Comparison of morphological changes of corneal collagen fibers treated with collagen crosslinking agents using second harmonic generation images

Corneal cross-linking (CXL) is a common surgical procedure used to modify corneal biomechanics and stabilize keratoconus progression which is still under discussion. Its side effects, which are mostly related to anatomical unpredictability and stromal exposure, are the reason for the search for new CXL agents. In this work we have quantitatively evaluated the porcine corneal stroma architecture treated with collagen crosslinking agents such as riboflavin solutions and açai extract, using second harmonic generation microscopy. Aimed at evaluating the morphological changes in the corneal stroma after collagen crosslinking under a CXL chemical agent, a tubeness filter based Hessian matrix to obtain a 3D fiber characterization of the SHG images was applied. The results showed a curling effect and shortening of the collagen fibers treated with açai as compared to the control. They also showed a higher degree of clustering of the collagen fibers with larger empty spaces when compared to the other two groups. We believe that studies such as these presented in this paper are a good direct nondestructive and free labeling evaluation technique that allows the observation of morphologic features of corneas treated with new CXL agents.Fil: Zeitoune, Angel Alberto. Universidad Nacional de Entre Ríos. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática; ArgentinaFil: Bersanetti, Patrícia A.. Universidade Federal de Sao Paulo; BrasilFil: Schor, Paulo. Universidade Federal de Sao Paulo; BrasilFil: Erbes, Luciana Ariadna. Universidad Nacional de Entre Ríos. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática; ArgentinaFil: Cesar, Carlos L.. Instituto Nacional de Fotônica Aplicada à Biologia Celular; Brasil. Universidade Federal do Ceara; BrasilFil: Adur, Javier Fernando. Universidad Nacional de Entre Ríos. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática; Argentin

Turismo sustentável e desenvolvimento local: projecto da Mata de Sesimbra

A presente dissertação investiga o campo do Turismo Sustentável/Ecológico, caracterizando diferentes conceitos; enquadra a evolução da problemática ambiental; estuda a relação entre o Turismo e o Ambiente, os seus impactos (económicos, sócio-culturais e ambientais) e respectiva medição e controlo; e destaca a importância de um bom planeamento e gestão turísticos. Numa segunda fase, relativa à área dos Projectos Turísticos Sustentáveis, e de modo a consolidar esta dissertação toma-se o caso de estudo do Projecto da Mata de Sesimbra – o primeiro projecto integrado de construção, ecoturismo sustentável e conservação da natureza a nível nacional, levado a cabo pela imobiliária Pelicano em parceria com a ONG World Wildlife Fund e a BioRegional – à semelhança do bem sucedido Projecto BedZed no Reino Unido (uma eco-cidade que permite aos seus residentes terem um estilo de vida amigo do ambiente, com o nível de conforto e qualidade de vida de hoje em dia). Então, são apresentadas as entidades envolvidas e projectos semelhantes, antes da análise do plano de pormenor e plano de gestão ambiental do projecto em estudo. Esta investigação pretende demonstrar o mérito que projectos como este têm no desenvolvimento local, os seus impactos positivos e negativos, e indica os instrumentos que os norteiam e os obstáculos que se lhes colocam, como linhas de orientação para projectos semelhantes. Para o efeito, tendo por base a documentação relativa ao mesmo, o projecto é analisado na vertente territorial (desenvolvimento do projecto e planos de ordenamento do território seguidos), sócio-cultural (relação entre a população local e o empreendimento turístico) e económica (importância do turismo, em especial de projectos inovadores deste género, na actividade local), tendo em conta a problemática ambiental transversal às três vertentes, ao tentar responder às perguntas de partida: Como criar valor local com o Projecto da Mata de Sesimbra? Qual a gestão do projecto, barreiras e apoios inerentes ao desenvolvimento turístico sustentável em Sesimbra?This dissertation investigates the field of Sustainable/Ecological Tourism, characterizes different concepts, within the evolution of the environmental problematic; studies the relation between Tourism and the Environment, its impacts (economic, socialcultural and environmental), evaluation and control; and highligths the importance of an efficient tourism planning and management. In the second part, concerning Sustainable Touristic Projects, to consolidate this work the case-study “Mata de Sesimbra” Project, in Portugal, is presented and analysed – the first national project that combines building constrution, sustainable ecotourism and nature conservation, developed by the real estate company Pelicano in partnership with the ONG World Wildlife Fund and BioRegional – following the example of the well succeeded BedZed Project in the United Kingdom (an eco-village where its residents can have a ecofriendly style of life, with the comfort and quality of life of nowadays). Therefore, the entities involved and similar projects are presented, before the analysis of the project’s detail plan and environmental management plan. This investigation aim consists in demonstrating the merit of such projects in the local development, its positive and negative impacts, indicating the instruments that guide them and the obstacles in its implementation, as a guide line for similar iniciatives. Based on the documentation related to it, the project is analysed by three approaches: territorial (project development and land-use plans followed), social-cultural (the relationship between local community and the touristic endeavour) and economic (tourism importance to the local economy, specially of this kind of innovative projects), taking into account the environmental issue cross-cutting these approaches, trying to answer the starting point questions: How to create local value with the “Mata de Sesimbra” Project? Which are the project management, barriers and supports inherent to sustainable touristic development in Sesimbra

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates.

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra

Corneal Absorption of a New Riboflavin-Nanostructured System for Transepithelial Collagen Cross-Linking

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion. © 2013 Bottos et al

Characterization of Rabbit Corneas Subjected to Stromal Stiffening by the Açaí Extract (<i>Euterpe oleracea</i>)

<p><i>Purpose</i>: In this study, we characterized rabbit corneas subjected to corneal cross-linking (CXL) with açaí extract compared with a riboflavin photo-stimulated procedure.</p> <p><i>Materials and Methods</i>: The corneas of the slaughterhouse rabbits were divided into three groups: control, consisting of untreated corneal samples; riboflavin/UVA, where corneas were treated with 0.1% riboflavin photo-stimulated at 365 nm as the standard protocol; and açaí, where the samples were subjected to 4% açaí extract for 0.5–2 h. After the CXL procedure, corneas of the three groups were characterized by analyzing their elastic modulus and thermal denaturation profile.</p> <p><i>Results</i>: The elastic modulus at 3% strain showed an approximately threefold increase in the riboflavin/UVA group and 10.5 times in the corneas treated with 4% açaí extract for 2 h, compared with the control group (<i>p</i> < 0.01). The denaturation temperature values of the two groups of crosslinked corneas increased significantly (<i>p</i> < 0.05) and were more pronounced in the açaí group.</p> <p><i>Conclusions</i>: The açaí extract was effective in promoting CXL in rabbit corneas as characterized by the different techniques.</p

CD spectra of AngI (A), TOAC⁰-AngI (B), TOAC¹-AngI (C) TOAC³-AngI (D), TOAC⁵-AngI (E), TOAC⁸-AngI (F), TOAC⁹-AngI (G) and TOAC¹⁰-AngI (H) as a function of TFE concentration.

<p>CD spectra of AngI (A), TOAC⁰-AngI (B), TOAC¹-AngI (C) TOAC³-AngI (D), TOAC⁵-AngI (E), TOAC⁸-AngI (F), TOAC⁹-AngI (G) and TOAC¹⁰-AngI (H) as a function of TFE concentration.</p

EPR spectra of TOAC⁰-AngI (A), TOAC¹-AngI (B), TOAC³-AngI (C), TOAC⁵-AngI (D), TOAC⁸-AngI (E), TOAC⁹-AngI (F) and TOAC¹⁰-AngI (G) in 20 mM phosphate buffer, pH 7.0.

<p>Field scan: 100 Gauss.</p

Kinetic parameters for hydrolysis of AngI and its TOAC-analogues by purified rabbit lung ACE^a.

<p>^aExperimental conditions: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136608#sec002" target="_blank">Methods</a>.</p><p>^bFrom Teixeira et al., 2007 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136608#pone.0136608.ref031" target="_blank">31</a>].</p><p>^cNot hydrolyzed by ACE.</p><p>Kinetic parameters for hydrolysis of AngI and its TOAC-analogues by purified rabbit lung ACE<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136608#t001fn001" target="_blank">^a</a>.</p

Kinetics of ACE-catalyzed hydrolysis of TOAC⁰-AngI (A), TOAC¹-AngI (B), TOAC³-AngI (C), and TOAC⁵-AngI (D) monitored by the variation of the h₀/h_-1 values in the peptides EPR spectra as a function of time.

<p>Kinetics of ACE-catalyzed hydrolysis of TOAC⁰-AngI (A), TOAC¹-AngI (B), TOAC³-AngI (C), and TOAC⁵-AngI (D) monitored by the variation of the h₀/h_-1 values in the peptides EPR spectra as a function of time.</p