Detection of EdU incorporation in proliferating germinal cells of <i>in vitro</i> cultured <i>E. multilocularis</i> vesicles.

<p>EdU positive cells are shown in a whole small vesicle (A) or in a large vesicle (B). Inserts in (B) are magnified images of a developing protoscolex indicated by the arrow heads. Images of the differential interference contrast (DIC) bright field microscopy are shown. Bar = 125 μm for (A) and 250 μm for (B), respectively.</p

Immunofluorescence detection of beta tubulin (A) and phosphorylated ERK (p-ERK) (B) in the germinal layer of the whole-mount preparations of <i>in vitro</i> cultured <i>E. multilocularis</i> vesicles.

<p>Arrow heads in (A) indicate the neuron cells with long neurites. Arrow heads in (B) indicate the areas which are magnified in the inserts. Note the higher expression of p-ERK in the nucleolus of some cells indicated by the arrows in the inserts. Merges of the differential interference contrast (DIC) bright field microscopy with the fluorescence channels are shown. Bar = 125 μm.</p

A Rapid and Convenient Method for Fluorescence Analysis of <i>In Vitro</i> Cultivated Metacestode Vesicles from <i>Echinococcus multilocularis</i>

<div><p>We here describe a convenient method for preparation, fixation and fluorescence analysis of <i>in vitro</i> cultivated metacestode vesicles from <i>E. multilocularis</i>. Parasite materials could be prepared in one hour, did not need to be sectioned, and were subsequently utilized for further whole-mount staining assays directly. Using these preparations, in combination with conventional fluorescence staining techniques, we could detect the expression and subcellular localization of a specific protein and identify <i>in situ</i> proliferative or apoptotic cells in the germinal layer of metacestode vesicles. Based on this approach, future molecular and cellular analysis of <i>Echinococcus</i> metacestode vesicles in the <i>in vitro</i> system will be greatly facilitated.</p></div

Detection of apoptotic cells by TUNEL in <i>in vitro</i> cultured <i>E. multilocularis</i> vesicles.

<p>Apoptosis was induced by H2O2 treatment (H2O2 +). Note rare apoptosis accrued in the vesicle without H2O2 treatment (H2O2 -). DNase treatment was used as a positive control. Bar = 25 μm.</p

Asymmetric Synthesis of Scillascillin-Type Homoisoflavonoid

The first asymmetric synthesis of a scillascillin-type homoisoflavonoid was reported. Key reactions for the asymmetric synthesis of benzocyclobutene include catalytic reductive desymmetrization of malonic ester and an intramolecular C–H activation of the methyl group

EGF-mediated EGFR/ERK signaling pathway promotes germinative cell proliferation in <i>Echinococcus multilocularis</i> that contributes to larval growth and development

<div><p>Background</p><p>Larvae of the tapeworm <i>E</i>. <i>multilocularis</i> cause alveolar echinococcosis (AE), one of the most lethal helminthic infections in humans. A population of stem cell-like cells, the germinative cells, is considered to drive the larval growth and development within the host. The molecular mechanisms controlling the behavior of germinative cells are largely unknown.</p><p>Methodology/Principal findings</p><p>Using <i>in vitro</i> cultivation systems we show here that the EGFR/ERK signaling in the parasite can promote germinative cell proliferation in response to addition of human EGF, resulting in stimulated growth and development of the metacestode larvae. Inhibition of the signaling by either the EGFR inhibitors CI-1033 and BIBW2992 or the MEK/ERK inhibitor U0126 impairs germinative cell proliferation and larval growth.</p><p>Conclusions/Significance</p><p>These data demonstrate the contribution of EGF-mediated EGFR/ERK signaling to the regulation of germinative cells in <i>E</i>. <i>multilocularis</i>, and suggest the EGFR/ERK signaling as a potential therapeutic target for AE and perhaps other human cestodiasis.</p></div

EGFR/ERK signaling contributes to <i>E. multilocularis</i> germinative cell proliferation.

<p>(A) CI-1033 impairs <i>E</i>. <i>multilocularis</i> ERK phosphorylation. Vesicles were treated with 10 μM CI-1033 for indicated time. (B) CI-1033 inhibits EGF-stimulated <i>E</i>. <i>multilocularis</i> ERK phosphorylation. Vesicles were treated with 10 μM CI-1033 (+) or DMSO (-) for 6 h, followed by stimulation with EGF (+) or not (-) for 20 minutes. (C) U0126 impairs <i>E</i>. <i>multilocularis</i> ERK phosphorylation. Vesicles were treated with indicated concentrations of U0126 for 6 h. (D) U0126 inhibits EGF-stimulated <i>E</i>. <i>multilocularis</i> ERK phosphorylation. Vesicles were treated with 20 μM U0126 (+) or DMSO (-) for 6 h, followed by stimulation with EGF (+) or not (-) for 20 minutes. (E) U0126 reduces the number of EdU⁺ germinative cells. Vesicles were treated with 20 μM U0126 for 6 days. Representative images are shown on the left and the quantification is shown on the right. Values represent the mean ± SD of 4 separate labeling experiments. *** <i>P</i> < 0.001. Bar = 20 μm. (F) Effects of U0126 on germinative cell proliferation during the recovery from hydroxyurea treatment. Vesicles were allowed for recovery in conditioned medium supplemented with EGF and U0126 was added into the medium to a final concentration of 20 μM immediately after the initial EdU pulse. Germinative cell proliferation was analyzed after 4 days of recovery. Images show rare EdU⁺ and EdU⁺BrdU⁺ cells following U0126 treatment (see the text). Bar = 20 μm. (G) Effects of U0126 on the larval growth. Vesicles were incubated with 10 μM U0126 and parasite growth was analyzed after 28 days of cultivation. Data are shown as mean ± SD of triplicates. ** <i>P</i> < 0.01.</p

EGF activates <i>E. multilocularis</i> EGF receptor EmER in <i>Xenopus</i> oocyte expression system.

<p>(A) Membrane extracts were prepared from <i>Xenopus</i> oocytes expressing EmER or not (NI, noninjected) and immunoprecipitated and analyzed by western blot using the anti-flag antibody. The results of two independent injection experiments are presented (I-1 and I-2). The bands exhibited a molecular mass larger than expected (178 kDa), which could be attributable to glycosylation. (B) Oocytes expressing EmER were incubated with CI-1033 or DMSO for 4 h followed by 20 minutes of EGF stimulation. Membrane extracts were immunoprecipitated by the anti-flag antibody and analyzed by western blot using the anti-flag or anti-phospho-tyrosine antibodies. (C) Induction of GVBD (germinal vesicle breakdown) in EmER-expressing <i>Xenopus</i> oocytes. Oocytes that had been expressing EmER for 48 h were pretreated with CI-1033 or DMSO for 4 h and then incubated with EGF. GVBD was monitored after 16 h of EGF incubation and the mean percentages of oocytes exhibiting GVBD for three separate experiments are shown. Noninjected oocytes were used as controls. DMSO (final concentration 0.25%) was found no effects on GVBD. PG: progesterone; “/”: not tested.</p

EGF promotes <i>E. multilocularis</i> germinative cell proliferation.

<p>(A) Vesicles were incubated with BrdU for two days and chromosomal DNA was isolated for BrdU incorporation assay. Control was set to 1 and results were normalized against the control. Data are shown as mean ± SD of triplicates. ** <i>P</i> < 0.01. (B) Vesicles were <i>in vitro</i> cultivated under normal conditions and germinative cell proliferation was visualized by EdU-BrdU dual labeling. Insert shows the magnified view. Arrows indicate EdU⁺BrdU⁺ cells. Bar = 20 μm. (C) Vesicles were pretreated with 40 mM of hydroxyurea for three days and allowed for recovery in conditioned medium (control) supplemented with EGF. Germinative cell proliferation was analyzed after 4 days of removal of hydroxyurea and representative images are shown. Quantification of EdU⁺ and EdU⁺BrdU⁺ cells is shown in the right panel. Data are shown as mean ± SD of 3 separate labeling experiments. * <i>P</i> < 0.05; ** <i>P</i> < 0.01. Bar = 20 μm.</p

Inhibition of <i>E. multilocularis</i> EGFR impairs germinative cell proliferation.

<p>(A) Metacestode vesicles were treated with 10 μM CI-1033 and the representative images for day 0, 3 and 6 are shown on the left (red: EdU; blue: DAPI). Quantifications of the EdU⁺ germinative cells in the vesicles treated with 10 μM CI-1033 for indicated time (middle) and 5–10 μM CI-1033 or DMSO control (0) for 6 days (right) are shown. Values represent the mean ± SD of 5 separate labeling experiments. * <i>P</i> < 0.05; *** <i>P</i> < 0.001. (B) Effects of CI-1033 on germinative cell proliferation during the recovery from hydroxyurea treatment. Vesicles were allowed for recovery in conditioned medium supplemented with EGF, and CI-1033 was added into the medium to a final concentration of 10 μM immediately after the initial EdU pulse. Germinative cell proliferation was analyzed by EdU-BrdU dual labeling after 4 days of recovery. Images show rare EdU⁺ and EdU⁺BrdU⁺ cells following CI-1033 treatment (see the text). (C) Representative images of the accumulations of EdU⁺ germinative cells in some cell aggregates (indicated by the dashed-line boxes) in the vesicles treated with DMSO (control) or 10 μM CI-1033 for 6 days (red: EdU; blue: DAPI). (D) Effects of CI-1033 on the larval growth and development. Vesicles or protoscoleces were cultivated in DMSO-containing conditioned medium (control) supplemented with the ingredients as indicated. Vesicle growth (left) and vesicle formation from protoscoleces (right) were analyzed after 28 days and 18 days of cultivation, respectively. Data are shown as mean ± SD of triplicates, representative of 3 independent experiments. ** P < 0.01 and *** P < 0.001. Bar = 20 μm in (A), (B) and (C).</p