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Conventional antifungal treatments against Candida albicans in the oral cavity often result in increased cytotoxicity. The goal of this study was to determine the potential of starch Pickering emulsion as a delivery vehicle for an antifungal natural phenolic compound such as thymol in simulated saliva fluid (SSF) compared to amphotericin B. An oil-in-water (o/w) emulsion was stabilized using starch particles. Physical stability of the emulsion and disruption induced by α-amylase activity in SSF was evaluated. Encapsulated thymol in o/w emulsion was compared to encapsulated amphotericin B for antifungal activity against C. albicans in suspension using emulsions or zone inhibition assay on agar plates using emulsions dispersed in alginate films. Results showed that the emulsions were stable for at least three weeks. Digestion of the emulsion by α-amylase led to coalescence of emulsion droplets. The antifungal activity of thymol and amphotericin B in emulsion formulation was enhanced upon incubation with α-amylase. Results from the zone inhibition assay demonstrated efficacy of the emulsions dispersed in alginate films. Interestingly, addition of α-amylase to the alginate films resulted in a decreased inhibitory effect. Overall, this study showed that starch Pickering emulsions have a potential to deliver hydrophobic antifungal compounds to treat oral candidiasis. [ABSTRACT FROM AUTHOR]

Background: Overexpression and abnormal accumulation of aggregated a-synuclein (aS) have been linked to Parkinson's disease (PD) and other synucleinopathies. aS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase aS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models. Results: Here we show that curcumin can alleviate aS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of aS and extracellular addition of oligomeric aS increase ROS which induces apoptosis, suggesting that aggregated aS may induce similar toxic effects whether it is generated intra- or extracellulary. Conclusions: Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Abstract: The presence of neurofibrillary tangles (NFTs) is a hallmark feature of various neurodegenerative disorders including Alzheimer’s (AD) and Niemann-Pick type C (NPC) diseases. NFTs have been correlated with elevated cholesterol levels and a cholesterol-scavenging compound, cyclodextrin, effectively modulates and traffics cholesterol from cell bodies in NPC disease models. Cyclodextrins are also used as drug carriers to the blood–brain barrier (BBB) and other tissues. While cyclodextrins have potential value in treating brain diseases, it is important to determine how cyclodextrins affect natively unfolded proteins such as beta-amyloid (Aβ) whose aggregation has been correlated with AD. We show that cyclodextrins drastically alter Aβ aggregation kinetics and induce morphological changes to Aβ that can enhance toxicity towards SH-SY5Y human neuroblastoma cells. These results suggest that care must be taken when using cyclodextrins for BBB delivery or for treatment of brain disease because cyclodextrins can promote toxic aggregation of Aβ. [Copyright &y& Elsevier]

Abstract: The distal to proximal degeneration of axons, or “dying back” is a common pattern of neuropathology in many diseases of the PNS and CNS. A long-standing debate has centered on whether this pattern of neurodegeneration is due to an insult to the cell body or to the axon itself, although it is likely that mechanisms are different for specific disease entities. We have addressed this question in a model system of vincristine-induced axonal degeneration. Here, we created a novel experimental apparatus combining a microfluidic divider with a multielectrode array substrate to allow for independent monitoring of injury-induced electrical activity from dorsal root ganglion (DRG) cell bodies and axons while isolating them into their own culture microenvironments. At specified doses, exposure of the cell body to vincristine caused neither morphological neurodegeneration nor persistent hyperexcitablility. In comparison, exposure of the distal axon to the same dose of vincristine first caused a decrease in the excitability of the axon and then axonal degeneration in a dying back pattern. Additionally, exposure of axons to vincristine caused an initial period of hyperexcitability in the cell bodies, suggesting that a signal is transmitted from the distal axon to the soma during the progression of vincristine-induced axonal degeneration. These data support the proposition that vincristine has a direct neurotoxic effect on the axon. [Copyright &y& Elsevier]

Abstract: This paper describes a microfabricated compartmented culture system (μ-CCS) for studying the effects of drugs on cultured neurons. We describe the fabrication of the microsystem and show the ability to culture DRG neurons in the microsystem. Furthermore, we demonstrate the ability to culture neurons with extensions growing into adjoining compartments while maintaining fluid isolation between compartments. The axonal growth pattern was controlled along the surface of the glass microelectrode substrate using a micropatterned collagen film. The ability to isolate fluids to selected compartments while simultaneously allowing intercompartmental growth of the axons enables various studies in which selected segments of neurons or populations of neurons can be selectively exposed to biochemical treatment. The neurotoxin vincristine was used as the test vehicle to demonstrate the functionality of the μ-CCS. Vincristine was applied to the axonal compartment to show that the interaction of drugs with DRG neurons progresses in a way similar to neurons cultured/exposed using conventional techniques. [Copyright &y& Elsevier]

Abstract: A simple method to generate cell microarrays with high-percentage well occupancy and well-defined cell confinement is presented. This method uses a synergistic combination of vacuum degassing and coverslip sweeping. The vacuum degassing step dislodges air bubbles from the microwells, which in turn enables the cells to enter the microwells, while the physical sweeping step using a glass coverslip removes the excess cells outside the microwells. This low-cost preparation method provides a simple solution to generating cell microarrays that can be performed in basic research laboratories and point-of-care settings for routine cell-based screening assays. [Copyright &y& Elsevier]

Background Oxidation of encapsulated bioactive compounds is a key challenge that limits shelf-life of bioactive containing products. The objectives of this study were to compare differences between the oxidative barrier properties of biopolymer particle based encapsulation system (zein colloidal particles) and oil-in-water emulsions and evaluate the impact of these differences on oxidative stability of encapsulated bioactives. Methods Both zein colloidal particles and oil-in-water emulsions were stabilized by casein protein. The oxidative barrier properties of the selected encapsulation systems were determined by measuring the permeation rate of peroxyl radicals and oxygen across the interface. Peroxyl radical permeation rates were correlated with stability of a model bioactive, curcumin and oxygen permeation rates were correlated with stability of another model bioactive, retinol. Results Radical permeation rate was significantly higher in oil-in-water emulsions compared to zein colloidal particles, indicating enhanced barrier property of zein colloidal particles against peroxyl radical induced oxidation. Consistent with these results, stability of curcumin encapsulated in zein colloidal particles was significantly higher compared to that in oil-in-water emulsions. Oxygen permeation measurements showed no significant differences in the barrier properties of both encapsulation systems against oxygen permeation. Consistent with these results, the oxidative stability of retinol was similar in both encapsulation systems. Conclusions The results of this study demonstrate the advantages of biopolymer particle based encapsulation system in limiting free radical induced oxidation of encapsulated bioactives and also demonstrate the ineffectiveness of both encapsulation systems in limiting oxygen permeation. [ABSTRACT FROM AUTHOR]

Background: Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely a-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes. Methodology/Principal Findings: Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of α-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains. Conclusions/Significance: These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases. [ABSTRACT FROM AUTHOR]

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SYSY), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue. [ABSTRACT FROM AUTHOR]

Abstract: Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of α-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson''s Disease (PD). A natively unfolded protein, α-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic. Antibodies that neutralize the neurotoxic aggregates without interfering with beneficial functions of monomeric α-synuclein can be useful therapeutics. We were able to isolate single chain antibody fragments (scFvs) from a phage displayed antibody library against the target antigen morphology using a novel biopanning technique that utilizes atomic force microscopy (AFM) to image and immobilize specific morphologies of α-synuclein. The scFv described here binds only to an oligomeric form of α-synuclein and inhibits both aggregation and toxicity of α-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of α-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody. [Copyright &y& Elsevier]

Alzheimer's disease (AD) is characterized by the deposition of amyloid-β (Aβ) protein in the brain. Immunization studies have demonstrated that anti-Aft antibodies reduce Aft deposition and improve clinical symptoms seen in AD. However, conventional antibody-based therapies risk an inflammatory response that can result in meningoencephalitis and cerebral hemorrhage. Here we report on the development of human-based single chain variable domain antibody fragments (scFvs) directed against the Aβ 25–35 region as potential therapeutics for AD that do not risk an inflammatory response. The 25–35 region of Aft represents a promising therapeutic target since it promotes aggregation and is highly toxic. Two scFvs with differing affinities for Aft were studied, and both inhibited aggregation of Aβ42 as determined by thioflavin T binding assay and atomic force microscopy analysis and blocked Aft-induced toxicity toward human neuroblastoma SH-SY5Y cells as determined by MTT and LDH release assays. These results provide additional evidence that scFvs against Aβ provide an attractive alternative to more conventional antibody-based therapeutics for controlling aggregation and toxicity of Aβ. [ABSTRACT FROM AUTHOR]

Background: Overexpression and abnormal accumulation of aggregated alpha-synuclein (alphaS) have been linked to Parkinson's disease (PD) and other synucleinopathies. alphaS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase alphaS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models.Results: Here we show that curcumin can alleviate alphaS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of alphaS and extracellular addition of oligomeric alphaS increase ROS which induces apoptosis, suggesting that aggregated alphaS may induce similar toxic effects whether it is generated intra- or extracellulary.Conclusions: Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]