Aggregation of ␣-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored ␣-synuclein oligomer formation at the single particle level. Organic solvents were used to trigger aggregation, which resulted in small oligomers ("intermediate I"). Under these conditions, Fe 3؉ at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers ("intermediate II"). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation. Notably, only Fe 3؉ -induced oligomers were SDS-resistant and could form ion-permeable pores in a planar lipid bilayer, which were inhibited by the oligomerspecific A11 antibody. Moreover, baicalein and N-benzylidenebenzohydrazide derivatives inhibited oligomer formation. Baicalein also inhibited ␣-synuclein-dependent toxicity in neuronal cells. Our results may provide a potential disease mechanism regarding the role of ferric iron and of toxic oligomer species in Parkinson diseases. Moreover, scanning for intensely fluorescent targets allows high throughput screening for aggregation inhibitors and may provide new approaches for drug development and therapy.

No abstract

Pseudomonas aeruginosa persists in patients with cystic fibrosis (CF) and drives CF lung disease progression. P. aeruginosa potently activates the innate immune system, mainly mediated through pathogen-associated molecular patterns, such as flagellin. However, the host is unable to eradicate this flagellated bacterium efficiently. The underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells generated in cancer and proinflammatory microenvironments and are capable of suppressing T cell responses. We hypothesized that P. aeruginosa induces MDSCs to escape T cell immunity. In this article, we demonstrate that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa and correlate with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was 1) dose-dependently mimicked by purified flagellin protein, 2) significantly reduced using flagellin-deficient P. aeruginosa bacteria, and 3) corresponded to TLR5 expression on MDSCs in vitro and in vivo. Both purified flagellin and flagellated P. aeruginosa induced an MDSC phenotype distinct from that of the previously described MDSC-inducing cytokine GM-CSF, characterized by an upregulation of the chemokine receptor CXCR4 on the surface of MDSCs. Functionally, P. aeruginosa–infected CF patient ex vivo–isolated as well as flagellin or P. aeruginosa in vitro–generated MDSCs efficiently suppressed polyclonal T cell proliferation in a dose-dependent manner and modulated Th17 responses. These studies demonstrate that flagellin induces the generation of MDSCs and suggest that P. aeruginosa uses this mechanism to undermine T cell–mediated host defense in CF and other P. aeruginosa–associated chronic lung diseases.

Previous studies have shown that the rough endoplasmic reticulum (ER) contains nascent precursor polypeptide gated channels. Circumstantial evidence suggests that these channels are formed by the Sec61p complex. We reconstituted the purified Sec61p complex in a lipid bilayer and characterized its dynamics and regulation. The Sec61p complex is sufficient to form the precursor polypeptide activated channel under co- and posttranslational transport conditions. Activity of the Sec61p channel in both transport modes is induced by direct interaction with precursor protein. The Sec61p complex comprises a highly dynamic pore covering conductances corresponding to channel openings from approximately 6 to 60 A. Its properties are indistinguishable from those we observed with native ER channels, directly demonstrating that these channels are formed by the Sec61p complex.

Purpose-High resolution peripheral quantitative CT (HR-pQCT) is a new imaging technique that assesses trabecular and cortical bone microarchitecture of the radius and tibia in vivo. The purpose of this study was to determine the extent to which microarchitectural variables measured by HR-pQCT reflect those measured by the "gold standard", transiliac bone biopsy.Methods-HR-pQCT scans (Xtreme CT, Scanco Medical AG) and iliac crest bone biopsies were performed in 54 subjects (aged 39±10 years). Biopsies were analyzed by 2D quantitative histomorphometry and 3D microcomputed tomography (μCT). Apparent Young's modulus, an estimate of mechanical competence or strength, was determined by micro-finite element analysis (μFE) of biopsy μCT and HR-pQCT images.Results-The strongest correlations observed were between trabecular parameters (bone volume fraction, number, separation) measured by μCT of biopsies and HR-pQCT of the radius (R: 0.365-0.522; p<0.01). Cortical width of biopsies correlated with cortical thickness by HR-pQCT, but only at the tibia (R=0.360, p<0.01). Apparent Young's modulus calculated by μFE of biopsies correlated with that calculated for both radius (R=0.442; p<0.001) and tibia (R=0.380; p<0.001) HR-pQCT scans.Conclusions-The associations between peripheral (HR-pQCT) and axial (transiliac biopsy) measures of microarchitecture and estimated mechanical competence are significant but modest.