Additional file 3: of Elevated expression of hyaluronan synthase 2 associates with decreased survival in diffusely infiltrating astrocytomas

Table S1. p-values of the other coefficients used in multivariate analyses. (DOCX 15 kb

Additional file 1: of Elevated expression of hyaluronan synthase 2 associates with decreased survival in diffusely infiltrating astrocytomas

Figure S1. IDH1-R132H –immunostaining in grade II (a) and grade IV tumors (b). Grade II astrocytomas contain several IDH1-R132H –positive cells, while grade IV tumors, glioblastomas, are mostly negative for IDH1-R132H –immunostaining. Scale bar 50 μm. (TIF 33050 kb

Amine Surface Modifications and Fluorescent Labeling of Thermally Stabilized Mesoporous Silicon Nanoparticles

Mesoporous silicon (PSi) has been shown to have extensive application opportunities in biomedicine, whereas it has frequently failed to produce complex systems based on PSi due to the lack of surface functional groups or the instability of the unmodified PSi surface. In the present study, PSi nanoparticles, stabilized by thermal oxidation or thermal carbonization, were successfully modified by grafting aminosilanes on the surface. The modifications were performed by covalently bonding 3-triethoxysilylpropylamine (APTES) or 3-(2-aminoethylamino) propyldimethoxymethylsilane (AEAPMS) on thermally oxidized PSi (TOPSi) and thermally carbonized PSi (TCPSi). These materials were systematically characterized with N₂ ad/desorption, TEM, contact angle, zeta potential, FT-IR, ²⁹Si CP/MAS NMR, and elemental analysis. To evaluate their application potentials, a fluorescent dye, fluorescein 5-isothiocyanate (FITC), was coupled on the surface of amine-modified nanoparticles. The effects of PSi matrix and surface amino groups on FITC coupling efficiency, fluorescent intensity, and the stability of fluorescence in simulated body fluid (SBF) were investigated. The nanoparticles modified with AEAPMS had higher FITC coupling efficiency than those modified with APTES. FITC-coupled TOPSi nanoparticles also possessed brighter fluorescence and better fluorescent stability in SBF. Furthermore, due to the protection caused by the mesoporous structure of PSi nanoparticles, the FITC-coupled TOPSi nanoparticles showed superior photostability in photobleaching experiment

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

<div><p>Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates <i>SQSTM1/p62</i> mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.</p></div

Sections of eyes with clinically diagnosed AMD immunostained for ubiquitin.

<p>The extent of Bruch’s membrane immunopositivity of the retinal pigment epithelial cells (RPE, shown by arrows) and in the drusen (asterisks) was evaluated microscopically (no staining or positive staining) by selecting 5 mm long areas of foveomacular (A), perimacular (B) and peripheral (C) regions. The uniform staining of ubiquitin in RPE cells Bruch’s membrane was observed in all these regions (arrows). There were no differences in the extent of staining between these regions in each group. The nuclei of RPE cells were mostly ubiquitin negative. Most of the drusen were strongly ubiquitin-positive (asterisks). (Original magnifications of x 200 and in insets x 400; Bruch's membrane shown by arrow heads).</p

Sections of foveomacular areas of age-matched control eyes for SQSTM1/p62, ubiquitin and ELAVL1/HuR.

<p>The extent of cytoplasmic immunopositivity for SQSTM1/p62, Bruch’s membrane immunopositivity for ubiquitin and the immunopositivity of nuclei of RPE cells for ELAVL1/HuR (foveomacular areas shown; A, B and C respectively) in the RPE cells (shown by arrows) was evaluated microscopically (no staining or positive staining). For SQSTM1/p62 there were only a few immunopositive cytoplasm’s randomly distributed through-out the RPE cell layer and the nuclei were negative. Bruch’s membrane (shown by arrowheads) was immunopositive for ubiquitin occasionally through-out the RPE cell layer in very small amounts while all of the nuclei were negative. Half of the nuclei showed minor immunopositivity for ELAVL1/HuR while the cytoplasm’s of RPE cells were negative. The foveomacular areas showed no difference in immunohistochemical stainings when compared to areas of perimacular and peripheral retina in all of the proteins studied. (Original magnifications of x 200 and in insets x 400).</p

Transmission electron micrographs of ARPE-19 cells exposed to AICAR or/and MG-132 and autophagic vesicles quantification.

<p>(A) Representative transmission electron micrographs of ARPE-19 cells exposed to MG-132 5 µM solely and with AICAR 2 mM for 3 h, 12 h and 24 h. Autophagic vesicles are indicated by arrows. (B) Quantification of autophagic vesicles in ARPE-19 exposed to MG-132 5 µM solely and with AICAR 2 mM for 3 h, 12 h and 24 h. Six parallel samples were measured in all treatments. *p<0.05, **p<0.01, Mann–Whitney.</p

Transmission electron micrographs of ARPE-19 cells exposed to AICAR or/and MG-132 and aggregate quantification.

<p>(A) Representative transmission electron micrographs of ARPE-19 untreated control cells, cells exposed to AICAR 2 mM or/and MG-132 5 µM for 24 h. Aggregates are indicated by arrows. (B) Quantification of aggregates in ARPE-19 cells exposed to AICAR 2 mM or/and MG-132 5 µM for 24 h. Eight parallel samples were measured in all treatments. **p<0.01, Mann–Whitney.</p

Cytoplasmic SQSTM1/p62, ubiquitin and ELAVL1/HuR proteins in foveomacular, perimacular and peripheral RPE cells.

<p>Cytoplasmic SQSTM1/p62, ubiquitin and ELAVL1/HuR proteins in foveomacular, perimacular and peripheral RPE cells.</p

Summary of the results.

<p>The proteasome inhibitor MG-132 down-regulates (−) the ubiquitin-proteasome pathway (UPP). AMPK-activator AICAR and proteasome inhibitor MG-132 co-treatment activates (+) the autophagy. UPP inhibition significantly increases (+) SQSTM1/p62 protein levels, while autophagy induction during UPP inhibition robustly decreases (−) SQSTM1/p62 protein levels. Since SQSTM1/p62 localizes to aggregates, the decreasing of SQSTM1/p62 reveals its autophagy clearance. In addition, proteasome inhibition significantly increases ELAVL1/HuR protein levels by itself and during the autophagy induction (+). Proteasome inhibition induces a positive regulation of <i>SQSTM1/p62</i> expression that occurs also at post-transcriptional level via ELAVL1/HuR protein (+). Activated autophagy is able to completely abolish the MG-132-induced protein aggregation (−), which, in turn improves the cell vitality. Ubiquitin is also found in intracellular aggregates in ARPE-19 cells as well as from drusens. In contrast, SQSTM1/p62 is found only in the intracellular aggregates in ARPE-19 cells, but not in drusens. However, SQSTM1/p62 levels were high in macular area of RPE cells revealing impaired autophagy. What is the relation between drusen and intracellular aggregates remains still unknown. Red lines with minus symbol represent inhibiting and decreasing events in ARPE-19 cells. Black lines and plus symbol represent activating and increasing events in the ARPE-19 cell. Zero (0) and dark blue line indicate neutral effects in the ARPE-19 cell. UPP: ubiquitin-proteasome pathway.</p