Grading facial aging: Comparing the clinical assessments made by three dermatologists with those obtained by an AI-based scoring system that analyses selfie pictures. A focus on Chinese subjects of both genders.

OBJECTIVE: The objective of this study is to assess the correspondence, in live conditions, between clinical gradings of facial aging signs by three dermatologists and those afforded by an automatic AI-based algorithm that analyses smartphones' selfie images of Chinese subjects. METHODS: In total, 125 Chinese subjects of both genders, aged 18-62y, took a selfie using their own smartphones and were immediately viewed by three dermatologists. The latter graded the severity of 15 facial signs in women and 9 in men, using the standardized values afforded by a Skin Aging Atlas referential dedicated to Asian skin. The data issued by both methodologies were then statistically compared. RESULTS: The absolute gradings of the automatic system were found highly correlated with clinical assessments, with lower values in most cases. In women, large differences in absolute values were found on the gradings for size of isolated spot, cheek fold, spread macules, and texture of mouth contour women. Analysis of the Mean Absolute Errors (M.A.E) revealed that these rarely exceed 0.6 grading units in women and to a lesser extent in men. CONCLUSION: The present study confirmed the value of the automatic system towards an extended use towards large human cohorts as a surrogate of clinical evaluations and allowed to detect the points where improvements must be brought to the system.

OBJECTIF: évaluer la correspondance, en conditions réelles, entre les classements cliniques des signes de vieillissement du visage par trois dermatologues et ceux établis par un algorithme automatique d'IA qui analyse les images de selfie sur smartphone de sujets chinois. MÉTHODES: 125 sujets chinois des deux sexes, âgés de 18 à 62 ans, ont pris un selfie à l'aide de leurs smartphones et ont été immédiatement examinés par trois dermatologues. Ces derniers ont évalué la sévérité de 15 signes faciaux chez les femmes et 9 chez les hommes, en utilisant les valeurs standardisées établies par un référentiel de l'Atlas du vieillissement cutané dédié à la peau asiatique. Les données obtenues par les deux méthodologies ont ensuite été comparées statistiquement. RÉSULTATS: les classements absolus du système automatique étaient fortement corrélés aux évaluations cliniques, avec des valeurs plus faibles dans la plupart des cas. Chez les femmes, des différences importantes en termes de valeurs absolues ont été observées concernant les classements de la taille de la tache isolée, du pli de la joue, des macules diffuses et de la texture du contour de la bouche chez les femmes. L'analyse des erreurs absolues moyennes (EAM) variables a révélé que celles­ci dépassent rarement 0,6 unité de classement chez les femmes et, dans une moindre mesure, chez les hommes. CONCLUSION: la présente étude a confirmé la valeur du système automatique en vue d'une utilisation étendue à de grandes cohortes humaines en remplacement des évaluations cliniques et a permis de détecter les points où des améliorations doivent être apportées au système.

DFT Study on the Second-Order NLO Responses of 2-Phenyl Benzoquinoline Ir(III) Complexes by Substituents and Redox Effects.

Metal complexes have received extensive attention in nonlinear optical (NLO) materials because of their advantages, such as shorter response times and more flexible structural properties. Density functional theory is used in investigating the geometric structures, electronic structures, charge centroid, and first hyperpolarizability (ßtot) of a series of selected 15 coordinated Ir(III) complexes. The substitute effect and one-electron redox process effects on the structures and properties of 15 coordinated Ir(III) complexes are considered. When the electron-withdrawing group is introduced into the ligand, the HOMO-LUMO energy gap decreases and the ßtot value increases, positively correlating with the electron-withdrawing ability. The single electron redox process can also improve the NLO responses of complexes, especially the reduction process. The ßtot value of complex 4- is the largest, 2078 times higher than that of complex 4. The analysis shows that the variation of NLO responses of complexes is ascribed to the change of electronic structures and the charge transfer modes induced by the ligand modification and redox process, which are considered to be two effective methods in enhancing the NLO responses of 2-phenyl benzoquinoline Ir(III) complexes. This study aims to offer design insights into high-performance nonlinear optical materials.

Holistic investigation of the anti-wrinkle and repair efficacy of a facial cream enriched with C-xyloside.

OBJECTIVE: To investigate the repairing and anti-wrinkle efficacy of the facial cream enriched with C-xyloside, aiming at comprehensively evaluating its skin anti- aging effect and clarify its potential mechanism of action. METHODS: The repairing efficacy was studied on 3D epidermis skin model and the antiaging efficacy was studied on ex-vivo human skin. Two clinical studies were conducted with Chinese females. In the first study, 49 subjects aged between 30 and 50 with wrinkle concerns were recruited and instructed to apply the investigational cream containing C-xyloside for 8 weeks. Wrinkles attributes were assessed by dermatologist. Instrumental measurements on skin hydration, trans-epidermal water loss (TEWL), and skin elasticity were also conducted. In the second study, 30 subjects aged between 25 and 60 with self-declared sensitive skin and facial redness were recruited and instructed to apply the cream for 4 weeks. Biomarker analysis of the stratum corneum was conducted through facial tape strips. RESULTS: The cream improved the histomorphology of the 3D epidermis skin model after SLS stimulation, and significantly increase the expression of LOR and FLG. On human skin, the cream improved the histopathology induced by UV, and significantly increased the protein content of COL I and COL III, collagen density and the number of Ki-67 positive cell of skin compared with model group (n = 3, p < 0.01). The results from the first clinical study demonstrate a significant increased the skin hydration and elasticity by 21.90%, 13.08% (R2) and 12.30% (R5), respectively (n = 49, p < 0.05), and the TEWL values decreased by 33.94% (n = 49, p < 0.05), after 8 weeks application of the cream. In addition, the scores for nasolabial folds, glabellar wrinkle, underneath eye wrinkles, crow's feet wrinkle and forehead wrinkle in the volunteers exhibited a significant reduction of 34.02%, 43.34%, 50.03%, 33.64% and 55.81% respectively (n = 49, p < 0.05). The (rCE)/(fCE) ratio of volunteers based on tape stripping significant increased after using the sample cream (n = 30, p < 0.05). CONCLUSION: The cream containing C-xyloside showed improvement of skin wrinkles and enhancement of skin barrier function. These efficacies may be attributed to the fact that the sample cream can increase the expression of skin barrier related proteins LOR and FLG, promote the maturation of cornified envelope, enhance collagen I and III protein expression and stimulate skin cell proliferation, to provide sufficient evidence supporting its antiaging efficacy of skin.

A Bayesian network meta-analysis of 14 molecules inhibiting UV daylight-induced pigmentation.

INTRODUCTION: Hyperpigmentation disorders are very frequent, affect the quality of life and may become a psychological burden for afflicted patients. Many anti-pigmenting or depigmenting agents are available with various efficacy and almost no comparative data. 2-mercaptonicotinoyl glycine (2-MNG) was recently proposed as a viable candidate showing safe and effective results on hyperpigmentation control in vitro and in vivo. OBJECTIVES: A Bayesian network meta-analysis (BNMA) was conducted to map and rank the anti-pigmenting and depigmenting efficacy of 2-MNG 0.5% on UV daylight (UVDL)-induced pigmentation together with 13 other reference molecules. A comparison in the kinetics of 2-MNG 0.5% was also performed. METHODOLOGY: Fourteen studies were conducted, for each, on 15-30 women of skin phototype III in Shanghai, China and Paris, France. The products were applied on mini zone, in randomized and blinded protocol, on the back, 5 days a week during 6 weeks, at a dose of 4 mg/cm2. During the second week, volunteers were exposed under to varying minimum erythemal dose of UVDL during 4 consecutive days-adapted to obtain a similar induction of skin pigmentation regardless of the population. Assessments were performed instrumentally using Chromameter®. Ascorbic acid 7% was used as a positive control for all experiments. A Bayesian network meta-analysis was then established to map and follow the kinetics of 2-MNG 0.5% performance with 13 reference molecules (glutathione 2%, kojic acid 1%, hydroquinone 4%, ascorbyl glucoside 2%, niacinamide 4%, etc.). RESULTS: 2-MNG 0.5% dominated the ranking at all time points with a significant high probability of strong efficacy against UVDL-induced pigmentation. Ascorbic acid 7% ranks second after 4 days of irradiations (D12) whereas hydroquinone 4% ranks second 1 month after irradiations (D40). In the kinetics, 2-MNG at 0.5% was effective as from the end of irradiations (D12) to the study endpoint (D40). This suggested an immediate and persistent efficacy across all timepoints evaluated. CONCLUSION: The BNMA revealed a rapid and lasting efficacy of 2-MNG 0.5% on the anti-pigmenting and depigmenting phases of the clinical protocol. 2-MNG 0.5% ranked first, with immediate and lasting effect compared to 13 other references. This study is the first allowing comparison between reference anti-pigmenting and depigmenting agents and will help clinicians for proposing the most effective approach for their patients.

Does eating with others promote happiness among older adults living alone? A 3-year longitudinal study of the Japan gerontological evaluation study.

OBJECTIVE: Living a happy life is an essential issue for old adults. However, how eating with others contributes to happiness and whether this association is different by living arrangements or not is unknown. The current study examined the relationship between the frequency of eating with others and happiness among older adults according to their living arrangements using 3-year longitudinal data. METHODS: The analyzed sample comprised 18,727 people (10,920 males and 7807 females) with low happiness (0-7 points on score of 0-10 points) from Japan Gerontological Evaluation Study (JAGES) in 2016. Our exposure was the frequency of eating with others: rarely, a few times a year, a few times a month, and a few times a week or more. We performed Modified Poisson Regression to examine the association between the frequency of eating with others and high happiness (8-10 points) in 2019 stratified by living arrangement (living alone/with others). RESULTS: A total of 4352 (23.2%) people showed high happiness in 2019. After adjusting for age, sex, marital status, education, household income, social participation, illnesses under treatment, and depressive symptoms in 2016, the cumulative incidence ratio (CIR) for high happiness in 2019 among people living alone was more significant, that is, 1.28 (95% confidence intervals: 0.88-1.87), 1.50 (1.05-2.14), and 1.82 (1.26-2.63), than 1.28 (1.11-1.48), 1.30 (1.12-1.50), and 1.33 (1.16-1.52) among people living with others for those who ate with others a few times a year, a few times a month, and a few times a week or more compared to those who rarely ate with others, respectively. The interaction between the frequency of eating with others and living arrangements was statistically significant. The trend test showed that higher frequency of eating with others was significantly associated with high happiness. CONCLUSIONS: Eating with others was associated with improved happiness among older adults, with such an association being stronger among people living alone.

Efficacy and safety of a facial serum and a mask containing salicylic acid and lipohydroxy acid in acne management: A randomized controlled trial.

BACKGROUND: Inflammatory and non-inflammatory acne lesions constitute a significant clinical challenge in acne subjects. AIM: To evaluate the efficacy and safety of a facial serum and a mask containing salicylic acid and lipohydroxy acid for improving skin conditions. METHODS: This randomized controlled trial included adults with comedones, post-inflammatory erythema (PIE) and/or hyperpigmentation (PIH) in Shanghai, China in July 2021. Participants were randomly assigned 1:1 to receive the study Serum + Mask or serum alone for 8 weeks. Acne severity, comedones, papules, pustules, PIE, PIH, skin pores, skin tone evenness, sebum secretion, skin hydration, and trans-epidermal water loss were evaluated at T0d, T1d, T7d, T14d, T28d, and T56d. RESULTS: Eighty-three participants were included, including 41 and 42 in the Serum + Mask and Serum groups, respectively. Acne severity, density of skin pores, skin tone evenness, PIH foci on face, PIE foci on nose, intensity of PIE and PIH, closed comedones on face, open comedones on nose, sebum secretion, and skin hydration were significantly improved from baseline after 8 weeks of treatment in both groups (all p < 0.05). Addition of the mask improved the number of closed comedones (-6.56 ± 0.39 vs. -5.19 ± 0.44, p = 0.022) and acne severity (-0.39 ± 0.08 vs. -0.12 ± 0.09, p = 0.026) substantially more than using the serum alone. No adverse reaction was reported in either group. CONCLUSIONS: The study serum improved skin conditions by regulating skin barrier function and achieving a balance of skin hydration and sebum secretion, removing comedones and improving PIE and PIH. Addition of the mask accelerated the effects without compromising safety.

Hydrogen Isotope Separation Using Graphene-Based Membranes in Liquid Water.

Hydrogen isotope separation has been effectively achieved electrochemically by passage of gaseous H2/D2 through graphene/Nafion composite membranes. Nevertheless, deuteron nearly does not exist in the form of gaseous D2 in nature but as liquid water. Thus, it is a more feasible way to separate and enrich deuterium from water. Herein, we have successfully transferred monolayer graphene to a rigid and porous polymer substrate, PITEM (polyimide track-etched membrane), which could avoid the swelling problem of the Nafion substrate as well as keep the integrity of graphene. Meanwhile, defects in the large area of CVD graphene could be successfully repaired by interfacial polymerization resulting in a high separation factor. Moreover, a new model was proposed for the proton transport mechanism through monolayer graphene based on the kinetic isotope effect (KIE). In this model, graphene plays a significant role in the H/D separation process by completely breaking the O-H/O-D bond, which can maximize the KIE, leading to increased H/D separation performance. This work suggests a promising application for using monolayer graphene in the industry and proposes a pronounced understanding of proton transport in graphene.

Heart rate estimation from facial videos with motion interference using T-SNE-based signal separation.

Remote photoplethysmography (RPPG) can detect heart rate from facial videos in a non-contact way. However, head movement often affects its performance in the real world. In this paper, a novel anti-motion interference method named T-SNE-based signal separation (TSS) is proposed to solve this problem. TSS first decomposes the observed color traces into pulse-related vectors and noise vectors using the T-SNE algorithm. Then, it selects the vector with the most significant spectral peak as the pulse signal for heart rate measurement. The proposed method is tested on a self-collected dataset (17 males and 8 females) and two public datasets (UBFC-RPPG and VIPL-HR). Experimental results show that the proposed method outperforms state-of-the-art methods, especially on the videos containing head movements, improving the Pearson correlation coefficient by 5% compared with the best contrasting method. To summarize, this work significantly strengthens the motion robustness of RPPG, which makes a substantial contribution to the development of video-based heart rate detection.

[Promoting community gathering places "Kayoinoba" for healthy aging reduce health inequalities among communities: An eight-year ecological study].

Objectives　This study aimed to investigate whether health inequalities among communities would be reduced by intensively enhancing the "Kayoinoba" program in model communities where many high-risk, older adults live.Methods　Kobe City and the Japan Gerontological Evaluation Study created a mail survey for older adults in 78 communities (community ≈ junior high school district) to conduct community diagnosis. Sixteen communities showed poor values along multiple dimensions of risk and required priority measures. From 2014 to 2019, we designated these 16 communities as model communities. Then, municipal officials and researchers cooperated to support the establishment and management of "Kayoinoba." By using four-waves of mail survey data (in 2011, 2013, 2016, and 2019 with n=8,872, 10,572, 10,063, and 5,759, respectively), secular transitions of nine intermediate outcome indicators (three=social participation, two=social network, and four=social support) and five health outcome indicators (physical function, malnutrition, oral function, cognitive function, and depressive symptoms) were compared between model (n=16) and non-model (n=62) communities via multilevel mixed-effects linear regression analysis.Results　In the 2011 and 2013 surveys, model communities showed poor value compared to the non-model communities in 13 of the 14 indicators. A significant interaction between the year and model/non-model communities was confirmed for four intermediate outcome indicators (sports and hobby group participation, number of friends met, and providing emotional support) and three health outcome indicators (oral function, cognitive function, and depressive symptoms). The differences were reduced or eliminated in the 2016 and 2019 surveys. For example, hobby group participation in 2011 was 29.7% vs. 35.0% in model vs. non-model communities; the difference narrowed to 35.2% vs. 36.1% (P=0.008). Similarly, providing emotional support increased from 83.9% vs. 87.0% to 93.3% vs. 93.3% (P=0.007). Depressive symptoms decreased from 31.4% vs. 27.2% to 18.6% vs. 20.3% (P<0.001).Conclusions　Promoting community gathering places "Kayoinoba" for six years in communities where many high-risk older adults live may foster social participation, networking, and support and may help reduce health inequalities among communities.

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

A Predictive Self-Organizing Multicellular Computational Model of Infant Skin Permeability to Topically Applied Substances.

Computational models of skin permeability are typically based on assumptions of fixed geometry and homogeneity of the whole epidermis or of epidermal strata and are often limited to adult skin. Infant skin differs quantitatively from that of the adult in its structure and its functional properties, including its barrier function to permeation. To address this problem, we developed a self-organizing multicellular epidermis model of barrier formation with realistic cell morphology. By modulating the parameters relating to cell turnover reflecting those in adult or infant epidermis, we were able to generate accordingly two distinct models. Emerging properties of these models reflect the corresponding experimentally measured values of epidermal and stratum corneum thickness. Diffusion of an externally applied substance (e.g., caffeine) was simulated by a molecular exchange between the model agents, defined by the individual cells and their surrounding extracellular space. By adjusting the surface concentration and the intercellular exchange rate, the model can recapitulate experimental permeability data after topical exposure. By applying these parameters to an infant model, we were able to predict the caffeine concentration profile in infant skin, closely matching experimental results. This work paves the way for a better understanding of skin physiology and function during the first years of life.

Mechanism of mesenchymal stem cells in spinal cord injury repair through macrophage polarization.

Treatment and rehabilitation of spinal cord injury (SCI) is a major problem in clinical medicine. Modern medicine has achieved minimal progress in improving the functions of injured nerves in patients with SCI, mainly due to the complex pathophysiological changes that present after injury. Inflammatory reactions occurring after SCI are related to various functions of immune cells over time at different injury sites. Macrophages are important mediators of inflammatory reactions and are divided into two different subtypes (M1 and M2), which play important roles at different times after SCI. Mesenchymal stem cells (MSCs) are characterized by multi-differentiation and immunoregulatory potentials, and different treatments can have different effects on macrophage polarization. MSC transplantation has become a promising method for eliminating nerve injury caused by SCI and can help repair injured nerve tissues. Therapeutic effects are related to the induced formation of specific immune microenvironments, caused by influencing macrophage polarization, controlling the consequences of secondary injury after SCI, and assisting with function recovery. Herein, we review the mechanisms whereby MSCs affect macrophage-induced specific immune microenvironments, and discuss potential avenues of investigation for improving SCI treatment.

APETALA 2 transcription factor CBX1 is a regulator of mycorrhizal symbiosis and growth of Lotus japonicus.

KEY MESSAGE: An AP2 family gene CBX1 is involved in mycorrhizal symbiosis and growth of Lotus japonicus. APETALA 2 (AP2) transcriptional regulator is highly conserved in plants. CBX1 from Lotus japonicus is a member of AP2 family. AMF (Arbuscular mycorrhizal fungi) inoculation experiment demonstrated that expression of CBX1 was significantly induced by AMF. Further promoter analysis showed that the - 764 to - 498 bp region of the CBX1 promoter containing CTTC motif is the AMF responsive region. Functional analysis of cbx1 mutant suggested CBX1 is critical for mycorrhizal symbiosis, especially for arbuscule formation. Moreover, under noncolonized condition, overexpression of CBX1 reduced the root length of L. japonicus but increased the size of root system and shoot length, whereas cbx1 mutant reduced the root size and shoot length, but not effect on root length. In addition, cbx1 altered activity of monolignol biosynthetic gene and increased lignin levels. Collectively, these data indicated that CBX1 is a positive regulator of symbiotic activity and plays roles in the growth of L. japonicus.

A unique gel matrix moisturizer delivers deep hydration resulting in significant clinical improvement in radiance and texture.

Introduction: As skin ages, it loses its ability to retain moisture and becomes rough and dry. This results in a clinically dull appearance with a loss of radiance, firmness, and suppleness. Symptoms can be improved with use of a moisturizer that builds and maintains skin hydration over time; however, most moisturizers that occlude the skin surface are perceived as heavy and greasy and are not consumer preferred. Methods: A unique, consumer-preferred gel matrix formula was developed by combining liquid crystal structures, which mimic skin barrier lipid assembly, with specific emulsifiers that deliver water deep into skin. Ex vivo studies were conducted to investigate the superior hydrating effects of the gel matrix formula. Confocal Raman microscopy studies assessed the spatial distribution of water in ex vivo skin after application of the gel matrix formula. To determine the effects of the gel matrix formula on dry facial skin, a 12-week clinical study was conducted with subjects with self-perceived skin dryness and dullness. Results: The formulation significantly increased the relative water content throughout epidermal regions, which was not observed with the application of a competitive gel formula. Instrumental measurements assessed improvements in skin surface moisturization and barrier function. Clinical grading showed significant improvements in hydration-related endpoints including radiance, clarity, and texture. Subject self-agree assessment demonstrated that subjects observed improvements in the appearance of their facial skin. Conclusion: These studies demonstrated that the gel matrix formula increased skin water content in deeper layers, and resulted in significant clinical improvements in hydration, barrier function, and clinical appearance of radiance.

Novel confocal Raman microscopy method to investigate hydration mechanisms in human skin.

BACKGROUND: Skin hydration is essential for maintaining stratum corneum (SC) flexibility and facilitating maturation events. Moisturizers contain multiple ingredients to maintain and improve skin hydration although a complete understanding of hydration mechanisms is lacking. The ability to differentiate the source of the hydration (water from the environment or deeper skin regions) upon application of product will aid in designing more efficacious formulations. MATERIALS AND METHODS: Novel confocal Raman microscopy (CRM) experiments allow us to investigate mechanisms and levels of hydration in the SC. Using deuterium oxide (D2 O) as a probe permits the differentiation of endogenous water (H2 O) from exogenous D2 O. Following topical application of D2 O, we first compare in vivo skin depth profiles with those obtained using ex vivo skin. Additional ex vivo experiments are conducted to quantify the kinetics of D2 O diffusion in the epidermis by introducing D2 O under the dermis. RESULTS: Relative D2 O depth profiles from in vivo and ex vivo measurements compare well considering procedural and instrumental differences. Additional in vivo experiments where D2 O was applied following topical glycerin application increased the longevity of D2 O in the SC. Reproducible rates of D2 O diffusion as a function of depth have been established for experiments where D2 O is introduced under ex vivo skin. CONCLUSION: Unique information regarding hydration mechanisms are obtained from CRM experiments using D2 O as a probe. The source and relative rates of hydration can be delineated using ex vivo skin with D2 O underneath. One can envision comparing these depth-dependent rates in the presence and absence of topically applied hydrating agents to obtain mechanistic information.

Using elongated microparticles to enhance tailorable nanoemulsion delivery in excised human skin and volunteers.

This study demonstrates, for the first time, clinical testing of elongated silica microparticles (EMP) combined with tailorable nanoemulsions (TNE) to enhance topical delivery of hydrophobic drug surrogates. Likewise, this is the first report of 6-carboxyfluorescein (a model molecule for topically delivered hydrophobic drugs) AM1 & DAMP4 (novel short peptide surfactants) used in volunteers. The EMP penetrates through the epidermis and stop at the dermal-epidermal junction (DEJ). TNE are unusually stable and useful because the oil core allows high drug loading levels and the surface properties can be easily controlled. At first, we chose alginate as a crosslinking agent between EMP and TNE. We initially incorporated a fluorescent lipophilic dye, DiI, as a hydrophobic drug surrogate into TNE for visualization with microscopy. We compared four different coating approaches to combine EMP and TNE and tested these formulations in freshly excised human skin. The delivery profile characterisation was imaged by dye- free coherent anti-Stoke Raman scattering (CARS) microscopy to detect the core droplet of TNE that was packed with pharmaceutical grade lipid (glycerol) instead of DiI. These data show the EMP penetrating to the DEJ followed by controlled release of the TNE. Freeze-dried formulations with crosslinking resulted in a sustained release profile, whereas a freeze-dried formulation without crosslinking showed an immediate burst-type release profile. Finally, we tested the crosslinked TNE coated EMP formulation in volunteers using multiphoton microscopy (MPM) and fluorescence-lifetime imaging microscopy (FLIM) to document the penetration depth characteristics. These forms of microscopy have limitations in terms of image acquisition speed and imaging area coverage but can detect fluorescent drug delivery through the superficial skin in volunteers. 6-Carboxyfluorescein was selected as the fluorescent drug surrogate for the volunteer study based on the similarity of size, charge and hydrophobicity characteristics to small therapeutic drugs that are difficult to deliver through skin. The imaging data showed a 6-carboxyfluorescein signal deep in volunteer skin supporting the hypothesis that EMP can indeed enhance the delivery of TNE in human skin. There were no adverse events recorded at the time of the study or after the study, supporting the use of 6-carboxyfluorescein as a safe and detectable drug surrogate for topical drug research. In conclusion, dry formulations, with controllable release profiles can be obtained with TNE coated EMP that can effectively enhance hydrophobic payload delivery deep into the human epidermis.

Age-related morphological changes of the dermal matrix in human skin documented in vivo by multiphoton microscopy.

Two-photon fluorescence (TPF) and second harmonic generation (SHG) microscopy provide direct visualization of the skin dermal fibers in vivo. A typical method for analyzing TPF/SHG images involves averaging the image intensity and therefore disregarding the spatial distribution information. The goal of this study is to develop an algorithm to document age-related effects of the dermal matrix. TPF and SHG images were acquired from the upper inner arm, volar forearm, and cheek of female volunteers of two age groups: 20 to 30 and 60 to 80 years of age. The acquired images were analyzed for parameters relating to collagen and elastin fiber features, such as orientation and density. Both collagen and elastin fibers showed higher anisotropy in fiber orientation for the older group. The greatest difference in elastin fiber anisotropy between the two groups was found for the upper inner arm site. Elastin fiber density increased with age, whereas collagen fiber density decreased with age. The proposed analysis considers the spatial information inherent to the TPF and SHG images and provides additional insights into how the dermal fiber structure is affected by the aging process.

Characterizing stratum corneum structure, barrier function, and chemical content of human skin with coherent Raman scattering imaging.

The most superficial layer of the epidermis, the stratum corneum, plays a crucial role in retaining hydration; if its structure or composition is compromised, dry skin may result as a consequence of poor water retention. Dry skin is typically treated with topical application of humectant agents that attract water into the skin. Corneometry, the industry standard for measuring skin hydration, works by assessing the bulk electrical properties of skin. However, this technique samples a large volume of tissue and thus does not resolve the biochemical changes that occur at the cellular level that may underlie mechanisms of dry skin. These limitations can be addressed using coherent Raman scattering (CRS) microscopy to probe the intrinsic vibrational modes of chemical groups such as lipids and water. In the present study, ex vivo human skin explants undergoing dehydration and humectant-induced rehydration were measured via CRS imaging and corneometry. Corneometry data and chemically specific images were obtained from the stratum corneum of each patient sample at each timepoint. The resulting data was statistically analyzed using linear mixed effect model regression analysis. The cellular imaging data revealed water loss in the stratum corneum during dehydration that was correlated with corneometer readings. Interestingly, the imaging data and corneometer readings show differences under the experimental rehydration conditions. The rehydration results suggest that hydration restored by the humectant agents may not be retained by the corneocytes in the ex vivo model system. Given the complementary nature of corneometry, a bulk assessment tool, and CRS microscopy, a modality with subcellular resolution implemented here in an en-face tissue imaging setup, these techniques can be used to measure uptake and efficacy of topical compounds in order to better understand their mode of action and improve therapeutic applications.

Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy.

Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin.

In vivo coherent Raman imaging of the melanomagenesis-associated pigment pheomelanin.

Melanoma is the most deadly form of skin cancer with a yearly global incidence over 232,000 patients. Individuals with fair skin and red hair exhibit the highest risk for developing melanoma, with evidence suggesting the red/blond pigment known as pheomelanin may elevate melanoma risk through both UV radiation-dependent and -independent mechanisms. Although the ability to identify, characterize, and monitor pheomelanin within skin is vital for improving our understanding of the underlying biology of these lesions, no tools exist for real-time, in vivo detection of the pigment. Here we show that the distribution of pheomelanin in cells and tissues can be visually characterized non-destructively and noninvasively in vivo with coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. We validated our CARS imaging strategy in vitro to in vivo with synthetic pheomelanin, isolated melanocytes, and the Mc1re/e, red-haired mouse model. Nests of pheomelanotic melanocytes were observed in the red-haired animals, but not in the genetically matched Mc1re/e; Tyrc/c ("albino-red-haired") mice. Importantly, samples from human amelanotic melanomas subjected to CARS imaging exhibited strong pheomelanotic signals. This is the first time, to our knowledge, that pheomelanin has been visualized and spatially localized in melanocytes, skin, and human amelanotic melanomas.