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NADPH oxidase superoxide production is increased in <i>mdx</i> muscle.

<p>A. Lucigenin chemiluminescent assay was used to measure NADPH-dependent superoxide production from muscle homogenates of WT and <i>mdx</i> mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015354#s2" target="_blank">Methods</a> for details). Values from each experiment were pooled for WT and <i>mdx</i> muscles (n = 5 in both groups). B. Inhibitors of various sources of ROS were added to the assay and superoxide production was compared to the control level (no inhibitor). Pooled values for WT and <i>mdx</i> mice.</p

NADPH oxidase subunits are increased in 9 week old <i>mdx</i> mice.

<p>A. Representative western blots of gp91^phox and p67^phox from 9 week old WT and <i>mdx</i> TA muscles. B. Pooled results of densitometry analysis for gp91^phox and p67^phox from WT (n = 4) and <i>mdx</i> (n = 8) muscles.</p

NADPH oxidase immunostaining of single fibers from <i>mdx</i> mice.

<p>Isolated <i>mdx</i> muscle fibers from the FDB muscle were immunostained using antibodies against the various NADPH oxidase subunits. Nuclei are stained by DAPI (blue). In the bottom right panel, p22^phox (green) was co-immunostained with caveolin-3 (red) to demonstrate sarcolemmal localization (yellow).</p

NADPH oxidase subunits are increased in 18–19 day old (pre-necrotic) <i>mdx</i> mice.

<p>A. Representative western blots of all NADPH oxidase subunits from TA muscles of 18–19 day old WT and <i>mdx</i> mice. B. Pooled results from densitometry for the subunits showing <i>mdx</i> values normalized to WT (represented by the blue dotted line). The proteins gp91^phox, p67^phox and rac1 were all significantly increased in <i>mdx</i> muscle compared to WT, whereas p22^phox and p47^phox were not different (n = 4 samples for each group).</p

Muscles of 18–19 day old <i>mdx</i> mice show no evidence of inflammatory cells.

<p>A. Representative images of TA cross-sections from 19 day old WT and <i>mdx</i> mice stained with H&E. Note the normal histological appearance of the <i>mdx</i> muscle, which is devoid of necrotic fibers and infiltrating inflammatory cells. B. Sample western blot showing the macrophage protein, CD68, from TA homogenates of 18–19 day old WT and <i>mdx</i> mice. C. Pooled data of CD68 expression from western blots of 18–19 day old mice, showing no significant difference (NS) between WT and <i>mdx</i> muscles.</p

NADPH oxidase inhibition reduces stretch-induced Ca²⁺ influx and force loss in <i>mdx</i> fibers.

<p>A. Fluorescent confocal images showing intracellular Ca²⁺ (Fluo-4) for <i>mdx</i> single fibers taken 25 min after a series of 10 stretched contractions. Fibers were either untreated controls (<i>mdx</i> Con) or treated with the NADH oxidase inhibitor DPI (<i>mdx</i> DPI). B. Pooled data of the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) as measured before and after 10 stretched contractions (dotted line with arrow). Values are shown for control fibers (n = 9; blue) and fibers treated with 1 µM DPI (n = 5; red) for 15 min before the stretched contractions. C. Pooled values showing the significantly greater muscle force after the stretched contractions for DPI- treated fibers (n = 5; red) compared to control fibers (n = 9; blue). Note that pre-stretch force values are represented by 100%.</p

In Silico Prediction of Physicochemical Properties of Environmental Chemicals Using Molecular Fingerprints and Machine Learning

There are little available toxicity data on the vast majority of chemicals in commerce. High-throughput screening (HTS) studies, such as those being carried out by the U.S. Environmental Protection Agency (EPA) ToxCast program in partnership with the federal Tox21 research program, can generate biological data to inform models for predicting potential toxicity. However, physicochemical properties are also needed to model environmental fate and transport, as well as exposure potential. The purpose of the present study was to generate an open-source quantitative structure–property relationship (QSPR) workflow to predict a variety of physicochemical properties that would have cross-platform compatibility to integrate into existing cheminformatics workflows. In this effort, decades-old experimental property data sets available within the EPA EPI Suite were reanalyzed using modern cheminformatics workflows to develop updated QSPR models capable of supplying computationally efficient, open, and transparent HTS property predictions in support of environmental modeling efforts. Models were built using updated EPI Suite data sets for the prediction of six physicochemical properties: octanol–water partition coefficient (logP), water solubility (logS), boiling point (BP), melting point (MP), vapor pressure (logVP), and bioconcentration factor (logBCF). The coefficient of determination (<i>R</i>²) between the estimated values and experimental data for the six predicted properties ranged from 0.826 (MP) to 0.965 (BP), with model performance for five of the six properties exceeding those from the original EPI Suite models. The newly derived models can be employed for rapid estimation of physicochemical properties within an open-source HTS workflow to inform fate and toxicity prediction models of environmental chemicals