Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

CNT and PDCs: A fruitful association? Study of a polycarbosilane–MWCNT composite

The effect of MWCNT introduction in a polycarbosilane based ceramic on its electrical properties is presented. The electrical conductivity of two MWCNT powders was measured under dynamic compaction up to 20 MPa when it reached 3–5 S/cm. The compaction behavior was also analyzed and modeled. A composite was then realized using allylhydridopolycarbosilane SMP10® and divinylbenzene as matrix. Intact 10 mm MWCNT-SiC ceramic discs samples with 2 wt.% filler load were produced pressure-less via liquid route despite the linear shrinkage of about 30%. Nanotubes microstructure and distribution in the matrix were confirmed after pyrolysis with TEM and SEM analysis. Anyhow similar electrical conductivity values after pyrolysis between the loaded and unloaded samples were measured. The microstructure analysis via XRD and TEM revealed that the percolative carbon network formed through the use of divinylbenzene improves the electric conductivity more than that of MWCNT addition and also simplifies the whole process.

Impact of Hybrid Iterative Reconstruction on Agatston Coronary Artery Calcium Scores in Comparison to Filtered Back Projection in Native Cardiac CT

PURPOSE To investigate whether the effects of hybrid iterative reconstruction (HIR) on coronary artery calcium (CAC) measurements using the Agatston score lead to changes in assignment of patients to cardiovascular risk groups compared to filtered back projection (FBP). MATERIALS AND METHODS 68 patients (mean age 61.5 years; 48 male; 20 female) underwent prospectively ECG-gated, non-enhanced, cardiac 256-MSCT for coronary calcium scoring. Scanning parameters were as follows: Tube voltage, 120 kV; Mean tube current time-product 63.67 mAs (50 - 150 mAs); collimation, 2 × 128 × 0.625 mm. Images were reconstructed with FBP and with HIR at all levels (L1 to L7). Two independent readers measured Agatston scores of all reconstructions and assigned patients to cardiovascular risk groups. Scores of HIR and FBP reconstructions were correlated (Spearman). Interobserver agreement and variability was assessed with ĸ-statistics and Bland-Altmann-Plots. RESULTS Agatston scores of HIR reconstructions were closely correlated with FBP reconstructions (L1, R = 0.9996; L2, R = 0.9995; L3, R = 0.9991; L4, R = 0.986; L5, R = 0.9986; L6, R = 0.9987; and L7, R = 0.9986). In comparison to FBP, HIR led to reduced Agatston scores between 97 % (L1) and 87.4 % (L7) of the FBP values. Using HIR iterations L1 - L3, all patients were assigned to identical risk groups as after FPB reconstruction. In 5.4 % of patients the risk group after HIR with the maximum iteration level was different from the group after FBP reconstruction. CONCLUSION There was an excellent correlation of Agatston scores after HIR and FBP with identical risk group assignment at levels 1 - 3 for all patients. Hence it appears that the application of HIR in routine calcium scoring does not entail any disadvantages. Thus, future studies are needed to demonstrate whether HIR is a reliable method for reducing radiation dose in coronary calcium scoring.

Serological diagnosis of echinococcosis: the diagnostic potential of native antigens

PURPOSE: Human alveolar (AE) and cystic echinococcosis (CE) caused by the metacestode stages of Echinococcus multilocularis and E. granulosus, respectively, lack pathognomonic clinical signs. Diagnosis therefore relies on the results of imaging and serological studies. The primary goal of this study was to evaluate the efficacy of several easy-to-produce crude or partially purified E. granulosus and E. multilocularis metacestode-derived antigens as tools for the serological diagnosis and differential diagnosis of patients suspicious for AE or CE. METHODS: The sera of 51 treatment-naïve AE and 32 CE patients, 98 Swiss blood donors and 38 patients who were initially suspicious for echinococcosis but suffering from various other liver diseases (e.g., liver neoplasia, etc.) were analysed. RESULTS: According to the results of enzyme-linked immunosorbent assays (ELISA), metacestode-derived antigens of E. granulosus had sensitivities varying from 81 to 97% and >99.9% for the diagnosis of CE and AE, respectively. Antigens derived from E. multilocularis metacestodes had sensitivities ranging from 84 to 91% and >99.9% for the diagnosis of CE and AE, respectively. Specificities ranged from 92 to >99.9%. Post-test probabilities for the differential diagnosis of AE from liver neoplasias, CE from cystic liver lesions, and screening for AE in Switzerland were around 95, 86 and 2.2%, respectively. Cross-reactions with antibodies in sera of patients with other parasitic affections (fasciolosis, schistosomosis, amebosis, cysticercosis, and filarioses) did occur at variable frequencies, but could be eliminated through the use of confirmatory testing. CONCLUSIONS: Different metacestode-derived antigens of E. granulosus and E. multilocularis are valuable, widely accessible, and cost-efficient tools for the serological diagnosis of echinococcosis. However, confirmatory testing is necessary, due to the lack of species specificity and the occurrence of cross-reactions to other helminthic diseases.

Conservation of the oligomeric state of native VDAC1 in detergent micelles.

The voltage-dependent anion-selective channel (VDAC) is an intrinsic β-barrel membrane protein located within the mitochondrial outer membrane where it serves as a pore, connecting the mitochondria to the cytosol. The high-resolution structures of both the human and murine VDACs have been resolved by X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) in 2008. However, the structural data are not completely in line with the findings that were obtained after decades of research on biochemical and functional analysis of VDAC. This discrepancy may be related to the fact that structural biology studies of membrane proteins reveal specific static conformations that may not necessarily represent the physiological state. For example, overexpression of membrane proteins in bacterial inclusion bodies or simply the extraction from the native lipid environment using harsh purification methods (i.e. chaotropic agents) can disturb the physiological conformations and the supramolecular assemblies. To address these potential issues, we have developed a method, allowing rapid one step purification of endogenous VDAC expressed in the native mitochondrial membrane without overexpression of recombinant protein or usage of harsh chaotropic extraction procedures. Using the Saccharomyces cerevisiae isoform 1 of VDAC as a model, this method yields efficient purification, preserving VDAC in a more physiological, native state following extraction from mitochondria. Single particle analysis using transmission electron microscopy (TEM) demonstrated conservation of oligomeric assembly after purification. Maintenance of the native state was evaluated using functional assessment that involves an ATP-binding assay by micro-scale thermophoresis (MST). Using this approach, we were able to determine for the first time the apparent KD for ATP of 1.2 mM.