UV radiation (UVR) induces serious structural and functional alterations in human skin leading to skin aging and carcinogenesis. Reactive oxygen species are key players in UVR-mediated photodamage and induce the DNA-base-oxidized, intermediate 8-hydroxy-2'-deoxyguanosine (8-OHdG). Herein, we report the protective action of melatonin against UVR-induced 8-OHdG formation and depletion of antioxidative enzymes using ex vivo human full-thickness skin exposed to UVR in a dose (0, 100, 300 mJ/cm(2))- and time-dependent manner (0, 24, 48 hr post-UVR). Dynamics of depletion of antioxidative enzymes including catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), or 8-OHdG formation were studied by real-time PCR and immunofluorescence/immunohistochemical staining. UVR-treated skin revealed significant and immediate (0 hr 300 mJ/cm(2)) reduction of gene expression, and this effect intensified within 24 hr post-UVR. Simultaneous increase in 8-OHdG-positive keratinocytes occurred already after 0 hr post-UVR reaching 71% and 99% up-regulation at 100 and 300 mJ/cm(2), respectively (P < 0.001). Preincubation with melatonin (10(-3) M) led to 32% and 29% significant reductions in 8-OHdG-positive cells and the prevention of antioxidative enzyme gene and protein suppression. Thus, melatonin was shown to play a crucial role as a potent antioxidant and DNA protectant against UVR-induced oxidative damage in human skin.

Melatonin exhibits protective effects against ultraviolet radiation (UVR) via modulation of proinflammatory mediators and its free radical scavenging capacity. To date, several reports presented protective mechanisms of this agent against UVR-induced alterations in mitochondria and nuclei. This investigation evaluates the potent preventing action of melatonin regarding early-stage UVR-mediated perturbations in plasma membrane potential (mbΔψ) and intracellular (cytosolic) pH (pH i) analyzed by flow cytometry. Experiments were carried out in a dose- and time-dependent manner using human keratinocytes [HaCaT and normal human epidermal keratinocytes (NHEK)]. First investigations, which used viability/cytotoxicity assays, showed the gradual mortality with increasing UVR doses and cultivation time. Pre-incubation with melatonin (10(-3) m) prior to UVR exposure reduced lactate dehydrogenase release by 30% (HaCaT) and 28% (NHEK) at the dose of 50 mJ/cm(2) after 48 hr (P < 0.001). Furthermore, UVR caused hyperpolarization of mbΔψ immediately (0 hr) after irradiation (25 or 50 mJ/cm(2)). At the dose of 50 mJ/cm(2), cells cultivated for 48 hr manifested a marked increase in mbΔψ by 112% (HaCaT) and 123% (NHEK). The presence of melatonin significantly protected the cells by 12% (HaCaT) and 14% (NHEK) (P < 0.001). Simultaneously, 50 mJ/cm(2) induced dramatic acidification reaching after 24 hr the level of 6.40 (without melatonin), 6.56 (with melatonin) for HaCaT and 6.11 (without melatonin), 6.43 (with melatonin) for NHEK. The results presented provide information about the protective mechanisms of melatonin itself on one hand and, combined with data reported so far, confirm the potent antiapoptotic action of melatonin.

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Background Human hair is highly responsive to stress, and human scalp hair follicles (HFs) contain a peripheral neuroendocrine equivalent of the systemic hypothalamic-pituitary-adrenal (HPA) stress axis. Androgenetic alopecia (AGA) is supposed to be aggravated by stress. We used corticotropin-releasing hormone (CRH), which triggers the HPA axis, to induce a stress response in human ex vivo male AGA HFs. Caffeine is known to reverse testosterone-mediated hair growth inhibition in the same hair organ culture model. Objectives To investigate whether caffeine would antagonize CRH-mediated stress in these HFs. Methods HFs from balding vertex area scalp biopsies of men affected by AGA were incubated with CRH (10 À7 mol L À1) with or without caffeine (0Á001% or 0Á005%). Results Compared to controls, CRH significantly enhanced the expression of catagen-inducing transforming growth factor-b2 (TGF-b2) (P < 0Á001), CRH receptors 1 and 2 (CRH-R1/2) (P < 0Á01), adrenocorticotropic hormone (ACTH) (P < 0Á001) and melanocortin receptor 2 (MC-R2) (P < 0Á001), and additional stressassociated parameters, substance P and p75 neurotrophin receptor (p75 NTR). CRH inhibited matrix keratinocyte proliferation and expression of anagen-promoting insulin-like growth factor-1 (IGF-1) and the pro-proliferative nerve growth factor receptor NGF-tyrosine kinase receptor A (TrkA). Caffeine significantly counteracted all described stress effects and additionally enhanced inositol trisphosphate receptor (IP 3-R), for the first time detected in human HFs. Conclusions These findings provide the first evidence in ex vivo human AGA HFs that the stress mediator CRH induces not only a complex intrafollicular HPA response, but also a non-HPA-related stress response. Moreover, we show that these effects can be effectively antagonized by caffeine. Thus, these data strongly support the hypothesis that stress can impair human hair physiology and induce hair loss,

BackgroundDuplications at the Xp21.2 locus have previously been linked to 46,XY gonadal dysgenesis (GD), which is thought to result from gene dosage effects of NR0B1 (DAX1), but the exact disease mechanism remains unknown.MethodsPatients with 46,XY GD were analysed by whole genome sequencing. Identified structural variants were confirmed by array CGH and analysed by high-throughput chromosome conformation capture (Hi-C).ResultsWe identified two unrelated patients: one showing a complex rearrangement upstream of NR0B1 and a second harbouring a 1.2 Mb triplication, including NR0B1. Whole genome sequencing and Hi-C analysis revealed the rewiring of a topological-associated domain (TAD) boundary close to NR0B1 associated with neo-TAD formation and may cause enhancer hijacking and ectopic NR0B1 expression. Modelling of previous Xp21.2 structural variations associated with isolated GD support our hypothesis and predict similar neo-TAD formation as well as TAD fusion.ConclusionHere we present a general mechanism how deletions, duplications or inversions at the NR0B1 locus can lead to partial or complete GD by disrupting the cognate TAD in the vicinity of NR0B1. This model not only allows better diagnosis of GD with copy number variations (CNVs) at Xp21.2, but also gives deeper insight on how spatiotemporal activation of developmental genes can be disrupted by reorganised TADs causing impairment of gonadal development.

X-linked dystonia–parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of the TAF1 gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the TAF1 intron retention transcript TAF1-32i can be found in XDP cells as compared to healthy controls. Here, we investigate the sequence of the deep intronic region included in this transcript and show that it is also present in cells from healthy individuals, albeit in lower amounts than in XDP cells, and that it undergoes degradation by nonsense-mediated mRNA decay. Furthermore, we investigate epigenetic marks (e.g., DNA methylation and histone modifications) present in this intronic region and the spanning sequence. Finally, we show that the SVA evinces regulatory potential, as demonstrated by its ability to repress the TAF1 promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons.

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