Peptide and protein mimetics inhibiting amyloid beta-peptide aggregation

Protein misfolding is related to some fatal diseases including Alzheimer's disease (AD). Amyloid beta-peptide (Abeta) generated from amyloid precursor protein can aggregate into amyloid fibrils, which are known to be a major component of Abeta deposits (senile plaques). The fibril formation of Abeta is typical of a nucleation-dependent process through self-recognition. Moreover, during fibrillization, several metastable intermediates such as soluble oligomers, including Abeta-derived diffusible ligands (ADDLs) and Abeta*56, are produced, which are thought to be the most toxic species to neuronal cells. Therefore, construction of molecules that decrease the Abeta aggregates, including soluble oligomers, protofibrils, and amyloid fibrils, might further our understanding of the mechanism(s) behind fibril formation and enable targeted drug discovery against AD. To this aim, various peptides and peptide derivatives have been constructed using the "Abeta binding element" based on the structural models of Abeta amyloid fibrils and the mechanisms of self-assembly. The central hydrophobic amino acid sequence, LVFF, of Abeta is a key sequence to self-assemble into amyloid fibrils. By combination of this core sequence with a hydrophobic or hydrophilic moiety, such as cholic acid or aminoethoxy ethoxy acetic acid units, respectively, good inhibitors of Abeta aggregation can be designed and synthesized. A peptide, LF, consisting of the sequence Ac-KQKLLLFLEE-NH 2, was designed based on the core sequence of Abeta but with a simplified amino acid sequence. The LF peptide can form amyloid-like fibrils that efficiently coassemble with mature Abeta1-42 fibrils. The LF peptide was also observed to immediately transform the soluble oligomers of Abeta1-42, which are thought to pose toxicity in AD, into amyloid-like fibrils. On the other hand, two Abeta-like beta-strands with a parallel orientation were embedded in green fluorescent protein (GFP), comprised of a beta-barrel structure, to make pseudo-Abeta beta-sheets on its surface. The GFP variant P13H binds to Abeta1-42 and inhibits Abeta1-42 oligomerization effectively in a substoichiometric condition. Thus, molecules capable of binding to Abeta can be designed based on structural similarities with the Abeta molecule. The peptide and protein mimetics based on the structural features of Abeta might lead to the development of drug candidates against AD.     

Role of water in protein aggregation and amyloid polymorphism

A variety of neurodegenerative diseases are associated with amyloid plaques, which begin as soluble protein oligomers but develop into amyloid fibrils. Our incomplete understanding of this process underscores the need to decipher the principles governing protein aggregation. Mechanisms of in vivo amyloid formation involve a number of coconspirators and complex interactions with membranes. Nevertheless, understanding the biophysical basis of simpler in vitro amyloid formation is considered important for discovering ligands that preferentially bind regions harboring amyloidogenic tendencies. The determination of the fibril structure of many peptides has set the stage for probing the dynamics of oligomer formation and amyloid growth through computer simulations. Most experimental and simulation studies, however, have been interpreted largely from the perspective of proteins: the role of solvent has been relatively overlooked in oligomer formation and assembly to protofilaments and amyloid fibrils. In this Account, we provide a perspective on how interactions with water affect folding landscapes of amyloid beta (Aβ) monomers, oligomer formation in the Aβ16-22 fragment, and protofilament formation in a peptide from yeast prion Sup35. Explicit molecular dynamics simulations illustrate how water controls the self-assembly of higher order structures, providing a structural basis for understanding the kinetics of oligomer and fibril growth. Simulations show that monomers of Aβ peptides sample a number of compact conformations. The formation of aggregation-prone structures (N*) with a salt bridge, strikingly similar to the structure in the fibril, requires overcoming a high desolvation barrier. In general, sequences for which N* structures are not significantly populated are unlikely to aggregate. Oligomers and fibrils generally form in two steps. First, water is expelled from the region between peptides rich in hydrophobic residues (for example, Aβ16-22), resulting in disordered oligomers. Then the peptides align along a preferred axis to form ordered structures with anti-parallel β-strand arrangement. The rate-limiting step in the ordered assembly is the rearrangement of the peptides within a confining volume. The mechanism of protofilament formation in a polar peptide fragment from the yeast prion, in which the two sheets are packed against each other and create a dry interface, illustrates that water dramatically slows self-assembly. As the sheets approach each other, two perfectly ordered one-dimensional water wires form. They are stabilized by hydrogen bonds to the amide groups of the polar side chains, resulting in the formation of long-lived metastable structures. Release of trapped water from the pore creates a helically twisted protofilament with a dry interface. Similarly, the driving force for addition of a solvated monomer to a preformed fibril is water release; the entropy gain and favorable interpeptide hydrogen bond formation compensate for entropy loss in the peptides. We conclude by offering evidence that a two-step model, similar to that postulated for protein crystallization, must also hold for higher order amyloid structure formation starting from N*. Distinct water-laden polymorphic structures result from multiple N* structures. Water plays multifarious roles in all of these protein aggregations. In predominantly hydrophobic sequences, water accelerates fibril formation. In contrast, water-stabilized metastable intermediates dramatically slow fibril growth rates in hydrophilic sequences.     

Exploring the early steps of amyloid peptide aggregation by computers

The assembly of normally soluble proteins into amyloid fibrils is a hallmark of neurodegenerative diseases. Because protein aggregation is very complex, involving a variety of oligomeric metastable intermediates, the detailed aggregation paths and structural characterization of the intermediates remain to be determined. Yet, there is strong evidence that these oligomers, which form early in the process of fibrillogenesis, are cytotoxic. In this paper, we review our current understanding of the underlying factors that promote the aggregation of peptides into amyloid fibrils. We focus here on the structural and dynamic aspects of the aggregation as observed in state-of-the-art computer simulations of amyloid-forming peptides with an emphasis on the activation-relaxation technique.     

Reactive oxygen species may cause amyloid beta aggregation by free radical polymerization

Alzheimer's disease (AD), the most common form of dementia, is closely related to the overproduction of reactive oxygen species (ROS). A kinetic model based on free radical polymerization of amyloid beta (Aβ) utilizing ROS as an initiator is proposed. Kinetic parameters involved in the model are tuned with the reported experimental data on fibril molecular weight. The tuned model is used to simulate time evolution of fibril length and polydispersity of Aβ aggregates. The simulated values are compared with the reported experimental values for these fibril properties. A good agreement is observed between the model simulated fibril properties and the reported experimental data. This supports the hypothesis of ROS as the cause of Aβ aggregation using the free radical polymerization. The proposed model is also able to predict the sigmoidal growth of fibrils at different set of parameter values. Sensitivity analysis has been performed to check the reliability of the proposed model. It is envisaged that the proposed model will be helpful to elucidate ROS-based therapeutic strategies for AD treatment in near future.     

Membrane dipole potential of interaction between amyloid protein and phospholipid membranes is dependent on protein aggregation state

At least 20 different human proteins can fold abnormally resulting in the formation of pathological aggregates and several deadly degenerative diseases. Evidence also suggests that non-disease-associated proteins, under appropriate conditions, can aggregate in vitro to form amyloid fibrillar species. Numerous reports have shown that the interaction between cell membrane and amyloid proteins is of particular importance in the cytotoxic effects elicited by amyloid proteins. Despite the significant progress has been made, there are still large gaps in our knowledge of the disease mechanism(s) associated with this aforementioned interaction. In the current research, using a dual-wavelength fluorescence ratiometric method along with a voltage-sensitive dye, di-8-ANEPPS, we found that a decrease in intramembrane dipole potential was observed upon binding of amyloid proteins with phospholipids and this decrease became more dramatic when protein was in its aggregated form. Moreover, our data revealed that a correlation among the presence of cholesterol, the type of phospholipid, and the drop in dipole potential was evident. In comparison with the pure DPPC, the relative difference in dipole potential between fibrillar and freshly prepared samples attenuated with the addition of cholesterol while an increase in relative potential difference was observed in DPPG. Importantly, our results, for the first time, presented that the membrane dipole potential in amyloid protein-phospholipid interaction was dependent on the aggregation state of proteins, which is highly associated with the biological effects elicited by amyloid proteins.     

Reinforcement of polypropylene with lignocellulose nanofibrils and compatibilization with biobased polymers

Freeze‐dried and milled lignocellulose nanofibrils (LCNF) were used to reinforce polypropylene (PP) nanocomposites. The LCNF, containing up to 9% lignin, was obtained from residual Empty Palm Fruit Bunch (EPFB) fibers. Soy protein isolate (SPI) and hydroxypropyl cellulose (HPC) were tested as coupling agents as well as maleic anhydride grafted polypropylene (MAPP), which was used as a reference. A good level of dispersion of LCNF in the PP matrix while mechanical testing and thermal analyses indicated an improvement of the thermo‐mechanical behavior of the nanocomposites was revealed upon loading of the lignin‐containing nanofibrils. The tensile modulus of PP was increased by 15% upon the addition of 1% LCNF with SPI as a compatibilizer. Likewise, the thermal stability of the composites was most markedly enhanced. Overall, LCNF and SPI, two important bioresources, are introduced here for the development of novel and cost‐effective PP‐based composites. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43854.     

Preparation of cellulose nanofibrils by high-pressure homogenizer and cellulose-based composite films

Cellulose nanofibrils were prepared from starting material microcrystalline cellulose (MCC) by an application of a high-pressure homogenizer at 20,000 psi and treatment consisting of 0, 1, 2, 5, 10, 15 and 20 passes. Hydroxypropyl cellulose (HPC) films reinforced with those cellulose nanofibrils were prepared at different filler loading levels. According to the morphology study by scanning electron microscope (SEM), the complete filbrillation of the bulk cellulose fibrils to nanoscale and high aspect ratio was accomplished by the homogenization process. The HPC film reinforced with cellulose fibrils after the 5–10 passes through the homogenizer improved significantly the tensile modulus and strength values for the HPC films. The thermal stability of HPC films is not affected by the addition of the cellulose nanofibrils, although MCC has the lower thermal stability than neat HPC. The development of novel cellulose nanofibrils and composites with high strength can be achieved by an application of the homogenization process.     

Significance of xylan on the stability and water interactions of cellulosic nanofibrils

In this paper, the significance of xylan on the behaviour of kraft birch pulp based nanofibrillated cellulose (CNF) is discussed. The influence of CNF xylan content on fibril morphology, charge and stability as well as on the film formation ability was investigated, and the features detected on nanoscale and on macroscale are compared. In addition to this, the ability of fibrils to uptake water molecules were investigated by bulk and surface sensitive methods which are dynamic water sorption analysis (DVS) and quartz crystal microbalance with dissipation monitoring (QCM-D) equipped with the humidity module, respectively. Surface xylan plays a significant role as an electrosteric stabilizer in dilute CNF dispersions when the surface forces are dominant whereas the removal of xylan drastically changes the CNF dispersion properties. The settling of the unstable CNF dispersions displays behaviour which is typical for hindered sedimentation. When considering thin nanoscale layers of CNF, nanofibrillated cellulosic materials with high content of surface xylan has somewhat higher ability to bind water molecules. However, it seems that in more concentrated CNF dispersions where the fibrillar network itself plays also a decisive role, especially with respect to film formation ability, the impact of xylan diminishes. Solvent cast macroscale CNF films are still enough dense to maintain good oxygen barrier performance at higher humid conditions although agglomeration tendency of fibrils is higher due to the excessive xylan removal. These findings are of high relevance when considering nanocellulosic materials, especially in the form of gels and films, as templates for high added value material solutions.     

Cross-linking cellulose nanofibrils for potential elastic cryo-structured gels

Cellulose nanofibrils were produced from P. radiata kraft pulp fibers. The nanofibrillation was facilitated by applying 2,2,6,6-tetramethylpiperidinyl-1-oxyl-mediated oxidation as pretreatment. The oxidized nanofibrils were cross-linked with polyethyleneimine and poly N-isopropylacrylamide-co-allylamine-co-methylenebisacrylamide particles and were frozen to form cryo-structured gels. Samples of the gels were critical-point dried, and the corresponding structures were assessed with scanning electron microscopy. It appears that the aldehyde groups in the oxidized nanofibrils are suitable reaction sites for cross-linking. The cryo-structured materials were spongy, elastic, and thus capable of regaining their shape after a given pressure was released, indicating a successful cross-linking. These novel types of gels are considered potential candidates in biomedical and biotechnological applications.     

Cellulose nanofibrils in biobased multilayer films for food packaging

The aim of this study was to evaluate a thin, TEMPO‐oxidized (2,2,6,6‐tetramethylpiperidine‐1‐oxyl–mediated oxidation) cellulose nanofibril (CNF) coating as a barrier layer in multilayer packaging films together with biobased polyethylenes. The purpose was also to explore the possible interactions between food products and the biobased films, and to evaluate the feasibility of these films for packaging of dry foods. CNF provided the biobased multilayer films with an oxygen barrier suitable for both demanding food products and modified atmosphere packaging (MAP). The MAP pouches made of these multilayer films retained their atmosphere and shape and protected ground hazelnuts from further oxidation for the storage time used in this study. However, irradiation used to sterilize packed foods and aroma compounds from clove in particular impaired the oxygen barrier property of the CNF layer, while the water vapor barrier property of the multilayer films remained unaffected. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44830.     

Flax nanofibrils production via supercritical carbon dioxide pre-treatment and enzymatic hydrolysis

Flax fibres are an agro-industrial waste available in large quantities in several countries around the world. This resource can be properly used. The goal of this work was to extract lignocellulosic nanosized flax fibres using an environmentally friendly process based on a combination of supercritical carbon dioxide (SC-CO₂) pre-treatment and enzymatic hydrolysis. Raw flax fibres (RFF) were submitted to a SC-CO₂ pre-treatment at various temperatures (ie, 70°C and 80°C) and pressures (ie, 20 and 37.7 MPa) for 60 minutes. The enzymatic hydrolysis was performed at 40°C for 24 hours in a pH 4.0 buffer. Cellulase, xylanase, pectinase, and viscozyme were used as hydrolytic enzymes. The as-received raw flax fibres, SC-CO₂ pretreated flax fibres, and extracted lignocellulosic nanofibrils (LCNF) were characterized by Fourier transformed infrared spectroscopy (FTIR), x-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). It was shown that the effect of the SC-CO₂ pre-treatment of flax fibres was two-fold. It helped to disorganize biomass without changing its chemical composition and it increased access to enzymes to extract LCNF. The FTIR analysis showed no changes in the functional groups after SC-CO₂ pre-treatment. The XRD characterization revealed that the crystallinity increased with the SC-CO₂ pre-treatment and LCNF extraction. SEM images showed holes, cracks, and erosion on the surface of the SC-CO₂ pretreated flax fibres (SC-CO₂-PFF). TEM evidenced the production of nano/micro-sized fibril and fibril aggregates.     

竹基纤维素纳米纤丝交联改性后对水体中低浓度磷的吸附

通过优化制备条件,将竹基纤维素纳米纤丝（cellulose nanofibrils,CNFs）与聚酰胺环氧氯丙烷树脂（polyamide epichlorohydrin resin,PAE）进行交联,随后负载Fe(OH)₃,得到了吸附废水中低浓度磷的新型复合材料CNFs-PAE-Fe。与其他交联剂相比,竹基纤维素纳米纤丝与PAE交联并负载后展现出更好的机械强度和吸附性能。通过BET比表面积测量、热重分析、FTIR、扫描电镜、XPS能谱分析,探究了交联负载Fe(OH)₃前后材料的孔径、热稳定性、表面形貌、元素组成等。CNFs-PAE-Fe对磷的吸附符合准二级动力学模型和Langmuir等温吸附模型,这表明改性材料对磷的吸附过程是以化学吸附为主的单分子层吸附。CNFs-PAE-Fe在较宽的pH范围下对低浓度磷都保持着较好的吸附性能,当pH=4.0时吸附容量最高,达到9.11mg/g。对吸附饱和的改性材料进行再生研究,洗脱液V(NaCl)∶V(NaOH)配比为3∶2时再生效果最好。解吸后的材料经冷冻干燥,重复利用5次,仍保持较好的强度和完整的形貌。     

Electrochemical copolymerization of pyrrole and thiophene nanofibrils using template‐synthesis method

Copolymer nanofibrils composed of pyrrole and thiophene were prepared by synthesizing the desired polymer within the pores of microporous anodic aluminum oxide (AAO) template membranes. The copolymer nanofibrils were photographed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for microstructure analyses. The results of the SEM and TEM revealed that the copolymer nanofibrils obtained had uniform and well‐aligned arrays and their diameter and length could be controlled by changing the aspect ratios of the AAO membrane. The results of cyclic voltammetry and IR spectrometry indicated that polypyrrole and polythiophene were both involved in the copolymer. The nanofibrils that were obtained were identified as copolymers rather than composites. The influence of the applied polymerization potential on the synthesis of copolymer nanofibrils was investigated. The higher potential favored the incorporation of thiophene units into the copolymer nanofibrils. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 2403–2407, 2002     

Silk nanofibrils/chitosan composite fibers with enhanced mechanical properties

Chitosan (CS) fibers have been applied in various fields due to their biocompatibility, biodegradability, and antibacterial properties. However, weak mechanical properties remain as obstacles to further applications. Silk nanofibrils (SNFs) extracted from natural silk fibroin fibers preserve outstanding mechanical properties at the nanoscale, which are expected to impact structural programming and mechanical reinforcement for CS fibers. In this study, wet-spun CS/SNFs composite fibers were continuously collected from NaOH/ethanol coagulation. Scanning electron microscope (SEM) results showed that SNFs were uniformly distributed in the CS matrix, and obvious orientation was observed when the mass ratio of SNF/CS was 75/100. Tensile tests showed that the introduction of SNFs significantly enhanced the mechanical properties of CS fibers when the mass ratio of SNF/CS was more than 25/100. With the increasing of SNF content, the tensile strength gradually increased, and the tensile strength and modulus could be increased 2.9 times and 3.5 times, respectively, when 100% SNF was added. The improvement of mechanical properties was partially attributed to hydrogen bonding between SNF filaments and CS, which was confirmed by FTIR and XRD results. This study provides a facile and eco-friendly method to spin CS fibers with enhanced mechanical properties and a hierarchical structure.     

Polyaniline‐modified cellulose nanofibrils as reinforcement of a smart polyurethane

Segmented polyurethanes exhibiting shape memory properties were modified by the addition of polyaniline (PANI)‐coated cellulose nanofibrils (CNFs). The two‐phase structure of the polymer is responsible for the material's ability to ‘remember’ and autonomously recover its original shape after being deformed in response to an external thermal stimulus. PANI was grown on the surface of the CNFs via in situ polymerization. Modified nanocrystals were added to the segmented polyurethane in concentrations ranging from 0 to 15 wt%. The changes in the material properties associated with the percolation of the coated fibrils appear at higher concentrations than previously observed for non‐modified CNFs, which suggests that fibril agglomeration is occurring due to the PANI coating. The shape memory behavior of the composites is maintained at about the same level as that of the unfilled polyurethane only up to 4 wt% of fibrils. At higher concentrations, the rigidity of the nanofibrils as well as their interaction with the hard‐segment phase and the increasing difficulty of dispersing them in the polymer collaborate to produce early breakage of the specimens when stretched at temperatures above the melting point of the soft segments. Copyright © 2010 Society of Chemical Industry     

The non-trivial role of native xylans on the preparation of TEMPO-oxidized cellulose nanofibrils

Cellulose nanofibrils have become increasingly prized as a raw material toward the preparation of composites due to their specific surface character and biocompatibility. TEMPO-mediated oxidation with post-mechanical treatment has been proposed as a promising method for the preparation of cellulose nanofibrils from cellulosic raw materials. In the current study it was found that the native hemicellulosic components in the raw material played a pivotal chemical role on the kinetics of generation of TEMPO-oxidized cellulose nanofibrils (TOCNs), as well as on thermal stability, and transmittance. The removal of xylans from the original feedstock facilitated not only an increase in both the carboxylate content and water retention value of the TEMPO-oxidized fibers, but also improved the transmittance of subsequently obtained TOCNs suspensions. It was also determined that the presence of xylans in the cellulosic feedstock hindered chemical accessibility through a barrier mechanism in which the TEMPO-mediated oxidation reaction rate was reduced.     

Creation of a new material stream from Japanese cedar resources to cellulose nanofibrils

Japanese cedar is one of the most abundant plantation softwoods in Japan, although it is not effectively utilized as a wood resource. Japanese cedar cellulose was isolated and subjected to one-pot catalytic oxidation and reduction with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and NaBH₄, respectively. The TEMPO-oxidized and NaBH₄-reduced Japanese cedar celluloses (TOCs-NaBH₄) had carboxylate content of up to 1.4 mmol/g and viscosity-average degrees of polymerization from 2000 to 3000. The X-ray diffraction patterns of the TOCs-NaBH₄ showed that the crystal widths were ~ 3 nm, indicating that the C6-OH groups present on the crystalline cellulose microfibril surfaces were selectively oxidized to C6-carboxylate groups. When the TOCs-NaBH₄ with carboxylate content of 0.9–1.4 mmol/g were mechanically disintegrated in water, transparent TEMPO-oxidized cellulose nanofibril (TOCN) dispersions were obtained. The lengths of the TOCNs, determined from their atomic force microscopy images, varied from 800 to 1500 nm, depending on the oxidation conditions. The TOCNs prepared from Japanese cedar cellulose have an average of high aspect ratios (> 300), which is greater than that (~ 150) prepared from wood pulp and thus advantageous.     

Surface modification of cellulose nanofibrils for poly(lactic acid) composite application

3-Methacryloxypropyltrimethoxysilane (MEMO) was used to modify the surface of cellulose nanofibrils (CNF) to improve the interfacial adhesion between the hydrophilic CNF and the hydrophobic poly(lactic acid) (PLA). MEMO modified CNF (M-CNF) were characterized by means of Fourier transform infrared spectroscopy (FTIR), thermo gravimetric analysis (TGA), and atomic force microscope (AFM). Testing thin films with good transparency were obtained by casting the DMAC solutions of the composites onto glass plates and evaporating the solvent at 80°C. PLA/M-CNF composites were tested by tensile testing, scanning electron microscope (SEM), and AFM. The effect of MEMO and CNF on performance of PLA was investigated. The FTIR analysis successfully showed that coupling reaction has been successfully occurred and the hydroxyl groups of MEMO are strongly hydrogen bonded to that of CNF. The thermal stability of M-CNF was little decreased. The M-CNF kept their morphological integrity. The highest tensile strength of composites was obtained for PLA with 1.0% v/v MEMO and 1.0 wt % CNF. M-CNF disperse well and cross with each other in the PLA matrix. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Preparation and characterization of pyrrole/aniline copolymer nanofibrils using the template‐synthesis method

Copolymer nanofibrils composed of pyrrole and aniline had been prepared by synthesizing the desired polymer within the pores of microporous anodic aluminum oxide (AAO) template membrane. To analyze their structure and properties, FTIR spectra were taken and thermogravimetric analysis (TGA) was applied. Also, the copolymer nanofibrils were photographed under scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for microstructure analysis, and the conductivities were obtained by the four‐probe method. The result of SEM and TEM revealed that the obtained copolymer nanofibrils had uniform and well‐aligned array, and their diameter and length can be controlled by changing the aspect ratios of the AAO membrane. The result of IR spectrometry and TGA indicated that both polypyrrole and polyaniline were involved in the copolymer. The obtained nanofibrils were identified to be copolymer rather than composite. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 3002–3007, 2001