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.

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.

Self-Assembly of Artificial Sweetener Aspartame Yields Amyloid-like Cytotoxic Nanostructures.

Recent reports have revealed the intrinsic propensity of single aromatic metabolites to undergo self-assembly and form nanostructures of amyloid nature. Hence, identifying whether aspartame, a universally consumed artificial sweetener, is inherently aggregation prone becomes an important area of investigation. Although the reports on aspartame-linked side effects describe a multitude of metabolic disorders, the mechanistic understanding of such destructive effects is largely mysterious. Since aromaticity, an aggregation-promoting factor, is intrinsic to aspartame's chemistry, it is important to know whether aspartame can undergo self-association and if such a property can predispose any cytotoxicity to biological systems. Our study finds that aspartame molecules, under mimicked physiological conditions, undergo a spontaneous self-assembly process yielding regular ß-sheet-like cytotoxic nanofibrils of amyloid nature. The resultant aspartame fibrils were found to trigger amyloid cross-seeding and become a toxic aggregation trap for globular proteins, Aß peptides, and aromatic metabolites that convert native structures to ß-sheet-like fibrils. Aspartame fibrils were also found to induce hemolysis, causing DNA damage resulting in both apoptosis and necrosis-mediated cell death. Specific spatial arrangement between aspartame molecules is predicted to form a regular amyloid-like architecture with a sticky exterior that is capable of promoting viable H-bonds, electrostatic interactions, and hydrophobic contacts with biomolecules, leading to the onset of protein aggregation and cell death. Results reveal that the aspartame molecule is inherently amyloidogenic, and the self-assembly of aspartame becomes a toxic trap for proteins and cells, exposing the bitter side of such a ubiquitously used artificial sweetener.

Influence of food commodities on hangover based on alcohol dehydrogenase and aldehyde dehydrogenase activities.

Alcohol consumption often leads to hangover, a condition characterized by several symptoms, characteristically headache, nausea, fatigue and drowsiness. Hangover may be alleviated by altering the rate of alcohol metabolism and facilitating elimination of acetaldehyde by affecting the activity of alcohol dehydrogenase (ADH) and/or aldehyde dehydrogenase (ALDH) enzymes. In the present study, several food commodities like fruits, vegetables, cereals, pulses, dairy products, spices and other miscellaneous products (ascorbic acid, cocoa sample, tea, coffee, egg yolk and date samples) were investigated for their effect on the in vitro activities of the enzymes and their antioxidant properties. Of the many screened food commodities, few showed an increase in the activity of either one or both the enzymes, ADH and ALDH. Studies showed no correlation between ADH and ALDH enzyme activities and antioxidant property of the selected food commodities for anti-hangover effect. Further, an anti-hangover (AHO) product was developed using pear (65%), sweet lime (25%) and coconut water (10%) and, validated for in vitro ADH and ALDH enzyme activities. AHO product was found to enhance ADH and ALDH activities by 23.31% and 70.02%, respectively.

Evaluation and application of prebiotic and probiotic ingredients for development of ready to drink tea beverage.

Ready-to-drink (RTD) ice tea is a ready prepared tea, produced from green and black tea originating from same plant Camellia sinensis. The objective of this study was to determine the effect of prebiotics [galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS), and inulin] or synbiotic ingredients (GOS, FOS, inulin, and Lactobacillus acidophilus) on the sensory properties and consumer acceptability of RTD. The quality of green tea extract (GTE) and black tea extract (BTE) was improved with pretreatment of cellulase and pectinase enzymes. The combined enzymatic extraction amplified total extractives up to 76% in GTE and 72% in BTE. Total polyphenol was found to be enhanced to 24% in GTE and 19% in BTE. GTE was further selected for development of RTD in two different formats; synbiotic RTD and prebiotic RTD premix and analyzed for sensory attributes (colour, aroma, taste, and acceptability). Synbiotic RTD was also evaluated for stability over a period of 28 days at 4 °C. Synbiotic RTD developed an unpleasant flavor and aroma during the shelf life. Premix format of RTD developed using spray drying was reconstituted and found to be functionally and sensorially acceptable.

Strategically Designed Antifibrotic Gold Nanoparticles to Prevent Collagen Fibril Formation.

Because uncontrolled accumulation of collagen fibrils has been implicated in a series of pathologies, inhibition of collagen fibril formation has become one of the necessary strategies to target such collagen-linked complications. The presence of hydroxyproline (Hyp) at the Y position in (Gly-X-Y)n sequence pattern of collagen is known to facilitate crucial hydrophobic and hydration-linked interactions that promote collagen fibril formation. Here, to target such Hyp-mediated interactions, we have synthesized uniform, thermostable, and hemocompatible Hyp coated gold nanoparticles (AuNPsHYP) and have examined their inhibition effect on the fibril formation of type I collagen. We found that collagen fibril formation is strongly suppressed in the presence of AuNPsHYP and no such suppression effect was observed in the presence of free Hyp and control Gly-coated nanoparticles at similar concentrations. Both isothermal titration calorimetric studies and bioinformatics analysis reveal possible interaction between Hyp and (Gly-Pro-Hyp) stretches of collagen triple-helical model peptides. Further, gold nanoparticles coated with proline (AuNPsPRO) and tryptophan (AuNPsTRP) also suppressed collagen fibril formation, suggesting their ability to interfere with aromatic-proline as well as hydrophobic interactions between collagen molecules. The Hyp molecules, when surface functionalized, are predicted to interfere with the Hyp-mediated forces that drive collagen self-assembly, and such inhibition effect may help in targeting collagen linked 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.

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.

Capsaicin inhibits collagen fibril formation and increases the stability of collagen fibers.

Capsaicin is a versatile plant product which has been ascribed several health benefits and anti-inflammatory and analgesic properties. We have investigated the effect of capsaicin on the molecular stability, self-assembly, and fibril stability of type-I collagen. It was found that capsaicin suppresses collagen fibril formation, increases the stability of collagen fibers in tendons, and has no effect on the molecular stability of collagen. Turbidity assay data show that capsaicin does not promote disassembly of collagen fibrils. However, capsaicin moderately protects collagen fibrils from enzymatic degradation. Computational studies revealed the functions of the aromatic group and amide region of capsaicin in the collagen-capsaicin interaction. The results may have significant implications for capsaicin-based therapeutics that target excess collagen accumulation-linked pathology, for example thrombosis, fibrosis, and sclerosis.

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.

Type I collagen prevents amyloid aggregation of hen egg white lysozyme.

Both collagen and amyloidogenic proteins have an inherent ability to undergo a self-assembly process leading to formation of supramolecular structures. Though our understanding of collagen-amyloid link is very poor, a few experimental evidences have indicated the protective nature of collagen against amyloid fibril formation. To further our understanding of collagen-amyloid relationship, we have explored the role of type I collagen on amyloid-aggregation of lysozyme. Thioflavin-T assay data indicated strong inhibition of both spontaneous and seeded aggregation of lysozyme by collagen. Both chemical and thermal denaturation experiments have showed increased lysozyme stability in the presence of collagen. However, the presence of collagen did not alter lysozyme activity. These findings confirm that type I collagen is capable of blocking or interfering with the amyloid aggregation of lysozyme, and the results may have significant implications for the design of collagen based therapeutics against aggregation of disease linked amyloidogenic proteins.

Investigation of potential rhizospheric isolate for cypermethrin degradation.

Rhizoremediation is the use of plant-microbe interaction for the enhanced degradation of contaminants. Rhizosphere bioremediation of pyrethroid pesticides will offer an attractive and potentially inexpensive approach for remediation of contaminated soil. The present study was done with the aim of establishment of highly effective remediation method using plant with degradative rhizosphere and isolation of naturally occurring rhizosphere associated potential degrader providing the possibility of both environmental and insitu detoxification of cypermethrin contamination. The remediation efficacy of Pennisetum pedicellatum was investigated using green house pot culture experiments in cypermethrin amended potting soil mix (10, 25, 50, 75 and 100 mg/kg) for periodic evaluation of changes in concentration. Total proportion of cypermethrin degraders was found to be higher in rhizosphere soil compared to bulk soil. The cypermethrin degrading strain associated with rhizosphere capable of surviving at higher concentrations of cypermethrin was designated as potential degrader. On the basis of morphological characteristics, biochemical tests and 16S rDNA analysis, isolate was identified as Stenotrophomonas maltophilia MHF ENV 22. Bioremediation data of cypermethrin by strain MHF ENV22 examined by HPLC and mass spectroscopy, indicated 100, 50 and 58 % degradation within the time period of 72, 24 and 192 h at concentrations 25, 50 and 100 mg/kg, respectively. This is the first report of effective degradation of cypermethrin by Stenotrophomonas spp. isolated from rhizosphere of Pennisetum pedicellatum. Rhizoremediation strategy will be of immense importance in remediation of cypermethrin residues to a level permissible for technogenic and natural environment.

Chlorpyrifos bioremediation in Pennisetum rhizosphere by a novel potential degrader Stenotrophomonas maltophilia MHF ENV20.

Rhizoremediation is a specific type of phytoremediation involving both plants and their rhizosphere associated microbes. In the present study Pennisetum pedicellatum and rhizosphere associated degrading strains were evaluated for chlorpyrifos remediation. Time-course pot experiments were conducted in greenhouse with P. pedicellatum grown in soil amended with chlorpyrifos at the concentrations of 10, 25, 50, 75 and 100 mg/kg for 60 days. The half life of chlorpyrifos varied from 19.25 to 13.02 days in planted treatments. Residual concentrations of chlorpyrifos were negatively correlated with abundance of degrading microorganisms in rhizosphere. The isolated species of Bacillus, Rhodococcus and Stenotrophomonas were evaluated for their degrading potential in mineral medium. A novel isolated strain of potential degrader Stenotrophomonas maltophilia named as MHF ENV20 showed better survival and degradation at high concentration of chlorpyrifos. Degradation of chlorpyrifos by strain MHF ENV20, 100, 50 and 33.3% degradation within the time period of 48 h (h), 72 and 120 h at 50,100 and 150 mg/kg concentrations, further the gene encoding the organophosphorous hydrolase (mpd) was amplified using PCR amplification strategy and predesigned primers. Our findings indicate that rhizosphere remediation is effective bioremediation technique to remove chlorpyrifos residues from soil. P. pedicellatum itself, in addition to the rhizosphere bacterial consortium, seemed to play an important role in reducing chlorpyrifos level in soil. High chlorpyrifos tolerance and rhizospheric degradation capability of P. pedicellatum, makes this plant suitable for decontamination and remediation of contaminated sites. The ability to survive at higher concentration of chlorpyrifos and enhanced degrading potential due to presence of mpd gene make S. maltophilia MHF ENV20 an ideal candidate for its application in chlorpyrifos remediation.