<i>V. cholerae</i> aggregates are present in the lumen of the lower SI, often associated with mucus.

<p>(A) Each panel shows lumenal aggregates of <i>V. cholerae</i> (VcRed and VcGreen) from the medial or distal SI. Colonies in the right panel reside on the surface of digesta. Scale bar = 100 µm. (B) Confocal microscopy images of tissue sections from the lower SI of infant mice inoculated with LB (control), VcGreen or the GFP-labeled cholera toxin–deficient mutant <i>ΔctxAB</i>-GFP. Sections were counterstained with wheat germ agglutinin (red) to visualize mucus and DAPI (blue) to stain nuclei. Scale bar = 25 µm.</p

Detection of fluorescently labeled <i>V. cholerae</i> in the intact infant mouse small intestine by intravital two-photon microscopy.

<p>(a) Schematic representation (left) of the surgical intravital imaging approach to visualize intestinal tissue by two-photo microscopy in live anesthetized mice following orogastric inoculation with <i>V. cholerae</i>. Transverse view of exteriorized intestinal loop (right), depicting objective focal path from the serosal side through the various tissue layers towards the intestinal villus. The resulting focal plane is a tangential section along the intestine as depicted in (b–d). (b) Sequential two-photon micrographs (XY plane) taken from a 3-dimensional Z-stack of the SI, revealing penetration depths for two-photon excitation of up to 150 µm below the serosa layer. Depths (Z axis) 0 to 50 µm below the serosa include crypt regions, and >100 µm encompass the villi region. Blue denotes collagen fibers, Magenta denotes blood vessels. Scale bar 50 µm. (c) Differential localization of <i>V. cholerae</i> within the crypt or villus regions in the proximal, medial, and distal segments of the SI. Top row panel displays representative intravital two-photon micrographs of the crypt region for the proximal SI segment, and of the villus region for the medial and distal SI segments, of non-infected mice. Bottom row panel displays representative intravital two-photon micrographs of the crypt region and villus regions for the proximal, medial, and distal regions of the SI following orogastric inoculation with VcGreen and VcRed, respectively. Blue denotes collagen fibers, Green denotes colonies of VcGreen, Red denotes colonies of VcRed. Scale bar 10 µm. (d) Visualization of VcGreen in explanted distal SI segments. Representative two-photon micrographs taken from time-lapsed movies (see Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004405#ppat.1004405.s008" target="_blank">Video S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004405#ppat.1004405.s009" target="_blank">Video S2</a>) show single VcGreen cells in the intravillus space and discrete microcolonies of VcGreen associated with the villus epithelium. White/Yellow denotes autofluorescence, Green denotes VcGreen. Scale bar 10 µm.</p

Distribution of fluorescently labeled <i>V. cholerae</i> in the infant mouse small intestine.

<p>(A) The small intestines of infant mice co-inoculated with VcRed and VcGreen were divided into three equal parts and the central 1 cm segments of the proximal (P), medial (M) and distal (D) parts were used for plating and microscopic analyses. (B) Numbers of CFUs recovered from homogenates of the proximal (P), medial (M) and distal (D) segments. Bars represent the geometric mean. *, p<0.05, and **, p<0.01, based on ANOVA with Tukey's multiple comparison test. (CEG) Confocal micrographs showing VcRed and VcGreen distribution in the proximal (C), medial (E) and distal (G) segments. Tissue sections were counterstained with phalloidin (blue) to visualize the surface of the epithelium. White arrowheads mark clonal microcolonies. Scale bar = 100 µm. (DFH) The distance separating microcolonies from the base of the villi in the proximal (D), medial (F) and distal (H) SI segments was measured using confocal microscopy in tissue cross sections from mice co-inoculated with VcRed and VcGreen. Data represent the mean ±SD from three mice. The number (n) of microcolonies analyzed is indicated to the left of each panel. Black triangles indicate the average length of villi in each region. (I) Average length of intestinal villi (±SD) in the proximal (P), medial (M) and distal (D) segments. ***, p<0.001, based on ANOVA with Bonferroni's multiple comparison test.</p

The intensity of WGA staining of the intestinal epithelium decreases along the length of the SI and can be reduced by N-acetyl-L-cysteine (NAC) treatment.

<p>(A) Confocal micrographs of longitudinal sections of the proximal, medial and distal SI from infant mice treated with PBS or NAC after 6 h. The sections were stained with WGA (red) and DAPI (blue). Scale bar = 200 µm. (B,C) PAS stained sections of Carnoy's fixed proximal, medial and distal SI segments from untreated infant mice (B) and of the proximal SI from infant mice treated with PBS or NAC after 6 h (C). Scale bar = 100 µm. Arrow points to mucus layer.</p

NAC pretreatment promotes <i>V. cholerae</i> intestinal colonization and reduces the requirement for motility.

<p>(A) Numbers of CFUs recovered from homogenates of the proximal (P), medial (M) and distal (D) segments of infant mice treated with PBS (black) or NAC (red) 30 minutes prior to inoculation with C6706 or <i>ΔmotB</i>. Bars represent the geometric mean. *P<0.05; Mann-Whitney test. (B) Confocal micrographs of tissue sections of the proximal SI from infant mice treated with PBS or NAC, 30 minutes prior to co-inoculation with VcGreen and VcRed. Scale bar = 50 µm. (C) Competition assays between GFP-labeled <i>ΔmotB</i> and VcRed in the P, M and D segments from mice treated with PBS or NAC 6 h prior to bacterial inoculation. Bars represent the geometric mean. ***P<0.001; Mann-Whitney test.</p

Chemotaxis is not required from the fine localization of <i>V. cholerae</i> microcolonies along the villous axis.

<p>(A) Competition assays between GFP-labeled chemotaxis cluster deletion mutants <i>Δche2</i> (cluster 2), <i>Δche13</i> (clusters 1 and 3) or <i>Δche123</i> (all 3 clusters) and VcRed in the proximal (P), medial (M) and distal (D) segments and in vitro (I). Bars represent geometric means. *P<0.05, **P<0.001, based on ANOVA followed by Bonferroni's multiple comparison post test, comparing the data with the corresponding VcGreen/VcRed samples. (B–D) Distribution of <i>Δche2</i>-GFP and VcRed microcolonies along the axes of intestinal villi in the proximal (B), medial (C), and distal (D) intestine. The distance separating microcolonies from the base of the villi was measured by confocal microscopy in tissue cross sections from three mice co-inoculated with GFP-labeled <i>Δche2</i> and VcRed. Data represent the mean ±SD. The number (n) of microcolonies analyzed is indicated in the bottom right of each panel.</p

Differential requirements for flagellar-based motility along the SI.

<p>(A) Competitive indices (CI) from competition assays using GFP-labeled C6706, <i>ΔflaA</i>, <i>ΔmotB</i> or <i>ΔtcpA</i> and VcRed in the proximal (P), medial (M) and distal (D) SI segments and in vitro (I). Bars represent the geometric mean. *P<0.05, **P<0.01, #P<0.001, based on ANOVA followed by Bonferroni's multiple comparison post test, comparing the data with the corresponding VcGreen/VcRed samples. Open symbols mark the limit of detection for animals from which no mutants were recovered. (B,C) Distribution of <i>ΔflaA</i>-GFP (B) and <i>ΔmotB</i>-GFP (C) vs VcRed microcolonies along the axis of intestinal villi in the distal SI segment. The distance separating microcolonies from the base of the villi was measured by confocal microscopy in tissue cross sections from three mice. Data represent the mean ±SD. The number (n) of microcolonies analyzed is indicated in the bottom right of each panel.</p

Controlling Complex Nanoemulsion Morphology Using Asymmetric Cosurfactants for the Preparation of Polymer Nanocapsules

Complex nanoemulsions, comprising multiphase nanoscale droplets, hold considerable potential advantages as vehicles for encapsulation and delivery as well as templates for nanoparticle synthesis. Although methods exist to controllably produce complex emulsions on the microscale, very few methods exist to produce them on the nanoscale. Here, we examine a recently developed method involving a combination of high-energy emulsification with conventional cosurfactants to produce oil–water–oil (O/W/O) complex nanoemulsions. Specifically, we study in detail how the composition of conventional ethoxylated cosurfactants Span80 and Tween20 influences the morphology and structure of the resulting complex nanoemulsions in the water–cyclohexane system. Using a combination of small-angle neutron scattering and cryo-electron microscopy, we find that the cosurfactant composition controls the generation of complex droplet morphologies including core–shell and multicore–shell O/W/O nanodroplets, resulting in an effective state diagram for the selection of nanoemulsion morphology. Additionally, the cosurfactant composition can be used to control the thickness of the water shell contained within the complex nanodroplets. We hypothesize that this degree of control, despite the highly nonequilibrium nature of the nanoemulsions, is ultimately determined by a competition between the opposing spontaneous curvature of the two cosurfactants, which strongly influences the interfacial curvature of the nanodroplets as a result of their ultralow interfacial tension. This is supported by a correlation between cosurfactant compositions that produces complex nanoemulsions and those that produce homogeneous mixed micelles in equilibrium surfactant–cyclohexane solutions. Ultimately, we show that the formation of complex O/W/O nanoemulsions is weakly perturbed upon the addition of hydrophilic polymer precursors, facilitating their use as templates for the formation of polymer nanocapsules

Chemical structure of 1R-Chl, imatinib, dasatinib, and their effects on BCR-ABL unmutated and mutated genes transduced into murine BM cells.

<p>(A) 1R-Chl (left) targets the DNA sequences 5′-WGGWGW-3′. Bold, imidazole rings. imatinib (middle) targets Bcr-Abl kinase, and dasatinib (right) targets 14 out of 15 Bcr-Abl mutants. (B) Murine BM cells transduced with unmutated BCR-ABL and single point mutation Y253H, E255K, and T315I genes were tested for the effectiveness of 1R-Chl (125 nM to 1000 nM), 1S-Chl (500 nM and 1000 nM), imatinib (500 nM and 5000 nM; IC₅₀ = 260 nM for the native BCR-ABL transduced cells) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003593#pone.0003593-OHare1" target="_blank">[6]</a>, and dasatinib (10 nM and 100 nM; IC₅₀ = 0.8 nM for the native BCR-ABL transduced cells) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003593#pone.0003593-OHare1" target="_blank">[6]</a>. Triplicate experiments were done, and the numbers of colonies were quantified 7 days after initial plating.</p

Cytotoxicity (MTS) assays on resting murine BM cells and human MNCs.

<p>(A) Ficoll-Hypaque-separated murine BM cells were plated in triplicate with 1R-Chl (10, 100, 500, and 1000 nM) without any growth factors or mitogens. After 72 and 96 hours, 20 µL of Celltiter 96 Aqueous One solution (Promega, WI) was added. The absorbances of the MTS metabolites were read corresponding to the numbers of metabolically active cells. (B) Ficoll-Hypaque-separated human MNCs were treated as above, and the viabilities of the cells were assessed after 72 and 96 hours.</p