Carotenoids - Effective Radical Scavengers for Healthy and Beautiful Skin

Free radicals are involved in various diseases and skin aging. To reduce and prevent this risk, our body produces antioxidants that can neutralize free radicals. However, some antioxidants need to be taken up with food, so a balanced and varied diet is essential for human health and beauty, along with sufficient exercise. Vegetables, especially curly kale, show very good antioxidative capacity due to the presence of carotenoids. As the recommended daily intake of vegetables is usually not consumed, dietary supplements are a good possibility to ingest carotenoids in a controlled and natural way. The positive effect of carotenoid-based dietary supplements on the skin has already been shown in several studies on healthy volunteers. Innovative non-invasive measuring methods have shown that oil extracts from vegetables significantly reduce not only free radicals in the skin but also the age-related breakdown of collagen and have a positive effect on skin parameters such as wrinkle volume. Thus, a balanced mixture of different natural carotenoids contributes to maintaining health and beauty

Wavelength, dose, skin type and skin model related radical formation in skin

The exposure to sun radiation is indispensable to our health; however, a long-term and high exposure could lead to cell damage, erythema, premature skin aging, and promotion of skin tumors. An underlying pathomechanism is the formation of free radicals which may induce oxidative stress at elevated concentrations. Different skin models, such as porcine-, murine-, human- ex vivo skin, reconstructed human skin (RHS) and human skin in vivo, were investigated during and after irradiation using X- and L-band EPR spectroscopy within different spectral regions (UVC to NIR). The amount of radical formation was quantified with the spin probe PCA and the radical types were measured ex vivo with the spin trap DMPO. The radiation dose influences the types of radicals formed in the skin. While reactive oxygen species (ROS) are always pronounced at low doses, there is an increase in lipid oxygen species (LOS) at high doses. Furthermore, the radical types arise independent from the irradiation wavelength, whereas the general amount of radical formation differs with the irradiation wavelength. Heat pre-stressed porcine skin already starts with higher LOS values. Thus, the radical type ratio might be an indicator of stress and the reversal of ROS/LOS constitutes the point where positive stress turns into negative stress.Compared to light skin types, darker types produce less radicals in the ultraviolet, similar amounts in the visible and higher ones in the infrared spectral region, rendering skin type-specific sun protection a necessity

Radical Stability vs. Temporal Resolution of EPR-Spectroscopy on Biological Samples

Spin-labeling active compounds is a convenient way to prepare them for EPR spectroscopy with minimal alteration of the target molecule. In this study we present the labeling reaction of dexamethasone (Dx) with either TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PCA (3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) with high yields. According to NMR data, both labels are attached at the primary hydroxy group of the steroid. In subsequent spin-stability measurements both compounds were applied onto HaCaT cells. When the signal of Dx-TEMPO decreased below the detection limit within 3 h, the signal of Dx-PCA remained stable for the same period of time

Red- and Near-Infrared-Excited Autofluorescence as a Marker for Acute Oxidative Stress in Skin Exposed to Cigarette Smoke Ex Vivo and In Vivo

Air pollution is increasing worldwide and skin is exposed to high levels of pollution daily, causing oxidative stress and other negative consequences. The methods used to determine oxidative stress in the skin are invasive and non-invasive label-free in vivo methods, which are severely limited. Here, a non-invasive and label-free method to determine the effect of cigarette smoke (CS) exposure on skin ex vivo (porcine) and in vivo (human) was established. The method is based on the measurement of significant CS-exposure-induced enhancement in red- and near-infrared (NIR)-excited autofluorescence (AF) intensities in the skin. To understand the origin of red- and NIR-excited skin AF, the skin was exposed to several doses of CS in a smoking chamber. UVA irradiation was used as a positive control of oxidative stress in the skin. The skin was measured with confocal Raman microspectroscopy before CS exposure, immediately after CS exposure, and after skin cleaning. CS exposure significantly increased the intensity of red- and NIR-excited skin AF in a dose-dependent manner in the epidermis, as confirmed by laser scanning microscopy AF imaging and fluorescence spectroscopy measurements. UVA irradiation enhanced the intensity of AF, but to a lower extent than CS exposure. We concluded that the increase in red- and NIR-excited AF intensities of the skin after CS exposure could clearly be related to the induction of oxidative stress in skin, where skin surface lipids are mainly oxidized

Drug distribution in nanostructured lipid particles

The targeted design of nanoparticles for efficient drug loading and defined release profiles is even after 25 years of research on lipid-based nanoparticles still no routine procedure. It requires detailed knowledge about the interaction of the drug with the lipid compounds and about its localisation and distribution in the nanoparticle. We present here an investigation on nano-sized lipid particles (NLP) composed of Gelucire and Witepsol as solid lipids, and Capryol as liquid lipid, loaded with Dexamethasone, a glucocorticoid used in topical treatment of inflammatory dermal diseases. The interactions of Dexamethasone, which was spin-labelled by 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (DxPCA), with its microenvironment are monitored by EPR spectroscopy at 94 GHz at low temperatures. The mobility of the spin-labelled drug was probed by X-band EPR at room temperature. In order to relate the magnetic and dynamic parameters deduced from EPR to the local environment of the spin probe in the NLP, investigations of DxPCA in the individual lipid compounds were carried out. The magnetic parameters reflecting the polarity of DxPCA’s environment as well as the parameters describing the mobility of the drug reveal that in the case of colloidal dispersions of the lipids and also the NLP DxPCA is attached to the surface of the nanoparticles. Although the lipophilic drug is almost exclusively associated with the NLP in aqueous solution, dilution experiments show, that it can be easily released from the nanoparticle

Follicular Delivery of Caffeine from a Shampoo for Hair Retention

A key factor in the prevention of hair loss is the provision of optimal conditions on the scalp. In this regard, reduction of oxidative stress on the scalp is one critical requirement to support the hair follicles to function optimally. Recently, a novel shampoo formulation technology containing anti-oxidants such as piroctone olamine has been demonstrated to improve hair retention based on micellar degradation and coacervation effects. Caffeine has also been shown to exhibit anti-oxidant activity including the ability to inhibit lipid peroxidation. As with piroctone olamine, it is expected that follicular delivery of caffeine will enhance its anti-oxidant activity in a region that will be beneficial for hair retention. In this study, two shampoo formulations as well as a control formulation were applied to the calf area of n = 9 male participants. The technique of differential tape stripping was applied to obtain the caffeine penetrated to the stratum corneum and to the hair follicles. Isotope-dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to demonstrate caffeine follicular delivery from the shampoo formulas. The results showed that the percentage of caffeine recovered in the hair follicles was 8–9% of the caffeine absorbed into the skin and matched an existing caffeine-based shampoo. In conclusion, a novel shampoo formulation technology has been developed that effectively delivers beneficial anti-oxidants to improve hair retention. This new shampoo is expected to be especially useful in the goal of retaining hair during aging

Nanocrystals for Improved Drug Delivery of Dexamethasone in Skin Investigated by EPR Spectroscopy

Nanocrystals represent an improvement over the traditional nanocarriers for dermal application, providing the advantages of 100% drug loading, a large surface area, increased adhesion, and the potential for hair follicle targeting. To investigate their advantage for drug delivery, compared to a base cream formulation, dexamethasone (Dx), a synthetic glucocorticoid frequently used for the treatment of inflammatory skin diseases, was covalently linked with the paramagnetic probe 3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA) to DxPCA. To investigate the penetration efficiency between these two vehicles, electron paramagnetic resonance (EPR) spectroscopy was used, which allows the quantification of a spin-labeled drug in different skin layers and the monitoring of the drug release. The penetration behavior in excised healthy and barrier-disrupted porcine skin was monitored by EPR, and subsequently analyzed using a numerical diffusion model. As a result, diffusion constants and free energy values in the different layers of the skin were identified for both formulations. Dx-nanocrystals showed a significantly increased drug amount that penetrated into viable epidermis and dermis of intact (factor 3) and barrier-disrupted skin (factor 2.1) compared to the base cream formulation. Furthermore, the observed fast delivery of the spin-labeled drug into the skin (80% DxPCA within 30 min) and a successive release from the aggregate unit into the viable tissue makes these nanocrystals very attractive for clinical applications

Skin tolerant inactivation of multiresistant pathogens using far-UVC LEDs

Multiresistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) cause serious postoperative infections. A skin tolerant far-UVC (< 240 nm) irradiation system for their inactivation is presented here. It uses UVC LEDs in combination with a spectral filter and provides a peak wavelength of 233 nm, with a full width at half maximum of 12 nm, and an irradiance of 44 µW/cm2. MRSA bacteria in different concentrations on blood agar plates were inactivated with irradiation doses in the range of 15–40 mJ/cm2. Porcine skin irradiated with a dose of 40 mJ/cm2 at 233 nm showed only 3.7% CPD and 2.3% 6-4PP DNA damage. Corresponding irradiation at 254 nm caused 11–14 times higher damage. Thus, the skin damage caused by the disinfectant doses is so small that it can be expected to be compensated by the skin's natural repair mechanisms. LED-based far-UVC lamps could therefore soon be used in everyday clinical practice to eradicate multiresistant pathogens directly on humans

Brazilian adolescents' knowledge and beliefs about abortion methods: A school-based internet inquiry

Background: Internet surveys that draw from traditionally generated samples provide the unique conditions to engage adolescents in exploration of sensitive health topics.Methods: We examined awareness of unwanted pregnancy, abortion behaviour, methods, and attitudes toward specific legal indications for abortion via a school-based internet survey among 378 adolescents aged 12-21 years in three Rio de Janeiro public schools.Results: Forty-five percent knew peers who had undergone an abortion. Most students (66.0%) did not disclose abortion method knowledge. However, girls (aOR 4.2, 95% CI 2.4-7.2), those who had experienced their sexual debut (aOR1.76, 95% CI 1.1-3.0), and those attending a prestigious magnet school (aOR 2.7 95% CI 1.4-6.3) were more likely to report methods. Most abortion methods (79.3%) reported were ineffective, obsolete, and/or unsafe. Herbs (e.g. marijuana tea), over-the-counter medications, surgical procedures, foreign objects and blunt trauma were reported. Most techniques (85.2%) were perceived to be dangerous, including methods recommended by the World Health Organization. A majority (61.4%) supported Brazil's existing law permitting abortion in the case of rape. There was no association between gender, age, sexual debut, parental education or socioeconomic status and attitudes toward legal abortion. However, students at the magnet school supported twice as many legal indications (2.7, SE.27) suggesting a likely role of peers and/or educators in shaping abortion views.Conclusions: Abortion knowledge and attitudes are not driven simply by age, religion or class, but rather a complex interplay that includes both social spaces and gender. Prevention of abortion morbidity and mortality among adolescents requires comprehensive sexuality and reproductive health education that includes factual distinctions between safe and unsafe abortion methods

Analysis of genomic aberrations with high resolution array-CGH for patients with malformations of the extremities

Veränderungen im Genom fördern die genomische Vielfalt zwischen Individuen und Spezies, können aber auch Fehlentwicklungen während der frühen Embryogenese hervorrufen. In den letzten Jahren konnte eindrücklich gezeigt werden, dass strukturelle Aberrationen (Deletionen, Duplikationen, Inversionen, Translokationen) für die Entstehung von verschiedenen Krankheiten und Fehlbildungen verantwortlich sein können. In dieser Arbeit wurden Patienten mit verschiedenen angeborenen Fehlbildungen der Extremitäten mittels hochauflösender Array-CGH untersucht, um genomische Aberrationen im Genom als mögliche Ursache für die Fehlbildungen zu detektieren. Da die Entwicklung des Skelettsystems bei Vertebraten ein sehr komplexer Prozess ist, der die Beteiligung und korrekte Interaktion einer Vielzahl an Signalmolekülen erfordert, stellen die Extremitäten ein gutes Modellsystem für die Untersuchung von zellulären Interaktionen und interzellulären Signalübertragungen dar. Fgf8, Shh und Ihh sind wichtige Signalmoleküle während der frühen Extremitätenentwicklung. Sie beeinflussen das distale Längenwachstum, die anterior-posteriore Musterausbildung und den Prozess der Verknöcherung (endochondrale Ossifikation). Konzentrations- oder Strukturänderungen von Molekülen, die während der Gliedmaßenentstehung von Bedeutung sind, können einzelne Signalwege in ihrer Aktivität so beeinflussen, dass Fehlentwicklungen von einzelnen Skelettelementen oder von der gesamten Extremität die Folge sind. In Projekt 1 wurden Patienten mit dem klinischen Erscheinungsbild einer Polysyndaktylie, einer Syndaktylie Typ IV (SD IV) sowie mit dem klinischen Erscheinungsbild des Laurin Sandrow Syndroms (LSS) auf genomische Aberrationen analysiert. Es konnten Duplikationen mit nicht- rekurrenten Bruchpunkten auf dem Lokus 7q36.3 nachgewiesen werden. Diese überlappenden Aberrationen befanden sich innerhalb der cis-regulatorischen Enhancer-Region (ZRS-Region) des SHH-Gens, das für die korrekte Expression von SHH während der Extremitätenentwicklung verantwortlich ist. Aus vorangegangenen Arbeiten ist bekannt, dass Punktmutationen, aber auch strukturelle Veränderungen innerhalb dieser Region, mit Handfehlbildungen (Polysyndaktylie und triphalangealer Daumen) assoziiert sind. Im Rahmen dieser Arbeit konnten weitere Handfehlbildungen mit einer Fehlexpression von SHH in Verbindung gebracht werden, sowie erstmalig die genetische Ursache für das LSS identifiziert werden. In Projekt 2 wurden Patienten mit einer Syndaktylie der Hände und Füße zum Teil in Kombination mit einer sagittalen Kraniosynostose (Kraniosynostose Philadelphia Typ) mit der Array-CGH Methode untersucht. Es konnten überlappende nicht-rekurrente Duplikationen detektiert werden, die am IHH-Lokus auf Chromosom 2 lokalisiert waren. Der Überlappungsbereich beinhaltete konservierte Regionen, für die in in vivo -Experimenten eine regulatorische Funktion gezeigt werden konnte. Diese Elemente induzieren eine mit dem Ihh-Wildtyp überlappende Expression des Reportergens in den Extremitäten und an den Schädelnähten. Es konnte erstmalig eine Enhancer- Region für Ihh ausfindig gemacht werden, die vermutlich die orthologe Region zu der ZRS-Region darstellt und für eine korrekte Expression von Ihh verantwortlich ist. Die Ergebnisse sprechen für eine wichtige Rolle von IHH während der Schädelentwicklung und erweitern die Bedeutung von IHH auf die desmale Ossifikation. Das Projekt 3 beschäftigte sich mit Patienten, die den klinischen Phänotyp der Ektrodaktylie in Kombination mit tibialen Defekten (SHFLD) aufwiesen. Eine Untersuchung von 17 Familien deckte überlappende Duplikationen auf dem Chromosom 17p13.3 mit nicht-rekurrenten Bruchpunkten auf. Der gemeinsame Überlappungsbereich, d.h. die kritische Region, beinhaltete das Gen BHLHA9, welches bis jetzt in seiner Funktion nicht näher definiert war. Das Expressionsmuster während der Embryonalentwicklung bei Mäusen, Hühnchen und Zebrafisch ergab eine extremitätenspezifische Expression in der Progresszone der Extremitätenknospe. In vivo Analysen dieses Gens im Modellsystem Zebrafisch zeigten dessen Relevanz während der Extremitätenentwicklung. Die Stammbaumanalyse der 17 Familien demonstriert eindrücklich das Vorliegen einer unvollständigen Penetranz und eine stark ausgeprägte Verschiebung des Geschlechterverhältnisses, eine sogenannte Geschlechterwendigkeit. Diese Beobachtungen lassen den Schluss zu, dass zusätzlich zu der Duplikation auf dem Chromosom 17p13.3 ein modifizierender Faktor benötigt wird. Um diesen Faktor zu identifizieren sind weiterführende Untersuchungen notwendig. Die Ergebnisse dieser Arbeit belegen, dass strukturelle Aberrationen für eine Vielzahl von angeborenen Fehlentwicklungen verantwortlich sind. Sie können, wie im Fall der Spalthand- Spaltfußfehlbildungen, die Funktionalität eines Gens direkt beeinflussen oder aber, wenn sie nicht-kodierende Bereiche des Genoms betreffen, durch die Veränderung von regulatorischen Elementen und der Interaktion zwischen Regulatoren und dem Zielgen, indirekt auf die Zielgene wirken.Genomic rearrangements lead to genomic diversity between individuals and species, however, they can also cause developmental defects during the early embryogenesis. In the last years structural variations (deletions, duplications, inversions, translocations) have been shown to cause human developmental defects such as limb malformations. The complex process of skeletal development in vertebrates needs a multitude of different signaling molecules, interacting in the right and specific manner. In this context, the limbs represent a good model to examine cellular interactions and intercellular communication. Signaling molecules like Fgf8, Shh and Ihh are very important during this process. They influence the proximal-distal outgrowth, the anterior-posterior patterning and the process of endochondral ossification. Changes in the concentration or structure of these signaling molecules can influence downstream pathways negatively and can cause malformations of single skeleton elements and the whole extremity. In this study patients with different congenital limb malformations were analyzed by high resolution array CGH for genomic aberrations i.e. deletions (losses) and duplications (gains). In the first project a patient cohort with the clinical phenotypes of polysyndactyly, syndactyly type IV (SD IV) and the Laurin- Sandrow syndrome (LSS) were analyzed. Duplications with non recurrent breakpoints were detected in all affected patients at the SHH locus on chromosome 7q36.3. These aberrations were encompassing the limb specific enhancer of SHH (ZRS) which promotes the expression of SHH during early limb development. It is known that point mutations but also structural aberrations at the ZRS region lead to limb malformations like polysyndactyly and the clinical phenotype of a triphalangeal thumb. We could show that a range of hand anomalies, including the LSS, are associated with microduplications most likely causing a misexpression of SHH. It was the first time that the genetic cause of LSS could be identified. The focus of the second project was on patients with syndactyly of the hands and feet partly in combination with sagittal craniosynostosis (craniosynostosis type Philadelphia). Non-recurrent duplications at the IHH locus were identified in all affected patients. The critical region contained highly conserved non-coding elements which have regulatory functions in limb as well as skull development as demonstrated by in vivo experimental approaches. The expression pattern of a reporter gene showed an overlapping expression pattern with the wildtype Ihh expression in the limb and skull sutures. It was the first time that an enhancer region for Ihh was identified which could represents the equivalent to the ZRS-region of Shh gene. Furthermore our results demonstrate the importance of Ihh not only during the endochondral ossification, but also in the process of desmal ossification. In the third project 17 families with ectrodactyly in combination with tibial defects (SHFLD) were analyzed by array CGH. All of the affected individuals carried non-recurrent duplications on 17p13.3 which overlap in a 9,1kb region containing a single gene BHLHA9. The function of BHLHA9 was so far unknown. RNA in situ hybridization for this gene in mouse, chicken and zebrafish showed an expression pattern within the progress zone of the limb bud. In vivo analyses of this gene by Morpholino knock-down in the model system zebrafish resulted in severe reduction of fin outgrowth, thus, revealing the functional relevance of Bhlha9 during early fin development. Based on the pedigrees of the 17 families an incomplete penetrance and a strong sex bias regarding the clinical phenotype were shown. One can speculate that in addition to duplications on chromosome 17p13.3 a modifying factor is needed for expressivity of phenotype. For this approach additional experiments are required. Based on these results it was further strengthened that genomic aberrations can cause a number of different congenital malformations. These aberrations can alter the function of one gene in a direct way (like in our patient cohort with SHFLD) or can influence the regulatory function of enhancer elements. Such aberrations are located within conserved noncoding regions and can influence the right interaction between the enhancer-element and its target gene