Thioflavin Research Articles

Sarkosyl differentially solubilizes patient-derived alpha-synuclein fibril strains.

Insoluble α-synuclein (αSyn) filaments in brain tissue are a hallmark of Parkinson's disease (PD) and Multiple system atrophy (MSA), and for structural studies, they have for decades been extracted using the detergent sarkosyl. We asked if PD and MSA patient-derived αSyn filament strains display different stability to sarkosyl extraction as this may confound our interpretation of the landscape of structural strains present in patients' tissue. We compared the stability of cerebrospinal fluid-derived strains from four PD and four MSA patients using sedimentation and immunoassays and tested the seeding competence and strain-specific characteristics of the sarkosyl-soluble fractions using a seed amplification assay (SAA) and Thioflavin T (ThT) fluorescence. We demonstrate that filaments from PD are less resistant to sarkosyl than from MSA after they have been subjected to freezing and sonication. An enhanced release of monomers from PD filaments was the major difference between PD and MSA, but the sarkosyl-soluble fraction released from both PD and MSA filaments contained aggregates that displayed aggregate-specific epitopes and seeding activity with preserved disease-specific strain characteristics. Our results demonstrate that sarkosyl differentially destabilizes patient derived αSyn filament strains, which may compromise our ability to fully appreciate the landscape of αSyn filament currently being uncovered by high resolution cryoEM analyses. This should motivate an effort to develop more gentle extraction protocols.

Non-micellar ganglioside GM1 induces an instantaneous conformational change in Aβ42 leading to the modulation of the peptide amyloid-fibril pathway

Alzheimer's disease is a progressive degenerative condition that mainly affects cognition and memory. Recently, distinct clinical and neuropathological phenotypes have been identified in AD. Studies revealed that structural variation in Aβ fibrillar aggregates correlates with distinct disease phenotypes. Moreover, environmental surroundings, including other biomolecules such as proteins and lipids, have been shown to interact and modulate Aβ aggregation. Model membranes containing ganglioside (GM1) clusters are specifically known to promote Aβ fibrillogenesis. This study unravels the modulatory effect of non-micellar GM1, a glycosphingolipid frequently released from the damaged neuronal membranes, on Aβ42 amyloid fibril formation. Using far-UV circular dichroism experiments, we observed a change in the peptide secondary structure from random-coil to β-turn structures with subsequent generation of predominantly β-sheet-rich species upon interaction with GM1. Thioflavin-T (ThT) fluorescence assays further indicated that GM1 likely interacts with an amyloidogenic Aβ42 intermediate species leading to a possible formation of GM1-modified Aβ42 fibril. Statistically, no significant difference in toxicity to RA-differentiated SH-SY5Y cells was observed between Aβ42 fibrils and GM1-tweaked Aβ42 aggregates. Moreover, GM1-modified Aβ42 aggregates exhibited prion-like properties in catalyzing the amyloid fibril formation of both major isomers of Aβ, Aβ40, and Aβ42.

High-frequency terahertz waves disrupt Alzheimer’s β-amyloid fibril formation

The accumulation and deposition of amyloid can cause a variety of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. The degradation or clearance of this accumulation is currently the most widely accepted therapeutic strategy for intervention in these pathologies. Our study on amyloid-β (Aβ) oligomers in vitro revealed that high-frequency terahertz (THz) waves at a specific frequency of 34.88 THz could serve as a physical, efficient, non-thermal denaturation technique to delay the fibrotic process by 80%, as monitored by a thioflavine T (ThT) binding assay and Fourier transform infrared (FTIR) spectroscopy. Additionally, THz waves of this frequency have been shown to have no side effects on normal cells, as confirmed by cell viability and mitochondrial membrane potential assays. Furthermore, molecular dynamic (MD) simulations revealed that the THz waves could resonate with Aβ fibrils, disrupting the dense conformation by breaking the β-sheet structure and promoting the formation of abundant coil and bend structures. This study uses the amyloid of Aβ as an example, and the results will further guide interventions for the accumulation of other amyloids, which may provide new ideas for the remission of related diseases.

Structure-switching aptasensors for sensitive detection of ochratoxin A.

Ochratoxin A (OTA) is a toxic metabolite commonly found in various foods and feedstuffs. Accurate and sensitive detection of OTA is needed for food safety and human health. Based on a common OTA-binding aptamer (OTABA), two structure-switching OTABAs, namely OTABA4 and OTABA3, were designed by configuring a split G-quadruplex and a split G-triplex, respectively, at the two ends of OTABA to construct aptasensors for the detection of OTA. The OTABA, G-quadruplex, and G-triplex all can capture the thioflavin T (ThT) probe, thereby enhancing the fluorescence intensity of ThT. Bonding with OTA could change the conformations of OTABA and G-quadruplex or G-triplex regions, resulting in the release of the captured ThT and diminution of its fluorescence intensity. Dual conformation changes in structure-switching OTABA synergistically amplified the fluorescence signal and improved the sensitivity of the aptasensor, especially for that with OTABA3. The detection limits of the OTABA4-ThT and OTABA3-ThT systems for OTA were 0.28 and 0.059 ng ml-1 , with a 1.4-fold and 6.7-fold higher sensitivity than that of the original OTABA-ThT system, respectively. They performed well in corn and peanut samples and met the requirements of the food safety inspections.

Construction of a Label-Free Ratiometric Biosensor Based on Target Recycling Amplification and Hg-ZnSe QDs for Assay of BChE and OPs.

Herein, we constructed a label-free ratiometric fluorescence biosensing strategy for the determination of butyrylcholinesterase (BChE) activity and organophosphorus (OPs) concentration. BChE promoted the hydrolysis of iodized s-butyrylthiocholine (BTCh) into a reducing substance thiocholine, which can decompose CoOOH nanosheets (CoOOH NSs) to Co2+. Subsequently, the single-stranded DNA (ssDNA) on the surface of CoOOH NSs was released. Then, ssDNA hybridized with hairpin DNA (h-DNA) and triggered the target recycling amplification process, producing large amounts of G-quadruplex. After adding thioflavin T (ThT), the target BChE was converted into activatable G-quadruplex/ThT with an amplified yellow fluorescence signal. The addition of OPs could significantly inhibit the hydrolysis of BTCh by BChE and thus unable to produce the yellow fluorescence G-quadruplex/ThT complex. Throughout the entire process, the fluorescence intensity of Hg-ZnSe QDs as a reference signal remained unchanged at 630 nm. Furthermore, this work provided an effective approach for detecting the BChE activity in serum samples and OPs in fruits and vegetables.

Inhibition of amyloid β1–42 peptide aggregation by newly designed cyclometallated palladium complexes

Uncontrolled amyloid aggregation is a frequent cause of neurodegenerative disorders such as prions and Alzheimer's disease (AD). As a result, many drug development approaches focus on evaluating novel molecules that can alter self-recognition pathways. Herein, we designed and synthesized the cyclometallated pyrene (Pd-1 and Pd-3) and anthracene (Pd-2) based palladium complexes ([Pd((L1)Cl] Pd-1, [Pd(L2)Cl](Pd-2), and [Pd(L3)Cl] (Pd-3)). This study explores the effect of these complexes on the aggregation, fibrillation, and amyloid formation of bovine serum albumin (BSA) and Aβ1–42 peptide. Several spectroscopic methods were used to characterize all the Pd-complexes, and the molecular structure of Pd-3 was determined by X-ray crystallography. The secondary structures were studied using circular dichroism (CD) and transmission electron microscopy (TEM), while amyloid aggregation and inhibitory activities were investigated using the Thioflavin-T (ThT) fluorescence assay. Molecular docking of the Pd-complex (Pd-3) was done using fibril (PDB: 2BEG) and monomeric (PDB: 1IYT) peptides using Auto-dock Vina. As a result, the hydrogen bonding and hydrophobic interaction between the aromatic rings of the Pd-complexes and the amino acids of amyloid-β peptides significantly reduced the production of ordered β-sheets of amyloid fibrils and protein aggregation in the presence of Pd-2 and Pd-3 complexes.

Vitamin E modified polyamidoamine dendrimer for piperine delivery to alleviate Aβ1–42 induced neurotoxicity in Balb/c mice model

In Alzheimer’s disease (AD), amyloid beta (Aβ1-42) aggregate formation and neurofibrillary tangles are major pathological hallmarks which are related to neurodegeneration in the brain. To alleviate Aβ1-42 fibrils toxicity vitamin E derivative tocopheryl polyethylene glycol succinate (TPGS) was conjugated with polyamidoamine (PAMAM) dendrimer through carbodiimide reaction to synthesize TPGS-PAMAM. This TPGS-PAMAM was employed to entrap neuroprotective agent piperine (PIP) through an anti-solvent technique to prepare PIP-TPGS-PAMAM. The dendrimer conjugate was prepared to reduce Aβ1-42 induced neurotoxicity and increase acetylcholine levels in AD mice models. The synthesis of dendrimer conjugate was characterized through proton nuclear magnetic resonance (NMR) and Trinitrobenzene sulphonic acid assay (TNBS). Physical characterization of dendrimers conjugates were done through various spectroscopic, thermal and microscopy based techniques. PIP-TPGS-PAMAM showed 43.25 nm particle size with PIP percentage encapsulation efficiency of 80.35%. Further Aβ1-42 fibril disaggregation effect of nanocarrier was evaluated using Thioflavin–T (ThT) assay and circular dichroism (CD). The neuroprotection studies for PIP-TPGS-PAMAM was evaluated against neurotoxicity induced using Aβ1–42 intracerebroventricular (ICV) injected in Balb/c mice. The group of mice administered with PIP-TPGS-PAMAM exhibited an increase in the proportion of random alternations in T-maze test and novel object recognition test (NORT) exhibited an increase in working memory cognitive functions. The biochemical and histopathological analysis revealed PIP-TPGS-PAMAM treated groups enhanced acetylcholine levels, reduced ROS and Aβ1-42 content significantly. Our findings imply that PIP-TPGS-PAMAM enhanced memory and reduced cognitive deficit in mice brain induced by Aβ1-42 toxicity.

Perturbation of surfactant-induced amyloids by abolishing electrostatic interactions

Protein amyloid fibrils are supramolecular homopolymers that play a role in numerous biological processes and illnesses. The link between type 2 diabetes and insulin-induced amyloidosis has prompted much research on the production of insulin amyloid fibrils. Many in vitro investigations have shown that insulin can turn into amyloid fibrils under certain conditions. Insulin was used as a model protein in this study to determine the role of electrostatic interactions in surfactant-induced amyloids. Our results showed that SDS-induced amyloid formation was nucleation-independent at pH 2.0, and the solubilization was also instantaneous upon electrostatic interaction disruption. Far UV circular dichroism (CD), turbidity, Rayleigh light scattering (RLS), thioflavin T (ThT), and transmission electron microscopy (TEM) imaging showed the attainment of the different structural forms of insulin due to varying degrees of electrostatic interactions between the anionic surfactant (SDS) and the cationic to the anionic state of insulin. Treatment of SDS with the cationic state of insulin strongly induces amyloid-like aggregate while anionic state insulin reverts to native-like alpha-helical structures. In all samples, SDS concentration remained unchanged. Thus, hydrophobicity remained constant; only the electrostatic interaction between anionic SDS and cationic insulin was abolished as pH increased.

Metal-Complexes Bearing Releasable CO Differently Modulate Amyloid Aggregation.

Neurodegenerative diseases are often associated with an uncontrolled amyloid aggregation. Hence, many studies are oriented to discover new compounds that are able to modulate self-recognition mechanisms of proteins involved in the development of these pathologies. Herein, three metal-complexes able to release carbon monoxide (CORMs) were analyzed for their ability to affect the self-aggregation of the amyloidogenic fragment of nucleophosmin 1, corresponding to the second helix of the three-helix bundle located in the C-terminal domain of the protein, i.e., NPM1264-277, peptide. These complexes were two cymantrenes coordinated to the nucleobase adenine (Cym-Ade) and to the antibiotic ciprofloxacin (Cym-Cipro) and a Re(I)-compound containing 1,10-phenanthroline and 3-CCCH2NHCOCH2CH2-6-bromo-chromone as ligands (Re-Flavo). Thioflavin T (ThT) assay, UV-vis absorption and fluorescence spectroscopies, scanning electron microscopy (SEM), and electrospray ionization mass spectrometry (ESI-MS) indicated that the three compounds have different effects on the peptide aggregation. Cym-Ade and Cym-Cipro act as aggregating agents. Cym-Ade induces the formation of NPM1264-277 fibers longer and stiffer than that formed by NPM1264-277 alone; irradiation of complexes speeds the formation of fibers that are more flexible and thicker than those found without irradiation. Cym-Cipro induces the formation of longer fibers, although slightly thinner in diameter. Conversely, Re-Flavo acts as an antiaggregating agent. Overall, these results indicate that metal-based CORMs with diverse structural features can have a different effect on the formation of amyloid fibers. A proper choice of ligands attached to metal can allow the development of metal-based drugs with potential application as antiamyloidogenic agents.

Fabrication of on-bead functional nucleic acid nanowires based on cyclic ligation reaction-driven rolling circle amplification for label-free detection of long noncoding RNAs in breast and lung tissues

Long noncoding RNAs (lncRNAs) act as the essential regulators in various biological processes, and are becoming potential diagnostic and prognostic biomarkers. Herein, we demonstrate the fabrication of on-bead functional nucleic acid nanowires based on cyclic ligation reaction-driven rolling circle amplification for label-free detection of long noncoding RNAs in breast and lung tissues. The presence of lncRNA activates the cyclic ligation reaction, converting low-abundance target to numerous biotinylated ligation products through repetitive denaturation-hybridization-ligation. The resulting ligation products immobilize on the magnetic bead to initiate the downstream rolling circle amplification (RCA), facilitating the synthesis of long G-quadruplex nanowires. The resultant nanowires can light up Thioflavin T (ThT) to generate a high fluorescence signal. This method displays ultrahigh sensitivity with a limit of detection of 9.97 aM, and it possesses excellent single-base mismatch discrimination capability. Moreover, it can accurately quantify cellular Homeobox (HOX) gene antisense intergenic RNA (HOTAIR) at single-cell level and distinguish the HOTAIR expression level in healthy person and lung/breast cancer patient tissues, holding great promise in clinical diagnosis.

Rolling-Circle-Amplification-Assisted DNA Biosensors for Sensitive and Specific Detection of Hypochlorous Acid and Myeloperoxidase

Hypochlorous acid (HClO) is a common reactive oxygen species (ROS), with a high chemical reactivity. Myeloperoxidase (MPO) is an enzyme that catalyzes in vivo redox reactions between H2O2 and Cl− to produce HClO. Abnormal levels of HClO and MPO may lead to oxidative stress, irreversible tissue damage and, thus, serious diseases; they are thus becoming important biomarkers and therapeutic targets. In this work, using HClO-induced site-specific cleavage of phosphorothioate-modified DNA to trigger rolling circle amplification (RCA), RCA-assisted biosensors have been developed for the highly sensitive and specific detection of HClO and MPO. Only two DNA oligonucleotides are used in the sensing systems. The powerful signal-amplification capability of RCA endows the sensing systems with a high sensitivity, and the specific fluorescent response of thioflavin T (ThT) to G-quadruplexes in RCA products makes a label-free signal output possible. The proposed biosensors were demonstrated to work well not only for the sensitive and specific quantitation of HClO and MPO with detection limits of 1.67 nM and 0.33 ng/mL, respectively, but also for the screening and inhibitory capacity evaluation of MPO inhibitors, thus holding great promise in disease diagnosis and drug analysis.

Molecular Docking Insight into the Label-Free Fluorescence Aptasensor for Ochratoxin A Detection.

Ochratoxin A (OTA) is the most common mycotoxin and can be found in wheat, corn and other grain products. As OTA pollution in these grain products is gaining prominence as a global issue, the demand to develop OTA detection technology has attracted increasing attention. Recently, a variety of label-free fluorescence biosensors based on aptamer have been established. However, the binding mechanisms of some aptasensors are still unclear. Herein, a label-free fluorescent aptasensor employing Thioflavin T (ThT) as donor for OTA detection was constructed based on the G-quadruplex aptamer of the OTA aptamer itself. The key binding region of aptamer was revealed by using molecular docking technology. In the absence of the OTA target, ThT fluorescent dye binds with the OTA aptamer to form an aptamer/ThT complex, and results in the fluorescence intensity being obviously enhanced. In the presence of OTA, the OTA aptamer binds to OTA because of its high affinity and specificity to form an aptamer/OTA complex, and the ThT fluorescent dye is released from the OTA aptamer into the solution. Therefore, the fluorescence intensity is significantly decreased. Molecular docking results revealed that OTA is binding to the pocket-like structure and surrounded by the A29-T3 base pair and C4, T30, G6 and G7 of the aptamer. Meanwhile, this aptasensor shows good selectivity, sensitivity and an excellent recovery rate of the wheat flour spiked experiment.

Amyloid fibrils degradation: the pathway to recovery or aggravation of the disease?

Background: The most obvious manifestation of amyloidoses is the accumulation of amyloid fibrils as plaques in tissues and organs, which always leads to a noticeable deterioration in the patients' condition and is the main marker of the disease. For this reason, early diagnosis of amyloidosis is difficult, and inhibition of fibrillogenesis, when mature amyloids are already accumulated in large quantities, is ineffective. A new direction for amyloidosis treatment is the development of approaches aimed at the degradation of mature amyloid fibrils. In the present work, we investigated possible consequences of amyloid's degradation. Methods: We analyzed the size and morphology of amyloid degradation products by transmission and confocal laser scanning microscopy, their secondary structure and spectral properties of aromatic amino acids, intrinsic chromophore sfGFP, and fibril-bound amyloid-specific probe thioflavin T (ThT) by the absorption, fluorescence and circular dichroism spectroscopy, as well as the cytotoxicity of the formed protein aggregates by MTT-test and their resistance to ionic detergents and boiling by SDS-PAGE. Results: On the example of sfGFP fibrils (model fibrils, structural rearrangements of which can be detected by a specific change in the spectral properties of their chromophore), and pathological Aβ-peptide (Aβ42) fibrils, leading to neuronal death in Alzheimer's disease, the possible mechanisms of amyloids degradation after exposure to factors of different nature (proteins with chaperone and protease activity, denaturant, and ultrasound) was demonstrated. Our study shows that, regardless of the method of fibril degradation, the resulting species retain some amyloid's properties, including cytotoxicity, which may even be higher than that of intact amyloids. Conclusion: The results of our work indicate that the degradation of amyloid fibrils in vivo should be treated with caution since such an approach can lead not to recovery, but to aggravation of the disease.

A G-triplex and G-quadruplex concatemer-enhanced fluorescence probe coupled with hybridization chain reaction for ultrasensitive aptasensing of ochratoxin A

Ochratoxin A (OTA), a typical mycotoxin contaminant found in various agricultural products and foods, poses a serious threat to human health. In this study, an aptasensor based on a novel fluorescence probe comprising a G-rich DNA sequence (G43) and thioflavin T (ThT) was designed via hybridization chain reaction (HCR) for the ultrasensitive detection of OTA. G43 is a concatemer of G-quadruplex and G-triplex (a G-quadruplex-like structure with one G-quartet removed), which can drastically enhance the fluorescence intensity of ThT. For this strategy to work, the OTA aptamer is pro-locked in a hairpin structure, denoted “hairpin-locked aptamer” (HL-Apt). OTA binds to HL-Apt, opens the hairpin structure, releases the trigger sequence, and initiates the HCR reaction to form a long DNA duplex and numerous side chains. The side chains combine entirely with the complementary DNA and liberate the pro-locked G43 DNA, dramatically enhancing the intensity of the ThT fluorescence signal. The fluorescence intensity correlates linearly with the OTA concentration between 0.02 and 2.00 ng mL−1, and the method has a detection limit of 0.008 ng mL−1. The developed aptasensor was used to detect OTA in foodstuffs with satisfactory results.

Superior Adsorption Efficiency of MIL‐101 (Cr) and Nano‐MIL‐101 (Cr) in Anionic and Cationic Dye Removal from Aqueous Solution

AbstractMetal‐organic frameworks (MOFs) as adsorbent have received considerable attention due to their remarkably high porosity, adjustable pore shape/size, and surface functionality. This study focusses on the adsorption performance and mechanism of two different typological and structural MOFs as superior adsorbents for the efficient removal of anionic and cationic dyes in an aqueous solution. MIL‐101(Cr) and nano‐MIL‐101(Cr) were successfully synthesized by the hydrothermal method using two different protocols and both materials were confirmed by XRD, TG‐DTA‐DTG, FTIR and SEM analyses. The adsorption kinetics and isotherms were determined from the experimental data obtained under optimal conditions. The adsorption kinetics suggests the adsorption process of Congo Red (CR) and Thioflavin T (TFT) onto MIL‐101 (Cr) and nano‐MIL‐101(Cr) conformed to the pseudo‐second‐order kinetic model. The Langmuir isotherm agrees well with the experimental data, and the adsorption capacities for MIL‐101 (Cr) and nano‐MIL‐101 (Cr) were found to be 986 mg g−1 (CR) and 863 mg g−1 (TFT), 1004 mg g−1 (TFT), respectively. The adsorption mechanism is to be related to π–π stacking, hydrogen bonding, pore filling, electrostatic interaction, and partial chemical interaction. This study demonstrates that MIL‐101 (Cr) and nano‐MIL‐101 (Cr) are promising reusable adsorbents with high adsorption capacity for removing dyes from aqueous solutions.

Quantitation of Tissue Amyloid via Fluorescence Spectroscopy Using Controlled Concentrations of Thioflavin-S.

Amyloids are misfolded proteins that aggregate into fibrillar structures, the accumulation of which is associated with the pathogenesis of many neurodegenerative diseases, such as Alzheimer's disease (AD). Early, sensitive detection of these misfolded aggregates is of great interest to the field, as amyloid deposition begins well before the presentation of clinical symptoms. Thioflavin-S (ThS) is a fluorescent probe commonly used to detect amyloid pathology. Protocols for ThS staining vary, but they often use high staining concentrations followed by differentiation, which causes varying levels of non-specific staining and potentially leaves more subtle amyloid deposition unidentified. In this study, we developed an optimized ThS staining protocol for the sensitive detection of β-amyloids in the widely used 5xFAD Alzheimer's mouse model. Controlled dye concentrations together with fluorescence spectroscopy and advanced analytical methods enabled not only the visualization of plaque pathology, but also the detection of subtle and widespread protein misfolding throughout the 5xFAD white matter and greater parenchyma. Together, these findings demonstrate the efficacy of a controlled ThS staining protocol and highlight the potential use of ThS for the detection of protein misfolding that precedes clinical manifestation of disease.

In vitro activity of polyphenol‐rich extract of sorghum relevant to treatment of Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder that accounts for most dementia cases. It has become a serious international health and economic concern as there is no effective treatment. Naturally derived products, particularly polyphenols (PP), have become very popular for developing effective therapeutic strategies for AD. PP apply their neuroprotective actions through targeting various pathological hallmarks of the disease, such as Aβ and/or aggregation, oxidative stress and mitochondrial dysfunction. Aβ aggregation is majorly implicated in the pathogenesis of AD. Therefore, finding a way to attenuate Aβ‐induced toxicity could be a potential approach for disease‐modifying treatments of AD. In the current study, the effect of polyphenolic extract from different sorghum varieties was assessed against Aβ42‐induced cytotoxicity.MethodA cell viability assay (MTS) was utilized to assess the protective effect of the extracts on Aβ‐induced cytotoxicity in human neuroblastoma BE (2)‐M17 cells. Following that, a thioflavin T (Th‐T) assay was performed to assess the anti‐Aβ aggregation effect of the extracts.ResultThe sorghum extracts significantly increased the cell viability. Of the six different tested extracts, QL33(2000 µg/ml) and B923296(1000 µg/ml) demonstrated the strongest and weakest protective effect in decreasing Aβ‐induced cell death (p < 0.01), respectively. In addition, the sorghum extracts were effective at inhibiting the Aβ aggregation.ConclusionSorghum polyphenolic extracts demonstrated a promising neuroprotective effect against Aβ‐induced cytotoxicity and therefore can be considered as a potential naturally derived agent for AD treatment. However, further studies are required to confirm the anti‐AD effects of these sorghum extracts.

Aggregation and antibody‐based inhibition of TDP‐43

AbstractBackgroundTAR DNA‐binding protein 43 (TDP‐43) is a highly conserved nuclear protein, predominantly associated with RNA metabolism. Aberrant TDP‐43 inclusions are present in over 95% of post‐mortem brain tissue samples from Amyotrophic Lateral Sclerosis (ALS) patients. The highly disordered TDP‐43 C‐Terminal Domain (CTD) is associated with the greatest amount of disease‐related mutations and phosphorylation sites. The exact mechanism of TDP‐43 aggregation and formation into a pathological state has yet to be elucidated but is an important therapeutic target to consider.MethodFull‐length phosphorylated TDP‐43 (pS410) protein was treated with 6 hours of daily agitation over 9 days in vitro. Agitation and aging took place with and without the presence of TDP‐43 RRM2‐CTD specific antibodies. Dot‐blot analysis was performed to confirm the binding of antibodies to TDP‐43. Aggregation was monitored and characterized using biophysical analysis; methods included Thioflavin T (ThT), turbidity, and transmission electron microscopy (TEM).ResultPhospho‐TDP‐43 that had undergone agitation and extensive aging formed insoluble aggregates and ThT positive fibrils. The incubation with antibodies specific to the RRM2‐CTD inhibited aggregation and fibril formation in a concentration‐dependent manner.ConclusionSimilar immunotherapies exist for other neurodegenerative disease‐related proteins; inhibition of TDP‐43 aggregation and fibril formation using antibodies demonstrates the potential for the epitope‐specific treatment of TDP‐43 proteinopathies.1) Esposto, J., & Martic‐Milne, S. (2021). Biochimica et Biophysica Acta (BBA) ‐ Molecular Basis of Disease, 1867, https://doi.org/10.1016/j.bbadis.2021.166234.

Chaperone Activity and Protective Effect against Aβ-Induced Cytotoxicity of Artocarpus camansi Blanco and Amaranthus dubius Mart. ex Thell Seed Protein Extracts.

Alzheimer's disease (AD) is the most common type of dementia and is listed as the sixth-leading cause of death in the United States. Recent findings have linked AD to the aggregation of amyloid beta peptides (Aβ), a proteolytic fragment of 39-43 amino acid residues derived from the amyloid precursor protein. AD has no cure; thus, new therapies to stop the progression of this deadly disease are constantly being searched for. In recent years, chaperone-based medications from medicinal plants have gained significant interest as an anti-AD therapy. Chaperones are responsible for maintaining the three-dimensional shape of proteins and play an important role against neurotoxicity induced by the aggregation of misfolded proteins. Therefore, we hypothesized that proteins extracted from the seeds of Artocarpus camansi Blanco (A. camansi) and Amaranthus dubius Mart. ex Thell (A. dubius) could possess chaperone activity and consequently may exhibit a protective effect against Aβ1-40-induced cytotoxicity. To test this hypothesis, the chaperone activity of these protein extracts was measured using the enzymatic reaction of citrate synthase (CS) under stress conditions. Then, their ability to inhibit the aggregation of Aβ1-40 using a thioflavin T (ThT) fluorescence assay and DLS measurements was determined. Finally, the neuroprotective effect against Aβ1-40 in SH-SY5Y neuroblastoma cells was evaluated. Our results demonstrated that A. camansi and A. dubius protein extracts exhibited chaperone activity and inhibited Aβ1-40 fibril formation, with A. dubius showing the highest chaperone activity and inhibition at the concentration assessed. Additionally, both protein extracts showed neuroprotective effects against Aβ1-40-induced toxicity. Overall, our data demonstrated that the plant-based proteins studied in this research work can effectively overcome one of the most important characteristics of AD.

Robust tag-free aptasensor for monitoring of tobramycin: Architecting of rolling circle amplification and fluorescence synergism

With the unpredictable risks on human health and ecological safety, tobramycin (TOB) as an extensively applied antibiotic has embraced global concern. Herein, a label-free fluorescent aptasensor was developed that opened up an innovative sensing strategy for monitoring trace TOB levels. Based on the rolling circle amplification (RCA) process, a giant DNA building was established by the catalytic action of T4 DNA ligase and Phi 29 DNA polymerase with the cooperation of the specific aptamer as a primer skeleton. By having the role of signal amplifier template, the RCA product with the G-quadruplex sequence duplications was decorated by a high number of the thioflavin T (ThT) fluorescent dyes. The aptasensor with good selectivity toward TOB achieved a detection limit as low as 150 pM. Thanks to its accurate target quantification, ease of operation, economic manufacture, as well as high potency for real-time and point-of-care testing, the represented aptasensor is superb for clinical application and food safety control.