Development of integument and cutaneous glands in larval,juvenile and adult toads (Rhinella granulosa): a morphological and morphometric study

In this study, the development of integument and cutaneous glands in the toad Rhinella granulosa (Bufonidae) at different larval stages and in postmetamorphic and adult forms was examined. The analyses were conducted using histological, ultrastructural and morphometric methods. The results showed that cellular aggregations of precursor epidermal glands start to appear in stage 31 of (Herpetologica, 16, 1960 and 183) and then proliferate and invade the dermis. After stage 41, granular and mucous glands are very similar to those found in adults. The granular glands are syncytial and are surrounded by a distinct layer of myoepithelial cells. In the region of parotoid macroglands, the granular glands accumulate and their alveoli progressively increase until they reach adult size. An analysis by scanning electron microscopy showed the inner distribution of the syncytial nuclei and the myoepithelial cells. The morphological changes observed in the integument of tadpoles are associated with the gradual adaptation to terrestrial environments by preparing the individual for future chemical defence against predators and micro‐organisms.     

Studying the structure of single-component ceramide bilayers with molecular dynamics simulations using different force fields

Molecular dynamics simulations are performed on a lipid bilayer that consists of ceramide NS 24:0 in an attempt to examine several structural and physicochemical properties of the specific system. The simulations are carried out with five different force fields (OPLS, GROMOS, BERGER, CHARMM and GAFF) in order to evaluate and compare their performance in modelling lipid systems that contain ceramides. The examined properties include bilayer thickness, chain tilt, density profiles, order parameters, chain conformation, area per lipid and (intermolecular or intramolecular) hydrogen bonding between the head groups. Special focus is given to the lateral lipid arrangement. To this purpose, a method is proposed that utilises the radial distribution functions of the alkyl chains to derive quantitative information about the lateral lipid packing. In most cases, all force fields lead to similar results. For a few properties (e.g. intramolecular hydrogen bonding), there is some discrepancy between the force fields but the lack of respective experimental data does not allow an unambiguous conclusion on which force field is the most reliable.     

The relationship between water loss,mechanical stress,and molecular structure of human stratum corneum ex vivo

Proper hydration of the stratum corneum (SC) is important for maintaining skin's vital functions. Water loss causes development of drying stresses, which can be perceived as ‘tightness’, and plays an important role in dry skin damage processes. However, molecular structure modifications arising from water loss and the subsequent development of stress has not been established. We investigated the drying stress mechanism by studying, ex vivo, the behaviors of the SC components during water desorption from initially fully hydrated samples using Raman spectroscopy. Simultaneously, we measure the SC mechanical stress with a substrate curvature instrument. Very good correlations of water loss to the mechanical stress of the stratum corneum were obtained, and the latter was found to depend mainly on the unbound water fraction. In addition to that, the water loss is accompanied with an increase of lipids matrix compactness characterized by lower chain freedom, while protein structure showed an increase in amount of α‐helices, a decline in α‐sheets, and an increase in folding in the tertiary structure of keratin. The drying process of SC involves a complex interplay of water binding, molecular modifications, and mechanical stress. This article provides a better understanding of the molecular mechanism associated to SC mechanics. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene‐350 and high‐performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole‐type pheomelanin) as well but not 4‐AHP (degradation product of benzothiazine‐type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.     

Assessment of ALA‐induced PpIX production in porcine skin pretreated with microneedles

Photodynamic therapy (PDT) is used for skin treatments of premalignant and cancer lesions and recognized as a non‐invasive technique that combines tissue photosensitization and subsequent exposure to light to induce cell death. However, it is limited to the treatment of superficial lesions, mainly due to the low cream penetration. Therefore, the improvement of transdermal distribution of aminolevulinic acid (ALA) is needed. In this study, the kinetics and homogeneity of production of ALA‐induced PpIX after the skin pre‐treatment with microneedles rollers of 0.5, 1.0 and 1.5 mm length were investigated. An improvement in homogeneity and production of PpIX was shown in a porcine model.

Widefield fluorescence imaging three hours after the topical application of ALA‐cream in the combined treatment with microeedles rollers.     

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ER^T2/UAS expression system¹ in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRAS^G12V expressing skin cells. The KalTA4-ER^T2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis^2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.     

Strategies for transformation of naturally-occurring amphibian antimicrobial peptides into therapeutically valuable anti-infective agents

The emergence of strains of pathogenic microorganisms with resistance to commonly used antibiotics has necessitated a search for novel types of antimicrobial agents. Many frog species produce amphipathic alpha-helical peptides with broad spectrum antimicrobial activity in the skin but their therapeutic potential is limited by varying degrees of cytolytic activity towards eukaryotic cells. Methods for development of such peptides into anti-infective drugs are illustrated by the example of temporin-1DRa (HFLGTLVNLAK KIL.NH(2)). Studies with model alpha-helical peptides have shown that increase in cationicity promotes antimicrobial activity whereas increases in hydrophobicity, helicity and amphipathicity promote hemolytic activity and loss of selectivity for microorganisms. Analogs of temporin-1DRa in which each amino acid is replaced by L-lysine and D-lysine were synthesized and their cytolytic activities tested against a range of microorganisms and human erythrocytes. Small changes in structure produced marked changes in conformation, as determined by retention time on reversed-phase HPLC, and in biological activity. However, peptides containing the substitutions (Val(7) -->L-Lys), (Thr(5)-->D-Lys) and (Asn(8)-->D-Lys) retained the high solubility and potent, broad spectrum antimicrobial activity of the naturally occurring peptide but were appreciably (up to 10-fold) less hemolytic. In contrast, analogs in which Leu(9) and Ile(13) were replaced by the more hydrophobic cyclohexylglycine residue showed slightly increased antimicrobial potencies (up to 2-fold) but a 4-fold increase in hemolytic activity. The data suggest a strategy of selective increases in cationicity concomitant with decreases in helicity and hydrophobicity in the transformation of naturally-occurring antimicrobial peptides into non-toxic therapeutic agents.     

Mexico in the International Reptile Skin Trade: a Case Study

This paper examines the role of Mexico as importer, manufacturer, producer and distributor centre of reptile skins from non-native and native species, through a combination of documentary research and survey methods. A number of key findings were derived from this study. Although Mexico has adopted the “System for the Conservation, Management and Sustainable Use of Wildlife” (SUMA), the country still relies on reptile skins from non-native species. In contrast, the smaller numbers of skins used from native species mainly derive from captive breeding schemes that although biologically sustainable, provide no incentive for habitat conservation. Sustainable use of reptile skins from native species could positively encourage conservation in Mexico. However, as a megadiverse country with potential to produce wildlife, Mexico will have to implement an appropriate regulatory framework to support local communities to promote the sustainable use of native species.     

Improvement of survival of skin flaps by combined gene transfer of hepatocyte growth factor and prostacyclin synthase

BACKGROUND: Increasing the local blood flow is a critical factor for long-term survival of skin flaps. Thus, a molecular therapy to increase the blood flow by means of an angiogenic factor is considered to be a useful strategy to improve skin flap survival. We focused on a combined strategy to stimulate not only angiogenesis, but also vasodilation of local microvessels, using co-transfection of the hepatocyte growth factor (HGF) and prostacyclin synthase (PGIS) genes to enhance the survival of random-pattern skin flaps. METHODS AND RESULTS: A 2 x 8 cm full thickness cranial pedicled random-pattern flap was made on the back of each 12-week-old male rat. At 3 days before operation, 400 microg of human HGF and PGIS naked plasmid DNA or control plasmid was transfected into the flaps by needle-less injection using a Shima Jet, resulting in successful expression of human HGF and PGIS in the skin flaps. Transfection of both genes into the distal half of skin flaps at 3 days prior to operation significantly increased the survival rate of skin flaps, while transfection all over the flaps did not. In addition, transfection prior to operation was more effective than simultaneous treatment. Moreover, co-transfection of these genes improved the survival area of skin flaps, accompanied by an increase in blood flow of skin flaps, even in a diabetic model. CONCLUSIONS: Overall, these results indicate that combination treatment with HGF and PGIS genes by Shima Jet could be an effective strategy to improve skin flap survival.     

Ultraviolet radiation exposure accelerates the accumulation of the aging-dependent T414G mitochondrial DNA mutation in human skin

The accumulation of mitochondrial DNA (mtDNA) mutations has been proposed as an underlying cause of the aging process. Such mutations are thought to be generated principally through mechanisms involving oxidative stress. Skin is frequently exposed to a potent mutagen in the form of ultraviolet (UV) radiation and mtDNA deletion mutations have previously been shown to accumulate with photoaging. Here we report that the age-related T414G point mutation originally identified in skin fibroblasts from donors over 65 years also accumulates with age in skin tissue. Moreover, there is a significantly greater incidence of this mutation in skin from sun-exposed sites (chi(2)= 6.8, P < 0.01). Identification and quantification of the T414G mutation in dermal skin tissue from 108 donors ranging from 8 to 97 years demonstrated both increased occurrence with photoaging as well as an increase in the proportion of molecules affected. In addition, we have discovered frequent genetic linkage between a common photoaging-associated mtDNA deletion and the T414G mutation. This linkage indicates that mtDNA mutations such as these are unlikely to be distributed equally across the mtDNA population within the skin tissue, increasing their likelihood of exerting focal effects at the cellular level. Taken together, these data significantly contribute to our understanding of the DNA damaging effects of UV exposure and how resultant mutations may ultimately contribute towards premature aging.