Circadian Oscillations in Skin and Their Interconnection with the Cycle of Life.

Periodically oscillating biological processes, such as circadian rhythms, are carefully concerted events that are only beginning to be understood in the context of tissue pathology and organismal health, as well as the molecular mechanisms underlying these interactions. Recent reports indicate that light can independently entrain peripheral circadian clocks, challenging the currently prevalent hierarchical model. Despite the recent progress that has been made, a comprehensive overview of these periodic processes in skin is lacking in the literature. In this review, molecular circadian clock machinery and the factors that govern it have been highlighted. Circadian rhythm is closely linked to immunological processes and skin homeostasis, and its desynchrony can be linked to the perturbation of the skin. The interplay between circadian rhythm and annual, seasonal oscillations, as well as the impact of these periodic events on the skin, is described. Finally, the changes that occur in the skin over a lifespan are presented. This work encourages further research into the oscillating biological processes occurring in the skin and lays the foundation for future strategies to combat the adverse effects of desynchrony, which would likely have implications in other tissues influenced by periodic oscillatory processes.

Carotenoids in Human SkinIn Vivo: Antioxidant and Photo-Protectant Role against External and Internal Stressors.

The antioxidant system of the human body plays a crucial role in maintaining redox homeostasis and has an important protective function. Carotenoids have pronounced antioxidant properties in the neutralization of free radicals. In human skin, carotenoids have a high concentration in the stratum corneum (SC)-the horny outermost layer of the epidermis, where they accumulate within lipid lamellae. Resonance Raman spectroscopy and diffuse reflectance spectroscopy are optical methods that are used to non-invasively determine the carotenoid concentration in the human SC in vivo. It was shown by electron paramagnetic resonance spectroscopy that carotenoids support the entire antioxidant status of the human SC in vivo by neutralizing free radicals and thus, counteracting the development of oxidative stress. This review is devoted to assembling the kinetics of the carotenoids in the human SC in vivo using non-invasive optical and spectroscopic methods. Factors contributing to the changes of the carotenoid concentration in the human SC and their influence on the antioxidant status of the SC in vivo are summarized. The effect of chemotherapy on the carotenoid concentration of the SC in cancer patients is presented. A potential antioxidant-based pathomechanism of chemotherapy-induced hand-foot syndrome and a method to reduce its frequency and severity are discussed.

Lateral Dermal Penetration Is Dependent on the Lipophilicity of Active Ingredients.

INTRODUCTION: With its large surface area, skin facilitates a topical administration of active ingredients, and thus percutaneous delivery to a specific target site. Due to its high barrier function and different diffusion characteristics, skin governs the efficacy of these active ingredients and a bioavailability in the epidermal and dermal tissue. OBJECTIVE: In order to characterize the vertical and lateral movement of molecules into and inside the skin, the diffusivity of active ingredients with different physicochemical properties and their penetration ability in different dermal skin layers was investigated. METHODS: A novel lateral dermal microdialysis (MD) penetration setup was used to compare the diffusion characteristics of active ingredients into superficial and deep-implanted MD membranes in porcine skin. The corresponding membrane depth was determined via ultrasound and the active ingredients concentration via high-pressure liquid chromatography measurement. RESULTS: The depth depended penetration of superficial and deep-implanted MD membranes and the quantitative diffusivity of two active ingredients was compared. An experimental lateral MD setup was used to determine the influence of percutaneous skin penetration characteristics of an active ingredient with different lipophilic and hydrophilic characteristics. Therefore, hydrophilic caffeine and lipophilic LIP1, which have an identical molecular weight but different lipophilic characteristics, were tested for their penetration ability inside a propylene glycol and oleic acid formulation. CONCLUSION: The vertical and lateral penetration movement of caffeine was found to exceed that of LIP1 through the hydrophilic dermal environment. The findings of this study show that the lipophilicity of active ingredients influences the penetration movement and that skin enables a conical increasing lateral diffusivity and transdermal delivery.

Microdialysis on Ex Vivo Porcine Ear Skin Can Validly Study Dermal Penetration including the Fraction of Transfollicular Penetration-Demonstrated on Caffeine Nanocrystals.

Common ex vivo methods for penetration investigations often fail to monitor transfollicular penetration appropriately. In the present investigation, the validity of dermal microdialysis on the ex vivo porcine ear skin to investigate penetration kinetics, including transfollicular penetration, was studied. In setup A, a caffeine nanocrystal formulation was compared to a non-particular caffeine gel formulation. In setup B, two caffeine nanocrystal formulations of different sizes (200 nm, 700 nm) were compared to each other. Microdialysis samples were collected for 46 h. After sampling, the skin layers were separated, homogenized, and caffeine was quantified in all samples. In setup A the area under the curve (AUC) after crystal gel formulation application was 12 times higher than after non-particular formulation application. Setup B showed an increased AUC of 42% in the microdialysis data when the 700 nm caffeine crystals were applied compared to the 200 nm crystals. The microdialysis data was supported by the separation, homogenization and extraction data. Microdialysis performed on ex vivo porcine ear skin is a novel experimental setup. It is of high interest for further investigations since it is able to also capture the impact of follicular and transfollicular penetration kinetics as no other ex vivo setup can.

Comparison of Membrane Depth Determination Techniques for Active Ingredient Skin Penetration Studies Using Microdialysis.

INTRODUCTION: The skin is a major physical barrier to the environment, and thus, percutaneous delivery of active ingredients to the dermal target site faces a unique set of hurdles. The efficacy of these active ingredients is governed by their release into the underlying epidermal and dermal tissue, especially when administered topically. OBJECTIVE: The aim of this study was to understand if different physicochemical properties influence the skin penetration of active ingredients and the depth to which they penetrate into the dermis. METHODS: A microdialysis (MD) setup was used to compare the percutaneous penetration in superficial and deep implanted MD membranes in porcine skin. The precise MD membrane depth was determined using histological sectioning paired with microscopy, ultrasound, and a novel computed tomographic approach. RESULTS: In study A, the measured depth of the superficial and deep implanted MD membranes was compared using histological sectioning, ultrasound, and computed tomography. Experimental determination of the depth up to which penetration occurs was found to be crucial to percutaneous penetration studies. In study B, the lipophilic differences of the active ingredients and its influences on the penetration was tested using hydrophilic caffeine and lipophilic LIP1 as model compounds, which have an identical molecular weight with different lipophilic characteristics. It is assumed that the lipophilic characteristics of active ingredients influence their penetration and thus governs the concentration of these molecules reaching their target site. CONCLUSION: The transdermal penetration of caffeine was found to exceed that of LIP1 through the hydrophilic environment of the dermis. Thus, the findings of this study show that the precise MD dermis localization and the physicochemical properties, such as lipophilicity, influence the penetration rate of active ingredients and lay the foundation for creating optimized transdermal delivery systems.

Retaining Skin Barrier Function Properties of the Stratum Corneum with Components of the Natural Moisturizing Factor-A Randomized, Placebo-Controlled Double-Blind In Vivo Study.

The influence of a topically applied formulation containing components of natural moisturizing factor (NMF) on barrier-related parameters of the stratum corneum (SC) was investigated in vivo using confocal Raman microspectroscopy in a randomized, placebo-controlled double-blind study on 12 volunteers for 14 days. This method allowed for the elucidation of subtle differences between the verum and the placebo even though the components of the verum naturally occur in the SC. This differentiation is not possible non-invasively by conventional methods. In this study, we found that the applied verum and placebo formulations disrupted the equilibrium of water, NMF and lipids in the SC. The adverse effects of the formulation could be mitigated by incorporating it into a simplified supplementation of NMF molecules. As a long-term effect, the amount of strongly bound water increases at 30-40% SC depth (p < 0.05) and the amount of weakly bound water decreases at 30-40% SC depth (p < 0.05) for the verum. This supplement was also unexpectedly able to prevent intercellular lipids (ICL) disorganization in selected depths. In the long term, the verum treatment limited the lateral disorganization of the ICL to the upper 20% SC depth. Further research is required to elucidate the interplay of these factors in the SC, to better understand their contribution to the equilibrium and barrier function of the skin. This understanding of the interaction of these naturally occurring components could help in the future to develop and optimize topical treatments for diseases like psoriasis, atopic dermatitis, ichthyosis where the skin barrier is disrupted.

Drug Development for Target Ribosomal Protein rpL35/uL29 for Repair of LAMB3R635X in Rare Skin Disease Epidermolysis Bullosa.

INTRODUCTION: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. METHODS: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. RESULTS: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. CONCLUSION: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.

Investigation of transfollicular caffeine penetration using microdialysis on ex vivo porcine ear skin.

The aim of this study was to develop an ex vivo method that allows to quantify the transfollicular penetration of topically applied substances by combining microdialysis and selective follicular closure with varnish. An experimental setup with three skin areas on ex vivo intact porcine ear skin was designed (varnish on hair follicle, varnish next to hair follicle, no varnish). On each area, 10 µl/cm2 caffeine-hydroxyethyl-cellulose-gel was applied. Samples were collected for 22 h by microdialysis. After sampling, the skin layers were separated, homogenized and caffeine was quantified by high pressure liquid chromatography (HPLC) in all samples. Potential impact of the varnish placed next to the follicle by tension on the follicle during the drying process was monitored by a microscopic setup and could be excluded. The microdialysis and homogenization study showed a significantly reduced penetration of caffeine when the hair follicles were closed. In areas with open hair follicles caffeine was detected already in the first ten minutes after application. The reported novel combination of two methods is suitable to investigate ex vivo transfollicular penetration. Possible impact of the closure material in the control area can be ruled out by adjusting the design of the control area in future studies.

Solvent-Containing Closure Material Can Be Used to Prevent Follicular Penetration of Caffeine and Fluorescein Sodium Salt on Porcine Ear Skin.

AIM: The skin represents a drug delivery portal. The establishment of a skin model capable of distinguishing between the follicular and intercellular penetration pathways remains a challenge. The study described herein was aimed at showing the influence of two nail varnishes as closure material and four application techniques to spread the active pharmaceutical ingredient (API) on a successful follicular closure without inducing penetration-enhancing effects. MATERIALS AND METHODS: For all experiments, ex vivo porcine ear skin was used. In study design A, a standard and a solvent-free nail varnish were compared. It was tested whether the different application techniques (spreading with pipette, careful finger massage, 5-Hz finger massage, 5-Hz automatic massage) potentially destroy an intact follicular closure. Laser scanning microscopy imaging was used to measure if the model drug (fluorescein sodium salt) penetrated into the hair follicles. Study design B investigated how the penetration is affected when applying standard nail varnish containing solvents to skin. It was tested if the varnish blocks the API (caffeine) on completely covered areas and if adjacent areas show increased penetration. Furthermore, lateral diffusion of the API was investigated. After 20 h, the skin layers were separated by tape stripping and heat separation. The tissue samples were homogenized. Caffeine was quantified by chromatography. RESULTS: In study design A, the standard nail varnish showed a secure follicular closure, while the solvent-free nail varnish was not able to prevent follicular penetration. Moreover, rapid application techniques were found to destroy an intact follicular closure. Only the two most gentle application techniques kept the follicular closing intact. In study design B, no caffeine was detected in both skin areas that were completely covered. Since no significant difference in caffeine penetration between the two uncovered groups was found, any influence of the applied closure material on adjacent areas was excluded. CONCLUSION: This study clearly demonstrates that a standard nail varnish in combination with a gentle application technique of the API provides a secure follicular closure. The presented study only investigated the closure for the substances caffeine and fluorescein sodium salt. The results might not be transferable to all kinds of APIs.

Amino acids in the cultivation of mammalian cells.

Amino acids are crucial for the cultivation of mammalian cells. This importance of amino acids was realized soon after the development of the first cell lines, and a solution of a mixture of amino acids has been supplied to cultured cells ever since. The importance of amino acids is further pronounced in chemically defined mammalian cell culture media, making the consideration of their biological and chemical properties necessary. Amino acids concentrations have been traditionally adjusted to their cellular consumption rates. However, since changes in the metabolic equilibrium of amino acids can be caused by changes in extracellular concentrations, metabolomics in conjunction with flux balance analysis is being used in the development of culture media. The study of amino acid transporters is also gaining importance since they control the intracellular concentrations of these molecules and are influenced by conditions in cell culture media. A better understanding of the solubility, stability, dissolution kinetics, and interactions of these molecules is needed for an exploitation of these properties in the development of dry powdered chemically defined media for mammalian cells. Due to the complexity of these mixtures however, this has proven to be challenging. Studying amino acids in mammalian cell culture media will help provide a better understanding of how mammalian cells in culture interact with their environment. It would also provide insight into the chemical behavior of these molecules in solutions of complex mixtures, which is important in the understanding of the contribution of individual amino acids to protein structure.

Isolation of subcellular organelles and structures.

One of the major challenges in functional proteomics is the separation of complex protein mixtures to allow detection of low abundance proteins and provide for reliable quantitative and qualitative analysis of proteins impacted by environmental parameters. Prerequisites for the success of such analyses are standardized and reproducible operating procedures for sample preparation prior to protein separation. Due to the complexity of total proteomes, especially of eukaryotic proteomes, and the divergence of protein properties, it is often beneficial to prepare standardized partial proteomes of a given organism to maximize the coverage of the proteome and to increase the chance to visualize low abundance proteins and make them accessible for subsequent analysis. In this chapter we will describe with detailed recipes procedures for the enrichment and isolation of the currently most investigated organelles and subcellular compartments in mammalian cells using classical centrifugation techniques to more sophisticated immunoaffinity-based procedures.