Influence of salt on the self-assembly of two model amyloid heptapeptides

We study the effects of NaCl on the self-assembly of AAKLVFF and beta A beta AKLVFF in solution. Both AAKLVFF and beta A beta AKLVFF self-assemble into twisted fibers in aqueous solution. The addition of NaCl to aqueous solutions of AAKLVFF produces large crystal-like nanotapes which eventually precipitate. In contrast, highly twisted fibrils were observed for beta A beta AKLVFF solutions at low salt concentration, while a coexistence of highly twisted fibers and nanotubes was observed for beta A beta AKLVFF at high salt concentration. The self-assembled structures observed for beta A beta AKLVFF in NaCl solutions were ascribed to the progressive screening of the beta A beta AKLVFF surface charge caused by the addition of salt.

Self-assembly of a designed amyloid peptide containing the functional thienylalanine unit

The self-assembly of a peptide based on a sequence from the amyloid beta peptide but incorporating the non-natural amino acid beta-2-thienylalanine (2-Thi) has been investigated in aqueous and methanol solutions. The peptide AAKLVFF was used as a design motif, replacing the phenylalanine residues (F) with 2-Thi units to yield (2-Thi)(2-Thi)VLKAA. The 2-Thi residues are expected to confer interesting electronic properties due to charge delocalization and pi-stacking. The peptide is shown to form beta-sheet-rich amyloid fibrils with a twisted morphology, in both water and methanol solutions at sufficiently high concentration. The formation of a self-assembling hydrogel is observed at high concentration. Detailed molecular modeling using molecular dynamics methods was performed using NOE constraints provided by 2D-NMR experiments. The conformational and charge properties of 2-Thi were modeled using quantum mechanical methods, and found to be similar to those previously reported for the beta-3-thienylalanine analogue. The molecular dynamics simulations reveal well-defined folded structures (turn-like) in dilute aqueous solution, driven by self-assembly of the hydrophobic aromatic units, with charged lysine groups exposed to water.

The role of seeds and airborne inoculum in the initiation of leaf blotch (Rhynchosporium secalis) epidemics in winter barley

Both airborne spores of Rhynchosporium secalis and seed infection have been implied as major sources of primary inoculum for barley leaf blotch (scald) epidemics in fields without previous history of barley cropping. However, little is known about their relative importance in the onset of disease. Results from both quantitative real-time PCR and visual assessments indicated that seed infection was the main source of inoculum in the field trial conducted in this study. Glasshouse studies established that the pathogen can be transmitted from infected seeds into roots, shoots and leaves without causing symptoms. Plants in the field trial remained symptomless for approximately four months before symptoms were observed in the crop. Covering the crop during part of the growing season was shown to prevent pathogen growth, despite the use of infected seed, indicating that changes in the physiological condition of the plant and/or environmental conditions may trigger disease development. However, once the disease appeared in the field it quickly became uniform throughout the cropping area. Only small amounts of R. secalis DNA were measured in 24 h spore-trap tape samples using PCR. Inoculum levels equivalent to spore concentrations between 30 and 60 spores per m3 of air were only detected on three occasions during the growing season. The temporal pattern and level of detection of R. secalis DNA in spore tape samples indicated that airborne inoculum was limited and most likely represented rain-splashed conidia rather than putative ascospores.

Self-assembly of a modified amyloid peptide fragment: pH-responsiveness and nematic phase formation

The self-assembly of peptide YYKLVFFC based on a fragment of the amyloid beta (A) peptide, A beta 16-20, KLVFF has been studied in aqueous solution. The peptide is designed with multiple functional residues to examine the interplay between aromatic interactions and charge on the self-assembly, as well as specific transformations such as the pH-induced phenol-phenolate transition of the tyrosine residue. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopies are used to investigate the conditions for beta-sheet self-assembly and the role of aromatic interactions in the CD spectrum as a function of pH and concentration. The formation of well-defined fibrils at pH 4.7 is confirmed by cryo-TEM (transmission electron microscope) and negative stain TEM. The morphology changes at higher pH, and aggregates of short twisted fibrils are observed at pH 11. Polarized optical microscopy shows birefringence at a low concentration (1 wt.-%) of YYKLVFFC in aqueous solution, and small-angle X-ray scattering was used to probe nematic phase formation in more detail. A pH-induced transition from nematic to isotropic phases is observed on increasing pH that appears to be correlated to a reduction in aggregate anisotropy upon increasing pH.

Influence of the solvent on the self-assembly of a modified amyloid beta peptide fragment. II. NMR and computer simulation investigation

The conformation of a model peptide AAKLVFF based on a fragment of the amyloid beta peptide A beta 16-20, KLVFF, is investigated in methanol and water via solution NMR experiments and Molecular dynamics computer simulations. In previous work, we have shown that AAKLVFF forms peptide nanotubes in methanol and twisted fibrils in water. Chemical shift measurements were used to investigate the solubility of the peptide as a function of concentration in methanol and water. This enabled the determination of critical aggregation concentrations, The Solubility was lower in water. In dilute solution, diffusion coefficients revealed the presence of intermediate aggregates in concentrated solution, coexisting with NMR-silent larger aggregates, presumed to be beta-sheets. In water, diffusion coefficients did not change appreciably with concentration, indicating the presence mainly of monomers, coexisting with larger aggregates in more concentrated solution. Concentration-dependent chemical shift measurements indicated a folded conformation for the monomers/intermediate aggregates in dilute methanol, with unfolding at higher concentration. In water, an antiparallel arrangement of strands was indicated by certain ROESY peak correlations. The temperature-dependent solubility of AAKLVFF in methanol was well described by a van't Hoff analysis, providing a solubilization enthalpy and entropy. This pointed to the importance of solvophobic interactions in the self-assembly process. Molecular dynamics Simulations constrained by NOE values from NMR suggested disordered reverse turn structures for the monomer, with an antiparallel twisted conformation for dimers. To model the beta-sheet structures formed at higher concentration, possible model arrangements of strands into beta-sheets with parallel and antiparallel configurations and different stacking sequences were used as the basis for MD simulations; two particular arrangements of antiparallel beta-sheets were found to be stable, one being linear and twisted and the other twisted in two directions. These structures Were used to simulate Circular dichroism spectra. The roles of aromatic stacking interactions and charge transfer effects were also examined. Simulated spectra were found to be similar to those observed experimentally.(in water or methanol) which show a maximum at 215 or 218 nm due to pi-pi* interactions, when allowance is made for a 15-18 nm red-shift that may be due to light scattering effects.

Fibrillar superstructure from extended nanotapes formed by a collagen-stimulating peptide

The nanostructure of a peptide amphiphile in commercial use in anti-wrinkle creams is investigated. The peptide contains a matrikine, collagen-stimulating, pentapeptide sequence. Selfassembly into giant nanotapes is observed and the internal structure was found to comprise bilayers parallel to the flat tape surfaces.

The climatology of the middle atmosphere in a vertically extended version of the Met Office's climate model. Part I: mean state

The climatology of a stratosphere-resolving version of the Met Office’s climate model is studied and validated against ECMWF reanalysis data. Ensemble integrations are carried out at two different horizontal resolutions. Along with a realistic climatology and annual cycle in zonal mean zonal wind and temperature, several physical effects are noted in the model. The time of final warming of the winter polar vortex is found to descend monotonically in the Southern Hemisphere, as would be expected for purely radiative forcing. In the Northern Hemisphere, however, the time of final warming is driven largely by dynamical effects in the lower stratosphere and radiative effects in the upper stratosphere, leading to the earliest transition to westward winds being seen in the midstratosphere. A realistic annual cycle in stratospheric water vapor concentrations—the tropical “tape recorder”—is captured. Tropical variability in the zonal mean zonal wind is found to be in better agreement with the reanalysis for the model run at higher horizontal resolution because the simulated quasi-biennial oscillation has a more realistic amplitude. Unexpectedly, variability in the extratropics becomes less realistic under increased resolution because of reduced resolved wave drag and increased orographic gravity wave drag. Overall, the differences in climatology between the simulations at high and moderate horizontal resolution are found to be small.

Molecular structures and electronic transitions of 3,6-Diphenyl-1,2-dithiin and its radical cation: a spectroelectrochemical and DFT study

One-electron oxidation of 3,6-diphenyl-1,2-dithiin yields the corresponding radical cation. The product is stable at low temperatures and can be distinguished by a triplet EPR signal. Cyclic voltammetric, UV-vis spectroelectrochemical, and DFT studies were performed to elucidate its molecular structure and electronic properties. Time-dependent DFT calculations reproduce appreciably well the UV-vis spectral changes observed during the oxidation. The results reveal a moderately twisted structure of the 1,2-dithiin heterocycle in the radical cation.

Influence of end-capping on the self-assembly of model amyloid peptide fragments

The influence of charge and aromatic stacking interactions on the self-assembly of a series of four model amyloid peptides has been examined. The four model peptides are based on the KLVFF motif from the amyloid Beta peptide, ABeta(16-20) extended at the N terminus with two Beta-alanine residues. We have studied NH2-BetaABetaAKLVFF-COOH (FF), NH2-BetaABetaAKLVFCOOH (F), CH3CONH-BetaABetaAKLVFF-CONH2 (CapF), and CH3CONH-BetaABetaAKLVFFCONH2 (CapFF). The former two are uncapped (net charge plus 2) and differ by one hydrophobic phenylalanine residue; the latter two are the analogous capped peptides (net charge plus 1). The self-assembly characteristics of these peptides are remarkably different and strongly dependent on concentration. NMR shows a shift from carboxylate to carboxylic acid forms upon increasing concentration. Saturation transfer measurements of solvent molecules indicate selective involvement of phenylalanine residues in driving the self-assembly process of CapFF due presumably to the effect of aromatic stacking interactions. FTIR spectroscopy reveals beta-sheet features for the two peptides containing two phenylalanine residues but not the single phenylalanine residue, pointing again to the driving force for self-assembly. Circular dichroism (CD) in dilute solution reveals the polyproline II conformation, except for F which is disordered. We discuss the relationship of this observation to the significant pH shift observed for this peptide when compared the calculated value. Atomic force microscopy and cryogenic-TEM reveals the formation of twisted fibrils for CapFF, as previously also observed for FF. The influence of salt on the self-assembly of the model beta-sheet forming capped peptide CapFF was investigated by FTIR. Cryo-TEM reveals that the extent of twisting decreases with increased salt concentration, leading to the formation of flat ribbon structures. These results highlight the important role of aggregation-induced pKa shifts in the self-assembly of model beta-sheet peptides.

Tuning the self-assembly of the bioactive dipeptide L-carnosine by incorporation of a bulky aromatic substituent

The dipeptide L-carnosine has a number of important biological properties. Here, we explore the effect of attachment of a bulky hydrophobic aromatic unit, Fmoc [N-(fluorenyl-9-methoxycarbonyl)] on the self-assembly of Fmoc-L-carnosine, i.e., Fmoc-Beta-alanine-histidine (Fmoc-BetaAH). It is shown that Fmoc-BetaAH forms well-defined amyloid fibril containing Beta sheets above a critical aggregation concentration, which is determined from pyrene and ThT fluorescence experiments. Twisted fibrils were imaged by cryogenic transmission electron microscopy. The zinc-binding properties of Fmoc-BetaAH were investigated by FTIR and Raman spectroscopy since the formation of metal ion complexes with the histidine residue in carnosine is well-known, and important to its biological roles. Observed changes in the spectra may reflect differences in the packing of the Fmoc-dipeptides due to electrostatic interactions. Cryo-TEM shows that this leads to changes in the fibril morphology. Hydrogelation is also induced by addition of an appropriate concentration of zinc ions. Our work shows that the Fmoc motif can be employed to drive the self-assembly of carnosine into amyloid fibrils.

Modulating self-assembly of a nanotape-forming peptide amphiphile with an oppositely charged surfactant

A peptide amphiphile (PA) C16-KTTKS, containing a pentapeptide headgroup based on a sequence from procollagen I attached to a hexadecyl lipid chain, self-assembles into extended nanotapes in aqueous solution. The tapes are based on bilayer structures, with a 5.2 nm spacing. Here, we investigate the effect of addition of the oppositely charged anionic surfactant sodium dodecyl sulfate (SDS) via AFM, electron microscopic methods, small-angle X-ray scattering and X-ray diffraction among other methods. We show that addition of SDS leads to a transition from tapes to fibrils, via intermediate states that include twisted ribbons. Addition of SDS is also shown to enhance the development of remarkable lateral ‘‘stripes’’ on the nanostructures, which have a 4 nm periodicity. This is ascribed to counterion condensation. The transition in the nanostructure leads to changes in macroscopic properties, in particular a transition from sol to gel is noted on increasing SDS (with a further reentrant transition to sol on further increase of SDS concentration). Formation of a gel may be useful in applications of this PA in skincare applications and we show that this can be controlled via development of a network of fine stranded fibrils.

Consumer perceptions of poultry meat: a qualitative analysis

Consumer studies of meat have tended to use quantitative methodologies providing a wealth of statistically malleable information, but little in-depth insight into consumer perceptions of meat. The aim of the present study was therefore, to understand factors perceived important in the selection of chicken meat, using qualitative methodology. Focus group discussions were tape recorded, transcribed verbatim and content analysed for major themes. Themes arising implied that “appearance” and “convenience” were the most important determinants of choice of chicken meat and these factors appeared to be associated with perceptions of freshness, healthiness, product versatility and concepts of value. A descriptive model has been developed to illustrate the interrelationship between factors affecting chicken meat choice. This study indicates that those involved in the production and retailing of chicken products should concentrate upon product appearance and convenience as market drivers for their products.

Structure property relationships in electrically conducting copolymers formed from pyrrole and N-methyl pyrrole

A series of copolymers containing differing proportions of pyrrole and N-methyl pyrrole were prepared electrochemically at various temperatures using acetonitrile as the solvent. The resultant electrical conductivity decreases universally with increasing fraction of N-methyl pyrrole. Films prepared with p-toluene sulfonate as the dopant show a marked variation in structural anisotropy as revealed by X-ray scattering with apparent copolymer content. There is a clear trend between the variation in electrical conductivity and this structural anisotropy. Different patterns of behaviour are observed for films prepared using perchlorate as the dopant and this is attributed to the role of the dopant and final structure in determining the relative reactivities of the pyrrole and N-methyl pyrrole monomers. These observations support the concept that the introduction of methyl substituents into a polypyrrole chain results in a twisted chain conformation. The structure and properties of the resultant copolymer films are particularly sensitive to the preparation conditions.

Fibrillisation of ring-closed amyloid peptides

Ring-closing olefin metathesis reactions are used to create intramolecularly ring closed peptides or inter-molecularly ring-closed peptide dimers based on a designed amyloid peptide sequence. The uncrosslinked peptide self-assembles into high aspect ratio nanotubes, however ring-closing leads to the formation of fibrillar and twisted/helical ribbon structures.

Standard cost model: three different paths and their common problems

Red tape is not desirable as it impedes business growth. Relief from the administrative burdens that businesses face due to legislation can benefit the whole economy, especially at times of recession. However, recent governmental initiatives aimed at reducing administrative burdens have encountered some success, but also failures. This article compares three national initiatives - in the Netherlands, UK and Italy - aimed at cutting red tape by using the Standard Cost Model. Findings highlight the factors affecting the outcomes of measurement and reduction plans and ways to improve the Standard Cost Model methodology.