Nanoscopic and photonic ultrastructural characterization of two distinct insulin amyloid States

Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO). This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM), we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM) and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states.     

Extraction Properties of 6,6′‐Bis‐(5,6‐dipentyl‐[1,2,4]triazin‐3‐yl)‐[2,2′]bipyridinyl (C5‐BTBP)

Abstract

The extraction of americium(III) and europium(III) into a variety of organic diluents by 6,6′‐bis‐(5,6,‐dipentyl‐[1,2,4]triazin‐3‐yl)‐[2,2′]bipyridinyl (C5‐BTBP) has been investigated. In addition to determining the stoichiometry for the extraction, the dependence of extraction on contact time and temperature was also studied. The resistance of the ligand to gamma irradiation and the possibility to recycle the organic phase after stripping were tested to determine how the molecule would perform in a radiochemical process. Different organic diluents gave different extraction results, ranging from no extraction to distribution ratios of over 1000 for americium(III). In 1,1,2,2‐tetrachloroethane, the extraction and separation of americium from europium and the extraction kinetics were good; a separation factor above 60 was obtained at equilibrium, ～5 min contact time. The extraction capabilities are adequate for C5‐BTBP to be used in a process for separating trivalent actinides from lanthanides. However, C5‐BTBP is susceptible to radiolysis (americium extraction decreases ～80% after a dose of 17 kGy) and may not be the best choice in the processing of spent nuclear fuel. Nonetheless it is a useful starting point for further development of this type of molecule. It could also prove useful for analytical scale separations for which radiolytic instability is less important.     

Flavour Instability of Pale Lager Beers: Determination of Analytical Markers in Relation to Sensory Ageing

Flavour changes of six Belgian pale lager beers were studied in order to estimate the importance of different parameters and reactions in relation to the ageing process. An attempt was made to link analytical data with sensory evaluation using multivariate statistical analysis. Partial least squares regression techniques (PLSR) were employed on the analytical and sensory data. As apparent from the PLSR model, significant indicators of lager beer ageing are aldehyde markers (especially total aldehydes, furfural, hexanal, 2‐methylpropanal, 2‐methylbutanal, and 3‐methylbutanal), cold and permanent haze, and beer colour. Conversely, compounds or parameters that load negatively in the PLSR model for beer ageing are trans‐isohumulones, cis‐isohumulones, total bitterness, the T/C‐ratio, polyphenolic markers (especially proanthocyanidins), the flavanoid content, and, to a lesser extent, the TB‐index and reducing power (TRAP). The integrated analytical‐sensorial methodology is proposed as a useful tool for evaluation of the flavour instability of pale lager beers.     

Development of a transformation and selection system for the glycolipid-producing yeast Candida bombicola

Sophorolipids are surface-active compounds synthesized by the non-pathogenic yeast Candida bombicola. Over recent decades much effort has been spent to optimize culture conditions in order to improve the yield and production process. As far as we know, however, hardly any attention has been given to the genetics of the producing yeast strain itself and there are no published results available on the genetic engineering of C. bombicola. Nevertheless, this can be a useful tool for the study of the sophorolipid synthesis pathway and open up perspectives for improved production. A first step is the development of a suitable transformation and selection method. This article describes the creation and selection of an uracil auxotrophic C. bombicola mutant, which can be transformed back to prototrophy with the species' own orotidine 5'-phosphate decarboxylase or URA3 gene. Successful transformation was confirmed by a PCR-based method discriminating between the wild-type and mutated URA3 gene.     

Affinity and Specificity for Binding to Glycosaminoglycans Can Be Tuned by Adapting Peptide Length and Sequence

Although glycosaminoglycan (GAG)–protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.     

Corpus-based vocabulary lists for language learners for nine languages

We present the KELLY project and its work on developing monolingual and bilingual word lists for language learning, using corpus methods, for nine languages and thirty-six language pairs. We describe the method and discuss the many challenges encountered. We have loaded the data into an online database to make it accessible for anyone to explore and we present our own first explorations of it. The focus of the paper is thus twofold, covering pedagogical and methodological aspects of the lists’ construction, and linguistic aspects of the by-product of the project, the KELLY database.     

Fabrication of Functionally Graded and Aligned Porosity in Thin Ceramic Substrates With the Novel Freeze–Tape-Casting Process

Functionally graded and continuously aligned pore structures have been fabricated by a modified tape-casting process for use as solid oxide fuel cell electrodes, catalysts, sensors, and filtration/separation devices. Pore gradients from <5 to 100 μm and aligned pore tubules have been directly fabricated in various ceramic materials with thin substrate sections approximately 500–1500 μm utilizing both low-toxicity aqueous-based slips and organic solvents. This process allows for the generation of pores without the use of thermally fugitive pore formers in a single processing step with no need for tape lamination. The incorporation of tape casting, unidirectional solidification, and the freeze-drying process results in uniformly acicular pores aligned with the direction of the moving carrier film. Processing and microstructure variability will be discussed as it pertains to the effects of solids loading, freezing temperatures, and solvent type. Applications for this ceramic processing technology will also be discussed.     

SALSA: QoS-aware load balancing for autonomous service brokering

The evolution towards “Software as a Service”, facilitated by various web service technologies, has led to applications composed of a number of service building blocks. These applications are dynamically composed by web service brokers, but rely critically on proper functioning of each of the composing subparts which is not entirely under control of the applications themselves. The problem at hand for the provider of the service is to guarantee non-functional requirements such as service access and performance to each customer. To this end, the service provider typically divides the load of incoming service requests across the available server infrastructure. In this paper we describe an adaptive load balancing strategy called SALSA (Simulated Annealing Load Spreading Algorithm), which is able to guarantee for different customer priorities, such as default and premium customers, that the services are handled in a given time and this without the need to adapt the servers executing the service logic themselves. It will be shown that by using SALSA, web service brokers are able to autonomously meet SLAs, without a priori over-dimensioning resources. This will be done by taking into account a real time view of the requests by measuring the Poisson arrival rates at that moment and selectively drop some requests from default customers. This way the web servers’ load is reduced in order to guarantee the service time for premium customers and provide best effort to default customers. We compared the results of SALSA with weighted round-robin (WRR), nowadays the most used load balancing strategy, and it was shown that the SALSA algorithm requires slightly more processing than WRR but is able to offer guarantees - contrary to WRR - by dynamically adapting its load balancing strategy.     

Unexpected analyte oxidation during desorption electrospray ionization-mass spectrometry

During the analysis of surface-spotted analytes using desorption electrospray ionization-mass spectrometry (DESI-MS), abundant ions are sometimes observed that appear to be the result of oxygen addition reactions. In this investigation, the effect of sample aging, the ambient lab environment, spray voltage, analyte surface concentration, and surface type on this oxidative modification of spotted analytes, exemplified by tamoxifen and reserpine, during analysis by DESI-MS was studied. Simple exposure of the samples to air and to ambient lighting increased the extent of oxidation. Increased spray voltage also led to increased analyte oxidation, possibly as a result of oxidative species formed electrochemically at the emitter electrode or in the gas phase by discharge processes. These oxidative species are carried by the spray and impinge on and react with the sampled analyte during desorption/ionization. The relative abundance of oxidized species was more significant for the analysis of deposited analyte having a relatively low surface concentration. Increasing the spray solvent flow rate and the addition of hydroquinone as a redox buffer to the spray solvent were found to decrease, but not entirely eliminate, analyte oxidation during analysis. The major parameters that both minimize and maximize analyte oxidation were identified, and DESI-MS operational recommendations to avoid these unwanted reactions are suggested.