Proteomic analysis of extracellular matrix proteins in exudates collected from mice injected with BaP1 or Leuc-a.

<p>*Values underlined correspond to proteins whose quantitative value differed by more than 3 fold when comparing the two SVMPs.</p

Histological and immunohistochemical analysis of the effects of BaP1 and leuc-a in skeletal muscle.

<p>Mice were injected with either 50 µL PBS as control (A, D), 50 µg of leuc-a (B, E) or 50 µg BaP1 (C, F). Tissue samples were collected 15 min after injection and processed for embedding in paraffin (see experimental details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028017#s2" target="_blank">Methods</a>). A, B and C: Hematoxylin-eosin staining. Hemorrhage, evidenced by the presence of erythrocytes in the interstitial space (arrows), occurred only in muscle injected with BaP1. D, E and F: Immunostaining with anti-type IV collagen (green) and anti-VEGFR2 (red). Arrows depict capillary vessels. There is a reduction in capillary vessels positive for type IV collagen and VEGFR2 in samples treated with BaP1, whereas no reduction in the staining of capillaries was observed in tissue injected with leuc-a. Bar corresponds to 50 µm. (G) The total number of capillaries and muscle cells were counted in various sections, and the capillary: muscle cell ratio was calculated. Results are presented as mean ± SD. A significant reduction in the ratio was observed only in muscle injected with BaP1, but not with leuc-a. *<i>P</i><0.05 when compared with capillary: muscle cell ratios of control and leuc-a-treated samples.</p

Hydrolysis of basement membrane components <i>in vitro</i>.

<p>Hydrolysis of laminin (A), nidogen (B), type IV collagen (C) and perlecan (D) by BaP1 and leuc-a, as detected by Western blotting of Matrigel. Matrigel was incubated with either BaP1, leuc-a or PBS (control, lane C), as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028017#pone-0028017-g003" target="_blank">Fig 3</a>. Matrigel preparations were separated by SDS-PAGE under reducing conditions using a 4-15% gradient gel and transferred to nitrocellulose membranes. Immunodetection was performed with either anti-laminin, anti-nidogen, anti-type IV collagen or anti-perlecan antibodies. Reaction was developed with a chemiluminiscent substrate.</p

Mol. mass of most abundant fragments of BM proteins generated by BaP1 and Leuc-a.

a<p>No degradation bands were detected by immunoblotting, and the intensity of the main bands of type IV collagen was not reduced.</p>b<p>No degradation bands were detected by immunoblotting, but the main laminin bands were reduced as compared to control samples.</p>c<p>No degradation bands were detected by immunoblotting.</p

Hydrolysis of Matrigel proteins by BaP1 and leuc-a.

<p>Matrigel was incubated at 37°C with each enzyme at a 1∶50 (w:w) enzyme:substrate ratio for 15 min, 1 h and 3 h. A control of Matrigel without enzymes (C) was included for each time interval. Matrigel solutions were separated by SDS–PAGE under reducing conditions using a 4–15% gradient gel, and transferred to nitrocellulose membrane and stained with Ponceau-S.</p

SDS-PAGE of exudates samples collected from mice injected with either BaP1 or leuc-a.

<p>Samples corresponding to 20 µg protein of exudates collected 15 min after injection of SVMPs were electrophoresed on a 4–20% gradient gel followed by staining with Coomassie Blue. Molecular mass markers are depicted to the left. Gel lanes were cut into ten equal size slices for further proteomic analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028017#s2" target="_blank">Methods</a> for details).</p

Hydrolysis of basement membrane components <i>in vivo</i>.

<p>Hydrolysis of laminin (A), nidogen (B), type IV collagen (C) and perlecan (D) by BaP1 and leuc-a, as detected by Western blotting of homogenates of injected mouse gastrocnemius muscle. Groups of mice were injected in the gastrocnemius muscle with either 50 µg BaP1, 50 µg leuc-a or PBS (lane C). After 15 min, mice were sacrificed and tissue was homogenized and centrifuged to obtain the supernatant. Supernatants of muscle homogenates were separated by SDS-PAGE under reducing conditions, using a 4–15% gradient gel, and transferred to nitrocellulose membranes. Immunodetection was performed with either anti-laminin, anti-nidogen, anti-type IV collagen or anti-endorepellin antibodies, and with anti-GAPDH as loading control in tissue homogenates. Reaction was developed with a chemiluminiscent substrate. Densitometry was carried out in blots of tissue homogenates with ImageLab software; a relative quantification was performed adjusting each sample to the corresponding control.</p

Legacy and Emerging Perfluoroalkyl Substances Are Important Drinking Water Contaminants in the Cape Fear River Watershed of North Carolina

Long-chain per- and polyfluoroalkyl substances (PFASs) are being replaced by short-chain PFASs and fluorinated alternatives. For ten legacy PFASs and seven recently discovered perfluoroalkyl ether carboxylic acids (PFECAs), we report (1) their occurrence in the Cape Fear River (CFR) watershed, (2) their fate in water treatment processes, and (3) their adsorbability on powdered activated carbon (PAC). In the headwater region of the CFR basin, PFECAs were not detected in raw water of a drinking water treatment plant (DWTP), but concentrations of legacy PFASs were high. The U.S. Environmental Protection Agency’s lifetime health advisory level (70 ng/L) for perfluorooctanesulfonic acid and perfluorooctanoic acid (PFOA) was exceeded on 57 of 127 sampling days. In raw water of a DWTP downstream of a PFAS manufacturer, the mean concentration of perfluoro-2-propoxypropanoic acid (PFPrOPrA), a replacement for PFOA, was 631 ng/L (<i>n</i> = 37). Six other PFECAs were detected, with three exhibiting chromatographic peak areas up to 15 times that of PFPrOPrA. At this DWTP, PFECA removal by coagulation, ozonation, biofiltration, and disinfection was negligible. The adsorbability of PFASs on PAC increased with increasing chain length. Replacing one CF₂ group with an ether oxygen decreased the affinity of PFASs for PAC, while replacing additional CF₂ groups did not lead to further affinity changes