8 research outputs found
Delayed Wallerian degeneration after proteasome inhibition is not reverted by PD184352 and BIX02189 combined.
<p>(A) Representative phase contrast images of transected wild-type neurites in SCG explant cultures treated with U0126 (50 µM), or PD184352 (5 µM) plus BIX02189 (10 µM) combined, after proteasome inhibition by MG-132 (20 µM) (untreated  =  DMSO). Images of the same field of distal neurites were captured just after transection and 6 hours later. (B) Quantification of neurite degeneration for transected neurites as in (A). Degeneration index (± SEM) was calculated from multiple fields in n = 3 independent experiments. ** <i>p</i><0.01, 2-way repeated measures ANOVA with Tukey's multiple comparisons post hoc tests. All statistically significant differences are marked. All other comparisons were not significant. (C) Representative immunoblots showing inhibition of ERK1/2 and ERK5 phosphorylation in whole wild-type SCG explant cultures (cell bodies and neurites combined) 9 hours after proteasome inhibition by MG-132 (20 µM) and 6 hours after treatment with U0126 (50 µM), or a combination of PD184352 (5 µM) plus BIX02189 (10 µM) (untreated  =  DMSO). Phosphorylated ERK1/2 was detected using a phosphorylation-dependent antibody. Phosphorylated ERK5 was detected as a slower migrating band (indicated by an arrow) using a phosphorylation-independent antibody. Total ERK1/2 and ERK5 levels act as sample references.</p
MEK1/2 inhibitor PD184352 and MEK5 inhibitor BIX02189 fail to revert the <i>Wld</i><sup>S</sup> phenotype.
<p>(A) Representative phase contrast images of transected neurites in <i>Wld</i><sup>S</sup> SCG explant cultures treated with U0126 (50 µM), PD184352 (5 µM) and/or BIX02189 (10 µM) as indicated (untreated  =  DMSO). Images of the same field of distal neurites were captured just after transection and 48 hours later. (B) Quantification of neurite degeneration for transected <i>Wld</i><sup>S</sup> neurites as in (A). Degeneration index (± SEM) was calculated from multiple fields in n = 3 or 4 independent experiments. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001, 2-way repeated measures ANOVA with Tukey's multiple comparisons post hoc tests. All statistically significant differences are marked. All other comparisons were not significant. (C) Representative immunoblots showing inhibition of ERK1/2 and ERK5 phosphorylation in whole <i>Wld</i><sup>S</sup> SCG explant cultures (cell bodies and neurites combined) after 48 hours treatment with U0126 (50 µM), PD184352 (5 µM) and/or BIX02189 (10 µM) as indicated (untreated  =  DMSO). Phosphorylated ERK1/2 was detected using a phosphorylation-dependent antibody. Phosphorylated ERK5 was detected as a slower migrating band (indicated by an arrow) using a phosphorylation-independent antibody. Total ERK1/2 and ERK5 levels act as sample references.</p
Reversion of the <i>Wld</i><sup>S</sup> phenotype by MEK inhibitor U0126.
<p>(A) Representative phase contrast images of transected neurites in <i>Wld</i><sup>S</sup> SCG explant cultures treated with different concentrations of U0126 as indicated (untreated  =  DMSO). Images of the same field of distal neurites were captured at the times after transection shown on the left. (B) Quantification of neurite degeneration for transected <i>Wld</i><sup>S</sup> neurites as in (A). Degeneration index (± SEM) was calculated from multiple fields in n = 4 independent experiments. * <i>p</i><0.05, ** <i>p</i><0.01, and *** <i>p</i><0.001, 2-way repeated measures ANOVA with Tukey's multiple comparisons post hoc tests. All statistically significant differences are marked. All other comparisons were not significant. (C) Representative immunoblots showing inhibition of ERK1/2 and ERK5 phosphorylation in whole <i>Wld</i><sup>S</sup> SCG explant cultures (cell bodies and neurites combined) 48 hours after treatment with different concentrations of U0126. Phosphorylated ERK1/2 was detected using a phosphorylation-dependent antibody. Phosphorylated ERK5 was detected as a slower migrating band (indicated by an arrow) using a phosphorylation-independent antibody. Total ERK1/2 and ERK5 levels act as sample references. (D) Representative phase contrast image (left) showing cut and uncut neurites in <i>Wld</i><sup>S</sup> SCG explant cultures 48 hours after transection and treatment with 50 µM U0126. Boxed regions are magnified to show morphology of cut and uncut neurites.</p
U0126 does not alter FLAG-NMNAT2 or FLAG-Wld<sup>S</sup> turnover in transfected HEK 293T cells.
<p>Immunoblot analyses assessing the effects of U0126 (50 µM) on natural turnover of FLAG-Wld<sup>S</sup> (A) and stabilization of FLAG-NMNAT2 after proteasome inhibition (B) in HEK 293T cells. Cells were co-transfected with FLAG-Wld<sup>S</sup> and FLAG-NMNAT2 expression constructs. 24 hours after transfection cells were treated with emetine (10 µM), together with proteasome inhibitor MG-132 (20 µM) and/or U0126 (50 µM), as indicated, for a further 24 hours. Control cells (–) treated with DMSO were collected at the time of emetine addition (0 hours) to act as a reference for expression levels before protein synthesis was blocked. ß-Actin acts as the sample reference. Blots of FLAG-NMNAT2 and FLAG-Wld<sup>S</sup> in (A) and (B) respectively (bottom panels) are included only as controls (for transfection efficiency or emetine efficacy). Representative images are shown. Relative mean levels (± SEM) of FLAG-Wld<sup>S</sup> (A) and FLAG-NMNAT2 (B) are shown below the relevant lanes on each blot after normalization to ß-Actin (based on data from n = 2 and n = 4 respectively). Data are presented relative to the DMSO controls (–) (set at 1).</p
Additional file 1: of Taking patient and public involvement online: qualitative evaluation of an online forum for palliative care and rehabilitation research
S1. COREQ checklist for qualitative studies, S2. Focus group topic guide (PPI members), S3. Focus group topic guide (Researchers), S4. GRIPP2-SF checklist for PPI in research, S5. Coding tree for analysis. (PDF 572 kb
Endometriosis and its influence on woman's life
This work is about gynecological disease, whose incidence is evergrowing, end metriosis. Endometriosis is in itself a very controversial disease, which is sign ificant intervening into life of a woman. Endometriosis is a common gynecological di sorder affecting fertile female population. It is the disease, that is presented by functional endometrial glandules and stroma outside their usuallocality within uterus (womb). General symptoms are pain and infertility. The diagnostic key is the cyclic character of symptom s and laparoscopy - histology testification presence of lesions. Tbe surgical treatment of endometriosis is considered as a golden standard of the therapy. My aim of this work is to have look up to aH sections of the disease. At the same time my work covers survey. I detected prospective abnormalities with theoretic knowledges via fonns distributed to patients with endometriosis. The fonn appraised frequen y oť symptoms in patiens. It presents the most frequent age group of disabled women as well. Fonns a1so outline dilernma of arguments about character of creation and it observes problems of indisposed women and possibility oftheir naturally gravidity. It also deals of possibilities of available therapy and its use in patients. ln no little importance my work adverts to next facts des rving next..
Additional file 2: of How integrated are neurology and palliative care services? Results of a multicentre mapping exercise
Catchment areas, population served and patients seen for neurology and palliative care services. This file contains in-depth information about the catchment areas, population served and patients seen for neurology and palliative care services, for the sites involved in the mapping exercise. (DOCX 22 kb