The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes

Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing.

Nuclear localization of HBD-1 in human keratinocytes.

OBJECTIVE: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. RESULTS: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. CONCLUSION: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions.

New insights into the pathobiology of Down syndrome--hyaluronan synthase-2 overexpression is regulated by collagen VI α2 chain.

Down syndrome (DS) is a common birth defect characterized by the trisomy of chromosome 21. DS-affected umbilical cords (UCs) of fetuses show altered architecture of the extracellular matrix. Overexpression of the chromosome 21 genes encoding the collagen type VI (COLVI) chains α1(VI) and α2(VI), COL6A1 and COL6A2, respectively, has also reported to occur in the nuchal skin of DS fetuses. The aim of this study was therefore to evaluate the COLVI content in euploid and DS-affected UCs and human skin fibroblasts, and to investigate the relationships between COLVI and hyaluronan (HA) and HA synthase-2 (HAS2). We found that the UCs of DS fetuses showed denser staining of COLVI and increased COL6A2 expression at both early and term gestational ages. In vitro expression studies in DS-derived fibroblasts showed similarly increased amounts of α1(VI) and α2(VI) chains at the protein and transcriptional level, supporting the hypothesis of the gene dosage effect. Furthermore, increased levels of HA and HAS2 were also found in DS-derived skin fibroblast cultures. Notably, silencing of COL6A2 in DS-derived cells resulted in downregulation of HAS2, with a simultaneous decrease in secreted HA. Exogenous addition of COLVI to normal fibroblasts did not have any effect on HAS2 expression. In conclusion, UCs and skin fibroblasts in DS show significant increases in COLVI and HA; the overexpression of COL6A2 in DS tissue and cells is closely related to the increased expression of HAS2. These data may explain the DS phenotypes and their effects in organ tissue maturation.

Groundwater dating with Atom Trap Trace Analysis of 39Ar

We report on the realization of Atom Trap Trace Analysis for39Ar and its first application to dating of groundwater samples. The presented system achieves an atmospheric39Ar count rate as high as 3.58 ± 0.10 atoms/h allowing for the determination of the39Ar concentration in less than a day. We demonstrate that the measured count rates are proportional to the39Ar concentration by intercomparison with Low-Level Counting results and by measurements on prepared argon samples with defined concentration. For a geophysical application, we degas three different groundwater samples and gas chromatographically extract the argon. The39Ar ages inferred from the count rates extend over the accessible dating range and are in agreement with the Low-Level Counting results as well as with complementary isotope data.

The canine neuronal ceroid-lipofuscinosis: a review.

The present article gives a survey over the current scientific knowledge of the canine neuronal ceroid-lipofuscinosis (NCL). NCL is a heterogenous group of lysosomal storage diseases in humans and animals. In consequence of a gene mutation, there is an accumulation of ceroid-lipofuscin in neurons, cells of the retina and the skin and other cells. The stored ceroid-lipofuscin in neurons leads to an impaired cell function and subsequently to cell death. Recently, the underlying genetic defect was discovered in several dog breeds. Genetic testing permits an ante mortem diagnosis of the disease, which up to now was only possible with a positive biopsy result. Another advantage is the identification of carrier animals to eliminate the deleterious alleles.

Oil-soluble vitamins: illegal use for lip augmentation.

Fillers for lip augmentation have become more and more popular in recent years and seem to be indispensable in the cosmetic market nowadays. A series of six young females is presented who developed massive swellings and pain after vitamins A and/or E lip augmentation. The vitamins were extracted from gelatinous capsules (Gericaps [Adipharm EAD, Sofia, Bulgaria], Geritamins [Actavis EAD, Balkanpharma-Dubnitsa AD, Bulgaria], or vitamin E yellow gel capsules) and injected by unprofessional physicians and beauticians in different cosmetic centers. Physical examination revealed firm indurations of the lips and perioral skin, tenderness, erythema, and hard dermal nodules. Histological analysis revealed numerous round-to-ovoid cavities of varying sizes, resulting in a Swiss cheese-like appearance, consistent with lipogranulomas. The patients were treated with systemic and intralesional triamcinolone injections and broad-spectrum antibiotics with good clinical response. In conclusion, these cases demonstrate the danger of the use of unregistered products as fillers injected by unprofessional physicians and beauticians.

First cases of besnoitiosis in cattle in Switzerland

Bovine besnoitiosis has been diagnosed in neighboring countries but not in Switzerland so far. This disease occurs endemically in France and focal outbreaks have been reported in Germany and Italy. To determine if Besnoitia besnoiti is introduced into Switzerland through the import of breeding cattle from France, a systematic serological survey was performed. A total of 412 breeding cattle (from 114 farms) imported from France into Switzerland between 2005 and 2011, were serologically examined for antibodies against B. besnoiti using a commercial ELISA kit (PrioCHECK© Besnoitia Ab 2.0, Prionics AG, Zurich, Switzerland). Sixty-four (15.5 %) animals reacted positive in ELISA. The serologic diagnosis was confirmed by an indirect immunfluorescence test (IFAT) and a Western blot (WB) in only 2 Limousin cows imported from France on a farm in Eastern Switzerland. Subsequently, this whole herd (n = 16) was examined clinically and serologically and 2 additional Limousin cows imported from Germany also reacted positive in the three serological tests. One of these cows presented B. besnoiti tissue cysts in the scleral conjunctiva and typical skin lesions in the head region. The infection was further confirmed cytologically, histopathologically and by PCR. It can be concluded that the parasite is most likely being introduced into Switzerland through the import of infected animals.

Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris

The majority of pemphigus vulgaris (PV) patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis). The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg) 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG), PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice) as well as PV patients' biopsies (n=6). A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP) and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other diseases including cancer.

Molecular Consequences of the SERPINH1/HSP47 Mutation in the Dachshund Natural Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a heritable connective tissue disease characterized by bone fragility and increased risk of fractures. Up to now, mutations in at least 18 genes have been associated with dominant and recessive forms of OI that affect the production or post-translational processing of procollagen or alter bone homeostasis. Among those, SERPINH1 encoding heat shock protein 47 (HSP47), a chaperone exclusive for collagen folding in the ER, was identified to cause a severe form of OI in dachshunds (L326P) as well as in humans (one single case with a L78P mutation). To elucidate the disease mechanism underlying OI in the dog model, we applied a range of biochemical assays to mutant and control skin fibroblasts as well as on bone samples. These experiments revealed that type I collagen synthesized by mutant cells had decreased electrophoretic mobility. Procollagen was retained intracellularly with concomitant dilation of ER cisternae and activation of the ER stress response markers GRP78 and phospho-eIF2α, thus suggesting a defect in procollagen processing. In line with the migration shift detected on SDS-PAGE of cell culture collagen, extracts of bone collagen from the OI dog showed a similar mobility shift, and on tandem mass spectrometry, the chains were post-translationally overmodified. The bone collagen had a higher content of pyridinoline than control dog bone. We conclude that the SERPINH1 mutation in this naturally occurring model of OI impairs how HSP47 acts as a chaperone in the ER. This results in abnormal post-translational modification and cross-linking of the bone collagen.

Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs

BACKGROUND Pinschers and other dogs with coat color dilution show a characteristic pigmentation phenotype. The fur colors are a lighter shade, e.g. silvery grey (blue) instead of black and a sandy color (Isabella fawn) instead of red or brown. In some dogs the coat color dilution is sometimes accompanied by hair loss and recurrent skin inflammation, the so called color dilution alopecia (CDA) or black hair follicular dysplasia (BHFD). In humans and mice a comparable pigmentation phenotype without any documented hair loss is caused by mutations within the melanophilin gene (MLPH). RESULTS We sequenced the canine MLPH gene and performed a mutation analysis of the MLPH exons in 6 Doberman Pinschers and 5 German Pinschers. A total of 48 sequence variations was identified within and between the breeds. Three families of dogs showed co-segregation for at least one polymorphism in an MLPH exon and the dilute phenotype. No single polymorphism was identified in the coding sequences or at splice sites that is likely to be causative for the dilute phenotype of all dogs examined. In 18 German Pinschers a mutation in exon 7 (R199H) was consistently associated with the dilute phenotype. However, as this mutation was present in homozygous state in four dogs of other breeds with wildtype pigmentation, it seems unlikely that this mutation is truly causative for coat color dilution. In Doberman Pinschers as well as in Large Munsterlanders with BHFD, a set of single nucleotide polymorphisms (SNPs) around exon 2 was identified that show a highly significant association to the dilute phenotype. CONCLUSION This study provides evidence that coat color dilution is caused by one or more mutations within or near the MLPH gene in several dog breeds. The data on polymorphisms that are strongly associated with the dilute phenotype will allow the genetic testing of Pinschers to facilitate the breeding of dogs with defined coat colors and to select against Large Munsterlanders carrying BHFD.

Neuartiger knochenverankerter Hämodialysezugang

Für Patienten an der Hämodialyse ist nach Versagen der klassischen arterio-venösen Fisteln oder Shunts ein direkter Gefässzugang mittels Katheter lebensnotwendig. Permanente zentralvenöse Katheter penetrieren die Hals- und Thoraxweichteile und die Haut ohne rigide Befestigung. Die Infektionsrate ist hoch und führt oft zur Explantation. Knochenverankerte Hörgeräte sind zur Behandlung bei Schalleitungsschwerhörigkeit etabliert. Das Implantat sitzt fest im Felsenbein und der Aufsatz penetriert die Haut. Schwere Infektionen, die eine Explantation nötig machen, sind sehr selten. Wir nehmen an, dass einer der Hauptgründe für die tiefe Komplikationsrate die starke Befestigung des Implantats am Knochen ist, wodurch die Hautbewegungen relativ zum Knochen minimiert werden. Basierend auf den Erfahrungen mit implantierten Hörsystemen haben wir einen perkutanen knochenverankerten Hämodialysezugang im Bereich des Felsenbeins als vorteilhafte Alternative zum herkömmlichen zentralvenösen Katheterzugang entwickelt. Dabei wurde die Felsenbeinanatomie und Knochendicke zur Lokalisierung des idealen Implantationsortes untersucht; die Schraubenstabilität im Knochen getestet; ein Titanimplantat inklusive Ventile und Katheter, sowie chirurgische Instrumente zur sicheren Implantation entwickelt. Der knochenverankerte Hämodialysezugang wurde auf Flussrate, Dichtigkeit und Reinigung getestet; die Platzierung des Katheters mittels Seldingertechnik in die V. jugularis interna über eine Halsinzision festgelegt. Die Resultate unserer Arbeit zeigen die technische Machbarkeit eines im Felsenbein verankerten neuartigen Hämodialysezuganges und bilden die Grundlage einer inzwischen bewilligten klinischen Pilotstudie.

Durazneros bajo tela antigranizo : efecto sobre rendimiento y coloración de piel

Para evaluar la influencia de la tela antigranizo sobre los rendimientos y la coloración de piel de los frutos de duraznero Prunus persicae L. Batsch variedad Dr. Davis se utilizaron plantas testigo y bajo tela antigranizo seleccionadas por uniformidad de copa y de carga frutal. La cosecha se realizó en dos pasadas. En cada recolección se determinó peso individual y color de piel de los frutos. Se calcularon los rendimientos totales y por pasadas y los rendimientos por categoría de color de piel. La tela antigranizo no afectó los rendimientos totales. En cambio retrasó el desarrollo de color de fondo y, por lo tanto, redujo el rendimiento de la primera pasada de cosecha. Además aumentó la proporción de frutos con madurez mínima en la segunda recolección.

La tela antigranizo en la maduración de ciruelas japonesas : : Prunus salicina Lindl

Se evaluó el efecto de la tela antigranizo sobre la evolución de la maduración de las cv. Linda Rosa y Larry Ann de ciruelo japonés. Periódicamente se cosechó fruta de plantas uniformes: ambas situaciones bajo tela y sin protección. Los parámetros de madurez y calidad evaluados fueron: peso, diámetros, color de piel, firmeza de pulpa, contenido de sólidos solubles (CSS), acidez titulable (AT) y la relación CSS/AT. La tela afectó la calidad de los frutos. El contenido de solubles y el color piel de la cv. Linda Rosa y el tamaño de frutos de la cv. Larry Ann, atributos de calidad muy valorados, fueron menores en las plantas bajo tela.

La Casa Domínguez. Alejandro de la Sota: Construir - Habitar

La Tesis Doctoral nace con una intensa vocación pedagógica. La hipótesis de trabajo se establece en torno a una cuestión de interés personal, un tema sobre el que se vertebran, desde el comienzo del doctorado, los diferentes cursos y trabajos de investigación: LA CASA DOMÍNGUEZ como paradigma de la dialéctica en la obra de Alejandro de la Sota. La clasificación de la realidad en categorías antagónicas determina un orden conceptual polarizado, una red de filiaciones excluyentes sobre las que Sota construye su personal protocolo operativo: la arquitectura intelectual o popular, experimental o tradicional, universal o local, ligera o pesada, elevada o enterrada, etc. Se propone el abordaje de una cuestión latente en el conjunto de la obra ‘sotiana’, desde la disección y el análisis de una de sus obras más pequeñas: la casa Domínguez. Se trata de una organización sin precedentes, que eleva la estrategia dialéctica al paroxismo: la vivienda se separa en dos estratos independientes, la zona de día, elevada, y la zona de noche, enterrada; cada uno de los estratos establece su propio orden geométrico y constructivo, su propio lenguaje y carácter, su propia identidad e incluso su propio presupuesto. Las relaciones entre interior y exterior se especializan en función de la actividad o el reposo, estableciéndose una compleja red de relaciones, algunas evidentes y otras celosamente veladas, entre los diferentes niveles. La estancia destinada a las tareas activas se proyecta como un objeto de armazón ligero y piel fría; la precisa geometría del cubo delimita la estancia vigilante sobre el paisaje conquistado. La ladera habitada se destina al reposo y se configura como una topografía verde bajo la que se desarrollan los dormitorios en torno a patios, grietas y lucernarios, generando un paisaje propio: la construcción del objeto frente a la construcción del lugar La casa Domínguez constituye uno de los proyectos menos estudiados, y por lo tanto menos celebrados, de la obra de Don Alejandro. Las publicaciones sucesivas reproducen la documentación gráfica junto a la memoria (epopeya) que el propio Sota compone para la publicación del proyecto. Apenas un par de breves textos críticos de Miguel Ángel Baldellou y, recientemente de Moisés Puente, abordan la vivienda como tema monográfico. Sin embargo, la producción de proyecto y obra ocupó a De la Sota un periodo no inferior a diez años, con casi cien planos dibujados para dos versiones de proyecto, la primera de ellas, inédita. El empeño por determinar hasta el último detalle de la ‘pequeña’ obra, conduce a Sota a controlar incluso el mobiliario interior, como hiciera en otras obras ‘importantes’ como el Gobierno Civil de Tarragona, el colegio mayor César Carlos o el edificio de Correos y Telecomunicaciones de León. La complicidad del cliente, mantenida durante casi cuarenta años, habilita el despliegue de una importante colección de recursos y herramientas de proyecto. La elección de la casa Domínguez como tema central de la tesis persigue por lo tanto un triple objetivo: en primer lugar, el abordaje del proyecto como paradigma de la dialéctica ‘sotiana’, analizando la coherencia entre el discurso de carácter heroico y la obra finalmente construida; en segundo lugar, la investigación rigurosa, de corte científico, desde la disección y progresivo desmontaje del objeto arquitectónico; y por último, la reflexión sobre los temas y dispositivos de proyecto que codifican la identificación entre la acción de construir y el hecho de habitar, registrando los aciertos y valorando con actitud crítica aquellos elementos poco coherentes con el orden interno de la propuesta. This doctoral thesis is the fruit of a profound pedagogical vocation. The central hypothesis was inspired by a question of great personal interest, and this interest has, since the very beginning of the doctorate, been the driving force behind all subsequent lines of research and investigation. The “Casa Domínguez” represents a paradigm of the dialectics found in the work of Alejandro de la Sota. The perception of reality as antagonistic categories determines a polarized conceptual order, a network of mutually excluding associations upon which Sota builds his own personal operative protocol: intellectual or popular architecture, experimental or traditional, universal or local, heavy or light, above or below ground, etc. Through the analysis and dissection of the “Casa Domínguez”, one of Sota’s smallest projects, an attempt is made to approach the underlying question posed in “Sotian” work as a whole. This is about organization without precedent, raising the strategic dialectics to levels of paroxysm. The house is divided into two separate levels, the day-time level above ground, and the lower night-time level beneath the surface of the ground. Each level has its own geometrical and stuctural order, its own language and character, its own identity and even has its own construction budget. The interaction between the two areas is centered on the two functions of rest and activity, and this in turn establishes a complex relationship network between both, which is sometimes self-evident, but at other times jealously guarded. The living area designed for daily activity is presented as an object of light structure and delicate skin; the precise geometry of the cube delimiting the ever watchful living area’s domain over the land it has conquered. A green topography is created on the slope below which lies an area adapted for rest and relaxation. Two bedrooms, built around patios, skylights and light crevices, generate an entirely independent environment: the construction of an object as opposed to the creation of a landscape. The “Casa Domínguez” project has been subject to much less scrutiny and examination than Don Alejandro’s other works, and is consequently less well-known. A succession of journals have printed the blueprint document together with a poetic description (epopee), composed by Sota himself, to mark the project’s publication. There has, however, scarcely been more than two brief critical appraisals, those by Miguel Ángel Baldellou and more recently by Moisés Puente, that have regarded the project as a monographic work. The project and works nevertheless occupied no less than ten years of De La Sota’s life, with over a hundred draft drawings for two separate versions of the project, the first of which remains unpublished. The sheer determination to design this “small” work in the most meticulous detail, drove Sota to manage and select its interior furniture, as indeed he had previously done with more “important” works like the Tarragona Civil Government, César Carlos College, or the Post Office telecommunications building in León. Client collaboration, maintained over a period of almost forty years, has facilitated an impressive array of the project’s tools and resources. The choice of “Casa Domínguez” as the central subject matter of this thesis, was made in pursuance of a triple objective: firstly, to approach the project as a paradigm of the “Sotian” dialectic, the analysis of the discourse between the heroic character and the finished building; secondly, a rigorous scientific investigation, and progressive disassembling and dissecting of the architectonic object; and finally, a reflection on aspects of the project and its technology which codify the identification between the action of construction and the reality of living, thus marking its achievements, whilst at the same time subjecting incoherent elements of the proposal’s established order to a critical evaluation.

Secure access control by means of human stress detection

This paper proposes a stress detection system based on fuzzy logic and the physiological signals heart rate and galvanic skin response. The main contribution of this method relies on the creation of a stress template, collecting the behaviour of previous signals under situations with a different level of stress in each individual. The creation of this template provides an accuracy of 99.5% in stress detection, improving the results obtained by current pattern recognition techniques like GMM, k-NN, SVM or Fisher Linear Discriminant. In addition, this system can be embedded in security systems to detect critical situations in accesses as cross-border control. Furthermore, its applications can be extended to other fields as vehicle driver state-of-mind management, medicine or sport training.