Axial localization of luminophores by partial coherence interferometry

We propose a solution for increasing the axial resolution of confocal microscopes. In the experimental set-up described in this paper an interference phenomenon between two counterpropagating beams is used to determine the axial position of a luminophore. The optical path difference between the two waves, which is related to the position of the luminophore, is recovered thanks to a second interferometer by using partial coherence interferometry demodulation technique. The proposed solution can find applications in biology for localizing with nanometric resolution a small number of tagged species

Mechanical Strength of 17 134 Model Proteins and Cysteine Slipknots

A new theoretical survey of proteins' resistance to constant speed stretching is performed for a set of 17 134 proteins as described by a structure-based model. The proteins selected have no gaps in their structure determination and consist of no more than 250 amino acids. Our previous studies have dealt with 7510 proteins of no more than 150 amino acids. The proteins are ranked according to the strength of the resistance. Most of the predicted top-strength proteins have not yet been studied experimentally. Architectures and folds which are likely to yield large forces are identified. New types of potent force clamps are discovered. They involve disulphide bridges and, in particular, cysteine slipknots. An effective energy parameter of the model is estimated by comparing the theoretical data on characteristic forces to the corresponding experimental values combined with an extrapolation of the theoretical data to the experimental pulling speeds. These studies provide guidance for future experiments on single molecule manipulation and should lead to selection of proteins for applications. A new class of proteins, involving cystein slipknots, is identified as one that is expected to lead to the strongest force clamps known. This class is characterized through molecular dynamics simulations.Comment: 40 pages, 13 PostScript figure

Burden of paediatric Rotavirus Gastroenteritis (RVGE) and potential benefits of a universal Rotavirus vaccination programme with a pentavalent vaccine in Spain

<p>Abstract</p> <p>Background</p> <p>Rotavirus is the most common cause of gastroenteritis in young children worldwide. The aim of the study was to assess the health outcomes and the economic impact of a universal rotavirus vaccination programme with RotaTeq, the pentavalent rotavirus vaccine, versus no vaccination programme in Spain.</p> <p>Methods</p> <p>A birth cohort was followed up to the age of 5 using a cohort model. Epidemiological parameters were taken from the REVEAL study (a prospective epidemiological study conducted in Spain, 2004-2005) and from the literature. Direct and indirect costs were assessed from the national healthcare payer and societal perspectives by combining health care resource utilisation collected in REVEAL study and unit costs from official sources. RotaTeq per protocol efficacy data was taken from a large worldwide rotavirus clinical trial (70,000 children). Health outcomes included home care cases, General Practioner (GP)/Paediatrician, emergency department visits, hospitalisations and nosocomial infections.</p> <p>Results</p> <p>The model estimates that the introduction of a universal rotavirus vaccination programme with RotaTeq (90% coverage rate) would reduce the rotavirus gastroenteritis (RVGE) burden by 75% in Spain; 53,692 home care cases, 35,187 GP/Paediatrician visits, 34,287 emergency department visits, 10,987 hospitalisations and 2,053 nosocomial infections would be avoided. The introduction of RotaTeq would avoid about 76% of RVGE-related costs from both perspectives: €22 million from the national health system perspective and €38 million from the societal perspective.</p> <p>Conclusions</p> <p>A rotavirus vaccination programme with RotaTeq would reduce significantly the important medical and economic burden of RVGE in Spain.</p

Direct Observation of Single Amyloid-β(1-40) Oligomers on Live Cells: Binding and Growth at Physiological Concentrations

Understanding how amyloid-β peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric Aβ interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high Aβ concentrations required for most biochemical analyses, the metastable nature of Aβ aggregates, and the complex variety of Aβ species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small Aβ(1-40) oligomers bind to living neuroblastoma cells at physiological Aβ concentrations. Single particle fluorescence intensity measurements indicate that cell-bound Aβ species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound Aβ oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary Aβ species that interact with neurons at physiological concentrations

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM1 in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM1 complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes