<i>Tb</i>SAXO RNAi knockdowns exhibit impaired flagellar motility.

<p>Inducible RNAi<i>^TbSAXO</i> in PCF (<b>a, b, c, d</b>) and BSF (<b>e, f, g, h</b>) cells. Growth curves of PCF (<b>a</b>) and BSF (<b>e</b>) RNAi<i>^TbSAXO</i> cell lines. Corresponding WBs (PCF in <b>b</b>, BSF in <b>f</b>) of WT (parental), RNAi non-induced (-), and 24 h and 96 h induced cells probed with mAb25 and L8C4 (anti-PFR2). For PCF 5.10⁶ cells were used and 1.25×10⁵ cells for BSF. <b>c</b>. Sedimentation assay of PCF RNAi. WT (closed squares). RNAi non-induced (−TET) (closed triangles) and induced (+TET) (open circles). <b>d</b>. Mobility graph obtained from Movie S1. The positions of individual cells are plotted at 2.5 s intervals. Open circle: starting position of each cell. Arrowhead: ending position. Number in parentheses: time in seconds of a given cell was within the field of view. Bar, 10 µm. <b>g</b>. Graph of cell populations with orthodox and unorthodox kinetoplast number in BSF RNAi cultures (72 h of induction). K: kinetoplast. N: nucleus. Asterisks indicate statistical significance compared with the WT population, and −TET <i>versus</i> +TET condition (*<i>P<0.1</i>; ** <i>P<0.05</i>; ***<i>P<0.01</i>). <b>h</b> Electron-micrograph of a thin section of an aberrant BSF RNAi induced cell (72 h). (*) indicates a flagellum. Scale bar, 2 µm. Error bars in a, c, e, and g represent the standard error from 3 independent experiments.</p

<i>Tb</i>SAXO is an axoneme-associated protein in <i>T. brucei</i>.

<p><b>A</b>. Immuno-labeling and localization of <i>Tb</i>SAXO on PCF cytoskeletons. Left panel: <i>Tb</i>SAXO localization in the flagellum was identified by the mAb25 antibody (green). Labeling extends along the entire length of the flagellum from the flagellar transition zone (*, labeled with the FTZC antibody) to the distal tip. The PFR is labeled red and begins where the flagellum exits the cell (antibody L8C4). Right panel: a merge of IF and phase contrast. N: nucleus. K: kinetoplast. F: flagellum. Bar, 5 µm. <b>B</b>. Images of the proximal flagellar regions of PCF cytoskeletons from cells through mitosis and cytokinesis. In each row, the left panel shows the PFR and FTZC (*) (red), the center panel <i>Tb</i>SAXO (green), and the right panel shows merged images. The cell cycle stages are defined as 1K1N1F (1 Kinetoplast, 1 Nucleus, 1 Flagellum), 1K1N2F, 2K1N2F and 2K2N2F in rows a–d respectively. <i>Tb</i>SAXO labeling is present immediately distal to the FTZC and is clearly distinct from PFR staining. Bar, 1 µm. <b>C</b>. Immuno-gold electron microscopy reveals that <i>Tb</i>SAXO is localized in the axoneme. Mab25 immuno-gold particles can be seen mainly on the axoneme and not on the PFR of flagella of PCF WT cells. Bars, 100 nm.</p

Identification of SAXO proteins, a MAP6-related protein family.

<p><b>A</b>. Motif 1. The N-terminal domain and its cysteine consensus sequence. Left panel: alignment of the N-terminal sequences of the proteins used for the MEME analysis in C. The boxed sequences correspond to motif 1. Amino acids corresponding to the regular expression of motif 1 are shown in blue. Right panel: motif 1 is represented as a position-specific probability matrice derived from the MEME analysis in C. <b>B</b>. Motif 2. Mn domains in mouse Map6-1, Map6d1, and Mn-like domains and inter-repeats in mouse Saxo1, <i>Plasmodium</i> SAXO and <i>Trypanosoma</i> SAXO. Left panel: characters in blue correspond to the regular expression of the Mn and Mn-like domains identified by the MEME analysis in C; the Mn-like domains identified manually are in italics. The underlined sequences in Map6-1 and Map6d1 correspond to the experimentally identified Mn domains in mouse <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031344#pone.0031344-GoryFaure1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031344#pone.0031344-Bosc2" target="_blank">[6]</a>. CP motifs are boxed. IR: inter-repeat regions. Right panel: the Mn-like regular expression is represented as position-specific probability matrix derived from MEME analysis in C. <b>C</b>. Identification of a family of proteins containing Mn-Like domains. MEME analysis using mouse Map6s, mouse Saxo1, and only protozoan SAXO sequences identified a characteristic N-terminal motif (motif 1, dark blue boxes) in SAXO proteins and Mn-like domains (motif 2, light blue boxes) common to the SAXO and MAP6 proteins. Manually identified supplementary motifs 2 are in grey boxes (Motif 2 manual). The asterisk indicates a conserved CP sequence in the last Mn-like domain.</p

<i>Tb</i>SAXO is a microtubule-associated protein and a microtubule-stabilizing protein.

<p>Mammalian cells (U-2 OS) expressing either MAP6-1-GFP (row a), <i>Tb</i>SAXO-Myc (row b), or various truncated versions of <i>Tb</i>SAXO-Myc (rows c–j) (constructs are represented on the schemes on the right panel). In each case, the transfected cells were incubated at 37°C or 4°C to test for MTs cold stability. Anti-tubulin (TAT1) and anti-Myc antibodies provided the images in left and centre columns respectively at each temperature. The right columns for each temperature set are merged images. The cells were subjected to short extraction before fixation and immuno-labeling. <i>Tb</i>SAXO MT stabilization is seen in images b, c, e, h and j. MT stabilization is also observed in the positive control MAP6-1-GFP expressing cells (a). Nuclei were labeled with DAPI. Bar, 20 µm.</p

Diagnostic performance of intravoxel incoherent motion diffusion-weighted imaging and dynamic contrast-enhanced MRI for assessment of anal fistula activity

<div><p>Objective</p><p>To evaluate intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) sequences for quantitative characterization of anal fistula activity.</p><p>Methods</p><p>This retrospective study was approved by the institutional review board. One hundred and two patients underwent MRI for clinical suspicion of anal fistula. Forty-three patients with demonstrable anal fistulas met the inclusion criteria. Quantitative analysis included measurement of DCE and IVIM parameters. The reference standard was clinical activity based on medical records. Statistical analyses included Bayesian analysis with Markov Chain Monte Carlo, multivariable logistic regression, and receiver operating characteristic analyses.</p><p>Results</p><p>Brevity of enhancement, defined as the time difference between the wash-in and wash-out, was longer in active than inactive fistulas (<i>p</i> = 0.02). Regression coefficients of multivariable logistic regression analysis revealed that brevity of enhancement increased and normalized perfusion area under curve decreased with presence of active fistulas (<i>p</i> = 0.03 and <i>p</i> = 0.04, respectively). By cross-validation, a logistic regression model that included quantitative perfusion parameters (DCE and IVIM) performed significantly better than IVIM only (<i>p</i> < 0.001). Area under the curves for distinguishing patients with active from those with inactive fistulas were 0.669 (95% confidence interval [CI]: 0.500, 0.838) for a model with IVIM only, 0.860 (95% CI: 0.742, 0.977) for a model with IVIM and brevity of enhancement, and 0.921 (95% CI: 0.846, 0.997) for a model with IVIM and all DCE parameters.</p><p>Conclusion</p><p>The inclusion of brevity of enhancement measured by DCE-MRI improved assessment of anal fistula activity over IVIM-DWI only.</p></div

Multivariable logistic regression analysis.

<p>Multivariable logistic regression analysis.</p

Study flowchart.

<p>Study flowchart.</p

Representative images in 2 patients.

<p>Crohn's disease in a 21-year-old man with active fistula (arrows) shows (A) an inter-sphincteric abscess with marked hyperintense signal on the T2-weighted axial turbo spin echo sequence, (B) hyperintense signal on the IVIM-DWI sequence at <i>b</i> = 350 sec/mm², and (C) marked enhancement on the contrast-enhanced sequence. Crohn's disease in a 46-year-old woman with inactive fistula (arrow) show (D) mild T2 hyperintense on the T2-weighted axial turbo spin echo sequence, (E) hypointense signal on the IVIM-DWI sequence at <i>b</i> = 350 sec/mm², and (F) minimal enhancement on the contrast-enhanced sequence.</p

Summary of quantitative and Semi-quantitative analyses.

<p>Summary of quantitative and Semi-quantitative analyses.</p

MRI protocol.

<p>MRI protocol.</p