Ratiometric fluorescence nanoscopy and lifetime imaging of novel Nile Red analogs for analysis of membrane packing in living cells.

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MINFLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging.

From static to dynamic: The influence of mechanotransduction on skin equivalents analyzed by bioimaging and RNAseq.

In this study, we explore the impact of mechanical stimuli on skin models using an innovative skin-on-a-chip platform, addressing the limitations of conventional transwell-cultured skin equivalents. This platform facilitates cyclic mechanical stimulation through compression and stretching, combined with automated media perfusion. Our findings, using bioimaging and bulk RNA sequencing, reveal increased expression of Keratin 10 and Keratin 14, indicating enhanced skin differentiation and mechanical integrity. The increase in desmosomes and tight junctions, observed through Claudin-1 and Desmoplakin 1 & 2 analysis, suggests improved keratinocyte differentiation due to mechanical stimulation. Gene expression analyses reveal a nuanced regulatory response, suggesting a potential connection to the Hippo pathway, indicative of a significant cellular reaction to mechanical stimuli. The results show the important influence of mechanical stimulation on skin model integrity and differentiation, demonstrating the potential of our microfluidic platform in advancing skin biology research and pharmaceutical testing.

Structural characterization of solvent-based food preparation of jellyfish.

Jellyfish as a potential sustainable food material has recently gained increasing interest. However, with their soft gel-like texture and easy spoilage, it remains challenging to achieve desirable edible structures from jellyfish. The culinary preparation of jellyfish is a complex process and extends beyond conventional cooking methods. In this study, we investigate the transformation of jellyfish into crispy-like structures by manipulating their microstructural and mechanical properties through a solvent-based preparation. The study focuses on the use of "poor solvents", namely ethanol and acetone, and employs rheology measurements and quantitative microscopy techniques to analyze the effects of these solvents on the mechanical properties and microstructure of jellyfish. Our findings reveal that both ethanol and acetone lead to a significant increase in jellyfish hardness and deswelling. Notably, a micro-scale network is formed within the jellyfish matrix, and this network is then mechanically reinforced before a crispy-like texture can be obtained. Our study points to solvent polarity as also being a crucial factor for creating these effects and determines an upper polarity limit in the range of 12.2-12.9 MPa1/2 for added solvents, corresponding to approximately 60% of added ethanol or 70% of added acetone. Our study highlights that solvent-based preparation serves as a "reverse cooking" technique, where mechanical modification rather than traditional softening mechanisms are employed to stabilize and strengthen the microstructures and fibers of jellyfish. By elucidating the underlying mechanisms of solvent-induced stabilization, our findings may facilitate the development of innovative and sustainable culinary practices, paving the way for broader applications of jellyfish and other soft edible materials in the gastronomic landscape.

Novel Chip for Applying Mechanical Forces on Human Skin Models Under Dynamic Culture Conditions.

In recent years the need for in vitro skin models as a replacement for animal studies has resulted in significant progress in the development of skin-on-a-chip models. These devices allow the fine control of the microenvironment of the model and the incorporation of chemical and physical stimuli. In this study, we describe the development of an easy and low-budget open-top dynamic microfluidic device for skin-on-a-chip experiments using polydimethylsiloxane and a porous polyethylene terephthalate membrane. The chip allows the incorporation of compressive stimuli during the cultivation period by the use of syringe pumps. Proof-of-concept results show the successful differentiation of the cells and establishment of the skin structure in the chip. The microfluidic skin-on-a-chip models presented in this study can serve as a platform for future drug and feasibility studies.

pH-Dependence Cytotoxicity Evaluation of Artepillin C against Tumor Cells.

Brazilian green propolis is a well-known product that is consumed globally. Its major component, Artepillin C, showed potential as an antitumor product. This study explored the impact of Artepillin C on fibroblast and glioblastoma cell lines, used as healthy and very aggressive tumor cell lines, respectively. The focus of the study was to evaluate the pH-dependence of Artepillin C cytotoxicity, since tumor cells are known to have a more acidic extracellular microenvironment compared to healthy cells, and Artepillin C was shown to become more lipophilic at lower pH values. Investigations into the pH-dependency of Artepillin C (6.0-7.4), through viability assays and live cell imaging, revealed compelling insights. At pH 6.0, MTT assays showed the pronounced cytotoxic effects of Artepillin C, yielding a notable reduction in cell viability to less than 12% among glioblastoma cells following a 24 h exposure to 100 µM of Artepillin C. Concurrently, LDH assays indicated significant membrane damage, affecting approximately 50% of the total cells under the same conditions. Our Laurdan GP analysis suggests that Artepillin C induces autophagy, and notably, provokes a lipid membrane packing effect, contributing to cell death. These combined results affirm the selective cytotoxicity of Artepillin C within the acidic tumor microenvironment, emphasizing its potential as an effective antitumor agent. Furthermore, our findings suggest that Artepillin C holds promise for potential applications in the realm of anticancer therapies given its pH-dependence cytotoxicity.

Fate of microplastic captured in the marine demosponge Halichondria panicea.

Microplastic particles are widespread pollutants in the sea and filter-feeding sponges have recently been suggested as useful monitoring organisms. However, the fate of microplastic particles in sponges is poorly understood, yet crucial for interpreting monitoring data. The present study aims to help develop sponges as more useful monitoring organisms for microplastic in the sea. Here, we describe the fate of inedible (2 and 10 µm) plastic beads compared to that of edible bacteria and algal cells captured in the marine demosponge Halichondria panicea. Small Cyanobium bacillare cells entered the choanocyte chambers and were phagocytized by choanocytes, while larger Rhodomonas salina cells were captured in incurrent canals and phagocytized in the mesohyl. Small 2 µm-beads were captured by choanocytes and subsequently expelled into the excurrent canals after 58 ± 34 min. Larger 10 µm-beads were captured in the incurrent canals and transferred to the mesohyl, where amoeboid cells moved them across the mesohyl before they were expelled into the excurrent canal after 95 ± 36 min. SEM observations further indicated engulfment of plastic beads on the outer sponge surface. This insight provides useful information on how sponges, in general, treat microplastic particles of various sizes. It helps us understand actual measured sizes and concentrations of microplastic particles in sponges in relation to those in the ambient water.

Helium Ion Microscopy and Sectioning of Spider Silk.

Focused ion beams have recently emerged as a powerful tool for ultrastructural imaging of biological samples. In this article, we show that helium ion microscopy (HIM), in combination with ion milling, can be used to visualize the inner structure of both major and minor ampullate silk fibers of the orb-web weaving spider Nephila madagascariensis. The internal nanofibrils were imaged in pristine silk fibers, with little or no damage to the sample structure observed. Furthermore, a method to cut/rupture the fibers using He+ ions combined with internal sample tension is presented. This showed that the stretching and rupturing of spider silk is a highly dynamic process with considerable material reorganization.

Nanoscale imaging of major and minor ampullate silk from the orb-web spider Nephila Madagascariensis.

Spider silk fibres have unique mechanical properties due to their hierarchical structure and the nanoscale organization of their proteins. Novel imaging techniques reveal new insights into the macro- and nanoscopic structure of Major (MAS) and Minor (MiS) Ampullate silk fibres from pristine samples of the orb-web spider Nephila Madagascariensis. Untreated threads were imaged using Coherent Anti-Stokes Raman Scattering and Confocal Microscopy, which revealed an outer lipid layer surrounding an autofluorescent protein core, that is divided into two layers in both fibre types. Helium ion imaging shows the inner fibrils without chemical or mechanical modifications. The fibrils are arranged parallel to the long axis of the fibres with typical spacing between fibrils of 230 nm ± 22 nm in the MAS fibres and 99 nm ± 24 nm in the MiS fibres. Confocal Reflection Fluorescence Depletion (CRFD) microscopy imaged these nano-fibrils through the whole fibre and showed diameters of 145 nm ± 18 nm and 116 nm ± 12 nm for MAS and MiS, respectively. The combined data from HIM and CRFD suggests that the silk fibres consist of multiple nanoscale parallel protein fibrils with crystalline cores oriented along the fibre axes, surrounded by areas with less scattering and more amorphous protein structures.

Spectral Unmixing for Label-Free, In-Liquid Characterization of Biomass Microstructure and Biopolymer Content by Coherent Raman Imaging.

Characterization of lignocellulosic biomass microstructure with chemical specificity and under physiological conditions could provide invaluable insights to our understanding of plant tissue development, microstructure, origins of recalcitrance, degradation, and solubilization. However, most methods currently available are either destructive, are not compatible with hosting a physiological environment, or introduces exogenous probes, complicating their use for studying changes in microstructure and mechanisms of plant development, recalcitrance, or degradation in situ. To address these challenges, we here present a multi-modal chemically specific imaging technique based on coherent anti-Stokes Raman scattering (CARS) microspectroscopy with simplex maximization and entropy-based spectral unmixing enabling label-free, chemically specific characterization of plant microstructure in liquid. We describe how spatial drift of samples suspended in liquid can introduce artifacts in spectral unmixing procedures for single-frequency CARS and propose a mitigative strategy toward these effects using simultaneously acquired forward-scattered CARS signals and epi-detected autofluorescence. We further apply the technique for chemical and microstructural characterization of untreated and liquid hot water pretreated rapeseed straw by CARS and show how the framework can be extended for 3D imaging with chemical specificity. Finally, we provide examples of the intricate chemical and microstructural details recovered by this hybrid imaging technique, including discerning between primary and secondary cell walls, localization of aqueous components to cell lumina, and the presence of funnel-type pits in samples ofBrassica napus.

Dissolvable microneedles for transdermal drug delivery showing skin pentation and modified drug release.

Topical therapies for chronic skin diseases suffer from a low patient compliance due to the inconvenient treatment regimens of available products. Dissolvable microneedles (MN) with modified release offer an interesting possibility to increase the compliance by acting as a depot in the skin and thereby decreasing the dosing frequency. Furthermore, the bioavailability can be increased significantly by bypassing the barrier of the skin by the direct penetration of the MN into the skin. In this study the depot effect and skin penetration of an innovative dissolvable MN patch was assessed by insertion in ex vivo human skin and in vivo using minipigs. The MN patches are based on biodegradable polymers and the active pharmaceutical ingredients calcipotriol (Calci) and betamethasone-17-21-dipropionate (BDP) used to treat psoriasis. Using computed tomography (CT) and Coherent anti-Stokes Raman scattering (CARS) microscopy it was possible to visualize the skin penetration and follow the morphology of the MN as function of time in the skin. The depot effect was assessed by studying the modified in vitro release in an aqueous buffer and by comparing the drug release of a single application of a patch both ex vivo and in vivo to daily application of a marketed oleogel containing the same active pharmaceutical ingredients. The CT and CARS images showed efficient penetration of the MN patches into the upper dermis and a slow swelling process of the drug containing tip over a period of 8 days. Furthermore, CARS demonstrated that it can be used as a noninvasive technique with potential applicability in clinical settings. The in vitro release studies show a release of 54% over a time period of 30 days. The pharmacological relevance of MNs was confirmed in human skin explants and in vivo after single application and showed a similar response on calcipotriol and BDP mediated signaling events compared to daily application of the active oleogel. Altogether it was demonstrated that the MN can penetrate the skin and have the potential to provide a depot effect.

Evaluation of native and non-native biomaterials for engineering human skin tissue.

A variety of human skin models have been developed for applications in regenerative medicine and efficacy studies. Typically, these employ matrix molecules that are derived from non-human sources along with human cells. Key limitations of such models include a lack of cellular and tissue microenvironment that is representative of human physiology for efficacy studies, as well as the potential for adverse immune responses to animal products for regenerative medicine applications. The use of recombinant extracellular matrix proteins to fabricate tissues can overcome these limitations. We evaluated animal- and non-animal-derived scaffold proteins and glycosaminoglycans for the design of biomaterials for skin reconstruction in vitro. Screening of proteins from the dermal-epidermal junction (collagen IV, laminin 5, and fibronectin) demonstrated that certain protein combinations when used as substrates increase the proliferation and migration of keratinocytes compared to the control (no protein). In the investigation of the effect of components from the dermal layer (collagen types I and III, elastin, hyaluronic acid, and dermatan sulfate), the primary influence on the viability of fibroblasts was attributed to the source of type I collagen (rat tail, human, or bovine) used as scaffold. Furthermore, incorporation of dermatan sulfate in the dermal layer led to a reduction in the contraction of tissues compared to the control where the dermal scaffold was composed primarily of collagen type I. This work highlights the influence of the composition of biomaterials on the development of complex reconstructed skin models that are suitable for clinical translation and in vitro safety assessment.

The ROC skin model: A robust skin equivalent for permeation and live cell imaging studies.

Rat epidermal keratinocyte (REK) Organotypic culture (ROC) is an epidermis model that is robust and inexpensive to develop and maintain, and it has in previous studies been shown to have permeability characteristics close to those of human skin. Here, we characterize the model further by structural comparison to native human and rat skin and by investigating functional characteristics of lipid packing, polarity, and permeability coefficients. We show that the ROC model has structural similarities to native human skin and observe human skin-like permeability coefficients for testosterone and mannitol. We develop a transwell device that allows live cell microscopy of the tissue at the air-liquid interface and establish transgenic cell lines expressing different fluorescently tagged proteins. This enables showing the migration of keratinocytes during the first days after seeding, finding that keratinocytes have a higher mean migration rate in the earlier days of development. Collectively, our results show that the ROC model is an inexpensive and robust epidermis model that works reproducibly across laboratories.

Multilayer diffusion modeling and Coherent anti-Stokes Raman scattering microscopy for spatially resolved water diffusion measurements in human skin.

In this work using Coherent anti-Stokes Raman Scattering microscopy, it was possible to directly measure the time dependent, spatially resolved change in concentration of water (D2 O) in intact skin tissue with a spatial resolution of under 1 µm, and combined with a multilayer diffusion model, diffusion coefficients at different depths in the tissue were extracted. The results show that the diffusion varies at different layers throughout the Stratum Corneum (SC), indicating that the SC is not a homogeneous barrier but a complicated heterogeneous structure. Interestingly, averaging over the diffusion at the different depths and samples gave a relatively constant value of 0.047 ± 0.01 µm2 /second. Treating the skin with acetone or tape stripping led to an increased diffusion coefficient of 0.064 ± 0.02 µm2 /second and 0.079 ± 0.03 µm2 /second, respectively. The combined method and model presented here shows potential for wide applications for measuring spatially resolved diffusion of different substances in a variety of different samples.

Polarized α-synuclein trafficking and transcytosis across brain endothelial cells via Rab7-decorated carriers.

Parkinson's disease is mainly caused by aggregation of α-synuclein (α-syn) in the brain. Exchange of α-syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of α-syn across the blood brain-barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of α-syn monomers and oligomers across an in vitro BBB model system. Both α-syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric α-syn in brain endothelial cells, we investigated co-localization of α-syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric α-syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric α-syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric α-syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric α-syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric α-syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinson's disease and related synucleinopathies.

Fractional CO2 laser ablation leads to enhanced permeation of a fluorescent dye in healthy and mycotic nails-An imaging investigation of laser-tissue effects and their impact on ungual drug delivery.

PURPOSE: Conventional oral antifungal therapies for onychomycosis (OM) often do not achieve complete cure and may be associated with adverse effects, medical interactions, and compliance issues restricting their use in a large group of patients. Topical treatment can bypass the systemic side effects but is limited by the physical barrier of the nail plate. Ablative fractional laser (AFL) treatment can be used to improve the penetration of topical drugs into the nail. This study visualized the effects of laser ablation of nail tissue and assessed their impact on the biodistribution of a fluorescent dye in healthy and fungal nail tissue. METHODS: For the qualitative assessment of CO2 AFL effects on healthy nail tissue, scanning electron microscopy (SEM), coherent anti-Stokes Raman scattering microscopy (CARS-M), and widefield fluorescence microscopy (WFM) were used. To quantitate the effect of laser-pretreatment on the delivery of a fluorescent dye, ATTO-647N, into healthy and fungal nail tissue, ablation depth, nail plate thickness, and ATTO-647N fluorescence intensity in three nail plate layers were measured using WFM. A total of 30 nail clippings (healthy n = 18, fungal n = 12) were collected. An aqueous ATTO-647N solution was directly applied to the dorsal surface of 24 nail samples (healthy n = 12, fungal n = 12) and incubated for 4 hours, of which half (healthy n = 6, fungal n = 6) had been pretreated with AFL (30 mJ/mb, 15% density, 300 Hz, pulse duration <1 ms). RESULTS: Imaging revealed a three-layered nail structure, an AFL-induced porous ablation crater, and changes in autofluorescence. While intact fungal samples showed a 106% higher ATTO-647N signal intensity than healthy controls, microporation led to a significantly increased fluorophore permeation in all samples (p < 0.0001). AFL processing of nail tissue enhanced topical delivery of ATTO-647N in all layers, (average increase: healthy +108%, fungal +33%), most pronounced in the top nail layer (healthy +122%, fungal +68%). While proportionally deeper ablation craters correlated moderately with higher fluorescence intensities in healthy nail tissue, fungal samples showed no significant relationship. CONCLUSION: Fractional CO2 laser microporation is a simple way of enhancing the passive delivery of topically applied ATTO-647N. Although the impaired nail plate barrier in OM leads to greater diffusion of the aqueous solution, AFL can increase the permeability of both structurally deficient and intact nails.

Enhancing the sweat resistance of sunscreens.

BACKGROUND: While sunbathing of performing outdoor sport activities, sunscreens are important for protection of uncovered skin against ultraviolet (UV) radiation. However, perspiration negatively affects the performance of a sunscreen film by weakening its substantivity and uniformity through the activation of two mechanisms, namely sunscreen wash-off and sunscreen redistribution. MATERIAL AND METHODS: We used a perspiring skin simulator to investigate the effect of sunscreen formulation on its efficiency upon sweating. Specifically, we modified the sunscreen formulation by incorporating a hydrophobic film former and adding water-absorbing particles. Sunscreen performance before and after perspiration is assessed by in vitro sun protection factor measurements, direct detection of changes in the sunscreen distribution using UV reflectance imaging, and by coherent anti-Stokes Raman scattering (CARS) microscopy for microscopic characterization of the UV filter relocation. RESULTS: The results show that incorporating a hydrophobic film former can decrease sunscreen wash-off due to sweating, while an excessive amount of film former might negatively affect the sunscreen distribution. The addition of water-absorbing particles, on the other hand, had either a negative or positive impact on the sunscreen substantivity, depending on the particle properties. While the addition of large water-absorbing particles appeared to increase sunscreen redistribution, smaller particles that could form a gel-like structure upon contact with water, appeared to change sunscreen wetting and sweat droplet spreading, thereby decreasing sunscreen wash-off and sunscreen redistribution. CONCLUSIONS: We find that using a combination of hydrophobic film formers, which increase water resistance, and small water-absorbing particles, which change the wetting behavior, can make sunscreen formulations more sweat-resistant and less runny.

In vitro skin model for characterization of sunscreen substantivity upon perspiration.

OBJECTIVE: The resistance of sunscreens to the loss of ultraviolet (UV) protection upon perspiration is important for their practical efficacy. However, this topic is largely overlooked in evaluations of sunscreen substantivity due to the relatively few well-established protocols compared to those for water resistance and mechanical wear. METHODS: In an attempt to achieve a better fundamental understanding of sunscreen behaviour in response to sweat exposure, we have developed a perspiring skin simulator, containing a substrate surface that mimics sweating human skin. Using this perspiring skin simulator, we evaluated sunscreen performance upon perspiration by in vitro sun protection factor (SPF) measurements, optical microscopy, ultraviolet (UV) reflectance imaging and coherent anti-Stokes Raman scattering (CARS) microscopy. RESULTS AND CONCLUSION: Results indicated that perspiration reduced sunscreen efficiency through two mechanisms, namely sunscreen wash-off (impairing the film thickness) and sunscreen redistribution (impairing the film uniformity). Further, we investigated how the sweat rate affected these mechanisms and how sunscreen application dose influenced UV protection upon perspiration. As expected, higher sweat rates led to a large loss of UV protection, while a larger application dose led to larger amounts of sunscreen being washed-off and redistributed but also provided higher UV protection before and after sweating.

OBJECTIF: La résistance des écrans solaires à la perte de protection contre les ultraviolets (UV) à cause de la transpiration est importante quant à leur efficacité pratique. Cependant, ce point est généralement négligé dans les évaluations de la substantivité des écrans solaires en raison du nombre relativement faible de protocoles bien établis, en comparaison avec ceux pour la résistance à l'eau et l'usure mécanique. MÉTHODES: Dans le but de parvenir à une meilleure compréhension fondamentale du comportement des écrans solaires en cas d'exposition à la sueur, nous avons développé un simulateur de peau transpirante, dont la surface de substrat imite la transpiration de la peau humaine. À l'aide de ce simulateur, nous avons évalué les performances des écrans solaires lors de la transpiration par des mesures in vitro du facteur de protection solaire (FPS), par microscopie optique, par imagerie de la réflectance ultraviolette (UV) et par microscopie cohérente de diffusion Raman anti-Stokes (coherent anti-Stokes Raman scattering, CARS). RÉSULTATS ET CONCLUSION: Les résultats ont montré que la transpiration réduisait l'efficacité de l'écran solaire en raison de deux mécanismes, à savoir le lavage de l'écran solaire (altération de l'épaisseur du film) et la redistribution de l'écran solaire (altération de l'uniformité du film). De plus, nous avons étudié comment le taux de transpiration affectait ces mécanismes et comment la dose d'application d'écran solaire influençait la protection UV en cas de transpiration. Comme l'on pouvait s'y attendre, des taux de sueur plus élevés ont entraîné une perte importante de protection contre les UV, tandis qu'une dose d'application plus importante a conduit à des quantités plus importantes d'écran solaire lavé et redistribué, mais a également fourni une protection contre les UV plus élevée avant et après la transpiration.

Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells.

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, ß1, ß2, ß3, and phospholemman. The α1, α2, ß1, and ß2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.

Epidermal Acyl-CoA-binding protein is indispensable for systemic energy homeostasis.

OBJECTIVES: The skin is the largest sensory organ of the human body and plays a fundamental role in regulating body temperature. However, adaptive alterations in skin functions and morphology have only vaguely been associated with physiological responses to cold stress or sensation of ambient temperatures. We previously found that loss of acyl-CoA-binding protein (ACBP) in keratinocytes upregulates lipolysis in white adipose tissue and alters hepatic lipid metabolism, suggesting a link between epidermal barrier functions and systemic energy metabolism. METHODS: To assess the physiological responses to loss of ACBP in keratinocytes in detail, we used full-body ACBP-/- and skin-specific ACBP-/- knockout mice to clarify how loss of ACBP affects 1) energy expenditure by indirect calorimetry, 2) response to high-fat feeding and a high oral glucose load, and 3) expression of brown-selective gene programs by quantitative PCR in inguinal WAT (iWAT). To further elucidate the role of the epidermal barrier in systemic energy metabolism, we included mice with defects in skin structural proteins (ma/ma Flgft/ft) in these studies. RESULTS: We show that the ACBP-/- mice and skin-specific ACBP-/- knockout mice exhibited increased energy expenditure, increased food intake, browning of the iWAT, and resistance to diet-induced obesity. The metabolic phenotype, including browning of the iWAT, was reversed by housing the mice at thermoneutrality (30 °C) or pharmacological ß-adrenergic blocking. Interestingly, these findings were phenocopied in flaky tail mice (ma/ma Flgft/ft). Taken together, we demonstrate that a compromised epidermal barrier induces a ß-adrenergic response that increases energy expenditure and browning of the white adipose tissue to maintain a normal body temperature. CONCLUSIONS: Our findings show that the epidermal barrier plays a key role in maintaining systemic metabolic homeostasis. Thus, regulation of epidermal barrier functions warrants further attention to understand the regulation of systemic metabolism in further detail.

Substituted 9-Diethylaminobenzo[a]phenoxazin-5-ones (Nile Red Analogues): Synthesis and Photophysical Properties.

Nile Red is a benzo[a]phenoxazone dye containing a diethylamino substituent at the 9-position. In recent years, it has become a popular histological stain for cellular membranes and lipid droplets due to its unrivaled fluorescent properties in lipophilic environments. This makes it an attractive lead for chemical decoration to tweak its attributes and optimize it for more specialized microscopy techniques, e.g., fluorescence lifetime imaging or two-photon excited fluorescence microscopy, to which Nile Red has never been optimized. Herein, we present synthesis approaches to a series of monosubstituted Nile Red derivatives (9-diethylbenzo[a]phenoxazin-5-ones) starting from 1-naphthols or 1,3-naphthalenediols. The solvatochromic responsiveness of these fluorophores is reported with focus on how the substituents affect the absorption and emission spectra, luminosity, fluorescence lifetimes, and two-photon absorptivity. Several of the analogues emerge as strong candidates for reporting the polarity of their local environment. Specifically, the one- and two-photon excited fluorescence of Nile Red turns out to be very responsive to substitution, and the spectroscopic features can be finely tuned by judiciously introducing substituents of distinct electronic character at specific positions. This new toolkit of 9-diethylbenzo[a]phenoxazine-5-ones constitutes a step toward the next generation of optical molecular probes for advancing the understanding of lipid structures and cellular processes.