SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes

Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.

SNARE-fusion mediated inserton of membrane proteins into native and artificial membranes

Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE-protein based method for incorporation of multiple membrane proteins into membranes, and for delivery of large water-soluble substrates into closed membrane vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0-ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.

Vultures vs livestock: conservation relationships in an emerging conflict between humans and wildlife

Human–wildlife conflict is emerging as an important topic in conservation. Carnivores and birds of prey are responsible for most conflicts with livestock and game but since the mid 1990s a new conflict is emerging in south-west Europe: the presumed killing of livestock by griffon vultures Gyps fulvus. Lack of scientific data and magnification of the problem by the media are increasing alarm amongst the public, and political pressures to implement management decisions have not been based on scientific evidence. We compiled information on 1,793 complaints about attacks by griffon vultures on livestock, lodged with Spanish authorities from 1996 to 2010. Spain is home to the majority (95%) of griffon vultures and other scavengers in the European Union. Most of the cases occurred in areas of high livestock density, affected principally sheep (49%) and cows (31%), and were associated with spring birthing times (April–June). On average 69% of the complaints made annually were rejected because of a lack of evidence about whether the animal was alive before being eaten. The total economic cost of compensation was EUR 278,590 from 2004 to 2010. We discuss possible ways to mitigate this emerging human–wildlife conflict. These need to include the participation of livestock farmers, authorities, scientists and conservation groups.

3D morphometry using automated aortic segmentation in native MR angiography: an alternative to contrast enhanced MRA?

INTRODUCTION Native-MR angiography (N-MRA) is considered an imaging alternative to contrast enhanced MR angiography (CE-MRA) for patients with renal insufficiency. Lower intraluminal contrast in N-MRA often leads to failure of the segmentation process in commercial algorithms. This study introduces an in-house 3D model-based segmentation approach used to compare both sequences by automatic 3D lumen segmentation, allowing for evaluation of differences of aortic lumen diameters as well as differences in length comparing both acquisition techniques at every possible location. METHODS AND MATERIALS Sixteen healthy volunteers underwent 1.5-T-MR Angiography (MRA). For each volunteer, two different MR sequences were performed, CE-MRA: gradient echo Turbo FLASH sequence and N-MRA: respiratory-and-cardiac-gated, T2-weighted 3D SSFP. Datasets were segmented using a 3D model-based ellipse-fitting approach with a single seed point placed manually above the celiac trunk. The segmented volumes were manually cropped from left subclavian artery to celiac trunk to avoid error due to side branches. Diameters, volumes and centerline length were computed for intraindividual comparison. For statistical analysis the Wilcoxon-Signed-Ranked-Test was used. RESULTS Average centerline length obtained based on N-MRA was 239.0±23.4 mm compared to 238.6±23.5 mm for CE-MRA without significant difference (P=0.877). Average maximum diameter obtained based on N-MRA was 25.7±3.3 mm compared to 24.1±3.2 mm for CE-MRA (P<0.001). In agreement with the difference in diameters, volumes obtained based on N-MRA (100.1±35.4 cm(3)) were consistently and significantly larger compared to CE-MRA (89.2±30.0 cm(3)) (P<0.001). CONCLUSIONS 3D morphometry shows highly similar centerline lengths for N-MRA and CE-MRA, but systematically higher diameters and volumes for N-MRA.

The association between in-stent neoatherosclerosis and native coronary artery disease progression: a long-term angiographic and optical coherence tomography cohort study.

AIMS The purpose of the present study was to investigate the relationship between in-stent neoatherosclerosis (NA) and native atherosclerosis progression of untreated coronary segments. METHODS AND RESULTS In-stent NA was assessed by optical coherence tomography (OCT) among patients included in the SIRTAX-LATE OCT study 5 years after drug-eluting stent (DES) (sirolimus-eluting and paclitaxel-eluting stents) implantation. Neoatherosclerosis was defined as the presence of fibroatheroma or fibrocalcific plaque within the neointima of stented segments with a longitudinal extension >1.0 mm. Atherosclerosis progression in untreated native coronary segments was evaluated by serial quantitative coronary angiography (QCA). The change in minimal lumen diameter (MLD) was serially assessed within matched segments at baseline and 5-year angiographic follow-up. The key clinical endpoint was non-target lesion (non-TL) revascularization throughout 5 years. A total of 88 patients with 88 lesions were available for OCT analysis 5 years after DES implantation. In-stent NA was observed in 16% of lesions with the majority of plaques being fibroatheromas (11.4%) followed by fibrocalcific plaques (5.7%). A total of 704 non-TL segments were serially evaluated by QCA. Between baseline and 5-year follow-up, the reduction in MLD was significantly more pronounced in patients with NA (-0.25 mm, 95% CI -0.36 to -0.17 mm) when compared with patients without NA (-0.13 mm, 95% CI -0.17 to -0.10 mm, P = 0.002). Similarly, non-TL revascularization was more frequent in patients with NA (78.6%) when compared with patients without NA (44.6%, P = 0.028) throughout 5 years. CONCLUSIONS In-stent NA is more common among patients with angiographic and clinical evidence of native atherosclerosis progression suggesting similar pathophysiological mechanisms.SIRTAX trial is registered at http://www.clinicaltrials.gov/ct2/show/NCT00617084.

Dynamics of Traditional Ecological Knowledge, Local Use and Conservation of Native Trees and Shrubs in the Bolivian Andes

Native trees and shrubs are essential components of rural landscapes in the semi-arid inner-Andean valleys of Bolivia. They can be found as hedges and bushes in various agroecosystems such as terrace walls, slopes, field boundaries and fallow land. Their distribution and floristic composition are the result of dynamic spatial and temporal interactions between local farmers and the environment. Local uses of natural resources and biodiversity reflect the constantly evolving Andean culture, which can be generally characterised as an intertwining of the human, natural, and spiritual worlds. The aim of the present ethnobotanical study was to analyse the dynamics of traditional ecological knowledge, to ascertain local farmers’ perceptions and uses of native woody species in Andean communities and to associate the results with local conservation activities for the trees and shrubs concerned. Our case study was carried out within two communities of the Tunari National Park (Dept. Cochabamba) in Bolivia. For data collection, research methods from social science (semi-structured interviews, participative observation, participatory mapping) as well as vegetation surveys were combined. Local actors included women and men of all ages as well as families from different social categories and altitudinal levels of permanent residence. Our study indicates that, due to a multitude of socio-economic pressures (e.g. migration of young people) as well as changes in use of biodiversity (e.g. replacement of native by exotic introduced species), the traditional ecological knowledge base of native trees and shrubs and their respective uses has become diminished over time. In many cases it has led to a decline in people’s awareness of native species and as a consequence their practical, emotional and spiritual relationships with them have been lost. However, results also show that applied traditional ecological knowledge has led to local conservation strategies, which have succeeded in protecting those tree and shrub species which are most widely regarded for their multifunctional, constant and exclusive uses (e.g. Schinus molle, Prosopis laevigata, Baccharis dracunculifolia). The presentation will discuss the question if and how applied traditional ecological knowledge positively contributes to local initiatives of sustainable use and conservation of biodiversity in rural areas.

Inversion, Obviation, and Animacy in Native Languages of the Americas: Elements for a Cross-linguistic Survey

Native languages of the Americas whose predicate and clause structure reflect nominal hierarchies show an interesting range of structural diversity not only with respect to morphological makeup of their predicates and arguments but also with respect to the factors governing obviation status. The present article maps part of such diversity. The sample surveyed here includes languages with some sort of nonlocal (third person acting on third person) direction-marking system.

Visualization of cell microtubules in their native state.

BACKGROUND INFORMATION Over the past decades, cryo-electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy. RESULTS We used cryo-electron microscopy and cryo-electron tomography of vitreous sections to investigate the ultrastructure of microtubules in their cellular context. Vitreous sections were obtained from organotypic slices of rat hippocampus and from Chinese-hamster ovary cells in culture. Microtubules revealed their protofilament ultrastructure, polarity and, in the most favourable cases, molecular details comparable with those visualized in three-dimensional reconstructions of microtubules reassembled in vitro from purified tubulin. The resolution of the tomograms was estimated to be approx. 4 nm, which enabled the detection of luminal particles of approx. 6 nm in diameter inside microtubules. CONCLUSIONS The present study provides a first step towards a description of microtubules, in addition to other macromolecular assemblies, in an unperturbed cellular context at the molecular level. As the resolution appears to be similar to that obtainable with plunge-frozen samples, it should allow for the in vivo identification of larger macromolecular assemblies in vitreous sections of whole cells and tissues.

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.

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.

Towards harmonised procedures in wildlife epidemiological investigations: A serosurvey of infection with Mycobacterium bovis and closely related agents in wild boar (Sus scrofa) in Switzerland

Bovine tuberculosis (bTB) is a (re-)emerging disease in European countries, including Switzerland. This study assesses the seroprevalence of infection with Mycobacterium bovis and closely related agents in wild boar (Sus scrofa) in Switzerland, because wild boar are potential maintenance hosts of these pathogens. The study employs harmonised laboratory methods to facilitate comparison with the situation in other countries. Eighteen out of 743 blood samples tested seropositive (2.4%, CI: 1.5-3.9%) by ELISA, and the results for 61 animals previously assessed using culture and PCR indicated that this serological test was not 100% specific for M. bovis, cross-reacting with M. microti. Nevertheless, serology appears to be an appropriate test methodology in the harmonisation of wild boar testing throughout Europe. In accordance with previous findings, the low seroprevalence found in wild boar suggests wildlife is an unlikely source of the M. bovis infections recently detected in cattle in Switzerland. This finding contrasts with the epidemiological situation pertaining in southern Spain.

Joining the dots - understanding the complex interplay between the values we place on wildlife, biodiversity conservation, human and animal health: A review.

The value of wildlife has long been ignored or under-rated. However, growing concerns about biodiversity loss and emerging diseases of wildlife origin have enhanced debates about the importance of wildlife. Wildlife-related diseases are viewed through these debates as a potential threat to wildlife conservation and domestic animal and human health. This article provides an overview of the values we place on wildlife (positive: socio-cultural, nutritional, economic, ecological; and negative: damages, health issues) and of the significance of diseases for biodiversity conservation. It shows that the values of wildlife, the emergence of wildlife diseases and biodiversity conservation are closely linked. The article also illustrates why investigations into wildlife diseases are now recognized as an integral part of global health issues. The modern One Health concept requires multi-disciplinary research groups including veterinarians, human physicians, ecologists and other scientists collaborating towards a common goal: prevention of disease emergence and preservation of ecosystems, both of which are essential to protect human life and well-being.

National competence center for wildlife diseases in Switzerland: Mandate, development and current strategies.

The need for wildlife health surveillance has become increasingly recognized. However, comprehensive programs which cover a wide spectrum of species, pathogens and geographic areas are still lacking in most European countries and practical examples of systems in place remain scarce. This article provides an overview of the organization of wildlife health surveillance in Switzerland, with a focus on the development, current strategies and the activities of the national program carried out by the Centre for Fish and Wildlife Health (FIWI), University of Bern. This documentation may stimulate on-going discussions on the design and development of national wildlife health surveillance programs in other countries. Investigations into wildlife health in Switzerland date back to the 1950s. The FIWI acts as a national competence center for wildlife diseases on mandate of the Swiss federal authorities. The mandate includes four main activities: disease diagnostics, research, consulting and teaching. In line with this, the FIWI has made continuous efforts to strengthen a national network of field partners and implemented strategies to facilitate long-term and metastudies.

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.