Age-related changes in cheek skin movement: A case study of Japanese women.

BACKGROUND: The majority of conventional studies on skin aging have focused on static conditions. However, in daily life, the facial skin we encounter is constantly in motion due to conversational expressions and changes in facial expressions, causing the skin to alter its position and shape, resulting in a dynamic state. Consequently, it is hypothesized that characteristics of aging not apparent in static conditions may be present in the dynamic state of the skin. Therefore, this study investigates age-related changes in dynamic skin characteristics associated with facial expression alterations. METHODS: A motion capture system measured the dynamic characteristics (delay and stretchiness of skin movement associated with expression) of the cheek skin in response to facial expressions among 86 Japanese women aged between 20 and 69 years. RESULTS: The findings revealed an increase in the delay of cheek skin response to facial expressions (r = 0.24, p < 0.05) and a decrease in the stretchiness of the lower cheek area with age (r = 0.60, p < 0.01). An increasing variance in delay and stretchiness within the same age group was also observed with aging. CONCLUSION: The findings of this study revealed that skin aging encompasses both static characteristics, such as spots, wrinkles, and sagging, traditionally studied in aging research, and dynamic aging characteristics of the skin that emerge in response to facial expression changes. These dynamic aging characteristics could pave the way for the development of new methodologies in skin aging analysis and potentially improve our understanding and treatment of aging impressions that are visually perceptible in daily life but remain unexplored.

Quantitative analysis of age-related changes in vascular structure, oxygen saturation, and epidermal melanin structure using photoacoustic methods.

BACKGROUND: Vascular structure, blood oxygen saturation, and melanin status of the epidermis are chromophore factors related to light absorption. Therefore, they are likely to be related to skin appearance. Thus, it is important to measure these internal skin features and understand their characteristics. Thus, we aimed to analyze the individual differences and aging changes in the skin by measuring the internal skin characteristics, such as vascular structure, oxygen saturation, and the 3D distribution of melanin in the epidermis, using a noninvasive photoacoustic (PA) measurement method. MATERIALS AND METHODS: A PA measurement device was used as a noninvasive measurement method. Eighty Japanese women aged between 20 and 60 years were enrolled. The target area was the buccal region of the face. RESULTS: The blood vessel structure showed a decrease in fine vessels with age, with a stronger tendency observed in the dermis layer, and the volume of blood vessels was larger in the dermis layer than in the dermal-subcutaneous fat boundary layer. Oxygen saturation showed a similar decreasing trend with age in all depths examined. Melanin condition as the torus-like pattern structure tended to increase with age. CONCLUSION: PA measurements revealed the characteristics of several chromophores, providing a new skin aging mechanism.

Age-related changes in the fiber structure around adipocytes in the subcutaneous fat layer and their association with skin viscoelasticity.

OBJECTIVE: Age-related changes in the fiber structure around adipocytes were investigated via scanning electron microscopy (SEM) of excised skin tissues. In addition, the viscoelasticity of the subcutaneous fat layer was evaluated via elastography, and the association between the fiber structure and the viscoelastic properties was assessed. METHODS: Skin tissues excised from the facial cheek area were used. Then, SEM images of these tissues were obtained. The thickness and quantity of the fibers around adipocytes were assessed using a 5-point scale. The score was used to grade 18 tissue samples. Moreover, the viscoelasticity of the subcutaneous fat layer in the same samples was evaluated via ultrasound elastography. RESULTS: Based on the SEM image score, an association was observed between the fiber status score and age, thereby indicating a tendency toward age-related fibrosis. Fiber structures with high scores, which indicate fibrosis, had a significantly lower viscoelasticity based on ultrasound elastography. CONCLUSION: The thickness and quantity of fibrous structures around adipocytes in the subcutaneous fat layer increase with age, and these changes can be associated with decreased viscoelasticity in the subcutaneous fat layer.

The effect of observation angles on facial age perceptions: A case study of Japanese women.

Most conventional aging research has limited its approach concerning the head and face shape and skin condition to the frontal face. However, in our daily lives, we observe facial features from various angles, which may reveal or obscure aging features that could only be identified under limited conditions in the past. This study systematically investigates the effect of facial observation angles-specifically, of horizontal and vertical angles-on age impression. A total of 112 Japanese women aged 20-49 years participated as observers who evaluated the age impressions of 280 Japanese women aged 20-69 years. A two-way analysis of the variance of the age impression score was conducted for two factors: observation angle (five angles with yaw and pitch directions) and age group (five ages, from the 20s to the 60s). The results reveal that, as compared with frontal observation, the perceived age tended to decrease with the facial observation angles and that the effect of the angle on perceived age decreased with increasing age, especially for the profile face. Understanding the effect of the facial observation angle on age impression and clarifying the characteristics of the face and skin not perceived in the frontal face will provide useful knowledge to make people look youthful, look more beautiful, and be happier in all aspects of their lives.

Investigation of oxygen saturation in regions of skin by near infrared spectroscopy.

BACKGROUND: Oxygen is essential for life, and investigation of the skin's oxygen environment and identification of its effects on the skin may lead to the discovery of new antiaging targets. To understand individual skin differences and age-related changes, we developed a noninvasive method using near infrared spectroscopy (NIRS) to measure the regional saturation of oxygen (rSO2 ) of human skin. MATERIALS AND METHODS: To construct an NIRS sensor probe specialized for skin measurement, the distance between the sensor transmitter and receiver was optimized based on data for the thickness of the facial skin to the subcutaneous fat layer. To analyze the relationship between skin oxygen saturation and body oxygen saturation, rSO2 was measured by NIRS, oxygen saturation of peripheral artery (SpO2 ) was measured by pulse oximeter, and physical conditions were considered, such as body mass index (BMI) and muscle mass, in Japanese women (age 20s-60s). RESULTS: Both skin rSO2 and SpO2 varied among individuals and decreased with age. Only SpO2 showed a relationship with BMI and muscle mass, whereas rSO2 showed no relationship with these physical conditions. No relationship between rSO2 and SpO2 was observed. CONCLUSION: Individual and age-related differences in skin by rSO2 values were found by NIRS optimized for local skin; however, the factors affecting rSO2 differed from those affecting SpO2 , and further study is needed.

Does age-dynamic movement accelerate facial age impression? Perception of age from facial movement: Studies of Japanese women.

We form impressions of others by observing their constant and dynamically-shifting facial expressions during conversation and other daily life activities. However, conventional aging research has mainly considered the changing characteristics of the skin, such as wrinkles and age-spots, within very limited states of static faces. In order to elucidate the range of aging impressions that we make in daily life, it is necessary to consider the effects of facial movement. This study investigated the effects of facial movement on age impressions. An age perception test using Japanese women as face models was employed to verify the effects of the models' age-dependent facial movements on age impression in 112 participants (all women, aged 20-49 years) as observers. Further, the observers' gaze was analyzed to identify the facial areas of interests during age perception. The results showed that cheek movement affects age impressions, and that the impressions increase depending on the model's age. These findings will facilitate the development of new means of provoking a more youthful impression by approaching anti-aging from a different viewpoint of facial movement.

Examination of age-related changes of viscoelasticity in the dermis and subcutaneous fat layer using ultrasound elastography.

BACKGROUND: Using ultrasound elastography, the present study aimed to measure the viscoelasticity in each skin layer and to determine the relationship between the measured value, age, and body mass index (BMI). MATERIALS AND METHODS: The present study included 77 Japanese women. We calculated the BMI and measured the facial cheek via elastography. With the use of the elastographic image, the dermis was divided into two layers, and the subcutaneous fat layer was divided into five equal sections according to the depth, ultimately obtaining seven layers. Furthermore, the thickness and viscoelasticity of each divided layer were measured. RESULTS: The analysis of echo images revealed that the thickness of the upper dermis layer decreased with age, whereas that of the subcutaneous fat layer tended to increase with age and BMI. As measured by elastography, the viscoelasticity of both the lower dermis and the upper subcutaneous fat layer decreased with age. As the BMI increased, the viscoelasticity of the lower subcutaneous layer also increased, but that of the upper subcutaneous layer decreased. CONCLUSIONS: The present study revealed the relationship between aging and viscoelasticity in the lower dermis and the relationship between aging, BMI, and viscoelasticity in the upper subcutaneous fat layer.

Effects of lactic acid on the flexibility of the stratum corneum.

BACKGROUND: Lactic acid increases the flexibility of the stratum corneum by linking directly to keratin without water. However, another study reported the involvement of water. This study aimed to clarify how lactic acid contributes to the flexibility of the stratum corneum (SC) of the face. MATERIALS AND METHODS: A tactile sensor was used to measure the change in shared frequency (Δf) in the cheeks of 88 women of various ages. Amounts of water and lactic acid in the SC were measured during all four seasons, and correlations with Δf evaluated. RESULTS: Lactic acid and water levels in the SC were not correlated in any season. In the younger subjects only, the lactic acid content correlated with Δf in spring (when at its highest level in the SC), whereas the water content correlated with Δf in summer and autumn (when the water content was high). CONCLUSIONS: The overall lack of correlations between lactic acid and water content across all ages and seasons suggested that each contributes independently to the flexibility of the SC and that lactic acid softens the SC without water.

Convenience biosensing approach of lactic acid in stratum corneum for skin care assessment.

BACKGROUND: Lactic acid in the stratum corneum contributes to skin flexibility, making it a useful indicator of skin conditions. It is this useful to devise a simple technique to measure the lactic acid in the stratum corneum. To this aim, a printed chip biosensor was developed to analyze lactic acid in tape stripped (TS) stratum corneum samples. MATERIALS AND METHODS: Lactic acid was extracted from TS stratum corneum samples. A normal lactic acid sensor was prepared by applying lactate oxidase (LOD) to a printed chip. Another lactic acid sensor was prepared using LOD and a mediator osmium polymer immobilized on a printed chip. The amount of lactic acid in the extracted solutions was quantified using either the prepared biosensors or an existing analysis method. RESULTS: The results measured using the normal lactic acid sensor show low correlation with the results measured using an existing analytical method as a comparison, but those of the mediator osmium lactic acid sensor show high correlation. CONCLUSIONS: The amount of lactic acid in samples extracted from the stratum corneum using a simple TS technique can be simply analyzed with high accuracy using a printed chip biosensor.

Analysis of the skin surface and inner structure around pores on the face.

BACKGROUND/PURPOSE: Facial pores do not appear to close again in old skin. Therefore, the tissue structure around the pore has been speculated to keep the pore open. To elucidate the reason for pore enlargement, we examined the relationship between the skin surface and inner skin structural characteristics in the same regions especially around the pore. METHODS: Samples of the skin surface were obtained from the cheek and examined using a laser image processor to obtain three-dimensional (3D) data. The inner structure of the skin was analyzed using in vivo confocal laser scanning microscopy (CLSM). RESULTS: The conspicuous pore not only had a concave structure but also a discontinuous convex structure on the skin surface surrounding the pore. Furthermore, CLSM image indicated that the skin inner structure developed a discontinuous dermal papilla structure and isotropic dermal fiber structure. CONCLUSION: There were structural changes in the skin surface around conspicuous pores, including not only a concave structure but also a convex structure with skin inner structure changing.

The investigation of the skin characteristics of males focusing on gender differences, skin perception, and skin care habits.

BACKGROUND/PURPOSE: Various studies have examined the properties of male skin. However, because these studies mostly involved simple measurement with non-invasive devices, a lack of understanding of the properties of male skin remains. METHODS: In this study, we focused and investigated not only on simple instrumental measurements but also on gender differences and men's subjective perceptions of skin and daily skin care habits. RESULTS: Barrier function depends on corneocyte maturation level as well as sebum amount. Irrespective of the skin type, a high percentage of male subjects perceived a 'tacky feeling'. However, the percentage of men perceiving a 'shiny feeling' differed by skin type. Furthermore, there was a relationship between skin care habits and skin function. Men who did not perform a daily skincare regimen demonstrated a significantly higher sebum amount and transepidermal water loss value than those who did perform a daily skincare regimen. CONCLUSION: The results of this study indicate that male skin has two specific characteristics: impaired barrier function because of the excess amount of sebum and a lack of an appropriate skin care regimen because of the 'tacky feeling' caused by excess sebum.

Terahertz wave techniques using a metal mesh for evaluating the components of the stratum corneum.

BACKGROUND/PURPOSE: Terahertz waves are located in the region of the spectrum between milliwaves and infrared. We analyzed the feasibility of terahertz spectroscopy to inspect the compositional variations of the stratum corneum (SC). METHODS: We used a terahertz time-domain spectroscopy system with the metal mesh technique. To investigate whether terahertz can inspect compositional variation of SC, we measured the terahertz frequency spectra of the SC sheet that was treated with chloroform-methanol, lipid mixture, a denaturation agent, and heating with hot air. RESULTS: The chloroform-methanol treatment of the SC shifted the dip position, which represents a convex downward shape of the spectra, to a higher frequency. This dip shift was reversed to an untreated position by the dose-dependent application of a lipid mixture. The heating treatment of the SC shifted the dip position to a higher frequency. The same dip shift was also induced by the application of a denaturation agent to the SC. CONCLUSION: The technique using terahertz waves with a metal mesh is effective because of its simplicity and its high degree of accuracy in detecting the amount of lipid and the protein conformation state.

The essential structures of ISP-I that influence serine palmitoyltransferase inhibition in Chinese hamster ovary cells.

We investigated the structure-activity relationship between various ISP-I (myriocin, thermozymocidin) analogous which has sphingosine-like structure and serine palmitoyltransferase (SPT) in Chinese hamster ovary (CHO) cells utilizing sphingolipid production as a marker. Our data suggest that the double bond and/or ketone group within the alkyl chain as well as the alkyl chain are necessary for ISP-I to inhibit SPT. In addition, a serine structure is necessary for SPT inhibitory activity, which confirms previous findings.

Effects of serine palmitoyltransferase inhibitor ISP-I on the stratum corneum of intact mouse skin.

Serine palmitoyltransferase (SPT) is involved in the ceramide synthesis pathway. We investigated the effects of ISP-I, a potent inhibitor of SPT, on the stratum corneum (SC) of hairless mouse skin. Application of ISP-I for one week resulted in a significant decrease in the amount of ceramide, which was associated with a decrease in SC hydration. However, there was an increase in the number of SC layers and less transepidermal water loss than control. Transmission Electron Microscopy observation revealed that the number of desmosome-like structures in the layers immediately above the stratum granulosum (SG) was significantly increased in ISP-I-treated skin compared to vehicle-treated skin. The activity of serine protease-an enzyme associated with the process of desquamation-was lower in the SC of ISP-I-treated skin than control. Furthermore, immunoelectronmicroscopy revealed that glucosylceramide and corneodesmosin tended to remain in corneocytes and were not secreted into the intercellular spaces of the SC in the ISP-I-treated skin. These results indicate that the application of ISP-I decreases ceramide and skin hydration, while at the same time increases the number of SC layers. The accumulation of corneocyte layers may originate from an aberrant desquamation process related to the decrease in the serine protease activity as well as an alteration in the transport of desquamation-related proteases by lamellar bodies.

The effects of serine palmitoyltransferase inhibitor, ISP-I, on UV-induced barrier disruption in the stratum corneum.

We examined the effects of ISP-I (myriocin, thermozymocidin) - a potent inhibitor of serine palmitoyltransferase (SPT) which is involved in the ceramide synthetic pathway-on skin barrier function in post-UVB-irradiated hairless mouse skin. Disruption of the skin barrier function after UVB irradiation as represented by the increase in transepidermal water loss (TEWL) was significantly suppressed with ISP-I treatment. In the ISP-I-treated skin, the peak of cell proliferation was observed 24 h earlier than in vehicle-treated skin. In addition, the number of apoptotic cells in ISP-I-treated skin showed a sharp decrease at 48 and 72 h post-irradiation. The number of stratum corneum cell layers was increased in ISP-I-treated skin at 72 h after UVB irradiation; at this time, TEWL in ISP-I-treated skin was lower than that in the vehicle-treated skin. We suggest ISP-I treatment altered cell proliferation and apoptosis after UVB exposure by modulating ceramide synthesis in epidermal cells, resulting in an increase of stratum corneum layers which lessened the effects of irradiation-induced barrier disruption.

Perturbation of lamellar granule secretion by sodium caprate implicates epidermal tight junctions in lamellar granule function.

BACKGROUND: Polarized secretion of lamellar granules (LGs) delivers various lipids, proteases, and protease inhibitors into the stratum corneum (SC) of the epithelium. Disruption of LGs is associated with severe cutaneous diseases, but the mechanism of their polarized secretion is not known. On the other hand, recent study shows epidermal tight junctions (TJs) localize in stratum granulosum (SG), and TJs are involved in polarized molecule secretion. Thus, we hypothesized epidermal TJs relate to polarized LGs secretion. OBJECTIVE: To assess the possibility that epidermal TJs are involved in polarized LGs secretion. METHODS: In order to examine LGs secretion, we used fluorescent ceramide (BODIPY FL C(5)-ceramide) and a natural LG cargo, lympho-epithelial Kazal-type-related inhibitor (LEKTI), in cultured normal human epidermal keratinocytes and a reconstructed human epidermis. We investigated their alteration using the medium-chain fatty acid sodium caprate (C10), TJs inhibitor. In addition, LG distribution was observed by electron microscopy. RESULTS: C10 significantly inhibited secretion of both fluorescent ceramide and LEKTI in cultured normal human epidermal keratinocytes and a reconstructed human epidermis. C10 also disturbed the polarized localization of fluorescent ceramide and LEKTI in the reconstructed epidermis. Electron microscopy revealed that a large number of LGs remained in corneocytes in the C10-treated epidermis, rather than being secreted. CONCLUSION: Our data indicate that C10 perturbs the polarized secretion of LGs. Our study therefore suggests that epidermal TJs are possibly involved in polarized LG secretion and provides new insights into potential of treatments for skin diseases caused by abnormal LG secretion.

Effects of vitamin C on dark circles of the lower eyelids: quantitative evaluation using image analysis and echogram.

BACKGROUND/AIMS: The pathogenesis of dark circles of the lower eyelid (DCLE) has been considered to involve stasis and hyperpigmentation of the eyelids. We have already reported that dermal thickness of lower eyelid skin may represent another factor that affects the appearance of DCLE. The aim of this study was to evaluate the efficacy of vitamin C, which is known to increase collagen, on DCLE through a clinical trial. METHODS: Fourteen subjects with DCLE applied either 10% sodium ascorbate (ANa) or ascorbic acid glucoside (AG) lotion in split-face fashion (opposite side: vehicle only) for 6 months. Melanin index (MI), erythema index (EI), thickness and echogenicity of the dermis at bilateral lower eyelids was measured during this trial. RESULTS: Change in EI was significantly smaller on the ANa-treated side than on the vehicle-treated side. Dermal thickness tended to be thicker for the ANa-treated side than for the vehicle-treated side, although no significant difference was seen. Both EI and dermal thickness tended to change in parallel manner. On the other hand, no significant differences in changes of EI, MI, and dermal thickness were found between AG- and vehicle-treated sides. CONCLUSION: ANa may improve DCLE by thickening the eyelid dermis and concealing dark coloration due to congested blood.

Epidermal terminal differentiation depends on B lymphocyte-induced maturation protein-1.

The cornified layer is a compacted lattice of lipid-embedded corneocytes that provides an organism's barrier to the external environment. Cornification is the final differentiative step for epidermal keratinocytes and involves dramatic cell condensation before death. Using conditional gene deletion in mice, we identified the transcriptional repressor Blimp-1 (B lymphocyte-induced maturation protein-1) as an important regulator of keratinocyte transition from the granular to the cornified layer. More than 250 genes are misregulated in conditional knockout epidermis, including those encoding transcription factors, signal transduction components, proteinases, and enzymes involved in lipid metabolism. Steady-state mRNA and ChIP analyses of a subset of these genes provide evidence that nfat5, fos, prdm1, and dusp16 are novel direct targets of Blimp-1. Identifying nfat5 as a target of Blimp-1 repression indicates that cornification involves suppression of normal osmotic regulation in granular cells. Consistently, conditional knockout mice have delayed barrier formation as embryos, enlarged granular layer cells and corneocytes, and a morphologically abnormal cornified layer. These studies provide insight into cornification, identifying transcriptional regulatory circuitry and indicating the importance of blocking osmotic homeostasis.

Objective evaluation of the efficacy of daily topical applications of cosmetics bases using the hairless mouse model of atopic dermatitis.

BACKGROUND/AIMS: Dry skin (xerosis) is a characteristic change associated with atopic dermatitis (AD) and has often been treated with topical petrolatum applications despite its unfavorable feel. Recently, various therapeutically effective skin-care products with better feel have been introduced. To elucidate the mechanisms underlying the clinical effectiveness of these newer treatments, we used our recently established hairless mouse model of AD. METHODS: We produced AD-like skin lesions in hairless mice with repeated applications of 2,4,6-trinitro-1-chrolobenzene (TNCB) in acetone for 36 days as reported previously. Groups of five mice with AD-like skin were treated once daily with an emollient-type cream containing petrolatum, a moisturizer-type cream containing 10% glycerin, a solution of 0.01% dexamethasone in acetone, or were left untreated. Over the duration of these treatments, we conducted non-invasive measurements of skin surface condition with biophysical instruments and electron microscopic evaluation of the surface area size and density of rear surface villi of superficial corneocytes. We also obtained skin biopsy samples and blood samples at each time point for histopathological evaluation and to assess serum IgE levels, respectively. RESULTS: After cessation of topical TNCB applications, AD-like skin underwent spontaneous resolution with normalization of skin appearance. A similar reduction in skin fold thickness was observed in the cream-treated mice and in the untreated mouse group, whereas a significant decrease in skin thickness was observed in the dexamethasone-treated mice. Transepidermal water loss, a measure of stratum corneum barrier function, rapidly normalized in all groups, without any statistical differences noted among groups. In comparison with untreated skin, skin surface hydration markedly improved after repeated applications of the moisturizer-type cream, whereas it consistently remained low in dexamethasone-treated skin. The skin treated with emollient-type cream appeared similar to skin that received no treatment. Reduced corneocyte surface area size resulting from repeated applications of TNCB returned to control size with cream treatments, while the corneocyte surface area size became much larger following dexamethasone treatment. In addition, the density of villi on the rear surface of corneocytes decreased with application of the creams or dexamethasone. By contrast, no changes were observed in the number of leukocytes in the epidermis or in serum IgE levels among the different treatment groups. In all treatment groups, even after 32 days of treatment, reapplication of TNCB resulted in early-stage skin swelling, but only in the steroid-treated animals did this swelling show a remarkably prolonged time course. CONCLUSIONS: Our present results indicate that the efficacy of skin-care products containing no active ingredients in treating atopic xerosis can be objectively evaluated using the hairless mouse model of AD.

Establishment of an atopic dermatitis-like skin model in a hairless mouse by repeated elicitation of contact hypersensitivity that enables to conduct functional analyses of the stratum corneum with various non-invasive biophysical instruments.

BACKGROUND/AIMS: Pathogenesis of atopic dermatitis (AD) has been studied in animal models such as the NC/Nga mouse strain or Balb/C mice that are repeatedly treated with 2,4,6-trinitro-1-chrolobenzene (TNCB). These mice exhibit features of chronic contact dermatitis, including an intensified early type skin reaction, increased number of mast cells and elevated serum IgE levels with a shift of cutaneous cytokine expression from a type 1 to type 2 profile. However, it is difficult to investigate the unique skin changes of AD such as dry skin, barrier dysfunction, and increased turnover of the stratum corneum (SC) in these animals with biophysical instruments because of the presence of their fur coats. In this study, we succeeded in establishing a mouse model of AD in hairless mice that are suitable for various functional analyses of the SC as well as for examining the immunological characteristics of human AD by treating TNCB-contact sensitized hairless mice with 1% TNCB every other day for 36 days. METHODS: In hairless animals treated with TNCB every 2 days for 36 days, we measured time courses of skin swelling induced by contact hypersensitivity reaction on days 0, 6, 20 and 36. During the time course, non-invasive measurements for skin surface condition with biophysical instruments were conducted, and the area size and the rear surface villi of corneocytes obtained were measured. Also skin samples and blood samples were taken at each time point for histology and measurement of serum IgE level. RESULTS: A gradual intensification of an early type contact hypersensitivity reaction was observed over the treatment period. These mice exhibited reduced SC hydration, heightened trans-epidermal water loss, and increased skin thickness. These mice also showed a decrease in the surface area size of each corneocytes and marked villus formation on their rear surface. Histologically, there was an increase in the number of CD4 and CD8 positive T cells in the epidermis. Also observed was a marked increase in the number of dermal mast cells and eosinophils, which correlated with elevated serum IgE levels induced by TNCB treatments. CONCLUSIONS: From the results obtained we conclude that repeated treatments of TNCB-sensitized hairless mice with TNCB provides a useful means by which to study the pathological characteristics of AD skin lesions as well as their immunological characteristics.