Bioactivation of an orthodontic wire using multifunctional nanomaterials to prevent plaque accumulation.

Controlling the growth of biofilm on orthodontic material has become a difficult challenge in modern dentistry. The antibacterial efficacy of currently used orthodontic material becomes limited due to the higher affinity of oral microbial flora for plaque formation on the material surface. Thus it is crutial to device an efficient strategy to prevent plaque buildup caused by pathogenic microbiota. In this work, we have fabricated a bioactive orthodontic wire using titanium nanoparticles (TiO2NPs) and silver nanoparticles (AgNPs). AgNPs were synthesized from the extracts of Ocimum sanctum, Ocimum tenuiflorum, Solanum surattense, and Syzygium aromaticum, while the TiO2NPs were synthesized by the Sol-Gel method. The nanoparticles were characterized by various biophysical techniques. The surface of the dental wire was molded by functionalizing these AgNPs followed by an additional coating of TiO2NPs. Functionalized dental wires were found to counteract the formation of tenacious intraoral biofilm, and showed an enhanced anti-bacterial effect against Multi-Drug Resistant (MDR) bacteria isolated from patients with various dental ailments. Data revealed that such surface coating counteracts the bacterial pathogens by inducing the leakage of Ag ions which eventually disrupts the cell membrane as confirmed from TEM micrographs. The results offer a significant opportunity for innovations in developing nanoparticle-based formulations to modify or fabricate an effective orthodontic material.

Native PLGA nanoparticles regulate APP metabolism and protect neurons against ß-amyloid toxicity: Potential significance in Alzheimer's disease pathology.

Biodegradable poly(lactic-co-glycolic acid)(PLGA) nanoparticles have been used extensively in delivering drugs to target tissues due to their excellent biocompatibility. Evidence suggests that PLGA-conjugated drugs/agents can attenuate pathology in cellular/animal models of Alzheimer's disease (AD), which is initiated by increased level/aggregation of amyloid ß (Aß) peptide generated from amyloid precursor protein (APP). The beneficial effects were attributed to conjugated-drugs rather than to PLGA nanoparticles. Interestingly, we recently reported that PLGA without any drug/agent (native PLGA) can suppress Aß aggregation/toxicity. However, very little is known about the internalization, subcellular localization or effects of PLGA in neurons. In this study, using primary mouse cortical neurons, we first showed that native PLGA is internalized by an energy-mediated clathrin-dependent/-independent pathway and is localized in endosomal-lysosomal-autophagic vesicles. By attenuating internalization, PLGA can protect neurons against Aß-mediated toxicity. Additionally, PLGA treatment altered expression profiles of certain AD-associated genes and decreased the levels of APP, its cleaved products α-/ß-CTFs and Aß peptides in mouse as well as iPSC-derived neurons from control and AD patients. Collectively, these results suggest that native PLGA not only protects neurons against Aß-induced toxicity but also influences the expression of AD-related genes/proteins - highlighting PLGA's implication in normal and AD-related pathology.

Evidence of Anti-amyloid Characteristics of Plumbagin via Inhibition of Protein Aggregation and Disassembly of Protein Fibrils.

The biological consequences associated with the conversion of soluble proteins into insoluble toxic amyloids are not only limited to the onset of neurodegenerative diseases but also to the potential health risks associated with supplements of protein therapeutic agents as well. Hence, finding inhibitors against amyloid formation is important, and natural product-based anti-amyloid compounds have gained much interest because of their higher efficacy and biocompatibility. Plumbagin has been identified as a potential natural product with multiple medical benefits; however, it remains largely unclear whether plumbagin can act against amyloid formation of proteins. Here, we show that plumbagin can effectively inhibit the temperature-induced amyloid aggregation of important proteins (insulin and serum albumin). Both experimental and computational data revealed that the presence of plumbagin in protein solutions, under aggregating conditions, promotes a direct protein-plumbagin interaction, which is predominantly stabilized by stronger H-bonds and hydrophobic interactions. Plumbagin-mediated retention of the native structures of proteins appears to play a crucial role in preventing their conversion into insoluble ß-sheet-rich amyloid aggregates. More importantly, the addition of plumbagin into a suspension of protein fibrils triggered their spontaneous disassembly, promoting the release of soluble proteins. The results highlight that a possible synergistic effect via both the stabilization of protein structures and the restriction of the monomer recruitment at the fibril growth sites could be important for the mechanism of plumbagin's anti-aggregation effect. These findings may inspire the development of plumbagin-based formulations to benefit both the prevention and treatment of amyloid-related health complications.

The intrinsic amyloidogenic propensity of cofilin-1 is aggravated by Cys-80 oxidation: A possible link with neurodegenerative diseases.

Cofilin-1, an actin dynamizing protein, forms actin-cofilin rods, which is one of the major events that exacerbates the pathophysiology of amyloidogenic diseases. Cysteine oxidation in cofilin-1 under oxidative stress plays a crucial role in the formation of these rods. Others and we have reported that cofilin-1 possesses a self-oligomerization property in vitro and in vivo under physiological conditions. However, it remains elusive if cofilin-1 itself forms amyloid-like structures. We, therefore, hypothesized that cofilin-1 might form amyloid-like assemblies, with a potential to intensify the pathophysiology of amyloid-linked diseases. We used various in silico and in vitro techniques and examined the amyloid-forming propensity of cofilin-1. The study confirms that cofilin-1 possesses an intrinsic tendency of aggregation and forms amyloid-like structures in vitro. Further, we studied the effect of cysteine oxidation on the stability and structural features of cofilin-1. Our data show that oxidation at Cys-80 renders cofilin-1 unstable, leading to a partial loss of protein structure. The results substantiate our hypothesis and establish a strong possibility that cofilin-1 aggregation might play a role in cofilin-mediated pathology and the progression of several amyloid-linked diseases.

Mimosine functionalized gold nanoparticles (Mimo-AuNPs) suppress ß-amyloid aggregation and neuronal toxicity.

Evidence suggests that increased level/aggregation of beta-amyloid (Aß) peptides initiate neurodegeneration and subsequent development of Alzheimer's disease (AD). At present, there is no effective treatment for AD. In this study, we reported the effects of gold nanoparticles surface-functionalized with a plant-based amino acid mimosine (Mimo-AuNPs), which is found to cross the blood-brain barrier, on the Aß fibrillization process and toxicity. Thioflavin T kinetic assays, fluorescence imaging and electron microscopy data showed that Mimo-AuNPs were able to suppress the spontaneous and seed-induced Aß1-42 aggregation. Spectroscopic studies, molecular docking and biochemical analyses further revealed that Mimo-AuNPs stabilize Aß1-42 to remain in its monomeric state by interacting with the hydrophobic domain of Aß1-42 (i.e., Lys16 to Ala21) there by preventing a conformational shift towards the ß-sheet structure. Additionally, Mimo-AuNPs were found to trigger the disassembly of matured Aß1-42 fibers and increased neuronal viability by reducing phosphorylation of tau protein and the production of oxyradicals. Collectively, these results reveal that the surface-functionalization of gold nanoparticles with mimosine can attenuate Aß fibrillization and neuronal toxicity. Thus, we propose Mimo-AuNPs may be used as a potential treatment strategy towards AD-related pathologies.

Osmoprotectant Coated Thermostable Gold Nanoparticles Efficiently Restrict Temperature-Induced Amyloid Aggregation of Insulin.

Naturally occurring osmoprotectants are known to prevent aggregation of proteins under various stress factors including extreme pH and elevated temperature conditions. Here, we synthesized gold nanoparticles coated with selected osmolytes (proline, hydroxyproline, and glycine) and examined their effect on temperature-induced amyloid-formation of insulin hormone. These uniform, thermostable, and hemocompatible gold nanoparticles were capable of inhibiting both spontaneous and seed-induced amyloid aggregation of insulin. Both quenching and docking experiments suggest a direct interaction between the osmoprotectant-coated nanoparticles and aggregation-prone hydrophobic stretches of insulin. Circular-dichroism results confirmed the retention of insulin's native structure in the presence of these nanoparticles. Unlike the indirect solvent-mediated effect of free osmolytes, the inhibition effect of osmolyte-coated gold nanoparticles was observed to be mediated through their direct interaction with insulin. The results signify the protection of the exposed aggregation-prone domains of insulin from temperature-induced self-assembly through osmoprotectant-coated nanoparticles, and such effect may inspire the development of osmolyte-based antiamyloid nanoformulations.

Analyzing organophosphate pesticide-serum albumin binding interaction: a combined STD NMR and molecular docking study.

In Vitro analysis of the interaction of organophosphate pesticides (OP) with bovine serum albumin (BSA) is crucial to understand their potential effects at the molecular level. In this context, we have employed Saturation Transfer Difference (STD) NMR experiments in conjunction with molecular docking studies to unravel the binding interaction of the OP chlorpyrifos (CPF), diazinon (DZN) and parathion (PA) in solution. The relative STD (%) suggested the detailed epitope mapping of these OP with BSA while the concentration-dependent STD NMR studies were performed to obtain the complex dissociation constant (KD) of the OP-BSA complexes; KD=1.81 × 10-4 M, 1.30 × 10-3 M and 1.11 × 10-3 M for CPF, DZN and PA were extracted respectively. Similar binding modes were identified for all the three OP using STD site-marker experiment. ITC experiments were performed as a complementary method that revealed a high binding affinity of OP-BSA complexes through non-covalent interaction. Molecular docking confirmed the possible interacting chemical groups of OP-BSA complexes. These significant results furnish valuable information about the toxicity risk of OP to proteins.Communicated by Ramaswamy H. Sarma.

Piperine-Coated Gold Nanoparticles Alleviate Paraquat-Induced Neurotoxicity in Drosophila melanogaster.

Parkinson's disease (PD) is the most common progressive neurodegenerative disease known to impart bradykinesia leading to diverse metabolic complications. Currently, scarcity of effective drug candidates against this long-term devastating disorder poses a big therapeutic challenge. Here, we have synthesized biocompatible, polycrystalline, and uniform piperine-coated gold nanoparticles (AuNPspiperine) to specifically target paraquat-induced metabolic complications both in Drosophila melanogaster and SH-SY5Y cells. Our experimental evidence clearly revealed that AuNPspiperine can effectively reverse paraquat-induced lethal effects in both in vitro and in vivo model systems of PD. AuNPspiperine were found to suppress oxidative stress and mitochondrial dysfunction, leading to inhibition of apoptotic cell death in paraquat-treated flies. AuNPspiperine were also found to protect SH-SY5Y cells against paraquat-induced toxicity at the cellular level preferably by maintaining mitochondrial membrane potential. Both experimental and computational data point to the possible influence of AuNPspiperine in regulating the homeostasis of parkin and p53 which may turn out to be the key factors in reducing PD symptoms. The findings of this work may facilitate the development of piperine-based nanoformulations against PD.

In vitro interaction of organophosphate metabolites with bovine serum albumin: A comparative 1H NMR, fluorescence and molecular docking analysis.

Since the exposure of organophosphate pesticides are known to cause severe health consequences, it is important to understand the molecular interaction of these pesticides metabolites with vital biomolecules, especially with the proteins. Here, considering bovine serum albumin (BSA) as a model protein, we have examined its interaction with two selected organophosphate metabolites, 3,5,6-trichloro-2-pyridinol (TCPy) and paraoxon methyl (PM). TCPy and PM are resultant metabolites of two most widely used organophosphate pesticides chlorpyrifos and parathion respectively. 1H NMR line broadening, selective spin-lattice relaxation rate measurements, saturation transfer difference (STD) NMR of both TCPy and PM were carried out in the presence and absence of BSA. The obtained values of the affinity index (A), binding constants (Ka) and thermodynamic parameters indicated strong organophosphates-BSA interaction. Further, fluorescence quenching data on TCPy-BSA and PM-BSA interactions strongly supported the NMR results, besides providing the stoichiometry of these complexes. Molecular docking analysis unraveled viable, strong hydrogen bonds and electrostatic interactions in TCPy-BSA and PM-BSA complexes. This study also revealed substantial time-dependent changes in the 1H NMR intensity of PM in the presence of BSA, which suggests faster degradation of PM with increasing protein concentration during protein-metabolite interactions. The hydrolysis is attributed to the esterase-like action of BSA. The result provides key insights into the direct interaction of the organophosphate metabolites with a biologically important carrier protein, serum albumin.

Myricetin inhibits amyloid fibril formation of globular proteins by stabilizing the native structures.

Myricetin has been identified as a naturally occurring flavonoid class of polyphenolic compound which shows multiple medical benefits including antidiabetic, anticancerous and antioxidant properties. Here, we report the protective effect of myricetin against in vitro amyloid fibril formation of selected globular proteins. The results reveal that myricetin is capable of inhibiting amyloid fibril formation of both insulin and serum albumin. Seed-induced aggregation of both proteins was also substantially suppressed in the presence of myricetin. Fluorescence quenching data indicated binding of myricetin with protein monomers as well as fibrils. The molecular docking studies revealed strong affinity of myricetin for both the native and partially unfolded conformation of proteins mediated by H-bonds and hydrophobic interactions. Myricetin was also observed to promote disassembly of mature amyloid fibrils. The results reveal that myricetin molecule has the potential for suppressing amyloid formation and such an inherent property may help in developing myricetin-based antiamyloid drugs.

Amyloid cross-seeding raises new dimensions to understanding of amyloidogenesis mechanism.

Hallmarks of most of the amyloid pathologies are surprisingly found to be heterocomponent entities such as inclusions and plaques which contain diverse essential proteins and metabolites. Experimental studies have already revealed the occurrence of coaggregation and cross-seeding during amyloid formation of several proteins and peptides, yielding multicomponent assemblies of amyloid nature. Further, research reports on the co-occurrence of more than one type of amyloid-linked pathologies in the same individual suggest the possible cross-talk among the disease related amyloidogenic protein species during their amyloid growth. In this review paper, we have tried to gain more insight into the process of coaggregation and cross-seeding during amyloid aggregation of proteins, particularly focusing on their relevance to the pathogenesis of the protein misfolding diseases. Revelation of amyloid cross-seeding and coaggregation seems to open new dimensions in our mechanistic understanding of amyloidogenesis and such knowledge may possibly inspire better designing of anti-amyloid therapeutics.

Tyrosine-Generated Nanostructures Initiate Amyloid Cross-Seeding in Proteins Leading to a Lethal Aggregation Trap.

Here, we show that aromatic amino acid tyrosine, under a physiologically mimicking condition, readily forms amyloid-like entities that can effectively drive aggregation of different globular proteins and aromatic residues. Tyrosine self-assembly resulted in the formation of cross-ß rich regular fibrils as well as spheroidal oligomers. Computational data suggest intermolecular interaction between specifically oriented tyrosine molecules mediated through π-π stacking and H-bonding interactions, mimicking a cross-ß-like architecture. Both individual protein samples and mixed protein samples underwent aggregation in the presence of tyrosine fibrils, confirming the occurrence of amyloid cross-seeding. The surface of the tyrosine's amyloid like entities was predicted to trap native protein structures, preferably through hydrophobic and electrostatic interactions initiating an aggregation event. Because tyrosine is a precursor to vital neuromodulators, the inherent cross-seeding potential of the tyrosine fibrils may have direct relevance to amyloid-linked pathologies.

Aß1-40 mediated aggregation of proteins and metabolites unveils the relevance of amyloid cross-seeding in amyloidogenesis.

The multicomponent nature of neuronal plaques in Alzheimer's disease signifies the possible recruitment of non-Aß candidates during the amyloid growth of Aß peptides. Here, we show that amyloid fibrils of Aß1-40 peptide can effectively initiate amyloid formation in different globular proteins and metabolites, converting native structures into ß-sheet rich assemblies. Structural and biophysical properties of the resultant protein fibrils display amyloid like characteristic features. Viable contacts between Aß peptide's cross-ß architecture and the native structure of proteins, mediated through H-bonds and hydrophobic interactions seem crucial for the onset of amyloid cross-seeding. Results reveal the inherent cross-seeding potential of Aß amyloids to initiate amyloid formation process in proteins and metabolites and revelation of such a property may further our mechanistic understanding of amyloid pathologies.

Intrinsic property of phenylalanine to trigger protein aggregation and hemolysis has a direct relevance to phenylketonuria.

Excess accumulation of phenylalanine is the characteristic of untreated Phenylketonuria (PKU), a well-known genetic abnormality, which triggers several neurological, physical and developmental severities. However, the fundamental mechanism behind the origin of such diverse health problems, particularly the issue of how they are related to the build-up of phenylalanine molecules in the body, is largely unknown. Here, we show cross-seeding ability of phenylalanine fibrils that can effectively initiate an aggregation process in proteins under physiological conditions, converting native protein structures to ß-sheet assembly. The resultant fibrils were found to cause severe hemolysis, yielding a plethora of deformed erythrocytes that is highly relevant to phenylketonuria. Unique arrangement of zwitterionic phenylalanine molecules in their amyloid-like higher order entities is predicted to promote both hydrophobic and electrostatic interaction, sufficient enough to trap proteins and to preferentially interact with the membrane components of RBCs. Since the prevalence of hemolysis and amyloid related psychoneurological severities are mostly observed in PKU patients, we propose that the inherent property of phenylalanine fibrils to trigger hemolysis and to induce protein aggregation may have direct relevance to the disease mechanism of PKU.

Eugenol prevents amyloid formation of proteins and inhibits amyloid-induced hemolysis.

Eugenol has attracted considerable attention because of its potential for many pharmaceutical applications including anti-inflammatory, anti-tumorigenic and anti-oxidant properties. Here, we have investigated the effect of eugenol on amyloid formation of selected globular proteins. We find that both spontaneous and seed-induced aggregation processes of insulin and serum albumin (BSA) are significantly suppressed in the presence of eugenol. Isothermal titration calorimetric data predict a single binding site for eugenol-insulin complex confirming the affinity of eugenol for native soluble insulin species. We also find that eugenol suppresses amyloid-induced hemolysis. Our findings reveal the inherent ability of eugenol to stabilize native proteins and to delay the conversion of protein species of native conformation into ß-sheet assembled mature fibrils, which seems to be crucial for its inhibitory effect.

Uniform, Polycrystalline, and Thermostable Piperine-Coated Gold Nanoparticles to Target Insulin Fibril Assembly.

Because the process of insulin fibril assembly is linked to a multitude of medical problems, finding effective and biocompatible inhibitors against such an aggregation process could be beneficial. Targeting the aggregation-prone residues of insulin may perhaps work as an effective strategy to prevent the onset of insulin fibril assembly. In this work, we have synthesized uniform sized, thermostable gold nanoparticles (AuNPspiperine) surface-functionalized with piperine to target amyloid-prone residues of insulin. We found that the process of both spontaneous and seed-induced amyloid formation of insulin was strongly inhibited in the presence of AuNPspiperine. Surface functionalization of piperine was found to be critical to its inhibition effect because no such effect was observed for free piperine as well as for uncoated control gold nanoparticles. Fluorescence quenching data revealed binding of AuNPspiperine with insulin's native structure which was further validated by docking studies that predicted viable H-bond and CH-π interactions between piperine and key aggregation-prone residues of insulin's B-chain. Our hemolysis assay studies further confirmed that these piperine coated nanoparticles were hemocompatible. Data obtained from both experimental and computational studies suggest that the retention of native structure of insulin and the ability of the piperine molecule to interact with the aggregation-prone residues of insulin are the key factors for the inhibition mechanism. The findings of this work may help in the development of nanoparticle-based formulations to prevent medical problems linked to insulin aggregation.

Capsaicin-Coated Silver Nanoparticles Inhibit Amyloid Fibril Formation of Serum Albumin.

We have synthesized capsaicin-coated silver nanoparticles (AgNPs(Cap)) and have tested their anti-amyloid activity, considering serum albumin (BSA) as a model protein. We found that amyloid formation of BSA was strongly suppressed in the presence of AgNPs(Cap). However, isolated capsaicin and uncapped control nanoparticles did not show such an inhibition effect. Bioinformatics analysis reveals CH-π and H-bonding interactions between capsaicin and BSA in the formation of the protein-ligand complex. These results suggest the significance of surface functionalization of nanoparticles with capsaicin, which probably allows capsaicin to effectively interact with the key residues of the amyloidogenic core of BSA.

In vitro cytotoxicity and antimicrobial activity of Talaromyces flavus SP5 inhabited in the marine sediment of Southern Coast of India.

Marine sediment samples were collected from the coastal areas of Southern India, particularly in Kanyakumari District. Twenty-eight different fungal strains were isolated. The screening of fungi from marine sediment was done to isolate a potent fungus that can produce bioactive compounds for biomedical applications. Only three strains viz Trichoderma gamsii SP4, Talaromyces flavus SP5 and Aspergillus oryzae SP6 were screened for further studies. The intracellular bioactive compounds were extracted using solvent extraction method. The crude extracts were tested for its anti-microbial and anti-cancer properties and analytically characterized using Gas Chromatography Mass Spectrometry (GC-MS). All the three extracts were active, but the extract from T. flavus SP5 was found to be more active against various human pathogens, viz., Pseudomonas aeruginosa ATCC 27853 (17.8 ± 0.1), Escherichia coli ATCC 52922 (18.3 ± 0.3), and Candida tropicalis ATCC 750 (17.7 ± 0.4). It also exhibited cytotoxic activity against HEp2 carcinoma cell line with the LC50 value of 25.7 µg·L-1. The GC-MS data revealed the presence of effective bioactive compounds. These results revealed that the extract from isolated fungus T. flavus SP5 acted as a potent antimicrobial, antifungal, and anticancer agent, providing basic information on the potency of marine fungi towards biomedical applications; further investigation may lead to the development of novel anticancer drugs.

Tyrosine- and tryptophan-coated gold nanoparticles inhibit amyloid aggregation of insulin.

Here, we have strategically synthesized stable gold (AuNPs(Tyr), AuNPs(Trp)) and silver (AgNPs(Tyr)) nanoparticles which are surface functionalized with either tyrosine or tryptophan residues and have examined their potential to inhibit amyloid aggregation of insulin. Inhibition of both spontaneous and seed-induced aggregation of insulin was observed in the presence of AuNPs(Tyr), AgNPs(Tyr), and AuNPs(Trp) nanoparticles. These nanoparticles also triggered the disassembly of insulin amyloid fibrils. Surface functionalization of amino acids appears to be important for the inhibition effect since isolated tryptophan and tyrosine molecules did not prevent insulin aggregation. Bioinformatics analysis predicts involvement of tyrosine in H-bonding interactions mediated by its C=O, -NH2, and aromatic moiety. These results offer significant opportunities for developing nanoparticle-based therapeutics against diseases related to protein aggregation.

Evidence of rapid coaggregation of globular proteins during amyloid formation.

The question of how an aggregating protein can influence aggregation of other proteins located in its vicinity is particularly significant because many proteins coexist in cells. We demonstrate in vitro coaggregation and cross-seeding of lysozyme, bovine serum albumin, insulin, and cytochrome c during their amyloid formation. The coaggregation process seems to be more dependent on the temperature-induced intermediate species of these proteins and less dependent on their sequence identities. Because amyloid-linked inclusions and plaques are recognized as multicomponent entities originating from aggregation of the associated protein, these findings may add new insights into the mechanistic understanding of amyloid-related pathologies.