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Amyloid β peptide (Aβ) aggregation and deposition are considered the main causes of Alzheimer's disease. In a previous study, we demonstrated that anionic Zn-phthalocyanine (ZnPc) can interact with the Aβ peptide and inhibit the fibril-formation process. However, due to the inability of anionic ZnPc to cross the intact blood–brain barrier, we decided to explore the interaction of cationic methylated Zn-phthalocyanine (cZnPc) with the peptide. Using a ThT fluorescence assay, we observed that cZnPc dose-dependently and time-dependently inhibited Aβ1-42 fibril levels under in vitro fibril-formation conditions. Electron microscopy revealed that it caused Aβ1-42 peptides to form small aggregates. Western blotting and dot immunoblot oligomer experiments demonstrated that cZnPc increased rather than decreased the levels of oligomers from the very early stages of incubation. A binding assay confirmed that cZnPc could bind with the peptide. Docking simulations indicated that the oligomer species of Aβ1-42 had a higher ability to interact with cZnPc. ANS fluorescence assay results indicated that cZnPc did not affect the hydrophobicity of the peptide. However, cZnPc significantly increased intrinsic tyrosine fluorescence of the peptide after 8 h of incubation in fibril-formation conditions. Importantly, cell culture experiments demonstrated that cZnPc did not exhibit any toxicity up to a concentration of 10 µM. Instead, it protected a neuronal cell line from Aβ1-42-induced toxicity. Thus, our results suggest that cZnPc can affect the aggregation process of Aβ1-42, rendering it non-toxic, which could be crucial for the therapy of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Human amylin (hIAPP) is found in the form of amyloid deposits within the pancreatic cells of nearly all patients diagnosed with type 2 diabetes mellitus (T2DM). However, rat amylin (rIAPP) and pramlintide - hIAPP analogs - are both non-toxic and non-amyloidogenic. Their primary sequences exhibit only slight variations in a few amino acid residues, primarily concentrated in the central region, spanning residues 20 to 29. This inspired us to study this fragment and investigate the impact on the aggregation properties of substituting residues within the central region of amylin and its analogs. Six fragments derived from amylin have undergone comprehensive testing against various metal ions by implementing a range of analytical techniques, including Nuclear Magnetic Resonance (NMR) spectroscopy, Thioflavin T (ThT) assays, Atomic Force Microscopy (AFM), and cytotoxicity assays. These methodologies serve to provide a thorough understanding of how the substitutions and interactions with metal ions impact the aggregation behavior of amylin and its analogs. [ABSTRACT FROM AUTHOR]

Interfering with the aggregation of the protein alpha-synuclein (aSyn) has been a topic of research for > 25 years, as this protein is found in pathognomonic protein aggregates in the brain of patients with Parkinson's disease and related synucleinopathies. This study aimed to characterize the adsorption of aSyn and the inhibition of aSyn fibril formation by nanocellulose (NC) and NC coated with gold atoms (NCCGAs) using by both in silico and laboratory approaches. First, using the HDOCK server and Ascalaph designer software we assessed the possible molecular interaction of partial aSyn (36–55) and full length aSyn (1–140) when exposed to NC and NCCGAs. We then used acid hydrolysis to synthesize NC and then it reacted with HAuCl4 to generate NCCGAs. The adsorption of aSyn by NC and NCCGAs was analyzed at 280 nm and the inhibition of aSyn fibril formation was assessed using Thioflavin T. By comparing the docking results, we found that NCCGAs have more and better adsorption to partial/full length aSyn than NC. Also, molecular dynamics simulations showed that adsorbed partial/full length aSyn display stable interactions and that this increases over time. Experimentally, the adsorption of aSyn and the inhibition of aSyn fibril formation were observed upon exposure to NC or NCCGAs. Although both NC and NCCGAs adsorbed aSyn, NCCGAs had a higher capability (P > 0.05). The same pattern was observed for the inhibition of aSyn fibril formation. [ABSTRACT FROM AUTHOR]

Human amylin (hIAPP) is found in the form of amyloid deposits within the pancreatic cells of nearly all patients diagnosed with type 2 diabetes mellitus (T2DM). However, rat amylin (rIAPP) and pramlintide - hIAPP analogs - are both non-toxic and non-amyloidogenic. Their primary sequences exhibit only slight variations in a few amino acid residues, primarily concentrated in the central region, spanning residues 20 to 29. This inspired us to study this fragment and investigate the impact on the aggregation properties of substituting residues within the central region of amylin and its analogs. Six fragments derived from amylin have undergone comprehensive testing against various metal ions by implementing a range of analytical techniques, including Nuclear Magnetic Resonance (NMR) spectroscopy, Thioflavin T (ThT) assays, Atomic Force Microscopy (AFM), and cytotoxicity assays. These methodologies serve to provide a thorough understanding of how the substitutions and interactions with metal ions impact the aggregation behavior of amylin and its analogs.

Milk whey proteins, which are derived from skim milk through membrane filtration, exhibit valuable functional properties when transformed into a fibrillar form. This conversion enhances their suitability for various applications, including thickening, gelling, emulsification, and foaming. However, reported fibrillation methods have longer heating times, which may not be economical for the dairy industry. To address these challenges, the current study was undertaken with the objective of reducing the time required for fibril formation. In this study, 2% milk whey protein isolate (mWPI) solution at pH 2 was heated with static and stirring heating conditions at 80 °C for 20 h to convert milk whey proteins into fibrils. Fibrils were observed using the thioflavin T value, transmission electron microscopy, Tricine SDS-PAGE, rheology, and protein oxidation. Results suggest that stirring heating conditions with 14 h heating time produced fibrils with good morphology compared to static heating, showing a 6 h reduction compared to an earlier reported 80 °C for 20 h heating time. Also, stirring heating produced a uniform and homogeneous fibril solution compared to the static heating method. Gentle stirring during heating can also help to scale up fibril production in an industrial setup. The fibrillation method with processing intervention will help to produce fibrils with enhanced functionality at the pilot and industrial scales. [ABSTRACT FROM AUTHOR]

Collagen is an important material for biomedical research, but using mammalian tissue-derived collagen carries the risk of zoonotic disease transmission. Marine organisms, such as farmed tilapia, have emerged as a safe alternative source of collagen for biomedical research. However, the tilapia collagen products for biomedical research are rare, and their biological functions remain largely unexamined. In this study, we characterized a commercial tilapia skin collagen using SDS-PAGE and fibril formation assays and evaluated its effects on skin fibroblast adhesion, proliferation, and migration, comparing it with commercial collagen from rat tails, porcine skin, and bovine skin. The results showed that tilapia skin collagen is a type I collagen, similar to rat tail collagen, and has a faster fibril formation rate and better-promoting effects on cell migration than porcine and bovine skin collagen. We also confirmed its application in a 3D culture for kidney cells' spherical cyst formation, fibroblast-induced gel contraction, and tumor spheroid interfacial invasion. Furthermore, we demonstrated that the freeze-dried tilapia skin collagen scaffold improved wound closure in a mouse excisional wound model, similar to commercial porcine or bovine collagen wound dressings. In conclusion, tilapia skin collagen is an ideal biomaterial for biomedical research. [ABSTRACT FROM AUTHOR]

Amyloid β peptide (Aβ) aggregation and deposition are considered the main causes of Alzheimer’s disease. In a previous study, we demonstrated that anionic Zn-phthalocyanine (ZnPc) can interact with the Aβ peptide and inhibit the fibril-formation process. However, due to the inability of anionic ZnPc to cross the intact blood–brain barrier, we decided to explore the interaction of cationic methylated Zn-phthalocyanine (cZnPc) with the peptide. Using a ThT fluorescence assay, we observed that cZnPc dose-dependently and time-dependently inhibited Aβ1-42 fibril levels under in vitro fibril-formation conditions. Electron microscopy revealed that it caused Aβ1-42 peptides to form small aggregates. Western blotting and dot immunoblot oligomer experiments demonstrated that cZnPc increased rather than decreased the levels of oligomers from the very early stages of incubation. A binding assay confirmed that cZnPc could bind with the peptide. Docking simulations indicated that the oligomer species of Aβ1-42 had a higher ability to interact with cZnPc. ANS fluorescence assay results indicated that cZnPc did not affect the hydrophobicity of the peptide. However, cZnPc significantly increased intrinsic tyrosine fluorescence of the peptide after 8 h of incubation in fibril-formation conditions. Importantly, cell culture experiments demonstrated that cZnPc did not exhibit any toxicity up to a concentration of 10 µM. Instead, it protected a neuronal cell line from Aβ1-42-induced toxicity. Thus, our results suggest that cZnPc can affect the aggregation process of Aβ1-42, rendering it non-toxic, which could be crucial for the therapy of Alzheimer’s disease.

Milk whey proteins, which are derived from skim milk through membrane filtration, exhibit valuable functional properties when transformed into a fibrillar form. This conversion enhances their suitability for various applications, including thickening, gelling, emulsification, and foaming. However, reported fibrillation methods have longer heating times, which may not be economical for the dairy industry. To address these challenges, the current study was undertaken with the objective of reducing the time required for fibril formation. In this study, 2% milk whey protein isolate (mWPI) solution at pH 2 was heated with static and stirring heating conditions at 80 °C for 20 h to convert milk whey proteins into fibrils. Fibrils were observed using the thioflavin T value, transmission electron microscopy, Tricine SDS-PAGE, rheology, and protein oxidation. Results suggest that stirring heating conditions with 14 h heating time produced fibrils with good morphology compared to static heating, showing a 6 h reduction compared to an earlier reported 80 °C for 20 h heating time. Also, stirring heating produced a uniform and homogeneous fibril solution compared to the static heating method. Gentle stirring during heating can also help to scale up fibril production in an industrial setup. The fibrillation method with processing intervention will help to produce fibrils with enhanced functionality at the pilot and industrial scales.

Collagen is an important material for biomedical research, but using mammalian tissue-derived collagen carries the risk of zoonotic disease transmission. Marine organisms, such as farmed tilapia, have emerged as a safe alternative source of collagen for biomedical research. However, the tilapia collagen products for biomedical research are rare, and their biological functions remain largely unexamined. In this study, we characterized a commercial tilapia skin collagen using SDS-PAGE and fibril formation assays and evaluated its effects on skin fibroblast adhesion, proliferation, and migration, comparing it with commercial collagen from rat tails, porcine skin, and bovine skin. The results showed that tilapia skin collagen is a type I collagen, similar to rat tail collagen, and has a faster fibril formation rate and better-promoting effects on cell migration than porcine and bovine skin collagen. We also confirmed its application in a 3D culture for kidney cells’ spherical cyst formation, fibroblast-induced gel contraction, and tumor spheroid interfacial invasion. Furthermore, we demonstrated that the freeze-dried tilapia skin collagen scaffold improved wound closure in a mouse excisional wound model, similar to commercial porcine or bovine collagen wound dressings. In conclusion, tilapia skin collagen is an ideal biomaterial for biomedical research.

This paper proposes a new paradigm for the biophysical concept of measuring the affinity of molecular complexes, based on a matrix representation of biological interactions and subsequent numerical analysis of the stability of this matrix. Our numerical criterion of stability (lg(cond(W))) correlates well with experimental values such as K d and IC 5 0 as well as with experimental data of aggregation kinetics in studies of amyloid peptides. The main goal of this work is to reduce the cost of biochemical experiments by obtaining preliminary information on the interaction of chemical compounds. The paper also presents our numerical calculations in comparison with a large amount of experimental data on the examples of binding of small chemical molecules gefitinib, erlotinib, imatinib, naquatinib, and CO-1686 with proteins, protein–peptide interactions of the Bcl-2 protein family, antibody–antigen CD20–rituximab, and aggregation of amyloid peptides. The description of the software package that implements the presented algorithm is given on the website: https://binomlabs.com/. [ABSTRACT FROM AUTHOR]

Acid-solubilized (ASC) and pepsin-solubilized collagen (PSC) extracted at 4 °C (ASC-4 and PSC-4), 12 °C (ASC-12 and PSC-12), and 20 °C (ASC-20 and PSC-20) from the skin of farmed pufferfish (Takifugu obscurus) was characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier-transform infrared spectroscopy (FTIR), and fibril-forming tests. The results indicate that extraction at 12 °C can effectively improve the extraction efficiency of natural collagen compared with extraction at 4 °C. However, extraction at 20 °C results in a decrease in molecular integrity, thus, inducing the resultant collagen to degrade or even lose fibril-forming ability. Transmission electron microscope (TEM) images revealed that ASC-4, PSC-4, ASC-12, and PSC-12 can assemble into fibrils with D-periodicities, and ASC-20 associated into molecular aggregates alongside partial D-banded fibrils, while no well-defined fibrils were observed in PSC-20. Scanning electron microscope (SEM) analysis confirmed the well-defined fibril morphologies of ASC-4, PSC-4, ASC-12, and PSC-12 with imino acid contents between 190.0 and 197.8 residues/1000 residues. The denaturation temperature of ASC-4, PSC-4, ASC-12 and PSC-12 was 30.0, 27.6, 25.9 and 22.7 °C, respectively. This study indicates that ASC and PSC extracted at 4 °C and 12 °C could be alternatives to terrestrial collagens for industrial applications. [ABSTRACT FROM AUTHOR]

The possibility of replacing the very time and resource demanding salting out (SO) method with isoelectric precipitation (IP) during collagen extraction from common starfish and lumpfish was investigated. The effect of IP on yield, structural and functional properties of the collagens was therefore compared with SO. Application of IP resulted in a higher or similar collagen mass yield compared with SO from starfish and lumpfish, respectively. However, the purity of collagens recovered with IP was lower than those recovered with SO. Replacing SO with IP did not affect polypeptide pattern and tropohelical structural integrity of collagen from the two resources as revealed with SDS-PAGE and FTIR analysis. Thermal stability and fibril formation capacity of collagens recovered with IP were also well preserved. Overall, the results showed that the IP can be a promising resource smart alternative for the classic SO precipitation during collagen extraction from marine resources.

Many proteins can undergo pathological conformational changes that result in the formation of amyloidogenic fibril structures. Various neurodegenerative diseases are associated with such pathological fibril formation of specific proteins. Transthyretin (TTR) is a tetrameric globular transport protein in the blood plasma that can dissociate, unfold, and form long and stable fibrils. Many TTR mutations are known that promote (TTR) amyloidosis and cause severe diseases. TTR amyloidosis has been studied extensively using biochemical methods and structures of various mutations in the globular form have been characterized. Recently, also the structure of a TTR fibril has been determined. In an effort to better understand why some mutations increase or decrease the tendency of amyloid formation, we have applied a combined molecular dynamics and continuum solvent approach to calculate the energetic influence of residue changes in the globular versus fibril form. For 29 out of 36 tested TTR single residue mutations, the approach correctly predicts the increased or decreased tendency for amyloidosis allowing us also to elucidate the origins of the tendency. We find that indeed the destabilization of the globular monomer or changes in dimer and tetramer stability due to mutation has a dominant influence on the amyloidogenic tendency. The continuum solvent model predicts a significantly more favorable mean energy per residue of the fibril form compared to the globular form. This effect is only slightly modulated by single‐point mutations preserving the energetic preference for fibril formation upon protein unfolding. It explains why no correlation between experimental amyloidosis and calculated change in fibril stability was observed. [ABSTRACT FROM AUTHOR]

The study of protein aggregation, and amyloidosis in particular, has gained considerable interest in recent times. Several neurodegenerative diseases, such as Alzheimer's (AD) and Parkinson's (PD) show a characteristic buildup of proteinaceous aggregates in several organs, especially the brain. Despite the enormous upsurge in research articles in this arena, it would not be incorrect to say that we still lack a crystal-clear idea surrounding these notorious aggregates. In this review, we attempt to present a holistic picture on protein aggregation and amyloids in particular. Using a chronological order of discoveries, we present the case of amyloids right from the onset of their discovery, various biophysical techniques, including analysis of the structure, the mechanisms and kinetics of the formation of amyloids. We have discussed important questions on whether aggregation and amyloidosis are restricted to a subset of specific proteins or more broadly influenced by the biophysiochemical and cellular environment. The therapeutic strategies and the significant failure rate of drugs in clinical trials pertaining to these neurodegenerative diseases have been also discussed at length. At a time when the COVID-19 pandemic has hit the globe hard, the review also discusses the plausibility of the far-reaching consequences posed by the virus, such as triggering early onset of amyloidosis. Finally, the application(s) of amyloids as useful biomaterials has also been discussed briefly in this review. [ABSTRACT FROM AUTHOR]

We analyzed the kinetics and thermodynamics of amyloid fibril formation by the amyloidogenic variant of apolipoprotein A-I and αsynuclein based on the Finke-Watzky two-step model of a homogeneous nucleation followed by autocatalytic fibril growth. The results demonstrated that in apoA-I, the nucleation process is enthalpically unfavorable but entropically favorable likely because of the desolvation of hydrophobic regions in the molecule, whereas the nucleation of α-synuclein is enthalpically and entropically unfavorable. Interestingly, Parkinson’s disease-related mutation and C-terminal truncation in α-synuclein were found to decrease the enthalpic barrier for nucleation, thereby promoting autocatalytic nucleation in fibril formation. [ABSTRACT FROM AUTHOR]

Amyloid-β peptide (Aβ) aggregates are known to be correlated with pathological neurodegenerative diseases. The fibril formation process of such peptides in solution is influenced by several factors, such as the ionic strength of the buffer, concentration, pH, and presence of other molecules, just to mention a few. In this paper, we report a detailed analysis of in vitro Aβ42 fibril formation in the presence of cortisol at different relative concentrations. The thioflavin T fluorescence assay allowed us to monitor the fibril formation kinetics, while a morphological characterization of the aggregates was obtained by atomic force microscopy. Moreover, infrared absorption spectroscopy was exploited to investigate the secondary structure changes along the fibril formation path. Molecular dynamics calculations allowed us to understand the intermolecular interactions with cortisol. The combined results demonstrated the influence of cortisol on the fibril formation process: indeed, at cortisol-Aβ42 concentration ratio (ρ) close to 0.1 a faster organization of Aβ42 fragments into fibrils is promoted, while for ρ = 1 the formation of fibrils is completely inhibited. [ABSTRACT FROM AUTHOR]

Islet amyloid polypeptide (IAPP), or amylin, has been identified as a key factor in the development of type 2 diabetes (T2D). IAPP aggregates, which form amyloid fibrils, contribute to cytotoxicity of the pancreatic β-cells, resulting in loss of function and subsequent reduction in insulin production. As a result, surviving β-cells overcompensate for this reduction of insulin production, further contributing to the loss of function because of increased stress, thus leading to insulin resistance. Endogenously, IAPP monomers function in a variety of roles; however, aggregation renders them non-functional. The use of naturally occurring compounds, including peptides and phytochemicals, has been explored as a way to mitigate or inhibit IAPP fibril formation. This review discusses the structure, endogenous roles and mechanism of IAPP fibril formation, recent advances on inhibitors of IAPP fibril formation, and new insights on the future development and application of food-derived inhibitors towards T2D management.

The fungal cell wall and cell membrane are an important target for antifungal therapies, and a needle-like cell wall or membrane disruption may be an entirely novel antifungal mode of action. In this work, we show how the coordination of Zn(II) triggers the antifungal properties of shepherin II, a glycine- and histidine-rich antimicrobial peptide from the root of Capsella bursa-pastoris. We analyze Cu(II) and Zn(II) complexes of this peptide using experimental and theoretical methods, such as: mass spectrometry, potentiometry, UV–Vis and CD spectroscopies, AFM imaging, biological activity tests and DFT calculations in order to understand the correlation between their metal binding mode, structure, morphology and biological activity. We observe that Zn(II) coordinates to Shep II and causes a structural change, resulting in fibril formation, what has a pronounced biological consequence – a strong anticandidal activity. This phenomenon was observed neither for the peptide itself, nor for its copper(II) complex. The Zn(II) – shepherin II complex can be considered as a starting point for further anticandidal drug discovery, which is extremely important in the era of increasing antifungal drug resistance. The fungal cell wall and cell membrane are an important target for antifungal therapies and a needle-like cell wall or membrane disruption may be an entirely novel antifungal mode of action. The coordination of Zn(II) to shepherin II, a glycine- and histidine-rich peptide from the Capsella bursa-pastoris roots, causes a structural and morphological change of the peptide, leading to fibril formation that results in anticandidal activity of Zn(II) – Shep II complex. [Display omitted] • Shep II needs Zn(II) as a trigger of its morphological change and antifungal activity. • An entirely novel mode of action of antifungal drugs - the mechanism of the fibrils. • The Zn(II) - Shep II complex as a starting point for further anticandidal drug discovery examination. • Metal complexes of AMPs are promising candidates for antimicrobial therapies. [ABSTRACT FROM AUTHOR]

Serum amyloid A (SAA) is actively involved in such pathological processes as atherosclerosis, rheumatoid arthritis, cancer and Alzheimer's disease by its aggregation. One of the factors that can attenuate its aggregation and so affects its physiological role is its interactions with glycosminoglycans (GAGs), linear anionic periodic polysaccharides. These molecules located in the extracellular matrix of the cell are highly variable in their chemical composition and sulfation patterns. Despite the available experimental evidence of SAA-GAG interactions, no mechanistic details at atomic level have been reported for these systems so far. In our work we aimed to apply diverse computational tools to characterize SAA-GAG complexes formation and to answer questions about their potential specificity, energetic patterns, particular SAA residues involved in these interactions, favourable oligomeric state of the protein and the potential influence of GAGs on SAA aggregation. Molecular docking, conventional and replica exchange molecular dynamics approaches were applied to corroborate the experimental knowledge and to propose the corresponding molecular models. SAA-GAG complex formation was found to be electrostatics-driven and rather unspecific of a GAG sulfation pattern, more favorable for the dimer than for the monomer when binding to a short GAG oligosaccharide through its N-terminal helix, potentially contributing to the unfolding of this helix, which could lead to the promotion of the protein aggregation. The data obtained add to the specific knowledge on SAA-GAG systems and deepen the general understanding of protein-GAG interactions that is of a considerable value for the development of GAG-based approaches in a broad theurapeutic context.

Acid-solubilized (ASC) and pepsin-solubilized collagen (PSC) extracted at 4 °C (ASC-4 and PSC-4), 12 °C (ASC-12 and PSC-12), and 20 °C (ASC-20 and PSC-20) from the skin of farmed pufferfish (Takifugu obscurus) was characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier-transform infrared spectroscopy (FTIR), and fibril-forming tests. The results indicate that extraction at 12 °C can effectively improve the extraction efficiency of natural collagen compared with extraction at 4 °C. However, extraction at 20 °C results in a decrease in molecular integrity, thus, inducing the resultant collagen to degrade or even lose fibril-forming ability. Transmission electron microscope (TEM) images revealed that ASC-4, PSC-4, ASC-12, and PSC-12 can assemble into fibrils with D-periodicities, and ASC-20 associated into molecular aggregates alongside partial D-banded fibrils, while no well-defined fibrils were observed in PSC-20. Scanning electron microscope (SEM) analysis confirmed the well-defined fibril morphologies of ASC-4, PSC-4, ASC-12, and PSC-12 with imino acid contents between 190.0 and 197.8 residues/1000 residues. The denaturation temperature of ASC-4, PSC-4, ASC-12 and PSC-12 was 30.0, 27.6, 25.9 and 22.7 °C, respectively. This study indicates that ASC and PSC extracted at 4 °C and 12 °C could be alternatives to terrestrial collagens for industrial applications.

The study of protein aggregation, and amyloidosis in particular, has gained considerable interest in recent times. Several neurodegenerative diseases, such as Alzheimer’s (AD) and Parkinson’s (PD) show a characteristic buildup of proteinaceous aggregates in several organs, especially the brain. Despite the enormous upsurge in research articles in this arena, it would not be incorrect to say that we still lack a crystal-clear idea surrounding these notorious aggregates. In this review, we attempt to present a holistic picture on protein aggregation and amyloids in particular. Using a chronological order of discoveries, we present the case of amyloids right from the onset of their discovery, various biophysical techniques, including analysis of the structure, the mechanisms and kinetics of the formation of amyloids. We have discussed important questions on whether aggregation and amyloidosis are restricted to a subset of specific proteins or more broadly influenced by the biophysiochemical and cellular environment. The therapeutic strategies and the significant failure rate of drugs in clinical trials pertaining to these neurodegenerative diseases have been also discussed at length. At a time when the COVID-19 pandemic has hit the globe hard, the review also discusses the plausibility of the far-reaching consequences posed by the virus, such as triggering early onset of amyloidosis. Finally, the application(s) of amyloids as useful biomaterials has also been discussed briefly in this review.

Objectives: The heterogeneity of Amyloid‐beta (Aβ) plaque load in patients with Alzheimer's disease (AD) has puzzled neuropathology. Since brain Aβ plaque load does not correlate with cognitive decline, neurotoxic soluble Aβ oligomers have been championed as disease‐causing agents in early AD. So far, investigating molecular interactions between soluble oligomeric Aβ and insoluble Aβ in vivo has been difficult because of the abundance of Aβ oligomer species and the kinetic equilibrium in which they coexist. Here, we investigated whether Aβ plaque heterogeneity relates to interactions of different Aβ conformers. Materials and Methods: We took advantage of transgenic mice that generate exclusively Aβ dimers (tgDimer mice) but do not develop Aβ plaques or neuroinflammation during their lifetime, crossed them to the transgenic CRND8 mice that develop plaques after 90 days and measured Aβ plaque load using immunohistochemical and biochemical assays. Furthermore, we performed in vitro thioflavin T (ThT) aggregation assays titrating synthetic Aβ42‐S8C dimers into fibril‐forming synthetic Aβ42. Results: We observed a lower number of Aβ plaques in the brain of double transgenic mice compared to tgCRND8 mice alone while the average plaque size remained unaltered. Corroborating these in vivo findings, synthetic Aβ‐S8C dimers inhibited fibril formation of wild‐type Aβ also in vitro, seen by an increased half‐time in the ThT assay. Conclusions: Our study indicates that Aβ dimers directly interfere with Aβ fibril formation in vivo and in vitro. The variable interaction of Aβ dimers with insoluble Aβ seeds could thus contribute to the heterogeneity of Aβ plaque load in AD patients. [ABSTRACT FROM AUTHOR]

One of the earliest events in amyloid β-protein (Aβ) self-association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used "click peptide" chemistry to quasi-synchronously create Aβ42 from 26-O-acyliso-Aβ42 (iAβ42) through a pH jump from 3 to 7.4. We also synthesized Nα-acetyl-Ser26-iAβ42 (Ac-iAβ42), which cannot undergo O→N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (thioflavin T, CD), hydrodynamic radius (RH), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy-mass spectrometry) were quite consistent. A rank order of Ac-iAβ42>iAβ42>Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into "pure" Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies.

Amyloid-β peptide (Aβ) aggregates are known to be correlated with pathological neurodegenerative diseases. The fibril formation process of such peptides in solution is influenced by several factors, such as the ionic strength of the buffer, concentration, pH, and presence of other molecules, just to mention a few. In this paper, we report a detailed analysis of in vitro Aβ42 fibril formation in the presence of cortisol at different relative concentrations. The thioflavin T fluorescence assay allowed us to monitor the fibril formation kinetics, while a morphological characterization of the aggregates was obtained by atomic force microscopy. Moreover, infrared absorption spectroscopy was exploited to investigate the secondary structure changes along the fibril formation path. Molecular dynamics calculations allowed us to understand the intermolecular interactions with cortisol. The combined results demonstrated the influence of cortisol on the fibril formation process: indeed, at cortisol-Aβ42 concentration ratio (ρ) close to 0.1 a faster organization of Aβ42 fragments into fibrils is promoted, while for ρ = 1 the formation of fibrils is completely inhibited.

Islet amyloid polypeptide (IAPP) fibrillation has been commonly associated with the exacerbation of type 2 diabetes prognosis. Consequently, inhibition of IAPP fibrillation to minimize β-cell cytotoxicity is an important approach towards β-cell preservation and type 2 diabetes management. In this study, we identified three tetrapeptides, TNGQ, MANT, and YMSV, that inhibited IAPP fibrillation. Using thioflavin T (ThT) fluorescence assay, circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and molecular docking, we evaluated the potential anti-fibrillation mechanism of the tetrapeptides. ThT fluorescence kinetics and microscopy as well as transmission electron microscopy showed that TNGQ was the most effective inhibitor based on the absence of normal IAPP fibrillar morphology. CD spectroscopy showed that TNGQ maintained the α-helical conformation of monomeric IAPP, while DLS confirmed the presence of varying fibrillation species. Molecular docking showed that TNGQ and MANT interact with monomeric IAPP mainly by hydrogen bonding and electrostatic interaction, with TNGQ binding at IAPP surface compared to YMSV, which had the highest docking score, but interact mainly through hydrophobic interaction in IAPP core. The highly polar TNGQ was the most active and appeared to inhibit IAPP fibrillation by disaggregation of preformed IAPP fibrils. These findings indicate the potential of TNGQ in the development of peptide-based anti-fibrillation and antidiabetic nutraceuticals.

A familial form of Alzheimer disease recently was described in a kindred in Osaka, Japan. This kindred possesses an amyloid β-protein (Aβ) precursor mutation within the Aβ coding region that results in the deletion of Glu22 (ΔE22). We report here results of studies of [ΔE22]Aβ40 and [ΔE22]Aβ42 that sought to elucidate the conformational dynamics, oligomerization behavior, fibril formation kinetics, fibril morphology, and fibril stability of these mutant peptides. Both [ΔE22]Aβ peptides had extraordinary β-sheet formation propensities. The [ΔE22]Aβ40 mutant formed β-sheet secondary structure elements ≈400-fold faster. Studies of β-sheet stability in the presence of fluorinated alcohol cosolvents or high pH revealed that the ΔE22 mutation substantially increased stability, producing a rank order of [ΔE22]Aβ42 >>Aβ42 > [ΔE22]Aβ40 > Aβ40. The mutation facilitated formation of oligomers by [ΔE22]Aβ42 (dodecamers and octadecamers) that were not observed with Aβ42. Both Aβ40 and Aβ42 peptides formed nebulous globular and small string-like structures immediately upon solvation from lyophilizates, whereas short protofibrillar and fibrillar structures were evident immediately in the ΔE22 samples. Determination of the critical concentration for fibril formation for the [ΔE22]Aβ peptides showed it to be ≈1/2 that of the wild type homologues, demonstrating that the mutations causes a modest increase in fibril stability. The magnitude of this increase, when considered in the context of the extraordinary increase in β-sheet propensity for the ΔE22 peptides, suggests that the primary biophysical effect of the mutation is to accelerate conformational changes in the peptide monomer that facilitate oligomerization and higher-order assembly.

The nucleation of Alzheimer-associated Aβ peptide monomers can be catalyzed by preexisting Aβ fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aβ42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the wellordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 μM Aβ42, 20 mM sodium phosphate, 200 μM EDTA, pH 6.8). [ABSTRACT FROM AUTHOR]

Nonmammalian collagens have attracted significant attention owing to their potential for use as a source of cell scaffolds for tissue engineering. Since the morphology of collagen fibrils controls cell proliferation and differentiation, its regulation is essential for fabricating scaffolds with desirable characteristics. In this study, we evaluated the effects of the phosphate ion (Pi) concentration on the characteristics of fibrils formed from swim bladder type I collagen (SBC) and skin type I collagen (SC) from the Bester sturgeon. An increase in the Pi concentration decreased the fibril formation rate, promoted the formation of thick fibrils, and increased the thermal stability of the fibrils for both SBC and SC. However, the SBC and SC fibrils exhibited different fibril formation rates, degrees of fibrillogenesis, morphologies, and denaturation temperatures for the same reaction conditions. Finally, by regulating the Pi concentration, various types of SBC and SC fibrils could be coated on cell culture wells, and fibroblasts could be cultured on them. The results showed that thin fibrils enhance fibroblast extension and proliferation, whereas thick fibrils restrain fibroblast extension but orient them in the same direction. The results of this study suggest that SBC fibrils, which exhibit diverse morphologies, are suitable for use as a novel scaffold material, whose characteristics can be tailored readily by varying the Pi concentration. • We evaluated the effects of Pi on in-vitro fibrillogenesis of sturgeon type I collagen. • Higher Pi concentrations limit the rate and degree of fibrillogenesis. • Thicker fibrils are formed at higher Pi concentrations. • Thin fibrils aid fibroblast extension while thick fibrils align them. • Both types of fibrils retard fibroblast proliferation. [ABSTRACT FROM AUTHOR]

Quasielastic light scattering spectroscopy (QLS) is an optical method for the determination of diffusion coefficients of particles in solution. Here we discuss the principles of QLS and explain how the distribution of particle sizes can be reconstructed from the measured correlation function of scattered light. Non-invasive observation of the temporal evolution of particle sizes provides a powerful tool for studying protein assembly. We illustrate practical applications of QLS with examples from studies of fibril formation of the amyloid beta-protein.

The self-assembly of amyloidogenic peptides and proteins into fibrillar structures has been intensively studied for several decades, because it seems to be associated with a number of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. Therefore, understanding the molecular mechanisms of this phenomenon is important for identifying an effective therapy for the corresponding diseases. Protein aggregation in living organisms very often takes place on surfaces like membranes and the impact of a surface on this process depends not only on the surface chemistry but also on its topology. Our goal was to develop a simple lattice model for studying the role of surface roughness in the aggregation kinetics of polypeptide chains and the morphology of aggregates. We showed that, consistent with the experiment, an increase in roughness slows down the fibril formation, and this process becomes inhibited at a very highly level of roughness. We predicted a subtle catalytic effect that a slightly rough surface promotes the self-assembly of polypeptide chains but does not delay it. This effect occurs when the interaction between the surface and polypeptide chains is moderate and can be explained by taking into account the competition between energy and entropy factors.

Takefumi Narita,1 Shunji Yunoki,1 Yoshimi Ohyabu,1 Naohisa Yahagi,2 Toshio Uraoka3 1Biotechnology Group, Tokyo Metropolitan Industrial Technology Research Institute, Koto-ku, 2Division of Research and Development for Minimally Invasive Treatment, Cancer Center, Keio University School of Medicine, Shinjuku-ku, 3Department of Gastroenterology, National Hospital Organization Tokyo Medical Center, Meguro-ku, Tokyo, Japan Abstract: We investigated the potential of collagen–genipin sols as biomaterials for treating artificial ulcers following endoscopic submucosal dissection. Collagen sol viscosity increased with condensation, allowing retention on tilted ulcers before gelation and resulting in collagen gel deposition on whole ulcers. The 1.44% collagen sols containing genipin as a crosslinker retained sol fluidity at 23°C for >20 min, facilitating endoscopic use. Collagen sols formed gel depositions on artificial ulcers in response to body temperature, and high temperature responsiveness of gelation because of increased neutral phosphate buffer concentration allowed for thick gel deposition on tilted ulcers. Finally, histological observations showed infiltration of gels into submucosal layers. Taken together, the present data show that genipin-induced crosslinking significantly improves the mechanical properties of collagen gels even at low genipin concentrations of 0.2–1 mM, warranting the use of in situ gelling collagen–genipin sols for endoscopic treatments of gastrointestinal ulcers. Keywords: collagen, genipin, in-situ gel, fibril formation, ulcer dressing

The interaction of reducing sugars with proteins leads to the formation of advanced glycation end products (AGE) and reactive oxidative species (ROS). ROS peroxidise free or membrane included unsaturated fatty acids, leading to generate reactive aldehydes as advanced lipid peroxidation end products (ALE). Aldehydes from lipid peroxidation (LPO) react with proteins to cause alteration of protein structure to exacerbate complication of diseases. Here we studied serum albumin glycation in the presence and absence of liposomes as a bio-membrane model to investigate protein structural changes using various techniques including intrinsic and extrinsic fluorescence spectroscopies and electron microscopy analysis. Accordingly, serum albumin glycation and fibrillation were accelerated and intensified in the presence of liposomes through a hypothesized glycation-peroxidation loop (GPL). Together, our results shed light on the necessity of reconsidering diabetic protein glycation to make it close to physiological conditions mimicry, more importantly, proteins structural change due to diabetic glycation is intensified in the proximity of cell membranes which probably potentiates programmed cell death distinct from apoptosis. Image 1 • Lipid peroxidation (LPO) of liposomes produces reactive carbonyl compounds that induce fibrillogenesis in serum albumin. • Glycation of serum albumin induces generation of reactive oxygen species (ROS). • The generated ROSs induce LPO in the presence of liposome that accelerates fibrillation of the glycated serum albumin. • The synergistic effect of protein glycation and LPO on fibrillogenesis was explained by a glycation peroxidation loop (GPL). [ABSTRACT FROM AUTHOR]

Abstract: The abnormal aggregation and deposition of human islet amyloid polypeptide (hIAPP) are implicated in the pathogeny of type 2 diabetes mellitus (T2DM). Many aromatic ring-containing Ru complexes inhibit the aggregation of hIAPP. A new Ru complex Ru(bipy)(met)2·3H2O (1), where bipy is 2,2ʹ-bipyridine and met is methionine, was synthesized and employed to resist the fibril formation of hIAPP and to promote the biocompatibility of metal complexes. Two polypyridyl Ru complexes, namely [Ru(bipy)3]Cl2(2) and Ru(bipy)2Cl2(3), were used for comparison. Results reveal that the three Ru complexes can inhibit hIAPP aggregation and depolymerize mature hIAPP fibrils. Interaction studies show that Ru complexes bind to hIAPP through metal coordination, hydrophobic interaction, and other intermolecular forces. The binding of the three compounds is spontaneous and exothermic. The compounds also rescue peptide-induced cytotoxicity to some extent. Similar to 3, the novel methionine-Ru complex 1 exhibits an enhanced inhibitory effect and binding affinity to hIAPP possibly because of the smaller steric hindrance and more profitable molecular configuration of 1 than those of 2. The newly designed amino acid-Ru complex may provide new insights into the treatment of T2DM and related amyloidosis diseases.Graphical abstract: Methionine-Ru complex effectively impedes the fibril formation of human islet amyloid polypeptide. [ABSTRACT FROM AUTHOR]

Abstract d -amino acid-containing proteins have been found in several human tissues, and the spontaneous accumulation of d -amino acids in proteins is thought to be involved in age-dependent diseases including dementia. Tau, a microtubule-associated protein, is a major component of neurofibrillary tangles in Alzheimer's disease. Site-specific amino acid D-isomerization in Tau has been observed in the brains of patients with Alzheimer's disease. Here, we conducted amino acid D-isomerization at specific sites in microtubule-binding repeat peptides of Tau (Tau R2 and R3) and examined the effects on Tau structure and fibril formation. Our results demonstrate that amino acid D-isomerization in Tau R2 peptides decreased the rates of β-sheet transition and fibril formation compared with those of the wild-type peptide composed of all l -amino acids. In contrast, Tau R3 peptides that had undergone amino acid D-isomerization at either Asp314, Ser316, or Ser324 showed increased rates of β-sheet transition and fibril formation compared with those of the wild-type Tau R3 peptide. Highlights • Amino acid D-isomerization in Tau R2 and R3 controls β-sheet transition rate. • Fibril formation rate also depends on amino acid D-isomerization in Tau R2 and R3. • D-isomerization at Asp283 and/or Asp295 in Tau R2 reduces both these rates. • D-isomerization at Asp314, Ser316, or Ser324 in Tau R3 increases both these rates. • D-isomerization at Ser320 in Tau R3 reduces both these rates. [ABSTRACT FROM AUTHOR]

Abstract The deposition of human islet amyloid polypeptide (hIAPP) is considered as a causative factor of type 2 diabetes mellitus (T2DM). Developing effective inhibitors against the fibril formation of hIAPP is a potential way to treat T2DM. Recent studies indicate that various metal complexes including homo-binuclear Ru complexes can inhibit hIAPP aggregation. Hetero-multinuclear Pt Ru metal complexes exhibit multiple bioactivities, but their roles in reversing amyloidosis remain unclear. In this work, we synthesized and identified a new hetero-binuclear Pt Ru metal complex Na{[RuCl 4 (DMSO-S)](bpy)[Pt(DMSO-S)Cl 2 ]} (bpy: 4,4′-bipyridyl). We studied the inhibitory effect of the compound on hIAPP aggregation together with K{[RuCl 4 (DMSO-S)](pyz)[Pt (DMSO-S)Cl 2 ]} (pyz: pyrazine) through diverse biophysical methods. Results showed that two Pt Ru metal complexes can remarkably reverse hIAPP aggregation and scatter the fibrils into nanoscale particles. Thermodynamic and spectrometric studies revealed that the binding of metal complexes with hIAPP was a spontaneous, enthalpy-driven process resulting from the predominant hydrophobic interaction and metal coordination. Two hetero-binuclear Pt Ru metal complexes showed stronger binding affinity and better inhibitory effects against peptide fibril formation than homo-binuclear Ru complexes and corresponding mononuclear Ru complexes. The compounds also regulated the peptide-induced cytotoxicity against Insulinoma β-cells and significantly increased the cell viability. This work shed light on a potential strategy for designing hetero-multinuclear metal complexes against amyloidosis-related diseases. Graphical abstract Heteronuclear Pt Ru complexes inhibit the fibril formation of human islet amyloid polypeptide. Unlabelled Image Highlights • Heteronuclear Pt Ru compounds reversed the aggregation of human islet amyloid polypeptide. • The complexes scattered the aggregates into nanoscale particles. • The complexes bound to the peptide by metal coordination and hydrophobic interaction. • The complexes rescued the peptide-caused cytotoxicity. [ABSTRACT FROM AUTHOR]

The precise kinetic pathways of peptide clustering and fibril formation are not fully understood. Here we study the initial clustering kinetics and transient cluster morphologies during aggregation of the heptapeptide fragment GNNQQNY from the yeast prion protein Sup35. We use a mid-resolution coarse-grained molecular dynamics model of Bereau and Deserno to explore the aggregation pathways from the initial random distribution of free monomers to the formation of large clusters. By increasing the system size to 72 peptides we could follow directly the molecular events leading to the formation of stable fibril-like structures. To quantify those structures we developed a new cluster helicity parameter. We found that the formation of fibril-like structures is a cooperative processes that requires a critical number of monomers, M⋆≈25, in a cluster. The terminal tyrosine residue is the structural determinant in the formation of helical fibril-like structures. This work supports and quantifies the two-step aggregation model where the initially formed amorphous clusters grow and, when they are large enough, rearrange into mature twisted structures. However, in addition to the nucleated fibrillation, growing aggregates undergo further internal reorganization, which leads to more compact structures of large aggregates.

The 3,3′,5,5′-tetrachloro-2-iodo-4,4′-bipyridine structure is proposed as a novel chemical scaffold for the design of new transthyretin (TTR) fibrillogenesis inhibitors. In the frame of a proof-of-principle exploration, four chiral 3,3′,5,5′-tetrachloro-2-iodo-2′-substituted-4,4′- bipyridines were rationally designed and prepared from a simple trihalopyridine in three steps, including a Cu-catalysed Finkelstein reaction to introduce iodine atoms on the heteroaromatic scaffold, and a Pd-catalysed coupling reaction to install the 2′-substituent. The corresponding racemates, along with other five chiral 4,4′-bipyridines containing halogens as substituents, were enantioseparated by high-performance liquid chromatography in order to obtain pure enantiomer pairs. All stereoisomers were tested against the amyloid fibril formation (FF) of wild type (WT)-TTR and two mutant variants, V30M and Y78F, in acid mediated aggregation experiments. Among the 4,4′-bipyridine derivatives, interesting inhibition activity was obtained for both enantiomers of the 3,3′,5,5′-tetrachloro-2′-(4-hydroxyphenyl)-2-iodo-4,4′-bipyridine. In silico docking studies were carried out in order to explore possible binding modes of the 4,4′-bipyridine derivatives into the TTR. The gained results point out the importance of the right combination of H-bond sites and the presence of iodine as halogen-bond donor. Both experimental and theoretical evidences pave the way for the utilization of the iodinated 4,4′-bipyridine core as template to design new promising inhibitors of TTR amyloidogenesis.

Hydrophobins are fungal proteins that can selfassemble into amphiphilic films at hydrophobic-hydrophilic interfaces. Class I hydrophobin aggregates resemble amyloid fibrils, sharing some features with them. Here, five site-directed mutants of Vmh2, a member of basidiomycota class I hydrophobins, were designed and characterized to elucidate the molecular determinants playing a key role in class I hydrophobin self-assembly. The mechanism of fibril formation proposed for Vmh2 foresees that the triggering event is the destabilization of a specific loop (L1), leading to the formation of a β-hairpin, which in turn generates the β-spine of the amyloid fibril. [ABSTRACT FROM AUTHOR]

Self-assembled environment of collagen is one of the important factors for improving and regulating the properties of collagen-based biomaterials. This study aimed to investigate the effect of ionic liquids (ILs) on the fibril-formation and gel properties of grass carp (Ctenopharyngodon idellus) skin collagen. Fibrillogenic kinetics analysis showed that the collagen self-assembly can be suppressed by the introduction of ILs, and the inhibitory effect is influenced by concentration and types of ILs. Scanning electron microscopy test indicated that the assembled collagen fibrils in the presence of ILs had bigger diameters than that in the conventional buffer. Differential scanning calorimetry analysis revealed that the thermal stability of collagen fibrils can be significantly increased when self-assembly is performed in the presence of ILs. Moreover, the introduction of ILs enhanced the mechanical strength of collagen gels. Finding from this work provides a new idea for improving the performance of fish-sourced collagen biomaterials. [ABSTRACT FROM AUTHOR]

Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid‐forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation‐prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo‐designed polypeptide β17. The fusion protein NT*‐β17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that β17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, β17 adopts a β‐sheet conformation in a pH‐ and salt‐dependent manner and assembles into amyloid‐like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases. [ABSTRACT FROM AUTHOR]

The present study describes the development and characterization of strontium(II)-modified biomimetic scaffolds based on mineralized collagen type I as potential biomaterial for the local treatment of defects in systemically impaired (e.g. osteoporotic) bone. In contrast to already described collagen/hydroxyapatite nanocomposites calcium was substituted with strontium to the extent of 25, 50, 75 and 100 mol% by substituting the CaCl 2 -stock solution (0.1 M) with SrCl 2 (0.1 M) during the scaffold synthesis. Simultaneous fibrillation and mineralization of collagen led to the formation of collagen-mineral nanocomposites with mineral phases shifting from nanocrystalline hydroxyapatite (Sr0) over poorly crystalline Sr-rich phases towards a mixed mineral phase (Sr100), consisting of an amorphous strontium phosphate (identified as Collin's salt, Sr 6 H 3 (PO 4 ) 5 ∗ 2 H 2 O, CS) and highly crystalline strontium hydroxyapatite (Sr 5 (PO 4 ) 3 OH, SrHA). The formed mineral phases were characterized by transmission electron microscopy (TEM) and RAMAN spectroscopy. All collagen/mineral nanocomposites with graded strontium content were processed to scaffolds exhibiting an interconnected porosity suitable for homogenous cell seeding in vitro. Strontium ions (Sr 2 + ) were released in a sustained manner from the modified scaffolds, with a clear correlation between the released Sr 2 + concentration and the degree of Sr-substitution. The accumulated specific Sr 2 + release over the course of 28 days reached 141.2 μg (~ 27 μg mg − 1 ) from Sr50 and 266.1 μg (~ 35 μg mg − 1 ) from Sr100, respectively. Under cell culture conditions this led to maximum Sr 2 + concentrations of 0.41 mM (Sr50) and 0.73 mM (Sr100) measured on day 1, which declined to 0.08 mM and 0.16 mM, respectively, at day 28. Since Sr 2 + concentrations in this range are known to have an osteo-anabolic effect, these scaffolds are promising biomaterials for the clinical treatment of defects in systemically impaired bone. [ABSTRACT FROM AUTHOR]

The effect of carboxymethylcellulose (CMC) on the fibril formation of collagen in vitro was studied by turbidity measurements and atomic force microscopy (AFM). The kinetics curves of fibril formation indicated that the rate of collagen fibrillogenesis was decreased with the addition of CMC, meanwhile the final turbidity was obviously increased as the CMC/collagen ratio reached 30%. The AFM images of collagen-CMC solutions showed that the number of nucleation sites of collagen fibrillogenesis was significantly increased with the presence of CMC, while the diameter of immature collagen fibrils was obviously decreased. Moreover, the thermal stability of collagen fibril hydrogels was obviously improved with the presence of CMC. In addition, the morphologies of collagen fibrils observed by AFM revealed that the adjacent fibril segments or fibrils were intertwisted and even tightly merged, probably due to the hydrogen bonding and molecular entanglement interactions between CMC and collagen molecules. [ABSTRACT FROM AUTHOR]

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) from golden pompano skins were extracted and characterized. The molecular weight of ASC was about 130 kDa for α1and 115 kDa for α2, which were slightly higher than those of PSC. Similar amino acid composition and Fourier transform infrared (FTIR) spectra were observed in both collagens, but slight differences were found in the peptide maps of collagen digested by V8 protease and trypsin. The denaturation temperatures (Tds) of ASC and PSC calculated from the reduced viscosity were 31.8 and 30.0°C, while the transition temperature (Tm) of ASC and PSC analyzed by DSC were 33.0 and 32.0°C, respectively. ASC has a lag phase, a growth phase, and a plateau phase in the turbidity–time curve, while PSC does not have similar phenomenon. It was found that the fibril gel of ASC could be formed at 25°C, leading to improved thermal stability. [ABSTRACT FROM PUBLISHER]

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from the skin of river puffer (ASC-RP and PSC-RP) and tiger puffer (ASC-TP and PSC-TP) were extracted and physicochemically examined. Denaturation temperature (Td) for all the collagens was found to be 25.5−29.5 °C, which was lower than that of calf skin collagen (35.9 °C). Electrophoretic patterns indicated all four samples were type I collagen with molecular form of (α1)2α2. FTIR spectra confirmed the extracted collagens had a triple-helical structure, and that the degree of hydrogen bonding in ASC was higher than PSC. All the extracted collagens could aggregate into fibrils with D-periodicity. The fibril formation rate of ASC-RP and PSC-RP was slightly higher than ASC-TP and PSC-TP. Turbidity analysis revealed an increase in fibril formation rate when adding a low concentration of NaCl (less than 300 mM). The fibril formation ability was suppressed with further increasing of NaCl concentration, as illustrated by a reduction in the turbidity and formation degree. SEM analysis confirmed the well-formed interwoven structure of collagen fibrils after 24 h of incubation. Summarizing the experimental results suggested that the extracted collagens from the skin of river puffer and tiger puffer could be considered a viable substitute to mammalian-derived collagens for further use in biomaterial applications.

BACKGROUND The fish swim bladder is considered as a potential source of realistic collagen. Currently, processing of the Indian major carp rohu ( Labeo rohita) generates an enormous quantity of non-edible by-products, including swim bladders, which are discarded as waste with no commercial value. In the present study, collagen was prepared from rohu swim bladder and its physicochemical and fibril-forming capacities were assessed. RESULTS The collagen isolated from rohu swim bladder was characterised as type I, containing α1 and α2 chains with triple helical structure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fourier transform infrared spectroscopy and amino acid analysis. The extracted collagen denaturation temperature was found to be 42.16 °C by differential scanning calorimetry analysis and also exhibited a high solubility in the presence of low NaCl concentrations (0-0.6 mol L−1). The extracted collagen exhibited a high fibril-formation capacity at a NaCl concentration of 1.5 mol L−1. Examination of the microstructure of collagen by scanning electron microscopy ( SEM) showed a porous, sheet-like film and a multilayered structure. The fibril formation capacity of collagen was also confirmed using SEM analysis. CONCLUSION The rohu swim bladder type I collagen was successfully extracted using an enzymatic method with a yield of 465.2 g kg−1 (dry weight basis) and was characterised as a well maintained triple helical structure. The extracted collagen exhibited a high fibril-forming ability. The results of the present study confirm that utilisation of rohu swim bladder will open up a new avenue for the better disposal of by-products and also help to minimise environmental pollution issues. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Collagen and collagen fibers have been widely documented as a potential and competitive biomaterial for medical applications. However, the searches for safe and realistic new collagen sources are still underway. Currently, fishery by-products (scales), a promising collagen source are usually discarded. In the present study, in vitro fibril-forming ability of the extracted fish scale collagen is reported. The aim of the investigation was to evaluate the concomitant comparison of fibril-forming abilities and characteristics of acid and pepsin soluble collagens from the scales of Indian major carp catla ( Catla catla ) and rohu ( Labeo rohita ). The extracted collagens were characterized as type I, with a total yield of 2.80–4.11% (w/w). Denaturation temperature determined for all collagens were between 35.9 and 37.7 °C. All collagens exhibited high solubility in acidic pH and low NaCl concentrations. SEM clarified the lyophilized collagens and their fibril-forming capacity. Amino acid content and radical scavenging efficacy were also analyzed for the extracted collagen. The results revealed that extracted scale collagen from a renewable biological source could be used as biomaterials in various sectors. It might be suitable for preparing collagen gel for biomedical devices or as a scaffold for cell culture because of its high stability and fibril formation capacity. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is associated with the aggregation of the misfolded neuronal peptide, amyloid-β42 (Aβ42). Evidence has suggested that several reasons are responsible for the toxicity caused by the aggregation of Aβ42, including the conformational restriction of Aβ42. In this study, one of the toxic conformers of Aβ42, which contains a Glu-to-Pro substitution (E22P-Aβ42), was explored using atomic force microscopy and molecular docking to study the aggregation dynamics. We proposed a systematic model of fibril formation to better understand the molecular basis of conformational transitions in the Aβ42 species. Our results demonstrated the formation of amorphous aggregates in E22P-Aβ42 that are stem-based, network-like structures, while the formation of mature fibrils occurred in the less toxic conformer of Aβ42, E22-Aβ42, that are sphere-like flexible structures. A comparison was made between the biophysical properties of E22P-Aβ42 and E22-Aβ42 that revealed that E22P-Aβ42 had greater stiffness, dihedral angle, number of β sheets involved, and elasticity, compared with E22-Aβ42. These findings will have considerable implications toward our understanding of the structural basis of the toxicity caused by conformational diversity in Aβ42 species.

We investigated the potential of collagen-genipin sols as biomaterials for treating artificial ulcers following endoscopic submucosal dissection. Collagen sol viscosity increased with condensation, allowing retention on tilted ulcers before gelation and resulting in collagen gel deposition on whole ulcers. The 1.44% collagen sols containing genipin as a crosslinker retained sol fluidity at 23°C for >20 min, facilitating endoscopic use. Collagen sols formed gel depositions on artificial ulcers in response to body temperature, and high temperature responsiveness of gelation because of increased neutral phosphate buffer concentration allowed for thick gel deposition on tilted ulcers. Finally, histological observations showed infiltration of gels into submucosal layers. Taken together, the present data show that genipin-induced crosslinking significantly improves the mechanical properties of collagen gels even at low genipin concentrations of 0.2-1 mM, warranting the use of in situ gelling collagen-genipin sols for endoscopic treatments of gastrointestinal ulcers. [ABSTRACT FROM AUTHOR]

A number of amyloidogenic variants of apoA-I have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased β-strand structure, increased N-terminal protease susceptibility, and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ∼90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposition. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to wild-type protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37°C with a half time (t1/2) of about 12 days. Upon prolonged incubation, the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.