 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH2- or -N(R 2 )-, R 2 is a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group. and retinol or a retinol derivative.

[16] In the manufacture of a photoaging inhibitor, a skin barrier function improver, or a skin roughness inhibitor, 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, comprising the following: The following formula: [ka] (In the formula, n is an integer from 1 to 3, R 1 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH2- or -N(R 2 )-, R 2 is a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group. and retinol or a retinol derivative. [Effects of the Invention]

[0007] The combination of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof with retinol or a retinol derivative promotes the expression of cornulin. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows immunohistochemical staining photographs showing changes in cornulin expression in non-exposed and exposed areas of young individuals in their 20s and 30s, and in elderly individuals in their 40s and older. [Figure 2] Figure 2 shows cornulin mRNA expression levels in the epidermis of a 3D cultured skin model when control, retinol (RO), 1-(2-hydroxyethyl)-2-imidazolidinone (HEI), or retinol and 1-(2-hydroxyethyl)-2-imidazolidinone (RO + HEI) were added. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention provides 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, which is as follows: The following formula: [ka] (In the formula, n is an integer from 1 to 3, R 1 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH2- or -N(R 2 )-, R 2 is a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group. and retinol or a retinol derivative.

[0010] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) alone failed to significantly promote cornulin gene expression (Figure 2). Retinol (RO) alone tended to increase cornulin expression, but the difference was not significant (Figure 2). The combination of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) and retinol (RO) significantly increased cornulin gene expression in the epidermis. While retinol alone tended to increase cornulin expression, the difference was not significant, and 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) did not affect cornulin expression. Therefore, the combined effect of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) and retinol (RO) is synergistic (Figure 2). Therefore, the combination of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) and retinol can be used as a cornulin expression promoter.

[0011] Cornulin expression is lower in sun-exposed skin compared to non-sun-exposed skin (Figure 1). Although there is a tendency for cornulin expression to decrease with aging in non-sun-exposed skin as well, the difference in expression is small (Figure 1). Cornulin can be said to be an epidermal differentiation marker that is significantly reduced by photoaging.

[0012] Cornulin is a protein that constitutes the cornified cell envelope, which lines the cell membrane of keratinocytes. Like other cornified cell envelope proteins, such as involucrin and loricrin, cornulin is a gene located on chromosome 1q21 and is expressed in the spinous and granular layers. Cornulin is used as an epidermal differentiation marker. High cornulin expression indicates proper differentiation of the epidermis into the stratum corneum, indicating healthy skin. Disruption of proper differentiation can lead to thickening of the epidermal layer and symptoms such as desquamation. Proteins that constitute the cornified cell envelope, along with keratin aggregated by the action of filaggrin, are involved in the skin's barrier function. Therefore, cornulin expression promoters have the effect of improving skin barrier function and can alleviate dryness and rough skin. Therefore, cornulin expression promoters may be related to skin barrier function improvers or rough skin inhibitors. Furthermore, cornulin expression promoters maintain the health of the epidermis, thereby improving or preventing photoaging. Therefore, the cornulin expression promoter can also be said to be a photoaging inhibitor.

[0013] Cornulin is also known as a late differentiation marker factor for the epidermis. Cornulin is a member of the same S100 fused-type protein family as filaggrin, and is a Ca 2+ binds to Ca and contributes to differentiation 2+ It is thought to be involved in the control of Ca 2+ It has been reported that the localization of Ca weakens with age (Applied Mathematics Vol. 27, No. 2, 18-24). The discovery that the amount of cornulin decreases with photoaging suggests that the localization of Ca 2+ The decrease in localization of Ca is thought to be the result of photoaging. 2+The localization of cornulin in the spinous to granular layers is important for the formation of skin barrier function, and may be involved in the formation of the cornified envelope through the activation of calcium-dependent enzymes, such as transglutaminase I (K) (Denda, M., Hosoi, J. and Ashida, Y., Visual imaging of ion distribution in human epidermis, Biochem. Biophys. Res. Commun., 272 (2000)). Therefore, cornulin is a Ca 2+ It is believed that cornulin expression promoters have an effect of combating photoaging through the control of cornulin expression, and therefore, cornulin expression promoters can also be called photoaging inhibitors.

[0014] A cornulin expression promoter is a component that, when applied, promotes cornulin expression in any cell, particularly epidermal cells. By promoting cornulin expression in epidermal cells, it contributes to the prevention of photoaging, improvement of skin barrier function, and prevention of rough skin.

[0015] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, has the following formula: [ka] (In the formula, n is an integer from 1 to 3, R 1 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH2- or -N(R 2 )-, R 2 is a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group. It is a compound represented by the formula:

[0016] Derivatives of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) include: These include, but are not limited to, 2-imidazolidinone, 1-methyl-2-imidazolidinone, 1,3-dimethyl-2-imidazolidinone, 1-(2-hydroxyethyl)-2-imidazolidinone, 1,3-bis-(2-hydroxyethyl)-2-imidazolidinone, and the like.

[0017] If the 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) derivative is a known substance, it can be easily synthesized by a known method or can be easily purchased as a commercial product. Even if it is a novel compound, a person skilled in the art can easily synthesize it by a known method.

[0018] 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof may be in the form of a salt. The type of salt is not particularly limited as long as it is pharmacologically acceptable, and may be an inorganic salt or an organic salt. Examples of inorganic salts include hydrochloride, sulfate, phosphate, hydrobromide, sodium salt, potassium salt, magnesium salt, calcium salt, magnesium salt, and ammonium salt. Examples of organic salts include acetate, lactate, maleate, fumarate, tartrate, methanesulfonate, p-toluenesulfonate, triethanolamine salt, and amino acid salt.

[0019] There are no particular limitations on the retinol or retinol derivatives, as long as they can be used in the fields of cosmetics, pharmaceuticals, quasi-drugs, etc. Examples of retinol derivatives include retinol palmitate, retinol acetate, retinal, tretinoin, isotretinoin, retinoic acid, retinol propionate, retinol linoleate, and hydrogenated retinol.

[0020] Combinations of 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, with retinol or a retinol derivative include, but are not limited to, HEI and retinol, HEI and retinol palmitate, and HEI and retinol acetate.

[0021] The route of administration of the cornulin expression promoter according to the present disclosure may be any route, including oral, transdermal, subcutaneous, intramuscular, intravenous, intraarterial, oral, and transmucosal administration. From the viewpoint of inhibiting photoaging, improving skin barrier function, and inhibiting rough skin, transdermal administration is preferred.

[0022] The cornulin expression promoter may be incorporated into a composition. The composition may be a transdermal composition. The transdermal composition is not particularly limited as long as it is applicable to the skin, and any dosage form may be used, such as a solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, or stick. In addition to 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, the transdermal composition may contain bases and excipients commonly used in transdermal compositions, such as preservatives, emulsifiers, and pH adjusters.

[0023] The composition may be a cosmetic composition, and when incorporated into a cosmetic composition, it may be incorporated into face or body cosmetics such as lotions, emulsions, serums, creams, lotions, packs, essences, gels, etc., makeup cosmetics such as foundations, makeup bases, concealers, etc., or even bath additives.

[0024] The composition may be a pharmaceutical composition. When formulated into a pharmaceutical composition, it can be formulated in a pharmaceutically acceptable carrier or the like.

[0025] The concentration of the cornulin expression promoter of the present disclosure can be selected arbitrarily based on the desired effect, such as suppressing photoaging, improving skin barrier function, and suppressing rough skin. When formulated as a transdermal composition, the cornulin expression promoter of the present disclosure can be formulated at 0.01% to 5% for HEI or a derivative thereof, and at 0.0001% to 1% for retinol or a derivative thereof. To fully exert its effects, HEI or a derivative thereof can be formulated at a concentration of preferably 0.01% or more, more preferably 1% or more, and retinol or a derivative thereof can be formulated at a concentration of 0.002% or more, even more preferably 0.01% or more. In terms of skin irritation, HEI or a derivative thereof can be formulated at a concentration of preferably 3% or less, even more preferably 2% or less, and retinol or a derivative thereof can be formulated at a concentration of 0.5% or less, even more preferably 0.3% or less.

[0026] The HEI or a derivative thereof and retinol or a derivative thereof can be blended at a blending ratio of 100:1 to 20:1. From the viewpoint of fully exerting the effect, they are preferably blended at a blending ratio of 35:1.

[0027] The cornulin expression promoter of the present disclosure can be applied to the skin and used in cosmetic methods to inhibit photoaging, improve skin barrier function, and prevent rough skin. The method of the present disclosure does not include medical procedures, i.e., surgery, treatment, or diagnosis performed on humans by a doctor or someone acting under his / her instructions, and can therefore be considered a non-medical procedure. The method and dosage of the transdermal composition of the present disclosure in such cosmetic methods are not particularly limited. Typically, an appropriate amount, e.g., 0.5 ml to 2 ml, can be directly rubbed into the skin of the entire face several times a day, for example, once or twice, or an appropriate amount can be soaked in gauze or the like and then applied to the skin.

[0028] The present disclosure also relates to a method for screening for a photoaging inhibitor using the expression of cornulin according to the present disclosure in keratinocytes as an index. More specifically, the screening method of the present disclosure includes the following steps: culturing a culture comprising keratinocytes in a medium containing a candidate drug; After culturing in a medium containing a candidate drug, measuring the expression of cornulin in the culture; and A step of comparing the measured expression level with a control to determine the photoaging inhibitory effect of the candidate drug. By carrying out this screening method, a combination of retinol and HEI is screened as a photoaging inhibitor.

[0029] The culture containing keratinocytes may be a culture of established keratinocytes, a culture of keratinocytes isolated from skin, an organ culture, or a three-dimensional culture. The three-dimensional culture may be a three-dimensional cultured skin model with dermis.

[0030] The control cornulin expression can be the expression of cornulin in keratinocytes cultured in a different process except that it does not contain the candidate component. A threshold may be set based on the cornulin expression in a control group obtained by a prior experiment, or the culture and measurement process may be carried out in parallel. Cornulin expression can be the amount of cornulin protein or mRNA in cultured keratinocytes, and can be measured using methods well known in the art, such as immunological techniques or quantitative PCR.

[0031] The candidate components used in the screening method of the present disclosure can be any library of cosmetic materials, food materials, pharmaceutical materials, etc. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or may be synthesized compounds and prepared extracts.

[0032] All documents mentioned herein are incorporated by reference in their entirety.

[0033] The examples of the present disclosure described below are for illustrative purposes only and do not limit the technical scope of the present disclosure. The technical scope of the present disclosure is limited only by the claims. The present disclosure may be modified, for example, by adding, deleting, or substituting components of the present disclosure, provided that the modifications do not depart from the spirit of the present disclosure. [Example]

[0034] Example 1: Immunohistochemical staining Skin tissue collected from the buttocks or corners of the eyes of individuals in their 20s to 80s was fixed using the AMeX fixation method, embedded in paraffin, and then sectioned at 4 μm. Immunostaining was performed using a rabbit polyclonal anti-cornulin antibody (Abcam, ab224122). Visualization was performed using a fluorescently labeled secondary antibody, Alexa 594 anti-rabbit IgG (Lifetechnologies), and the sections were mounted using Vectasheeld with DAPI (VECTOR) and observed under a fluorescence microscope (BX51, Evident). The results are shown in Figure 1.

[0035] Example 2: Cultivation of a 3D skin model A 3D cultured skin model with dermis (EFT-400, Mattek Life Science) was cultured for two days with or without 0.01% 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) in the medium. After that, the model was cultured for another two days with or without 10 μM retinol (RO) (Sigma Aldorich, R7632) in the medium, continuing the HEI-containing conditions. To create the following groups, a control group was cultured without any addition to the medium during the culture period; an HEI group containing only HEI; an RO group containing only RO; and an HEI+RO group containing both HEI and RO.

[0036] Example 3: Measurement of cornulin mRNA by quantitative real-time RT-PCR After culturing the skin model under the above conditions for a total of 4 days, the epidermis and dermis were separated with tweezers, immersed in Trizol reagent (Invitrogen), and disrupted using zirconia balls and a TissueLyser (Qiagen). RNA was extracted from the disrupted samples according to the Trizol reagent manual. After quality inspection using TapeStation, RNA sequencing analysis was performed using SMART-Seq v4 Ultra Low Input RNA and NovaSeq 6000. Expression levels of known genes were compared, and it was found that the combination of retinol and HEI showed specific changes in cornulin gene expression. Furthermore, the extracted RNA was reverse transcribed using SuperScript VILO (Thermo Fisher Scientific). The reverse-transcribed nucleic acid was subjected to quantitative PCR using the following primer set, Platinum® SYBR® green qRCR SuperMix-UDG with ROX (Thermo Fisher Scientific), and StepOne® plus (Applied Biosystems). Cornulin expression levels in the epidermis were quantified relative to the expression level of MRPL19 as an internal standard gene, and the expression levels of each group were compared. The results are shown in Figure 2. The primer combinations listed in Table 1 below were used. [Table 1]

Claims

1. 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) or a derivative thereof, or a salt thereof, which is The following formula: 【Chemical 1】 (In the formula, n is an integer from 1 to 3, R １ represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X is -CH ２ - or -N(R ２ )-, R ２ is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydroxyl group. A cornulin expression promoter comprising a compound represented by the formula (I) and retinol or a retinol derivative.

2. The cornulin expression promoter according to claim 1, wherein the retinol derivative is selected from the group consisting of retinol palmitate, retinol acetate, retinal, tretinoin, isotretinoin, retinoic acid, retinol propionate, retinol linoleate, and hydrogenated retinol.

3. A photoaging inhibitor comprising the cornulin expression promoter according to claim 1 or 2.

4. A skin barrier function improving agent comprising the cornulin expression promoter according to claim 1 or 2.

5. A rough skin inhibitor comprising the cornulin expression promoter according to claim 1 or 2.

6. A transdermal composition containing the cornulin expression promoter according to claim 1 or 2.

7. The composition of claim 6, which is a cosmetic composition.

8. culturing a culture comprising keratinocytes in a medium containing a candidate agent; After culturing in a medium containing a candidate drug, measuring the expression of cornulin in the culture; and A step of comparing the measured expression level with a control to determine the photoaging inhibitory effect of the candidate drug. A method for screening a photoaging inhibitor, comprising:

9. The method according to claim 8, wherein the control is the expression level of cornulin in a culture containing keratinocytes cultured in a medium containing no candidate drug, or a threshold determined from the expression level.

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