Thioflavin Research Articles

Surfactant-Assisted Label-Free Fluorescent Aptamer Biosensors and Binding Assays.

Using DNA staining dyes such as SYBR Green I (SGI) and thioflavin T (ThT) to perform label-free detection of aptamer binding has been performed for a long time for both binding assays and biosensor development. Since these dyes are cationic, they can also adsorb to the wall of reaction vessels leading to unstable signals and even false interpretations of the results. In this work, the stability of the signal was first evaluated using ThT and the classic adenosine aptamer. In a polystyrene microplate, a drop in fluorescence was observed even when non-binding targets or water were added, whereas a more stable signal was achieved in a quartz cuvette. Equilibrating the system can also improve signal stability. In addition, a few polymers and surfactants were also screened, and 0.01% Triton X-100 was found to have the best protection effect against fluorescence signal decrease due to dye adsorption. Three aptamers for Hg2+, adenosine, and cortisol were tested for their sensitivity and signal stability in the absence and presence of Triton X-100. In each case, the sensitivity was similar, whereas the signal stability was better for the surfactant. This study indicates that careful control experiments need to be designed to ensure reliable results and that the reliability can be improved by using Triton X-100 and a long equilibration time.

Tuning the aggregation behavior of human insulin in the presence of luteolin: An in vitro and in silico approach

Protein misfolding and related formation of amyloid fibrils are associated with several conformational diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), prion diseases, and Diabetes mellitus, Type 2 (DM-II). Several molecules including antibiotics, polyphenols, flavonoids, anthraquinones, and other small molecules are implicated to modulate amyloid assembly. The stabilization of the native forms of the polypeptides and prevention of their misfolding and aggregation are of clinical and biotechnological importance. Among the natural flavonoids, luteolin is of great importance because of its therapeutic role against neuroinflammation. Herein, we have explored the inhibitory effect of luteolin (LUT) on aggregation of a model protein, human insulin (HI). To understand the molecular mechanism of the inhibition of aggregation of HI by LUT, we employed molecular simulation, UV–Vis, fluorescence, and circular dichroism (CD) spectroscopies along with the dynamic light scattering (DLS). The analysis of the tuning of the HI aggregation process by luteolin revealed that interaction of HI with LUT resulted in the decrease in binding of the various fluorescent dyes, such as thioflavin T (ThT) and 8-anilinonaphthalene-1-sulfonic acid (ANS) to this protein. Retention of the native-like CD spectra and resistance to the aggregation in the presence of LUT has confirmed the aggregation inhibitory potential of LUT. The maximum inhibitory effect was found at the protein-to-drug ratio of 1:12, and no significant change was observed beyond this concentration.

Design and synthesis of piano-stool ruthenium(II) complexes and their studies on the inhibition of amyloid β (1–42) peptide aggregation

Misfolding and protein aggregation have been linked to numerous human neurodegenerative disorders such as Alzheimer's, prion, and Parkinson's diseases. Ruthenium (Ru) complexes have received considerable attention in studying protein aggregation due to their interesting photophysical and photo properties. In this study, we have synthesized the novel Ru complexes ([Ru(p-cymene)Cl(L-1)][PF6](Ru-1), and [Ru(p-cymene)Cl(L-2)][PF6](Ru-2)) and investigated their inhibitory activity against the bovine serum albumin (BSA) aggregation and the Aβ1–42 peptides amyloid formation. Several spectroscopic methods were used to characterize these complexes, and the molecular structure of the complex was determined by X-ray crystallography. Amyloid aggregation and inhibition activities were examined using the Thioflavin-T (ThT) assay, and the secondary structures of the protein were analyzed by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). The cell viability assay was carried out on the neuroblastoma cell line, revealing that the complex Ru-2 showed better protective effects against Aβ1–42 peptide toxicity on neuro-2a cells than the complex Ru-1. Molecular docking studies elucidate the binding sites and interactions between the Ru-complexes and Aβ1–42 peptides. The experimental studies revealed that these complexes significantly inhibited the BSA aggregation and Aβ1–42 amyloid fibril formation at 1:3 and 1:1 molar concentrations, respectively. Antioxidant assays demonstrated that these complexes act as antioxidants, protecting from amyloid-induced oxidative stress. Molecular docking studies with the monomeric Aβ1–42 (PDB: 1IYT) show hydrophobic interaction, and both complexes bind preferably in the central region of the peptide and coordinate with two binding sites of the peptide. Hence, we suggest that the Ru-based complexes could be applied as a potential agent in metallopharmaceutical research against Alzheimer's disease.

Cavity hairpin ThT-light nucleic acid switches: the construction of label- and enzyme-free sensing and imaging platforms.

Thioflavin T (ThT) is a classical fluorescent dye gaining prominence in current research regarding nucleic acid conformations (NACs). However, most NACs with the ability to excite ThT fluorescent are unique or form in demanding conditions, limiting the extensiveness and depth of ThT application in sensing and imaging. Therefore, this study proposed CGG-AAA mismatched cavity hairpin ThT-light nucleic acid switches (CHTLNAS) with excellent fluorescence excitation over 500-fold higher than spontaneous, 17∼20-fold higher than ssDNA and 2.5∼5-fold higher than complementary duplex. Based on the excellent fluorescence excitation, convenient conformation formation, good sequence programmability, and flexible allosteric ability (known as the Worm-crack pod mechanism mediated by the target), it achieved the label- and enzyme-free detection of tetracycline (TET) and berberine (BB) at the pM level within 10 min. Moreover, it was found enable to realize the sensitive tracking of intracellular carriers at the nM level of ThT entry concentration, and prolongated its cell nuclear-entry time of ThT over 8 h, overcoming the non-specific high background signal interference of ThT in the nuclear region, and expanding the diversified application of ThT in cell biology research. Therefore, CHTLNAS is a more universal, practical tool than G-quadruplex or other kinds of NACs for ThT development and utilization in sensing and imaging platforms.

Dual fluorescence output aptamer sensing of cortisol in Cushing syndrome samples assisted by tandem hybridization chain reaction

Accurate and sensitive detection of serum free cortisol has been a challenge for precise diagnosis and assessment of Cushing syndrome (CS). The present study described a dual fluorescent homogeneous cortisol aptasensor based on the tandem hybridization chain reaction (HCR) amplification strategy. The system used split G-quadruplex (G4)/thioflavin T (ThT) complexes and dsDNA-templated copper nanoparticles (Cu NPs) as signal reporters. The cortisol binds its aptamer, inhibiting the triggering of HCRs between the aptamer and the four hairpin structures (H1-H4), thereby inhibiting the generation of the dsDNA template and G4 structure. Under optimized conditions, the limit of detection (LOD) was 3 fg/mL for G4/ThT and 2.5 fg/mL for Cu NPs, with a linear range of 1 fg/mL-100 pg/mL, indicating high specificity to various potential interfering substances. This method was used to determine cortisol levels in 54 clinical blood samples. Cortisol levels in clinical samples detected by this method agreed well with those obtained by the electrochemiluminescence immunoassay (ECL-IA) kit and radiological findings. We could distinguish CS from healthy controls and other patients using this approach, with 0.94 of the area under the receiver operating characteristic curve (AUC). This cortisol quantification method was 100% specific and 83.3% sensitive. This sensing strategy has shown great potential as an additional diagnostic tool for cortisol-related disorders.

Substantial dimerized G-quadruplex signal units engineered by cutting-mediated exponential rolling circle amplification for ultrasensitive and label-free detection of exosomes

Sensitive and accurate determination of tumor-derived exosomes from complicated biofluids is an important prerequisite for early tumor diagnosis through exosome-based liquid biopsy. Herein, a label-free fluorescence immunoassay protocol for ultrasensitive detection of exosomes was developed by engineering substantial dimerized guanine-quadruplex (Dimer-G4) signal units via in situ cutting-mediated exponential rolling circle amplification (CM-ERCA). First, exosomes were captured and enriched via immunomagnetic separation. Then, molecular recognition was built by the formation of antibody−aptamer sandwich immunocomplex through the specific binding of the designed aptamer-primers with the targeted exosomes. The accuracy of exosome detection was significantly improved by the specific recognition of two typical exosomal protein markers simultaneously. Eventually, in situ CM-ERCA was triggered by a perfect match between the multifunctional circular DNA template and the aptamer-primer on exosomal surface. Amplicons of CM-ERCA loaded with Dimer-G4 were exponentially accumulated during continuous cyclic amplification, dramatically lighting up the thioflavin T (ThT) and generating substantial Dimer-G4 signal units. As a result, ultrasensitive detection of exosomes with the detection limit down to 2.4 × 102 particles/mL was achieved due to the fluorescence enhancement of substantial Dimer-G4 signal units, which is ahead of most of available fluorescence-based methods reported currently. In addition, the intense fluorescence emission and favorable anti-interference of the proposed immunoassay supports identification of exosomes direct in human serums, overcoming the limitations of conventional G4/ThT in serum analysis and revealing its potential for exosome-based liquid biopsy.

Effect of Ovocystatin on Amyloid β 1-42 Aggregation-In Vitro Studies.

Amyloid β peptides (Aβ) aggregating in the brain have a potential neurotoxic effect and are believed to be a major cause of Alzheimer's disease (AD) development. Thus, inhibiting amyloid polypeptide aggregation seems to be a promising approach to the therapy and prevention of this neurodegenerative disease. The research presented here is directed at the determination of the inhibitory activity of ovocystatin, the cysteine protease inhibitor isolated from egg white, on Aβ42 fibril genesis in vitro. Thioflavin-T (ThT) assays, which determine the degree of aggregation of amyloid peptides based on fluorescence measurement, circular dichroism spectroscopy (CD), and transmission electron microscopy (TEM) have been used to assess the inhibition of amyloid fibril formation by ovocystatin. Amyloid beta 42 oligomer toxicity was measured using the MTT test. The results have shown that ovocystatin possesses Aβ42 anti-aggregation activity and inhibits Aβ42 oligomer toxicity in PC12 cells. The results of this work may help in the development of potential substances able to prevent or delay the process of beta-amyloid aggregation-one of the main reasons for Alzheimer's disease.

Antiepileptic Drugs Modulate Alzheimer-Related Tau Aggregation in a Neuronal Activity-Independent Manner

Introduction: A rapidly increasing number of patients with dementia present a serious social problem. Recently, the incidence of epilepsy in patients with Alzheimer’s disease (AD) is increasing, drawing attention to the pathological relationship between the two conditions. Clinical studies have suggested the protective action of antiepileptic agents on dementia; however, the underlying mechanism remains unknown. We evaluated the effects of multiple antiepileptic drugs using tau aggregation assay systems to determine the effects of antiepileptic agents on tau aggregation, a major neuropathological finding associated with AD. Methods: We evaluated the effects of seven antiepileptic agents on intracellular tau aggregation using a tau-biosensor cell-based high-throughput assay. Next, we tested these agents in a cell-free tau aggregation assay using thioflavin T (ThT). Results: The assay results revealed that phenobarbital inhibited tau aggregation, whereas sodium valproate, gabapentin, and piracetam promoted tau aggregation. In the cell-free tau aggregation assay using ThT, we confirmed that phenobarbital significantly inhibited tau aggregation. Conclusion: Antiepileptic drugs may modify the tau pathology in AD in a neural activity-independent manner. Our finding may provide an important insight into the optimization of antiepileptic drug therapy in older adults with dementia.

Modulation of Aβ42 Aggregation Kinetics and Pathway by Low-Molecular-Weight Inhibitors.

The aggregation of amyloid-β 42 (Aβ42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aβ42 aggregation kinetics, characterized by the quantification of Aβ42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aβ42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.

Spectroscopic analysis of recombinant human growth hormone in the presence of sucrose and trehalose.

Recombinant human growth hormone (rhGH) is a therapeutic protein, associated with various human diseases, such as growth hormone deficiency. One of the interesting issues in the formulation of therapeutic proteins is excipients like disaccharides. In the current study, we try to compare the effect of sucrose and trehalose on the structure of rhGH in the liquid state at 25°C and 55°C. We use spectroscopic techniques including intrinsic and extrinsic fluorescence, Fourier-transform infrared (FTIR), circular dichroism (CD), dynamic light scattering (DLS), and time-resolved fluorescence. FTIR shows a slight change in the secondary structure of rhGH in presence of the sugars as sucrose is more effective than trehalose. Fluorescence investigations also confirm the enhancements of folding of rhGH and fluorescein isothiocyanate (FITC)-rhGH in presence of sucrose (1.5-fold more than trehalose). Also, we studied sucrose's effect on the rete of aggregation of rhGH using spectroscopy of Congo red, and fluorescence imaging of thioflavin T (ThT)-treated samples. It can be suggested that sucrose facilitates the amyloid formation of rhGH during 20days of incubation at 37°C. This study will help to understand the growth hormone structural behavior in the liquid state in the presence of sucrose and trehalose in vitro.

Cycloartane triterpenoid from Euphorbia macrostegia modulates ER stress signaling pathways to induce apoptosis in MDA-MB231 and MCF-7 breast cancer cell lines.

Unfolded protein response (UPR) is involved in breast cancer (BC) progression and drug resistance. Many natural products (NPs) could modulate UPR and used for therapeutic purposes. Herein, we aimed to investigate the molecular mechanism of Cycloart-23E-ene-3β, 25-diol (Cycloart-E25), cytotoxicity, as a NP extracted from Euphorbia macrostegia and focused on endoplasmic-reticulum stress (ERS) and UPR signaling pathways. Reactive oxygen species (ROS) were probed by DCFDA fluorescence dye. Apoptosis was assayed by annexin V/propidium iodide (PI), immunoblotting of anti- and proapoptotic, Bcl-2 and Bax proteins, and mitochondrial transmembrane potential (ΔΨm) changes. Thioflavin T (ThT) staining and immunoblotting of UPR signaling components (CHOP, PERK, ATF6, BiP, and XBP1) were recruited for the assessment of ERS. Our results indicated that Cycloart-E25 noticeably increases ROS levels in both MB-231 MDA-MB-231 and MCF-7 cell lines, p>0.05. Flow cytometry assessments revealed an increase in the cell population undergoing apoptosis. Also, the Bax/Bcl-2 ratio increased in a dose-dependent manner following Cycloart-E25 treatment, significantly, p>0.05. Mitochondrial involvement could be deduced by significant decreases in ΔΨm, p>0.05. Cycloart-E25 potently induces protein aggregation and upregulated CHOP, PERK, ATF6, BiP, and XBP1 factors in both MDA-MB-231 MB-231 and MCF-7 cell lines, indicating the involvement of ERS in Cycloart-E25-mediated apoptosis. In conclusion, Cycloart-E25 increased the accumulation of misfolded proteins and upregulated UPR components. Therefore, induction of ERS may be involved in the trigger of apoptosis in BC cell lines. Cycloart-E25 induced apoptosis in breast cancer cell lines through ERS. More assessments are needed to confirm its in vivo anti-tumoral effects.

Controlling Amyloid Beta Peptide Aggregation and Toxicity by Protease-Stable Ligands.

Polymerization of soluble amyloid beta (Aβ) peptide into protease-stable insoluble fibrillary aggregates is a critical step in the pathogenesis of Alzheimer's disease (AD). The N-terminal (NT) hydrophobic central domain fragment 16KLVFF20 plays an important role in the formation and stabilization of β-sheets by self-recognition of the parent Aβ peptide, followed by aggregation of Aβ in the AD brain. Here, we analyze the effect of the NT region inducing β-sheet formation in the Aβ peptide by a single amino acid mutation in the native Aβ peptide fragment. We designed 14 hydrophobic peptides (NT-01 to NT-14) by a single mutation at 18Val by using hydrophobic residues leucine and proline in the natural Aβ peptide fragment (KLVFFAE) and analyzed its effect on the formation of Aβ aggregates. Among all these peptides, NT-02, NT-03, and NT-13 significantly affected the Aβ aggregate formation. When the NT peptides were coincubated with the Aβ peptide, a significant reduction in β-sheet formation and increment in random coil content of Aβ was seen, confirmed by circular dichroism spectroscopy and Fourier transform infrared spectroscopy, followed by the reduction of fibril formation measured by the thioflavin-T (ThT) binding assay. The aggregation inhibition was monitored by Congo red and ThT staining and electron microscopic examination. Moreover, the NT peptides protect the PC-12 differentiated neurons from Aβ-induced toxicity and apoptosis in vitro. Thus, manipulation of the Aβ secondary structure with protease-stable ligands that promote the random coil conformation may provide a tool to control the Aβ aggregates observed in AD patients.

A label-free ThT-assisted fluorescence detection strategy of alkaline phosphatase activity based on MnO2 nanosheets

Alkaline phosphatase (ALP) is a metalloenzyme, the level of which is clinically significant as an abnormality of ALP activity results in several diseases. In the present study, we introduced a MnO2 nanosheet-based assay for ALP detection employing the adsorption and reduction characteristics of G-rich DNA probes and ascorbic acid (AA), respectively. Ascorbic acid 2-phosphate (AAP) was utilized to act as a substrate for ALP which hydrolyzes AAP generating AA. In the absence of ALP, MnO2 nanosheets adsorb the DNA probe destructing the G-quadruplex formation and showing no fluorescence emission. On the contrary, being present in the reaction mixture ALP hydrolyzes AAP yielding AA, then the AA reduce the MnO2 nanosheets into Mn2+, hence, the probe is free to react with a dye, thioflavin T (ThT), and synthesizes ThT/G-quadruplex to spark high fluorescence intensity. Therefore, under optimized conditions (250 nM DNA probe, 8 μM ThT, 96 μg/mL MnO2 nanosheets, and 1 mM AAP) the sensitive and selective measurement of ALP activity can be achieved through the change of fluorescence intensity, with a linear range and a limit of detection of 0.1–5 U/L and 0.045 U/L. Our assay exhibited its potential to assess the ALP inhibitor when in an inhibition assay Na3VO4 inhibited ALP with an IC50 value of 0.137 mM and also was validated in clinical samples.

An efficient DNAzyme-based DNA scaffold for label-free and sensitive bacterial pathogen detection

Even though it is very important, it is still rather difficult to detect minuscule levels of the bacterial pathogen in clinical practice, such as samples from dental implants. We construct here an efficient scaffold for label-free and sensitive Staphylococcus aureus (S. aureus) detection. The precise recognition of target bacteria by the detection scaffold leads to the self-assembly of Chain i and DNAzyme based cleavage of Chain iii. In detail, active DNAzyme conformation is formed based on the hybridization of Chain iii and Chain ii, and a nicking site is generated in Chain iii, making it possible to form a self-primer in Chain i. With the assistance of DNA polymerase, a single-strand DNA chain is added to the 3′ terminal of Chain i, in which process the bacteria is released for the complex to bind with a next detection scaffold, forming a signal recycle. Following DNAzyme-based cleavage, the liberated sequences unroll MB and release G-rich sequences that can specifically bind with the fluorescent dye Thioflavin T (ThT), initiating ThT's fluorescence signal production. The approach demonstrates a wide detection range of 102 CFU/mL and 106 CFU/mL with a low limit of detection of 45 CFU/mL based on the developed detection scaffold, offering good prospects in the diagnosis of bacterial illnesses.

Electroabsorption and Stark Fluorescence Spectroscopies of Thioflavin T.

Thioflavin T (ThT) is a typical fluorescent marker for detecting the formation of amyloid fibrils, because its fluorescence intensity increases by more than 2 orders of magnitude upon complexation with the fibrils. Strong electrostatic fields on protein surfaces are known to be a significant factor in chemical reactions and biological functions. Therefore, ThT bound to amyloid fibrils must experience strong electric fields. This study employed electroabsorption and Stark fluorescence spectroscopies to clarify the effects of external electric fields on the photophysics of ThT. The absorption spectrum shows two bands ascribed to locally excited (LE) and charge transfer (CT) states. Coupling between the LE and CT states is enhanced in the presence of an external electric field, resulting in fluorescence quenching. The electric field strength of the amyloid fibril surface was inferred from the fluorescence quenching efficiency of ThT.

Label-free and highly sensitive fluorescent detection of bleomycin based on CRISPR-Cas12a and G-quadruplex-thioflavin T

CRISPR-Cas12a system opens up a new avenue for biosensing due to its flexible programmability and excellent target recognition accuracy. In this work, a novel label-free fluorescent biosensor for highly sensitive detection of bleomycin (BLM) was developed based on BLM-mediated inactivation of CRISPR-Cas12a and thioflavin T (ThT)-induced G-quadruplex formation. We designed a single-stranded DNA (ssDNA) probe containing the scission site of BLM as a DNA activator for Cas12a-crRNA. In the absence of BLM, the DNA activator binds to and activates the Cas12a, the activated CRISPR-Cas12a system trans-cleaves the ThT-induced G-quadruplex formation. Upon the addition of BLM, the DNA activator undergoes an irreversible cleavage event at the scission site via the oxidative effect of BLM with Fe(Ⅱ) as a cofactor. As a result, the trans-cleavage activity of Cas12a cannot be activated so the G-quadruplex-ThT complex remains intact, resulting in a significant increase of fluorescence signal. Attributing to the specific recognition of BLM and the excellent cleavage activity of Cas12a on DNA G-quadruplexes, the fluorescent biosensor exhibits high sensitivity and selectivity for detection of BLM in human serum samples, illustrating a promising tool for BLM assay in biomedical research.

Design of Fmoc-Phenylalanine Nanofibrillar Hydrogel and Mechanistic Studies of Its Antimicrobial Action against Both Gram-Positive and Gram-Negative Bacteria.

In pursuit of efficient antimicrobial agents, biomaterials such as hydrogels have drawn a considerable amount of attention due to their numerous advantages such as a high degree of hydration, biocompatibility, stability, and direct application at an infectious site. Particularly, biomaterials such as hydrogels based on Fmoc-protected peptides and amino acids have proven to be immensely advantageous. Such biomaterials can undergo gelation by simple pH modulation and can be used for various biological applications. Keeping this in mind, in this work, we reported the synthesis of Fmoc-phenylalanine (Fmoc-F)-based hydrogels using trisodium citrate as a pH modulator and compared them with the previously reported pH modulator glucono-δ-lactone. The gels were compared using various characterization techniques such as rheometry, field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), small angle X-ray scattering (SAXS), FT-IR, thioflavin T (ThT) binding assay, and zeta potential studies. These studies highlighted the role of pH modulators in affecting various parameters such as the ability to alter the zeta potential of the nanofibrils, improve their bactericidal action, reduce the amyloidic characters, shift the lattice packing from amorphous to crystalline, and introduce fluorescence and thermoreversibility. Interestingly, this is the first report where the Fmoc-F-based hydrogel has been shown to be effective against Gram-negative bacteria along with Gram-positive bacteria as well. Additionally, the mechanism of antimicrobial action was investigated using docking and antioxidant studies.

Anti-Amyloidogenic Effects of Metasequoia glyptostroboides Fruits and Its Active Constituents.

Alzheimer's disease (AD) is a serious neurodegenerative brain disease that interferes with daily life. The accumulation of beta-amyloid (Aβ), along with oxidative stress-inducing neurocellular apoptosis, has been considered one of the causes of AD. Thus, the purpose of this study is to find natural products that can reduce Aβ accumulation. The ethanol extract of Metasequoia glyptostroboides Hu & Cheng fruits (Cupressaceae) significantly reduced the aggregation of Aβ into oligomers and fibrils determined by Thioflavin T (ThT) assay. The solvent-partitioned ethyl acetate layer was further separated based on the bioassay-guided isolation method combined with the ThT assay. As a result, five compounds were isolated and elucidated as taxoquinone (1), sugiol (2), suginal (3), sandaracopimarinol (4), and sandaracopimaradien-19-ol (5) by comparing NMR data with references. All the compounds significantly reduced the aggregation of Aβ and enhanced the disaggregation of pre-formed Aβ aggregates in a dose-dependent manner. Furthermore, the inhibition of Aβ aggregation by the compounds protected PC12 cells from Aβ aggregate-induced toxicity. Among the five compounds, sandaracopimarinol (4) and sandaracopimaradien-19-ol (5) were the most effective. These results suggest that M. glyptostroboides and isolated five compounds have a potential for further study to be developed as anti-AD agents.

Interaction of Thioflavin T (ThT) and 8-anilino-1-naphthalene sulfonic acid (ANS) with macromolecular crowding agents and their monomers: Biophysical analysis using in vitro and computational approaches

Protein aggregation is a major side reaction that may be observed during protein folding and may prevent proteins from performing their intended functions. It is a hallmark of various human diseases, specifically neurodegenerative diseases. Fluorescence spectroscopy is one such technique in which extrinsic fluorescent dyes are used to monitor protein aggregation in vitro. These dyes are known to get bound to the aggregated portion of proteins, which thereby leads to an enhancement of fluorescence intensity. In this study, we have uniquely investigated with a battery of evidence to show that extrinsic fluorescent dyes bind to macromolecular crowding agents and their monomers even in absence of protein. The additives used in this study are dextran-70 and ficoll-70 and their monomers, glucose and sucrose, respectively. Here, with the help of various biophysical techniques such as extrinsic fluorescent dye-binding assays, molecular docking, ITC and TEM, we have explored the interactions that exist between these extrinsic fluorescent dyes and the macromolecular crowding agents along with their monomers. This study inferred that there is a high possibility of interaction between dyes and additives, as these dyes showed strong binding with above-mentioned crowding agents and their monomers.

Plant Protein Amyloid Fibrils for Multifunctional Sustainable Materials

AbstractArtificial functional materials based on amyloid fibrils are proven to be a promising strategy toward functional materials. However, scaling‐up applications present sustainability concerns, as animal proteins are the main sources for fabricating amyloid fibrils. Plant‐protein‐based amyloid fibrils, a more sustainable alternative to animal proteins, are attracting increasing interests as building blocks in functional materials. Herein, 11 different sources from a wide range of plants are evaluated, and a comprehensive analysis of seven species of plant proteins, including kidney bean, black bean, cowpea, mung bean, chickpea, lentil, and pumpkin seed, with an excellent ability to form fibrils, is presented. A universal strategy for a diversity of plant protein extraction and fibrillization is applied. Flexible fibrils with a persistence length of ≈100 nm and rigid fibrils of several micrometers are discovered in 7S/8S and 11S subunits dominated protein, respectively. Structural evolution toward the β‐sheet content on these proteinaceous assemblies is characterized by thioflavin T (ThT) intensity, circular dichroism (CD) spectra, attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectra, and typical wide angle X‐ray scattering (WAXS) spectra. Finally, their multifunctional applications are further explored and proven that these sustainable protein amyloids demonstrate excellent performance in renewable and degradable bioplastics, and in water purification membranes for heavy metal removal.