An innervated skin 3D in vitro model for dermatological research

A 3D in vitro model of innervated skin would be a useful tool in dermatological research to study the effect of different chemicals and compounds on the sensory properties of skin. Current innervated skin models are limited in composition and often composed of ex vivo skin explants and/or animal-derived material. In this study, our aim was to develop a human innervated skin model with a better biomimicry composition for in vitro research. Fibrin hydrogel and aligned electrospun fibers of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) were used as a scaffold to generate the 3D in vitro model. The skin component was made of primary human keratinocytes and primary human fibroblasts, while the neuronal component was composed of iPSC-derived sensory neurons. Our results showed that the dermal component consisted of fibroblasts and synthesized collagen. The epidermal component was characterized by the expression of keratins 10 and 14, and involucrin. Finally, sensory neurons extended axons throughout the scaffold and reached the epidermis. Treating the model with a capsaicin solution for 30 min, which was performed as a proof of concept test for sensitization studies, resulted into partial depletion of substance P and tubulin β3. This model could be used for studying skin-neuron interactions and cutaneous toxicity.</p

Insight into the antioxidant effect of fermented and non-fermented Spirulina water and ethanol extracts at the proteome level using a yeast cell model

Spirulina is rich in various antioxidants and nutraceuticals and it has proven to be effective in the treatment of various pathological conditions. This study explores the antioxidant effect of fermented and non-fermented Spirulina extracts on the proteome level using the yeast Saccharomyces cerevisiae as a model organism. Yeast cells were treated with fermented Spirulina water extract (SV), non-fermented Spirulina water extract (NFV), fermented Spirulina ethanol extract (SE), and nonfermented Spirulina ethanol extract (NFE). Cell lysates were prepared, and label-free quantitative proteome analysis was performed. In SV, when compared to NFV samples, the levels of most differentially expressed proteins were upregulated. Alternatively, SE compared to NFE samples showed a significant downregulation for the majority of the analyzed proteins involved in different cellular processes. Additionally, a higher downregulation of stress response related proteins was observed in SE compared to NFE samples, while their abundance in SV samples increased compared to NFV. This study provided a global view, on a proteome level, of how cells cope with exogenous antioxidants and remodel their cellular processes to maintain metabolic and redox balance. Furthermore, it combined for the first time the analysis of different extract effect, including the contribution of lactic acid fermentation to the cell activity

Short-term high fat-feeding results in morphological and metabolic adaptations in the skeletal muscle of C57BL/6J mice

The prevalence of the metabolic syndrome (MS) is rapidly increasing all over the world. Consequently, there is an urgent need for more effective intervention strategies. Both animal and human studies indicate that lipid oversupply to skeletal muscle can result in insulin resistance, which is one of the characteristics of the MS. C57BL/6J mice were fed a low-fat (10 kcal%) palm oil diet or a high-fat (45 kcal%; HF) palm oil diet for 3 or 28 days. By combining transcriptomics with protein and lipid analyses we aimed to better understand the molecular events underlying the early onset of the MS. Short-term HF feeding led to altered expression levels of genes involved in a variety of biological processes including morphogenesis, energy metabolism, lipogenesis, and immune function. Protein analysis showed increased levels of the myosin heavy chain, slow fiber type protein, and the complexes I, II, III, IV, and V of the oxidative phosphorylation. Furthermore, we observed that the main mitochondrial membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine, contained more saturated fatty acids. Altogether, these results point to a morphological as well as a metabolic adaptation by promoting a more oxidative fiber type. We hypothesize that after this early positive adaptation, a continued transcriptional downregulation of genes involved in oxidative phosphorylation will result in decreased oxidative capacity at a later stage. Together with increased saturation of phospholipids of the mitochondrial membrane this can result in decreased mitochondrial function, which is a hallmark observed in insulin resistance and Type 2 diabetes

Inhibition of Extracellular Cathepsin D Reduces Hepatic Lipid Accumulation and Leads to Mild Changes in Inflammationin NASH Mice

Background &amp; AimsThe lysosomal enzyme, cathepsin D (CTSD) has been implicated in the pathogenesis of non-alcoholic steatohepatitis (NASH), a disease characterised by hepatic steatosis and inflammation. We have previously demonstrated that specific inhibition of the extracellular CTSD leads to improved metabolic features in Sprague-Dawley rats with steatosis. However, the individual roles of extracellular and intracellular CTSD in NASH are not yet known. In the current study, we evaluated the underlying mechanisms of extracellular and intracellular CTSD fractions in NASH-related metabolic inflammation using specific small-molecule inhibitors.MethodsLow-density lipoprotein receptor knock out (Ldlr-/-) mice were fed a high-fat, high cholesterol (HFC) diet for ten weeks to induce NASH. Further, to investigate the effects of CTSD inhibition, mice were injected either with an intracellular (GA-12) or extracellular (CTD-002) CTSD inhibitor or vehicle control at doses of 50 mg/kg body weight subcutaneously once in two days for ten weeks.ResultsLdlr-/- mice treated with extracellular CTSD inhibitor showed reduced hepatic lipid accumulation and an associated increase in faecal bile acid levels as compared to intracellular CTSD inhibitor-treated mice. Furthermore, in contrast to intracellular CTSD inhibition, extracellular CTSD inhibition switched the systemic immune status of the mice to an anti-inflammatory profile. In line, label-free mass spectrometry-based proteomics revealed that extra- and intracellular CTSD fractions modulate proteins belonging to distinct metabolic pathways.ConclusionWe have provided clinically translatable evidence that extracellular CTSD inhibition shows some beneficial metabolic and systemic inflammatory effects which are distinct from intracellular CTSD inhibition. Considering that intracellular CTSD inhibition is involved in essential physiological processes, specific inhibitors capable of blocking extracellular CTSD activity, can be promising and safe NASH drugs

AntagomiR-103 and -107 Treatment Affects Cardiac Function and Metabolism

MicroRNA-103/107 regulate systemic glucose metabolism and insulin sensitivity. For this reason, inhibitory strategies for these microRNAs are currently being tested in clinical trials. Given the high metabolic demands of the heart and the abundant cardiac expression of miR-103/107, we questioned whether antagomiR-mediated inhibition of miR-103/107 in C57BL/6J mice impacts on cardiac function. Notably, fractional shortening decreased after 6 weeks of antagomiR-103 and -107 treatment. This was paralleled by a prolonged systolic radial and circumferential time to peak and by a decreased global strain rate. Histology and electron microscopy showed reduced cardiomyocyte area and decreased mitochondrial volume and mitochondrial cristae density following antagomiR-103 and -107. In line, antagomiR-103 and -107 treatment decreased mitochondrial OXPHOS complexes’ protein levels compared to scrambled, as assessed by mass spectrometry-based label-free quantitative proteomics. MiR-103/107 inhibition in primary cardiomyocytes did not affect glycolysis rates, but it decreased mitochondrial reserve capacity, reduced mitochondrial membrane potential, and altered mitochondrial network morphology, as assessed by live-cell imaging. Our data indicate that antagomiR-103 and -107 decrease cardiac function, cardiomyocyte size, and mitochondrial oxidative capacity in the absence of pathological stimuli. These data raise concern about the possible cardiac implications of the systemic use of antagomiR-103 and -107 in the clinical setting, and careful cardiac phenotyping within ongoing trials is highly recommended. Keywords: Cardiomyocyte, cardiac, microRNA, miR-103, miR-107, antagomiR, mitochondria, metabolism, mass spectrometry, electron microscop

An 8-Week high-fat diet induces obesity and insulin resistance with small changes in the muscle transcriptome of C57BL/6J mice

Background: Skeletal muscle is responsible for most of the insulin-stimulated glucose uptake and metabolism. Therefore, it plays an important role in the development of insulin resistance, one of the characteristics of the metabolic syndrome (MS). As the prevalence of the MS is increasing, there is an urgent need for more effective intervention strategies. Methods: C57BL/6J mice were fed an 8-week low-fat diet (10 kcal%; LFD) or high-fat diet (45 kcal%; HFD). Microarray analysis was performed by using two comparisons: (1) 8-week HFD transcriptome versus 8-week LFD transcriptome and (2) transcriptome of mice sacrificed at the start of the intervention versus 8-week LFD transcriptome and 8-week HFD transcriptome, respectively. Results: Although an 8-week HFD induced obesity and impaired insulin sensitivity, HFD-responsive changes in the muscle transcriptome were relatively small (<1.3-fold). In fact, 8-weeks of aging induced more pronounced changes than an HFD. One comparison revealed the transcriptional downregulation of the mito- gen-activated protein kinase cascade, whereas both comparisons showed the upregulation of fatty acid oxidation, demonstrating that the two comparison strategies are confirmative as well as complementary. Conclusion: We suggest using complementary analysis strategies in the genome-wide search for gene expression changes induced by mild interventions, such as an HFD