Amyloid ß 42 fibril structure based on small-angle scattering.

Amyloid fibrils are associated with a number of neurodegenerative diseases, including fibrils of amyloid ß42 peptide (Aß42) in Alzheimer's disease. These fibrils are a source of toxicity to neuronal cells through surface-catalyzed generation of toxic oligomers. Detailed knowledge of the fibril structure may thus facilitate therapeutic development. We use small-angle scattering to provide information on the fibril cross-section dimension and shape for Aß42 fibrils prepared in aqueous phosphate buffer at pH = 7.4 and pH 8.0 under quiescent conditions at 37 °C from pure recombinant Aß42 peptide. Fitting the data using a continuum model reveals an elliptical cross-section and a peptide mass-per-unit length compatible with two filaments of two monomers, four monomers per plane. To provide a more detailed atomistic model, the data were fitted using as a starting state a high-resolution structure of the two-monomer arrangement in filaments from solid-state NMR (Protein Data Bank ID 5kk3). First, a twofold symmetric model including residues 11 to 42 of two monomers in the filament was optimized in terms of twist angle and local packing using Rosetta. A two-filament model was then built and optimized through fitting to the scattering data allowing the two N-termini in each filament to take different conformations, with the same conformation in each of the two filaments. This provides an atomistic model of the fibril with twofold rotation symmetry around the fibril axis. Intriguingly, no polydispersity as regards the number of filaments was observed in our system over separate samples, suggesting that the two-filament arrangement represents a free energy minimum for the Aß42 fibril.

X-Ray Scattering Reveals Two Mechanisms of Cellulose Microfibril Degradation by Filamentous Fungi.

Mushroom-forming fungi (Agaricomycetes) employ enzymatic and nonenzymatic cellulose degradation mechanisms, the latter presumably relying on Fenton-generated radicals. The effects of the two mechanisms on the cellulose microfibrils structure remain poorly understood. We examined cellulose degradation caused by litter decomposers and wood decomposers, including brown-rot and white-rot fungi and one fungus with uncertain wood decay type, by combining small- and wide-angle X-ray scattering. We also examined the effects of commercial enzymes and Fenton-generated radicals on cellulose using the same method. We detected two main degradation or modification mechanisms. The first characterized the mechanism used by most fungi and resembled enzymatic cellulose degradation, causing simultaneous microfibril thinning and decreased crystalline cellulose. The second mechanism was detected in one brown-rot fungus and one litter decomposer and was characterized by patchy amorphogenesis of crystalline cellulose without substantial thinning of the fibers. This pattern did not resemble the effect of Fenton-generated radicals, suggesting a more complex mechanism is involved in the destruction of cellulose crystallinity by fungi. Furthermore, our results showed a mismatch between decay classifications and cellulose degradation patterns and that even within litter decomposers two degradation mechanisms were found, suggesting higher functional diversity under current ecological classifications of fungi. IMPORTANCE Cellulose degradation by fungi plays a fundamental role in terrestrial carbon cycling, but the mechanisms by which fungi cope with the crystallinity of cellulose are not fully understood. We used X-ray scattering to analyze how fungi, a commercial enzyme mix, and a Fenton reaction-generated radical alter the crystalline structure of cellulose. Our data revealed two mechanisms involved in crystalline cellulose degradation by fungi: one that results in the thinning of the cellulose fibers, resembling the enzymatic degradation of cellulose, and one that involves amorphogenesis of crystalline cellulose by yet-unknown pathways, resulting in a patchy-like degradation pattern. These results pave the way to a deeper understanding of cellulose degradation and the development of novel ways to utilize crystalline cellulose.

α-Synuclein Interaction with Lipid Bilayer Discs.

α-Synuclein (aSyn) is a 140 residue long protein present in presynaptic termini of nerve cells. The protein is associated with Parkinson's disease, in which case it has been found to self-assemble into long amyloid fibrils forming intracellular inclusions that are also rich in lipids. Furthermore, its synaptic function is proposed to involve interaction with lipid membranes, and hence, it is of interest to understand aSyn-lipid membrane interactions in detail. In this paper we report on the interaction of aSyn with model membranes in the form of lipid bilayer discs. Using a combination of cryogenic transmission electron microscopy and small-angle neutron scattering, we show that circular discs undergo a significant shape transition after the adsorption of aSyn. When aSyn self-assembles into fibrils, aSyn molecules desorb from the bilayer discs, allowing them to recover to their original shape. Interestingly, the desorption process has an all-or-none character, resulting in a binary coexistence of circular bilayer discs with no adsorbed aSyn and deformed bilayer discs having a maximum amount of adsorbed protein. The observed coexistence is consistent with the recent finding of cooperative aSyn adsorption to anionic lipid bilayers.

Strong inhibition of peptide amyloid formation by a fatty acid.

The aggregation of peptides into amyloid fibrils is associated with several diseases, including Alzheimer's and Parkinson's disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic molecules, such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68-78 of the protein α-synuclein involved in Parkinson's disease. Based primarily on circular dichroism measurements, we found that even a very small amount of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the observed inhibitory effect on fibrillation is due to co-association of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that associate with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.

Adsorption of Anionic Dyes Using a Poly(styrene-block-4-vinylpyridine) Block Copolymer Organogel.

An organogel was prepared by chemically cross-linking a poly(styrene-block-4-vinylpyridine) diblock copolymer using dibromododecane in dimethylformamide. Analysis of the prominent structure peak in small-angle X-ray scattering along with the results of light scattering and rheological profile suggests the bridging of the spherical micelles to one another to form an interconnected network after gelation. The use of this organogel as a selective adsorbent for removing anionic dyes from individual aqueous dye solutions and in a mixture of cationic and anionic dye solutions has shown more than 90% removal of the anionic dyes within 2 h. The regeneration and reusability studies showed that even after 20 cycles, the adsorption property of the organogel holds extremely well still beyond 90%. These results are indicative of the potential use of poly(styrene-block-4-vinylpyridine) organogel for the anionic ions removal in wastewater treatment.

Two Dimensional Oblique Molecular Packing within a Model Peptide Ribbon Aggregate.

A10 K (A=alanine, K=lysine) model peptides self-assemble into ribbon-like ß-sheet aggregates. Here, we report an X-ray diffraction investigation on a flow-aligned dispersion of these self-assembly structures. The two-dimensional wide-angle X-ray scattering pattern suggests that peptide pack in a two-dimensional oblique lattice, essentially identical to the crystalline packing of polyalanine, An (for n>4). One side of the oblique unit cell, corresponding to the anti-parallel ß-sheet, is oriented along the ribbon's axis. Together with recently published small angle X-ray scattering data of the same system, this work thus yields a detailed description of the self-assembled ribbon aggregates, down to the molecular length scale. Notably, our results highlight the importance of the crystalline peptide packing within its self-assembly aggregates, which is often neglected.

Self-Assembly of Model Amphiphilic Peptides in Nonaqueous Solvents: Changing the Driving Force for Aggregation Does Not Change the Fibril Structure.

Within the homologous series of amphiphilic peptides AnK, both A8K and A10K self-assemble in water to form twisted ribbon fibrils with lengths around 100 nm. The structure of the fibrils can be described in terms of twisted ß-sheets extending in the direction of the fibrils, laminated to give a constant cross section of 4 nm by 8 nm. The finite width of the twisted ribbons can be reasonably explained within a simple thermodynamic model, considering a free energy penalty for the stretching of hydrogen bonds along the twisted ß-sheets and an interfacial free energy gain for the lamination of the hydrophobic ß-sheets. In this study, we characterize the self-assembly behavior of these peptides in nonaqueous solutions as a route to probe the role of hydrophobic interaction in fibril stabilization. Both peptides, in methanol and N,N-dimethylformamide, were found to form fibrillar aggregates with the same ß-sheet structure as in water but with slightly smaller cross-sectional sizes. However, the gel-like texture, the slow relaxation in dynamic light scattering experiments, and a correlation peak in the small-angle X-ray scattering pattern highlighted enhanced interfibril interactions in the nonaqueous solvents in the same concentration range. This could be ascribed to a higher effective volume of the aggregates because of enhanced fibril growth and length, as suggested by light scattering and cryogenic transmission electron microscopy analyses. These effects can be discussed considering how the solvent properties affect the different energetic contributions (hydrophobic, electrostatic, and hydrogen bonding) to fibril formation. In the analyzed case, the decreased hydrogen bonding propensity of the nonaqueous solvents makes the hydrogen bond formation along the fibril a key driving force for peptide assembly, whereas it represents a nonrelevant contribution in water.

Arrested dynamics in a model peptide hydrogel system.

We report here on a peptide hydrogel system, which in contrast to most other such systems, is made up of relatively short fibrillar aggregates, discussing resemblance with colloidal rods. The synthetic model peptides A8K and A10K, where A denotes alanine and K lysine, self-assemble in aqueous solutions into ribbon-like aggregates having an average length 〈L〉 on the order of 100 nm and with a diameter d≈ 6 nm. The aggregates can be seen as weakly charged rigid rods and they undergo an isotropic to nematic phase transition at higher concentrations. Translational motion perpendicular to the rod axis gets strongly hindered when the concentration is increased above the overlap concentration. Similarly, the rotational motion is hindered, leading to very long stress relaxation times. The peptide self-assembly is driven by hydrophobic interactions and due to a net peptide charge the system is colloidally stable. However, at the same time short range, presumably hydrophobic, attractive interactions appear to affect the rheology of the system. Upon screening the long range electrostatic repulsion, with the addition of salt, the hydrophobic attraction becomes more dominant and we observe a transition from a repulsive glassy state to an attractive gel-state of the rod-like peptide aggregates.

Tube to ribbon transition in a self-assembling model peptide system.

Peptides that self-assemble into ß-sheet rich aggregates are known to form a large variety of supramolecular shapes, such as ribbons, tubes or sheets. However, the underlying thermodynamic driving forces for such different structures are still not fully understood, limiting their potential applications. In the AnK peptide system (A = alanine, K = lysine), a structural transition from tubes to ribbons has been shown to occur upon an increase of the peptide length, n, from 6 to 8. In this work we analyze this transition by means of a simple thermodynamic model. We consider three energy contributions to the total free energy: an interfacial tension, a penalty for deviating from the optimal ß-sheet twist angle, and a hydrogen bond deformation when the ß-sheets adopt a specific self-assembled structure. Whilst the first two contributions merely provide similar constant energy offsets, the hydrogen bond deformations differ depending on the studied structure. Consequently, the tube structure is thermodynamically favored for shorter AnK peptides, with a crossover at n≈ 13. This qualitative agreement of the model with the experimental observations shows, that we have achieved a good understanding of the underlying thermodynamic features within the self-assembling AnK system.

Slow Dissolution Kinetics of Model Peptide Fibrils.

Understanding the kinetics of peptide self-assembly is important because of the involvement of peptide amyloid fibrils in several neurodegenerative diseases. In this paper, we have studied the dissolution kinetics of self-assembled model peptide fibrils after a dilution quench. Due to the low concentrations involved, the experimental method of choice was isothermal titration calorimetry (ITC). We show that the dissolution is a strikingly slow and reaction-limited process, that can be timescale separated from other rapid processes associated with dilution in the ITC experiment. We argue that the rate-limiting step of dissolution involves the breaking up of inter-peptide ß-sheet hydrogen bonds, replacing them with peptide-water hydrogen bonds. Complementary pH experiments revealed that the self-assembly involves partial deprotonation of the peptide molecules.

Fibril Charge Affects α-Synuclein Hydrogel Rheological Properties.

In this paper, we have investigated the interactions between α-synuclein fibrils at different pH values and how this relates to hydrogel formation and gel properties. Using a combination of rheology, small-angle X-ray scattering, Raman spectroscopy, and cryo-transmission electron microscopy (cryo-TEM) experiments, we have been able to investigate the relationship between protein net charge, fibril-fibril interactions, and hydrogel properties, and have explored the potential for α-synuclein to form hydrogels at various conditions. We have found that α-synuclein can form hydrogels at lower concentrations (50-300 µM) and over a wider pH range (6.0-7.5) than previously reported. Over this pH range and at 300 µM, the fibril network is electrostatically stabilized. Decreasing the pH to 5.5 results in the precipitation of fibrils. A maximum in gel stiffness was observed at pH 6.5 (∼1300 Pa), which indicates that significant attractive interactions operate at this pH and cause an increase in the density of hydrophobic contacts between the otherwise negatively charged fibrils. We conclude that fibril-fibril interactions under these conditions involve both long-range electrostatic repulsion and a short-range hydrophobic attractive (sticky) component. These results may provide a basis for potential applications and add to the understanding of amyloids.

Twisted Ribbon Aggregates in a Model Peptide System.

The model peptides A8K and A10K self-assemble in water into ca. 100 nm long ribbon-like aggregates. These structures can be described as ß-sheets laminated into a ribbon structure with a constant elliptical cross-section of 4 by 8 nm, where the longer axis corresponds to a finite number, N ≈ 15, of laminated sheets, and 4 nm corresponds to a stretched peptide length. The ribbon cross-section is strikingly constant and independent of the peptide concentration. High-contrast transmission electron microscopy shows that the ribbons are twisted with a pitch λ ≈ 15 nm. The self-assembly is analyzed within a simple model taking into account the interfacial free energy of the hydrophobic ß-sheets and a free energy penalty arising from an increased stretching of hydrogen bonds within the laminated ß-sheets, arising from the twist of the ribbons. The model predicts an optimal value N, in agreement with the experimental observations.

Shape and Phase Transitions in a PEGylated Phospholipid System.

Poly(ethylene glycol) (PEG) polymers and PEG-conjugated lipids are widely used in bioengineering and drug transport applications. A PEG layer in a drug carrier increases hydrophilic repulsion, inhibits membrane fusion and serum opsonin interactions, and prolongs the storage and circulation time. It can also change the carrier shape and have an influence on many properties related to the content release of the carrier. In this paper, we focus on the physicochemical effects of PEGylation in the lipid bilayer. We introduce laurdanC as a fluorophore for shape recognition and phase transition detection. Together with laurdanC, cryogenic transmission electron microscopy, differential scanning calorimetry, molecular dynamics simulations, and small-angle X-ray scattering/wide-angle X-ray scattering, we acquire information of the particle/bilayer morphology and phase behavior in systems containing 1,2-dipalmitoyl- sn-glycero-3-phosphocholine:1,2-distearoyl- sn-glycero-3-phosphoethanolamine-PEG(2000) with different fractions. We find that PEGylation leads to two important and potentially usable features of the system. (1) Spherical vesicles present a window of elevated chain-melting temperatures and (2) lipid packing shape-controlled liposome-to-bicelle transition. The first finding is significant for targets requiring multiple release sequences and the second enables tuning the release by composition and the PEG polymer length. Besides drug delivery systems, the findings can be used in other smart soft materials with trigger-polymers as well.

Renal ultrasonographic abnormalities are associated with low glomerular filtration rate calculated by scintigraphy in dogs.

Ultrasound provides information on kidney morphology, but studies relating structural and functional abnormalities in chronic kidney disease (CKD) are lacking. The aim of this descriptive cross-sectional study was to compare individual kidney (IK) B-mode ultrasound abnormalities to IK glomerular filtration rate (GFR) estimated by scintigraphy normalized to plasma volume (PV) in dogs, to evaluate if ultrasonographic findings were associated with low IKGFR/PV. Eighty privately owned dogs with and without clinical suspicion of CKD were prospectively enrolled, and kidney ultrasound and IKGFR/PV were evaluated independently. Ultrasound images were assessed retrospectively for subjective abnormalities (shape, cortical, and medullary hyperechogenicity), and kidney size was measured. The normal IKGFR/PV cutoff was derived from dogs in the study group with no history and clinical signs of kidney disease and normal blood and urine results (n = 28) and was 16.84 mL/min/L. Kidneys were categorized into normal, mild, moderate, and severe ultrasound changes according to subjective ultrasound grades. Associations were found between low IKGFR/PV and abnormal kidney shape (P = .0004), cortical hyperechogenicity (P = .0008), medullary hyperechogenicity (P < .0001), and low kidney volume (P = .0092). Apart from the moderate and severe category comparison, IKGFR/PV value significantly decreased with increasing severity of category. The combination of ultrasonographic subjective abnormalities had a high sensitivity (93.8%) and moderate specificity (65.7%) for detecting low IKGFR/PV. Kidneys with normal IKGFR/PV had a low frequency of mild ultrasound changes. Findings indicate kidneys with increasing number and grade of subjective ultrasound abnormalities are more likely to have a lower IKGFR/PV.

Multilamellar Vesicle Formation Probed by Rheo-NMR and Rheo-SALS under Large Amplitude Oscillatory Shear.

The formation of multilamellar vesicles (MLVs) in the lyotropic lamellar phase of the system triethylene glycol mono n-decyl ether (C10E3)/water is investigated under large amplitude oscillatory shear (LAOS) using spatially resolved rheo-NMR spectroscopy and a combination of rheo-small angle light scattering (rheo-SALS) and conventional rheology. Recent advances in rheo-NMR hardware development facilitated the application of LAOS deformations in high-field NMR magnets. For the range of investigated strain amplitudes (10-50) and frequencies (1 and 2 rad s-1), MLV formation is observed in all NMR and most SALS experiments. It is found that the MLV size depends on the applied frequency in contrast to previous steady shear experiments where the shear rate is the controlling parameter. The onset of MLV formation, however, is found to vary with the shear amplitude. The LAOS measurements bear no indication of the intermediate structures resembling aligned multilamellar cylinders observed in steady shear experiments. Lissajous curves of stress vs strain reveal a transition from a viscoelastic solid material to a pseudoplastic material.

Ferrihydrite Nanoparticle Aggregation Induced by Dissolved Organic Matter.

Ferrihydrite (Fh) nanoparticles are omnipresent in nature and often highly mobile because of their colloidal stability. Thus, Fh serves as a vector for iron as well as associated nutrients and contaminants. Here, we demonstrate, using small-angle X-ray scattering combined with cryo-transmission electron microscopy (cryo-TEM), that dissolved organic matter (DOM), extracted from a boreal forest soil, induce aggregation of Fh nanoparticles, of radius 3 nm, into fractal aggregates, having a fractal dimension D = 1.7. The DOM consists of both fractal-like colloids (>100 nm) and small molecular DOM, but the attractive Fh interparticle interaction was mediated by molecular DOM alone as shown by cryo-TEM. This highlights the importance of using soil extracts, including all size fractions, in studies of the colloidal behavior of DOM-mineral aggregates. The Fh nanoparticles also self-assemble during synthesis into aggregates with the same fractal dimension as the DOM-Fh aggregates. We propose that, in both the absence and presence of DOM, the aggregation is controlled by the Fh particle charge, and the process can be viewed as a linear polymerization into a self-avoiding random walk structure. The theoretical D value for this is 5/3, which is in close agreement with our Fh and DOM-Fh results.

Bone mineral crystal size and organization vary across mature rat bone cortex.

The macro- and micro-features of bone can be assessed by using imaging methods. However, nano- and molecular features require more detailed characterization, such as use of e.g., vibrational spectroscopy and X-ray scattering. Nano- and molecular features also affect the mechanical competence of bone tissue. The aim of the present study was to reveal the effects of mineralization and its alterations on the mineral crystal scale, by investigating the spatial variation of molecular composition and mineral crystal structure across the cross-section of femur diaphyses in young rats, and healthy and osteoporotic mature rats (N=5). Fourier transform infrared spectroscopy and scanning small- and wide-angle X-ray scattering (SAXS/WAXS) techniques with high spatial resolution were used at identical locations over the whole cross-section. This allowed quantification of point-by-point information about the spatial distribution of mineral crystal volume. All measured parameters (crystal dimensions, degree of orientation and predominant orientation) varied across the cortex. Specifically, the crystal dimensions were lower in the central cortex than in the endosteal and periosteal regions. Mineral crystal orientation followed the cortical circumference in the periosteal and endosteal regions, but was less well-oriented in the central regions. Central cortex is formed rapidly during development through endochondral ossification. Since rats possess no osteonal remodeling, this bone remains (until old age). Significant linear correlations were observed between the dimensional and organizational parameters, e.g., between crystal length and degree of orientation (R(2)=0.83, p<0.001). Application of SAXS/WAXS provides valuable information on bone nanostructure and its constituents, effects of diseases and, prospectively, mechanical competence.

Small-Angle X-ray Scattering Demonstrates Similar Nanostructure in Cortical Bone from Young Adult Animals of Different Species.

Despite the vast amount of studies focusing on bone nanostructure that have been performed for several decades, doubts regarding the detailed structure of the constituting hydroxyapatite crystal still exist. Different experimental techniques report somewhat different sizes and locations, possibly due to different requirements for the sample preparation. In this study, small- and wide-angle X-ray scattering is used to investigate the nanostructure of femur samples from young adult ovine, bovine, porcine, and murine cortical bone, including three different orthogonal directions relative to the long axis of the bone. The radially averaged scattering from all samples reveals a remarkable similarity in the entire q range, which indicates that the nanostructure is essentially the same in all species. Small differences in the data from different directions confirm that the crystals are elongated in the [001] direction and that this direction is parallel to the long axis of the bone. A model consisting of thin plates is successfully employed to describe the scattering and extract the plate thicknesses, which are found to be in the range of 20-40 Å for most samples but 40-60 Å for the cow samples. It is demonstrated that the mineral plates have a large degree of polydispersity in plate thickness. Additionally, and equally importantly, the scattering data and the model are critically evaluated in terms of model uncertainties and overall information content.

On cellulose dissolution and aggregation in aqueous tetrabutylammonium hydroxide.

Aqueous tetrabutylammonium hydroxide, TBAH(aq), has been found to dissolve cellulose and to be a potential solvent for chemical processing or fiber spinning. In this paper, we have investigated the dissolution state of cellulose in 40 wt % TBAH(aq) solvent, and present an extensive study of rheology, combined with static light and small-angle X-ray scattering, to correlate cellulose aggregation with changes in the rheological parameters. Two cellulose molecular weights are compared. Microcrystalline cellulose (MCC), with a degree of polymerization of ca. 260, and a dissolving pulp with an approximately ten times higher molecular weight. Scattering data demonstrate that cellulose is molecularly dissolved at lower cellulose concentrations, while aggregates are present when the concentration exceeds a certain value. The onset of the aggregate formation is marked by a pronounced increase in the scattering intensity at low q, shear thinning behavior and violation of the empirical Cox-Merz rule. Additionally, the SAXS data suggest the presence of a solvation shell enriched in TBA(+) ions, compared to the bulk solvent. The results are consistent with the recent suggestion that while native cellulose I may still dissolve, solutions are, above a particular concentration, becoming supersaturated with respect to the more stable crystal form cellulose II.

A Simple Simulation Technique for Nonnormal Data with Prespecified Skewness, Kurtosis, and Covariance Matrix.

We present and investigate a simple way to generate nonnormal data using linear combinations of independent generator (IG) variables. The simulated data have prespecified univariate skewness and kurtosis and a given covariance matrix. In contrast to the widely used Vale-Maurelli (VM) transform, the obtained data are shown to have a non-Gaussian copula. We analytically obtain asymptotic robustness conditions for the IG distribution. We show empirically that popular test statistics in covariance analysis tend to reject true models more often under the IG transform than under the VM transform. This implies that overly optimistic evaluations of estimators and fit statistics in covariance structure analysis may be tempered by including the IG transform for nonnormal data generation. We provide an implementation of the IG transform in the R environment.