Large plasmid profiles of 70 <i>Salmonella</i> isolates.

<p>These isolates consisted of 25 isolates harboring <i>bla</i>_CMY-2 (23 <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Infantis, one <i>S</i>. Manhattan, and one O-untypeable:r:1,5), nine isolates harboring other <i>bla</i> genes (three <i>S</i>. Infantis and six <i>S.</i> Manhattan), and 36 isolates susceptible to 11 antibiotics (18 <i>S</i>. Infantis and 18 <i>S.</i> Manhattan). The 70 isolates generated eight large plasmid profiles (LPPs). The <i>S.</i> Infantis isolates generated LPPs 1 (n = 41), 2 (n = 1), 3 (n = 1), and 4 (n = 1). All <i>S.</i> Infantis isolates harboring <i>bla</i> genes showed LPP 1, except for one <i>S</i>. Infantis isolate carrying <i>bla</i>_CTX-M-2 that was classified as LPP 2. All 18 susceptible <i>S.</i> Infantis isolates showed LPP 1, except for two isolates that were collected in 2009 and 2008 showing LPP 3 and LPP 4, respectively. One O-untypeable:r:1,5 isolate was classified as LPP 1. <i>S.</i> Manhattan isolates generated four different LPPs (LPP 5-LPP 8). LPPs 5, 6, 7, and 8 were found in one, 21, one, and two <i>S.</i> Manhattan isolate(s), respectively. <i>S.</i> Infantis, O-untypeable:r:1,5, and <i>S.</i> Manhattan are expressed as <i>S.</i> I, OUT, and <i>S.</i> M in the figure, respectively.</p

Pulsed-field gel electrophoresis profiles (PFPs) of 70 <i>Salmonella</i> isolates harboring <i>bla</i> and non-<i>bla</i> genes.

<p>The isolates consisted of 25 isolates harboring <i>bla</i>_CMY-2 (23 <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Infantis, one <i>S</i>. Manhattan, and one O-untypeable:r:1,5), nine isolates harboring other <i>bla</i> genes (three <i>S</i>. Infantis and six <i>S.</i> Manhattan), and 36 isolates susceptible to 11 antibiotics (18 <i>S</i>. Infantis and 18 <i>S.</i> Manhattan). One <i>S</i>. Infantis isolate that harbored <i>bla</i>_CMY-2 could not be analyzed by PFGE because of damage that occurred during storage. The letters, names, and figures in parentheses on the right of the dendrogram are PFPs, resistance genes, and sampling year of isolates, respectively. The 44 <i>S</i>. Infantis isolates were subtyped as: PFP A (n = 1), PFP B (n = 3), PFP C (n = 12), PFP D (n = 1), PFP E (n = 1), PFP F (n = 1), PFP G (n = 1), PFP H (n = 6), PFP I (n = 3), PFP J (n = 1), PFP K (n = 1), PFP L (n = 6), PFP M (n = 1), PFP N (n = 3), PFP P (n = 2), and Q (n = 1). The 25 <i>S.</i> Manhattan isolates belonged to subtypes PFP R (n = 24) and PFP S (n = 1). One O-untypeable:r:1,5 isolate was PFP E. The scale indicates the percent similarity, as determined by the Dice coefficients.</p

Pulsed-field gel electrophoresis (PFGE) and Southern hybridization images of <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Infantis.

<p>Selected <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar (<i>S</i>.) Infantis isolates were selected to demonstrate the plasmid location of <i>bla</i>_CMY-2. (A) PFGE separation of S1 nuclease-digested genomic DNA from selected <i>S.</i> Infantis isolates, followed by Southern hybridization with a <i>bla</i>_CMY-2 probe. Lane 1, Lambda ladder marker; lane 2, isolate 1993; lane 3, isolate 2127; lane 4, isolate 2150; and lane 5, isolate 1737, which does not harbor <i>bla</i>_CMY-2. (B) <i>Bln</i>I-digested whole-genomic DNA from selected <i>S.</i> Infantis isolates, followed by Southern hybridization with a <i>bla</i>_CMY-2 probe. Lane 1, Lambda ladder marker; lane 2, isolate 1993; lane 3, isolate 2127; lane 4, isolate 2150; and lane 5, isolate 1737, which does not harbor <i>bla</i>_CMY-2.</p

Location of <i>bla</i>_CMY-2 in <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar (<i>S</i>.) Manhattan.

<p>(A) Pulsed-field gel electrophoresis (PFGE) separation of S1 nuclease- or <i>Bln</i>I-digested genomic DNA from <i>S.</i> Manhattan isolates, followed by Southern hybridization with a <i>bla</i>_CMY-2 probe. Lane 1, Lambda ladder marker; lanes 2 and 4, isolate 2179, which harbors <i>bla</i>_CMY-2; lanes 3 and 5, isolate 2129, which does not harbor <i>bla</i>_CMY-2. Lanes 2 and 3, S1 nuclease-digested genomic DNA; lanes 4 and 5, <i>Bln</i>I-digested genomic DNA. (B) Densitometric curves of PFGE separation with S1 nuclease- and <i>Bln</i>I-digested genomic DNA from <i>S.</i> Manhattan isolate 2179. The arrows show hybridization signals and corresponding positions of densitometric curves. Lambda ladder marker consisted of concatemers starting at 48.5 kb.</p

Increased ischemia-induced angiogenesis by <i>in vivo</i> shRNA targeting <i>Sprouty2 and Sprouty4</i>.

<p>(A) Representative laser Doppler images for each group are depicted. Arrowheads indicate ischemic limbs. The interval of low perfusion is displayed as dark blue; the highest perfusion interval is displayed as red. (B) Recovery of limb perfusion in C57BL/6J mice (8 weeks old) injected with the control shRNA (n = 10) or <i>Sprouty2/Sprouty4</i> shRNA vectors (n = 12) after hind limb ischemia as assessed by laser Doppler blood flow analysis on day 14. Data shown are means±SD. *: <i>P</i><0.05. (C) Blood vessels (green) in the non-ischemic or ischemic adductor muscle injected with the control shRNA or <i>Sprouty2/Sprouty4</i> shRNA vectors stained with anti-PECAM-1/CD31Ab. Nuclei were stained with Hoechst 33342 dye (blue). The CD31-positive vessel area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. Scale bars (C): 100 µm.</p

Blood and lymphatic vessels of <i>Sprouty4</i> single KO mice.

<p>(A) Blood vessels (green) and lymphatic vessels (red) in the ears of WT and <i>Sprouty4</i> KO mice (8 weeks old) were analyzed by whole-mount immunohistochemical staining with anti-PECAM-1/CD31Ab and anti-LYVE-1 Ab, respectively. (B) CD31-positive vessel area or LYVE1-positive area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. (C) Blood vessels (green) and lymphatic vessels (red) in the dorsal skin of WT and <i>Sprouty4</i> KO mice (8 weeks old) were analyzed by immunohistochemical staining with anti-PECAM-1/CD31Ab and anti-LYVE-1 Ab, respectively. Nuclei were stained with Hoechst 33342 dye (Blue). (D) CD31-positive vessel area or LYVE1-positive area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. (E) FITC-dextran-perfused flat-mounted retinal samples of WT and <i>Sprouty4</i> KO mice at postnatal day 3. Scale bars (A, C): 100 µm.</p

Characterization of <i>Sprouty2/Sprouty4</i> DKO mice.

<p>(A, B) Gross appearance of wild-type (WT) (A) and <i>Sprouty2/Sprouty4</i> DKO (B) embryos at embryonic day 12.5. The arrow and arrowheads indicate hemorrhage and edema, respectively. (C, D) Hematoxylin-eosin (H&E) staining of sections of WT (C) and <i>Sprouty2/Sprouty4</i> DKO (D) skin. (E, F) H&E staining and immunohistochemical staining with von Willebrand factor (vWF) of sections of hepatic hemangiomas in <i>Sprouty2/Sprouty4</i> DKO liver. vWF was used as a blood vessel marker. (G) Expression of <i>Sproutys</i> in endothelial cells. About 5.0×10⁴ BECs and LECs were FACS-sorted at embryonic day 14.5, and were used for RT-PCR analysis. <i>GAPDH</i> served as a loading control. Good separation of BECs and LECs was confirmed by BEC markers (<i>Nrp1</i>, <i>CD44</i>) and LEC markers (<i>LYVE1</i>, <i>Prox1</i>). Scale bars (C–F): 100 µm.</p

<i>In vivo</i> effects of shRNA targeting <i>Sprouty2</i> and <i>Sprouty4</i> in corneal micropocket assay.

<p>(A) Corneal neovascularization was induced by mouse VEGF-A (200 ng) on day 12 after hydron pellets had been implanted into male BALB/c mouse corneas. After implantation, 10 µg shRNA plasmids per eye were delivered by subconjunctival injection. Representative photos are shown. (B) Quantitative analysis of neovascularization on day 12. Areas are expressed in mm². Bars show the mean±SEM (n = 5). *: <i>P</i><0.05. (C) Sections of corneas implanted with VEGF-A stained by anti-PECAM-1/CD31Ab on day 12. Scale bars (C): 100 µm.</p

<i>Sprouty4</i> KO mice are also more resistant in a soft tissue ischemia model.

<p>(A) Representative photos of ischemic dorsal skin of male WT and <i>Sprouty4</i> KO mice (8–10 weeks old). Arrows indicate necrotic skin. (B) Left: Blood vessels (green) in the ischemic dorsal skin of male WT and <i>Sprouty4</i> KO mice were analyzed by immunohistochemical staining with anti-PECAM-1/CD31Ab. Nuclei were stained with Hoechst 33342 dye (blue). Right: The CD31-positive vessel area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. Scale bars (B): 100 µm.</p

<i>Sprouty4</i> KO mice are more resistant in a hind-limb ischemia model.

<p>(A) Representative photos of ischemic limbs, indicated by arrows. (B) Representative laser Doppler images for each group are depicted. Arrowheads indicate ischemic limbs. The interval of low perfusion is displayed as dark blue; the highest perfusion interval is displayed as red. (C) Recovery of limb perfusion in WT (n = 10) and <i>Sprouty4</i> KO (n = 7) mice after hind limb ischemia as assessed by laser Doppler blood flow analysis on day 14. Data shown are means±SD. *: <i>P</i><0.001. (D) Blood vessels (green) in the non-ischemic or ischemic adductor muscles of male WT and <i>Sprouty4</i> KO mice (8–10 weeks old) were analyzed by immunohistochemical staining with anti-PECAM-1/CD31Ab. Nuclei were stained with Hoechst 33342 dye (blue). The CD31-positive vessel area was quantified. Data shown are means±SEM. *: <i>P</i><0.05. Scale bars: (D) 100 µm.</p