<i>Wolbachia</i> density (<i>Wolbachia</i> genomes/host genome) in the germline (ovaries and testes) of <i>An. gambiae</i>, <i>An. stephensi</i>, <i>C. tarsalis</i>, <i>Ae. aegypti</i> and <i>D. melanogaster</i> 2 days post infection.

<p>Two replicate experiments were completed to infect the germline of insects with wAlbB 8.5×10⁴ live cells inoculated into each well for replicate 1 (R1), while 5.5×10⁵ live cells were added to each well in replicate 2 (R2). For each replicate, five pools, each containing five pairs of ovaries or testes, were evaluated for <i>Wolbachia</i> density. Asterisk(s) denote significance (** P<0.01, * P<0.05). Error bars indicated SEM.</p

Comparison of the density (<i>Wolbachia</i> genomes/host genome) of multiple <i>Wolbachia</i> strains cultured in the <i>ex vivo</i> setting.

<p><i>Wolbachia</i> strains: <i>w</i>AlbB (checkered) and <i>w</i>MelPop (solid) in ovaries of <i>An. gambiae</i>, <i>An. stephensi</i>, <i>C. tarsalis Ae. aegypti</i>, and <i>D. melanogaster</i> 2 days post infection. Ovaries naturally infected with <i>w</i>MelPop from <i>D. melanogaster</i> were cultured <i>ex vivo</i> for 2 days (yellow hatched) and are compared to the density in the fly (yellow horizontal lines). As the density of <i>Wolbachia</i> used as the inoculum for uninfected ovaries differed between strains, the final <i>Wolbachia</i> genomes:host genomes value were normalized to 10⁶ bacterial cells to compare between strains. The <i>w</i>MelPop strain infects <i>D. melanogaster</i> ovaries approximately 500 to 3000 fold higher, while <i>w</i>AlbB infects <i>Ae. aegypti</i> ovaries 1000 to 7000 fold greater than the other insect species. Asterisk(s) denote significance (** P<0.01, * P<0.05). There was no significant difference in <i>w</i>MelPop titer after two days of <i>ex vivo</i> culture compared to <i>in vivo</i>. For each insect species and <i>Wolbachia</i> strain, five pools, each containing five pairs of ovaries, were evaluated for <i>Wolbachia</i> density. Error bars indicated SEM.</p

The effect of culturing before inoculation of <i>Wolbachia</i> on ovary density.

<p>Ovaries were inoculated with <i>Wolbachia</i> immediately after culture (day 0) or 6 days post culture for <i>An. gambiae</i> (Ag), <i>An. stephensi</i> (As), <i>C. tarsalis</i> (Ct), <i>Ae. aegypti</i> (Aa) and <i>D. melanogaster</i> (Dm). Two days post-infection, the density of <i>Wolbachia</i> was compared in these two treatments. Density of <i>Wolbachia</i> in ovaries was significantly different (P<0.007) between the <i>Ae. aegypti</i> day 0 and day 6 treatments. For each insect species, five pools, each containing five pairs of ovaries, were evaluated for <i>Wolbachia</i> density. Error bars indicate SEM.</p

FISH images of <i>w</i>AlbB infection in insect ovaries captured using a confocal microscope for <i>An. gambiae</i> (A–C), <i>An. stephensi</i> (D–F), <i>Ae. aegypti</i> (G–H) <i>C. tarsalis</i> (J–L) and <i>D. melanogaster</i> (M–O).

<p>Images in the left column are the red channel only (<i>Wolbachia</i>), images in the center column are blue channel only (host nuclei) and images in the right column are the merged image (Red: <i>Wolbachia</i>, Blue: host nuclei) The Z-stacks of the ovaries are available as supplementary videos (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036277#pone.0036277.s001" target="_blank">Movie S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036277#pone.0036277.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036277#pone.0036277.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036277#pone.0036277.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036277#pone.0036277.s005" target="_blank">S5</a>). The scale bar represents 20 µm. Images are compiled from compression of Z stack with a depth of 5 µm.</p

Density of <i>Wolbachia</i> in insect ovaries measured over time. The left axis indicates the ratio of <i>Wolbachia</i> genomes/host genome (red line).

<p>The right axis indicates the fold change for <i>Wolbachia</i> (green) and host (blue) single copy gene for each species: <i>An. gambiae</i> (A; <i>Ag</i>), <i>An. stephensi</i> (B; <i>As</i>), <i>C. tarsalis</i> (C; <i>Ct</i>) <i>Ae. aegypti</i> (D; <i>Aa</i>) and <i>D. melanogaster</i> (E; <i>Dm</i>) infected with <i>w</i>AlbB. <i>D. melanogaster</i> ovaries naturally infected with <i>w</i>MelPop were also cultured (F; <i>Dm</i>). For each insect species infected with <i>w</i>AlbB, three pools, each containing five pairs of ovaries, were evaluated for <i>Wolbachia</i> density. Four pools each containing five pairs of ovaries were completed for naturally infected <i>D. melanogaster</i> ovaries. Error bars indicate SEM.</p

Enantioselective Preparation of <i>cis</i>-β-Azidocyclopropane Esters by Cyclopropanation of Azido Alkenes Using a Chiral Dirhodium Catalyst

A diastereo- and enantiocontrolled preparation of the conformationally restricted <i>cis</i>-β-azidocyclopropane esters have been developed. The Rh₂(<i>S</i>-DOSP)₄ was found to be an efficient catalyst in hexane for the cyclopropanation of azido alkenes with diazo esters, and 19 <i>cis</i>-β-azidocyclopropane esters were prepared in excellent yields. The value of the diastereomer ratio was up to 99:1, and the enantiomeric excess was up to 95%. Furthermore, the relative and absolute configuration was confirmed by X-ray analysis

Differentially expressed proteins identified by MALDI-TOF-MS in liver biopsy samples from BA and NC patients.

<p>BA: biliary atresia; NC: non-BA neonatal cholestasis infants.</p

Representative 2-DE gel images.

<p>Identical images are shown in A and B. (A) Highlighted spots are more abundant in BA than NC controls. (B) Highlighted spots are more abundant in NC than BA patients. Numbers beside the spots correspond to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068602#pone-0068602-t001" target="_blank">Table 2</a>. (C) and (D) Representative Spot 10, cropped 2-DE gel images of HSP90 in BA patients and NC controls. (E) HSP90 showed the most significant differences in expression between BA (101.0±2.3) and NC controls (2060.0±8.6) (<i>p</i><0.0001). HSP90 was down-regulated more than 20-fold in BA patients when compared to NC controls. BA: biliary atresia; NC: non-BA neonatal cholestasis infants.</p

Enantioselective Preparation of <i>cis</i>-β-Azidocyclopropane Esters by Cyclopropanation of Azido Alkenes Using a Chiral Dirhodium Catalyst

A diastereo- and enantiocontrolled preparation of the conformationally restricted <i>cis</i>-β-azidocyclopropane esters have been developed. The Rh₂(<i>S</i>-DOSP)₄ was found to be an efficient catalyst in hexane for the cyclopropanation of azido alkenes with diazo esters, and 19 <i>cis</i>-β-azidocyclopropane esters were prepared in excellent yields. The value of the diastereomer ratio was up to 99:1, and the enantiomeric excess was up to 95%. Furthermore, the relative and absolute configuration was confirmed by X-ray analysis

Table 1. Distribution of study subjects and liver function tests.

<p>1. Type III biliary atresia refers to the discontinuity of both right and left hepatic ducts to the level of the porta hepatis. Unfortunately, type III BA is common, accounting for >90% of cases. *at liver biopsy sample day.</p><p>BA: biliary atresia; NC: non-BA neonatal cholestasis infants.</p><p>ALT: Alanine transaminase; AST: Aspartate transaminase; DB: Direct bilirubin; TB: Total bilirubin; γ-GGT: Gamma glutamyl transpeptidase.</p