Managerial lead of an independent school refectory - allowance organization

Phylogeny of Forkhead (FOX) transcription factors. The FKH domain of forkhead proteins of H. sapiens, D. melanogaster, E. multilocularis, H. microstoma, and published FoxQ2 homologs from other animals were aligned, and a phylogeny was estimated by Maximum Likelihood analysis (with a JTT model) using MEGA 5.2 [85]. Bootstrap support values from 1,000 replicates are indicated next to the nodes. Nodes with lower than 50 % support were collapsed. GeneDB accession codes are given for E. multilocularis and H. microstoma and Genbank accession codes are given for all other sequences. The FoxQ2 group is outlined, and the foxQ2 genes of E. multilocularis and H. microstoma are indicated by arrows. (PDF 33 kb

Additional file 5: of Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms

Alkaline phosphatase-based development of whole-mount in situ hybridization in H. microstoma : Wnt2 and Wnt5. Bars: 50 Οm. (PDF 1653 kb

Analysis of EmPlk1 activity in <i>Xenopus</i> oocytes.

<p>Wild-type (wt) and mutant forms (T₁₇₉D, constitutively active; T₁₇₉V, non-activatable; wt^KD, T₁₇₉D^KD, kinase dead mutants) of EmPlk1 were expressed in <i>Xenopus</i> oocytes (20 oocytes per set) and germinal vesicle breakdown (GVBD) assays were carried out in the presence of different concentrations of BI 2536 as indicated to the left. Treatment by the natural inducer, progesterone (PG), served as a positive control. Displayed are the percentages of <i>Xenopus</i> oocytes that underwent GVBD in the different experimental settings (mean of three independent experiments). ‘-’ indicates that this condition has not been tested.</p

WISH detection of <i>emplk1</i> transcripts.

<p>(A) General view of a metacestode hybridized with the <i>emplk1</i> antisense probe. Note the accumulation of <i>emplk1</i> positive cells in the developing protoscoleces (arrows). (B) General view of a metacestode hybridized with the <i>emplk1</i> sense control probe. No signal is detected. (C–F) Details of <i>emplk1</i> WISH detection in different developmental stages. (C) Detail of the metacestode germinative layer, in which dispersed <i>emplk1</i> positive cells can be distinguished (inset: higher magnification of a positive cell, combined with 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining). (D) Accumulation of <i>emplk1</i> positive cells in the early brood capsule bud (arrow). (E) Early protoscolex development, with abundant <i>emplk1</i> positive cells in the interior. (F) Late protoscolex development. <i>emplk1</i> positive cells accumulate at the base of the developing suckers (arrows). Bars represent 200 µm (A, B) and 50 µm (C–F).</p

BI 2536 inhibits the formation of metacestode vesicles from <i>E. multilocularis</i> primary cells.

<p>Primary cell cultures were established from metacestode vesicles and incubated under ideal growth conditions (A4 medium) in the presence of different concentrations of BI 2536 (5, 10, 25, 50, 100 nM) as indicated. After 14 days of incubation, fully formed (mature) metacestode vesicles were counted microscopically. ‘Control’ indicates cultures with A4 medium, ‘mock’ indicates cultures with DMSO (without inhibitor). All conditions were at least assessed in three different biological replicates with at least three technical replicates. *(p<0.05); **(p = 0.001–0.01) (Student's t-test).</p

Short-term treatment of metacestode vesicles with BI 2536.

<p>Metacestode vesicles were treated for 24, 48 or 72(as indicated) with 50 nM (light grey) or 100 nM (dark grey) of BI 2536, followed by one day of recovery without inhibitor. EdU labeling (5 h, 50 µM EdU) was carried out and labeled cells were microscopically counted. Shown are EdU-positive cell counts per mm² of GL. ‘Control’ indicates vesicles cultivated in A4 medium alone. ‘Mock’ indicates the DMSO control. ***(p<0.001) (Student's t-test).</p

Expression of <i>emplk1</i> in <i>E. multilocularis</i> larval stages.

<p>Total RNA was isolated from primary cell cultures (Pc) after 2, 5 and 11 days (d2, d5, d11) of development towards the metacestode stage, from dormant (Ps_non) and pepsin/low pH-activated protoscoleces (Ps_act), as well as from mature metacestode vesicles (Mc). The isolated RNA was reverse transcribed to cDNA and equal amounts of serial (10-fold) dilutions of cDNA were subjected to gene-specific RT-PCR for <i>emplk1</i> and the constitutively expressed gene <i>elp</i> (control) as indicated to the right. PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. gDNA indicates positive control lanes where PCR has been performed on genomic DNA. ‘RT-neg’ indicates the negative control where reverse transcriptase has been omitted. Marker sizes are indicated to the left.</p

Amino acid sequence and structural analysis of EmPlk1.

<p>The EmPlk1 amino acid sequence was aligned to Plk1-like enzymes of <i>S. mansoni</i> (SmPlk1; GenBank accession number: AY747306), <i>Homo sapiens</i> (Plk1; P53350); <i>D. melanogaster</i> (Polo; P52304) and <i>X. laevis</i> (Plx1; P70032). The predicted kinase domain is boxed, predicted Polo-boxes (Polo-box 1; Polo-box 2) are indicated above the alignment. Residues identical in at least four of the sequences are shown in white on black background. The eleven sub-domains known to be highly conserved in kinases are indicated above (I–XI). Highly conserved amino acid residues and sequence motifs are indicated above the alignment. The highly conserved threonine residue which is critical for activation is marked by (T*) above the alignment. Residues important for phosphosubstrate binding of the C-terminal domain are marked by (#) below the alignment. Residues involved in binding of BI 2536 to Plk1 are indicated by grey dots below the alignment.</p

Long-term BI 2536 effects on <i>E. multilocularis</i> metacestode vesicles.

<p>Metacestode vesicles were treated for 21 days with different concentrations (10, 25, 50 and 100 nM) of BI 2536, followed by three days of recovery without inhibitor. On day three of recovery, EdU labeling (5 h, 50 µM EdU) was carried out and labeled cells were microscopically counted. (A) Microscopic images of the GL of BI 2536 treated (10, 25, 50, 100 nM) and non-treated <i>E. multilocularis</i> metacestode vesicles, stained with EdU. Bar represents 25 µm. Blue, nuclear staining, DAPI; red, EdU staining. (B) EdU-positive cell counts per mm² of GL. ‘Control’ indicates vesicles cultivated in A4 medium alone. ‘Mock’ indicates the DMSO control. ***(p<0.001) (Student's t-test). Please note that the specific examples shown in (A) display slightly different total cell numbers but that the overall cell numbers in metacestode vesicles were not altered by drug treatment, as already outlined in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002870#pntd.0002870-Koziol1" target="_blank">[14]</a> for HU treatment.</p