Oxidation is an important method for the synthesis of chemical intermediates in the manufacture of high-tonnage commodities, high-value fine chemicals, agrochemicals and pharmaceuticals: but oxidations are often inefficient. The introduction of catalytic systems using oxygen from air is preferred for 'green' processing. Gold catalysis is now showing potential in selective redox processes, particularly for alcohol oxidation and the direct synthesis of hydrogen peroxide. However, a major challenge that persists is the synthesis of an epoxide by the direct electrophilic addition of oxygen to an alkene. Although ethene is epoxidized efficiently using molecular oxygen with silver catalysts in a large-scale industrial process, this is unique because higher alkenes can only be effectively epoxidized using hydrogen peroxide, hydroperoxides or stoichiometric oxygen donors. Here we show that nanocrystalline gold catalysts can provide tunable active catalysts for the oxidation of alkenes using air, with exceptionally high selectivity to partial oxidation products ( approximately 98%) and significant conversions. Our finding significantly extends the discovery by Haruta that nanocrystalline gold can epoxidize alkenes when hydrogen is used to activate the molecular oxygen; in our case, no sacrificial reductant is needed. We anticipate that our finding will initiate attempts to understand more fully the mechanism of oxygen activation at gold surfaces, which might lead to commercial exploitation of the high redox activity of gold nanocrystals.

SummaryIn order to investigate the possibility that qualitative type 2 defects in von Willebrand factor (VWF) occurred in patients previously diagnosed with quantitative type 1 von Willebrand disease (VWD), the phenotypes and genotypes were reanalysed in 30 patients who exhibited discrepant VWF activity/VWF:Ag ratios of less than 0.7. The capacity of VWF to bind to glycoprotein Ib (GpIb) was reassessed using the ristocetin co-factor activity (VWF:RiCo) assay compared to an in-house and a commercial ELISA assay (based on a mAb directed against the GpIb binding site on VWF). This was supplemented by multimeric analysis and the amplification and sequencing of a 936 bp fragment of exon 28 of the VWF gene with the aim of identifying mutations in the A1 domain. On reappraisal, using the VWF:RiCo assay all patients demonstrated a disproportionately reduced VWF:RiCo/VWF:Ag ratio, indicative of a qualitative defect, while abnormal ratios were detected in only seven kindreds using the in-house ELISA assay and in only one kindred with the commercial ELISA assay. Eight single amino acid substitutions were found in nine kindreds, four of which were novel candidate VWF mutations and four previously described in association with type 2 VWD. In agreement with the phenotype, the novel VWF mutations were located in the VWF-A1 crystal structure at positions that corresponded to potential type 2M defects. This study underlines the difficulties of correct diagnosis of the subtype of VWD and emphasises the importance of using sensitive phenotypic assays, the relevance of the VWF:RiCo/ VWF:Ag ratio, multimeric analysis and molecular modelling analysis.

Summary. This clinical retrospective study investigated the dif®culties in diagnosing type 1 von Willebrand disease (VWD). A total of 246 patients previously diagnosed with type 1 VWD were reclassi®ed into`possible' type 1 VWD (patients with low levels of VWF adjusted for the blood group and either a signi®cant bleeding history or family history) and`de®nite' type 1 VWD, requiring low levels of von Willebrand factor (VWF), a bleeding history and inheritance. On reclassi®cation, only 144/246 (59%) patients had low VWF levels adjusted for blood group, 88/246 (36%) patients met all the criteria for`de®nite' type 1 VWD and 51/246 (21%) patients were`possible' type 1 VWD. A signi®cant proportion of patients, 102/246 (42%), remained an indeterminate group with blood type O, VWF levels between 35 and 50 U/dl and personal and/or family bleeding history. This subgroup might require reclassi®cation as`not VWD'.However, a similar bleeding tendency was found in two matched groups of patients of blood groups O and non-O and VWF levels between 35 and 50 U/dl. These results suggest that the use of ABO adjusted ranges for VWF levels might not be essential for diagnosis, because bleeding symptoms may depend on the VWF level regardless of the ABO type. Of the diagnostic criteria, the bleeding history was of prime importance in the clinical decision to diagnose and treat type 1 VWD. These observations could help in the reconsideration of how the criteria for diagnosing type 1 VWD could be adjusted in order to maximize their clinical relevance.

Abstractvon Willebrand factor (vWF) is a large multimeric, multidomain glycoprotein found in platelets, endothelial cells and plasma. The A1, A2, and A3 domains in vWF mediate binding to glycoprotein Ib, ristocetin, botrocetin, collagen, sulphatides, and heparin and provide a protease cleavage site. Mutations causing types 2B, 2M, and 2A von Willebrand's disease (vWD) are located in the A1 and A2 domains. Homology modeling was performed to provide a molecular interpretation of vWF function and mutation sites. This was based on our previous alignment of 75 vWF-A sequences, the doubly wound α/β fold seen in recent vWF-A crystal structures from complement receptor type 3 and lymphocyte function-associated antigen-1, and our new alignment of 28 vWF A1 and A2 sequences from different species. The active site in doubly-wound α/β folds forms a crevice that is located at the switch point between the two halves of the central β-sheet, and usually contains two metal-binding Asp residues in the vWF-A superfamily. Although one of these Asp residues is absent from the A1, A2, and A3 domains, this crevice is shown to correspond to the ristocetin binding site in the A1 domain and the protease cleavage site in the A2 domain. The residues R571-K572-R578-R579-K585 are found to be conserved in 28 A1 sequences and are predicted to constitute the heparin binding site in the A1 domain. Inspection of the type 2M vWD mutation sites that are involved in downregulation of glycoprotein Ib (GpIb) binding to vWF shows that these are spatially clustered at the carboxyl-edge of the β-sheet and above it in the A1 domain and may directly perturb GpIb binding. In contrast, the type 2B vWD mutation sites that are involved in upregulation of GpIb binding to vWF are spatially clustered at the amino edge of this β-sheet and below it and are located on the opposite side of the A1 domain from the type 2M mutation sites. The type 2B mutations are located between the heparin and GpIb binding sites. Because heparin binding inhibits the interaction with GpIb, this provides an explanation of vWF upregulation. The type 2A vWD mutation sites in the A2 domain correspond to buried residues that are otherwise 100% conserved across all 28 species, and are likely to be important for the correct folding of the A2 domain and its physiologically important protease site.

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Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime.

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