Optimized sonoreactor for accelerative amyloid-fibril assays through enhancement of primary nucleation and fragmentation

Nakajima, Katsuaki; Noi, Kentaro; Yamaguchi, Keiichi; So, Masatomo; Ikenaka, Kazuhiro; Mochizuki, Hideki; Ogi, Hirotsugu; Goto, Yuji

doi:10.1016/j.ultsonch.2021.105508

Cited by 15 publications

(35 citation statements)

References 49 publications

(43 reference statements)

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“…S1 ). First, we monitored the kinetics of fibril amplification by our HANABI assay 30,31 ( Figure 1.A ), an instrument that consists of an optimized sonoreactor equipment developed by our group, that applies ultrasonic irradiation pulses individually to each one of the wells of a 96-wells microplate, and records the ThT fluorescence in function of time, named HANABI-2000 31 . This instrument accelerates substantially the rate of fibril formation reaction compared with the traditional RT-QuIC methods 32,33 , from several days to a few hours.…”

Section: Resultsmentioning

confidence: 99%

“…The ThT fluorescence intensity was monitored using a HANdai Amyloid Burst Inducer (HANABI-2000) equipment, developed by our group in collaboration with CORONA ELECTRIC, Ibaraki, Japan, consisting of a transducer driving system, an acoustic-intensity measurement system, and a fluorescence measure system, in which a microplate reader was combined with a multichannel ultrasonication system that applies ultrasonic irradiation independently to each individual well of the microplate via a miniaturized ultrasonic resonator attached to each well. Ultrasonication was applied to accelerate amyloid formation at an optimized frequency of 30 kHz 31,47 in cycles of 300 ms of irradiation and 500 ms of quiescence. The kinetic parameters of fibril formation was determined by fitting the experimental data to empirical equations 48 .…”

Section: Kinetics Of Fibril Formation Followed By Thioflavin T (Tht)mentioning

confidence: 99%

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Conformational switch in the alpha-synuclein C-terminus domain directs its fibril polymorphs

Aguirre

Ikenaka

et al. 2022

Preprint

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α-Synuclein inclusion bodies are a pathological hallmark of several neurodegenerative diseases. Although it has been hypothesized a relationship between fibril polymorphism and different pathologies, the molecular origins of polymorphism are not understood. Here, employing biophysical approaches, we demonstrate that the conformational state of the monomeric αSyn is responsible for fibril polymorphism: αSyn can exist as a compact monomer that produces rod fibrils, and as extended monomers that generate twisted fibrils. Using NMR, we found that the compaction relies on a polar interaction between the initial part of the NAC region and a wide section of the C-terminus domain. The compaction can be commonly affected by changes in the chemical environment, like NaCl, the presence of Ca2+ or cellular components, like endotoxins, that alter the interaction NAC/C-terminus domain. Our compaction model also provides mechanistic insights that explain how the behavior of the C-terminus domain imparts the polymorphism during the fibril formation.

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Section: Resultsmentioning

confidence: 99%

Section: Kinetics Of Fibril Formation Followed By Thioflavin T (Tht)mentioning

confidence: 99%

Conformational switch in the alpha-synuclein C-terminus domain directs its fibril polymorphs

Aguirre

Ikenaka

et al. 2022

Preprint

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“…Thus, control reactions were performed at 1.0 mg/mL 2m at pH 7.4 and 60 °C. To apply further effective agitation to induce amyloid fibril formation, we adopted an originally developed ultrasonic instrument, HANABI-2000 [27][28][29] , which accelerates amyloid fibril formation 10,11,31,32 by the effects of ultrasonic cavitation 33,34 and monitors amyloid formation by amyloid-specific fluorescence dye, thioflavin-T (ThT) 35 . Scheme of the experiment is described in Figure S2.…”

Section: Hanabi Assay For 2m Amyloid Fibril Formationmentioning

confidence: 99%

“…It should be noted that, although previous studies [23][24][25][26] investigated amyloid fibril formation in a crowded milieux (e.g., serum milieu), there is no consensus on the effects of macromolecular crowding in a serum milieux. With HANABI-2000 [27][28][29] , an ultrasonication-forced amyloid fibril inducer, the effects of sera from hemodialysis patients on 2m amyloid fibril formation were investigated. To perform a systematic investigation, we collected over 100 sera from individuals with or without hemodialysis therapy.…”

Section: Introductionmentioning

confidence: 99%

Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

Nakajima¹,

Yamaguchi²,

Noji

et al. 2022

Preprint

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Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of β2-microglobulin (β2m). Although high serum β2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigated the effects of human sera on β2m amyloid fibril formation. Although sera markedly inhibited amyloid fibril formation, the inhibitory effects were weaker for sera collected from dialysis patients than for control sera. When sera collected before and after maintenance dialysis treatments were compared, the latter inhibited amyloid fibril formation more than the former. These results indicate that, although the inhibitory effects of sera were deteriorated in long-term dialysis patients, they were ameliorated by maintenance dialysis treatments in the short term. Maintenance dialysis decreases the body weight by 5% and consequently increases the concentrations of serum components. Among these components, we found that serum albumin prevented amyloid fibril formation based on macromolecular crowding effects, and that the decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. The model was constructed assuming accumulative effects of three risk factors and may be useful for predicting the onset of DRA. Furthermore, the model suggested the importance of monitoring temporary and accumulated risks to prevent the development of amyloidoses in general, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu.

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“…As a result, we successfully detected the amyloid fibril formation of β2-microglobulin (β2m) and the low concentration seed of β2m. 37,38 We name this improved HANABI system "HANABI-2000". In the previous study, 36 we have shown that the developed multichannel ultrasound reactor is capable of inducing fibrillation reactions of Aβ 1−40 and αsynuclein solutions.…”

Section: Introductionmentioning

confidence: 99%

Acceleration of amyloid fibril formation by multichannel sonochemical reactor

Noi,

Nakajima,

Yamaguchi

et al. 2021

Preprint

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Formation of amyloid fibrils of various amyloidogenic proteins is dramatically enhanced by ultrasound irradiation. For applying this phenomenon to the study of protein aggregation science and diagnosis of neurodegenerative diseases, a multichannel ultrasound irradiation system with individually adjustable ultrasound-irradiation conditions is necessary. Here, we develop a sonochemical reaction system, where an ultrasonic transducer is placed in each well of a 96-well microplate to perform ultrasonic irradiation of sample solutions under various conditions with high reproducibility, and applied it for studying amyloidfibril formation of amyloid β, α-synuclein, β2-microglobulin, and lysozyme. The results clearly show that our instrument is superior to conventional shaking method in terms of degree of acceleration and reproducibility of fibril formation reaction. The acceleration degree is controllable by controlling the driving voltage applied to each transducer. We have thus succeeded in developing a useful tool for the study of amyloid fibril formation in various proteins.

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Optimized sonoreactor for accelerative amyloid-fibril assays through enhancement of primary nucleation and fragmentation

Abstract: Graphical abstract

Cited by 15 publications

References 49 publications

Conformational switch in the alpha-synuclein C-terminus domain directs its fibril polymorphs

Conformational switch in the alpha-synuclein C-terminus domain directs its fibril polymorphs

Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

Acceleration of amyloid fibril formation by multichannel sonochemical reactor

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