Electrophysiological characterization of ClC-7.

<p>If not otherwise indicated conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012585#pone-0012585-g003" target="_blank">Fig. 3</a>. <b>A:</b> pH dependence of ClC-7 transport activity. Transport was initiated by a rapid concentration jump of 30 mM NaCl. Solutions: (NA) 330 mM Na-aspartate, 3 mM Ca-gluconate, 60 mM HEPES, (A) 30 mM NaCl, 300 mM Na-aspartate, 3 mM Ca-gluconate, 60 mM HEPES. HEPES buffer was replaced by MES or TRIS for acidic and basic conditions, respectively. After buffer exchange to a different pH, sensors were incubated for 15 min to allow equilibration to new conditions. <b>B:</b> Specificity of ClC-7 anion transport with Cl⁻>Br⁻>I⁻. Transport was initiated by a rapid pH jump (establishing a proton gradient pH 5.0 inside, pH 7.2 outside of the vesicle) with different anions present in all solutions. Solutions: (NA) 30 mM NaCl (NaBr, NaI), 300 mM Na-aspartate, 3 mM Ca-gluconate, 30 mM MES, pH 5.0. (A) 30 mM NaCl (NaBr, NaI), 300 mM Na-aspartate, 3 mM Ca-gluconate, 30 mM MOPS, pH 7.2). pH artifacts (same protocol, but substrate substituted by 30 mM Na-aspartate in A and NA, amplitude: ∼1 nA) were subtracted and the resulting currents were normalized to currents in the presence of NaCl. <b>C:</b> H⁺-coupled transporter activity of ClC-7 compared to other CLC proteins (ClC-Ka+barttin and EcClC-1). Transport was initiated by a simultaneous NaCl concentration (30 mM) and pH (ΔpH∼2) jump. Solutions pH 5.0_inside–7.2_outside: (NA) 30 mM Na-aspartate, 30 mM MES, pH 5.0, (A) 30 mM NaCl, 30 mM MOPS, pH 7.2. Solutions pH 9.0_inside–7.2_outside: (NA) 30 mM Na-aspartate, 30 mM TRIS, pH 9.0, (A) 30 mM NaCl, 30 mM MOPS, pH 7.2. In addition all buffers contained 300 mM Na-aspartate and 3 mM Ca-gluconate. pH artifacts (same protocol, substrate in A substituted by 30 mM Na-aspartate, amplitude: ∼1 nA) were subtracted and resulting currents were normalized to NaCl initiated currents in the absence of a pH gradient (pH 7.2). P = Student's <i>t</i>-test, significance level = 0.05.</p

Inhibition of ClC-7.

<p>Several putative Cl⁻ channel inhibitors were tested for inhibition potency (Tab.1). ClC-7 was activated with 30 mM Cl⁻ concentration jump while different concentrations of inhibitors (1–1000 µM) were present in all buffers. The plots show mean inhibition values and standard deviation of at least 3 independent experiments (5 measurements per concentration each). The data was fitted with a Hill function, yielding IC₅₀ values of 39±3.8 µM (Hill coefficient n = 0.6) for DIDS and 156±7.8 µM (Hill coefficient n = 1.0) for NPPB.</p

Localization of wild type and G213R ClC-7 in CHO cells.

<p><b>A:</b> Confocal images of wt ClC-7-EGFP expressing living CHO cells stained with LysoTracker®-Red (top row). Images of EGFP fluorescence, LysoTracker®-Red fluorescence and the merge of both are shown. G213R ClC-7-EGFP stained with LysoTracker®-Red (middle row) and ER-Tracker™-Red (lower row). Individual fluorescence images and merge are shown. <b>B:</b> Co-expression of wt ClC-7-EGFP with its ß-subunit Ostm1. Compared images were recorded with fixed hardware settings and at constant experimental conditions. Localization of ClC-7 in Ostm1 expressing cells was not affected but a significantly increased EGFP fluorescence could be detected. <b>C:</b> Co-expression of G213R ClC-7-EGFP with Ostm1 partly (∼50%) restored lysosomal localization.</p

Analyzed Cl⁻ Channel inhibitors.

<p>16 different putative Cl⁻ channel inhibitors were tested. Concentration range (µM), maximum inhibition (%) and IC₅₀ value (if determinable) is shown. *Could not be investigated (see text), #Precipitates at high concentrations (>100 µM).</p

Fold expression (mean(95%CI)) of pre-hypertrophic marker in response to 1 and 10 nM sCT stimulation of T3-induced explants compared with T3-induced alone (T3).

§<p>P-value of ANOVA test,</p>#<p>P-value of Dunnett’s post-test.</p

Expression and induction of the calcitonin receptor (CTR) as a result of T3 stimulation.

<p>The level of cAMP produced by the chondrocytes two hours after application of A) salmon calcitonin (sCT) or B) human calcitonin (hCT). The measurements were taken at day 0, 4 and 8 in explants with or without T3. The measurements were normalized to vehicle control for each time point, giving the fold induction. Significance level; *p<0.05, and ***p<0.001, data shown as geometric mean with 95%-CI (N = 2, n = 10).</p

The effect of co-stimulation with salmon calcitonin (sCT) on cartilage turnover.

<p>A) Cell viability, measured by Alamar blue, of the cartilage explants. B) Induction of alkaline phosphatase (ALP) activity in the conditioned medium. C) Measurement of matrix metalloproteinase (MMP)-derived type II collagen degradation fragments (C2M) released to the conditioned medium. D) Measurement of type II procollagen (P2NP) released to the conditioned medium. E) Measurement of MMP-derived aggrecan collagen degradation fragments (AGNx-II) released to the conditioned medium. Comparisons were performed by one-way ANOVA with Dunnetts post test. Significance levels; *p<0.05 and **p<0.01, data shown as mean with CI-95% n = 6.</p

Does exposure to phthalates influence thyroid function and growth hormone homeostasis? The Taiwan Environmental Survey for Toxicants (TEST) 2013

[[abstract]]BACKGROUND: Previous epidemiologic and toxicological studies provide some inconsistent evidence that exposure to phthalates may affect thyroid function and growth hormone homeostasis. OBJECTIVE: To assess the relations between exposure to phthalates and indicators of thyroid function and growth hormone homeostasis disturbances both among adults and minors. METHODS: We conducted a population-based cross-sectional study of 279 Taiwanese adults (>/=18 years old) and 79 minors (<18 years old) in 2013. Exposure assessment was based on urinary biomarkers, 11 phthalate metabolites measured by using online liquid chromatography/tandem mass spectrometry. Indicators of thyroid function included serum levels of thyroxine (T4), free T4, triiodothyronine, thyroid-stimulating hormone, and thyroxine-binding globulin (TBG). Growth hormone homeostasis was measured as the serum levels of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP3). We applied multivariate linear regression models to examine these associations after adjusting for covariates. RESULTS: Among adults, serum T4 levels were negatively associated with urinary mono-(2-ethyl-5-hydroxyhexyl) phthalate (beta=-0.028, P=0.043) and the sum of urinary di-(2-ethylhexyl) phthalate (DEHP) metabolite (beta=-0.045, P=0.017) levels. Free T4 levels were negatively associated with urinary mono-ethylhexyl phthalate (MEHP) (beta=-0.013, P=0.042) and mono-(2-ethyl-5-oxohexyl) phthalate (beta=-0.030, P=0.003) levels, but positively associated with urinary monoethyl phthalate (beta=0.014, P=0.037) after adjustment for age, BMI, gender, urinary creatinine levels, and TBG levels. Postive associations between urinary MEHP levels and IGF-1 levels (beta=0.033, P=0.006) were observed. Among minors, free T4 was positively associated with urinary mono benzyl phthalate levels (beta=0.044, P=0.001), and IGF-1 levels were negatively associated with the sum of urinary DEHP metabolite levels (beta=-0.166, P=0.041) after adjustment for significant covariance and IGFBP3. CONCLUSIONS: Our results are consistent with the hypothesis that exposure to phthalates influences thyroid function and growth hormone homeostasis

A new method for dynamic studies, in situ, of water infiltration in the unsaturated zone of soil.

A agricultura é a atividade humana que mais afeta o meio ambiente. Imensas quantidades de insumos agrícolas são aplicados sobre o solo e grande parte destes degrada os recursos hídricos. Para uma investigação adequada do efeito destes insumos, estudam-se as propriedades hidráulicas do solo, que influem no transporte de solutos neste meio. Medir tais propriedades e modelar os parâmetros correlatos são tarefas extremamente complexas, devido ao tempo requerido, dinheiro, instrumentação e escala. As metodologias convencionais inferem as propriedades hidráulicas em amostras que estão em equilíbrio, através de técnicas invasivas e sob restrições especiais. Esta tese contribui com a ciência do ambiente, via ciência do solo, propondo um novo método de estudo da infiltração da água na região não-saturada do solo, utilizando a tomografia computadorizada (TC). O tomógrafo foi aqui desenvolvido e construído. A TC, neste trabalho, mediu a umidade (teta) durante o fluxo não-saturado e, através da solução numérica da equação de Richards e do modelo de Rossi-Nimmo, obtiveram-se a curva de retenção, a sortividade, k(teta) e a difusividade D(teta). Resultados qualitativos, como imagens 2D e 3D, e resultados quantitativos demonstraram a boa correlação do método proposto com o método tradicional de medida da curva de retenção. Amostras de solo estrurado foram analisadas em laboratório e em campo.Agriculture is the human activity that most affects the environment. Huge amounts of chemicals are applied on the soil. Pesticides percolation and runoff degrades water resources. Thus, soil hydraulic properties must be known due to their influence on solute transport. The measurement of these properties and the modelling of related parameters are often difficult, if not impossible, due to the involved time, money, instrumentation, and scale. Traditional methodologies infer hydraulic properties in samples that are in equilibrium, through invasive techniques and under some special constraints. This thesis contributes with environmental science, via soil science, as it proposes a new method to study the infiltration in the unsaturated zone of soil, by means of CT. The scanner was developed and constructed in this work. The proposed methodology uses profiling CT to measure the water content (theta) during the water flow, and by means of numerical solution of Richards equation and Rossi-Nimmo model water retention, sorptivity, hydraulic conductivity k(theta), and diffusivity D(theta) are obtained. Qualitative results, as 2D and 3D images, are presented and the quantitative results of water retention show good correlation of the proposed method with the conventional tensiometers method. Structured soil column samples are analyzed in the field and in laboratory

Improvement in risk prediction with the addition of Tau-A, Tau-C or Tau-A and Tau-C.

<p>Improvement in risk prediction with the addition of Tau-A, Tau-C or Tau-A and Tau-C.</p