Thioflavin Research Articles

Cold atmospheric pressure plasma assisted rapid assembly of peptide-based structures: a molecular scaffold to form supramolecular architectures

The use of cold atmospheric pressure plasma (CAPP) technology in the production of peptide-based materials has shown great potential in modern technology. Herein, two aggregation-prone oligopeptides, GNNQQNY and KLVFFA, were subjected to CAPP treatment to form supramolecular assemblies/aggregates. Through peptide engineering and biophysical techniques, the effect of CAPP-generated reactive oxygen and nitrogen species on the oligopeptides were investigated for different treatment times revealing that the formation of these aggregates were primarily driven by electrostatic interactions without any chemical modifications. Field emission-scanning electron microscopy and Thioflavin T (ThT) binding assay confirmed the presence of distinct β-strands, particularly in the aggregates of the KLVFFA peptide upon CAPP irradiation. The combination of CAPP technology with peptide self-assembly process and the characterization techniques employed in this study holds promise for the development of such peptide supramolecular structures based on the specific amino acid sequences.

Engineering of a DNAzyme-Based dimeric G-quadruplex rolling circle amplification for robust analysis of lead ion

Detecting heavy metal pollution, particularly lead ion (Pb2⁺) contamination, is imperative for safeguarding public health. In this study, we introduced an innovative approach by integrating DNAzyme with rolling circle amplification (RCA) to propose an amplification sensing method termed DNAzyme-based dimeric-G-quadruplex (dimer-G4) RCA. This sensing approach allows for precise and high-fidelity Pb2⁺ detection. Strategically, in the presence of Pb2⁺, the DNAzyme undergoes substrate strand (S-DNA) cleavage, liberating its enzyme strand (E-DNA) to prime isothermal amplification. This initiates the RCA process, producing numerous dimer-G-Quadruplexes (dimer-G4) as the signal reporting transducers. Compared to conventional strategies using monomeric G-quadruplex (mono-G4) as the reporting transducers, these dimer-G4 structures exhibit significantly enhanced fluorescence when bound with Thioflavin T (ThT), offering superior target signaling ability for even detection of Pb2⁺ at low concentration. Conversely, in the absence of Pb2⁺, the DNAzyme structure remains intact so that no primers can be produced to cause the RCA initiation. This nucleic acid amplification-based Pb2⁺ detection method combing with the high specificity of DNAzymes for Pb2⁺ recognition ensures highly sensitive detection of Pb2+ with a detection limit of 0.058 nM, providing a robust tool for food safety analysis and environmental monitoring.

Design, synthesis, and applications of nucleic acid-specific benzoxazole-N,N-dialkylphenylamines derivatives for nucleolus imaging in the cells

Considered the brain of the nucleus, alterations in the structure and function of the nucleolus are linked to numerous cellular functions and, consequently, contribute to several diseases. The identification of nucleolar morphology and activity via novel biomarkers presents new avenues for the development of therapeutic approaches for a variety of human diseases, including cancer, neurodegeneration, and aging. Therefore, specific detection of the nucleolus with fluorescence probes is of critical importance for clinical applications. In the present study, a series of benzoxazole-N,N-dialkylphenylamines derivative compounds were designed and synthesized based on the benzothiazole-based fluorescence probe Thioflavin T (ThT). Among the compounds, BX-3 and BX-16, which carry electron-withdrawing substituents in the benzoxazole ring, were observed to have higher fluorescence emission at wavelengths of 470 and 465 nM, respectively. The general morphology and divisions of the cells were examined under inverted and light microscopes, respectively, and the fluorescence potentials of selected compounds were determined using immunofluorescence microscopy. The fluorescence intensity of molecules and 3D interactive surface plot images of cells were analyzed using ImageJ software. Cell imaging analyses showed that BX-6 and BX-13, like ThT, specifically stain the cell nucleolus. Moreover, molecular docking studies showed that the compounds could identify the RNA-rich nucleolus by binding with high affinity to the guanine region in the RNA structure. The results suggest that the compounds may be an initial route in developing specific biosensor compounds for nucleolus imaging.

Anion–Induced Amyloid Fibrillation of Human Insulin In vitro

AbstractIn the present study, we have focused on the effect of three biologically important salt anions on insulin aggregation. The result of the present study reveals that salts differing in their anions (NaI, NaOAc, and NaNO3) induce the self–assembly formation of insulin even at low physiological (in the micromolar range) salt concentration with efficacy that follows the order I−>CH3COO−>NO3−. Here, the anion–driven aggregation of insulin does not follow either the Hofmeister series or electroselectivity series at very low salt concentrations; instead, the binding of anions at low pH to the positively charged residues of insulin is determined by a mechanism where the salt anions promote the fibrillation of insulin and modify the morphology of the monomeric precursor molecule. Both the nucleation and fibril elongation are controlled by the electrostatic forces and hydrophobic interactions. Aggregation mechanism and aggregate morphology were investigated employing a combination of Thioflavin T (ThT) and ANS fluorescence, circular dichroism (CD), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Overall, the present data will enhance the idea to rationalize the anion effects on the aggregation of amyloid‐prone protein and to understand the influence of charged biomolecules in cellular compartments.

Plasmonic colorimetry and G-quadruplex fluorescence-based aptasensor: A dual-mode, protein-free and label-free detection for OTA

G-quadruplexes (G4) have received significant attention in the field of aptasensors owing to their unique physicochemical characteristics. A dual-mode, protein-free and label-free aptamer sensor based on plasmonic colorimetry and G4 fluorescence (PC@GF-aptasensor) was proposed for ochratoxin A (OTA). Colorimetry mode was achieved through the surface plasmon resonance (SPR) effect, which related to the OTA-Apt-based G4–OTA. The fluorescence mode was reflected by the insertion of thioflavin T (ThT) into G4–OTA. The OTA could be interpreted via three readouts: (1) naked eye (LOD of 2.0 ng mL−1), (2) smartphone (LOD of 1.65 ng mL−1), and (3) spectrofluorometer (LOD of 0.93 ng mL−1). The PC@GF-aptasensor exhibited several advantages, such as a standardised recognition group, simplified operation, low background signal, and practicality. The proposed PC@GF-aptasensor integrated SPR-based multicolour interpretation and ThT-inserted fluorescence reflection to obtain a dual-mode optical biosensor, which may provide valuable insights for the development of other targets with G4-based aptamers.

Construction of a fluorescent and colorimetric multi-mode aptasensor for label-free and sensitive detection of ochratoxin A in agricultural products

Ochratoxin A (OTA) can induce a serious danger to human beings, and it is crucial to develop a sensitive and simple strategy for visual detection of OTA. Herein, we construct a fluorescent and colorimetric multi-mode aptasensor for label-free and sensitive detection of OTA on the basis of target-initiated assembly of functional G-quadruplex (G4) DNAzyme nanostructures. When OTA is present, it specifically recognizes the aptamer and disintegrates the aptamer-cDNA duplex structure, resulting in the liberation of intact cDNA and the subsequent initiation of terminal deoxynucleotidyl transferase (TdT) assisted-extension reaction. The resultant G4 DNAzyme nanostructures can light up Thioflavin T (ThT) to produce a high fluorescence signal. Meanwhile, the addition of hemin can trigger the self-assembly of hemin/G4 DNAzymes that can catalyze the 3,3′,5,5′-tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) system to generate a distinct colorimetric signal. Consequently, the introduction of G4 DNAzyme/ThT and hemin/G4 DNAzyme can efficiently transduce the OTA signal into both a fluorescence signal and a colorimetric signal. The further utilization of a smartphone facilitates the development of a simple method based on red-green-blue (RGB) digital analysis for OTA assay. The proposed multi-mode aptasensor displays ultrahigh sensitivity with a detection limit of 2 pg/mL for the fluorescent mode, 25 pg/mL for the colorimetric mode, and 6 pg/mL for the RGB mode. Moreover, this multi-mode aptasensor can be applied to accurately detect OTA in real samples, providing a new platform for on-site monitoring of OTA in agricultural products.

Delineating the impact of N21D mutation on the conformational preferences and structural transitions in human islet amyloid polypeptide

The amyloid deposits of human islet amyloid polypeptide (hIAPP) in the pancreatic β-cells are linked to a chronic metabolic disease known as type 2 diabetes (T2D). Recent studies reported that N21D mutation in hIAPP inhibited the formation of fibrillar aggregates, however, the underlying molecular mechanism of fibril inhibition in the N21D mutant remains largely unclear. Thus, molecular dynamics (MD) simulations have been employed to illuminate the effect of N21D mutation on the conformational dynamics and structural transformations in hIAPP at the atomistic level. Notably, the structural stability of WT-hIAPP (wild type-hIAPP) was enhanced upon N21D mutation. Interestingly, the sampling of helical conformation in hIAPP was significantly increased from 12.60 to 19.80% with a concomitant decrease in β-sheet from 7.40 to 1.20% upon N21D mutation that corroborates with the circular dichroism (CD). The dihedral principal component analysis (dPCA) depicted a notable reduction in the conformational phase space of [N21D]hIAPP as compared to WT-hIAPP, which highlights the prevention of conformational transitions and enhanced structural stability. Remarkably, a complete loss in the β-sheet content was noted in the conformations extracted from the free energy landscape of [N21D]hIAPP as compared to 24–25% sampling of β-sheets in minimum energy conformations of WT-hIAPP consistent with the transmission electron microscopy (TEM) and thioflavin T (ThT) fluorescence assay. The present study provided key insights into the N21D mutation-induced conformational changes in hIAPP and highlighted that Asn21 plays a key role in hIAPP aggregation. The insights from this work will be beneficial in the design of drug candidates by selectively targeting Asn21 of hIAPP which could interfere with its aggregation and associated cytotoxicity.

G-Quadruplex Structure in the ATP-Binding DNA Aptamer Strongly Modulates Ligand Binding Activity.

Secondary structures formed by single-stranded DNA aptamers can allow for the binding of small-molecule ligands. Some of these secondary structures are highly stable in solution and are great candidates for use in the development of molecular tools for biomarker detection, environmental monitoring, and others. In this paper, we explored adenosine triphosphate (ATP)-binding aptamers for the simultaneous detection of two small-molecule ligands: adenosine triphosphate (ATP) and thioflavin T (ThT). The aptamer can form a G-quadruplex (G4) structure with two G-quartets, and our results show that each of these quartets is equally involved in binding. Using fluorescently labeled and label-free methods, we further explored the role of the G4 motif in modulating the ligand binding property of the aptamer by making two extended variants that can form three or four G-quartet G4 structures. Through equilibrium binding and electrospray ionization mass spectrometry (ESI-MS) analysis, we observed a stronger affinity of aptamers to ATP by the variant G4 constructs relative to the native aptamer (Kd range of 0.040-0.042 μM for variants as compared to 0.15 μM for the native ATP aptamer). Additionally, we observed a dual binding of ThT and ATP to the G4 constructs in the label-free and ESI-MS analyses. These findings together suggest that the G4 motif in the ATP aptamer is a critical structural element that is required for optimum ATP binding and can be modulated for the binding of multiple ligands. These findings are instrumental for designing smart molecular tools for a wide range of applications, including biomarker monitoring and ligand binding studies.

Flavones in pomelo peel resist fibril formation of human islet amyloid polypeptide

ObjectiveExploring the formation and aggregation of human islet amyloid polypeptide (hIAPP) (amylin) fibers is significant for promoting the prevention and treatment of type II diabetes mellitus (T2DM). Flavones in pomelo peel have visible biological activity in the anti-diabetes aspect. The present study aimed to investigate the effects of five flavones [naringin (NRG), narirutin (NRR), nobiletin (NOB), sinensetin (SIN), and neohesperidin (NHP)] in pomelo peel on peptide aggregation and explore its possible mechanisms. The cell viability of flavones against peptide aggregation was also evaluated. MethodsThe thioflavin T (ThT) assay and transmission electron microscopy (TEM) were used for evaluating the inhibition and disaggregation of flavones on peptide aggregation. The interaction mechanism was analyzed by endogenous fluorescence, molecular dynamics (MD) simulations, ultraviolet spectroscopy (UV) and isothermal titration calorimetry (ITC) experiments. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and immune assays were performed to characterize the cell viability of flavones against peptide aggregation. ResultsThe five flavones showed a decrease in fluorescence intensity, fiber number and size under incubation with different molar ratios of hIAPP. The compounds can bind to the aromatic tyrosine (Tyr) residueTyr 37, resulting in the intrinsic fluorescence quenching of the peptides. Five flavones can form hydrogen bonds with hIAPP, which is likely to be based on their phenolic hydroxyl structure. They showed strong binding affinity with peptides. The reaction system of NRG and NRR observed an exothermic reaction, and the others were endothermic reactions. The absorption peaks of the compounds with hIAPP changed and showed hypochromic effects, indicating that there may be π-π stacking interaction. Flavones noticeably increased the cell viability in the presence of amyloid peptides and reduced the absorption intensity induced by peptide oligomers. ConclusionFive flavones in pomelo peel have inhibitory and depolymerization effects on amyloid fibrils, and can significantly protect cells from the toxic effect of hIAPP and reduce the production of toxic oligomers.

Understanding the Modulation of α-Synuclein Fibrillation by N-Acetyl Aspartate: A Brain Metabolite.

α-Synuclein (α-Syn) fibrillation is a prominent contributor to neuronal deterioration and plays a significant role in the advancement of Parkinson's Disease (PD). Considering this, the exploration of novel compounds that can inhibit or modulate the aggregation of α-Syn is a topic of significant research. This study, for the first time, elucidated the effect of N-acetyl aspartate (NAA), a brain osmolyte, on α-Syn aggregation using spectroscopic and microscopic approaches. Thioflavin T (ThT) assay revealed that a lower concentration of NAA inhibits α-Syn aggregation, whereas higher concentrations of NAA accelerate the aggregation. Further, this paradoxical effect of NAA was complemented by ANS, RLS, and the turbidity assay. The secondary structure transition was more pronounced at higher concentrations of NAA by circular dichroism, corroborating the fluorescence spectroscopic observations. Confocal microscopy also confirmed the paradoxical effect of NAA on α-Syn aggregation. Interaction studies including fluorescence quenching and molecular docking were employed to determine the binding affinity and critical residues involved in the α-Syn-NAA interaction. The explanation for this paradoxical nature of NAA could be a solvophobic effect. The results offer a profound understanding of the modulatory mechanism of α-Syn aggregation by NAA, thereby suggesting the potential role of NAA at lower concentrations in therapeutics against α-Syn aggregation-related disorders.

Amyloidogenic Propensity of Metabolites in the Uric Acid Pathway and Urea Cycle Critically Impacts the Etiology of Metabolic Disorders.

Novel insights into the etiology of metabolic disorders have recently been uncovered through the study of metabolite amyloids. In particular, inborn errors of metabolism (IEMs), including gout, Lesch-Nyhan syndrome (LNS), xanthinuria, citrullinemia, and hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, are attributed to the dysfunction of the urea cycle and uric acid pathway. In this study, we endeavored to understand and mechanistically characterize the aggregative property exhibited by the principal metabolites of the urea cycle and uric acid pathway, specifically hypoxanthine, xanthine, citrulline, and ornithine. Employing scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM), we studied the aggregation profiles of the metabolites. Insights obtained through molecular dynamics (MD) simulation underscore the vital roles of π-π stacking and hydrogen bonding interactions in the self-assembly process, and thioflavin T (ThT) assays further corroborate the amyloid nature of these metabolites. The in vitro MTT assay revealed the cytotoxic trait of these assemblies, a finding that was substantiated by in vivo assays employing the Caenorhabditis elegans (C. elegans) model, which revealed that the toxic effects were more pronounced and dose-specific in the case of metabolites that had aged via longer preincubation. We hence report a compelling phenomenon wherein these metabolites not only aggregate but transform into a soft, ordered assembly over time, eventually crystallizing upon extended incubation, leading to pathological implications. Our study suggests that the amyloidogenic nature of the involved metabolites could be a common etiological link in IEMs, potentially providing a unified perspective to study their pathophysiology, thus offering exciting insights into the development of targeted interventions for these metabolic disorders.

Early events during the aggregation of Aβ16-22-derived switch-peptides tracked using Protein Charge Transfer Spectra

BackgroundUnderstanding Aβ aggregation and inhibiting it at early stages is of utmost importance in treating Alzheimer's and other related amyloidogenic diseases. However, majority of the techniques to study Aβ aggregation mainly target the late stages; while those used to monitor early stages are either expensive, use extrinsic dyes, or do not provide information on molecular level interactions. Here, we investigate the early events of Aβ16-22(KLVFFAE) aggregation using Aβ16-22 derived switch-peptides (SwPs) through a novel label-free approach employing Protein Charge Transfer Spectra (ProCharTS). ResultsWhen pH is increased from 2 to 7.2, the Aβ-derived switch peptides undergo controlled self-assembly, where the initial random coil peptides convert into β-sheet. We leveraged the intrinsic absorbance/luminescence arising from ProCharTS among growing peptide oligomers to observe the aggregation kinetics in real-time. In comparison to monomer, the lysine and glutamate headgroups in the peptide oligomer are expected to come in proximity enhancing ProCharTS intensity due to photoinduced electron transfer. With a combination of Aβ-derived switch-peptides and ProCharTS, we obtained structural insights on the early stages of Aβ-derived SwP aggregation in four unique peptides. Increase in scatter corrected ProCharTS absorbance (250–500 nm) and luminescence (320–720 nm) along with decreased mean luminescence lifetime (2.3–0.8 ns) characterize the initial stages of aggregation monitored for 1–96 h depending on the peptide. We correlated the results with Circular Dichroism (CD), 8-anilino-1-naphthalenesulfonic acid (ANS) and Thioflavin T (ThT) measurements. SignificanceWe demonstrate ProCharTS as an intrinsic analytical probe with following advantages over other conventional methods to track aggregation: it is a label-free probe; it's intensity can be measured using a UV–Vis spectrophotometer; it is more sensitive in detecting the early molecular events in aggregation compared to ANS and ThT; and it can provide information on specific contacts made between charged headgroups of Lysine/Glutamate in the oligomer.

Intrinsically disordered Pseudomonas chaperone FapA slows down the fibrillation of major biofilm-forming functional amyloid FapC.

Functional bacterial amyloids play a crucial role in the formation of biofilms, which mediate chronic infections and contribute to antimicrobial resistance. This study focuses on the FapC amyloid fibrillar protein from Pseudomonas, a major contributor to biofilm formation. We investigate the initial steps of FapC amyloid formation and the impact of the chaperone-like protein FapA on this process. Using solution nuclear magnetic resonance (NMR), we recently showed that both FapC and FapA are intrinsically disordered proteins (IDPs). Here, the secondary structure propensities (SSPs) are compared to alphafold (DeepMind, protein structure prediction tool/algorithm: https://alphafold.ebi.ac.uk/) models. We further demonstrate that the FapA chaperone interacts with FapC and significantly slows down the formation of FapC fibrils. Our NMR titrations reveal ~ 18% of the resonances show FapA-induced chemical shift perturbations (CSPs), which has not been previously observed, the largest being for A82, N201, C237, C240, A241, and G245. These sites may suggest a specific interaction site and/or hotspots of fibrillation inhibition/control interface at the repeat-1 (R1)/loop-2 (L2) and L2/R3 transition areas and at the C-terminus of FapC. Remarkably, ~ 90% of FapA NMR signals exhibit substantial CSPs upon titration with FapC, the largest being for S63, A69, A80, and I92. A temperature-dependent effect of FapA was observed on FapC by thioflavin T (ThT) and NMR experiments. This study provides a detailed understanding of the interaction between the FapA and FapC, shedding light on the regulation and slowing down of amyloid formation, and has important implications for the development of therapeutic strategies targeting biofilms and associated infections.

Single-Particle Photoluminescence Measures a Heterogeneous Distribution of Differential Circular Absorbance of Gold Nanoparticle Aggregates near Constricted Thioflavin T Molecules.

The chirality of biomacromolecules is critical for their function, but the optical signal of this chirality is small in the visible range. Plasmonic nanoparticles are antennas that can couple to this chiral signal. Here, we examine the molecular-scale mechanism behind the induced circular dichroism of gold nanorods (AuNRs) in solution with insulin fibrils and the fibril-intercalating dye thioflavin T (ThT) with polarization-resolved single-molecule fluorescence and single-particle photoluminescence (PL) imaging. We compared the PL upon excitation by left- and right-handed circularly polarized light to calculate the differential absorbance of AuNRs near insulin fibrils with and without ThT. Overall, our results indicate that AuNRs do not act as chiral absorbers near constricted ThT molecules. Instead, we hypothesize that fibrils promote AuNR aggregation, and this templating is mediated by subtle changes in the solution conditions; under the right conditions, only a few chiral aggregates with significantly higher circular dichroism signal contribute to a large net circular dichroism.

The Structural Features of Skeletal Muscle Titin Aggregates

Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are β-sandwiches with a predominant β-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction, and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphologies in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The "KCl-aggregates" according to AFM data had the form of a "sponge"-like structure, while amorphous "glycine-aggregates" of titin formed "branching" structures. Spectrofluorometry revealed the ability of "glycine-aggregates" of titin to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross β-structure, a reflection of ~4.6 Å, in these aggregates. These data indicate that "glycine-aggregates" of titin are amyloid or amyloid-like. No similar structural features were found in "KCl-aggregates" of titin; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data we obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.

Mirror-Image Phage Display for the Selection of D-Amino Acid Peptide Ligands as Potential Therapeutics.

In neurodegenerative diseases like Alzheimer's disease (AD), endogenous proteins or peptides aggregate with themselves. These proteins may lose their function or aggregates and/or oligomers can obtain toxicity, causing injury or death to cells. Aggregation of two major proteins characterizes AD. Amyloid-β peptide (Aβ) is deposited in amyloid plaques within the extracellular space of the brain and Tau in so-called neurofibrillary tangles in neurons. Finding peptide ligands to halt protein aggregation is a promising therapeutical approach. Using mirror-image phage display with a commercially available, randomized 12-mer peptide library, we have selected D-amino acid peptides, which bind to the Tau protein and modulate its aggregation in vitro. Peptides can bind specifically and selectively to a target molecule, but natural L-amino acid peptides may have crucial disadvantages for in vivo applications, as they are sensitive to protease degradation and may elicit immune responses. One strategy to circumvent these disadvantages is the use of non-naturally occurring D-amino acid peptides as they exhibit increased protease resistance and generally do not activate the immune system. To perform mirror-image phage display, the target protein needs to be synthesized as D-amino acid version. If the target protein sequence is too long to be synthesized properly, smaller peptides derived from the full length protein can be used for the selection process. This also offers the possibility to influence the binding region of the selected D-peptides in the full-length target protein. Here we provide the protocols for mirror-image phage display selection on the PHF6* peptide of Tau, based on the commercially available Ph.D.™-12 Phage Display Peptide Library Kit, leading to D-peptides that also bind the full length Tau protein (Tau441), next to PHF6*. In addition, we provide protocols and data for the first characterization of those D-peptides that inhibit Tau aggregation in vitro. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mirror image phage display selection against D-PHF6* fibrils Support Protocol 1: Single phage ELISA Basic Protocol 2: Sequencing and D-peptide generation Basic Protocol 3: Thioflavin-T (ThT) test to control inhibition of Tau aggregation Support Protocol 2: Purification of full-length Tau protein Basic Protocol 4: ELISA to demonstrate the binding of the generated D-peptides to PHF6* and full-length Tau fibrils.

Prediction of the Feasibility of Using the ≪Gold Standard≫ Thioflavin T to Detect Amyloid Fibril in Acidic Media.

Ordered protein aggregates, amyloid fibrils, form toxic plaques in the human body in amyloidosis and neurodegenerative diseases and provide adaptive benefits to pathogens and to reduce the nutritional value of legumes. To identify the amyloidogenic properties of proteins and study the processes of amyloid fibril formation and degradation, the cationic dye thioflavin T (ThT) is the most commonly used. However, its use in acidic environments that induce amyloid formation in vitro can sometimes lead to misinterpretation of experimental results due to electrostatic repulsion. In this work, we show that calculating the net charge per residue of amyloidogenic proteins or peptides is a simple and effective approach for predicting whether their fibrils will interact with ThT at acidic pH. In particular, it was shown that at pH 2, proteins and peptides with a net charge per residue > +0.18 are virtually unstained by this fluorescent probe. The applicability of the proposed approach was demonstrated by predicting and experimentally confirming the absence of ThT interaction with amyloids formed from green fluorescent (sfGFP) and odorant-binding (bOBP) proteins, whose fibrillogenesis was first carried out in an acidic environment. Correct experimental evidence that the inability to detect these fibrils under acidic conditions is precisely because of the lack of dye binding to amyloids (and not their specific structure or the low fluorescence quantum yield of the bound dye) and that the number of ThT molecules associated with fibrils increases with decreasing acidity of the medium was obtained by using the equilibrium microdialysis approach.

Advanced technique of myocardial no-reflow quantification using indocyanine green.

The post-ischemic no-reflow phenomenon after primary percutaneous coronary intervention (PCI) is observed in more than half of subjects and is defined as the absence or marked slowing of distal coronary blood flow despite removal of the arterial occlusion. To visualize no-reflow in experimental studies, the fluorescent dye thioflavin S (ThS) is often used, which allows for the estimation of the size of microvascular obstruction by staining the endothelial lining of vessels. Based on the ability of indocyanine green (ICG) to be retained in tissues with increased vascular permeability, we proposed the possibility of using it to assess not only the severity of microvascular obstruction but also the degree of vascular permeability in the zone of myocardial infarction. The aim of our study was to investigate the possibility of using ICG to visualize no-reflow zones after ischemia-reperfusion injury of rat myocardium. Using dual ICG and ThS staining and the FLUM multispectral fluorescence organoscope, we recorded ICG and ThS fluorescence within the zone of myocardial necrosis, identifying ICG-negative zones whose size correlated with the size of the no-reflow zones detected by ThS. It is also shown that the contrast change between the no-reflow zone and nonischemic myocardium reflects the severity of blood stasis, indicating that ICG-negative zones are no-reflow zones. The described method can be an addition or alternative to the traditional method of measuring the size of no-reflow zones in the experiment.

A background correctable fluorescence sensing strategy for adenosine triphosphate evaluation based on aptamer configuration alteration

Ratiometric sensors with background correction are expected to get around the interference from complicated systems. However, there is currently a scarcity of viable carriers for quickly and easily assembling fluorophores with varying fluorescence emission. In this work, we developed a novel aptamer-based ratiometric fluorescence sensor for analysis of adenosine triphosphate (ATP) based on the properties of aptamer that enable conformational transformations upon binding to the target. The carbon dots (CDs) and thioflavin T (ThT) are employed as the reference and detection signal for the ratio-dependent sensor, which are both integrated with the aptamer. The green fluorescence of ThT was initially turn-on after binding with aptamer and then quenched in the presence of ATP, whereas the red fluorescence of CDs remained practically steady. This approach exhibits remarkable selectivity and can detect ATP in the range of 0.5–60 μM with a detection limit of 0.167 μM. Moreover, the method was successfully applied to the determination of ATP in human serum as well as in A549 cells, demonstrating the potential for medical diagnostic applications.

High-Throughput Screening of pH-Dependent β-sheet Self-Assembling Peptide.

pH-dependent peptide biomaterials hold tremendous potential for cell delivery and tissue engineering. However, identification of responsive self-assembling sequences with specified secondary structure remains a challenge. In this work, An experimental procedure based on the one-bead one-compound (OBOC) combinatorial library is developed to rapidly screen self-assembling β-sheet peptides at neutral aqueous solution (pH 7.5) and disassemble at weak acidic condition (pH 6.5). Using the hydrophobic fluorescent molecule thioflavin T (ThT) as a probe, resin beads displaying self-assembling peptides show fluorescence under pH 7.5 due to the insertion of ThT into the hydrophobic domain, and are further cultured in pH 6.5 solution. The beads with extinguished fluorescence are selected. Three heptapeptides are identified that can self-assemble into nanofibers or nanoparticles at pH 7.5 and disassemble at pH 6.5. P1 (LVEFRHY) shows a rapid acid response and morphology transformation with pH modulation. Changes in the charges of histidine and hydrophobic phenyl motif of phenylalanine may play important roles in the formation of pH-responsive β-sheet nanofiber. This high-throughput screening method provides an efficient way to identify pH-dependent β-sheet self-assembling peptide and gain insights into structural design of such nanomaterials.