Medical classification in sport of disabled people

Background: In the live of disabled people sport is very important not only in terms of health, but often becomes a passion that gives the life meaning. For many disabled athletes this passion becomes their profession. Medico-sport classification divides athletes into groups and classes based on the type of the physical and mental dysfunction in order to equalize the odds in a sports competition. Material and methods: Analysis of available literature, articles in the Google Scholar and PubMed database using keywords: disabled, sport, classification, dysfunction. Results: Medical-sport classification distinguishes groups of disability of: sight, hearing, limb dismemberment, paraplegia, diseases of the musculoskeletal system, cerebral palsy and intellectual disability. In the professional sport the athletes classification is done by medical doctors in cooperation with physiotherapists under the supervision of the Central Classification Committee of the “Start” organization, which verifies and approves the athletes membership to a certain group or class. During the athletes World Championships decision about the classification is done by the International Paralympic Committee (IPC), Cerebral Palsy International Sports and Recreations Association (CP – ISRA) and International Wheelchair and Amputee Sports Federation (IWAS). The group/class examination is carried out during the most important sports events like Paralympic Games, World Championships and European Athletics Championships, and remains valid for the next four years. Conclusions: The aim of this classification is to make the athlete’s achievement in a sport competition be a result of the athlete’s physical and psychological training rather than the level of his or hers disability. The subject of grouping and classification of the disabled athletes still remains an open topic for discussion

Endocytic Adaptor Protein Tollip Inhibits Canonical Wnt Signaling

Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery

zTollip is broadly expressed during early zebrafish development.

<p>Immunocytochemistry analysis of zTollip protein localization in early embryonic development of zebrafish. Embryos fixed at various stages (indicated below the images) were incubated with primary antibodies against human Tollip, followed by secondary antibodies and chromogenic peroxidase-based detection. As controls for staining specificity, embryos at tail bud stage were processed omitting the primary antibody (G) or secondary antibody (H). zTollip expression is uniform from 1-cell stage until tail bud (A-F), but its expression increases in the head (arrowhead in I) and intersomitic regions (black arrows in I) at 24 hpf. At 3 dpf, zTollip is maintained in the intersomitic regions (arrows in J), but it is also expressed in the tissue surrounding the heart (arrowhead in J). Lateral views (A-H). Anterior to the left (I, J).</p

Tollip requires an intact ubiquitin-binding domain to function in canonical Wnt signaling.

<p>(A) Schematic representation of deletion and point mutants of human Tollip. (B-D) myc-tagged Tollip wild-type (wt) and mutants were tested in the Super8xTOPFlash reporter assay (upper panels; B, deletion mutants; C, ubiquitin-binding deficient M240A/F241A mutant; D, phosphoinositide-binding deficient point mutants). Increasing amounts of mutant-encoding plasmids were transfected. Ctrl, cells transfected with an empty pcDNA plasmid instead of a Tollip-encoding construct. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium (CM) and transfected with an empty plasmid and the Super8xTOPFlash reporter. Data are mean ± SEM from 3 (C) or 4 (B, D) independent experiments; *<i>P</i>≤0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (Mann-Whitney U test). Expression of mutated Tollip proteins was verified by Western blotting (WB) using anti-myc antibodies, with α-tubulin as a loading control (lower panels). (E) Expression of <i>FGF9</i> and <i>NRP1</i> genes upon overexpression of Tollip wt, K150E and M240A/F241A mutants in Wnt3a-stimulated HEK293 cells (Ctrl), measured by qPCR. All values are relative expression levels, compared to controls from cells incubated with control-conditioned medium and transfected with an empty plasmid (normalized to 1 for each gene; not shown). Data are mean ± SEM from 3 independent experiments; *<i>P</i>≤0.05 (Mann-Whitney U test). (F) Immunoprecipitation of β-catenin from lysates of HEK293 cells transfected with an empty plasmid (Ctrl) or with constructs expressing wild-type Tollip (wt), M240A/F241A mutant or deletion 1 (del.1) mutant. Cells were either untreated or incubated with MG132 for 4 h before lysis. Upper panel; immunoprecipitates were probed with antibodies against β-catenin, total ubiquitin (Ub), its K48-linked (Ub-K48) or K63-linked (Ub-K63) chains. Lower panel; 10% of starting lysates taken for immunoprecipitation (input) were blotted against total ubiquitin (Ub), β-catenin and Tollip, with actin as a loading control.</p

Tollip inhibits ligand-independent Wnt signaling in HEK293 and cancer cell lines.

<p>(A) Overexpression of Tollip inhibits the activity of Super8xTOPFlash luciferase reporter in HEK293 cells stimulated by overexpression of Wnt1 or Disheveled 2 (Dvl2), or LiCl treatment (left panel). Two amounts of Tollip-encoding plasmids were tested. All values are expressed as fold of untreated control, i.e. the reporter activity in cells without any stimulant which was normalized to 1 (Ctrl NS). Ctrl denotes the reporter activity in cells treated with a given stimulant but transfected with an empty plasmid instead of Tollip. Data are mean ± SEM from 3 independent experiments; *<i>P</i>≤0.05 (Wilcoxon signed-rank test). Right panel: Levels of active and total β-catenin were investigated in lysates of cells tested in the reported assay. NS, non-stimulated cells. Immunoblotting was performed with the indicated antibodies, with Ponceau staining shown as a loading control. (B) Tollip inhibits Wnt signaling in HEK293 cells stimulated by overexpression of β-catenin-S33Y mutant. Overexpression of Tollip (left panel) or its siRNA-mediated silencing (right panel) alters the activity of Super8xTOPFlash luciferase reporter. All values are expressed as fold of untreated control, i.e. the reporter activity in cells without β-catenin-S33Y overexpression which was normalized to 1 (Ctrl NS). Ctrl denotes the reporter activity in cells treated with β-catenin-S33Y overexpression and transfected with an empty plasmid instead of Tollip (left panel) or non-targeting siRNA control (right panel). Data are mean ± SEM from 3 independent experiments; *<i>P</i>≤0.05 (Wilcoxon signed-rank test). (C) Levels of active and total β-catenin, and of Tollip were investigated in HEK293 cells treated with Wnt3a- or control conditioned (CM) medium for 6 h, and in unstimulated DLD1 and HCT-116 cells. Immunoblotting was performed with the indicated antibodies, with actin as a loading control. (D) Overexpression of Tollip suppresses the activity of Super8xTOPFlash luciferase reporter in cancer cell lines DLD1 and HCT116 without exogenous stimulation (upper panel). Increasing amounts of Tollip-encoding plasmid were tested. The reporter activity in cells transfected with an empty plasmid was normalized to 1 for each line (Ctrl). Data are mean ± SEM from 3 independent experiments; *<i>P</i>≤0.05 (Mann-Whitney U test). Lower panel: the levels of Tollip overexpression were verified by Western blotting (WB), with α-tubulin serving as a loading control. (E) Levels of active and total β-catenin, and of Tollip were investigated in DLD1 and HCT-116 cells overexpressing Tollip or transfected with an empty plasmid (Ctrl). Immunoblotting was performed with the indicated antibodies, with actin as a loading control.</p

Tollip functions in Wnt signaling independently of dynamin-mediated endocytosis.

<p>(A) Expression of dynamin2-K44A mutant (Dnm2 K44A) inhibits the activity of Super8xTOPFlash luciferase reporter in a dose-dependent manner in HEK293 cells stimulated with Wnt3a-conditioned medium. No effects were observed in cells treated with control-conditioned medium (CM) or in Wnt3a-stimulated cells expressing mutated Super8xFOPFlash reporter. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium and transfected with an empty plasmid and the Super8xTOPFlash reporter. Protein levels of overexpressed Dnm2-K44A were verified by Western blotting, with GAPDH as a loading control (lower panel). (B) Overexpression of Tollip further represses the Super8xTOPFlash reporter activity in Wnt3a-stimulated cells HEK293 cells overproducing Dnm2-K44A. Ctrl, cells transfected with an empty pcDNA plasmid instead of Tollip-encoding construct. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium (CM) and transfected with an empty plasmid and the Super8xTOPFlash reporter. Protein levels of overexpressed Dnm2-K44A and Tollip (detected via its myc tag) were verified by Western blotting, with GAPDH as a loading control (lower panel). (C) Overexpression of Tollip further represses the Super8xTOPFlash reporter activity in HEK293 cells stimulated with Wnt3a for 5 h and concomitantly treated with dynasore or DMSO as vehicle control. Ctrl, cells transfected with an empty pcDNA plasmid instead of Tollip-encoding construct. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium (CM) and DMSO, and transfected with the Super8xTOPFlash reporter. Protein levels of overexpressed Tollip (detected via its myc tag) were verified by Western blotting, with vinculin as a loading control (lower panel). (D) The activity of Super8xTOPFlash luciferase reporter in HEK293 cells depleted of Tollip (by siRNA #1), stimulated with Wnt3a and treated with dynasore for 5 h. Ctrl, cells transfected with non-targeting siRNA. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium (CM) and DMSO, and transfected with the Super8xTOPFlash reporter. Knockdown efficiency of Tollip was verified by Western blotting, with actin as a loading control (lower panel). (A-D) Data are mean ± SEM from 3 independent experiments; *<i>P</i>≤0.05 (Mann-Whitney U test or Wilcoxon signed-rank test in B).</p

Tollip inhibits canonical Wnt signaling.

<p>(A) Depletion of Tollip potentiates the activity of Super8xTOPFlash luciferase reporter in HEK293 cells stimulated with Wnt3a-conditioned medium (left bars). Three RNAi reagents were used for Tollip silencing (esiRNA#1, siRNA #1 and #2) with appropriate non-targeting controls (Ctrl). No effects were observed in cells treated with control-conditioned medium (CM; middle bars and magnification above) or in Wnt3a-stimulated cells expressing mutated Super8xFOPFlash reporter (right bars and magnification above). The luciferase activity was measured as described in Materials and Methods. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium and transfected with non-targeting RNAi reagents and the Super8xTOPFlash reporter. Data are mean ± SEM from 3 independent experiments; *<i>P</i>≤0.05, **<i>P</i><0.01 (Wilcoxon signed-rank test). (B) Efficiency of Tollip silencing with RNAi reagents used in A was verified by Western blotting (WB), with α-tubulin serving as a loading control. (C) Overexpression of Tollip in Wnt3a-stimulated HEK293 cells inhibits the activity of Super8xTOPFlash reporter. Cells treated with control-conditioned medium (CM) or bearing mutated Super8xFOPFlash reporter served as controls, as in panel A. Ctrl, cells transfected with an empty pcDNA plasmid instead of Tollip-encoding construct. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium and transfected with an empty plasmid and the Super8xTOPFlash reporter. (D) Overexpression of Tollip represses the activity of <i>AXIN2</i> and <i>CCND1</i> promoters (driving expression of luciferase) in HEK293 cells stimulated with Wnt3a. CM, cells treated with control medium. Ctrl, cells transfected with an empty pcDNA plasmid instead of Tollip-encoding construct. All values are expressed as fold of untreated control, i.e. cells incubated with control-conditioned medium and transfected with an empty plasmid and the respective luciferase reporter. (E) Overexpression of Tollip decreases mRNA levels of <i>FGF9</i> and <i>NRP1</i> genes (but not of <i>NKD1</i> or <i>DKK1</i>) in HEK293 cells stimulated with Wnt3a, measured by qPCR. Ctrl, cells transfected with an empty pcDNA plasmid instead of Tollip-encoding construct. All values are relative expression levels, compared to controls from cells incubated with control-conditioned medium and transfected with an empty plasmid (normalized to 1 for each gene; not shown). (C-E) Data are mean ± SEM from 3 independent experiments; *<i>P</i>≤0.05 (Mann-Whitney U test).</p

Tollip affects a pool of active β-catenin.

<p>(A) Levels of active and total β-catenin were investigated in HEK293 cells treated with Wnt3a- or control conditioned (CM) medium for 1.5 h upon overexpression of myc-Tollip (left panel) or siRNA-mediated depletion of Tollip (right panel). Immunoblotting was performed with the indicated antibodies, with actin as a loading control. (B-C) HEK293 cells were transfected with siRNA silencing Tollip (siRNA #1 or #2) or with appropriate non-targeting control (Ctrl). After 72 h they were stimulated with control medium (CM; B) or Wnt3a-conditioned medium (C) for 10 h. Cells were immunostained for β-catenin and Tollip, with DAPI indicating cell nuclei. Residual nuclear staining visible in Tollip-depleted cells represents an unspecific signal of the Tollip antibody (compare silencing efficiency visualized by immunoblotting in F). Scale bar 20 μm. (D-E) Acquired images were analyzed with ImageJ to calculate the percentage of β-catenin-positive nuclei (D) or mean fluorescence intensity of nuclear β-catenin (expressed in arbitrary units, AU; shown in E), as described in Materials and Methods. Data are mean ± SEM from 3 independent experiments; *P≤0.05 (Mann-Whitney U test). (F) Efficiency of Tollip silencing with RNAi reagents used in (A) was verified by Western blotting (WB), with actin serving as a loading control.</p