Amorphous Aggregates Research Articles

Tau protein- induced sequestration of the eukaryotic ribosome: Implications in neurodegenerative disease

The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer’s disease, ribosomal dysfunction might be a crucial event in the disease pathology. Our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. Our in vitro studies show that incubation of Ht40 and the K18 tau variants with isolated non-translating yeast ribosome can induce a loss of ribosome physical integrity resulting in formation of tau-rRNA-ribosomal protein aggregates. Incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lysate and suppression of translation in the human in vitro translation system. The incubation of tau protein with the ribosomal RNA leads to the formation of tau-rRNA aggregates. The effect of K18 on the yeast ribosome can be mitigated in the presence of cellular polyanions like heparin and tRNA, thereby indicating the electrostatic nature of the aggregation process.

The C291R Tau Variant Forms Different Types of Protofibrils.

Mutations in the MAPT gene can lead to disease-associated variants of tau. However, the pathological mechanisms behind these genetic tauopathies are poorly understood. Here, we characterized the aggregation stages and conformational changes of tau C291R, a recently described MAPT mutation with potential pathogenic functions. The C291R variant of the tau four-repeat domain (tau-K18; a functional fragment with increased aggregation propensity compared with the full-length protein), aggregated into a mix of granular oligomers, amorphous and annular pore-like aggregates, in native-state and heparin-treated reactions as observed using atomic force microscopy (AFM) and negative-stained electron microscopy. On extended incubation in the native-state, tau-K18 C291R oligomers, unlike wild type (WT) tau-K18, aggregated to form protofibrils of four different phenotypes: (1) spherical annular; (2) spherical annular encapsulating granular oligomers; (3) ring-like annular but non-spherical; and (4) linear protofibrils. The ring-like tau-K18 C291R aggregates shared key properties of annular protofibrils previously described for other amyloidogenic proteins, in addition to two unique features: irregular/non-spherical-shaped annular protofibrils, and spherical protofibrils encapsulating granular oligomers. Tau-K18 C291R monomers had a circular dichroism (CD) peak at ~210 nm compared with ~199 nm for tau-K18 WT. These data suggest mutation-enhanced β-sheet propensity. Together, we describe the characterization of tau-K18 C291R, the first genetic mutation substituting a cysteine residue. The aggregation mechanism of tau-K18 C291R appears to involve β-sheet-rich granular oligomers which rearrange to form unique protofibrillar structures.

Effect of additives on liquid droplets and aggregates of proteins.

This review briefly summarizes the effect of additives on the formation of liquid droplets and aggregates of proteins. Proteins have the property of forming liquid droplets and aggregates both in vivo and in vitro. The liquid droplets of proteins are mainly stabilized by electrostatic and cation-π interactions, whereas the amorphous aggregates are mainly stabilized by hydrophobic interactions. Crowders usually stabilize liquid droplets, whereas ions and hexandiols destabilize the droplets. Additives such as kosmotropes, sugars, osmolytes, and crowders promote the formation of amorphous aggregates, whereas additives such as arginine and chaotropes can prevent the formation of amorphous aggregates. Further, amyloid has a different mechanism for its formation from amorphous aggregates because it is primarily stabilized by a cross-β structure. These systematic analyses of additives will provide clues to controlling protein aggregations and will aid the true understanding of the transition of proteins from liquid droplets and aggregates.

In-situ micro-Raman spectroscopy study of gypsum crystallization driven by chemical reaction

As an industry useful mineral, calcium sulfate crystal system including gypsum, bassanite, and anhydride, has numerous storage and widespread applications in our society. Controlling the process of gypsum formation and crystallization still remains a certain challenge under different conditions. In this work, an in-situ micro-Raman spectroscopy was used to explore the effect of chemical reaction on the crystallization process of gypsum (CaSO4·2H2O). Under the conditions of equal molar volume reaction, it was found that the formation time and crystallization rate of gypsum were related to the reactant concentration, which showed us the driving force of chemical reaction kinetics and the chemical potential of crystallization. Excess reactants in the non-isomolecular reaction clearly modified the precipitation reaction and crystallization of gypsum, which were recorded by in-situ Raman spectral variations at molecular level. The whole process involves chemical reaction, amorphous precursor aggregation, nucleation and crystal growth, which may provide us solid evidences towards deeply clarifying the microscopic effect of chemical reaction on gypsum crystallization at molecular level.

Membrane distillation of a silver leaching solution: Role of the coexisting aluminum ions on silica scaling

Silver, with excellent physicochemical properties, has been widely used in modern industry. Silver recovery from end-of-life product is economically significant for sustainability. However, silver leaching solution (SLS) containing diverse heavy metal ions as well as dissolved silica is a challenging waste stream for treatment and silver recovery. In this work we explored treatment of a real SLS via direct contact membrane distillation (DCMD). Results showed that silica scaling in DCMD process was closely related to the coexisting aluminum ions (Al3+) in SLS. The amount of Al(OH)3 formed was dependent on the initial pH of SLS and was effective to reduce concentration of the total dissolved silica in SLS, which was the key factor that influenced particle size distribution of SLS and degree of silica scaling. The highest silica removal rate of 83.7% was achieved at pH of 11. Moreover, it was found that the Al(OH)3 with positive charge accelerated mono-silicic acids aggregation, leading to prompt silica polymerization. This hypothesis was underpinned by evidence that amorphous yet compact aggregates formed on membrane surface and trimer silicates formed in the concentrated SLS with initial pH of 11. As a result, the most severe silica scaling occurred during DCMD treatment of SLS, resulting in the lowest initial permeate flux and water recovery. Our results shed light on prevention and management of silica scaling in treatment of waste stream.

Alpha-Synuclein Amyloid Aggregation Is Inhibited by Sulfated Aromatic Polymers and Pyridinium Polycation.

The effect of a range of synthetic charged polymers on alpha-synuclein aggregation and amyloid formation was tested. Sulfated aromatic polymers, poly(styrene sulfonate) and poly(anethole sulfonate), have been found to suppress the fibril formation. In this case, small soluble complexes, which do not bind with thioflavin T, have been formed in contrast to the large stick-type fibrils of free alpha-synuclein. Sulfated polysaccharide (dextran sulfate), as well as sulfated vinylic polymer (poly(vinyl sulfate)) and polycarboxylate (poly(methacrylic acid)), enhanced amyloid aggregation. Conversely, pyridinium polycation, poly(N-ethylvinylpyridinium), switched the mechanism of alpha-synuclein aggregation from amyloidogenic to amorphous, which resulted in the formation of large amorphous aggregates that do not bind with thioflavin T. The obtained results are relevant as a model of charged macromolecules influence on amyloidosis development in humans. In addition, these results may be helpful in searching for new approaches for synucleinopathies treatment with the use of natural polymers.

Microwave-assisted extraction of an acidic polysaccharide from Ribes nigrum L.: Structural characteristics and biological activities

Microwave-assisted extraction of the polysaccharide from blackcurrant (Ribes nigrum L.) fruits (MBP) was optimized using response surface methodology (RSM). The yield of MBP was 10.59 % (w/w) under the optimal conditions of power (414 W), liquid to material ratio (31/1 mL/g), and the time (41 min). After purification using D4006 macroporous resin and Sepharose-6B column chromatography, a water-soluble acidic heteropolysaccharide (PMBP) was obtained with a molecular weight of 1.99 × 106 g/mol. PMBP was thermally stable and exhibited excellent rheological properties. Fourier transform infrared spectroscopy, gas chromatography and nuclear magnetic resonance analysis confirmed the presence of α-, β-pyranose and six main glycosidic linkages in PMBP. Scanning electron microscopy, atomic force microscopy, and X-ray diffraction studies demonstrated that PMBP was amorphous aggregates with irregular shape, porous, and smooth surface. Bioactivity assays revealed the notable antioxidant and hypoglycemic activities of PMBP. Moreover, PMBP was kinetically characterized as a competitive inhibitor for α-amylase and α-glucosidase.

Synthesis and multicolor emission properties of polystyrene with difluoroboron avobenzone complexes at side chains

Random copolymers (poly(ABSt-co-St)) consisting of avobenzone-BF2 complex monomer (ABSt) and styrene (St) were synthesized and used for emission color tuning including white-like emission in the solid state using St units as a spacer. The emission color of ABSt in the resulting copolymers changed from green to blue via bluish-white by changing the content of St. The fluorescence quantum yields of the polymers in the solid state were relatively high (Φf = 0.30–0.51) independent of the composition contents. The polymers have two main emission bands around 440 and 540 nm, which were derived from the emission of single-molecule and amorphous aggregation, respectively. The fluorescence color changed according to a change in the ratio of the fluorescence intensity at 440 and 540 nm (F440/F540). The absorption and fluorescence properties of the polymers in tetrahydrofuran were independent of the composition ratio of ABSt and St, which indicates no intermolecular interaction between avobenzone-BF2 (AB) fluorophores in the intra-polymer chain. The emission color of all polymers in the solution was blue and the fluorescence spectra were the same as that of ABSt in the solution. Thus, emission color changes observed in the solid state were mainly derived from the intermolecular interaction between AB fluorophores in inter-polymer chains because the polymers intertwined each other in the solid state. When the polymers in the solid state were annealed, F440/F540 increased and all of the fluorescence spectra became similar to that of monomer emission according to the arrangement change of the polymer chain. It is concluded that emission color changes of the polymers are mainly derived from intermolecular interaction between AB fluorophores in the inter-polymer chains. We have demonstrated that poly(ABSt-co-St) forms excellent homogeneous fluorescence polymer films. In contrast, the mixture of methoxy AB (ABOMe) and poly(St) undergoes crystallization even in the low content of ABOMe in poly(St).

The molecular chaperone β-casein prevents amorphous and fibrillar aggregation of α-lactalbumin by stabilisation of dynamic disorder

Deficits in protein homeostasis (proteostasis) are typified by the partial unfolding or misfolding of native proteins leading to amorphous or fibrillar aggregation, events that have been closely associated with diseases including Alzheimer's and Parkinson's diseases. Molecular chaperones are intimately involved in maintaining proteostasis, and their mechanisms of action are in part dependent on the morphology of aggregation-prone proteins. This study utilised native ion mobility–mass spectrometry to provide molecular insights into the conformational properties and dynamics of a model protein, α-lactalbumin (α-LA), which aggregates in an amorphous or amyloid fibrillar manner controlled by appropriate selection of experimental conditions. The molecular chaperone β-casein (β-CN) is effective at inhibiting amorphous and fibrillar aggregation of α-LA at sub-stoichiometric ratios, with greater efficiency against fibril formation. Analytical size-exclusion chromatography demonstrates the interaction between β-CN and amorphously aggregating α-LA is stable, forming a soluble high molecular weight complex, whilst with fibril-forming α-LA the interaction is transient. Moreover, ion mobility–mass spectrometry (IM-MS) coupled with collision-induced unfolding (CIU) revealed that α-LA monomers undergo distinct conformational transitions during the initial stages of amorphous (order to disorder) and fibrillar (disorder to order) aggregation. The structural heterogeneity of monomeric α-LA during fibrillation is reduced in the presence of β-CN along with an enhancement in stability, which provides a potential means for preventing fibril formation. Together, this study demonstrates how IM-MS and CIU can investigate the unfolding of proteins as well as examine transient and dynamic protein–chaperone interactions, and thereby provides detailed insight into the mechanism of chaperone action and proteostasis mechanisms.

Recycling of groundwater treatment sludge to prepare nano-rod erdite particles for tetracycline adsorption

Groundwater treatment sludge was a ferrihydrite-rich solid waste and abundantly generated for potable water production in groundwater treatment plant. Here we demonstrated an alternative strategy to reuse the sludge for preparing nano-rod erdite particles (EPs) via a facile hydrothermal method with only adding Na2S. The produced EPs were characterized by scanning electron microscopy (SEM), X-ray diffractometer (XRD) and X-ray photoelectron spectroscopy (XPS). The SEM results showed that with the molar ratio of N2S to Fe in the sludge increasing from 1 to 2, the product EPs grew long from 0.5-1 μm to 1–4 μm. The product EP-0.2, prepared at the molar ratio of 0.2, was amorphous aggregates with dotted distribution of hexahedral pyrite. With the molar ratio increasing to 2, the product EP-2 was in the form of nanorod, and exhibited high TC adsorption capacity of 1960.8 mg/g, approximately 15 times of that of EP-0.2. By adding 0.02g EP-2, the removal rate of TC was 92.5% at the initial TC concentration of 1000 mg/L, higher than that of active carbon, diatomite, polyaluminium chloride and polyferric sulfate, and nearly 100% TC and 51.5% total organic carbon were removed from pharmaceutical wastewater. EP-2 was completely hydrolyzed to yield Fe oxyhydroxide in TC-containing solution, which exhibited adequate hydroxyl groups to link the –NH2 group at the side chain of TC molecules, resulting in high TC adsorption. This is the first report of recycling groundwater treatment sludge for preparing nano-rod erdite particles, which can be serve as low-cost materials in wastewater treatment.

Exploring the interplay between fibrillization and amorphous aggregation channels on the energy landscapes of tau repeat isoforms

Filaments made up of different isoforms of tau protein are associated with a variety of neurodegenerative diseases. Filaments made up of the 4R-tau isoform, which has four repeat regions (R1 to R4), are found in patients suffering from Alzheimer's disease, while filaments made of the 3R-tau isoform, which contains only three repeat units (R1, R3, and R4), are found in patients with Pick's disease (frontotemporal dementia). In this work, a predictive coarse-grained protein force field, the associative memory water-mediated structure and energy model (AWSEM), is used to study the energy landscapes of nucleation of the two different fibrils derived from patients with Pick's and Alzheimer's diseases. The landscapes for nucleating both fibril types contain amorphous oligomers leading to branched structures as well as prefibrillar oligomers. These two classes of oligomers differ in their structural details: The prefibrillar oligomers have more parallel in-register β-strands, which ultimately lead to amyloid fibrils, while the amorphous oligomers are characterized by a near random β-strand stacking, leading to a distinct amorphous phase. The landscape topography suggests that there must be significant structural reordering, or "backtracking," to transit from the amorphous aggregation channel to the fibrillization channel. Statistical mechanical perturbation theory allows us to evaluate the effects of changing concentration on the aggregation free-energy landscapes and to predict the effects of phosphorylation, which is known to facilitate the aggregation of tau repeats.

Resolving the nanostructure of sodium carbonate extracted pectins (DASP) from apple cell walls with atomic force microscopy and molecular dynamics

The diluted alkali-soluble fraction of the cell wall pectic matrix extracted with sodium carbonate (DASP) from apple fruit (M. domestica cv. Golden Delicious) was subjected to extensive analysis aimed at refining the current view concerning its molecular structure. Pectins were examined by means of atomic force microscopy (AFM) and HPLC chemical analysis. In addition to this, computational chemistry was employed to characterize the structural features of the basic plant cell wall polysaccharides and their possible assemblies. The AFM imaging revealed that DASP molecules deposited on mica structurally resembled rod-like objects, consisting of relatively long linear sections separated by bend points or branches. Locally molecules also formed aggregates, which were visible as an amorphous phase, concentrated around linear sections. Based on chemical analysis combined with molecular dynamics (MD) the linear chains were identified as sections of unbranched homogalacturonan, while the second most abundant polysaccharide present in DASP – arabinose – was considered to constitute the amorphous aggregates. The study showed that despite being present in small amounts, rhamnose is an important factor affecting the formation of the characteristic shape of DASP molecules on mica. The structural characterization of the bend points showed that linear molecules oriented at specific angles are most likely to be formed by two sections of homogalacturonan separated by a single rhamnose unit. AFM images confirmed that this is a common feature of the molecular structure of the DASP fraction, suggesting a possible role in the networking of pectic polysaccharides. Rhamnose interspersions within the homogalacturonan chains lead to the formation of spatial structures, which resemble kinked rods with segments having the ability to change their relative positions. The higher mobility of the linear sections as well as the higher number of segments increases the number of possible interactions with surrounding molecules and thus increases its ability to form gels. Therefore, it is expected that the number of rhamnose units and the length of the linear homogalacturonan sections will be one of the factors that influence the gelling abilities of pectin. Furthermore, it was proposed that branch points are created, similar to bend points, by single interspersions of rhamnose, which connect three homogalacturonan chains or short sections (two or three dimers) of rhamnogalacturonan-I with a homogalacturonan side branch. The third proposed alternative was a complex of two adjacent homogalacturonan chains, one of them having a single rhamnose insertion causing a bend in the structure.

Hazard identification of pyrogenic synthetic amorphous silica (NM-203) after sub-chronic oral exposure in rat: A multitarget approach

Food additive E551 consists of synthetic amorphous silica (SAS), comprising agglomerates and aggregates of primary particles in the nanorange (<100 nm), which potential nanospecific risks for humans associated to dietary exposure are not yet completely assessed. In NANoREG project, aim of the study was to identify potential hazards of pyrogenic SAS nanomaterial NM-203 by a 90-day oral toxicity study (OECD test guideline 408). Adult Sprague-Dawley rats of both sexes were orally treated with 0, 2, 5, 10, 20 and 50 mg SAS/kg bw per day; dose levels were selected to be as close as possible to E551 dietary exposure. Several endpoints were investigated, the whole integrative study is presented here along with the results of dispersion characterization, tissue distribution, general toxicity, blood/serum biomarkers, histopathological and immunotoxicity endpoints. No mortality, general toxicity and limited deposition in target tissues were observed. NM-203 affected liver and spleen in both sexes. Proposed NOAEL 5 mg/kg bw per day in male rats for enlarged sinusoids in liver. In female rats, TSH and creatinine levels were affected, proposed LOAEL 2 mg/kg bw per day. Overall, these data provide new insight for a comprehensive risk assessment of SAS exposure by the oral route.

Effect of tetracycline family of antibiotics on actin aggregation, resulting in the formation of Hirano bodies responsible for neuropathological disorders

Actin, an ATPase superfamily protein, regulates some vital biological functions like cell locomotion, cytokinesis, synaptic plasticity and cell signaling in higher eukaryotes, and is dependent on the dynamics of actin polymerization process. Impaired regulation of actin polymerization has been implicated in the formation and deposition of rod-like paracrystalline structures called as Hirano bodies in neuronal cells of patients suffering from Alzheimer’s disease, Pick’s disease, Guam amyotrophic lateral sclerosis and parkinsonism–dementia complex. Aggregation of actin forming amorphous deposition in the brain cells is also associated with chronic alcoholism and aging of the neurons. In the current article, we propose the breaking of the highly amorphous and dysregulated actin aggregates using generic compounds like tetracycline, oxytetracycline, doxycycline and minocycline which are used as antibiotics against tuberculosis and infection caused due to various Gram-negative bacteria. We have investigated the effect and affinity of binding of these four compounds to that of actin aggregates using 90° light scattering, size exclusion chromatography, dynamic light scattering, circular dichroism, scanning electron microscopy, transmission electron microscopy imaging and kinetic analysis. The isothermal calorimetric measurements showed that the binding constant for the cycline family molecules used in this study range from 9.8 E4 M−1 to 1.3 E4 M−1. To understand the in vivo effect, we also studied the effect of these drugs on Saccharomyces cerevisiae Δend3 mutant cells. Our data suggest that these generic compounds can plausibly be used for the treatment of various neurodegenerative diseases occurring due to Hirano body formation in brain cells.Communicated by Ramaswamy H. Sarma

Impact of metalloporphyrin‐based porous coordination polymers on catalytic activities for the oxidation of alkylbenzene

Seven metalloporphyrin‐based porous coordination polymers: Feш (TZP)Poly (CP1), CoII (TZP)Poly (CP2), NiII (TZP)Poly (CP3), CuII (TZP)Poly (CP4), ZnII (TZP)Poly (CP5), MnII (TZP)Poly (CP6), PbII (TZP)Poly (CP7) (TZP = 5,10,15,20‐ tetrakis[4‐(2,3,4,5‐tetrazolylphenyl)] porphyrin) were prepared and characterized. CP1−CP7 are amorphous aggregation supported with lower crystallinity by scanning electron microscopy, Brunauer−Emmett−Teller and powder X‐ray diffraction. These coordination polymers exhibit effective dye scavenging and catalytic activities toward the oxidation of alkylbenzene to ketones and can be reused by filtration with a slight decreasing of catalytic activities. Metal atoms metalloporphyrin polymers have a great influence on the catalytic activities of metalloporphyrin polymers.

Flavonoid-Derived Human Phenyl-γ-Valerolactone Metabolites Selectively Detoxify Amyloid-β Oligomers and Prevent Memory Impairment in a Mouse Model of Alzheimer's Disease.

Amyloid-β oligomers (AβO) are causally related to Alzheimer's disease (AD). Dietary natural compounds, especially flavonoids and flavan-3-ols, hold great promise as potential AD-preventive agents but their host and gut microbiota metabolism complicates identification of the most relevant bioactive species. This study aims to investigate the ability of a comprehensive set of phenyl-γ-valerolactones (PVL), the main circulating metabolites of flavan-3-ols and related dietary compounds in humans, to prevent AβO-mediated toxicity. The anti-AβO activity of PVLs is examined in different cell model systems using a highly toxic β-oligomer-forming polypeptide (β23) as target toxicant. Multiple PVLs, and particularly the monohydroxylated 5-(4'-hydroxyphenyl)-γ-valerolactone metabolite [(4'-OH)-PVL], relieve β-oligomer-induced cytotoxicity in yeast and mammalian cells. As revealed by atomic force microscopy (AFM) and other in vitro assays, (4'-OH)-PVL interferes with AβO (but not fibril) assembly and actively remodels preformed AβOs into nontoxic amorphous aggregates. In keeping with the latter mode of action, treatment of AβOs with (4'-OH)-PVL prior to brain injection strongly reduces memory deterioration as well as neuroinflammation in a mouse model of AβO-induced memory impairment. PVLs, which have been validated as biomarkers of the dietary intake of flavan-3-ols, lend themselves as novel AβO-selective, candidate AD-preventing compounds.

Nucleation-dependent amyloid fibrillation of human GRASP55 in aqueous solution.

GRASP55, one of the two human GRASP proteins, has been implicated in the organization of Golgi stacks and in unconventional protein secretion. However, the detailed molecular mechanisms supporting GRASP55 participation in those processes remain mostly unclear. We have shown that GRASP55 exists as monomers in solution, which transitions to amorphous aggregates with increasing temperatures. Here, we further investigated the formation of higher order structures of GRASP55 by exploring its amyloid fibrillation at 37°C. Sequence-based AGGRESCAN analysis revealed that GRASP55 has ten aggregation "hot spots", preferentially concentrated in its N-terminal half. Congo Red, ThT, and circular dichroism assays suggested GRASP55 formed amyloid-like fibrils in a time-dependent manner at 37°C. Dynamic light scattering showed the mean hydrodynamic radius of GRASP55 amyloid-like fibrils increased with increasing incubation times at 37°C. Transmission electron microscopy and intrinsic fluorescence lifetime imaging showed that, upon increasing incubation time at 37°C, GRASP55 yielded amyloid-like fibrils in a nucleation-dependent process via a sequence of events: lag-phase (monomers to oligomers), growth phase (oligomers to organized protofibrils), and plateau phase (protofibrils to amyloid-like fibrils). The insights gained herein may help in better understanding the mechanisms of GRASP55 amyloid fibrillation in vivo and its potential association with neurological disorders.

The Significance of Utilizing In Vitro Transfer Model and Media Selection to Study the Dissolution Performance of Weak Ionizable Bases: Investigation Using Saquinavir as a Model Drug.

This study investigated the dissolution behavior of BCS class II ionizable weak base Saquinavir and its mesylate salt in the multi-compartment transfer setup employing different composition of dissolution media. The dissolution behavior of Saquinavir was studied by using a two-compartment transfer model representing the transfer of drug from the stomach (donor compartment) to the upper intestine (acceptor compartment). Various buffers like phosphate, bicarbonate, FaSSIF, and FeSSIF were employed. The dissolution was also studied in the concomitant presence of the additional solute, i.e., Quercetin. Further, the dissolution profiles of Saquinavir and its mesylate salt were simulated by GastroPlusTM, and the simulated dissolution profiles were compared against the experimental ones. The formation of in situ HCl salt and water-soluble amorphous phosphate aggregates was confirmed in the donor and acceptor compartments of the transfer setup, respectively. As the consequence of the lower solubility product of HCl salt of Saquinavir, the solubility advantage of mesylate salt was vanished leading to the lower than the predicted dissolution in the acceptor compartment. However, the formation of water-soluble aggregates in the presence of the phosphate salts was observed leading to the higher than the predicted dissolution of the free base in the transfer setup. Interestingly, the formation of such water-soluble aggregates was found to be hindered in the concomitant presence of an ionic solute resulting in the lower dissolution rates. The in situ generation of salts and aggregates in the transfer model lead to the inconsistent prediction of dissolution profiles by GastroPlusTM.

Kinetic Variability in Seeded Formation of ALS-Linked SOD1 Fibrils Across Multiple Generations.

The unseeded aggregation of superoxide dismutase-1 (SOD1) into amyloid-like fibrils occurs stochastically in vitro and in vivo, that is, isolated populations of SOD1 proteins (within microplate wells or living cells) self-assemble into amyloid at rates that span a probability distribution. This stochasticity has been attributed to variable degrees of monomer depletion by competing pathways of amorphous and fibrillar aggregation (inter alia). Here, microplate-based thioflavin-T (ThT) fluorescence assays were performed at high iteration (∼300) to establish whether this observed stochasticity persists when progenitor ("parent") SOD1 fibrils are used to seed the formation of multiple generations of progeny fibrils (daughter, granddaughter, and great-granddaughter fibrils). Populations of progenitor fibrils formed stochastically at different rates and fluorescence intensity, however, progeny fibrils formed at more similar rates regardless of the formation rate of the progenitor fibril. For example, populations of progenitor fibrils that formed with a lag time of ∼30 h or ∼15 h both produced progeny fibrils with lag times of ∼8 h. Likewise, populations of progenitor fibrils with high or low maximum fluorescence (e.g., ∼450 or ∼75 A.U.) both produced progeny fibrils with more similar maximum fluorescence (∼125 A.U.). The rate of propagation was found to be more dependent on monomer concentration than seed concentration. These results can be rationalized by classical rate laws for primary nucleation and monomer-dependent secondary nucleation. We also find that the seeding propensity of some "families" of in vitro grown fibrils exhibit a finite lifetime (similar to that observed in the seeding of small molecule crystals and colloids). The single biological takeaway of this study is that the concentration of native SOD1 in a cell can have a stronger effect on rates of seeded aggregation than the concentration of prion-like seed that infected the cell.

Cascade Energy Transfer in Insulin Amyloid Fibrils Doped by Thioflavin T, Benzanthrone and Squarine Dyes

The three-step Förster resonance energy transfer (FRET) within the cascade of four dyes, including the classical amyloid marker Thioflavin T as a primary donor, two jumper dyes, benzanthrone ABM and squaraine SQ4, and terminal acceptor SQ1, was tested as a possible tool for detection and characterization of insulin amyloid fibrils. The results obtained confirm the occurrence of highly efficient multistep FRET (msFRET) in the chromophore ensemble in the presence of insulin fibrils formed at elevated temperature under pH 2 (InsF1) or pH 7.4, 0.15 M NaCl (InsF2), while negligible FRET efficiencies were obtained for the control unfibrillized protein, suggesting the specificity of msFRET to cross-β-sheet architecture characteristic of amyloid fibrils. Specifically, the efficiencies of FRET for the donor-acceptor pairs ThT-ABM, ABM-SQ4 and SQ4-SQ1 at maximum acceptor concentrations (~0.4 µM – 1.6 µM) were estimated to be 86%/94%, 48%/34% and 66%/32%, respectively, in the presence of InsF1/InsF2. The most significant differences between InsF1/InsF2 and the control protein were observed for the donor-acceptor pair ThT-ABM, suggesting that ABM is the key mediator in the whole process of msFRET. Assuming the isotropic rotation of the fluorophores, the average donor-acceptor distances were estimated in the presence of InsF1, yielding the values 1.3 nm, 5.3 nm, and 3.9 nm for the ThT-ABM, ABM-SQ4 and SQ4-SQ1 pairs, respectively. The obtained distances are indicative of different fibril binding sites for the chromophores in the insulin fibrils, although due to their high specificity to the fibrillar structure, the dyes are most likely to localize in the surface grooves of β-sheets running along the main axis of amyloid fibril. Remarkably, the differences in the insulin amyloid morphology can be clearly distinguished using msFRET. As evidenced from TEM, InsF2 were thinner, shorter and contained amorphous aggregates, as compared to InsF1. Thus, different amyloid formation pathways under neutral and acidic pH resulted in the changes in the dye affinity for to the fibril binding sites, and, as a consequence, in the distinct msFRET efficiencies, especially for the pair SQ4-SQ1. The ability of ThT to serve as an efficient amplifier for the two near-infrared dyes, SQ4 and SQ1, with the benzanthrone fluorophore ABM as a jumper dye, allows detection of fibrillar insulin in the optical window of the biological samples, with the Stokes shift of the four-chromophore being ca. 240 nm. The proposed msFRET-based approach can be employed not only for insulin amyloid detection but also for distinguishing between different amyloid fibril morphologies and gaining further insights into the mechanisms involved in the development of the injection-localized insulin amyloidosis.