Pharmacological MT stabilization rescues fibroblast phenotype.

<p>(A) Representative immunoblot of Triton X-100-soluble (free α-tubulin, S) and -insoluble fraction (α-tubulin incorporated into MTs, I) of patients fibroblasts treated with paclitaxel (Tax) or solvent (Met). Morphometric analyses showing the ratio between maximum and minimum axes (B), the area (C) and the number of overlapping regions between cells (D) of paclitaxel (TAX) or solvent (Met)-treated patient fibroblasts. ns = not significant, *<i>p</i><0.05, **<i>p</i><0.02 and ***<i>p</i><0.005 according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM. PARK = patients with mutations of <i>parkin</i> (N = 4); LRRK2 = patients carrying mutations in <i>LRRK2</i> (N = 3); PD = idiopathic Parkinson’s disease patients (N = 3). (E) Representative immunoblot of Triton X-100-soluble (free α-tubulin, S) and -insoluble fraction (α-tubulin incorporated into MTs, I) of control fibroblasts treated with colchicine (COLC), nocodazole (NOC) or solvents (DMSO or Met). Morphometric analyses showing the ratio between maximum and minimum axes (F), the area (G) and the number of overlapping regions between cells (H) of colchine (COLC, N = 5), nocodazole (NOC, N = 5) or solvent (DMSO or Met, N = 5 respectively)-treated control fibroblasts. ns = not significant and ***<i>p</i><0.005 according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM.</p

Impairment of MT stability is shared by PD fibroblasts.

<p>(A) Immunoblot and (B) densitometric analyses of Tyr, deTyr, and Ac tubulin, were performed in whole cell extracts from human fibroblasts deriving from control (white bars), mutated <i>parkin</i> (dark grey bars), mutated <i>LRRK2</i> (light grey bars) and idiopathic PD (black bars). For the quantitation, values of each α-tubulin PTM were normalized on the level of α-tubulin of the relative sample. Triton X-100-soluble (free α-tubulin, Dim) and -insoluble fraction (α-tubulin incorporated into MTs, MT) of human fibroblasts were analyzed by (C) immunoblot and (D) densitometric analyses and are shown as ratio. *<i>p</i><0.05 and ***<i>p</i><0.005 vs control, ##<i>p</i><0.02 vs PD, according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM. CONT = control (N = 10); PARK = patients with mutations of <i>parkin</i> (N = 6); LRRK2 = patients carrying mutations in <i>LRRK2</i> (N = 6); PD = idiopathic Parkinson’s disease patients (N = 3).</p

Phenotype and genotype characterisation of investigated individuals.

a<p>Age at time of skin biopsy and establishment of fibroblast cell line.</p>b<p>The age at which the patient first noticed a PD-related symptom was considered the age of onset of the disease.</p

Stress-induced pathways are not activated in PD fibroblasts.

<p>(A) Immunoblot and (B) densitometric analyses of caspase 3 (CASP 3), heat shock protein 70 (HSP 70) and microtubule-associated protein 1 light chain 3 (LC3) I and II were performed in whole cell extracts from human fibroblasts deriving from control (CONT, white bars, N = 10), mutated <i>parkin</i> (PARK, dark grey bars, N = 6), mutated <i>LRRK2</i> (LRRK2, light grey bars, N = 6) and idiopathic PD (PD, black bars, N = 3). For the quantitation, values of each protein were normalized on the level of GAPDH of the relative sample. All values are expressed as mean ± SEM. *<i>p</i><0.05 and ***<i>p</i><0.005 vs control, ##<i>p</i><0.02 vs PD according to ANOVA, Tukey HSD <i>post hoc</i> test.</p

Morphological alterations characterize PD fibroblasts.

<p>(A) Representative phase contrast micrographs of cultured human fibroblasts of healthy and PD affected people. Scale bar: 25 µm. Morphometric analysis showed reduced ratio between maximum and minimum axes in parkinsonian fibroblast (B) and increased area in the presence of mutated parkin or LRRK2 (C). (D) Histogram showing the increased number of overlapping regions between cells in patient fibroblasts. *<i>p</i><0.05 and ***<i>p</i><0.005 vs control according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM. CONT = control (N = 10); PARK = patients with mutations of <i>parkin</i> (N = 6); LRRK2 = patients carrying mutations in <i>LRRK2</i> (N = 6); PD = idiopathic Parkinson’s disease patients (N = 3).</p

Genetic manipulation restores MT stability and rescues fibroblast phenotype.

<p>(A) Representative immunoblot of Triton X-100-soluble (free α-tubulin, S) and -insoluble fraction (α-tubulin incorporated into MTs, I) of fibroblasts collected from patients with parkin mutations (PARK) transfected with control plasmid (VEC) or WT parkin (WT), and of control fibroblasts (CONT) transfected with short hairpin RNA, sh-183 (183) or control shRNA (VEC). (B-D) Morphometric analyses of patients fibroblasts expressing control plasmid (PARK-VEC, N = 4) or WT parkin (PARK-WT, N = 4), and control fibroblasts transfected with control shRNA (CONT-VEC, N = 4) or silenced with sh-183 (CONT-183, N = 4), showing the ratio between maximum and minimum axes (B), the area (C) and the number of overlapping regions between cells (D). ns = not significant, **<i>p</i><0.02 and ***<i>p</i><0.005 according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM. (E) Representative immunoblot of Triton X-100-soluble (free α-tubulin, S) and -insoluble fraction (α-tubulin incorporated into MTs, I) of fibroblasts collected from patients with LRRK2 mutations (LRRK2) transfected with control plasmid (VEC) or WT LRRK2 (WT), and of control fibroblasts (CONT) expressing control plasmid (VEC) or G2019S mutant LRRK2 (MUT). Morphometric analyses of patients fibroblasts expressing control plasmid (LRRK2-VEC, N = 3) or WT LRRK2 (LRRK2-WT, N = 3), or of control fibroblasts transfected with control plasmid (CONT-VEC, N = 3) or G2019S mutant LRRK2 (CONT-MUT, N = 3), showing the ratio between maximum and minimum axes (F), the area (G) and the number of overlapping regions between cells (H). ns = not significant, *<i>p</i><0.05, **<i>p</i><0.02 and ***<i>p</i><0.005 according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM.</p

PD fibroblasts show subtle cytoskeleton differences.

<p>(A) Immunoblot and (B) densitometric analyses of vimentin (Vim), α-tubulin (α-Tub), β-tubulin (β-Tub) and actin (Actin) were performed in whole cell extracts from human fibroblasts deriving from control (CONT, white bars, N = 10), mutated <i>parkin</i> (PARK, dark grey bars, N = 6), mutated <i>LRRK2</i> (LRRK2, light grey bars, N = 6) and idiopathic PD (PD, black bars, N = 3). For the quantitation, values of each protein were normalized on the level of GAPDH of the relative sample. All values are expressed as mean ± SEM. *<i>p</i><0.05 and ***<i>p</i><0.005 vs control, ##<i>p</i><0.02 vs PD, according to ANOVA, Tukey HSD <i>post hoc</i> test. (C) Cultured human fibroblasts were stained with anti-vimentin and anti-α-tubulin primary antibodies or with TRITC-conjugated phalloidin to reveal the organization of intermediate filaments (Vim, top), microtubules (α-Tub, middle) and actin fibers (Actin, bottom), respectively. Concurrent nuclear staining was made by using DAPI (Blue). Scale bar: 20 µm.</p

GSK3β phosphorylation is reduced in PD fibroblasts.

<p>(A) Immunoblot and densitometric analyses of (B) total and (C) phosphorylated glycogen synthase kinase 3 beta (GSK3β), p38 MAP Kinase (p38) and p44/42 MAPK (Erk) were performed in whole cell extracts from human fibroblasts deriving from control (CONT, white bars, N = 3), mutated <i>parkin</i> (PARK, dark grey bars, N = 3), mutated <i>LRRK2</i> (LRRK2, light grey bars, N = 3) and idiopathic PD (PD, black bars, N = 3). For the quantitation, values of total protein were normalized on the level of GAPDH of the relative sample, whereas the levels of phosphorylated form were normalized on the values of total protein. All values are expressed as mean ± SEM. *<i>p</i><0.05 and **<i>p</i><0.02 vs control, #<i>p</i><0.05 vs PD according to ANOVA, Tukey HSD <i>post hoc</i> test.</p

Table_1_Sex-specific extracerebral complications in patients with aneurysmal subarachnoid hemorrhage.docx

BackgroundExtracerebral complications in patients with aneurysmal subarachnoid hemorrhage (aSAH) often occur during their stay at the neurocritical care unit (NCCU). Their influence on outcomes is poorly studied. The identification of sex-specific extracerebral complications in patients with aSAH and their impact on outcomes might aid more personalized monitoring and therapy strategies, aiming to improve outcomes.MethodsConsecutive patients with aSAH admitted to the NCCU over a 6-year period were evaluated for the occurrence of extracerebral complications (according to prespecified criteria). Outcomes were assessed with the Glasgow Outcome Scale Extended (GOSE) at 3 months and dichotomized as favorable (GOSE 5–8) and unfavorable (GOSE 1–4). Sex-specific extracerebral complications and their impact on outcomes were investigated. Based on the results of the univariate analysis, a multivariate analysis with unfavorable outcomes or the occurrence of certain complications as dependent variables was performed.ResultsOverall, 343 patients were included. Most of them were women (63.6%), and they were older than men. Demographics, presence of comorbidities, radiological findings, severity of bleeding, and aneurysm-securing strategies were compared among the sexes. More women than men suffered from cardiac complications (p = 0.013) and infection (p = 0.048). Patients with unfavorable outcomes were more likely to suffer from cardiac (p ConclusionExtracerebral complications after aSAH are frequent. Cardiac and pulmonary complications are independent predictors of unfavorable outcomes. Sex-specific extracerebral complications in patients with aSAH exist. Women suffered more frequently from cardiac and infectious complications potentially explaining the worse outcomes.</p

Additional file 1 of SMARCA5 interacts with NUP98-NSD1 oncofusion protein and sustains hematopoietic cells transformation

Additional file 1