Surface Functionalization of Gold Nanorods Improves Nanostructure Assemblies on Amyloid Fibril Scaffolds.

A hybrid scaffold containing gold nanorods and lysozyme amyloid fibrils has been fabricated, and the effect of surface modification on improving nanostructure assembly on the biological template has been investigated. The nanohybrid system was characterized by monitoring surface plasmon resonance bands, dynamic light scattering spectroscopy, Thioflavin-T assay, and transmission electron microscopy. Surface of gold nanorods (GNRs) was modified with polystyrene sulfonate (PSS), and possible difference in assembly of the pristine and modified nanostructures was compared upon interaction with amyloid fibrils. Analysis of transmission electron microscopy showed that changing the surface charge of GNRs with biocompatible polymer improved electrostatic interactions between the nanostructures and amyloid fibril templates. Analysis of cell viability assays also showed that surface functionalization of GNRs remarkably improved biocompatibility of the nanoscaffold. Results of this study encourage utilization of modification strategies to fabricate a new generation of nanoscaffolds with fruitful applications in regenerative medicine.

Antiviral effects of nisin, lysozyme, lactoferrin and their mixtures against bovine viral diarrhoea virus.

BACKGROUND: Bovine viral diarrhoea virus (BVDV), an enveloped, single-stranded, positive-sense RNA virus from the Flaviviridae family, is a globally distributed bovine pathogen. BVDV infection in cattle, despite having a wide range of clinical manifestations, is invariably responsible for significant economic losses. To counteract these losses, various schemes to control and eradicate BVDV have been implemented, although safe drugs effectively inhibiting the replication of the virus are still lacking. The purpose of this study was to characterize the antiviral effect of naturally occurring proteins and peptide, such as bovine lactoferrin, chicken egg lysozyme, and nisin from Lactococcus lactis, used both individually and in combination, against the cytopathic NADL strain of BVDV in vitro. After determining the cytotoxicity level of each protein or peptide to MDBK cells, its antiviral effects were evaluated using virucidal, cytopathic effect inhibition and viral yield reduction assays. In addition, the influence of the tested compounds on the intracellular viral RNA level was determined. RESULTS: The highest efficacy among the single treatments was achieved by bovine lactoferrin, which was effective both at the early stages of viral infection and during its entire course, although the effect weakened over time. Nisin and lysozyme were effective at later stages of infection, and the intensity of their effect did not diminish with time. Nisin+lactoferrin and lysozyme+lactoferrin combinations demonstrated stronger antiviral effects than did the single substances. The nisin+lactoferrin mixture present during the whole period of infection produced the strongest anti-BVDV effect in our entire research on both the extracellular viral titre (titre reduction up to 2.875 log ≈ 99.9%) and the intracellular viral RNA level (reduction up to 89%), and this effect intensified over the incubation time. CONCLUSIONS: The tested substances could be applied in bovine viral diarrhoea prevention and therapy, especially when used in combination.

Exploring the influence of brilliant blue G on amyloid fibril formation of lysozyme.

Evidence suggests that amyloid fibril mitigation/inhibition is considered a promising approach toward treating amyloid diseases. In this work, we first examined how amyloid fibrillogenesis of lysozyme was affected by BBG, a safe triphenylmethane compound with nice blood-brain-barrier-permeability, and found that shorter fibrillar species were formed in the lysozyme samples treated with BBG. Next, alterations in the features including the secondary as well as tertiary structure, extent of aggregation, and molecular distribution of lysozyme triggered by the addition of BBG were examined by various spectroscopic techniques, right-angle light scattering, dynamic light scattering, and SDS-PAGE. In addition, we have investigated how BBG affected the lysozyme fibril-induced cytotoxicity in SH-SY5Y cells. We found that a large quantity of shorter fibrillar species and more lysozyme monomers were present in the samples treated with BBG. Also, the addition of BBG rescued SH-SY5Y cells from cell death induced by amyloid fibrils of lysozyme. Finally, information about the binding sites and interacting forces involved in the BBG-lysozyme interaction was further explored using synchronous fluorescence and molecular docking approaches. Molecular docking results revealed that, apart from the hydrophobic interaction(s), hydrogen bonding, electrostatic interactions, and van der Waal forces may also be involved in the binding interaction.

The hepatic-targeted, resveratrol loaded nanoparticles for relief of high fat diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the early stage of many metabolic syndromes. The intervention of NAFLD can prevent its further development into severe metabolic syndromes. Given the inefficiency and side effects of chemical drugs for treating NAFLD, the hepatic-targeted nanocarriers loaded with bioactive compounds may offer a more effective and acceptable strategy for eliminating NAFLD. Here we developed hepatic-targeted oxidized starch-lysozyme (OSL) nanocarriers to specifically deliver resveratrol (Res) to liver tissue in order to maximize its therapeutic efficiency. The hepatic targeting was achieved using covalently conjugated galactose (Gal), which is recognized by the asialoglycoprotein receptors specifically expressed in hepatocytes. In steatotic HepG2 cell model, treatment with hepatic-targeted Gal-OSL/Res nanocarriers enhanced the cellular Res uptake and anti-lipogenesis capabilities, and effectively decreased triglyceride accumulation by modulating AMP-activated protein kinase (AMPK)/silent information regulation 2 homolog 1(SIRT1)/fatty acid synthase (FAS)/sterol regulatory element-binding protein-1c (SREBP1c) signaling pathway. In mice, Gal-OSL increased Res delivery into liver tissues and increased their hepatic effective concentration in liver. Most importantly, Gal-OSL/Res nanocarriers effectively reversed NAFLD and recovered hepatic insulin sensitivity of NAFLD mice to the healthy state. Furthermore, Gal-OSL/Res efficiently ameliorated lipid deposition and insulin resistance by modulating AMPK/SIRT1/FAS/SREBP1c signaling pathway and downregulated insulin receptor substrate-1 (IRS-1) phosphorylation at serine 307 in liver. These findings suggested that the hepatic-targeted Gal-OSL nanocarriers delivering Res could potentially serve as a safe and promising platform for NAFLD and other liver related diseases.

Effect of fibrillation conditions on the anti-amyloidogenic properties of polyphenols and their involved mechanisms.

In the present study, we have investigated the effects of protein concentration and stirring on the in vitro assembly of Hen Egg White Lysozyme (HEWL), particularly with regard to the aggregate morphology and anti-amyloidogenic properties of two naturally occurring polyphenols, taxifolin and silibinin. The results obtained clearly demonstrated that applying stirring and concentration enhancement alter the amount as well as morphology of amyloid fibrils formed. Additionally, latter aggregates exhibited higher affinity for amyloid-specific dyes. The second part of the present investigation was devoted to studies involving anti-amyloidogenic properties of selected polyphenols. Importantly, we found that the potency of polyphenols to inhibit HEWL amyloid fibrillation and related toxicity is strongly dependent on the amyloidogenic conditions in which amyloid fibrils are produced. Based on obtained data, under condition where the rate of protein assembly is high (higher protein concentration and stirring), the capacity of polyphenols to inhibit HEWL fibrillogenesis and related cytotoxicity may dramatically decrease. Similar results were obtained when we used taxifolin to inhibit bovine insulin amyloid fibrillation. Additionally, amyloidogenic conditions may also affect the mechanism by which these molecules inhibit HEWL fibrillation. The possible mechanism by which selected polyphenols exert their inhibitory effects, under various experimental conditions, is also discussed.

Effects of dissolving microneedle fabrication parameters on the activity of encapsulated lysozyme.

Dissolving microneedle (DMN) is referred to a microscale needle that encapsulates drug(s) within a biodegradable polymer matrix and delivers it into the skin in a minimally invasive manner. Although vast majority of studies have emphasized DMN as an efficient drug delivery system, the activity of DMN-encapsulated proteins or antigens can be significantly affected due to a series of thermal, physical and chemical stress factors during DMN fabrication process and storage period. The objective of this study is to evaluate the effects of DMN fabrication parameters including polymer type, polymer concentration, fabrication and storage temperature, and drying conditions on the activity of the encapsulated therapeutic proteins by employing lysozyme (LYS) as a model protein. Our results indicate that a combination of low temperature fabrication, mild drying condition, specific polymer concentration, and addition of protein stabilizer can maintain the activity of encapsulated LYS up to 99.8 ±â€¯3.8%. Overall, findings of this study highlight the importance of optimizing DMN fabrication parameters and paves way for the commercialization of an efficient delivery system for therapeutics.

Safety evaluation of a novel muramidase for feed application.

Safety evaluation of a muramidase produced by a Trichoderma reesei strain (safe lineage), expressing a muramidase gene isolated from Acremonium alcalophilum is presented. Intended use in feed of this enzyme is as digestive aid in broiler chickens. Muramidase 007, was non-mutagenic and non-clastogenic in vitro, and no adverse effects were observed in 90-day subchronic toxicity studies in rats at doses up to 1132 mg TOS/kg body weight/day. The enzyme did not exhibit, in vitro, skin, nor eye irritation potential. Acute aquatic toxicity evaluated on daphnia and algae showed absence of effect of the enzyme at the standard doses tested. Muramidase 007 was fully tolerated by broiler chickens in a 6-weeks tolerance study showing no adverse effects in any of the dietary treatments (0, 1×, 5× and 10× maximum recommended dose). In conclusion, Muramidase 007 is found to be toxicologically inert, and there are no worker's safety concerns if standard precautions are instituted and a non-dusty formulation is employed. Muramidase 007 is well tolerated by the target species (broiler chickens) and cause no harm to the environment. The beneficial safety evaluation of Muramidase 007 is in line with this type of enzyme that is found ubiquitously in nature.

Analysis of Toxic Amyloid Fibril Interactions at Natively Derived Membranes by Ellipsometry.

There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity.

Oligomers, protofibrils and amyloid fibrils from recombinant human lysozyme (rHL): fibrillation process and cytotoxicity evaluation for ARPE-19 cell line.

Amyloid-associated diseases, such Alzheimer's, Huntington's, Parkinson's, and type II diabetes, are related to protein misfolding and aggregation. Herein, the time evolution of scattered light intensity, hydrophobic properties, and conformational changes during fibrillation processes of rHL solutions at 55 °C and pH 2.0 were used to monitor the aggregation process of recombinant human lysozyme (rHL). Dynamic light scattering (DLS), thioflavin T (ThT) fluorescence, and surface tension (ST) at the air-water interface were used to analyze the hydrophobic properties of pre-amyloid aggregates involved in the fibrillation process of rHL to find a correlation between the hydrophobic character of oligomers, protofibrils and amyloid aggregates with the gain in cross-ß-sheet structure, depending on the increase in the incubation periods. The ability of the different aggregates of rHL isolated during the fibrillation process to be adsorbed at the air-water interface can provide important information about the hydrophobic properties of the protein, which can be related to changes in the secondary structure of rHL, resulting in cytotoxic or non-cytotoxic species. Thus, we evaluated the cytotoxic effect of oligomers, protofibrils and amyloid fibrils on the cell line ARPE-19 using the MTT reduction test. The more cytotoxic protein species arose after a 600-min incubation time, suggesting that the hydrophobic character of pre-amyloid fibrils, in addition to the high prevalence of the cross-ß-sheet conformation, can become toxic for the cell line ARPE-19.

Protein microgels from amyloid fibril networks.

Nanofibrillar forms of proteins were initially recognized in the context of pathology, but more recently have been discovered in a range of functional roles in nature, including as active catalytic scaffolds and bacterial coatings. Here we show that protein nanofibrils can be used to form the basis of monodisperse microgels and gel shells composed of naturally occurring proteins. We explore the potential of these protein microgels to act as drug carrier agents, and demonstrate the controlled release of four different encapsulated drug-like small molecules, as well as the component proteins themselves. Furthermore, we show that protein nanofibril self-assembly can continue after the initial formation of the microgel particles, and that this process results in active materials with network densities that can be modulated in situ. We demonstrate that these materials are nontoxic to human cells and that they can be used to enhance the efficacy of antibiotics relative to delivery in homogeneous solution. Because of the biocompatibility and biodegradability of natural proteins used in the fabrication of the microgels, as well as their ability to control the release of small molecules and biopolymers, protein nanofibril microgels represent a promising class of functional artificial multiscale materials generated from natural building blocks.

Polymorphism of hen egg white lysozyme amyloid fibrils influences the cytotoxicity in LLC-PK1 epithelial kidney cells.

The polymorphism of amyloid fibrils is potentially crucial as it may underlie the natural variability of amyloid diseases and could be important in developing a fuller understanding of the molecular basis of protein deposition disorders. This study examines morphological differences in lysozyme fibrils and the implications of these differences in terms of cytotoxicity. The structural characteristics of amyloid fibrils formed under two different experimental conditions (acidic and neutral) were evaluated using spectroscopic methods, atomic force microscopy and image analysis. Growth curves and apoptotic/necrotic assays were used to determine the cytotoxic effect of fibrils on the LLC-PK1 renal cells. The results reveal that both types of mature lysozyme amyloid fibrils are actively involved in the cytotoxic process, however each exhibit different levels of cytotoxicity. Fibrils formed at acidic pH affect cell growth in a dose-dependent manner, but a threshold-dependent inhibition of cell growth was observed in the case of lysozyme fibrils prepared at neutral pH. Experiments examining the mechanism of the cell death suggest that both types of mature lysozyme fibrils trigger late apoptosis/necrosis at different fibril concentrations. Our findings clearly indicate that the intrinsic differences between amyloid fibrils due to their polymorphism result in different degrees of cytotoxicity.

Carnosine's effect on amyloid fibril formation and induced cytotoxicity of lysozyme.

Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.

Mixed protein-DNA gel particles for DNA delivery: role of protein composition and preparation method on biocompatibility.

Mixtures of two cationic proteins were used to prepare protein-DNA gel particles, employing associative phase separation and interfacial diffusion (Morán et al., 2009a). By mixing the two proteins, we have obtained particles that displayed higher loading efficiency and loading capacity values than those obtained in single-protein systems. However, nothing is known about the adverse effects on haemocompatibility and cytotoxicity of these protein-DNA gel particles. Here, we examined the interaction of protein-DNA gel particles obtained by two different preparation methods, and their components, with red blood cells and established cells. From a haemolytic point of view, these protein-DNA gel particles were demonstrated to be promising long-term blood-contacting medical devices. Safety evaluation with the established cell lines revealed that, in comparison with proteins in solution, the cytotoxicity was reduced when administered in the protein-DNA systems. In comparison with large-sized particles, the cytotoxic responses of small-sized protein-DNA gel particles showed to be strongly dependent of both the protein composition and the cell line being the tumour cell line HeLa more sensitive to the deleterious effects of the mixed protein-based particles. The observed trends in haemolysis and cell viabilities were in agreement with the degree of complexation values obtained for the protein-DNA gel particles prepared by both preparation methods.

Antigen-specific regulation of IgE antibodies by non-antigen-specific γδ T cells.

We re-examined the observation that γδ T cells, when transferred from mice tolerized to an inhaled conventional Ag, suppress the allergic IgE response to this Ag specifically. Using OVA and hen egg lysozyme in crisscross fashion, we confirmed the Ag-specific IgE-regulatory effect of the γδ T cells. Although only Vγ4(+) γδ T cells are regulators, the Ag specificity does not stem from specificity of their γδ TCRs. Instead, the Vγ4(+) γδ T cells failed to respond to either Ag, but rapidly acquired Ag-specific regulatory function in vivo following i.v. injection of non-T cells derived from the spleen of Ag-tolerized mice. This correlated with their in vivo Ag acquisition from i.v. injected Ag-loaded splenic non-T cells, and in vivo transfer of membrane label provided evidence for direct contact between the injected splenic non-T cells and the Vγ4(+) γδ T cells. Together, our data suggest that Ag itself, when acquired by γδ T cells, directs the specificity of their IgE suppression.

The non-core regions of human lysozyme amyloid fibrils influence cytotoxicity.

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-ß core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.

28-Day repeated dose oral toxicity of recombinant human apo-lactoferrin or recombinant human lysozyme in rats.

Lactoferrin and lysozyme are important proteins of the human innate immune system. These proteins are found in breast milk and have been associated with improved infant health. Recombinant human apo-lactoferrin (apo-rhLF), 1800 and 180mg/kg bw/day, and recombinant human lysozyme (rhLZ), 360 and 36mg/kg bw/day, were orally administered to Wistar rats for 28 days. Apo-rhLF and rhLZ were expressed in rice grain, extracted, purified; the lactoferrin was iron desaturated. The animals were examined for evidence of toxicity; there were no deaths and in-life physical signs were normal. Transient differences in mean food consumption occurred in high dose apo-rhLF and low dose LZ females at week three. There were no biologically significant differences in hematological or clinical chemistry parameters. Necropsy results were normal and microscopic evaluation showed no treatment related changes in animals dosed with 1800mg/kg/day apo-rhLF or 360mg/kg/day rhLZ. The results of the 28-day oral administration demonstrate a lack of toxicity of apo-rhLF and rhLZ in rats. There were no treatment related, toxicologically relevant changes in clinical signs, growth, food consumption, hematology, clinical chemistry, organ weight and pathology. The no observed adverse effect level (NOAEL) is greater than 1800mg/kg/day for apo-rhLF and 360mg/kg/day for rhLZ.

The toxicity study on marine low-temperature lysozyme.

Marine low-temperature lysozyme is purified from a marine bacterium. The lysozyme can keep high activity at low-temperature and has broad-spectrum antibiotic reaction. This study was undertaken to investigate the major characteristics, acute and subchronic toxicity of marine low-temperature lysozyme. The relative molecular weight of this lysozyme was determined as approximate 16 kD; its optimum pH value and temperature towards Micrococcus lysodleikticus were pH 6.5 and 35 degrees C, respectively. The lysozyme activity was slightly enhanced by Zn(2+) and Cu(2+) and slightly inhibited by Mn(2+) and Ag(+). The lysozyme showed good compatibility to many common chemical agents such as EDTA (0.1%), KH(2)PO(4) (1.0%), etc. In experiments on acute toxicity, the drug was injected through the tail vein of mice, and intoxication symptoms and date of death were recorded. The 50% lethal dose (LD(50)) of Marine low-temperature lysozyme and 95%, 99% confidence interval (CI) was calculated. The subchronic study was designed to determine whether effects progressed with repeated Marine low-temperature lysozyme exposure. Wistar rats were tested by daily intragastric administration of Marine low-temperature lysozyme at the doses of 1.0; 0.5; 0.25 g/kg bw for 90 days. The LD(50) value of lysozyme was 4530 mg/kg bw; 90 days of Marine low-temperature lysozyme treatment at three doses, and there is no significant difference on blood biochemistry and organ index in drug treatment groups compared to saline treatment group. There is no affirmative pathologic change of all the observed organs in this study. The present results suggest that Marine low-temperature lysozyme can be safely used at the dose of experiment applied.

Soluble oligomers from a non-disease related protein mimic Abeta-induced tau hyperphosphorylation and neurodegeneration.

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.

The anti-proliferative effects of recombinant human lysozyme on human gastric cancer cells.

We investigated the anti-proliferative effects of recombinant human lysozyme (rHlys) on gastric cancer cell lines and normal human lung fibroblasts. Using conventional molecular cloning techniques we purified rHlys, which we incubated with cultured cells and measured the effects on cell proliferation and viability. At concentrations of 100 and 1000 microg/l, rHlys significantly inhibited the growth of human gastric cancer cell lines. In contrast, 10 and 50 microg/l of rHlys stimulated gastric cancer cell growth. None of the concentrations of rHlys affected cell viability. Only the highest concentration of rHlys (1000 microg/l) inhibited human lung fibroblast growth. Our results suggest that 100 microg/l is the optimum growth inhibiting concentration, which inhibited cancer cell growth but not normal cell growth. Our in vitro findings suggest that genetically engineered rHlys might inhibit human gastric cancer cell proliferation in vivo, so it might warrant further investigation as a potential novel anti-cancer agent.