A mechanism of melanogenesis mediated by E-cadherin downregulation and its involvement in solar lentigines.

OBJECTIVE: Intensive studies have revealed that pleiotropic melanocytic factors are associated with age-spot formation. Dysfunctional keratinocyte differentiation is thought to be an upstream cause of age-spot formation. Although it has been shown that keratinocyte differentiation is mediated by the cell-cell contact factor E-cadherin, its involvement in age-spot formation remains unknown. Thus, to determine the origin of age-spots and an integrated solution for the same, we focused on E-cadherin expression in the present study. METHODS: First, we assessed the solar lentigines in cutaneous and cultured cells by means of immunofluorescence staining. Following that, keratinocytes treated with siRNAs against E-cadherin were co-cultured with melanocytes, and the secreted factors were identified by means of proteomic analysis of the culture supernatants. We also performed quantitative PCR to assess melanogenesis activity and screen ingredients. For behavioural analysis of melanocytes, we performed time-lapse imaging using confocal laser scanning microscopy. RESULTS: E-cadherin expression was downregulated in the epidermis of the solar lentigines, suggesting its involvement in age-spot formation. E-cadherin knocked down keratinocytes not only promoted the secretion of melanocytic/inflammatory factors but also increased melanogenesis by upregulating the expression of melanogenesis factors. Furthermore, live-imaging showed that the downregulation of E-cadherin inhibited melanocyte dynamics and accelerated melanin uptake. Finally, we identified Rosa multiflora fruit extract as a solution that can upregulate E-cadherin expression in keratinocytes. CONCLUSION: Our findings showed that E-cadherin downregulation triggers various downstream melanocytic processes, such as the secretion of melanocytic factors and melanogenesis. Additionally, we showed that the Rosa multiflora fruit extract upregulated E-cadherin expression in keratinocytes.

OBJECTIF: Des études intensives ont révélé que les facteurs mélanocytaires pléiotropiques sont associés à la formation de taches de vieillesse. On pense que la différenciation des kératinocytes dysfonctionnels est une cause en amont de la formation des taches de vieillesse. Bien qu'il ait été démontré que la différenciation des kératinocytes est médiée par le facteur de contact cellule-cellule E-cadhérine, son implication dans la formation des taches de vieillesse reste inconnue. Ainsi, pour déterminer l'origine des taches de vieillesse et une solution intégrée pour celles-ci, nous nous sommes concentrés sur l'expression de la E-cadhérine dans la présente étude. MÉTHODES: Tout d'abord, nous avons évalué les lentigines solaires dans les cellules cutanées et cultivées au moyen d'une coloration par immunofluorescence. Par la suite, les kératinocytes traités avec des siRNA contre l'E-cadhérine ont été co-cultivés avec des mélanocytes, et les facteurs sécrétés ont été identifiés au moyen d'une analyse protéomique des surnageants de culture. Nous avons également effectué une PCR quantitative pour évaluer l'activité de la mélanogénèse et dépister les ingrédients. Pour l'analyse comportementale des mélanocytes, nous avons réalisé une imagerie accélérée à l'aide de la microscopie confocale à balayage laser. RÉSULTATS: L'expression de l'E-cadhérine a été régulée à la baisse dans l'épiderme des lentigines solaires, suggérant son implication dans la formation des taches de vieillesse. Les kératinocytes dans lesquels l'E-cadhérine a été réduite non seulement ont favorisé la sécrétion de facteurs mélanocytaires/inflammatoires, mais ont également accru la mélanogenèse en régulant à la hausse l'expression de facteurs de mélanogenèse. De plus, l'imagerie en direct a montré que la régulation négative de l'E-cadhérine inhibait la dynamique des mélanocytes et accélérait l'absorption de la mélanine. Enfin, nous avons identifié l'extrait de fruit de Rosa multiflora comme une solution capable de réguler positivement l'expression de l'E-cadhérine dans les kératinocytes. CONCLUSION: Nos résultats ont montré que la régulation négative de la E-cadhérine déclenche divers processus mélanocytaires en aval, tels que la sécrétion de facteurs mélanocytaires et la mélanogénèse. De plus, nous avons montré que l'extrait de fruit de Rosa multiflora régulait à la hausse l'expression de l'E-cadhérine dans les kératinocytes.

Direct Quantification of Natural Moisturizing Factors in Stratum Corneum using Direct Analysis in Real Time Mass Spectrometry with Inkjet-Printing Technique.

Proper hydration of the stratum corneum, the skin's outermost layer, is essential for healthy skin. Water-soluble substances called natural moisturizing factors (NMF) are responsible for maintaining adequate moisture in the skin and are closely associated with a variety of the skin's functions. Therefore, quantitative analysis methods for NMF are indispensable when attempting to clarify one of the mechanisms of hydration and its effect on the skin. This study sought to develop a quick and simple analytical technique, which can quantify NMF from the skin without the need for extraction or separation, using direct analysis in real time-mass spectrometry (DART-MS). The goal was to deliver a high quantitative capability, so a unique inkjet printing technique was employed to evenly coat a stable isotope-labeled internal standard (SIL-IS) on tape-stripped skin. This technique allowed for the quantification of 26 NMF with established calibration curves and comparatively high linear correlations. The speed of measurement was found to be advantageous as 100 strips of tape can be measured in roughly 2 hours. The effectiveness of the inkjet coating was also verified by comparing its precision with that of conventional pipetting. This new technique can be an alternative method to quantify NMF rapidly and perhaps allow for a clearer elucidation of their function in skin.

High-resolution Kendrick Mass Defect Analysis of Poly(ethylene oxide)-based Non-ionic Surfactants and Their Degradation Products.

Matrix assisted laser desorption ionization (MALDI) high-resolution mass spectrometry (HRMS) and the recently introduced high-resolution Kendrick mass defect (HRKMD) analysis are combined to thoroughly characterize non-ionic surfactants made of a poly(ethylene oxide) (PEO) core capped by esters of fatty acids. A PEO monostearate surfactant is first analyzed as a proof of principle of the HRKMD analysis conducted with a fraction of EO as the base unit (EO/X with X being an integer) in lieu of EO for a regular KMD analysis. Data visualization is greatly enhanced and the distributions detected in the MALDI mass spectrum are assigned to a pristine (H, OH)-PEO as well as mono- and di-esterified PEO chains with palmitate and stearate end-groups in HRKMD plots computed with EO/45. The MALDI-HRMS/HRKMD analysis is then successfully applied to the more complex case of ethoxylated hydrogenated castor oil (EHCO) found to contain a large number of hydrogenated ricinoleate moieties (up to 14) in its HRKMD plot computed with EO/43, departing from the expected triglyceride structure. The exhaustiveness of the MALDI-HRMS/HRKMD strategy is validated by comparing the so-obtained fingerprints with results from alternative techniques (electrospray ionization MS, size exclusion and liquid adsorption chromatography, ion mobility spectrometry). Finally, aged non-ionic surfactants formed upon hydrolytic degradation are analyzed by MALDI-HRMS/HRKMD to easily assign the degradation products and infer the associated degradation routes. In addition to the hydrolysis of the ester groups observed for EHCO, chain scissions and new polar end-groups are observed in the HRKMD plot of PEO monostearate arising from a competitive oxidative ageing.

Rapid Analysis of Ingredients in Cream Using Ultrasonic Mist-Direct Analysis in Real-Time Time-of-Flight Mass Spectrometry.

A novel method for the simultaneous detection of ingredients in pharmaceutical applications such as creams and lotions was developed. An ultrasonic atomizer has been used to produce a mist containing ingredients. The analyte molecules in the mist can be ionized by using direct analysis in real time (DART) at lower temperature than traditionally used, and we thus solved the problem of normal DART-MS measurement using a high-temperature gas. Thereby, molecular-related ions of heat-unstable components and nonvolatile components became detectable. The deprotonated molecular ion of glycyrrhizic acid (m/z 821), which is unstable at high temperatures, was detected without pyrolysis by ultrasonic mist-DART-MS using unheated helium gas, although it was not detected by normal DART-MS using heated helium gas. The cationized molecular ions of derivatives of polyethylene glycol fatty acid monoesters, which are nonvolatile compounds, were also detected as m/z peaks observed from 800 to 2300. Although the protonated molecular ion of tocopherol acetate was not detected in ionization by ultrasonic mist, it was detected by ultrasonic mist-DART-MS even in the emulsion. It was not necessary to dissolve a sample completely to detect its ions. This method enabled us to obtain the composition of pharmaceutical applications simply and rapidly. Graphical Abstract ᅟ.

Integration of uniform porous shell layers in very long pillar array columns using electrochemical anodization for liquid chromatography.

Electrochemical anodization has been applied to grow porous shell layers of 300 nm (30 nm pores) in 5 µm diameter pillar array columns (PACs) with a spacing of 2.5 µm. Using turn structures preceded and followed by the flow distributor structures recently introduced by our group and filled with radially elongated pillars, columns with quasi unlimited channel lengths could be conceived. The uniformity of the porous PAC was assessed by determining local plate heights along the channel, which appeared to be constant. Minimal (absolute) plate heights (H) between 4 and 6 µm were obtained at optimal flow rates when imposing increasing retention factors. Upon measuring the surface area involved in chromatographic retention as an indicator of the available surface area, an increase in the surface area by a factor of about 30 compared to that of non-anodized pillars was found. On reconfiguring a commercial HPLC instrument to enable on-chip injections, 90% of the performance (expressed in theoretical plates) could be maintained for a 1 m column, while for a 25 cm column severe losses were still observed. As the corresponding pressure drop for optimal operation of retained components is on the order of 10 bar per m only, portable and cheaper HPLC devices with high efficiencies become realistically conceivable.

Phosphorylcholine (PC)-bonded protein A affinity chromatographic medium for high-throughput purification with reduced non-specific protein adsorption.

A protein A affinity chromatographic medium based on porous silica modified with phosphorylcholine (PC) groups and amino groups (PNSP) was synthesized. The PC groups functioned as suppressors of non-specific protein adsorption. Recombinant protein A was bound to the amino groups on PNSP with a glutaraldehyde used as a spacer (PNSP-PA). The PC groups and amino groups were immobilized on porous-silica particles using two silane coupling reagents, PC-bound silane, and 3-aminopropyltrimethoxysilane. After optimizing various factors in the synthetic process, the resultant protein A medium showed improvements in non-specific protein adsorption, dynamic binding capacity, and chemical stability under basic conditions compared with conventional protein A affinity media.

An activated medium with high durability and low nonspecific adsorption: application to protein A chromatography.

Activated media allow the user to easily synthesize a variety of affinity media. We have developed a novel activated medium based on porous silica modified with phosphorylcholine (PC) and N-hydroxysuccinimide (NHS) groups for the purpose of high-throughput purification and reducing nonspecific protein adsorption. The PC groups function as suppressors of nonspecific protein adsorption, whereas the NHS groups are able to covalently bind to the primary amino groups of ligands. Because protein A affinity medium is the most frequently used affinity medium, we prepared protein A media in which a recombinant protein A was bound to the NHS groups of the activated media and evaluated its utility. After optimizing various factors in the synthetic process, the resultant protein A medium showed improved durability at a high flow rate over 300 purification cycles and reduced nonspecific protein adsorption compared with commercially available protein A media.