Methods of determining moisture content of skin

Abstract

To provide new biomarkers that are specifically associated with skin dryness.SOLUTION: Biomarkers, namely extended synaptotagmin-3 (ESYT3), ubiquinin-carboxyl terminal hydrolase 7 (USP7), periplakine (PPL) and late cornified envelope protein 1C (LCE1C), are provided to enable accurate evaluation of moisture content of the skin, in particular, diagnosis of dry skin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for determining the moisture content of skin based on the expression levels of certain biomarkers. [Background technology]

[0002] Skin consists of three main layers: the epidermis, the dermis, and the subcutaneous tissue. The epidermis, the outer layer of skin, is made up of keratinocytes (the majority), melanocytes (responsible for skin pigmentation), and Langerhans cells. Its function is to protect the body from the external environment and ensure its integrity, in particular to slow the invasion of microorganisms or chemicals, and to prevent evaporation of the water contained in the skin.

[0003] For this purpose, keratinocytes undergo a continuous, directed maturation process in which keratinocytes located in the basal layer of the epidermis form, at the final stage of their differentiation, fully cornified dead cells in the form of a cornified envelope composed of proteins and lipids such as ceramides. This top layer of the epidermis is called the stratum corneum and is an essential component of the epidermal barrier function.

[0004] However, the epidermal barrier function can be disrupted under certain climatic conditions (e.g., under the influence of cold and / or wind), or under the influence of stress, fatigue, or aging. The breakdown of the skin barrier function facilitates the penetration of allergens, irritants, or microorganisms, and directly leads to dry skin, which is often accompanied by little discomfort such as tightness or redness.

[0005] To prevent or correct this phenomenon, it is known practice to apply to the skin cosmetic compositions containing hygroscopic agents such as sugars or polyols intended to capture the moisture present on the skin and thereby slow its evaporation.

[0006] Several moisturizing compounds are known and available. However, these compounds work through different mechanisms of action and are therefore suited to different skin types (dry skin, sensitive skin, combination skin, etc.). Unfortunately, most people do not know their skin type and therefore use inappropriate products.

[0007] Skin dryness is usually assessed clinically, visually, or by using a stratum corneum moisture analyzer, which is designed to measure the dielectric constant of the upper layers of the skin. Unfortunately, both of these methods face significant limitations. Visual assessment of the skin depends on the expert performing the assessment and can vary from one assessor to another. Similarly, assessments using a stratum corneum moisture analyzer are also limited in terms of reproducibility; the results are closely related to the applied pressure and the atmospheric conditions under which the measurements are performed.

[0008] Therefore, new assessment methods are needed that allow for the correct determination of skin type. In particular, it would be advantageous to identify specific markers associated with dry skin and to indicate potential targets for preventing / reversing the skin dryness process. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Khazaka G., Bioengineering of the skin: water and stratum corneum (2005): 249 [Non-patent document 2] Mohamad M. et al. Journal of Physics: Conference Series. Vol. 546. No. 1. IOP Publishing, 2014 [Non-patent document 3] Michaels, Barry. (2002). Chapter 26 - Skin Sampling Techniques: Handbook of Topical Antimicrobials and Their Applications [Non-patent document 4] Hughes, AJ, et al. British Journal of Dermatology 185.1 (2021): 26-35 [Non-patent document 5] Brohem et al. Pigment cell & melanoma research 24.1 (2011): 35-50 [Non-patent document 6] Yun et al. Journal of Pharmaceutical Investigation 48.2 (2018): 215-223 Summary of the Invention

[0010] The invention is defined by the claims.

[0011] Using tape-stripped stratum corneum samples, the inventors identified highly specific biomarkers associated with the dry skin phenotype. The inventors further demonstrated that these specific biomarkers can actually be directly targeted to prevent, limit, or even reverse the skin dryness process. Therefore, these biomarkers are highly promising tools for assessing and treating dry skin. Furthermore, these biomarkers can also be used to identify interesting candidate cosmetic compounds for hydrating the skin and evaluate their effectiveness. The biomarkers identified in the context of the present invention are extended synaptotagmin-3 (ESYT3), ubiquinin carboxyl-terminal hydrolase 7 (USP7), periplakin (PPL), and late cornified envelope protein 1C (LCE1C).

[0012] Therefore, a first aspect of the present invention relates to a method for determining the moisture content of a subject's skin, said method comprising measuring the expression level of at least one protein selected from the group consisting of ESYT3, USP7, PPL and LCE1C in a skin sample obtained from said subject. Such a method particularly allows for the detection of dry skin and therefore allows for the appropriate treatment of such conditions.

[0013] According to a second aspect, the present invention also relates to a method for assessing the effectiveness of a cosmetic treatment for hydrating the skin of a subject, said method comprising the steps of: a) measuring the expression level of at least one protein selected from the group consisting of ESYT3, USP7, PPL and LCE1C in a skin sample obtained from said subject before undergoing said cosmetic treatment; b) measuring the expression level of said at least one protein in a skin sample obtained from said subject after said cosmetic treatment; c) comparing the expression levels measured in steps a) and b); and d) determining that the cosmetic treatment is efficient if the expression level measured in step b) is lower than the expression level measured in step a), or determining that the treatment is inefficient if the expression level measured in step b) is equal to or higher than the expression level measured in step a).

[0014] Finally, the present invention further relates to a method for screening candidate cosmetic compounds for hydrating the skin, said method comprising the steps of: a) measuring the expression level of at least one protein selected from the group consisting of ESYT3, USP7, PPL and LCE1C in a skin sample; b) contacting the skin sample with a test compound; c) measuring the expression level of said at least one protein in said skin sample; and d) selecting the compound if the expression level measured in step c) is lower than the expression level measured in step a). Includes. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates to the identification of novel biomarkers specifically associated with dry skin. These biomarkers, extended synaptotagmin-3 (ESYT3), ubiquinin carboxyl-terminal hydrolase 7 (USP7), periplakin (PPL), and late cornified envelope protein 1C (LCE1C), enable accurate assessment of skin moisture content and, in particular, the diagnosis of dry skin. The inventors have specifically demonstrated that these biomarkers are significantly overexpressed in dry skin compared to normal skin, and that their expression levels are directly associated with skin dryness. The inventors have further demonstrated that moisturizing treatments have a direct effect on the expression levels of the biomarkers according to the present invention, thus demonstrating that they are highly promising tools for the treatment of dry skin and the identification of novel moisturizers.

[0016] The results disclosed herein are: - the expression levels of ESYT3, USP7, PPL and LCE1C in a skin sample can be used to assess whether the skin is hydrated; - that these biomarkers are directly involved in the process of dry skin and therefore may be suitable targets for preventing / treating dry skin; and - The expression levels of ESYT3, USP7, PPL and LCE1C can be used as markers to identify interesting cosmetic compounds for hydrating the skin. Shows.

[0017] Therefore, a first aspect of the present invention relates to a method for determining the moisture content of skin, and in particular a method for diagnosing dry skin in a subject, said method comprising measuring the expression level of at least one protein selected from the group consisting of ESYT3, USP7, PPL and LCE1C in a skin sample obtained from said subject. Such a method in particular allows for the detection of dry skin and therefore allows for the appropriate treatment of such conditions.

[0018] ESYT3, USP7, PPL and LCE1C are known proteins with which those skilled in the art are thoroughly familiar.

[0019] Extended synaptotagmin-3 (ESYT3) is a member of the extended synaptotagmin (Esyt) family, which includes multidomain C2 membrane proteins whose orthologs are found in organisms ranging from yeast to humans. There are three Esyt genes in mice and humans. These genes are involved in the formation of junctions between the endoplasmic reticulum and the plasma membrane, as well as Ca transport. 2+ It is known to be involved in the maintenance of membrane phospholipids in a phospholipid-dependent manner. ESYT3 binds to glycerophospholipids through its barrel-like domain and may be involved in intracellular lipid transport. The sequence of human ESYT3 is accessible in the Uniprot database entry A0FGR9.

[0020] Ubikinin carboxyl-terminal hydrolase 7 (USP7) has been shown to regulate the renewal of many signaling molecules, including p53 and PTEN. (EMBO Rep. 2020 May 6; 21(5)) showed that deletion of USP7 in keratinocytes results in increased ubiquitination of IKKα (IκB kinase α), which in turn leads to decreased levels of IKKα during cell differentiation. Skin transplanted with USP7 KO cells exhibits significant epidermal abnormalities, including epidermal thickening and basal cell expansion. Deletion of USP7 in epidermal keratinocytes results in decreased IKKα levels and abnormal differentiation. The sequence of human USP7 is available in the Uniprot database under entry Q93009.

[0021] Periplakin (PPL) is a component of the keratinocyte cornified envelope. The cornified envelope is assembled from a series of precursor proteins that are cross-linked under the action of transglutaminase. The resulting structure is an insoluble layer of proteins covalently bound to lipids, approximately 15 nm thick, deposited on the inner surface of the keratinocyte plasma membrane. Members of the plakin protein family, to which periplakin belongs, function as cell-cell junctions in the epidermis and as components of cell-cell and cell-matrix adhesion complexes, namely desmosomes and hemidesmosomes, respectively. The sequence of human PPL is available in the Uniprot database entry O60437.

[0022] Late cornified envelope protein 1C (LCE1C) is a precursor of the cornified envelope of the stratum corneum. LCE1C belongs to the late cornified envelope (LCE) family, whose members are integrated into the cornified envelope by transglutaminase-mediated cross-linking during the process of envelope maturation. The sequence of human LCE1C is available in the Uniprot database entry Q5T751.

[0023] In the context of the present invention, the expression level of at least one of these biomarkers is measured.The expression level of one of these biomarkers is sufficient to determine the moisture content of skin.Nevertheless, the method according to the present invention can comprise measuring the expression levels of one, two, three or all of these four biomarkers.

[0024] Those skilled in the art are familiar with the many techniques routinely used to determine the expression level of a protein.

[0025] The expression level of a protein can be assessed by measuring the expression level of the gene encoding it. The expression level of a gene is typically determined by analyzing the mRNA(s) transcribed from that gene. These nucleic acid molecules are typically extracted from a biological sample obtained from a subject and can be analyzed by standard methods. One commonly used method is to subject isolated mRNA to reverse transcription ("RT") and polymerase chain reaction ("PCR") amplification using oligonucleotide primers specific to the gene of interest. Quantification of mRNA can typically be performed using one of two real-time quantification technologies called SYBR Green® or TaqMan®.

[0026] Alternatively, the expression level of a protein can be directly assessed by measuring the amount of the protein detected in a sample. Such methods typically involve contacting the biological sample to be analyzed with an agent capable of specifically binding the target protein. This agent is usually a polyclonal or monoclonal antibody. The presence of the protein is then detected by standard immunodetection methods, typically by separating the proteins by electrophoresis (a technique also known as "Western blotting") or by immunoassays using direct, indirect, competitive, or immunocapture methods (a technique also known as "ELISA"). The formation of a complex between the protein of interest and an antibody(ies) targeting the protein is typically detected and quantified by measuring an enzymatic reaction that generates a colored, chemiluminescent, or fluorescent product.

[0027] "Skin moisture content," as used herein, refers to the amount of moisture contained in the skin, and particularly in the stratum corneum. Our skin is composed of 70% water, distributed in a gradient from 70% deep down to 15% at the surface. This natural phenomenon, known as cutaneous sweating, is made possible by a good skin barrier that allows moisture retention within the skin. A healthy skin barrier is based on a delicate balance between water (15%) and lipids (10-15%). A healthy, normal stratum corneum contains approximately 15-20% water. Under these normal conditions, skin is supple and soft. Skin moisture content allows classification according to its moisture content. Generally, skin is classified into four groups: well-moisturized skin, normal / moisturized skin, dry skin, and very dry skin. Dry skin is defined as skin with a moisture content of less than 15%, especially less than 10%, in the stratum corneum, while very dry skin is defined as skin with a moisture content of 5% or less. Similarly, normal / moist skin has a moisture content of the stratum corneum between 15 and 20%, while fully moist skin has a moisture content of over 20%. Dry skin is defined by an imbalance of the above, particularly a lack of lipids and a disruption of the physical barrier, which leads to moisture loss and a decrease in cellular maturity. This permanent skin condition is characterized by tightness, redness, and scaling. Moisturizing compounds generally aim to compensate for this lack of surface lipids with ceramides or urea, reducing transepidermal water loss.

[0028] Skin moisture content is the standard that dermatologists and skin specialists routinely evaluate.Currently, several techniques are used to determine skin moisture content.These techniques include, for example, using a stratum corneum moisture meter, visual assessment or near-infrared (NIR) spectroscopy (for review, see, for example, Khazaka G., Bioengineering of the skin: water and stratum corneum (2005): 249; or Mohamad M. et al. Journal of Physics: Conference Series. Vol. 546. No. 1. IOP Publishing, 2014).

[0029] The method according to the invention makes it possible to determine the moisture content of the skin. Thus, in the context of the present invention, the method advantageously comprises a step of classifying the skin as very dry, dry, normal / moisturized or well-moisturized based on the expression level of the biomarker.

[0030] As mentioned above, the present inventors have found that the biomarkers according to the present invention are overexpressed in dry skin. The present inventors have particularly shown that the higher the expression level of the biomarkers in a skin sample, the drier the skin. Therefore, the method according to the present invention particularly enables the diagnosis, i.e., detection, of dry skin (including very dry skin). Thus, according to a particular embodiment, the method according to the present invention comprises determining that the higher the expression level of the at least one protein, the drier the subject's skin.

[0031] Advantageously, the expression level of the biomarker according to the present invention can be compared with a reference value. This reference value is determined for each biomarker. It can be determined experimentally, empirically, or theoretically and is set to obtain optimal specificity and selectivity. This reference value can be determined based on the results obtained with a control population, for example, normal skin / moist skin or dry skin. An expression level equivalent to the threshold determined based on normal skin / moist skin will be associated with a normal skin profile, while an expression level significantly different from this value, and in particular an expression level higher than this reference value, will be associated with a dry skin profile. Conversely, an expression level equivalent to the threshold determined based on dry skin will be associated with a dry skin profile, while an expression level significantly different, in particular an expression level lower than such a reference value, will be associated with a normal skin / moist skin profile.

[0032] According to a preferred embodiment, the reference value is determined based on the expression level of the biomarker according to the present invention measured in normal / moist skin. According to such an embodiment, the present invention relates to a method for determining the moisture content of the skin of a subject, said method comprising the following steps: a) measuring the expression level of at least one protein selected from the group consisting of ESYT3, USP7, PPL and LCE1C in a skin sample obtained from the subject; b) comparing the expression level of said at least one protein with a reference value determined in normal / moist skin; and c) concluding that the subject's skin is dry or very dry if the expression level of said at least one protein is higher than the reference value. Includes.

[0033] The "skin sample" according to the present invention can be any skin sample in which the expression level of the protein according to the present invention can be detected and measured. Typically, the skin sample according to the present invention is a stratum corneum sample. Stratum corneum samples can be obtained by various techniques, including biopsy, skin scraping, or tape stripping (for a review, see Michaels, Barry. (2002). Chapter 26 - Skin Sampling Techniques: Handbook of Topical Antimicrobials and Their Applications). The sample according to the present invention is preferably obtained by using a non-invasive technique. According to a preferred embodiment, the stratum corneum sample is a tape-stripped stratum corneum sample. Typically, adhesive tape is pressed against the surface of the skin and then suddenly removed. This technique is routinely used in the field of dermatological research (for a review, see Hughes, AJ, et al. British Journal of Dermatology 185.1 (2021): 26-35).

[0034] A "subject" according to the present invention is a mammal, preferably a human.

[0035] According to a specific embodiment, the method of the present invention further comprises measuring the expression level of interleukin-1 receptor antagonist protein (IL1RN). According to this specific embodiment, the method of the present invention comprises measuring the expression level of one, two, three, or four proteins selected from ESYT3, USP7, PPL, and LCE1C, and further comprises measuring the expression level of IL1RN. IL1RN is one of the inhibitory cytokines of the IL-1 family. It is expressed in keratinocytes and has anti-inflammatory effects. The sequence of human IL1RN is available in the Uniprot database entry P18510. The inventors have shown that the expression level of IL1RN also correlates with dry / very dry skin.

[0036] As described throughout this specification, the expression level of the protein according to the present invention is related to the moisture content of skin.Therefore, these proteins are good markers for evaluating the effect of compound on the moisture content of skin, and particularly for evaluating whether compound can improve the moisture content of skin.Therefore, the expression level of these proteins can be used to evaluate whether moisturizing treatment is effective, or alternatively, to screen candidate cosmetic compounds.

[0037] Therefore, according to a further embodiment, the present invention also relates to a method for assessing the effectiveness of a cosmetic treatment for hydrating the skin of a subject, said method comprising the following steps: a) measuring the expression level of at least one protein selected from the group consisting of extended synaptotagmin-3 (ESYT3), ubiquinone-carboxyl terminal hydrolase 7 (USP7), periplakin (PPL) and late cornified envelope protein 1C (LCE1C) in a skin sample obtained from the subject before the cosmetic treatment; b) measuring the expression level of said at least one protein in a skin sample obtained from said subject after said cosmetic treatment; c) comparing the expression levels measured in steps a) and b); and d) determining that the cosmetic treatment is efficient if the expression level measured in step b) is lower than the expression level measured in step a), or that the treatment is inefficient if the expression level measured in step b) is equal to or higher than the expression level measured in step a). Includes.

[0038] The present invention also relates to a method for screening candidate cosmetic compounds for hydrating the skin, said method comprising the steps of: a) measuring the expression level of at least one protein selected from the group consisting of extended synaptotagmin-3 (ESYT3), ubiquinin carboxyl-terminal hydrolase 7 (USP7), periplakin (PPL) and late cornified envelope protein 1C (LCE1C) in a skin sample; b) contacting a test compound with said skin sample; c) measuring the expression level of said at least one protein in said skin sample; and d) selecting the compound if the expression level measured in step c) is lower than the expression level measured in step a). Includes.

[0039] The candidate cosmetic compound can be of any type, it can be of natural origin or produced by chemical synthesis, it can include libraries of structurally defined chemical compounds, unidentified compounds or substances, or mixtures of compounds.

[0040] Natural compounds include compounds of botanical origin, such as plants. Preferably, the candidate cosmetic compounds are botanical, and preferably selected from plant extracts.

[0041] According to a preferred embodiment, the skin sample used in the context of the screening method according to the present invention is an artificial skin sample. Artificial skin is mainly used in the field of cosmetic research (see, for example, Brohem et al. Pigment cell & melanoma research 24.1 (2011): 35-50; or Yun et al. Journal of Pharmaceutical Investigation 48.2 (2018): 215-223) and is easily available from specialized manufacturers.

[0042] The present disclosure also provides a method for treating dry skin, said method comprising treating skin identified as dry by the method of the present invention with an effective amount of a hydrating compound.

[0043] The present invention is further illustrated by the following non-limiting examples. [Example]

[0044] [Example 1] Identifying biomarkers of dry skin: The study was conducted on 37 women aged 35–40 years with normal (17) or severely dry (20) leg skin. The women's skin condition was classified using different parameters: visually under the supervision of a dermatologist, using a dry skin grade scale, by TWEL (Telescopic Field of Effect) and capacitance measurements, and by sampling continuous tape strips. Staining of the tape strips allows for classification of different grades of dry skin based on the distribution, number, and color intensity of keratinocytes on the tape strips. Twelve tape strips were dissolved and subjected to proteomic identification and quantification. This proteomic characterization allowed for the identification of four novel biomarkers preferentially expressed on the surface of dry skin.

[0045] 1- Materials and Methods Cohort Description: The purpose of this study was to identify targets specifically related to dry skin. To identify these targets, a cohort of women with normal versus severely dry skin was selected. Thirty-seven volunteers, aged 35-40 years, ranging from phototype 1 to 3, were recruited and divided into two subpopulations: 17 women with normal skin and 20 women with visibly severely dry skin. This distribution was determined by dermatologists assessing the sampled area, the anterior legs, in relation to visual appearance on the day of the study. Photographic confirmation was correlated with biophysical measurements such as TWEL and capacitance.

[0046] Description of sampling method: Tape stripping is a non-invasive method that allows the removal of the first layer of the stratum corneum. The first tape strip is placed on the front of the leg, and then a standardized weight is applied with constant and repeatable pressure. The weight is applied for 10 seconds and then lifted. The tape strip is outlined, and the next tape strip is applied in the same position. The tape strip is then removed in one swoop with a clip, with a steady hand gesture. The first tape strip is discarded to standardize the amount of material collected next. The next tape strip is then applied and removed in the same manner as the first tape strip. The tape strips are then stored at -80°C. A total of 15 tape strips from three separate areas were sampled. 12 of these were used for protein extraction and one for morphological staining.

[0047] Staining of tape strips: Tape strips were stained to highlight the distribution and morphology of keratinocytes in each volunteer. Tape strips were thawed at room temperature and placed on a hot plate at 37°C before being covered with a mixture of Stain PMS: basic fuchsin and toluidine blue for 1 hour. The stained tape strips were then rinsed in successive tap water baths, allowed to dry in open air, and then placed between slides and lamellae for further analysis.

[0048] Image analysis: Slide scans were performed using a ScanScope® AT Turbo slide scanner at x40 magnification for a resolution of 0.25 μm / px. Based on color deconvolution, two algorithms were developed for: 1) The tissue surface positive for staining is measured and the surface of the positive keratinocytes is classified according to three classes of colorimetric intensity. The algorithm applies a colorimetric intensity gradient to measure the positive tissue surface. This is sufficient to classify the surface of the keratinocytes into three groups: weak, medium, and strong keratinocyte labeling. Finally, the algorithm determines a normalized mean optical density value for each tape strip, which corresponds to the mean intensity of the keratinocytes. The thresholds and parameters used for measuring the surface of the keratinocytes and classifying them by color gradient are the same for each tape strip. 2) Count the islets formed by keratinocytes and measure their area. First, the analysis area and possible exclusion areas are determined. Next, color deconvolution is applied to extract positive tissue from the rest of the image, freeing it from artifacts that could alter the analysis of the image. Finally, an algorithm segments and individualizes the islets, allowing their number to be counted and their area to be measured. The ratio between the total analyzed area and the area occupied by the islets allows the islet density per tape strip to be quantified as a percentage.

[0049] Protein extraction: Each tape was placed sticky-side up in its own 20 mL borosilicate scintillation vial (Wheaton; VWR). The tape was immersed in PBS buffer containing SDS (0.2%), propylene glycol (0.5%), and HALT antiprotease cocktail (1X). The vial was placed in a sonication bath for 60 minutes. Cell lysates were collected from each vial and pooled according to donor number. Proteins were precipitated overnight in 90% methanol at -20°C. The precipitated proteins were collected by centrifugation (15,000 g, 20 minutes, 4°C) and dissolved in Tris-HCl buffer containing urea (8 M). Protein concentration was determined using the BCA method.

[0050] Protein purification and concentration: Peptide extracts were prepared using the FASP (Filter-Aided Sample Preparation) method. 50 μg of protein was loaded onto an ultrafiltration device (Amicon Ultra 0.5, 10 kDa MWCO) and then subjected to reduction (dithiothreitol), alkylation (iodoacetamide), and trypsin digestion. Peptides were purified by SPE chromatography (C18), dried, and solubilized in 100 μl of 0.1% formic acid. Peptide concentrations were determined using the BCA method.

[0051] Protein analysis LC-MS / MS <<Shotgun Proteomics>>: When possible, 250 ng of peptides were injected in triplicate for each sample. For samples with unknown peptide concentrations, 10 μl of undiluted sample was used. Chromatography was performed using an Ultimate3000 (Dionex) instrument with a C18 (75 μm x 50 cm, 2 μm material) column, followed by a 3-minute precolumn trapping step followed by a 60-minute gradient of 2.5% to 35% acetonitrile at a flow rate of 300 nl / min. Data were acquired using a Q-Exactive Plus (Thermo) mass spectrometer. MS scans were performed at a resolution of 70,000 and an accumulation time of 60 ms. MS / MS scans were performed on the 10 most intense ions of each cycle at a resolution of 17,500 and an accumulation time of 60 ms. 5,600 cycles were performed, resulting in an average of 14 cycles per chromatographic peak. The following experimental settings were applied: power supply voltage (2300 V), sweep gas (0 psi), transfer tube temperature (275 °C), normalized collision energy (28 * ( * The collision energy is determined automatically depending on the mass and charge of the analyte compound).

[0052] Protein Identification: Proteins were identified using the SEQUEST-HT algorithm and a database compiled from the Human Reference Proteome mined from UNIPROT. Search parameters were: enzyme = trypsin (full); allowed iscleavage = 2; precursor error tolerance = 10 ppm; fragment error tolerance = 0.02 Da; dynamic modifications = oxidation (M), deamidation (N / Q); protein terminal modification = acetylation; static modification = carbamidomethyl (C). False discovery rate (FDR) was assessed using the Percolator algorithm.

[0053] Protein quantification: Data were processed using Minora and the feature mapper in Proteome Discoverer 2.2 software. Peak integration parameters were as follows: post-acquisition recalibration = True (fine parameters); minimum trace length = 5; minimum number of isotopes = 2; maximum delta RT for isotopes = 0.2 min for integration; PSM confidence level = high. Chromatographic alignment parameters were as follows: RT alignment = TRUE; parameter adjustment = fine; maximum RT shift = 5 min; mass tolerance = 10 ppm. Feature mapping parameters were as follows: RT tolerance = auto; mass tolerance = auto; S / N threshold = 2. Statistical analysis was performed using the Precursors Ions quantifier node in Proteome Discoverer 2.2 software. General Quantification Settings were as follows: Peptide to use = Unique + RAZOR (Unique = peptide not shared by different proteins or protein groups; RAZOR = peptide shared by multiple protein groups but used only for quantification of proteins with the highest number of unique peptides and the longest amino acid sequence); Consider Proteins Groups for Peptide Uniqueness = True; Reject Quan Results with Missing Channels = False. Precursor Quantification Settings were as follows: Precursor Abundance Based on Area; Min number Replicate feature = 50% (peptides used for quantification must be detected in at least 50% of the samples in a group). Normalization Settings: Total Peptide Abundance (for all identified peptides, the sum of the abundance values ​​for each injection is calculated, and the injection with the highest total abundance is used as the reference, and the abundance values ​​of all other injections are corrected by a constant factor for each injection, so that the final total abundance is the same for all injections).Quan Rollup Hypothesis Testing settings: Ratio Calculation = Summed abundance based (Protein Ratio is calculated from the median of the sum of sample abundances of the replicates). Imputation Mode = Replicate Based Resampling (Missing values ​​are replaced by random values ​​sampled from a distribution centered on the median of the detection values ​​of the (technical and biological) replicates). Hypothesis Test=ANOVA (individual proteins).

[0054] 2 / Results: Tape strip staining: morphological analysis. Tape strip staining provided a wide range of information, from the surface area occupied by corneocytes on the entire tape strip to the number of islets formed by them and the associated colorimetric intensity. Indeed, dry skin was associated with a gradual increase in the surface area occupied by corneocytes on the tape strip. Dry skin was also associated with a gradual increase in the number of islets with high colorimetric density. The colorimetric density and dense distribution of corneocytes directly correlated with the degree of skin dryness.

[0055] Quantitative analysis of dyed tape strips In addition to the analysis performed by dermatologists and the correlation of said analysis with biophysical measurements, a population of people with normal skin and a population of people with dry skin was determined, and after applying the same distribution, these results correlated with the results obtained regarding the average size of the islets obtained for the entire tape strip.

[0056] Dry skin corneocytes occupy 43% more of the tape strip surface compared to normal skin. The islet surface increases by an average of 28% in dry skin. Dry skin exhibits a colorimetric intensity 255% higher than that observed in normal skin.

[0057] Proteomic characterization of dry skin: Analysis of dry and normal skin revealed that of the 613 proteins identified, 340 were differentially regulated in dry skin. An average of 227 and 212 proteins were identified in samples from the dry and normal skin groups, respectively. From a quantitative perspective, of the 340 quantifiable proteins (after curation), 88 were considered overexpressed and 77 were considered underexpressed in sensitive skin (threshold: p-value < 0.05, fold change > 1.5 or < 0.67).

[0058] Specific protein biomarkers for dry skin: By applying a 100% imputation rate to normal skin, in contrast, a very low percentage is obtained in dry skin. This low percentage of imputation is inversely proportional to the expression of the target in dry skin. Consequently, this means that the lower the percentage of imputation, the more the target is expressed in the skin. This experiment allowed us to select targets that are preferentially expressed in dry skin. The five main targets that are only present in dry skin are: periplakin, ESYT3, LCE1C, USP7, and IL1RN.

[0059] Overexpression of these different targets at the keratinocyte level allows the creation of new models for the study of dry skin. The action of selected active ingredients on these targets can then be evaluated to assess their moisturizing effects.

[0060] [Example 2] Effect of moisturizers on selected biomarkers: Overexpression of the four proteins mentioned characterizes dry skin. Consequently, active ingredients that reduce their overexpression would allow dry skin to return to a normal state. This hypothesis was tested for LCE1C. The effects of systemic treatment with active Anthyllis at two different concentrations, 0.1% and 0.033%, on melanized reconstructed epidermis were determined. After five days of treatment, LCE1C expression was reduced 4.1-fold and 11.8-fold, respectively, compared to untreated controls.

[0061] The biomarkers according to the invention therefore make it possible to accurately diagnose dry skin and represent highly relevant targets for assessing the effectiveness of hydrating compounds or for monitoring the effectiveness of hydrating treatments.

Claims

1. A method for determining the water content of the skin of a subject, comprising the step of measuring the expression level of at least one protein selected from the group consisting of extended synaptotagmin-3 (ESYT3), ubiquinine carboxyl-terminal hydrolase 7 (USP7), periplakin (PPL), and late cornified envelope protein 1C (LCE1C) in a skin sample obtained from the subject.

2. The method according to claim 1, further comprising the step of classifying the skin as severely dry, dry, moist, or well moist based on the measured expression level.

3. The method according to claim 1, wherein the higher the expression level of the at least one protein, the drier the skin of the subject is determined to be.

4. The method according to claim 1, further comprising the step of measuring the level of interleukin-1 receptor antagonist protein (IL1RN) in the skin sample obtained from the subject.

5. The method according to any one of claims 1 to 4, wherein the skin sample is a stratum corneum sample.

6. The method according to claim 5, wherein the stratum corneum sample is a tape-stripped stratum corneum sample.

7. A method for evaluating the effectiveness of a cosmetic treatment for hydrating the skin in a subject, comprising the following steps: a) A step of measuring the expression level of at least one protein selected from the group consisting of extended synaptotagmin-3 (ESYT3), ubiquinine carboxyl-terminal hydrolase 7 (USP7), periplakin (PPL), and late cornified envelope protein 1C (LCE1C) in a skin sample obtained from the subject before the cosmetic treatment; b) A step of measuring the expression level of the at least one protein in a skin sample obtained from the subject after the cosmetic treatment; c) A step of comparing the expression levels measured in steps a) and b); and d) A step to determine that the cosmetic treatment is efficient if the expression level measured in step b) is lower than the expression level measured in step a), or that the treatment is inefficient if the expression level measured in step b) is equal to or higher than the expression level measured in step a). Methods that include...

8. A method for screening candidate cosmetic compounds for hydrating the skin, comprising the following steps: a) A step of measuring the expression level of at least one protein selected from the group consisting of extended synaptotagmin-3 (ESYT3), ubicinine carboxyl-terminal hydrolase 7 (USP7), periplakin (PPL), and late cornified envelope protein 1C (LCE1C) in a skin sample; b) The step of bringing the test compound into contact with the skin sample; c) A step of measuring the expression level of the at least one protein in the skin sample; and d) If the expression level measured in step c) is lower than the expression level measured in step a), the step of selecting the compound. Methods that include...

9. The method according to claim 8, wherein the candidate cosmetic compound is a plant extract.

10. The method according to claim 8 or 9, wherein the skin sample is an artificial skin sample.