β-Carboline Alkaloids Resist the Aggregation and Cytotoxicity of Human Islet Amyloid Polypeptide

β-Carboline alkaloids have a variety of pharmacological activities, such as antitumor, antibiosis and antidiabetes. Harmine and harmol are two structurally similar β-carbolines that occur in many medicinal plants. In this work, we chose harmine and harmol to impede the amyloid fibril formation of human islet amyloid polypeptide (hIAPP) associated with type 2 diabetes mellitus (T2DM), by a series of physicochemical and biochemical methods. The results indicate that harmine and harmol effectively prevent peptide fibril formation and alleviate toxic oligomer species. In addition, both small molecules exhibit strong binding affinities with hIAPP mainly through hydrophobic and hydrogen bonding interactions, thus reducing the cytotoxicity induced by hIAPP. Their distinct binding pattern with hIAPP is closely linked to the molecular configuration of the two small molecules, affecting their ability to impede peptide aggregation. The study is of great significance for the application and development of β-carboline alkaloids against T2DM.     

Nanoscale Surface Topography Modulates hIAPP Aggregation Pathways at Solid–Liquid Interfaces

The effects that solid–liquid interfaces exert on the aggregation of proteins and peptides are of high relevance for various fields of basic and applied research, ranging from molecular biology and biomedicine to nanotechnology. While the influence of surface chemistry has received a lot of attention in this context, the role of surface topography has mostly been neglected so far. In this work, therefore, we investigate the aggregation of the type 2 diabetes-associated peptide hormone hIAPP in contact with flat and nanopatterned silicon oxide surfaces. The nanopatterned surfaces are produced by ion beam irradiation, resulting in well-defined anisotropic ripple patterns with heights and periodicities of about 1.5 and 30 nm, respectively. Using time-lapse atomic force microscopy, the morphology of the hIAPP aggregates is characterized quantitatively. Aggregation results in both amorphous aggregates and amyloid fibrils, with the presence of the nanopatterns leading to retarded fibrillization and stronger amorphous aggregation. This is attributed to structural differences in the amorphous aggregates formed at the nanopatterned surface, which result in a lower propensity for nucleating amyloid fibrillization. Our results demonstrate that nanoscale surface topography may modulate peptide and protein aggregation pathways in complex and intricate ways.     

The Impact of Halogenated Phenylalanine Derivatives on NFGAIL Amyloid Formation

The hexapeptide hIAPP_22–27 (NFGAIL) is known as a crucial amyloid core sequence of the human islet amyloid polypeptide (hIAPP) whose aggregates can be used to better understand the wild-type hIAPP′s toxicity to β-cell death. In amyloid research, the role of hydrophobic and aromatic-aromatic interactions as potential driving forces during the aggregation process is controversially discussed not only in case of NFGAIL, but also for amyloidogenic peptides in general. We have used halogenation of the aromatic residue as a strategy to modulate hydrophobic and aromatic-aromatic interactions and prepared a library of NFGAIL variants containing fluorinated and iodinated phenylalanine analogues. We used thioflavin T staining, transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) to study the impact of side-chain halogenation on NFGAIL amyloid formation kinetics. Our data revealed a synergy between aggregation behavior and hydrophobicity of the phenylalanine residue. This study introduces systematic fluorination as a toolbox to further investigate the nature of the amyloid self-assembly process.     

Interfacial Interactions within Amyloid Protein Corona Based on 2D MoS2 Nanosheets

The interfacial interaction within the amyloid protein corona based on MoS₂ nanomaterial is crucial, both for understanding the biological effects of MoS₂ nanomaterial and the evolution of amyloid diseases. The specific nano-bio interface phenomenon of human islet amyloid peptide (hIAPP) and MoS₂ nanosheet was investigated by using theoretical and experimental methods. The MoS₂ nanosheet enables the attraction of hIAPP monomer, dimer, and oligomer on its surface through van der Waals forces. Especially, the means of interaction between two hIAPP peptides might be changed by MoS₂ nanosheet. In addition, it is interesting to find that the hIAPP oligomer can stably interact with the MoS₂ nanosheet in one unique “standing” binding mode with an entire exposed β-sheet surface. All the interaction modes on the surface of MoS₂ nanosheet can be the essence of amyloid protein corona that may provide the venue to facilitate the fibrillation of hIAPP proteins. Further, it was verified experimentally that MoS₂ nanosheets could accelerate the fibrillation of hIAPP at a certain concentration mainly based on the newly formed nano-bio interface. In general, our results provide insight into the molecular interaction mechanism of the nano-bio interface within the amyloid protein corona, and shed light on the pathway of amyloid protein aggregation that is related to the evolution of amyloid diseases.     

Quantifying Prefibrillar Amyloids in vitro by Using a “Thioflavin‐Like” Spectroscopic Method

In Alzheimer's disease (AD) and other neurodegenerative disorders, proteins accumulate into ordered aggregates, called amyloids. Recent evidence suggests that these structures include both large, insoluble fibrils and smaller, prefibrillar structures, such as dimers, oligomers, and protofibrils. Recently, focus has shifted to the prefibrillar aggregates because they are highly neurotoxic and their levels appear to correlate with cognitive impairment. Thus, there is interest in finding methods for specifically quantifying these structures. One of the classic ways of detecting amyloid formation is through the fluorescence of the benzothiazole dye, thioflavin T (ThT). This reagent has been a “workhorse” of the amyloid field because it is robust and inexpensive. However, one of its limitations is that it does not distinguish between prefibrillar and fibrillar aggregates. We screened a library of 37 indoles for those that selectively change fluorescence in the presence of prefibrillar amyloid‐β (Aβ). From this process, we selected the most promising example, tryptophanol (TROL), to use in a quantitative “thioflavin‐like” assay. Using this probe in combination with electron microscopy, we found that prefibrils are largely depleted during Aβ aggregation in vitro but that they remain present after the apparent saturation of the ThT signal. These results suggest that a combination of TROL and ThT provides greater insight into the process of amyloid formation by Aβ. In addition, we found that TROL also recognizes other amyloid‐prone proteins, including ataxin‐3, amylin, and CsgA. Thus, this assay might be an inexpensive spectroscopic method for quantifying amyloid prefibrils in vitro.     

A β‐Hairpin‐Binding Protein for Three Different Disease‐Related Amyloidogenic Proteins

Amyloidogenic proteins share a propensity to convert to the β‐structure‐rich amyloid state that is associated with the progression of several protein‐misfolding disorders. Here we show that a single engineered β‐hairpin‐binding protein, the β‐wrapin AS10, binds monomers of three different amyloidogenic proteins, that is, amyloid‐β peptide, α‐synuclein, and islet amyloid polypeptide, with sub‐micromolar affinity. AS10 binding inhibits the aggregation and toxicity of all three proteins. The results demonstrate common conformational preferences and related binding sites in a subset of the amyloidogenic proteins. These commonalities enable the generation of multispecific monomer‐binding agents.     

Stereoselective Behavior of Nafion® Membranes towards (+)‐α‐Pinene and (−)‐α‐Pinene

α‐Pinene enantiomers were sorbed in Nafion® membranes. The membranes included a commercial extruded Nafion® 115 membrane as well as membranes prepared by casting a Nafion® solution, evaporating the solvent, and a thermal treatment at different temperatures. The microstructure of membranes was studied by small‐angle and wide‐angle X‐ray scattering, and magic‐angle spinning nuclear magnetic resonance spectroscopy. The change of membrane weight during the sorption process was determined with a sorption microbalance. Noticeable differences concerning the sorption behavior of the various membranes could be stated. The sorption of (+)‐α‐pinene and (?)‐α‐pinene in an extruded Nafion® membrane turned out to be rather low.     

Simultaneous IR‐Spectroscopic Observation of α‐Synuclein,Lipids, and Solvent Reveals an Alternative Membrane‐Induced Oligomerization Pathway

The intrinsically disordered protein α‐synuclein (αS), a known pathogenic factor for Parkinson's disease, can adopt defined secondary structures when interacting with membranes or during fibrillation. The αS–lipid interaction and the implications of this process for aggregation and damage to membranes are still poorly understood. Therefore, we established a label‐free infrared (IR) spectroscopic approach to allow simultaneous monitoring of αS conformation and membrane integrity. IR showed its unique sensitivity for identifying distinct β‐structured aggregates. A comparative study of wild‐type αS and the naturally occurring splicing variant αS Δexon3 yielded new insights into the membrane's capability for altering aggregation pathways.     

Ac‐LPFFD‐Th: A Trehalose‐Conjugated Peptidomimetic as a Strong Suppressor of Amyloid‐β Oligomer Formation and Cytotoxicity

The inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Peptide‐based inhibitors, which have been widely investigated, are generally derived from original amyloid sequences. Most interestingly, trehalose, a nonreducing disaccharide of α‐glucose, is effective in preventing the aggregation of numerous proteins. We have determined that the development of hybrid compounds could provide new molecules with improved properties that might synergically increase the potency of their single moieties. In this work, the ability of Ac‐LPFFD‐Th, a C‐terminally trehalose‐conjugated derivative, to slow down the Aβ aggregation process was investigated by means of different biophysical techniques, including thioflavin T fluorescence, dynamic light scattering, ESI‐MS, and NMR spectroscopy. Moreover, we demonstrate that Ac‐LPFFD‐Th modifies the aggregation features of Aβ and protects neurons from Aβ oligomers' toxic insult.     

A Cyclic KLVFF‐Derived Peptide Aggregation Inhibitor Induces the Formation of Less‐Toxic Off‐Pathway Amyloid‐β Oligomers

Inhibition of amyloid‐β (Aβ) aggregation could be a target of drug development for the treatment of currently incurable Alzheimer's disease. We previously reported that a head‐to‐tail cyclic peptide of KLVFF (cyclic‐KLVFF), a pentapeptide fragment corresponding to the Aβ16–20 region (which plays a critical role in the generating Aβ fibrils), possesses potent inhibitory activity against Aβ aggregation. Here we found that the inhibitory activity of cyclic‐KLVFF was significantly improved by incorporating an additional phenyl group at the β‐position of the Phe4 side chain (inhibitor 3 ). Biophysical and biochemical analyses revealed the rapid formation of 3 ‐embedded oligomer species when Aβ1–42 was mixed with 3 . The oligomer species is an “off‐pathway” species with low affinity for cross‐β‐sheet‐specific dye thioflavin T and oligomer‐specific A11 antibodies. The oligomer species had a sub‐nanometer height and little capability of aggregation to amyloid fibrils. Importantly, the toxicity of the oligomer species was significantly lower than that of native Aβ oligomers. These insights will be useful for further refinement of cyclic‐KLVFF‐based aggregation inhibitors.     

Membrane reactor immobilized with palladium‐loaded polymer nanogel for continuous‐flow Suzuki coupling reaction

A catalytic membrane reactor, which was immobilized with palladium‐loaded nanogel particles (NPs), was developed for continuous‐flow Suzuki coupling reaction. Palladium‐loaded membranes were prepared by immobilization of NPs, adsorption of palladium ions, and reduction into palladium(0). The presence of palladium in the membrane was confirmed by the scanning electron microscopy; palladium aggregation was not observed. The catalytic activity of the membrane reactor in continuous‐flow Suzuki coupling reaction was approximately double that of a comparable reactor in which palladium ions were directly adsorbed onto an aminated membrane. This was attributed to the formation of small palladium particles. The reusability in the continuous‐flow system was higher than that in a batch system, and the palladium‐loaded membrane reactor had high long‐term stability. © 2014 American Institute of Chemical Engineers AIChE J, 61: 582–589, 2015     

Not Oligomers but Amyloids are Cytotoxic in the Membrane‐Mediated Amyloidogenesis of Amyloid‐β Peptides

The formation of neurotoxic aggregates by amyloid‐β peptide (Aβ) is considered to be a key step in the onset of Alzheimer's disease. It is widely accepted that oligomers are more neurotoxic than amyloid fibrils in the aqueous‐phase aggregation of Aβ. Membrane‐mediated amyloidogenesis is also relevant to the pathology, although the relationship between the aggregate size and cytotoxicity has remained elusive. Here, aggregation processes of Aβ on living cells and cytotoxic events were monitored by fluorescence techniques. Aβ formed amyloids after forming oligomers composed of ≈10 Aβ molecules. The formation of amyloids was necessary to activate apoptotic caspase‐3 and reduce the ability of the cell to proliferate; this indicated that amyloid formation is a key event in Aβ‐induced cytotoxicity.     

Studies on the δ‐form complex in the syndiotactic polystyrene–organic molecule system. II. Structural investigation of the δ‐form with p‐ and m‐xylene isomers

Syndiotactic polystyrene (sPS) membranes containing different mole fractions of p‐xylene were prepared by a solution‐casting procedure. Complex formation between sPS and xylene was studied by thermogravimetric analysis and Fourier transform infrared spectroscopy. The stability and desorption behavior of the sPS–guest solvent and phase transitions were studied by differential scanning calorimetry. The formation of the δ‐form complex in the presence of different mole fractions of xylene isomers was analyzed and confirmed. The mole fraction of p‐xylene in the dried membrane was found to be higher than that of the corresponding mole fraction in the isomer solvent solution used for casting. This was attributed to the preferential complexing ability of p‐xylene with sPS. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 2882–2887, 2003     

Cholesterol: A Key in the Pathogenesis of Alzheimer's Disease

Herein we highlight recent advances in our understanding of the role of cholesterol in Alzheimer′s disease (AD). It has been proposed that cholesterol could enhance the risk of AD, and the interaction between cholesterol and amyloid‐β peptide 42 (Aβ42) has been studied extensively, yet until recently, the specific interaction mechanisms between them and how this affects Aβ42 aggregation had not yet been fully explored and had remained ambiguous. Vendruscolo and co‐workers addressed these issues in their recent article entitled “Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes” (Habchi et al., Nat. Chem. 2018 , 10, 673). In this article, the authors revealed the mechanism behind cholesterol‐catalyzed Aβ42 aggregation, providing the potential to address the molecular origins of AD, thereby opening a new avenue for effective AD therapy.     

Super‐Resolution Imaging and Quantitative Analysis of Membrane Protein/Lipid Raft Clustering Mediated by Cell‐Surface Self‐Assembly of Hybrid Nanoconjugates

Super‐resolution imaging was used to quantify organizational changes in the plasma membrane after treatment with hybrid nanoconjugates. The nanoconjugates crosslinked CD20 on the surface of malignant B cells, thereby inducing apoptosis. Super‐resolution images were analyzed by using pair‐correlation analysis to determine cluster size and to count the average number of molecules in the clusters. The role of lipid rafts was investigated by pre‐treating cells with a cholesterol chelator and actin destabilizer to prevent lipid raft formation. Lipid raft cluster size correlated with apoptosis induction after treatment with the nanoconjugates. Lipid raft clusters had radii of ～200 nm in cells treated with the hybrid nanoconjugates. Super‐resolution images provided precise molecule location coordinates that could be used to determine density of bound conjugates, cluster size, and number of molecules per cluster.     

Effects of γ‐aminopropyltriethoxylsilane on morphological characteristics of hybrid nylon‐66‐based membranes before electron beam irradiation

Nylon‐66 is a typical semicrystalline polymer that can be crosslinked using crosslinking agents and electron beam irradiation. Hybrid nylon‐66‐based membranes are more porous but denser compared to the pure nylon‐66 membrane. Besides that, hybrid nylon‐66 membranes exhibit higher water uptake and severe swelling in water. Si/nylon‐66 membranes were prepared by adding γ‐aminopropyltriethoxylsilane (APTEOS). Crosslinked silica in nylon‐66 membranes is confirmed with high gel content and Fourier transform infrared peaks, but XRD results showed that there is a low crystalline degree in these membranes. The thermal stability of hybrid nylon‐66 membranes is also less affected by APTEOS. The crosslinking agent only improves storage modulus in hybrid nylon‐66 membranes. After irradiation, it is learned that APTEOS improves separation performance of nylon‐66 membranes. However, excessive APTEOS causes the ratio of effective thickness over porosity (Δx/A_k) reduces significantly resulting a lower permeability membrane. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Structural Features and Toxicity of α-Synuclein Oligomers Grown in the Presence of DOPAC

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson’s disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.     

Polymer electrolyte membranes prepared by EB‐crosslinking of sulfonated poly(ether ether ketone) with 1,4‐butanediol

Crosslinked sulfonated poly(ether ether ketone) (SPEEK) membranes were prepared through the electron beam (EB)‐irradiation crosslinking of SPEEK/1,4‐butanediol under various irradiation conditions and used as a proton exchange membrane (PEM) for fuel cell applications. The crosslinked membranes were characterized by gel fraction, a universal testing machine (UTM), dynamic mechanical analysis (DMA), and small‐angle X‐ray scattering (SAXS). The gel fraction of the crosslinked membranes was used to estimate the degree of crosslinking, and the gel fraction was found to be increased with an increase of the crosslinker content and EB‐absorbed dose. The UTM results indicate that a brittle EB‐crosslinked membrane becomes more flexible with an increase in the crosslinker content. The DMA results show that the EB‐crosslinked membranes have well‐developed ionic aggregation regions and the cluster T_g of membranes decrease with an increase in the 1,4‐butanediol crosslinker content. The SAXS results show that the Bragg and persistence distance of crosslinked membranes increase with an increase in the crosslinker content. The proton conductivities of the EB‐crosslinked membranes were more than 9 × 10^?2 S/cm. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41760.     

A Combined High‐Resolution Mass Spectrometric and in silico Approach for the Characterisation of Small Ligands of β2‐Microglobulin

β₂‐Microglobulin (β₂‐m) is a protein responsible for a severe complication of long‐term hemodialysis, known as dialysis‐related amyloidosis, in which initial β₂‐m misfolding leads to amyloid fibril deposition, mainly in the skeletal tissue. Whereas much attention is paid to understanding the complex mechanism of amyloid formation, the evaluation of small molecules that may bind β₂‐m and possibly inhibit the aggregation process is still largely unexplored mainly because the protein lacks a specific active site. Based on our previous findings, we selected a pilot set of sulfonated molecules that are known to either bind or not to the protein, including binders that are anti‐amyloidogenic. We show how a complementary approach, using high‐resolution mass spectrometry and in silico studies, can offer rapid and precise information on affinity, as well as insight into the structural requisites that favour or disfavour the inhibitory activity. Overall, this approach can be used for predictive purposes and for a rapid screening of fibrillogenesis inhibitors.     

Selective separation of water from aqueous alcohol mixtures through functionalized syndiotactic poly(styrene‐co‐4‐methylstyrene) membranes by pervaporation

The pervaporation performances of a series of functionalized syndiotactic poly(styrene‐co‐4‐methylstyrene) (SPSM) membranes for various alcohol mixtures were investigated. The syndiotactic polystyrene copolymers, poly(styrene‐co‐4‐methylstyrene) (SPSM), were prepared by styrene with 4‐methylstyrene using a Cp*Ti(OCH₃)₃/methyl aluminoxane (metallocene/MAO) catalyst. The effect of functionalization on the thermal properties and polymer structure of the SPSM membranes were also investigated. The crystallinity of the functionalized SPSM membrane is lower than that of the unfunctionalized SPSM membranes. The water molecules preferentially permeate through the SPSM membranes. Compared with unfunctionalized SPSM membranes, the functionalized SPSM membrane effectively increases the membrane formation performances and the pervaporation performances. The optimun pervaporation performance (a separation factor of 510 and permeation rate of 220 g/m²h) was obtained by the bromination of SPSM (SPSMBr) membrane with a 90 wt % aqueous ethanol solution. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 2247–2254, 2002