Thioflavin Research Articles

Selection of DNA aptamers for detecting metronidazole and ibuprofen: two common additives in soft drinks.

To enhance the effects of some functional soft drinks, drugs, especially metronidazole (MNZ) and ibuprofen (IBF), are often illegally added. This poses a serious threat to the health of consumers. Therefore, developing simple and rapid detection methods for these additives is crucial. In this study, DNA aptamers of metronidazole and ibuprofen were selected using the library-immobilized method. The best aptamer for metronidazole, named MNZ-1, has a dissociation constant (Kd) value of 4.9 μM and the aptamer for ibuprofen, named IBF-1, shows a Kd of 9.3 μM, as determined by the thioflavin T (ThT) fluorescence assay. The Kd values measured using isothermal titration calorimetry (ITC) were 17.0 μM and 66.7 μM for these two aptamers, respectively. Selectivity experiments indicate that MNZ-1 demonstrates very weak binding to structurally similar drugs, whereas IBF-1 exhibits binding capability to some structurally similar compounds comparable to ibuprofen, enabling the simultaneous detection of these types of drugs. Neither MNZ-1 nor IBF-1 binds to other common drugs. Using ThT, a label-free fluorescent detection method was developed for metronidazole and ibuprofen in soft drinks, showing limits of detection (LODs) of 0.6 μM and 4.7 μM, respectively. Owing to their small size and well-defined secondary structures, these aptamers are expected to be utilized in analytical applications for food and environmental monitoring.

Novel photocatalytic carbon dots: efficiently inhibiting amyloid aggregation and quickly disaggregating amyloid aggregates.

Amyloid aggregation is implicated in the pathogenesis of various neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD). It is critical to develop high-performance drugs to combat amyloid-related diseases. Most identified nanomaterials exhibit limited biocompatibility and therapeutic efficacy. In this work, we used a solvent-free carbonization process to prepare new photo-responsive carbon nanodots (CNDs). The surface of the CNDs is densely packed with chemical groups. CNDs with large, conjugated domains can interact with proteins through π-π stacking and hydrophobic interactions. Furthermore, CNDs possess the ability to generate singlet oxygen species (1O2) and can be used to oxidize amyloid. The hydrophobic interaction and photo-oxidation can both influence amyloid aggregation and disaggregation. Thioflavin T (ThT) fluorescence analysis and circular dichroism (CD) spectroscopy indicate that CNDs can block the transition of amyloid from an α-helix structure to a β-sheet structure. CNDs demonstrate efficacy in alleviating cytotoxicity induced by Aβ42 and exhibit promising blood-brain barrier (BBB) permeability. CNDs have small size, low biotoxicity, good fluorescence and photocatalytic properties, and provide new ideas for the diagnosis and treatment of amyloid-related diseases.

Inhibition of cytotoxic self-assembly of HEWL through promoting fibrillation by new synthesized α-hydroxycarbamoylphosphinic acids.

The main objective of the present study is to investigate the potency of new synthesized hydroxycarbamoyl phosphinic acid derivatives in modulating cytotoxic fibrillogenesis of hen egg white lysozyme (HEWL), as a common model in protein aggregation studies. Hydroxycarbamoyl phosphinic acid derivatives were prepared by the reaction of α-hydroxyalkylphosphinic acids with isocyanates (or isothiocyanates) in the presence of trimethylsilyl chloride (TMSCl). The designed process involves the condensation reaction leading to formation of new C sp2-P bond formation. The synthesis and purity of novel designed compounds were confirmed by NMR, LC-MS, and HPLC techniques. A range of experiments, including thioflavin T (ThT) and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence assays, Congo red binding measurement, atomic force microscopy imaging, MTT-based cell viability and hemolysis assays were employed to investigate anti-amyloidogenic effects of tested compounds. The obtained results demonstrate that these compounds are able to significantly modulate the self-assembly process of HEWL via shortening of nucleation phase leading to the acceleration of fibrillation and appearance of very large and thick fibrils with decreased surface hydrophobicity and cytotoxicity. Based on ANS binding data, we suggest that increased exposure of hydrophobic patches of oligomeric species is the possible mechanism by which tested compounds promote self-assembly process of HEWL. Fluorescence anisotropy and molecular docking studies indicate the interaction of both synthesized compounds with HEWL, and more specifically with residues that are situated in the highly aggregation-prone β-domain region of protein. This study unveils the potential of hydroxyalkylphosphinic acids as modulators of amyloid fibrillation highlighting these compounds as a promising approach for targeting protein aggregates associated with neurodegenerative diseases.

De novo designed aliphatic and aromatic peptides assemble into amyloid-like cytotoxic supramolecular nanofibrils.

Peptides are very interesting biomolecules that upon self-association form a variety of thermodynamically stable supramolecular structures of nanometric dimension e.g. nanotubes, nanorods, nanovesicles, nanofibrils, nanowires and many others. Herein, we report six peptide molecules having a general chemical structure, H-Gaba-X-X-OH (Gaba: γ-aminobutyric acid, X: amino acid). Out of these six peptides, three are aromatic and the others are aliphatic. Atomic force microscopic (AFM) studies reveal that except peptide 6 (H-Gaba-Trp-Trp-OH), all the reported peptides adopt nanofibrillar morphology upon aggregation in aqueous medium. These supramolecular assemblies can recognize amyloid-specific molecular probe congo red (CR) and thioflavine t (ThT) and exhibit all the characteristic properties of amyloids. The MTT cell viability assay reveals that the toxicity of both aliphatic and aromatic peptides increases with increasing concentration of the peptides to both cancer (HeLa) and non-cancer (HEK 293) cells. Of note, the aromatic peptides show a slightly higher cytotoxic effect compared to the aliphatic peptides. Overall, the studies highlight the self-assembling nature of the de novo designed aliphatic and aromatic peptides and pave the way towards elucidating the intricacies of pathogenic amyloid assemblies.

DNA aptamers for common buffer molecules: possibility of buffer interference in SELEX.

During a typical aptamer selection experiment, buffer molecules are used at the 10 to 50 mM range, whereas target molecules could be used at much lower concentrations even in low μM levels. Therefore, doubts existed regarding the potential enrichment of buffer binding aptamers, particularly for failed selections that cannot validate binding of enriched sequences. In this study, we used two common buffer molecules, Tris and HEPES, as target molecules. While we successfully isolated aptamers for Tris buffer, our attempts to generate aptamers for HEPES buffer failed. Thioflavin T (ThT) fluorescence spectroscopy showed the dissociation constant (Kd) of the Tris buffer aptamer to be 2.9 mM, while isothermal titration calorimetry showed a Kd of 43 μM. NMR spectroscopy also confirmed aptamer binding. Finally, we discussed the implications of this buffer selection work and recommended the use of certain buffers.

Target-initiated triplex signal amplification cascades for non-label and sensitive fluorescence sensing of microRNA.

The aberrant expression of microRNAs (miRs) in cells is closely linked to the initiation and progression of various diseases. Sensitive monitoring of their level is hence vital for biomedical research and disease diagnosis. Herein, a highly sensitive and non-label fluorescence sensor based on multiple recycling signal amplification cascades is constructed for the detection of miR-21 in human sera. The presence of miR-21 initiates the primer-fueled target recycling process for the generation of many primer/hairpin templates for the subsequent auto-cycling primer extension (APE) amplification cycles, which result in the formation of lots of long-stem hairpins. The enzyme-based cleavage of such hairpins via polymerization/excision cycles further leads to the generation of abundant G-quadruplex strands, which associate with the thioflavin T (ThT) dye to emit remarkably magnified fluorescence for detecting miR-21 in the range of 1 pM-100 nM with a 0.32 pM detection limit without labeling the probes. Besides, the proposed assay can selectively discriminate miR-21 against other control molecules and realize the sensing of low levels of miR-21 in diluted sera. With features of high sensitivity via the triplex signal amplification cycles and simplicity in a non-label homogeneous manner, our miR sensing protocol can be a robust means for detecting various nucleic acids for the early diagnosis of diseases.

Interactions of Turmeric- and Curcumin-Functionalized Gold Nanoparticles with Human Serum Albumin: Exploration of Protein Corona Formation, Binding, Thermodynamics, and Antifibrillation Studies.

In order to better understand the bioavailability and biocompatibility of polyphenol-assisted surface-modified bioengineered nanoparticles in nanomedicine applications, here, we address a series of photophysical experiments to quantify the binding affinity of serum albumin toward polyphenol-capped gold nanoparticles. For this, two different gold nanoparticles (AuNPs) were synthesized via the green synthesis approach, where curcumin and turmeric extract act as reducing as well as capping agents. The size, surface charge, and surface plasmon bands of the AuNPs were highly affected by the adsorption of human serum albumin (HSA) during protein corona formation, which was investigated using dynamic light scattering (DLS), ξ-potential, ultraviolet-visible (UV-vis) spectroscopy, and transmission electron microscopy (TEM) measurements. Fluorescence-based methods, absorbance, and SERS experiments were carried out to evaluate the binding aspects of AuNPs with HSA. We found that the AuNPs show moderate binding affinity toward HSA (Kb ∼ 104 M-1), irrespective of the capping agents on the surface. Hydrophobic association, along with some contribution of electrostatic interaction, played a key role in the binding process. The binding interaction was more toward the subdomain IIA region of HSA, as indicated by the competitive displacement studies using site-specific binders (warfarin and flufenamic acid). Because of the large surface curvature of small-sized AuNPs, the secondary structural conformations of HSA were slightly altered, as revealed by circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy, and surface-enhanced Raman scattering (SERS) measurements. Additionally, the findings of the binding interactions were re-evaluated using molecular dynamics (MD) simulation studies by determining the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), and changes in the binding energy of HSA upon complexation with AuNPs. To determine the tentative evidence for pharmacokinetic administration, these biocompatible AuNPs were applied to inhibit the amyloid fibril formation of HSA and monitored by using the thioflavin T (ThT) assay, ANS fluorescence assay, fluorescence microscopic imaging, and FESEM. AuNPs were found to show better resistance toward fibrillation of the adsorbed protein.

Construction of Fluorescent G-Quadruplex Nanowires for Label-Free and Accurate Monitoring of Circular RNAs in Breast Cancer Cells and Tissues with Low Background.

Circular RNAs (circRNAs) represent an emerging category of endogenous transcripts characterized by long half-life time, covalently closed structures, and cell-/tissue-specific expression patterns, making them potential disease biomarkers. Herein, we demonstrate the construction of fluorescent G-quadruplex nanowires for label-free and accurate monitoring of circular RNAs in breast cancer cells and tissues by integrating proximity ligation-rolling circle amplification cascade with lighting up G-quadruplex. The presence of target circRNA facilitates the SplintR ligase-mediated ligation of the padlock probe. Upon the addition of primers, the ligated padlock probe can serve as a template to initiate subsequent rolling circle amplification (RCA), generating numerous long G-quadruplex nanowires that can incorporate with thioflavin T (ThT) to generate a remarkably improved fluorescence signal. Benefiting from good specificity of SplintR ligase-mediated ligation reaction and exponential amplification efficiency of RCA, this strategy can sensitively detect target circRNA with a limit of detection of 4.65 × 10-18 M. Furthermore, this method can accurately measure cellular circRNA expression with single-cell sensitivity and discriminate the circRNA expression between healthy para-carcinoma tissues and breast cancer tissues, holding great potential in studying the pathological roles of circRNA and clinic diagnostics.

PCR-Free, Label-Free, and Centrifugation-Free Diagnosis of Multiplex Antibiotic Resistance Genes by Combining mDNA-Au@Fe3O4 from Heating Dry and DNA Concatamers with G-Triplex.

Accurate identification of antibiotic resistance genes (ARGs) is crucial for improving treatment and controlling the spread of antibiotic-resistant bacteria (ARB). Herein, a novel PCR-free, centrifugation-free, and label-free magnetic fluorescent biosensor (MFB) was developed by combining polyA-medium DNA-polyT (mDNA, which contained a partial sequence of a target DNA), gold nanoparticle (AuNP)-anchored magnetic nanoparticle (Au@Fe3O4), complementary strand DNA (CS) of the target DNA, DNA concatamer with G-triplex (G3), and thioflavin T (ThT). Thereinto, Au@Fe3O4 nanoparticles were first capped by mDNA strands within 20 min using a simple hot drying method, and then CS was added and hybridized with mDNA on Au@Fe3O4. Second, a DNA concatamer was used to bind with CS on Au@Fe3O4. When an ARG was present in the sample, the CS would recognize it and release the DNA concatamer into solution by a toehold-mediated strand displacement reaction. Finally, under magnetic separation, the free DNA concatamers with G3 were taken out easily and bound with ThT, resulting in strong fluorescence signals. The fluorescence intensity of ThT was positively correlated with the concentration of the ARG. The whole analysis was accomplished within 1.5 h using 96-well plates. Remarkably, our MFB was universal; eight ARGs were detected by replacing the corresponding mDNA and CS in this study. To verify the practicability of our method, 12 clinically isolated strains were analyzed. The results of the MFB method were in good agreement with those of the quantitative real-time PCR method with an area under the curve of 0.92 (95% confidence interval: 0.8479 to 0.9932), sensitivity of 92.00%, and specificity of 91.55%. Above all, the MFB assay established here is simple, low-cost, and universal and has great potential for applications in the identification of ARGs.

1,4-Diurea- and 1,4-Dithiourea-Substituted Aromatic Derivatives Selectively Inhibit α-Synuclein Oligomer Formation In Vitro.

Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting the elderly population worldwide. In PD, the misfolding of α-synuclein (α-syn) results in the formation of inclusions referred to as Lewy bodies (LB) in midbrain neurons of the substantia nigra and other specific brain localizations, which is associated with neurodegeneration. There are no approved strategies to reduce the formation of LB in the neurons of patients with PD. Our drug discovery program focuses on the synthesis of urea and thiourea compounds coupled with aminoindole moieties to abrogate α-syn aggregation and to slow down the progression of PD. We synthesized several urea and thiourea analogues with a central 1,4-phenyl diurea/thiourea linkage and evaluated their effectiveness in reducing α-syn aggregation with a special focus on the selective inhibition of oligomer formation among other proteins. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), as well as M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. Our results identified compound 1 as the best compound in reducing α-syn fibril formation via ThT assays. The antioligomer formation of compound 1 was subsequently superseded by compound 2. Both compounds selectively curtailed the oligomer formation of α-syn but not tau 4R isoforms (0N4R, 2N4R) or p-tau (isoform 1N4R). Compounds 1 and 2 failed to abrogate tau 0N3R fibril formation by ThT and atomic force microscopy. Compound 2 was best at reducing the formation of recombinant α-syn fibrils by TEM. In contrast to compound 2, compound 1 reduced the formation of α-syn inclusions in M17D neuroblastoma cells in a dose-dependent manner. Compound 1 may provide molecular scaffolds for the optimization of symmetric molecules for its α-syn antiaggregation activity with potential therapeutic applications and development of small molecules in PD.

Sensitized Emission Imaging Allows Nanoscale Surface Polarity Mapping of α-Synuclein Amyloid Fibrils.

When misfolded, α-Synuclein (α-Syn), a natively disordered protein, aggregates to form amyloid fibrils responsible for the neurodegeneration observed in Parkinson's disease. Structural studies revealed distinct molecular packing of α-Syn in different fibril polymorphs and variations of interprotofilament connections in the fibrillar architecture. Fibril polymorphs have been hypothesized to exhibit diverse surface polarities depending on the folding state of the protein during aggregation; however, the spatial variation of surface polarity in amyloid fibrils remains unexplored. To map the local polarity (or hydrophobicity) along α-Syn fibrils, we visualized the spectral characteristics of two dyes with distinct polarities-hydrophilic Thioflavin T (ThT) and hydrophobic Nile red (NR)─when both are bound to α-Syn fibrils. Dual-channel fluorescence imaging reveals uneven partitioning of ThT and NR along individual fibrils, implying that relatively more polar/hydrophobic patches are spread over a few hundred nanometers. Remarkably, spectrally resolved sensitized emission imaging of α-Syn fibrils provides unambiguous evidence of energy transfer from ThT to NR, implying that dyes of dissimilar polarity are in close proximity. Furthermore, spatially resolved fluorescence spectroscopy of the solvatochromic probe NR allowed us to quantitatively map the range and variation of the polarity parameter ET30 along individual fibrils. Our results suggest the existence of interlaced polar and nonpolar nanoscale domains throughout the fibrils; however, the relative populations of these patches vary considerably over larger length scales likely due to heterogeneous packing of α-Syn during fibrilization and dissimilar exposed polarities of polymorphic segments. The employed method may provide a foundation for imaging modalities of other similar structurally unresolved systems with diverse hydrophobic-hydrophilic topology.

Biosorptive removal of cationic dyes from ternary system using a magnetic nanocomposite hydrogel based on modified tragacanth gum

Performance of a magnetic tragacanth gum-crosslinked-poly(acrylic acid) (TG-cl-PAA/Fe3O4) nanocomposite hydrogel in removal of malachite green (MG), thioflavin T (ThT), and rhodamine B (RhB) cationic dyes from single, binary, and ternary systemes were investigated through the batch method. The adsorption parameters, including adsorbent amount, initial concentrations of dyes, pH, and contact time were optimized for all systems. Langmuir and Freundlich models were used for equilibrium adsorption isotherm investigation, and it was found that the Langmuir model is more fitted than those of the Freundlich model, which proved the linearity of the adsorption process. Maximum adsorption capacities (Qm) of TG-cl-PAA/Fe3O4 were calculated for all systems. The Qm for MG, RhB, and ThT dyes in ternary system were obtained as 642.9, 552.6, and 413.6 mgg−1, resepectively. The removal of all dyes in single, binary as well as ternary systems were well-fitted with the pseudo-second-order model that confirm the rate-limiting step might be the chemical adsorption. The developed adsorbent was regenerated through desorption process and reused for several times. It was found that the removal efficiency of adsorbent approximately remains the same for the first three cycles, and after which decreased gradually.

Evaluation of the effect of phenylpropanoids on the binding of heparin to human serum albumin and glycosylated human serum albumin concerning anticoagulant activity: A comparison study

The nonenzymatic advanced glycation end products (AGEs) and the accumulation of AGEs are the two main factors associated with the long-term pathogenesis of diabetes. Human serum albumin (HSA) as the most abundant serum protein has a higher fortuity to be modified by nonenzymatic glycation. In this study, the interaction of three phenylpropanoids (caffeic acid (Caf), p-coumaric acid (Cou), and cinnamic acid (Cin)) toward HSA and glycosylated HSA (gHSA) was analyzed by multiple spectroscopic techniques combined with molecular docking. The formation of fibrils in HSA and gHSA was confirmed by the Thioflavin T (ThT) assay. The phenylpropanoids have shown anti-fibrillation properties in vitro. The obtained thermodynamic parameters indicated that hydrogen bonding and van der Waals forces are the main forces in the binding interaction, and the quenching mechanism of the protein fluorescence is static. Molecular docking results, as well as the in vitro results, showed that Caf, Cou, and Cin exhibit more stable interactions with HSA, respectively. In addition, molecular docking analysis showed that Caf and Cou interact well with K199. Given the critical role of K199 in HSA glycosylation in diabetic patients, this process inhibits the interaction of stabilizer compounds and thus accelerates gHSA aggregation.

Triple cycling integrating with primer exchange reaction (TCiPER) for ultra-sensitive and label-free detection of MicroRNA

Primer exchange reaction (PER), a novel and effective isothermal amplification method, is extensively employed in the development of microRNAs (miRNAs) detection methods. Nevertheless, the PER-based miRNA detection methods necessitate enhanced sensitivity and interference resistance. Here, we propose a fluorescence biosensor, referred to as Triple Cycling integrating with Primer Exchange Reaction (TCiPER), for miRNA detection with straightforward operation, reduced interferences, and significantly increased sensitivity. This method has a high sensitivity with a low limit of detection of 56 aM because the target miRNA recognizes the detection probe and initiates triple signal cycles. PER template (PT) is initially occupied by the hairpin probe (HP), which reduces the possibility of fault initiation of PER by the primer similar sequences, and ii) the produced G-quadruplex sequences combine with thioflavin T (ThT) to produce significant fluorescence, which is superior to traditional fluorescent methods that are developed on the basis of costly and carefully selected fluorescent probes. In conclusion, this strategy substantially enhanced the convenience of sensors fabricated using PER strategies and provided a new perspective for the future development of extensive and sensitive nucleic acid detection analysis tools.

Characterizing cannabis-prevalent terpenes for neuroprotection reveal a role for α and β-pinenes in mitigating amyloid β-evoked neurotoxicity and aggregation in vitro

BackgroundCannabis sativa L. (C. sativa) can efficiently synthesize of over 200 terpenes, including monoterpenes, sesquiterpenes and triterpenes that may contribute to the known biological activities of phytocannabinoids of relevance for the burgeoning access to medicinal cannabis formulations globally; however, to date have been uncharacterized. We assessed twelve predominant terpenes in C. sativa for neuroprotective and anti-aggregative properties in semi-differentiated PC12 neuronal cell line that is robust and validated as a cell model responsive to amyloid β (Aβ1–42) protein exposure and oxidative stress. MethodsCell viability was assessed biochemically using the MTT assay in the presence of myrcene, β-caryophyllene, terpinolene, limonene, linalool, humulene, α-pinene, nerolidol, β-pinene, terpineol, citronellol and friedelin (1–200 μM) for 24 hr. Sub-toxic threshold test concentrations of each terpene were then applied to cells, alone or with concomitant incubation with the lipid peroxidant tert-butyl hyrdroperoxide (t-BHP; 0–250 μM) or amyloid β (Aβ1–42; 0–1 μM) to assess neuroprotective effects. Direct effects of each terpene on Aβ fibril formation and aggregation were also evaluated using the Thioflavin T (ThT) fluorometric kinetic assay and transmission electron microscopy (TEM) to visualize fibril and aggregate morphology. ResultsTerpenes were intrinsically benign to PC12 cells up to 50 μM, with higher concentrations of β-caryophyllene, humulene and nerolidol inducing some loss of PC12 cell viability. No significant protective effects of terpenes were observed following t-BHP (0–200 µM) administration, with some enhanced toxicity instead demonstrated from both β-caryophyllene and humulene treatment (each at 50 µM). α-pinene and β-pinene demonstrated a significant neuroprotective effect against amyloid β exposure. α-pinene, β-pinene, terpineol, terpinolene and friedelin were associated with a variable inhibition of Aβ1–42 fibril and aggregate density. ConclusionsThe outcomes of this study underline a neuroprotective role of α-pinene and β-pinene against Aβ-mediated neurotoxicity associated with an inhibition of Aβ1–42 fibrilization and density. This demonstrates the bioactive potential of selected terpenes for consideration in the development of medicinal cannabis formulations targeting neurodegenerative diseases.

Fluorometric and colorimetric platforms for rapid and sensitive hydroxychloroquine detection in aqueous samples

The detection of pharmaceuticals has been an active area of research with numerous application areas ranging from therapeutic and environmental monitoring to pharmaceutical manufacturing and diagnostics. And, the emergence of COVID-19 pandemic has increased the demand for detection of certain active pharmaceutical ingredients such as Hydroxychloroquine (HCQ) mainly due to their increased manufacturing and usage. In this study, we present two optical, fluorometric and colorimetric, detection platforms for the rapid and sensitive detection of HCQ. These platforms take advantage of the interactions between the highly fluorescent dye Thioflavin T (ThT) and Tel24 G-quadruplex (G4) DNA structure, as well as the salt-induced aggregation behavior of negatively charged citrate-capped silver nanoparticles (Cit-AgNPs) in the presence of HCQ. In the fluorometric method, the addition of HCQ led to a significant and rapid decrease in the fluorescence signal of the ThT + Tel24 probe. In the colorimetric method, HCQ induced the aggregation of Cit-AgNPs in the presence of NaCl, resulting in a noticeable color change from yellowish-gray to colorless. Under the optimized conditions, the colorimetric platform exhibited a linear range of 18.0–240.0 nM and a detection limit of 9.2 nM, while the fluorometric platform showed a linear range of 0.24–5.17 μM and a detection limit of 120 nM. The selectivity of the proposed optical methods towards the target analyte was demonstrated by evaluating the response to other structurally similar small molecules. Finally, the practical applicability of both detection systems was confirmed by analyzing HCQ-spiked human urine samples that yielded average recoveries ranging from 75.4 to 110.2 % for the fluorometric platform and 86.9–98.2 % for the colorimetric platform. These results indicate the potential of the developed methods for HCQ detection in complex matrices.

Plasma Exchange Reduces Aβ Levels in Plasma and Decreases Amyloid Plaques in the Brain in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common type of dementia, characterized by the abnormal accumulation of protein aggregates in the brain, known as neurofibrillary tangles and amyloid-β (Aβ) plaques. It is believed that an imbalance between cerebral and peripheral pools of Aβ may play a relevant role in the deposition of Aβ aggregates. Therefore, in this study, we aimed to evaluate the effect of the removal of Aβ from blood plasma on the accumulation of amyloid plaques in the brain. We performed monthly plasma exchange with a 5% mouse albumin solution in the APP/PS1 mouse model from 3 to 7 months old. At the endpoint, total Aβ levels were measured in the plasma, and soluble and insoluble brain fractions were analyzed using ELISA. Brains were also analyzed histologically for amyloid plaque burden, plaque size distributions, and gliosis. Our results showed a reduction in the levels of Aβ in the plasma and insoluble brain fractions. Interestingly, histological analysis showed a reduction in thioflavin-S (ThS) and amyloid immunoreactivity in the cortex and hippocampus, accompanied by a change in the size distribution of amyloid plaques, and a reduction in Iba1-positive cells. Our results provide preclinical evidence supporting the relevance of targeting Aβ in the periphery and reinforcing the potential use of plasma exchange as an alternative non-pharmacological strategy for slowing down AD pathogenesis.

Structural implications of amyloidogenic rare variants Ser282Leu and Gln356Arg identified in h-BRCA1.

Preliminary studies have shown BRCA1 (170-1600) residues to be intrinsically disordered with unknown structural details. However, thousands of clinically reported variants have been identified in this central region of BRCA1. Therefore, we aimed to characterize h-BRCA1(260-553) to assess the structural basis for pathogenicity of two rare missense variants Ser282Leu, Gln356Arg identified from the Indian and Russian populations respectively. Small-angle X-ray scattering analysis revealed WT scores Rg -32 Å, Dmax -93 Å, and Rflex-51% which are partially disordered, whereas Ser282Leu variant displayed a higher degree of disorderedness and Gln356Arg was observed to be aggregated. WT protein also possesses an inherent propensity to undergo a disorder-to-order transition in the presence of cruciform DNA and 2,2,2-Trifluoroethanol (TFE). An increased alpha-helical pattern was observed with increasing concentration of TFE for the Gln356Arg mutant whereas Ser282Leu mutant showed significant differences only at the highest TFE concentration. Furthermore, higher thermal shift was observed for WT-DNA complex compared to the Gln356Arg and Ser282Leu protein-DNA complex. Moreover, mature amyloid-like fibrils were observed with 30 μM thioflavin T (ThT) at 37°C for Ser282Leu and Gln356Arg proteins while the WT protein exists in a protofibril state as observed by TEM. Gln356Arg formed higher-order aggregates with amyloidogenesis over time as monitored by ThT fluorescence. In addition, computational analyses confirmed larger conformational fluctuations for Ser282Leu and Gln356Arg mutants than for the WT. The global structural alterations caused by these variants provide a mechanistic approach for further classification of the variants of uncertain clinical significance in BRCA1 into amyloidogenic variants which may have a significant role in disease pathogenesis.

Multi-Target Actions of Flavonoid Derivatives from Mesua ferrea Linn Flower against Alzheimer’s disease Pathogenesis

OBJECTIVE Kaempferol-3-O-rhamnoside (compound 1) and quercetin- 3-O-rhamnoside (compound 2), two flavonoids isolated from Mesua ferrea L. flowers, were examined for their activities related Alzheimer’s disease (AD) pathogenesis including antioxidant, acetylcholinesterase (AChE) inhibition, anti-beta amyloid (Ab) aggregation and neuroprotection. METHODS The two flavonoids were isolated from M. ferrea L. flowers using the column chromatography technique. Both compounds were evaluated for their effects on AD pathogenesis, including antioxidant action by ABTS assay, AChE inhibition by Ellman’s method, and anti-Ab aggregation by thioflavin T (ThT) assay and neuroprotection by cell base assay. To explain the mechanism of AChE inhibition and anti-Ab aggregation, binding interactions between the test compounds and AChE and Ab were studied in-silico. RESULTS Compounds 1 and 2 showed an ability to scavenge ABTS radicals, with IC50 values of 424.57±2.97 and 308.67±9.90 µM, respectively, and to inhibit AChE function with IC50 values of 769.23±6.23 and 520.64±5.94, respectively. ThT assay indicated that both compounds inhibited Ab aggregation with IC50 values of 406.43±9.95 and 300.69 ±1.18 µM, respectively. The neuroprotection study revealed that the two flavonoids could reduce human neuroblastoma (SH-SY5Y) cell death induced by H2O2. The in-silico study showed that both compounds bound AChE at catalytic anionic and peripheral anionic sites. In addition, the test compounds prevented Ab aggregation by interacting at the central hydrophobic core, the C-terminal hydrophobic region, and the important residues of Ile41. CONCLUSIONS Together, the results showed that kaempferol-3-O- rhamnoside and quercetin-3-O- rhamnoside exhibit multiple mechanisms of action that are involved in the pathogenesis of AD including antioxidant, AChE inhibition, anti-Ab aggregation, and neuroprotection. KEYWORDS flavonoid rhamnosides, Alzheimer’s disease, oxidation, beta amyloid, acetylcholinesterase, molecular docking

The role of the biofilm of Porphyromonas gingivalis in driving amyloid beta secretion and aggregation

AbstractBackgroundOver the past five years, the hypothesis linking periodontitis to Alzheimer’s disease (AD) has gained significant traction even though little is known about the underlying mechanisms driving this association. Periodontitis is caused by the dysbiosis of the dental biofilm, an extra‐cellular matrix produced by bacteria, which is mostly caused by the Gram‐negative bacterium Porphyromonas gingivalis. P. gingivalis has been found to infect the brain of AD patients and drive amyloid beta (Aβ) production while biofilm components can be identified interwoven with Aβ plaques1,2. We therefore hypothesize that P. gingivalis drives Aβ production in neural cells which co‐aggregates with bacterial biofilm to expedite plaque formation and downstream neuroinflammation.MethodTo assess if the biofilm of co‐aggregation of Aβ and the matrix was then analyzed by fluorescence microscopy, atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assays. To examine whether P. gingivalis stimulates Aβ production, we infected BE(2)‐M17 human neuroblastomas at a mean of infection of 1:100 before collecting cells and supernatant proteins to observe the cell response through protein and transcription assays.ResultsWhen co‐cultured, P. gingivalis and Aβ1‐42 co‐aggregated in plaque‐like structures, Interestingly, colocalization analysis revealed that Aβ1‐42 particularly associate with the extracellular component of biofilm (Figure 1). ThT assays and AFM imaging demonstrated faster aggregation of Aβ1‐42 when co‐incubated with fragments of P. gingivalis biofilm for 8 h, which suggests a significant cross‐seeding phenomenon between the two components (Figure 2). Furthermore, infection of human neuroblastomas resulted in a significant increase in Aβ production that could be detected by western blot, thus offering a workable model to study the link between AD and periodontitis (Figure 3).ConclusionOur project offers significant insights into the relationship between periodontitis and AD, allowing us to understand how P. gingivalis triggers Aβ production and how its biofilm drives plaque aggregation. This research greatly contributes to a better understanding of external AD risk factors and how to mediate their impacts on neurodegeneration. 1Dominy et al. Science Advances (2019) 2Mirzaei et al. Microbial Pathogenesis (2020)