Fibril Morphology Research Articles

Effect of Thermal Treatment on the Self-Assembly of Wheat Gluten Polypeptide.

Self-assembled fibrillation of wheat gluten is a common phenomenon in the daily production and processing of wheat flour products. The driving forces for its formation and the factors that influence the morphology of fibrils have not been thoroughly investigated. In this study, the effect of three bonding changes (breaking hydrogen bonds, strengthening hydrophobic interactions, and SH-SS exchange reactions) on gluten polypeptide (GP) fibrillation was simulated by adjusting the heating temperature (room temperature (RT), 45 °C, 65 °C, and 95 °C). The results showed that the breakage of hydrogen bonds could induce conformational transitions in GPs and help to excite fibrillation in GPs. Strengthened hydrophobic interactions significantly contributed to the fibrillation of GPs. Covalent crosslinks generated by SH-SS exchange reactions might also promote the fibrillation of GPs. GPs with different degrees of hydrolysis (4.0%, 6.0%, and 10.0%, represented by DH 4, DH 6, and DH 10, respectively) presented different extents of fibrillation, with DH 10 GPs having a higher propensity to fibrillation than DH 4 and DH 6 GPs. The results of Fourier's transform infrared spectroscopy indicated that hydrophobic interactions drive the transition from a random coil and α-helix to a β-sheet. In addition, hydrophobic interactions also drive the intermolecular polymerization of GPs, resulting in larger molecular weight aggregates. The morphology presented by transmission electron microscopy showed that the greater the DH, the stronger the tendency for the worm-like aggregation of GPs.

The role of glycosylation in amyloid fibril formation of bovine κ-casein

In order to explore the functions of glycosylation of κ-Casein (κ-CN) in bovine milk, unglycosylated (UG) and twice glycosylated (2G) forms of κ-CN B were purified by selective precipitation followed by anion exchange chromatography from κ-CN BB milk and tested for their amyloid fibril formation and morphology, oligomerisation states and protein structure. The diameter of self-assembled κ-CN B aggregates of both glyco-form were shown for the first time to be in the same 26.0–28.7 nm range for a 1 mg mL−1 solution. The presence of two bound glycans in the protein structure of 2G κ-CN B led to a greater increase in the maximum amyloid fibril formation rate with increasing protein concentration and a difference in both length (82.0 ± 29.9 vs 50.3 ± 13.7 nm) and width (8.6 ± 2.1 vs 13.9 ± 2.5 nm) for fibril morphology compared to UG κ-CN B. The present results suggest that amyloid fibril formation proceeds at a slow but steady rate via the self-assembly of dissociated, monomeric κ-CN B proteins at concentrations of 0.22–0.44 mg mL−1. However amyloid fibril formation proceeds more rapidly via the assembly of either aggregated κ-CN present in a micelle-like form or dissociated monomeric κ-CN, packed into reorganised formational structures above the critical micellar concentration to form fibrils of differing width. The degree of glycosylation has no effect on the polarity of the adjacent environment, nor non-covalent and disulphide interactions between protein molecules when in the native form. Yet glycosylation can influence protein folding patterns of κ-CN B leading to a reduced tryptophan intrinsic fluorescence intensity for 2G compared to UG κ-CN B. These results demonstrate that glycosylation plays an important role in the modulation of aggregation states of κ-CN and contributes to a better understanding of the role of glycosylation in the formation of amyloid fibrils from intrinsically disordered proteins.

Atrial Natriuretic Peptide Associated with Cardiovascular Diseases Inhibits Amyloid-β Aggregation via Cross-Seeding.

Both cardiovascular diseases (CVDs) and Alzheimer's disease (AD) share some common risk factors (e.g., age, obesity, oxidative stress, inflammation, hypertension) that contribute to their overlapping pathogenesis, indicating a "head-to-heart" pathological connection between CVDs and AD. To explore this potential connection at the protein level, we study the potential cross-seeding (heterotypic interactions) between CVD-associated atrial natriuretic peptide (ANP) and AD-associated β-amyloid (Aβ). Collective aggregation and cell assays demonstrate the cross-seeding of ANP with different Aβ species including monomers, oligomers, and fibrils with high binding affinity (KD = 1.234-1.797 μM) in a dose-dependent manner. Such ANP-induced cross-seeding also modifies the Aβ aggregation pathway, fibril morphology, and cell deposition pattern by inhibiting Aβ fibrillization from small aggregates, disassembling preformed Aβ fibrils, and alleviating Aβ-associated cytotoxicity. Finally, using transgenic C. elegans worms that express the human muscle-specific Aβ1-42, ANP can also effectively delay Aβ-induced worm paralysis, decrease Aβ plaques in worm brains, and reduce reactive oxygen species (ROS) production, confirming its in vivo inhibition ability to prevent neurodevelopmental toxicity in worms. This work discovers not only a new cross-seeding system between the two disease-related proteins but also a new finding that ANP possesses a new biological function as an Aβ inhibitor in the nonaggregated state.

Physicochemical and Fibril Formation Properties of Pufferfish (Takifugu obscurus) Skin Collagen from Solvent Extraction in Different Conditions.

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 residual structure of acid-denatured β2 -microglobulin is relevant to an ordered fibril morphology.

β2 -Microglobulin (β2m) forms amyloid fibrils in vitro under acidic conditions. Under these conditions, the residual structure of acid-denatured β2m is relevant to seeding and fibril extension processes. Disulfide (SS) bond-oxidized β2m has been shown to form rigid, ordered fibrils, whereas SS bond-reduced β2m forms curvy, less-ordered fibrils. These findings suggest that the presence of an SS bond affects the residual structure of the monomer, which subsequently influences the fibril morphology. To clarify this process, we herein performed NMR experiments. The results obtained revealed that oxidized β2m contained a residual structure throughout the molecule, including the N- and C-termini, whereas the residual structure of the reduced form was localized and other regions had a random coil structure. The range of the residual structure in the oxidized form was wider than that of the fibril core. These results indicate that acid-denatured β2m has variable conformations. Most conformations in the ensemble cannot participate in fibril formation because their core residues are hidden by residual structures. However, when hydrophobic residues are exposed, polypeptides competently form an ordered fibril. This conformational selection phase may be needed for the ordered assembly of amyloid fibrils.

Site-Specific C-Terminal Fluorescent Labeling of Tau Protein.

Formation of Tau protein aggregates in neurons is a pathological hallmark of several neurodegenerative diseases, including Alzheimer's disease. Fluorescently labeled Tau protein is therefore useful to study the aggregation of these pathological proteins and to identify potential therapeutic targets. Conventionally, cysteine residues are used for labeling Tau proteins; however, the full-length Tau isoform contains two cysteine residues in the microtubule-binding region, which are implicated in Tau aggregation by forming intermolecular disulfide bonds. To prevent the fluorescent label from disturbing the microtubule binding region, we developed a strategy to fluorescently label Tau at its C-terminus while leaving cysteine residues unperturbed. We took advantage of a Sortase A-mediated transpeptidation approach to bind a short peptide (GGGH6-Alexa647) with a His-tag and a covalently attached Alexa 647 fluorophore to the C-terminus of Tau. This reaction relies on the presence of a Sortase recognition motif (LPXTG), which we attached to the C-terminus of recombinantly expressed Tau. We demonstrate that C-terminal modification of Tau protein results in no significant differences between the native and C-terminally labeled Tau monomer with regard to aggregation kinetics, secondary structure, and fibril morphology.

Modulation Effects of Fe3+, Zn2+, and Cu2+ Ions on the Amyloid Fibrillation of α-Synuclein: Insights from a FTIR Investigation

Amyloid fibrillation of α-synuclein is implicated in the pathogenesis of Parkinson's disease and heavy metal ions such as Fe3+, Zn2+, and Cu2+ are known to be involved in the process. In this work, we explored the use of FTIR spectroscopy to look into the modulation effects of Fe3+, Zn2+, and Cu2+ on the amyloid fibrillation of α-synuclein. We performed a curve-fitting analysis on the FTIR amide I bands of these α-synuclein fibril systems, namely, the pristine fibril and the fibrils prepared in the presence of Fe3+, Zn2+, and Cu2+. We found that the α-synuclein fibrils under the influences of metal ions all possessed a parallel β-sheet structure, turn structure, and disordered structure, similar to that of pristine α-synuclein fibril. We also observed metal-induced increases in the proportions of the β-sheet secondary structure within the α-synuclein fibrils, with Fe3+ being the most effective inducer. We performed second derivative analysis of the side chain carboxylic groups of α-synuclein fibrils and found that the side chain microenvironment of the α-synuclein fibrils was more influenced by Fe3+ than Zn2+, and Cu2+. In addition, our atomic force microscopic study revealed that the morphologies of α-synuclein fibrils under the influence of Fe3+ was quite different from that of the Zn2+ and Cu2+ systems. Our FTIR results suggested that the modulation effects of Fe3+, Zn2+, and Cu2+ on α-synuclein fibrillation occurred at both secondary and quaternary structural levels. At last, we proposed a mechanistic hypothesis to interpret how metal ions could affect the morphology of α-synuclein amyloid fibril based on the conformational plasticity properties of intrinsically disordered proteins.

Amyloid fibril structure from the vascular variant of systemic AA amyloidosis

Systemic AA amyloidosis is a debilitating protein misfolding disease in humans and animals. In humans, it occurs in two variants that are called ‘vascular’ and ‘glomerular’, depending on the main amyloid deposition site in the kidneys. Using cryo electron microscopy, we here show the amyloid fibril structure underlying the vascular disease variant. Fibrils purified from the tissue of such patients are mainly left-hand twisted and contain two non-equal stacks of fibril proteins. They contrast in these properties to the fibrils from the glomerular disease variant which are right-hand twisted and consist of two structurally equal stacks of fibril proteins. Our data demonstrate that the different disease variants in systemic AA amyloidosis are associated with different fibril morphologies.

Photoinduced Amyloid Fibril Degradation for Controlled Cell Patterning.

Amyloid-like fibrils are a special class of self-assembling peptides that emerge as a promising nanomaterial with rich bioactivity for applications such as cell adhesion and growth. Unlike the extracellular matrix, the intrinsically stable amyloid-like fibrils do not respond nor adapt to stimuli of their natural environment. Here, a self-assembling motif (CKFKFQF), in which a photosensitive o-nitrobenzyl linker (PCL) is inserted, is designed. This peptide (CKFK-PCL-FQF) assembles into amyloid-like fibrils comparable to the unsubstituted CKFKFQF and reveals a strong response to UV-light. After UV irradiation, the secondary structure of the fibrils, fibril morphology, and bioactivity are lost. Thus, coating surfaces with the pre-formed fibrils and exposing them to UV-light through a photomask generate well-defined areas with patterns of intact and destroyed fibrillar morphology. The unexposed, fibril-coated surface areas retain their ability to support cell adhesion in culture, in contrast to the light-exposed regions, where the cell-supportive fibril morphology is destroyed. Consequently, the photoresponsive peptide nanofibrils provide a facile and efficient way of cell patterning, exemplarily demonstrated for A549, Chinese Hamster Ovary, and Raw Dual type cells. This study introduces photoresponsive amyloid-like fibrils as adaptive functional materials to precisely arrange cells on surfaces.

FapA is an Intrinsically Disordered Chaperone for Pseudomonas Functional Amyloid FapC

Bacterial functional amyloids contribute to biofilm development by bacteria and provide protection from the immune system and prevent antibiotic treatment. Strategies to target amyloid formation and interrupt biofilm formation have attracted recent interest due to their antimicrobial potential. Functional amyloid in Pseudomonas (Fap) includes FapC as the major component of the fibril while FapB is a minor component suggested to function as a nucleator of FapC. The system also includes the small periplasmic protein FapA, which has been shown to regulate fibril composition and morphology. The interplay between these three components is central in Fap fibril biogenesis. Here we present a comprehensive biophysical and spectroscopy analysis of FapA, FapB and FapC and provide insight into their molecular interactions. We show that all three proteins are primarily disordered with some regions with structural propensities for α-helix and β-sheet. FapA inhibits FapC fibrillation by targeting the nucleation step, whereas for FapB the elongation step is modulated. Furthermore, FapA alters the morphology of FapC (more than FapB) fibrils. Complex formation is observed between FapA and FapC, but not between FapA and FapB, and likely involves the N-terminus of FapA. We conclude that FapA is an intrinsically disordered chaperone for FapC that guards against fibrillation within the periplasm. This new understanding of a natural protective mechanism of Pseudomonas against amyloid formations can serve as inspiration for strategies blocking biofilm formation in infections.

Effects of carbodiimide combined with ethanol-wet bonding pretreatment on dentin bonding properties: an in vitro study.

PurposeThis study evaluated the combined effects of Carbodiimide (EDC) and ethanol–wet bonding (EWB) pretreatment on the bond strength and resin–dentin surface.MethodsPhosphoric acid-etched dentin specimens were randomly divided into five groups based on the following pretreatments: deionized water (control), EWB, 0.3M EDC in water (EDCw), EDC water solution combined EWB (EDCw + EWB), and 0.3M EDC in ethanol (EDCe). A scanning electron microscope (SEM) was used to observe the morphology of collagen fibrils on the demineralized dentin matrix in each group after pretreatment. The adhesives Prime & Bond NT (PB) (Dentsply De trey, Konstanz, Germany) or Single bond 2 (SB) (3M ESPE, St. Paul, MN, USA) was applied after pretreatments, and a confocal laser scanning microscope (CLSM) was used to evaluate the quality of resin tags. The degree of conversion (DC) of the adhesive was investigated by Fourier transform infrared spectroscopy (ATR-FTIR). The dentin was first bonded with resin and bathed in water at 37 °C for 24 h. Half of them were subjected to 10, 000 cycles in a thermocycler between 5 °C and 55 °C before a microshear bond strength (µSBS) test. The statistical methods were Analysis of Variance (ANOVA) and a Tukey post hoc test at α = 0.05.ResultsThe µSBS was significantly affected by pretreatments (p < 0.001), adhesives (p < 0.001), and aging conditions (p < 0.001) as revealed by the three-way ANOVA. The EDCw, EDCw + EWB, and EDCe groups significantly increased the µSBS; the EDCw + EWB and EDCe groups produced the highest µSBS. In the EDC-containing groups, the SEM showed at the collagen fibrils in the dentin matrix formed a three-dimensional network structure in the tubules after cross-linking into sheets, and the hybrid layer formed thicker resin tags under a CLSM. In the EDC-containing groups, the CLSM observed an increase in the length of resin tags. PB showed a higher DC and bonding strength than SB, and the five pretreatment groups tested did not affect the DC of the two adhesives.ConclusionsIn etch–and–rinse bonding system, EDC combined with EWB pretreatment can improve the quality of the hybrid layer and enhance the mechanical properties of demineralized dentin matrix. Pretreatment with EDC–ethanol solution may be a new clinically friendly option for enhancing dentin bonding durability.

Structural consequences of sequence variation in mammalian prion β2α2 loop segments.

Sequence variation in the β2α2 loop, residues 165-175 of the mammalian prion protein (PrP), influences its structure. To better understand the consequences of sequence variation in this region of the protein, we biochemically and biophysically interrogate natural and artificial sequence variants of the β2α2 loop of mammalian PrP. Using microcrystal electron diffraction (MicroED), we determine atomic resolution structures of segments encompassing residues 168-176 from the β2α2 loop of PrP with sequences corresponding to human, mouse/cow, bank vole/hamster, rabbit/pig/guinea pig, and naked mole rat (elk-T174S) β2α2 loops, as well as synthetic β2α2 loop sequences. This collection of structures presents two dominant amyloid packing polymorphisms. In the first polymorph, denoted "clasped", side chains within a sheet form polar clasps by facing each other on the same strand, exemplified by the mouse/cow, human, and bank vole/hamster sequences. Because its stability is derived from within a strand and through polar ladders within a sheet, the sequence requirements for the mating strand are less restrictive. A second polymorph, denoted "interdigitated," has sidechains interdigitate across mating sheets, exemplified by the elk, naked mole rat (elk T174S), and rabbit sequences. The two types of packing present distinct networks of stabilizing hydrogen bonds. The identity of residue 174 appears to strongly influence the packing adopted in these peptides, but consideration of the overall sequence of a given segment is needed to understand the stability of its assemblies. Incorporation of these β2α2 loop sequences into an 85 residue recombinant segment encoding wild-type bank vole PrP94-178 demonstrates that even single residue substitutions could impact fibril morphology as evaluated by negative stain electron microscopy. This is in line with recent findings supporting the accessibility of different structural geometries by varied mammalian prion sequences, and indicates that sequence-specific polymorphisms may be influenced by residues in the β2α2 loop.

Anionic lipid vesicles have differential effects on the aggregation of early onset-associated α-synuclein missense mutants

α-synuclein (αS) is the key component of synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. αS was first linked to PD through the identification of point mutations in the SNCA gene, causing single amino acid substitutions within αS and familial autosomal dominant forms of PD that profoundly accelerated disease onset by up to several decades. At least eight single-point mutations linked to familial PD (A30G/P, E46K, H50Q, G51D, and A53T/E/V) are located in proximity of the region preceding the non-β amyloid component (preNAC) region, strongly implicating its pathogenic role in αS-mediated cytotoxicity. Furthermore, lipids are known to be important for native αS function, where they play a key role in the regulation of synaptic vesicle docking to presynaptic membranes and dopamine transmission. However, the role of lipids in the function of mutant αS is unclear. Here, we studied αS aggregation properties of WT αS and five of the most predominant single-point missense mutants associated with early onset PD in the presence of anionic 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine lipid vesicles. Our results highlight significant differences between aggregation rates, the number of aggregates produced, and overall fibril morphologies of WT αS and the A30P, E46K, H50Q, G51D, and A53T missense mutants in the presence of lipid vesicles. These findings have important implications regarding the interplay between the lipids required for αS function and the individual point mutations known to accelerate PD and related diseases.

Collagen V deficiency during murine tendon healing results in distinct healing outcomes based on knockdown severity

Tendon function is dependent on proper organization and maintenance of the collagen I tissue matrix. Collagen V is a critical regulator of collagen I fibrils, and while prior studies have shown a negative impact of collagen V deficiency on tendon healing outcomes, these studies are confounded by collagen V deficiency through tendon development. The specific role of collagen V in regulating healing tendon properties is therefore unknown. By using inducible Col5a1 knockdown models and analyzing gene expression, fibril and histological tendon morphology, and tendon mechanical properties, this study defines the isolated role of collagen V through tendon healing. Patellar tendon injury caused large changes in tendon gene expression, and Col5a1 knockdown resulted in dysregulated expression of several genes through tendon healing. Col5a1 knockdown also impacted collagen fibril size and shape without observable changes in scar tissue formation. Surprisingly, heterozygous Col5a1 knockdown resulted in improved stiffness of healing tendons that was not observed with homozygous Col5a1 knockdown. Together, these results present an unexpected and dynamic role of collagen V deficiency on tendon healing outcomes following injury. This work suggests a model of tendon healing in which quasi-static mechanics may be improved through titration of collagen fibril size and shape with modulation of collagen V expression and activity.

Morphological integrity of insulin amyloid-like aggregates depends on preparation methods and post-production treatments

Protein aggregates are often varying extensively in their morphological characteristics, which may lead to various biological outcomes, such as increased immunogenicity risk. However, isolation of aggregates with a specific morphology within an ensemble is often challenging. To gain vital knowledge on the effects of aggregate characteristics, samples containing a single morphology must be produced by direct control of the aggregation process. Moreover, the formed aggregates need to be in an aqueous solution suitable for biological assays, while keeping their morphology intact. Here we evaluated the dependence of morphology and integrity of amyloid-like fibrils and spherulites on preparation conditions and post-treatment methods. Samples containing either amyloid-like fibrils or spherulites produced from human insulin in acetic acid solutions are dependent on the presence of salt (NaCl). Moreover, mechanical shaking (600 rpm) inhibits spherulite formation, while only affecting the length of the formed fibrils compared to quiescent conditions. Besides shaking, the initial protein concentration in the formulation was found to control fibril length. Surprisingly, exchanging the solution used for aggregate formation to a physiologically relevant buffer, had a striking effect on the morphological integrity of the fibril and spherulite samples. Especially the secondary structure of one of our spherulite samples presented dramatic changes of the aggregated β-sheet content after exchanging the solution, emphasizing the importance of the aggregate stability. These results and considerations have profound implications on the data interpretation and should be implemented in the workflow for both fundamental characterization of aggregates as well as assays for evaluation of their corresponding biological effects.

Bivalent metal ions induce formation of α-synuclein fibril polymorphs with different cytotoxicities

α-Synuclein (α-Syn) aggregates are key components of intracellular inclusion bodies characteristic of Parkinson’s disease (PD) and other synucleinopathies. Metal ions have been considered as the important etiological factors in PD since their interactions with α-Syn alter the kinetics of fibrillation. In the present study, we have systematically explored the effects of Zn2+, Cu2+, Ca2+, and Mg2+ cations on α-Syn fibril formation. Specifically, we determined fibrillation kinetics, size, morphology, and secondary structure of the fibrils and their cytotoxic activity. While all cations accelerate fibrillation, we observed distinct effects of the different ions. For example, Zn2+ induced fibrillation by lower tlag and higher kapp and formation of shorter fibrils, while Ca2+ ions lead to formation of longer fibrils, as evidenced by dynamic light scattering and atomic force microscopy studies. Additionally, the morphology of formed fibrils was different. Circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopies revealed higher contents of β-sheets in fibrils. Interestingly, cell viability studies indicated nontoxicity of α-Syn fibrils formed in the presence of Zn2+ ions, while the fibrils formed in the presence of Cu2+, Ca2+, and Mg2+ were cytotoxic. Our results revealed that α-Syn fibrils formed in the presence of different divalent cations have distinct structural and cytotoxic features.

SERF, a family of tiny highly conserved, highly charged proteins with enigmatic functions.

Amyloid formation is a misfolding process that has been linked to age-related diseases, including Alzheimer's and Huntington's. Understanding how cellular factors affect this process in vivo is vital in realizing the dream of controlling this insidious process that robs so many people of their humanity. SERF (small EDRK-rich factor) was initially isolated as a factor that accelerated polyglutamine amyloid formation in a C. elegans model. SERF knockouts inhibit amyloid formation of a number of proteins that include huntingtin, α-synuclein and β-amyloid which are associated with Huntington's, Parkinson's and Alzheimer's disease, respectively, and purified SERF protein speeds their amyloid formation in vitro. SERF proteins are highly conserved, highly charged and conformationally dynamic proteins that form a fuzzy complex with amyloid precursors. They appear to act by specifically accelerating the primary step of amyloid nucleation. Brain-specific SERF knockout mice, though viable, appear to be more prone to deposition of amyloids, and show modified fibril morphology. Whole-body knockouts are perinatally lethal due to an apparently unrelated developmental issue. Recently, it was found that SERF binds RNA and is localized to nucleic acid-rich membraneless compartments. SERF-related sequences are commonly found fused to zinc finger sequences. These results point towards a nucleic acid-binding function. How this function relates to their ability to accelerate amyloid formation is currently obscure. In this review, we discuss the possible biological functions of SERF family proteins in the context of their structural fuzziness, modulation of amyloid pathway, nucleic acid binding and their fusion to folded proteins.

Decellularized Human Umbilical Tissue-Derived Hydrogels Promote Proliferation and Chondrogenic Differentiation of Mesenchymal Stem Cells

Tissue engineering is a promising approach for the repair and regeneration of cartilaginous tissue. Appropriate three-dimensional scaffolding materials that mimic cartilage are ideal for the repair of chondral defects. The emerging decellularized tissue-based scaffolds have the potential to provide essential biochemical signals and structural integrity, which mimics the natural tissue environment and directs cellular fate. Umbilical cord-derived hydrogels function as 3D scaffolding material, which support adherence, proliferation, migration, and differentiation of cells due to their similar biochemical composition to cartilage. Therefore, the present study aimed to establish a protocol for the formulation of a hydrogel from decellularized human umbilical cord (DUC) tissue, and assess its application in the proliferation and differentiation of UC-MSCs along chondrogenic lineage. The results showed that the umbilical cord was efficiently decellularized. Subsequently, DUC hydrogel was prepared, and in vitro chondral differentiation of MSCs seeded on the scaffold was determined. The developed protocol efficiently removed the cellular and nuclear content while retaining the extracellular matrix (ECM). DUC tissue, pre-gel, and hydrogels were evaluated by FTIR spectroscopy, which confirmed the gelation from pre-gel to hydrogel. SEM analysis revealed the fibril morphology and porosity of the DUC hydrogel. Calcein AM and Alamar blue assays confirmed the MSC survival, attachment, and proliferation in the DUC hydrogels. Following seeding of UC-MSCs in the hydrogels, they were cultured in stromal or chondrogenic media for 28 days, and the expression of chondrogenic marker genes including TGF-β1, BMP2, SOX-9, SIX-1, GDF-5, and AGGRECAN was significantly increased (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Moreover, the hydrogel concentration was found to significantly affect the expression of chondrogenic marker genes. The overall results indicate that the DUC-hydrogel is compatible with MSCs and supports their chondrogenic differentiation in vitro.

Cross-seeding of WT amyloid-β with Arctic but not Italian familial mutants accelerates fibril formation in Alzheimer's disease

Alzheimer’s disease (AD) involves the neurotoxic self-assembly of a 40 and 42 residue peptide, Amyloid-β (Aβ). Inherited early-onset AD can be caused by single point mutations within the Aβ sequence, including Arctic (E22G) and Italian (E22K) familial mutants. These mutations are heterozygous, resulting in an equal proportion of the WT and mutant Aβ isoform expression. It is therefore important to understand how these mixtures of Aβ isoforms interact with each other and influence the kinetics and morphology of their assembly into oligomers and fibrils. Using small amounts of nucleating fibril seeds, here, we systematically monitored the kinetics of fibril formation, comparing self-seeding with cross-seeding behavior of a range of isoform mixtures of Aβ42 and Aβ40. We confirm that Aβ40(WT) does not readily cross-seed Aβ42(WT) fibril formation. In contrast, fibril formation of Aβ40(Arctic) is hugely accelerated by Aβ42(WT) fibrils, causing an eight-fold reduction in the lag-time to fibrillization. We propose that cross-seeding between the more abundant Aβ40(Arctic) and Aβ42(WT) may be important for driving early-onset AD and will propagate fibril morphology as indicated by fibril twist periodicity. This kinetic behavior is not emulated by the Italian mutant, where minimal cross-seeding is observed. In addition, we studied the cross-seeding behavior of a C-terminal-amidated Aβ42 analog to probe the coulombic charge interplay between Glu22/Asp23/Lys28 and the C-terminal carboxylate. Overall, these studies highlight the role of cross-seeding between WT and mutant Aβ40/42 isoforms, which can impact the rate and structure of fibril assembly.

One- and Two-Photon Excited Autofluorescence of Lysozyme Amyloids.

Autofluorescence properties of amyloid fibrils are of much interest but, to date, the attention has been given mostly to one-photon excited fluorescence (1PEF), while the two-photon excited fluorescence (2PEF) properties of amyloids are much less explored. We investigate 1PEF and 2PEF of hen egg-white lysozyme (HEWL) in the form of monomers and fibrils. HEWL monomers feature some autofluorescence, which is enhanced in the case of fibrils. Moreover, by varying NaCl content, we introduce changes to fibrils morphology and show how the increase of the salt concentration is linked with an increase of 1PEF and 2PEF intensities. Interestingly, we observe 2PEF emission red-shifted in comparison to 1PEF. We confirm the presence of different relaxation pathways upon one- or two-photon excitation by different lifetimes of the fluorescence decays. Finally, we correlate the changes in optical properties of HEWL fibrils and monomers with salt-mediated changes in their morphology and the secondary structure.