Biophysical Characterization of Pro-apoptotic BimBH3 Peptides Reveals an Unexpected Capacity for Self-Association

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome.This work was funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC-2033—Projektnummer 390677874, the DFG Priority Program SPP1601 “New Frontiers in Sensitivity in EPR Spectroscopy” (to E.B.), DFG BO 3000/5-1 (to E.B.), SFB958 – Z04 (to E.B.), DFG grant INST 130/972-1 FUGG (to E.B.). P.E.C. is supported by an Australian NHMRC fellowship (1079700

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Platinum corrosion products from electrode contacts of human cochlear implants induce cell death in cell culture models

Despite the technological progress made with cochlear implants (CI), impedances and their diagnosis remain a focus of interest. Increases in impedance have been related to technical defects of the electrode as well as inflammatory and/or fibrosis along the electrode. Recent studies have demonstrated highly increased impedances as the result of corroded platinum (Pt) electrode contacts. This in vitro study examined the effects of Pt ions and compounds generated by corrosion of the electrode contacts of a human CI on cell metabolism. Since traces of solid Pt in surrounding cochlear tissues have been reported, the impact of commercially available Pt nanoparticles (Pt-NP, size 3 nm) on the cell culture model was also determined. For this purpose, the electrode contacts were electrically stimulated in a 0.5% aqueous NaCl solution for four weeks and the mass fraction of the platinum dissolute (Pt-Diss) was determined by mass spectrometry (ICP-MS). Metabolic activity of the murine fibroblasts (NIH 3T3) and the human neuroblastoma (SH-SY5Y) cells was determined using the WST-1 assay following exposure to Pt-Diss and Pt-NP. It was found that 5-50 μg/ml of the Pt-NP did not affect the viability of both cell types. In contrast, 100 μg/ml of the nanoparticles caused significant loss in metabolic activity. Furthermore, transmission electron microscopy (TEM) revealed mitochondrial swelling in both cell types indicating cytotoxicity. Additionally, TEM demonstrated internalized Pt-NP in NIH 3T3 cells in a concentration dependent manner, whereas endocytosis in SH-SY5Y cells was virtually absent. In comparison with the Pt-NP, the corrosion products (Pt-Diss) with concentrations between 1.64 μg/ml and 8.2 μg/ml induced cell death in both cell lines in a concentration dependent manner. TEM imaging revealed both mitochondrial disintegration and swelling of the endoplasmic reticulum, suggesting that Pt ions trigger cytotoxicity in both NIH 3T3 and SH-SY5Y cell lines by interacting with the respiratory chain

Gewalt und Gesundheit Violence and health

Gewalt hat viele Formen und zieht bei Betroffenen meist eine Vielzahl von Folgen nach sich. Bisher gibt es Hinweise darauf, dass Männer und Frauen unterschiedliche Arten und unterschiedliche Folgen von Gewalt erleben. Im klinischen Kontext fehlen Daten zu genauen diesbezüglichen Prävalenzen bei stationären und ambulanten Patientinnen und Patienten. Wir haben uns dieser Frage im Rahmen einer Prävalenzerhebung an einer Uniklinik gewidmet und auch geprüft, inwiefern eine psychologische Beratung bei Gewalterlebnissen positive Effekte erzielen kan

Determination of the effects of Pt-NP with varying concentrations in cell cultivation.

<p>Metabolic activity of both NIH 3T3 (<b>A</b>) and SH-SY5Y cells (<b>B</b>) grown in culture medium supplied with 5 μg/ml– 100 μg/ml Pt-NP was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 12–14; SH-SY5Y, n = 11–15). The resulting formazan dye intensities were related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SE_M. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).</p

Microscopic characterization of the morphology of SH-SY5Y cells following exposure to varying NaCl concentrations.

<p>SH-SY5Y cells were cultivated either in normal cell culture medium containing 6.4 mg/ml NaCl as reference (<b>A</b>) or in culture medium containing 6.26 mg/ml (1:10) (<b>B</b>), 6.12 mg/ml (1:5) (<b>C</b>) and 5.93 mg/ml (1:3) NaCl (<b>D</b>). Microscopic images demonstrated reduced cell attachment and growth in a concentration dependent manner without any signs of cytotoxicity.</p

Microscopic characterization of the morphology of NIH 3T3 cells following exposure to Pt-NP.

<p>NIH 3T3 cells were cultivated either without any additional Pt particles as reference (<b>A</b>) or in culture medium containing 25 μg/ml (<b>B</b>), 50 μg/ml (<b>C</b>) and 100 μg/ml (<b>D</b>) of the Pt-NP. The images demonstrated highly uniform cell adhesion without any morphological impairment throughout the cell cultures assays with varying Pt-NP concentrations. Size of bars: 200 μm.</p

Determination of the effects of Pt-Diss with varying Pt-Diss concentrations in cell cultivation.

<p>Metabolic activity of both NIH 3T3 (<b>A</b>) and SH-SY5Y cells (<b>B</b>) grown in culture medium supplied with 0.82 μg/ml– 8.2 μg/ml Pt-Diss concentration was determined by indirect reduction of WST-1 by mitochondrial dehydrogenases to a formazan dye. Optical densities (OD) were measured in 48 h and 6 d cultivation assays (NIH 3T3, n = 12–16; SH-SY5Y, n = 10). The resulting formazan dye intensities were related to those obtained from the reference and calculated as a percentage [%]. Each data point is presented as mean and SE_M. ANOVA with Newman-Keuls multiple comparison test was performed for statistical assessment (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).</p

Ultrastructural morphology of NIH 3T3 cells following exposure to Pt-Diss.

<p>After cultivation either without Pt as reference (<b>A</b>) or in culture medium containing 6.0 μg/ml (<b>B</b>), 0.11 μg/ml (<b>C</b>) or 0.02 μg/ml Pt (<b>D</b>) the ultrastructure of 3T3 fibroblasts was compared. They demonstrated mitochondrial swelling (arrow in <b>B</b>) only at the highest tested Pt concentration. At 0.11 μg/ml Pt the cells showed greater phagocytic activity (arrowhead in <b>C</b>). Lower amount of Pt in the culture medium induced no morphological changes in comparison with the control. Size of bars: 2 μm.</p