Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation

The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils. In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.</p

The Stokes-Einstein-Sutherland equation at the nanoscale revisited

The Stokes-Einstein-Sutherland (SES) equation is at the foundation of statistical physics, relating a particle's diffusion coefficient and size with the fluid viscosity, temperature and the boundary condition for the particle-solvent interface. It is assumed that it relies on the separation of scales between the particle and the solvent, hence it is expected to break down for diffusive transport on the molecular scale. However, a number of experimental studies showed a remarkable small, if any, violation of this equation down to the size of a nm, where there is no scale separation. To resolve this puzzle we combine analytical ultracentrifugation experiments and molecular dynamics simulations to study the transport of buckminsterfullerene C$_{60}$ suspended in toluene at infinite dilution. We show that this system clearly violates the conditions of slow momentum relaxation. Yet, through a linear response to a constant force, we show both in experiments and in simulations that the SES equation can be recovered in the long time limit with no more than 4% uncertainty. This nonetheless requires partial slip on the particle interface, extracted consistently from all the data. Our results, thus, resolve a long-standing discussion on the validity and limits of the SES equation at the molecular scale

Examination of effects of GSK3β phosphorylation, β-catenin phosphorylation, and β-catenin degradation on kinetics of Wnt signaling pathway using computational method

<p>Abstract</p> <p>Background</p> <p>Recent experiments have explored effects of activities of kinases other than the well-studied GSK3β, in wnt pathway signaling, particularly at the level of β-catenin. It has also been found that the kinase PKA attenuates β-catenin degradation. However, the effects of these kinases on the level and degradation of β-catenin and the resulting downstream transcription activity remain to be clarified. Furthermore, the effect of GSK3β phosphorylation on the β-catenin level has not been examined computationally. In the present study, the effects of phosphorylation of GSK3β and of phosphorylations and degradation of β-catenin on the kinetics of the wnt signaling pathway were examined computationally.</p> <p>Methods</p> <p>The well-known computational Lee-Heinrich kinetic model of the wnt pathway was modified to include these effects. The rate laws of reactions in the modified model were solved numerically to examine these effects on β-catenin level.</p> <p>Results</p> <p>The computations showed that the β-catenin level is almost linearly proportional to the phosphorylation activity of GSK3β. The dependence of β-catenin level on the phosphorylation and degradation of free β-catenin and downstream TCF activity can be analyzed with an approximate, simple function of kinetic parameters for added reaction steps associated with effects examined, rationalizing the experimental results.</p> <p>Conclusion</p> <p>The phosphorylations of β-catenin by kinases other than GSK3β involve free unphorphorylated β-catenin rather than GSK3β-phosphorylated β-catenin*. In order to account for the observed enhancement of TCF activity, the β-catenin dephosphorylation step is essential, and the kinetic parameters of β-catenin phosphorylation and degradation need to meet a condition described in the main text. These findings should be useful for future experiments.</p

Cell entry of a host targeting protein of oomycetes requires gp96

This work is supported by the [European Community’s] Seventh Framework Programme [FP7/2007–2013] under grant agreement no. [238550] (L.L., J.D.-U., C.J.S., P.v.W.); BBSRC [BBE007120/1, BB/J018333/1 and BB/G012075/1] (F.T., I.d.B., C.J.S., S.W., P.v.W.); Newton Global Partnership Award [BB/N005058/1] (F.T., P.v.W.), the University of Aberdeen (A.D.T., T.R., C.J.S., P.v.W.) and Deutsche Forschungsgemeinschaft [CRC1093] (P.B., T.S.). We would like to acknowledge the Ministry of Higher Education Malaysia for funding INA. We would like to thank Brian Haas for his bioinformatics support. We would like to acknowledge Neil Gow and Johannes van den Boom for critical reading of the manuscript. We would like to acknowledge Svetlana Rezinciuc for technical help with pH-studies.Peer reviewedPublisher PD

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

Background: Pythium ultimum (P. ultimum) is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. Results: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions although surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report in a genome outside the metazoans. Conclusions: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae

Dickkopf1 - A New Player in Modelling the Wnt Pathway

The Wnt signaling pathway transducing the stabilization of β-catenin is essential for metazoan embryo development and is misregulated in many diseases such as cancers. In recent years models have been proposed for the Wnt signaling pathway during the segmentation process in developing embryos. Many of these include negative feedback loops where Axin2 plays a key role. However, Axin2 null mice show no segmentation phenotype. We therefore propose a new model where the negative feedback involves Dkk1 rather than Axin2. We show that this model can exhibit the same type of oscillations as the previous models with Axin2 and as observed in experiments. We show that a spatial Wnt gradient can consistently convert this temporal periodicity into the spatial periodicity of somites, provided the oscillations in new cells arising in the presomitic mesoderm are synchronized with the oscillations of older cells. We further investigate the hypothesis that a change in the Wnt level in the tail bud during the later stages of somitogenesis can lengthen the time period of the oscillations and hence the size and separation of the later somites