Thioflavin Research Articles

Imaging G-Quadruplex Nucleic Acids in Live Cells Using Thioflavin T and Fluorescence Lifetime Imaging Microscopy.

Visualization of guanine-rich oligonucleotides that fold into G-quadruplex (G4) helical structures is of great interest in cell biology. There is a large body of evidence that suggests that these noncanonical structures form in vivo and play important biological roles. A promising recent development highlighted fluorescence lifetime imaging microscopy (FLIM) as a robust technique for the direct and quantitative imaging of G4s in live cells. However, this method requires specialized, bespoke synthetic dyes that are not widely available. Herein, we demonstrate that the fluorescence lifetime of commercially available environmentally sensitive dyes Thioflavin T (ThT) and Thiazole Orange (TO) is strongly dependent on the type of DNA topology they bind to, with G4s showing long and distinctive decay times that should allow G4 detection in the biological environment. We applied this observation to visualize G4s in live U2OS cells using FLIM of ThT, upon alteration in G4 levels due to competitive binding or nuclease treatment of cells.

Exploring Cimetidine as a Potential Therapeutic Attenuator against Amyloid Formation in Parkinson's Disease: Spectroscopic and Microscopic Insights into Alpha-Synuclein and Human Insulin.

Neurodegenerative diseases, notably Alzheimer's and Parkinson's, hallmark their progression through the formation of amyloid aggregates resulting from misfolding. While current therapeutics alleviate symptoms, they do not impede disease onset. In this context, repurposing existing drugs stands as a viable therapeutic strategy. Our study determines the antihistamine drug Cimetidine's potential as an inhibitor using diverse spectroscopic and microscopic methods on alpha-synuclein and human insulin amyloid formation, unveiling its efficacy. The thioflavin T (ThT) assay illustrated a dose-dependent reduction in amyloid formation with escalating concentrations of Cimetidine. Notably, the antihistamine drug maintained a helical structure and showed no significant conformational changes in the secondary structure. Confocal microscopy validated fewer fibrils in the Cimetidine-treated samples. Remarkably, Cimetidine interacted with pre-existing fibrils, leading to their disintegration. Further analyses (ThT, circular dichroism, and dynamic light scattering) showcased the conversion of fibrils into smaller aggregates upon Cimetidine addition. These findings signify the potential of this antihistamine drug as a plausible therapeutic option for Parkinson's disease. This study may open avenues for deeper investigations and possible therapeutic developments, emphasizing Cimetidine's promising role in mitigating neurodegenerative diseases like Parkinson's.

Quantification of Rapid Cooling of Glycerol/Water Solutions Based on Photoluminescence from Thioflavin T.

Rapid cooling to a solid state allows intermediates in chemical and biomolecular processes that occur in solution near room temperature to be trapped for subsequent measurements by magnetic resonance spectroscopies, electron microscopy, or other techniques. In time-resolved solid state nuclear magnetic resonance and rapid freeze-quench electron paramagnetic resonance studies, solutions are typically frozen by spraying into a cold bath or onto a cold metal surface. Although simulations suggest freezing on millisecond or submillisecond time scales, direct experimental measurements of cooling rates have been elusive. Here, we describe a method for quantification of rapid cooling rates based on measurements of temperature-dependent photoluminescence from thioflavin T (ThT). In our experiments, a jet of ThT solution in glycerol/water, with 10.8 m/s jet velocity and 30 μm diameter, freezes on a cold, rotating copper surface. Images of ThT photoluminescence on the copper surface indicate that the cooling rate of the solution increases linearly with the surface velocity over the 0.45-6.2 m/s range. At surface velocities greater than 3.8 m/s, the time to cool from 300 to 260 K or from 300 to 230 K is less than 100 μs or less than 700 μs. The experimental results do not agree quantitatively with calculations in which a layer of glycerol/water cools by thermal conduction when suddenly brought in contact with a cold copper surface. Discrepancies between experimental results and simplistic calculations illustrate the importance of direct measurements of cooling rates.

Protein misfolding by manganese (III) oxide nanoparticles generated by pulsed laser ablation in liquids

The high demand for metal oxide nanoparticles for applications in various fields has led to increased research and investigation. However, one tends to neglect the harmful effects of these synthesized nanoparticles on human health. Mn2O3 nanoparticles are a type of metal oxide nanoparticle that can have harmful effects if ingested or inhaled. In this study, Mn2O3 nanoparticles are synthesized using laser ablation at the manganese-water interface. The structural and optical properties of the nanoparticles are studied using HR-TEM, Micro-Raman, Ultraviolet–visible (UV–vis), and Photoluminescence spectroscopy. The cubic bixbyite α-Mn2O3 nanoparticles produced by laser ablation are used for studying their role in inhibiting or promoting protein fibrillation. Different concentrations of α-Mn2O3 nanoparticles are added to Human serum albumin (HSA) protein, where the fibrillation of the protein is investigated with the help of thioflavin T (ThT) dye using fluorescence spectroscopy. The protein misfolded on adding the nanoparticles that could be seen in the images obtained using fluorescence microscopic imaging, where the HSA forms a dense network of fibrils. In-vitro studies reveal that the increasing concentration of the nanoparticles increases the fibrillation of the HSA protein.

Unveiling molecular mechanism underlying inhibition of human islet amyloid polypeptide fibrillation by benzene carboxylic acid-peptide conjugate

Type 2 diabetes (T2D) is linked to the apoptosis of insulin-producing β-cells due to the aberrant fibrillation of the human islet amyloid polypeptide (hIAPP or amylin) to cytotoxic aggregates. Profit et al. generated conjugates (C1–C7) by appending benzene carboxylic acids of varying charges to the N-terminal of hIAPP22-29 fragment peptide NFGAILSS to modulate hIAPP fibrillation and cytotoxicity. C5 (4 µM) derived by conjugating low-cost, commercially available benzene-1,2,4,5-tetracarboxylic acid known as pyromellitic acid to NFGAILSS completely abolishes the hIAPP (40 µM) self-assembly as noted in the thioflavin T (ThT) fluorescence assay. The circular dichroism (CD) spectra highlighted that C5 stabilized hIAPP in a distinctive conformation and blocked its conformational switching to amyloidogenic β-sheet structure. C5 possessing a charge-dense pyromellitic acid moiety appended on the N-terminal region of self-recognition hIAPP fragment sequence NFGAILSS created significant interest as it effectively inhibited hIAPP fibrillation and possessed lower molecular mass, smaller size, and charge as compared to hIAPP aggregation inhibitor EEEENFGAILSS (P10). However, it remains unclear how C5 traps hIAPP into a unique conformation that abolishes its self-aggregation propensity. Thus, molecular dynamics (MD) simulations have been employed to illuminate the conformational transitions and structural changes in hIAPP on the inclusion of C5. C5 displayed high-affinity binding interactions to hIAPP (ΔGbinding = −37.11 ± 3.72 kcal/mol) with major contributions from the van der Waals and electrostatic interactions. Furthermore, residue-specific binding free energy analysis depicted high-affinity binding interactions of C5 with Phe15, His18, Asp21, and Tyr37 of hIAPP that play a crucial role in hIAPP fibrillation. The negatively charged carboxylate groups of pyromellitic acid moiety of C5 displayed interactions with the key residues of hIAPP as noted in the conformational clustering analysis. Notably higher sampling of helix from 24.00 ± 0.84 % in hIAPP to 34.20 ± 1.92 % in hIAPP–C5 is consistent with the CD studies, which depicted that C5 trapped hIAPP monomer in a conformation that blocked its self-association. MD simulations illuminated the molecular mechanism and atomistic details of the C5 interactions with hIAPP, which are responsible for its inhibitory activity against hIAPP fibrillation and alleviating hIAPP aggregates-induced cytotoxicity.

Exploring the rhodanine universe: Design and synthesis of fluorescent rhodanine-based derivatives as anti-fibrillar and anti-oligomer agents against α-synuclein and 2N4R tau

Tau and α-synuclein (α-syn) are prone-to-aggregate proteins that coprecipitate in the brains of Alzheimer’s disease (AD) and Parkinson’s disease (PD) patients. The early-stage oligomers and protofibrils of tau are believed to be strongly linked to human cognitive impairment while the toxic α-syn oligomers are associated with behavioral motor deficits. Therefore, concurrent targeting of both proteinaceous aggregates and their lower dense oligomers are very challenging. Herein, rhodanine-based compounds were designed and synthesized to tackle the fibrils and oligomers of tau and α-syn proteins. In particular, the indole-containing rhodanines 5l and 5r displayed significantly high anti-aggregation activity towards α-syn fibrils by reduction of the thioflavin-T (ThT) fluorescence to lower than 5 %. Moreover, 5r showed a remarkable decrease in the fluorescence of thioflavin-S (ThS) when incubated with the non-phosphorylated tau 0N4R and 2N4R as well as the hyperphosphorylated tau isoform 1N4R, respectively. Transmission electron microscopy (TEM) analyses validated the powerful anti-fibrillar activity of 5l and 5r towards both protein aggregates. In addition, both 5l and 5r highly suppressed 0N4R tau and α-syn oligomer formation using the photo-induced cross-linking of unmodified Protein (PICUP) assay. The fluorescence emission intensity of 5l was quenched to almost half in the presence of both protein fibrils at 510 nm. 5r showed a similar fluorescence response upon binding to 2N4R fibrils while no quenching effect was observed with α-syn aggregates. Ex vivo disaggregation assay using extracted human Aβ plaques was employed to confirm the ability of 5l and 5r to disaggregate the dense fibrils. Both inhibitors showed no promising activity towards the cell-based α-syn inclusion formation. However, another indolyl derivative 5j prevented the α-syn inclusion at 5 µM. Collectively, the indolyl-rhodanine scaffold could act as a building block for further structural optimization to obtain dual targeting disease-modifying candidates for AD and PD.

Highly Sensitive Naphthalene-Based Twisted Intramolecular Charge Transfer Molecules for the Detection of In Vitro and In Cellulo Protein Aggregates.

Newly synthesized naphthalene-based twisted intramolecular charge transfer (TICT) molecules show 8.5- and 2.6-fold increases in fluorescence intensity upon binding with protein aggregates in comparison with the fluorescence enhancement for thioflavin T (ThT). The dissociation constant (K d ) values of these compounds with bovine serum albumin (BSA) aggregates are in the 145-176 nM range, which is 103 times lower than that of ThT. Along with the strong binding propensity, these molecules are also capable of measuring protein aggregate (BSA) concentration in the 500 to 5 pM level. Interestingly, one of the synthesized molecules was also able to bind with the intracellular protein aggregates.

G-quadruplex fluorescent and lateral flow colorimetric aptasensor for the detection of capsaicin in illicit cooking oil

The safety of cooking oil is a major global concern and capsaicin (CAP) serves as a crucial marker for assessing its quality. To address this issue, we have devised an innovative biosensor using a truncated aptamer, Cap-1–2, as the foundation for a dual-mode aptasensor. The aptasensor employs a combination of Mg2+-dependent DNAzyme (MNAzyme) cleavage and hybridization chain reaction (HCR) to achieve highly sensitive CAP detection. Notably, the fluorescence signal originates from the inherent fluorescence of thioflavin T (ThT) embedded in a G-quadruplex (G4) structure, enabling the detection of CAP in high-throughput oil samples. Additionally, the biosensor employed the MNAzyme to cleave biotinylated DNA (S1-Biotin), which is subsequently ligated to functionalized gold nanoparticles (AuNPs-Poly A-cDNA), generating outstanding colorimetric signals on lateral flow test strips for immediate point-of-care testing (POCT). The linear detection ranges of the established fluorescence and colorimetric aptasensor were 0.15–80 ng mL−1 and 0.2–100 ng mL−1, respectively, and the limits of detection (LOD) were 0.054 ng mL−1 and 0.137 ng mL−1, respectively. These results demonstrate the practicality of the aptasensor for detecting CAP in real-world samples. Moreover, the aptasensor surpasses traditional biosensors in terms of high-throughput, portability, and sensitivity, making it highly promising for rapid detection in the food industry.

Cross-Interactions of Aβ Peptides Implicated in Alzheimer's Disease Shape Amyloid Oligomer Structures and Aggregation.

A defining hallmark of Alzheimer's disease (AD) is the synaptic aggregation of the amyloid β (Aβ) peptide. In vivo, Aβ production results in a diverse mixture of variants, of which Aβ40, Aβ42, and Aβ43 are profusely present in the AD brain, and their relative abundance is recognized to play a role in disease onset and progression. Nonetheless, the occurrence of Aβ40, Aβ42, and Aβ43 hetero-oligomerization and the subsequent effects on Aβ aggregation remain elusive and were investigated here. Using thioflavin-T (ThT)-monitored aggregation assays and native mass spectrometry coupled to ion mobility analysis (IM-MS), we first show that all Aβ peptides are aggregation-competent and self-assemble into homo-oligomers with distinct conformational populations, which are more pronounced between Aβ40 than the longer variants. ThT assays were then conducted on binary mixtures of Aβ variants, revealing that Aβ42 and Aβ43 aggregate independently from Aβ40 but significantly speed up Aβ40 fibrillation. Aβ42 and Aβ43 were observed to aggregate concurrently and mutually accelerate fibril formation, which likely involves hetero-oligomerization. Accordingly, native MS analysis revealed pairwise oligomerization between all variants, with the formation of heterodimers and heterotrimers. Interestingly, IM-MS indicates that hetero-oligomers containing longer Aβ variants are enriched in conformers with lower collision cross-sections when compared to their homo-oligomer counterparts. This suggests that Aβ42 and Aβ43 are capable of remodeling the oligomer structure toward a higher compaction level. Altogether, our findings provide a mechanistic description for the hetero-oligomerization of Aβ variants implicated in AD, contributing to rationalizing their in vivo proteotoxic interplay.

Construction of G-quadruplex/thioflavin T fluorescent probe for label-free detection of malachite green

Malachite green (MG), owing to its extreme toxicity, is strictly prohibited as a drug in aquaculture across numerous countries. To facilitate an economical and user-friendly strategy for MG detection, we rigorously screened c-MYC22 from a wide range of G-quadruplex (G4) DNAs, aiming to craft a fluorescent probe in tandem with the water-soluble fluorescent dye thioflavin T (ThT). This endeavor culminated in the development of a label-free, rapid and sensitive method for MG detection. The fluorescence of the G4/ThT complex dims significantly when MG interacts with G4, allowing for the quantitative analysis of MG. Our method boasts linear detection ranges between 50–1000 µg/L and 400–6000 µg/L, with an impressive limit of detection as low as 6.82 µg/L and a short detection time of 15 min. The fluorescent probe demonstrates excellent specificity, rendering it ideal for MG detection in real water samples. Furthermore, this method stands out for its simplicity and efficiency, the probe used does not need to be modified, and the detection takes only one step with a short detection time. These features make it a strong contender for commercialization as a method of MG detection, presenting significant potential for further advancement.

Effects of intracoronary supersaturated oxygen infusion on the time course of myocardial blood flow and extent of microvascular obstruction in an ischemia/reperfusion preclinical swine model

Abstract Introduction Despite successful epicardial reperfusion with primary PCI, reactive changes in microvascular function lead to impaired myocardial reperfusion. Intracoronary SuperSaturated Oxygen Therapy (SSO2) is the only approved therapy that improves myocardial salvage in STEMI pts through the delivery of hyperbaric levels of oxygen to viable ischemic vascular and myocardial cells. Several important questions remain unanswered regarding the potential mechanisms of this benefit. Therefore, the purpose of this study is to determine the effects of SSO2 on the time course of myocardial blood flow (MBF) and the extent of microvascular obstruction (MVO) in an ischemia/reperfusion model. Methods 10 swine (32-40 kg) surviving 90-minute balloon-induced anterior STEMI, and 15-min auto reperfusion, were assigned to 120-min of SSO2 (n=5) or control (n=5) with 120-min auto reperfusion. MBF was evaluated at various time points during the experiment by injection of 15–µm microspheres (BioPal, USA) through a catheter placed in the left ventricle. Before euthanasia, 4% thioflavin-S (TS) was injected into the left anterior descending artery. The left ventricle was sectioned into 10-mm thick slices, and the myocardial rings were photographed under UV light to determine areas at risk (TS positive), MVO (lack of TS staining within TS positive area), and remote zones (contiguous areas unstained by TS). MBF was calculated as the total blood flow within the areas at risk normalized to the total flow within the remote zones. Results All animals experienced significant reduction (p = 0.02) in MBF due to balloon occlusion at the 85-minute MI timepoint compared to baseline (Fig). Following the removal of occlusion, reactive hyperemic flows were observed for both groups at the 15-minute auto reperfusion time point (Fig). Starting at the 30-min time point of autoperfusion to 120-min, the MBF significantly decreased (p=0.02) in the controls (Fig). In contrast during these time points, for the SSO2 group the MBF values were numerically higher than the controls, with average MBF for the SSO2 group being significantly higher (p = 0.03) than the controls (1.1 ± 0.18 vs 0.6 ± 0.13, respectively) at the 120-min timepoint (Fig). MVO was found within the areas at risk in all animals, but the extent of MVO in relation to the area at risk was significantly reduced (p < 0.001) in the SSO2 group versus controls (15.2 ± 7.3% vs 41.8% ± 6.2%, respectively). Conclusions Results from this study showed that in a STEMI swine model, SSO2 prevented reduction in MBF during the 120-min reperfusion period, with significantly increased MBF at the end of the experiment compared to controls. This flow improvement translated to a 64% relative reduction in the extent of MVO compared to controls. These results suggest that one of the mechanisms by which SSO2 improves myocardial salvage may be by limiting early microcirculatory alterations set in motion by a cascade of reduced myocardial flow and MVO.Myocardial Blood Flow vs Time

Noradrenaline Protects Human Microglial Cells (HMC3) Against Apoptosis and DNA Damage Induced by LPS and Aβ1-42 Aggregates In Vitro.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, characterized by the accumulation of amyloid-beta (Aβ) plaques and neuroinflammation. This study investigates the protective effects of noradrenaline (NA) on human microglial cells exposed to lipopolysaccharides (LPS) and Aβ aggregates-major contributors to inflammation and cellular damage in AD. The reduced Aβ aggregation in the HMC3 human microglial cells co-treated with Aβ and NA was confirmed by thioflavin T (ThT) assay, fluorescent ThT staining, and immunocytochemistry (ICC). The significantly increased viability of HMC3 cells after 48 h of incubation with NA at 50 µM, 25 µM, and 10 µM, exposed to IC50 LPS and IC50 Aβ, was confirmed by XTT and LDH assays. Moreover, we found that NA treatment at 25 μM and 50 μM concentrations in HMC3 cells exposed to IC50 LPS or IC50 Aβ results in an increased proliferation of HMC3 cells, their return to normal morphology, decreased levels of DNA damage, reduced caspase-3 activity, decreased expression of pro-apoptotic DDIT3 and BAX, and increased expression of anti-apoptotic BCL-2 genes and proteins, leading to enhanced cell survival, when compared to that of the HMC3 cells treated only with IC50 LPS or IC50 Aβ. Furthermore, we showed that NA induces the degradation of both extracellular and intracellular Aβ deposits and downregulates hypoxia-inducible factor 1α (HIF-1α), which is linked to impaired Aβ clearance and AD progression. These findings indicate that NA holds promise as a therapeutic target to address microglial dysfunction and potentially slow the progression of AD. Its neuroprotective effects, particularly in reducing inflammation and regulating microglial activity, warrant further investigation into its broader role in mitigating neuroinflammation and preserving microglial function in AD.

Thioflavin-T: application as a neuronal body and nucleolar stain and the blue light photo enhancement effect

Thioflavin-T (THT) is a common and indispensable tool for the study of amyloid pathologies and protein aggregation, both in vitro and for histological samples. In this study we expand the use of THT beyond its canonical usage for staining amyloid plaques and demonstrate its novel use as an easy and rapid stain comparable to fluorescent Nissl staining, allowing for clear discernment of neuronal cell bodies and also nucleoli in fixed tissue and live cells. We believe that this is of value for any lab that studies central nervous system (CNS) tissues. Furthermore, we show that THT could potentially be used as a an alternative to the use of fluorescent reporters or other more costly RNA binding compounds in the study of nucleolar dynamics owing to its ability to clearly stain nucleoli in live cells. We also discovered the previously unreported effect of blue light exposure on the photo enhancement of THT excited by a 488 nm laser in stained tissue sample and how to avoid complications arising from this effect. Finally, we provide a simple protocol that can be easily adjusted either for using THT as a neuronal cell body and nucleoli stain, compatible with antibody based staining methods tested up to 4 fluorophores, or alternatively by using an additional washing step the protocol may be used for amyloid plaque detection in fixed brain tissue.

Evaluation of Alpha-Synuclein and Tau Antiaggregation Activity of Urea and Thiourea-Based Small Molecules for Neurodegenerative Disease Therapeutics.

Alzheimer's disease (AD) and Parkinson's disease (PD) are multifactorial, chronic diseases involving neurodegeneration. According to recent studies, it is hypothesized that the intraneuronal and postsynaptic accumulation of misfolded proteins such as α-synuclein (α-syn) and tau, responsible for Lewy bodies (LB) and tangles, respectively, disrupts neuron functions. Considering the co-occurrence of α-syn and tau inclusions in the brains of patients afflicted with subtypes of dementia and LB disorders, the discovery and development of small molecules for the inhibition of α-syn and tau aggregation can be a potentially effective strategy to delay neurodegeneration. Urea is a chaotropic agent that alters protein solubilization and hydrophobic interactions and inhibits protein aggregation and precipitation. The presence of three hetero atoms (O/S and N) in proximity can coordinate with neutral, mono, or dianionic groups to form stable complexes in the biological system. Therefore, in this study, we evaluated urea and thiourea linkers with various substitutions on either side of the carbamide or thiocarbamide functionality to compare the aggregation inhibition of α-syn and tau. A thioflavin-T (ThT) fluorescence assay was used to evaluate the level of fibril formation and monitor the anti-aggregation effect of the different compounds. We opted for transmission electron microscopy (TEM) as a direct means to confirm the anti-fibrillar effect. The oligomer formation was monitored via the photoinduced cross-linking of unmodified proteins (PICUP). The anti-inclusion and anti-seeding activities of the best compounds were evaluated using M17D intracellular inclusion and biosensor cell-based assays, respectively. Disaggregation experiments were performed with amyloid plaques extracted from AD brains. The analogues with indole, benzothiazole, or N,N-dimethylphenyl on one side with halo-substituted aromatic moieties had shown less than 15% cutoff fluorescence obtained with the ThT assay. Our lead molecules 6T and 14T reduced α-syn oligomerization dose-dependently based on the PICUP assays but failed at inhibiting tau oligomer formation. The anti-inclusion effect of our lead compounds was confirmed using the M17D neuroblastoma cell model. Compounds 6T and 14T exhibited an anti-seeding effect on tau using biosensor cells. In contrast to the control, disaggregation experiments showed fewer Aβ plaques with our lead molecules (compounds 6T and 14T). Pharmacokinetics (PK) mice studies demonstrated that these two thiourea-based small molecules have the potential to cross the blood-brain barrier in rodents. Urea and thiourea linkers could be further improved for their PK parameters and studied for the anti-inclusion, anti-seeding, and disaggregation effects using transgenic mice models of neurodegenerative diseases.

Dual-color fluorescent multi-component hybrid materials for copper ion detection, NADH regeneration and cell imaging

Gold nanoclusters (AuNCs) exhibit unique physical, chemical and photoelectric properties with various advantages. On the other hand, small organic fluorescent molecules show alternative specialized characteristics. To overcome their weaknesses and integrate strengths, we propose here a workable platform to fabricate protein-gold-dye multifunctional hybrid materials. Briefly, protein skeleton bovine serum albumin (BSA), AuNCs, and organic molecule dye Thioflavin T (ThT) are co-assembled mutually to form a dual-color fluorescent material BSA-AuNCs@ThT. This approach is simple, cost-effective, green and environmental friendly. To tackle their biological application, we coat BSA-AuNCs@ThT with cationic polyethyleneimine (PEI). The prepared BSA-AuNCs@ThT@PEI hybrid materials can be used to fluorescence imaging of microbial cells as well as HeLa cells. Due to the unique background of BSA-AuNCs@ThT@PEI, the hybrid materials work well in differentiate heavy metal ions such as Hg2+ and Cu2+ ions. Moreover, this system is also suitable to do the photocatalytic transformation and regeneration of coenzyme NADH. Therefore, we propose this platform to build new nano-dye hybrid materials for copper ion detection, NADH regeneration and cell imaging, or potentially other applications.

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 morphology 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 titin “glycine aggregates” 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 the “glycine-aggregates” of titin are amyloid or amyloid-like. No similar structural features were found in titin “KCl-aggregates”; 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 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.

Distance-Dependent Tryptophan-Induced Quenching of Thioflavin T Defines the Amyloid Core Architecture.

Thioflavin T (ThT) is widely employed as a fluorogenic marker for amyloid formation. ThT fluorescence is utilized to detect amyloid fibrils as well as to follow aggregation kinetics. Here, we make a unique case to demonstrate that site-specific tryptophan-induced fluorescence quenching of ThT bound to the α-synuclein amyloid can define the central amyloid core. We show that distance-dependent quenching of amyloid-bound ThT by site-specifically incorporated tryptophan maps the proximal and distal locations of the polypeptide chain within amyloid fibrils. Our studies indicate that tryptophan-induced fluorescence quenching is dominated by the static quenching mechanism. Our findings underscore the utility of site-specific amino acid-based quenching of ThT fluorescence to characterize the core architecture of amyloid derived from a wide range of proteins.

TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways.

Intrinsically disordered regions (IDRs) in proteins can undergo liquid-liquid phase separation (LLPS) for functional assembly, but this increases the chance of forming disease-associated amyloid fibrils. Not all amyloid fibrils form through LLPS however, and the importance of LLPS relative to other pathways in fibril formation remains unclear. We investigated this question in TDP-43, a motor neuron disease and dementia-causing protein that undergoes LLPS, using thioflavin T (ThT) fluorescence, NMR, transmission electron microscopy (TEM), and wide-angle X-ray scattering (WAXS) experiments. Using a fluorescence probe modified from ThT strategically designed for targeting protein assembly rather than β-sheets and supported by TEM images, we propose that the biphasic ThT signals observed under LLPS-favoring conditions are due to the presence of amorphous aggregates. These aggregates represent an intermediate state that diverges from the direct pathway to β-sheet-dominant fibrils. Under non-LLPS conditions in contrast (at low pH or at physiological conditions in a construct with key LLPS residues removed), the protein forms a hydrogel. Real-time WAXS data, ThT signals, and TEM images collectively demonstrate that the gelation process circumvents LLPS and yet still results in the formation of fibril-like structural networks. We suggest that the IDR of TDP-43 forms disease-causing amyloid fibrils regardless of the formation pathway. Our findings shed light on why both LLPS-promoting and LLPS-inhibiting mutants are found in TDP-43-related diseases.

A label-free strategy for direct detection of Staphylococcus aureus in complex matrixes based on RCA and aptamer

Given the significant threat to human health posed by Staphylococcus aureus, rapid and accurate quantification of S. aureus is essential to prevent and diagnose foodborne diseases and to ensure the safety of food consumption. Here, we developed a novel, simple，and label-free biosensor for quantitatively detecting S. aureus by integrating an allosteric aptamer probe and a rolling circle amplification technique. Pathogen S. aureus-specific binding could induce the structural change of the allosteric aptamer beacon to expose the primer for the padlock probe. Then, the primer could trigger the RCA reaction to produce a long-chain singles-tranded DNA harboring repeating Thioflavin T (ThT) aptamer, leading to powerful fluorescence signals. The assay demonstrates sensitivity, specificity, and accuracy, showing its potential as an alternative tool for pathogen detection in different fields, such as food safety monitoring.

Exploring the effects of 4-chloro-o-phenylenediamine on human fibrinogen: A comprehensive investigation via biochemical, biophysical and computational approaches

Fibrinogen (Fg), an essential plasma glycoprotein involved in the coagulation cascade, undergoes structural alterations upon exposure to various chemicals, impacting its functionality and contributing to pathological conditions. This research article explored the effects of 4-Chloro-o-phenylenediamine (4-Cl-o-PD), a common hair dye component (IUPAC = 1-Chloro-3,4-diaminobenzene), on human fibrinogen through comprehensive computational, biophysical, and biochemical approaches. The formation of a stable ligand-protein complex is confirmed through molecular docking and molecular dynamics simulations, revealing possible interaction having a favorable −4.8 kcal/mol binding energy. Biophysical results, including UV–vis and fluorescence spectroscopies, corroborated with the computational findings, whereas Fourier transform infrared spectroscopy (FT-IR) and circular dichroism spectroscopy (CD) provide insights into the alterations of secondary structures upon interaction with 4-Cl-o-PD. Anilinonaphthalene-sulfonic acid (ANS) fluorescence showed a partially unfolded protein, with enhanced α to β-sheet transition as evidenced by thioflavin T (ThT) spectroscopy and microscopy. Moreover, biochemical assays confirmed the formation of carbonyl compounds that may be responsible for the oxidation of methionine residues in fibrinogen. Electrophoresis and electron microscopy confirmed the formation of aggregates. Our findings elucidate the interaction pattern of 4-Cl-o-PD with Fg, leading to structural perturbation, which may have potential implications for fibrinogen misfolding or its aggregation. Protein aggregation or its misfolded products affect peripheral tissues and the central nervous system. Many chronic progressive diseases, like type II diabetes mellitus, Alzheimer's disease, Parkison's disease, and Creutzfeldt-Jakob disease are associated with intrinsically aberrant disordered proteins. Understanding these interactions may offer new perspectives on the safety and biocompatibility of dye compounds, which may contribute to developing improved strategies for acquired amyloidogenesis.