Photophysical Properties of Fluorescent Probe Thioflavin T in Crowded Milieu

Thioflavin T (ThT) is a widely used fluorescent probe of amyloid fibrils, which accompanies many serious neurodegenerative and other diseases. Until recently, examinations of processes of amyloid fibril formation in vitro were conducted in solutions whose properties were significantly different from those found inside the densely packed cells. Such crowded cellular milieu is typically simulated in vitro using concentrated solutions of inert polymers, which do not usually interact with proteins. However, these crowding agents can have a direct effect on the ThT molecule, and this effect must be taken into account. We examined the influence of PEG-400, PEG-12000, and Dextran-70 on the photophysical properties of ThT. It was shown that these crowding agents caused the red shift of the absorption, fluorescence excitation, and fluorescence spectra of ThT. Under these conditions, the increases of the molar extinction coefficient, fluorescence quantum yield, and excitation lifetime of ThT are also observed. However, these changes are significantly less pronounced than those observed for ThT bound to fibrils. It is concluded that, despite some effects of crowding agents on intrinsic fluorescent properties of ThT, this dye can be used as a probe of structure and formation of amyloid fibrils in crowded milieu in vitro

Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique

A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates

Point mutations affecting yeast prion propagation change the structure of its amyloid fibrils

We investigated the effect of the point substitutions in the N-terminal domain of the yeast prion protein Sup35 (Sup35NMp) on the structure of its amyloid fibrils. As the objects of the study, proteins with mutations that have different influence on the [PSI+] prion propagation, but do not prevent the aggregation of Sup35NMp in vitro were chosen. The use of the wide range of physico-chemical methods allowed us to show significant differences in the structure of these aggregates, their physical size, clumping tendency. Also we demonstrated that the fluorescent probe thioflavin T (ThT) can be successfully used for investigation of subtle changes in the structural organization of fibrils formed from various Sup35NMp. The obtained results and our theoretical predictions allowed us to conclude that some of selected amino acid substitutions delimit the region of the protein that forms the core of amyloid fibrils, and change the fibrils structure. The relationship of structural features of in vitro Sup35NMp amyloid aggregates with the stability of the [PSI+] prion in vivo allowed us to suggest that oligopeptide repeats (R) of the amyloidogenic N-terminal domain of Sup35NMp from R0 to R2 play a key role in protein aggregation. Their arrangement rather than just presence is critical for propagation of the strong [PSI+] prion variants. The results confirm the suitability of the proposed combination of theoretical and empirical approaches for identifying changes in the amyloid fibrils structure, which, in turn, can significantly affect both the functional stability of amyloid fibrils and their pathogenicity.Laboratorio de Investigación y Desarrollo de Bioactivo

Fluorescence Quantum Yield of Thioflavin T in Rigid Isotropic Solution and Incorporated into the Amyloid Fibrils

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils

ОПЫТ ПРИМЕНЕНИЯ ИНДИВИДУАЛЬНЫХ ТИТАНОВЫХ ИМПЛАНТАТОВ ДЛЯ ВОССТАНОВЛЕНИЯ АНАТОМИЧЕСКОЙ СТРУКТУРЫ НОСА

Aim. Clinical use of implants based on patient’s individual topographic anatomical features. Materials and methods. The paper describes two case reports involving the correction of nasal anatomical structure affected during the surgery for facial cancer. Using selective laser sintering technology we manufactured 2 intricate 3D-deformed individual implants from Ti70V30 alloy powders. Implant design was based on CAT/MRT data. Clinical use was conducted on the premises of the department of head and neck surgery in Chelyabinsk Regional Oncological Dispensary. Results. In the fi rst case we obtained an impressive functional result – huge and wide nasal cavity, nose skeleton, and easy nasal breathing. In 30 days we performed endoscopic video-analysis (via nasal passages) which showed that 65% of implant surface was covered with mucosa. In the second case, the designed implant allowed the combined surgery including excision of soft tissues, atypical resection of the upper jaw (left part) and the left cheek, left maxillary sinusotomy and reconstruction of the left part of the nose and the left cheek with implant and skin fl ap. Conclusion. Clinical use revealed pros and contras of the applied technology. Among contras there are relatively high cost of technology, high standards of competency of a surgeon, a programmer and engineers. Pros include free modeling and setting the implant parameters before operation, exact match between implant size/shape and topographic anatomical data of a certain patient. Цель. Клиническое применение имплантатов с индивидуальными топографо-анатомическими данными пациента. Материалы и методы. Описываются 2 клинических случая восстановления структуры носа, нарушенной в ходе операций по поводу рака области лица. При помощи технологии селективного лазерного сплавления по данным КТ/МРТ было изготовлено 2 сложных, деформированных в трехмерном пространстве индивидуальных имплантата из порошков сплава Ti70V30. Клиническое применение проходило в отделении хирургии головы и шеи Челябинского окружного онкологического диспансера. Результаты. В первом клиническом случае был достигнут отличный функциональный результат – большая и широкая полость носа, каркас носа и свободное носовое дыхание. Через 30 дней эндоскопический видеоанализ (через носовые отверстия) показал 65% покрытие поверхности имплантата слизистой оболочкой. Во втором случае проведена комбинированная операция по удалению мягких тканей, атипичная резекция верхней челюсти слева, левой щеки, гайморотомия слева и реконструкция левой половины носа и левой щеки с помощью имплантата и лучевого лоскута. Заключение. Клиническое применение показало плюсы и минусы технологии. К минусам относится относительно высокая стоимость и высокие профессиональные требования к врачу, программисту, инженерам. К плюсам – свобода моделирования и задание параметров будущего имплантата до операции и точное соответствие размеров и формы имплантата топографо-анатомическим данным конкретного пациента.

Two Novel Amyloid Proteins, RopA and RopB, from the Root Nodule Bacterium Rhizobium leguminosarum

Amyloids represent protein fibrils with a highly ordered spatial structure, which not only cause dozens of incurable human and animal diseases but also play vital biological roles in Archaea, Bacteria, and Eukarya. Despite the fact that association of bacterial amyloids with microbial pathogenesis and infectious diseases is well known, there is a lack of information concerning the amyloids of symbiotic bacteria. In this study, using the previously developed proteomic method for screening and identification of amyloids (PSIA), we identified amyloidogenic proteins in the proteome of the root nodule bacterium Rhizobium leguminosarum. Among 54 proteins identified, we selected two proteins, RopA and RopB, which are predicted to have β-barrel structure and are likely to be involved in the control of plant-microbial symbiosis. We demonstrated that the full-length RopA and RopB form bona fide amyloid fibrils in vitro. In particular, these fibrils are β-sheet-rich, bind Thioflavin T (ThT), exhibit green birefringence upon staining with Congo Red (CR), and resist treatment with ionic detergents and proteases. The heterologously expressed RopA and RopB intracellularly aggregate in yeast and assemble into amyloid fibrils at the surface of Escherichia coli. The capsules of the R. leguminosarum cells bind CR, exhibit green birefringence, and contain fibrils of RopA and RopB in vivo.This work was supported by the Russian Science Foundation, grant 17-16-01100

The molecular lifecycle of amyloid – Mechanism of assembly, mesoscopic organisation, polymorphism, suprastructures, and biological consequences

The formation of a diverse range of amyloid structures from normally soluble proteins and peptides is a hallmark of devastating human disorders as well as biological functions. The current molecular understanding of the amyloid lifecycle reveals four processes central to their growth and propagation: primary nucleation, elongation, secondary nucleation and division. However, these processes result in a wide range of cross-β packing and filament arrangements, including diverse assemblies formed from identical monomeric precursors with the same amino acid sequences. Here, we review current structural and mechanistic understanding of amyloid self-assembly, and discuss how mesoscopic, i.e. micrometre to nanometre, organisation of amyloid give rise to suprastructural features that may be the key link between the polymorphic amyloid structures and the biological response they elicit. A greater understanding of the mechanisms governing suprastructure formation will guide future strategies to combat amyloid associated disorders and to use and control the amyloid quaternary structure in synthetic biology and materials applications

β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis

Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another—followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis (“on-pathway state”), or can be formed as a result of an alternative assembly of partially unfolded monomers (“off-pathway state”). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only “the tip of the iceberg”. Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds