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Batrachochytrium salamandrivorans (Bsal), a species related to the destructive pathogen Batrachochytrium dendrobatidis (Bd), was found and identified in Europe in 2013. Now, a decade later, a large amount of information is available. This includes data from studies in the field, reports of infection in captive amphibians, laboratory studies testing host susceptibility, and data from prospective studies that test for Bsal’s presence in a location. We conducted a systematic review of the published literature and compiled a dataset of Bsal tests. We identified 67 species that have been reported positive for Bsal, 20 of which have a threatened conservation status. The distribution of species that have been found with infection encompasses 69 countries, highlighting the potential threat that Bsal poses. We point out where surveillance to detect Bsal have taken place and highlight areas that have not been well monitored. The large number of host species belonging to the families Plethodontidae and Salamandridae suggests a taxonomic pattern of susceptibility. Our results provide insight into the risk posed by Bsal and identifies vulnerable species and areas where surveillance is needed to fill existing knowledge gaps.

Producción Científica, Tree pollarding was a dominant management strategy of European forests for centuries creating open agroforestry landscapes with important cultural and environmental values. This traditional practice has been widely abandoned in last decades with a subsequent impact in terms of biodiversity and cultural loss. Central Spain hosts the largest and best-preserved area of pollarded narrow-leaved ash (Fraxinus angustifolia Vahl.) woodlands in Europe. The main aim of this research is to obtain rigorous historical records of pollarding frequency to get adequate information for traditional ash management. We used dendrochronological techniques to evaluate temporal changes of pollarding frequency and rotation length. We analysed the stand level synchrony and the effect of land property on pollarding activity from 322 trees growing in eight pollard stands in Central Spain. Pollarding events were unequivocally identified at tree level by a characteristic change in growth pattern. We identified 2426 tree-level pruning events with the first event dated in 1777. Historical pruning recurrence ranged between 5 and 10 years with higher pollarding frequency on private lands. Pruning events within each site were synchronous, suggesting the existence of a rotational schema within each stand. Pruning frequency decreased drastically in the 1970s matching with the depopulation of rural areas and the general abandonment of traditional practices. Pollarding practices have recovered in recent decades although with lower intensity and lacking the synchronic historical patterns. Providing technical and economic support to make this traditional activity profitably would have strong environmental revenue due the multiple ecosystem services provided by pollarded ashes., iuFOR Institute Unit of Excellence" of the University of Valladolid, funded by the Junta de Castilla y León and co-financed by the European Union (ERDF "Europe drives our growth") project (CLU-2019-01), Ministerio de Ciencia e Innovación y Agencia Estatal de Investigación - MCIN/AEI/10.13039/501100011033 project PROWARM (PID2020-118444GA-100),(IJC2019-040571-I),(grant PRE2018-084106), Junta de Castilla y León, projects, (VA171P20) and (IR2020-1-UVA08), Publicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCLE

Background Rapidly resolving acute subdural hematomas (RRASDHs) have been described in case reports and case series but are still poorly understood. We hypothesized that a cohort analysis would confirm previously reported predictors of RRASDH including coagulopathy, additional intracranial hemorrhage, and low-density band on imaging. We also hypothesized that rapid resolution would be associated with improved trauma outcomes. Methods We reviewed all nonoperative acute subdural hematomas (ASDHs) treated at our center from 2011 to 2015. Inclusion criteria were ASDH on computed tomography (CT), admission Glasgow coma score >7, and repeat CT to evaluate ASDH change. RRASDH was defined as reduced hematoma thickness by 50% within 72 h. Clinical data, CT findings, and trauma end points were analyzed for the RRASDH and nonresolving groups. Results There were 154 ASDH patients included, with 29 cases of RRASDH. The RRASDH group had a lower rate of comorbidities than the nonresolving group (58.6% versus 78.4%, P = 0.03) and a lower rate of prehospital anticoagulation (7.7% versus 37.1%, P = 0.004). Previously reported predictors of RRASDH did not differ between the groups, nor did any clinical outcome measures. When compared with patients who experienced rapid growth (>50% increased width in 72 h), the RRASDH group had lower mortality (3.4% versus 23.5%, P = 0.04). Conclusions To our knowledge, this is the largest review of RRASDHs. We identified two previously unrecognized factors that may predict resolution; however, previously reported predictors were not associated with resolution. We also found no relationship between RRASDHs and improved standard trauma outcomes, calling into question the clinical significance of RRASDH.

Se propone un cuadro de clasificación del archivo personal del artista Pedro Gharel, configurado como una sección del fondo del Archivo Espacio P, que permita organizar y recuperar la documentación generada por el artista en el contexto de un archivo no institucional con voluntad de actuar como centro de documentación de prácticas artísticas contemporáneas internacionales, pionero en España. Al tratarse de un fondo complejo, constituido por una documentación heterogénea y diversa, se ha estructurado mediante un cuadro de clasificación de amplia temática que contempla todas las actividades polifacéticas e interconectadas a las que se dedicaba el artista entre las artes plásticas, visuales, escénicas y sonoras.

Infection records of Batrachochytrium dendrobatidis (Bd), a pathogen that has devastated amphibian populations worldwide, have rapidly increased since the pathogen’s discovery. Dealing with so many records makes it difficult to (a) know where, when and in which species infections have been detected, (b) understand how widespread and pervasive Bd is and (c) prioritize study and management areas. We conducted a systematic review of papers and compiled a database with Bd infection records. Our dataset covers 71 amphibian families and 119 countries. The data revealed how widespread and adaptable Bd is, being able to infect over 50% of all tested amphibian species, with over 1000 confirmed host species and being present in 86 countries. The distribution of infected species is uneven among and within countries. Areas where the distributions of many infected species overlap are readily visible; these are regions where Bd likely develops well. Conversely, areas where the distributions of species that tested negative overlap, such as the Atlantic Coast in the USA, suggest the presence of Bd refuges. Finally, we report how the number of tested and infected species has changed through time, and provide a list of oldest detection records per country.

Whit the emergence of new materials and additives to improve the performance of road infrastructure it is necessary to evaluate, compare and validate the contribution of new materials in pavement performance. This document presents the evolution of the structural performance of a natural scale section constructed of concrete. The section is divided in two areas, the first area constructed with reinforced concrete with metal fibers and the second area constructed of plain concrete. The main purpose of the experiment is the knowledge about the contribution of the metallic fiber to the pavement performance. To accelerate the deterioration of the pavement, both areas were exposed to an accelerated application of damage with the HVS (Heavy Vehicle Simulator) device. To determine the structural evolution of the pavement, surface deformation sensors were installed and deflectometry tests were performed to determine the load transfer between slabs, maximum deflection, back calculation of elasticity modulus and others parameters, the evaluation of pavement performance was made every 500,000 equivalent axles of 80 kN.

TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc.

ResumenEn los últimos 20 años, las células troncales han sido del interés de muchos grupos de investigación, y el ámbito oftalmológico no es la excepción. Sin embargo, la gran cantidad de información existente y la existencia de resultados contradictorios pueden llegar a ser confusos, haciendo que a los profesionales de la salud les sea difícil mantenerse actualizados. En este trabajo se presentan algunos de los aspectos básicos que es necesario entender sobre las propiedades de las células troncales, así como las dificultades asociadas a su empleo en la terapia oftalmológica. Está claro que a pesar de que las células troncales pueden ser una herramienta muy útil en el tratamiento de diversas enfermedades, aún desconocemos muchas de sus propiedades y de los mecanismos que las regulan. Asimismo es notable la carencia de técnicas que permitan el aislamiento con certeza de una población de células troncales puras y el trasplante al paciente de poblaciones celulares completamente diferenciadas, hechos que implican un riesgo real para el candidato a ser tratado con este tipo celular. Por el momento, la terapia que implica el uso de células troncales se ha enfocado en el tratamiento de la DMRE húmeda o seca, así como en la LSCD. No obstante, al considerar las dificultades existentes, se puede concluir que tal vez sea el momento de hacer una pausa en las posibles aplicaciones terapéuticas adicionales, y dedicarnos a entender mucho mejor este tipo celular, antes de dar un paso más en el tratamiento de problemas oftalmológicos.AbstractDuring the last 20 years, stem cells became a central issue for many research groups, and the ophthalmologic field is not the exception. Under such scenario, the huge amount of available information together with contradictory results may lead to confusion; making the continuous updating by health professionals difficult and challenging. Here, we describe some of the essential properties of stem cells, as well as the intricacies associated with their use in ophthalmic therapy. It is clear that despite stem cells may be a very useful tool in the treatment of several diseases, we still ignore many of their properties as well as the mechanisms that regulate them. Also, it is significant the lack of procedures that could allow their isolation as a pure stem cell population and their subsequent controlled transplantation into patients as completely differentiated cells; facts that imply a real risk for the candidate to this kind of treatment. At present, therapies that involve the use of stem cells have focused on treatment of wet or dry AMD, as well as LSCD. However, considering the current difficulties, one can conclude that maybe it is time to pause possible additional therapeutic uses and gain a better understanding of stem cells before we continue treating ophthalmologic disease.

Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt+ /Vim+ cell number and ΔNp63α expression when cells reached confluence and formed a 4-5 layered epithelium, while there was a concomitant increase of both Pax-6 expression and Krt+ /Vim- cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co-linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim+ /Krt+ /ΔNp63α+ /Pax-6low /α6 integrin+ phenotype. J. Cell. Physiol. 232: 818-830, 2017. © 2016 Wiley Periodicals, Inc.

Lipid droplets are dynamic organelles that store triglycerides and participate in their mobilization in adipose cells. These organelles require the reorganization of some structural components, the cytoskeleton, and the activation of lipogenic enzymes. Using confocal microscopy, we analyzed the participation of cytoskeletal components and two lipogenic enzymes, fatty acid synthase and glycerophosphate dehydrogenase, during lipid droplet biogenesis in differentiating 3T3-F442A cells into adipocytes. We show that subcortical actin microfilaments are extended at the basal side of the cells in parallel arrangement to the culture dish substrate, and that the microtubule network traverses the cytoplasm as a scaffold that supports the round shape of the mature adipocyte. By immunoprecipitation, we show that vimentin and perilipin1a associate during the early stages of the differentiation process for lipid droplet formation. We also report that the antibody against perilipin1 detected a band that might correspond to a modified form of the molecule. Finally, the cytosolic distribution and punctate organization of lipogenic enzymes and their co-localization in the proximity of lipid droplets suggest the existence of dynamic protein complexes involved in synthesis and storage of triglycerides. J. Cell. Biochem. 117: 2315-2326, 2016. © 2016 Wiley Periodicals, Inc.

Using the rabbit corneal epithelial cell line RCE1(5T5) as a model, we analyzed three differentiation stages, distinguished on basis to the growth state of cultured cells and after studying the expression of transcription factors such as Oct4, Pax6 and ΔNp63α, selected differentiation markers, and signaling or epigenetic markers such as Notch receptors and Prdm3. Namely, proliferative non-differentiated cells, committed cells, and cells that constitute a stratified epithelium with a limbal epithelial-like structure. RNAseq based transcriptome analysis showed that 4891 genes were differentially expressed among these stages displaying distinctive gene signatures: proliferative cells had 1278 genes as gene signature, and seem to be early epithelial progenitors with an Oct4+, KLF4+, Myc+, ΔNp63α+, ABCG2+, Vimentin+, Zeb1+, VANGL1+, Krt3-, Krt12- phenotype. Committed cells had a gene signature with 417 genes and displayed markers indicative of the beginning of corneal differentiation, and genes characteristic of proliferative cells; we found the possible participation of Six3 and Six4 transcription factors along this stage. The third stage matches with a stratified corneal epithelium (gene signature comprising 979 genes) and is typified by an increase in the expression of WNT10A and NOTCH 2 and 3 signaling and Cux1 transcription factor, besides Pax6, KLF4 or Sox9. The differentiated cells express about 50% of the genes that belong to the Epidermal Differentiation Complex (EDC). Analysis of the differences between corneal epithelium and epidermis could be crucial to understand the regulatory mechanisms that lead to the expression of the differentiated phenotype.

Ecological effects of alien species can be dramatic, but management and prevention of negative impacts are often hindered by crypticity of the species or their ecological functions. Ecological functions can change dramatically over time, or manifest after long periods of an innocuous presence. Such cryptic processes may lead to an underestimation of long-term impacts and constrain management effectiveness. Here, we present a conceptual framework of crypticity in biological invasions. We identify the underlying mechanisms, provide evidence of their importance, and illustrate this phenomenon with case studies. This framework has potential to improve the recognition of the full risks and impacts of invasive species.

Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5 T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.

Ecological effects of alien species can be dramatic, but management and prevention of negative impacts are often hindered by crypticity of the species or their ecological functions. Ecological functions can change dramatically over time, or manifest after long periods of an innocuous presence. Such cryptic processes may lead to an underestimation of long-term impacts and constrain management effectiveness. Here, we present a conceptual framework of crypticity in biological invasions. We identify the underlying mechanisms, provide evidence of their importance, and illustrate this phenomenon with case studies. This framework has potential to improve the recognition of the full risks and impacts of invasive species.

El objetivo del estudio es proponer un cuadro de clasificación del fondo generado por Espacio P, especializado en videoarte, como instrumento pionero en su campo que permita organizar y recuperar la documentación que conserva este archivo mueístico y otros semejantes.Culminar su organización y digitalizar dicho fondo, contribuirá a completar el panorama cultural de los años iniciales de la Democracia en España, facilitando el estudio de la aportación de los artistas a la institucionalización del arte contemporáneo y el papel de las nuevas prácticas artísticas (performance, video y arte sonoro, multimedia y arte digital pionero) a cuya interconexión contribuyó decididamente Espacio P.

This study reports an efficient reduction of the drag exerted by a flow on a cylinder when the former is forced with a plasma actuator. A three-electrode plasma device (TED) disposed on the surface of the body is considered, and the effect of the actuation frequency and amplitude is studied. Particle image velocimetry (PIV) measurements provided a detailed information that was processed to obtain the time-averaged drag force and to compare the performances of TED actuator and the canonical dielectric discharge barrier actuator. For the Reynolds number considered (Re = 5500), excitations with the TED actuator were more efficient, achieving drag reductions that attained values close to 40% with high net energy savings. The reduction of coherent structures using the instantaneous vorticity fields and a clustering technique allowed us to gain insight into the physical mechanisms involved in these phenomena. This highlights that the symmetrical forcing of the wake flow at its resonant frequency with the TED promotes symmetrical vorticity patterns which favor drag reductions. Fil: D'adamo, Juan Gastón Leonel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Mecánica. Laboratorio de Fluidodinámica; Argentina Fil: Leonardo, Leandro. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Mecánica. Laboratorio de Fluidodinámica; Argentina Fil: Castro Hebrero, Federico Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Mecánica. Laboratorio de Fluidodinámica; Argentina Fil: Sosa, Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Mecánica. Laboratorio de Fluidodinámica; Argentina Fil: Duriez, Thomas Pierre Cornil. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Mecánica. Laboratorio de Fluidodinámica; Argentina Fil: Artana, Guillermo Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Departamento de Ingeniería Mecánica. Laboratorio de Fluidodinámica; Argentina

The role of vimentin (Vim) in diploid epithelial cells is not well known. To understand its biological function, we cultured human epidermal keratinocytes under conditions that support migration, proliferation, stratification and terminal differentiation. We identified a keratinocyte subpopulation that shows a p63(+)/α5β1(bright) phenotype and displays Vim intermediate filaments (IFs) besides their keratin IF network. These cells were mainly located at the proliferative/migratory rim of the growing colonies; but also, they were scarce and scattered or formed small groups of basal cells in confluent stratified epithelia. Stimulation of cells with EGF and wounding experiments in confluent arrested epithelia increased the number of Vim(+) keratinocytes in an extent higher to the expected for a cell population doubling. BrdU labeling demonstrated that most of the proliferative cells located at the migratory border of the colony have Vim, in contrast with proliferative cells located at the basal layer at the center of big colonies which lacked of Vim IFs, suggesting that Vim expression was not solely linked to proliferation. Therefore, we silenced Vim mRNA in the cultured keratinocytes and observed an inhibition of colony growth. Such results, together with long-term cultivation assays which showed that Vim might be associated to pattern formation in cultured epithelia, suggest that Vim expression is essential for a highly motile phenotype, which is necessary for keratinocyte colony growth and possibly for development and wound healing. Vim(+)/p63(+)/α5β1(bright) epithelial cells may play a significant physiological role in embryonic morphogenetic movements; wound healing and other pathologies such as carcinomas and hyperproliferative diseases.

Background The bone nonunion is an important complication of bone fracture repair. The existing models developed on small animal species prevent using osteosynthesis materials designed to be implanted in human bones. The goal of this study was to develop a nonunion process in a noncritical segmental tibial defect in sheep, a species analogous in size to humans. Materials and methods The animals were divided into two groups of four animals each. In Group 1 (experimental), the defect was created by surgically stripping the periosteum from the edges of a distal tibial osteotomy, keeping the edges 5 mm apart, and placing an incomplete O-shaped silicone ring in the gap. Group 2 (control) was intervened with a simple fracture at the distal end of the tibia. In both groups an interlocking nail was used as a fixation system. Over 8 wk after surgery, radiographs and histologic and histomorphometric analyses were performed. Results The control group showed a typical bone repair process. In contrast, the experimental group showed a fracture line with rounded edges and a scarce callus formation. The bone callus showed reduced amount of bone formation and large content of fibrous tissue ( P = 0.001). Conclusions These results indicate that our model developed an atrophic nonunion in sheep, a species having multiple similarities to humans, such as weight, size, bone structure, and bone remodeling process.

Stem cells emerged as a concept during the second half of 19(th) century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet's membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing.

TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca

Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt

Summary Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3–5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.

Aims A major objective in the treatment of obese individuals is the development of agents that reduce body fat and improve metabolic homeostasis. Among a variety of biological effects, green tea exerts an anti-obesity effect; however, the mechanisms behind its activity on adipose tissue are uncertain. Tea contains high levels of (−)-epigallocatechin-3-gallate (EGCG), which is one of its main bioactive substances. Therefore, we studied the effects of EGCG on mouse embryonic fibroblast-adipose like cell line (3T3-L1) preadipocyte proliferation and differentiation. Main methods 3T3-L1 cells were incubated with physiologically attainable (0.1–1 μM) and pharmacological (5 μM, 10 μM) concentrations of EGCG for various lengths of time. Cell proliferation was assessed by cell counting and cell cycle analysis. Adipose conversion was evaluated by lipid accumulation and expression of CCAAT/enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) transcription factors. Key findings A dose dependent suppressive effect on preadipocyte proliferation was observed, with the highest reduction in cell number at 10 μM EGCG. On the other hand, adipose conversion was fully inhibited with 10 μM EGCG. Flow-cytometric analysis showed that 3T3-L1 cells treated with EGCG underwent an arrest of cell cycle at G2/M. The inhibition of the expression of C/EBPα and PPARγ was accompanied by the inhibitory effect of EGCG. Microscopic observation showed that 3T3-L1 cells treated with EGCG maintained the fibroblastic shape and failed to accumulate cytoplasmic fat droplets even after the induction of differentiation. Significance Our results suggest that EGCG reduces adipogenesis through an arrest of cell cycle and a blockage of adipose phenotype expression. These results also suggest that the anti-obesity activity of green tea may be partially attributed to its suppressive effects in adipogenesis.

This article summarizes the findings from 3 recipients of hand allografts, including a description of the preparatory surgery and the transplant and secondary procedures to enhance the function of the hand, forearm, and arm allografts. The study focuses on the complications and disability reported by each patient, with a minimum follow-up of 2 years. The few complications were controlled successfully with medical treatment. Hand transplantation is a major reconstructive procedure that requires careful medical follow-up. The authors provide the first report of a significant improvement in disabilities of the upper limb as a result of hand allotransplantation.

In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme involved in several cellular functions including glycolysis, membrane transport, microtubule assembly, DNA replication and repair, nuclear RNA export, apoptosis, and the detection of nitric oxide stress. Therefore, modifications in the regulatory ability and function of GAPDH may alter cellular homeostasis. We report here that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone, which are well-known ligands for the aryl hydrocarbon receptor (AhR), increase GAPDH mRNA levels in vivo and in vitro, respectively. These compounds fail to induce GAPDH transcription in an AhR-null mouse model, suggesting that the increase in GAPDH level is dependent upon AhR activation. To analyse the consequences of AhR ligands on GAPDH function, mice were treated with TCDD and the level of liver activity of GAPDH was determined. The results showed that TCDD treatment increased GAPDH activity. On the other hand, treatment of Hepa-1 cells with beta-naphthoflavone leads to an increase in microfilament density when compared to untreated cultures. Collectively, these results suggest that AhR ligands, such as polycyclic hydrocarbons, can modify GAPDH expression and, therefore, have the potential to alter the multiple functions of this enzyme.

Pax-6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1-(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax-6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax-6-positive cells were also K3 keratin-positive, suggesting that Pax-6 is expressed in differentiating cells. Pax-6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT-qPCR. The raise in Pax-6 expression preceded for 9 h the increase in LDH-H and LDH-M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full-length mRNAs encoding for the two major Pax-6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax-6 mRNA was 2- to 2.5-fold more abundant than Pax-6(5a) mRNA. The ectopic expression of Pax-6 or Pax-6(5a) decreased proliferative ability leading to the formation of abortive, non-proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax-6. Collectively, results show that Pax-6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax-6 as the main driver of the differentiation of corneal epithelial cells.

Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-beta biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family.

Understanding of visual system function and the development of new therapies for corneal diseases and damages depend upon comprehension of the biological roles of the tissue. The in vitro cultivation of corneal epithelial cells and cell lines derived from them has become a powerful tool to analyze and understand such issues. Currently, researchers have developed well-defined and precisely described culture protocols and a collection of corneal epithelial cell lines. These cell lines have been obtained through different experimental approaches: (1) the ectopic expression of oncogenes, (2) the inactivation of p16 and p53 pathways and hTERT expression, and (3) the spontaneous establishment after serial cultivation of cells. The advantages or disadvantages for these approaches are discussed. In conclusion, the availability of several culture protocols and immortalized cell lines that express corneal epithelial phenotype will be useful for investigating issues such as gene regulation and tissue development, or for validating alternative methods in toxicology.

Lipid droplets are dynamic organelles that store triglycerides and participate in their mobilization in adipose cells. These organelles require the reorganization of some structural components, the cytoskeleton, and the activation of lipogenic enzymes. Using confocal microscopy, we analyzed the participation of cytoskeletal components and two lipogenic enzymes, fatty acid synthase and glycerophosphate dehydrogenase, during lipid droplet biogenesis in differentiating 3T3-F442A cells into adipocytes. We show that subcortical actin microfilaments are extended at the basal side of the cells in parallel arrangement to the culture dish substrate, and that the microtubule network traverses the cytoplasm as a scaffold that supports the round shape of the mature adipocyte. By immunoprecipitation, we show that vimentin and perilipin1a associate during the early stages of the differentiation process for lipid droplet formation. We also report that the antibody against perilipin1 detected a band that might correspond to a modified form of the molecule. Finally, the cytosolic distribution and punctate organization of lipogenic enzymes and their co-localization in the proximity of lipid droplets suggest the existence of dynamic protein complexes involved in synthesis and storage of triglycerides. J. Cell. Biochem. 117: 2315-2326, 2016. © 2016 Wiley Periodicals, Inc.

The distribution of fish in the water column, particularly of demersal species near the seabed, is relevant to the detection and assessment of fish populations. Here, acoustic data recorded during six consecutive seasons on the Patagonian stock of argentine hake (Merluccius hubbsi) provided detailed information about the vertical distribution of the species along the acoustic track. It was determined that during the day, about 95% of individuals are located within the first ten meters above the bottom, and that almost half of them are concentrated in the first meter from it. An exponential function was found to represent this near-bottom distribution, with a marked positive gradient in the vicinity of the seabed. The height of the Acoustic Dead Zone (ADZ) was estimated and employed together with the true vertical distribution of fish to calculate correction factors for the nautical area scattering coefficient (NASC). Considering the closeness of fish to the bottom, a distribution variable is proposed, the demersality coefficient, which allows mapping the behavior of fish over the area of distribution, providing a tool for the analysis of distributional aspects of ecological importance.

A method is described for assembling an evaporation manifold from a cell culture flask, which allows for efficient nitrogen evaporation of hexane from 24- and 96-well plates. The precursor parts are readily available in most research laboratories. The nitrogen evaporation manifold is inexpensive, could possibly be used with other organic solvents, and appears ideal for a small number of samples in a multi-well format.

We studied the commitment of 3T3-F442A cells during stimulation with adipogenic serum or growth hormone. Confluent 3T3-F442A preadipocytes were incubated with adipogenic medium for increasing times; the number of adipose clusters, GPDH activity, and lipid accumulation were evaluated. Results show that cell commitment took place during the first 24-36 h after stimulation under adipogenic conditions. Then, cultures underwent a 2-fold increase in total cell number through selective multiplication of committed cells, followed by a dramatic decrease in colony-forming ability and 300- to 1000-fold raise in GPDH activity. Cell commitment was not modulated by insulin, but this hormone stimulated clonal expansion of committed cells and lipogenesis. Commitment was inhibited by TNF-alpha at concentrations as low as 5 ng/ml, and by retinoic acid. The results show that TNF-alpha inhibits adipose conversion at two different levels: at concentrations as low as 5 ng/ml, it blocks commitment, and at concentrations of 100 ng/ml or higher the cytokine seems to block mitotic expansion and other steps of differentiation after cell commitment. The identification of a specific time for cell commitment would allow the study of the early genes that might regulate cell reprogramming into adipocytes.

Stem cell niche may be described as an anatomically defined and protected location that provides housing, positioning information and signaling inputs necessary to support normal stem cell activity. Based on the distribution of the differentiation-linked keratins, proliferative potential and wound healing abilities in ocular surface, it was proposed that an anatomical structure, the limbus, was the presumptive site of residence of corneal stem cells. Further analysis determined that the limbus contains a specific anatomical structure that probably provides the microenvironmental characteristics that correspond to the stem cell niche. This structure was termed as the Limbal Epithelial Crypt (LEC). Accumulated evidences show that the LEC is the site where stem cells interact directly and/or indirectly with at least six different cell types: epithelial, stromal, Langerhans cells, melanocytes, and telocytes. In addition, a rich and distinctive vasculature as well as an extensive neural network exist at limbal niches. These cell types, together with growth factors, cytokines and specific components of the Extracellular Matrix establish the conditions for the regulated growth, migration and delayed differentiation of the corneal stem cells. In spite of the wide variety of molecular markers described for limbal epithelial cells, it has been extremely difficult to isolate stem cells. This is explained by the persistence of stem cell markers in the transient amplifying cell population and in the early differentiating cells. Consequently, the use of stem cells in ophthalmic therapy shows variable outcomes and research must be increased before a formal clinical use. Nevertheless, the analysis of the different cells and factors involved in stem cell regulation, may help the development of new therapies based on the interference or stimulation of the signaling pathways and microenvironmental components that control limbal stem cells.

In a murine model of full-thickness wounds, healing is stimulated by the application of human frozen cultured epidermal sheets. With immunofluorescence techniques, we studied, during this process, the spatial and temporal pattern of expression of: transforming growth factor-alpha (TGF-alpha); transforming growth factor-beta (TGF-beta) isoforms 1, 2, and 3; platelet-derived growth factor (PDGF); and the extracellular matrix proteins fibronectin, collagen IV, and tenascin. The growth factors, with the exception of PDGF, were found to be located in the frozen cultured sheet of keratinocytes before and after its application to the wound, whereas collagen IV and tenascin were deposited in the connective tissue under the frozen cultures. None of these factors were detected in control wound beds. Monoclonal antibodies against collagen IV and tenascin showed that both were of murine origin. We propose that the frozen cultures of human keratinocytes promote faster reepithelialization through the release of growth factors such as TGF-alpha which directly enhance migration and proliferation of murine keratinocytes, and through the stimulation of murine subepithelial cells, by TGF-beta, to secrete basement membrane proteins such as collagen IV, laminin, and tenascin, which provide a provisional substrate that improves migration of the murine epidermal cells.

Interruption of the main descending respiratory drive to phrenic motoneurons by cold block or spinal cord hemisection results in morphological modifications of the ipsilateral phrenic nucleus in the rat. The modifications consist of an increase in the number of multiple synapses and dendrodendritic appositions and elongation of the asymmetric and symmetric synaptic active zones. Hemisection and hemispinalization by cold block cause not only "functional deafferentation" of the ipsilateral phrenic neurons (i.e., a loss of ipsilateral descending respiratory drive), but also an increase in the remaining contralateral descending respiratory drive. The contralateral respiratory pathways connect with phrenic motoneurons ipsilateral to cold block or hemisection by decussating collateral axons which cross the spinal cord midline below the hemisection/cold block site. Thus, the phrenic nucleus synaptic plasticity could possibly be induced by functional deafferentation or by an increase of the descending respiratory drive. To differentiate between these two possible inducers of the plasticity, we assessed the synaptic morphology of the phrenic nucleus of nonoperated rats exposed to 48 h of hypoxia in an atmosphere chamber. The hypoxia exposure produces an increased descending respiratory drive without functional deafferentation. The quantitative data extracted from electron micrographs of the phrenic nucleus from four experimental rats were compared with the data from four normal breathing animals. Phrenic nucleus morphometric analysis showed that there was no significant difference in the mean number of single synapses between the samples from control animals (141 +/- 12.12) and the experimental animals (156 +/- 26.73). Similarly, no significant difference was detected in the total number of synaptic active zones of control animals (178.25 +/- 11.13) and experimental animals (195.05 +/- 5.35). Furthermore, the length of synaptic active zones of asymmetrical synapses (0.21 +/- 0.024 micron) or symmetrical synapses (0.22 +/- 0.022 micron) did not change significantly compared to the synaptic active zone length in control animals (0.21 +/- 0.018 micron for asymmetrical and 0.21 +/- 0.010 micron for symmetrical). We conclude that no synaptic plasticity occurs in the phrenic nucleus without functional deafferentation in spite of an increase in descending respiratory drive. Therefore functional deafferentation may be the primary inducer of phrenic nucleus synaptic plasticity occurring after hemisection or cold block.

Diagnosis of myelopathies of vascular origin is difficult and they are probably underdiagnosed at this time because of the lack of diagnostic tools. A recent report of a 58 year old patient who developed ASAS after an episode of cardiac arrest pointed out the importance of MRI and somatosensory evoked potentials (SEP) to support the diagnosis. MRI with T2 weighted imaging demonstrated diffuse signal abnormalities in both gray matter and surrounding white matter below T7. Furthermore, SEP latencies showed a delay between T6 and T7. Therefore, new technologies including MRI and SEP may improve the diagnosis of spinal cord ischemic injuries. A brief discussion of the normal blood supply of the human spinal cord is presented in this review followed by new, pathophysiologically based classifications of the clinical syndromes of vascular myelopathies. A complete description of the clinical syndromes related to vascular myelopathies is included. Vascular myelopathies were divided into acute and chronic syndromes depending on the time at which the pathophysiological events take place. Subsequently, the two major groups of vascular myelopathies were divided depending on the type of vascular damage, e.g., arterial, venous and/or mixed origin. Posttraumatic spinal cord ischemia is included in the present classification because it is generally considered to be a significant factor contributing to secondary damage following blunt trauma. Since several new diagnostic techniques are now available to characterize the pathology of spinal cord injury, physicians involved in the diagnosis and treatment of vascular myelopathies may find the new classification useful in correlating clinical presentation with subjacent pathology. Identification of the correct pathology should result in more accurate treatment approaches.

Sin resumen.

To know whether 22.0-kDa human growth hormone (hGH22K) and the 20.0-kDa isoform (hGH20K) show different activities, we used 3T3-F442A and 3T3-F442A/C4 cells to evaluate their adipogenic and metabolic effects. Both isoforms had similar adipogenic and insulinlike activities. Lipolytic and diabetogenic effects of hGH22K were, respectively, 12.5 and 1.7-fold higher than those found for hGH20K. The 3T3-F442A/C4 clone was not responsive to insulinlike and diabetogenic effects of hGH. The results suggest that adipogenic and lipolytic effects of hGH are mediated by mechanisms different from those involved in insulin-like and diabetogenic activities.

We describe a new Swiss 3T3 preadipose clone, 3T3-F442A/C4, which shows higher sensitivity to serum adipogenic factors and to human growth hormone as compared to other 3T3 preadipose clones. The 3T3-F442A/C4 clone exhibited several characteristics different from the parental 3T3-F442A cells, mainly a high extent of adipose conversion under culture conditions that are non-adipogenic for the parental cells. The 3T3-F442A/C4 cells are not committed to undergo adipose differentiation, since they do not differentiate into adipocytes under serum-free or low-serum culture conditions, unless adipogenic factors or growth hormone are added into the culture medium. The 3T3-F442A/C4 cells showed 1.5- to 3.6-fold higher sensitivity to serum adipogenic factors and 5- to 6-fold higher sensitivity to human growth hormone as compared to the 3T3-F442A cells. The 3T3-F442A/C4 variant clone also differed from the parental clone by having a shorter population doubling time, an increased saturation density, and lower activity levels in some biochemical markers of adipose differentiation. On the other hand, the new variant clone has a similar proportion of cells susceptible to become adipocytes, and a similar response to insulin as compared to the parental cells. Our results show that the 3T3-F442A/C4 cells represent a new 3T3 preadipose clone that could be useful as a bioassay to evaluate growth hormone activity, as well as to purify and characterize hormones, adipogenic factors, and those compounds that affect mammalian adipogenesis.