Small molecule induced toxic human-IAPP species characterized by NMR.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective, which is reflected in a strong interference with hIAPP aggregation while showing weaker interactions with human-calcitonin and amyloid-ß1-40 in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.

High-Throughput Screening at the Membrane Interface Reveals Inhibitors of Amyloid-ß.

Aggregation and the formation of oligomeric intermediates of amyloid-ß (Aß) at the membrane interface of neuronal cells are implicated in the cellular toxicity and pathology of Alzheimer's disease. Small molecule compounds have been shown to suppress amyloid aggregation and cellular toxicity, but often the presence of a lipid membrane negates their activity. A high-throughput screen of 1800 small molecules was performed to search for membrane active inhibitors, and 21 primary hits were discovered. Through the use of fluorescence-based assays, transmission electron microscopy, and dot blot assays, the initial 21 primary hits were narrowed down to five lead compounds. Nuclear magnetic resonance and circular dichroism experiments were used for further confirmation of amyloid inhibition at the membrane interface and to obtain insights into the secondary structure of amyloid-ß, while size exclusion chromatography was used to characterize the size of Aß species. Lastly, dye-leakage assays allowed us to understand how the addition of the five lead compounds affected amyloid-ß's ability to permeate the lipid bilayer. These results provide insights into small molecules that stabilize small amyloid species in the presence of membranes for the development of tool compounds for deeper investigations of these transient species.

High-resolution probing of early events in amyloid-ß aggregation related to Alzheimer's disease.

In Alzheimer's disease (AD), soluble oligomers of amyloid-ß (Aß) are emerging as a crucial entity in driving disease progression as compared to insoluble amyloid deposits. The lacuna in establishing the structure to function relationship for Aß oligomers prevents the development of an effective treatment for AD. While the transient and heterogeneous properties of Aß oligomers impose many challenges for structural investigation, an effective use of a combination of NMR techniques has successfully identified and characterized them at atomic-resolution. Here, we review the successful utilization of solution and solid-state NMR techniques to probe the aggregation and structures of small and large oligomers of Aß. Biophysical studies utilizing the commonly used solution and 19F based NMR experiments to identify the formation of small size early intermediates and to obtain their structures, and dock-lock mechanism of fiber growth at atomic-resolution are discussed. In addition, the use of proton-detected magic angle spinning (MAS) solid-state NMR experiments to obtain high-resolution insights into the aggregation pathways and structures of large oligomers and other aggregates is also presented. We expect these NMR based studies to be valuable for real-time monitoring of the depletion of monomers and the formation of toxic oligomers and high-order aggregates under a variety of conditions, and to solve the high-resolution structures of small and large size oligomers for most amyloid proteins, and therefore to develop inhibitors and drugs.

Zinc boosts EGCG's hIAPP amyloid Inhibition both in solution and membrane.

Amyloid aggregation of human islet amyloid polypeptide (hIAPP) is linked to insulin-producing islet cell death in type II diabetes. Previous studies have shown that zinc (Zn(II)) and insulin, co-secreted with hIAPP, have an inhibition effect on hIAPP aggregation. Lipid membranes have also been shown to significantly influence the aggregation kinetics of hIAPP. An increasing number of studies report the importance of developing small molecule inhibitors to suppress the hIAPP's aggregation and subsequent toxicity. The ability of epigallocatechin-gallate (EGCG) to inhibit aggregation of a variety of amyloid peptide/proteins initiated numerous studies as well as the development of derivative compounds to potentially treat amyloid diseases. In this study, a combination of Thioflavin-T fluorescence kinetics, transmission electron microscopy, isothermal titration calorimetery, circular dicrosim and nucelar magnetic resonance experiments were used to demonstrate a significant enhancement in EGCG's efficiency when complexed with Zn(II). We demonstrate that the Zn-EGCG complex is able to significantly suppress hIAPP's amyloid aggregation both in presence and absence of lipid membrane. Circular dichroism experiments indicate the formation and stabilization of a helical structure of hIAPP in presence of the EGCG:Zn(II) complex. Our results also reveal the ability of EGCG or EGCG:Zn(II) to efficiently suppress hIAPP's cellular toxicity. We believe that the reported results could be useful to develop strategies to trap hIAPP intermediates for further biophysical and structural studies, and also to devise approaches to abolish amyloid aggregation and cellular toxicity.

Alzheimer's amyloid-beta intermediates generated using polymer-nanodiscs.

Polymethacrylate-copolymer (PMA) encased lipid-nanodiscs (∼10 nm) and macro-nanodiscs (>15 nm) are used to study Aß1-40 aggregation. We demonstrate that PMA-nanodiscs form a ternary association with Aß and regulate its aggregation kinetics by trapping intermediates. Results demonstrating the reduced neurotoxicity of nanodisc-bound Aß oligomers are also reported.

Nanodisc-Forming Scaffold Protein Promoted Retardation of Amyloid-Beta Aggregation.

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimer's amyloid-beta (Aß40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aß40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20 µM) and nanodisc size (<10 nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aß40 aggregation and were also found to be suitable to trap Aß40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aß40 conformation upon 4F binding through electrostatic and π-π interactions. Specifically, the 4F peptide was found to interfere with the central ß-sheet-forming residues of Aß40 through substantial hydrogen, π-π, and π-alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aß40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aß40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aß40 intermediates for high-resolution structural and neurobiological studies.

hIAPP forms toxic oligomers in plasma.

In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in ß-sheet rich oligomers with increased ß-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.

Real-time monitoring of the aggregation of Alzheimer's amyloid-ß via1H magic angle spinning NMR spectroscopy.

Proton magic-angle-spinning NMR used for real-time analysis of amyloid aggregation reveals that mechanical rotation of Aß1-40 monomers increases the rate of formation of aggregates, and that the increasing lag-time with peptide concentration suggests the formation of growth-incompetent species. EGCG's ability to shift off-pathway aggregation is also demonstrated.

Formation of pH-Resistant Monodispersed Polymer-Lipid Nanodiscs.

Polymer lipid nanodiscs are an invaluable system for structural and functional studies of membrane proteins in their near-native environment. Despite the recent advances in the development and usage of polymer lipid nanodisc systems, lack of control over size and poor tolerance to pH and divalent metal ions are major limitations for further applications. A facile modification of a low-molecular-weight styrene maleic acid copolymer is demonstrated to form monodispersed lipid bilayer nanodiscs that show ultra-stability towards divalent metal ion concentration over a pH range of 2.5 to 10. The macro-nanodiscs (>20 nm diameter) show magnetic alignment properties that can be exploited for high-resolution structural studies of membrane proteins and amyloid proteins using solid-state NMR techniques. The new polymer, SMA-QA, nanodisc is a robust membrane mimetic tool that offers significant advantages over currently reported nanodisc systems.

Developing a Gel-Based Sensor Using Crystal Morphology Prediction.

The stimuli-responsive nature of molecular gels makes them appealing platforms for sensing. The biggest challenge is in identifying an appropriate gelator for each specific chemical or biological target. Due to the similarities between crystallization and gel formation, we hypothesized that the tools used to predict crystal morphologies could be useful for identifying gelators. Herein, we demonstrate that new gelators can be discovered by focusing on scaffolds with predicted high aspect ratio crystals. Using this morphology prediction method, we identified two promising molecular scaffolds containing lead atoms. Because solvent is largely ignored in morphology prediction but can play a major role in gelation, each scaffold needed to be structurally modified before six new Pb-containing gelators were discovered. One of these new gelators was developed into a robust sensor capable of detecting lead at the U.S. Environmental Protection Agency limit for paint (5000 ppm).

Understanding foot-and-mouth disease virus transmission biology: identification of the indicators of infectiousness.

The control of foot-and-mouth disease virus (FMDV) outbreaks in non-endemic countries relies on the rapid detection and removal of infected animals. In this paper we use the observed relationship between the onset of clinical signs and direct contact transmission of FMDV to identify predictors for the onset of clinical signs and identify possible approaches to preclinical screening in the field. Threshold levels for various virological and immunological variables were determined using Receiver Operating Characteristic (ROC) curve analysis and then tested using generalized linear mixed models to determine their ability to predict the onset of clinical signs. In addition, concordance statistics between qualitative real time PCR test results and virus isolation results were evaluated. For the majority of animals (71%), the onset of clinical signs occurred 3-4 days post infection. The onset of clinical signs was associated with high levels of virus in the blood, oropharyngeal fluid and nasal fluid. Virus is first detectable in the oropharyngeal fluid, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Detection of virus in the air was also significantly associated with transmission. This study is the first to identify statistically significant indicators of infectiousness for FMDV at defined time periods during disease progression in a natural host species. Identifying factors associated with infectiousness will advance our understanding of transmission mechanisms and refine intra-herd and inter-herd disease transmission models.

IL-6 production following vaccination in pigs--an additional immune response parameter for assessing FMD vaccine efficacy?

Foot-and-mouth disease vaccine potency testing involving live virus challenge can be problematical in pigs. Alternative methods of assessing vaccine efficacy are therefore desirable. Here we investigate the link between IL-6 in blood at time of challenge and protection against challenge by carrying out statistical analyses utilising data from six separate potency tests performed in swine with the aim of assessing whether IL-6 could be exploited as an additional parameter for confirming vaccine efficacy in pigs. These analyses confirmed that systemic IL-6 levels increased when the administered vaccine dose increased and that the odds of protection against challenge increased as IL-6 levels increased. The link between increased protection and increased antibody was reaffirmed and a significant link between IL-6 levels and antibody levels was shown. We therefore conclude that quantifying the levels of IL-6 in serum could provide additional means of qualifying whether a vaccine will afford clinical protection or not in pigs, in the absence of an actual challenge, and thus offer the possibility of improved vaccine potency testing in pigs both in terms of animal welfare as well as cost.

Longevity of protection in cattle following immunisation with emergency FMD A22 serotype vaccine from the UK strategic reserve.

To determine the longevity of protective immunity following a single administration of emergency vaccine, and establish whether the immune response could be enhanced by increasing the antigen payload even further, cattle were vaccinated with an A22 Iraq vaccine containing either 1x antigen payload (field dose) or 5x antigen payload. Six months post-immunisation all cattle received a homologous virus challenge. The magnitude of the virus neutralising antibody response elicited was consistent with the response to similarly formulated A serotype vaccines with a PD(50) greater than 32. All the vaccinated cattle, regardless of antigen payload, were protected from clinical disease following challenge although some cattle in both groups became sub-clinically infected. We conclude that immunisation with a single inoculation of vaccine from the UK emergency reserve can protect cattle from clinical disease for at least 6 months post-vaccination and that a boost may be unnecessary in an outbreak situation. Some animals may become sub-clinically infected but this is likely to be dependent on the severity of challenge. The study confirmed that a booster at 21 days post-vaccination was not necessary to maintain a cell-mediated response in cattle for 6 months. No increased benefits were recognised by increasing the antigen payload of this vaccine 5x.

Experimental evaluation of foot-and-mouth disease vaccines for emergency use in ruminants and pigs: a review.

Changes to foot-and-mouth disease (FMD) control policies since 2001 mean that emergency vaccination must be considered more readily as a control measure in the future. Since field application of vaccine for emergency use has only rarely been applied, the effectiveness of single dose administration, as a control measure in an outbreak situation, is poorly understood. In this review we consider all the available experimental data from studies utilizing either experimental or readily available, commercially produced vaccines, in order to assess their likely effectiveness as an additional means of controlling FMD transmission and spread in an emergency. Overall it is concluded that such vaccines offer an additional and valuable means of FMD control for both ruminants and pigs. They are able to reduce clinical disease, sub-clinical infection and excretion and onward transmission of virus. However, to be most effective, vaccination should be rapidly applied to give maximum opportunity for immunity to develop. We also identify areas for future research and emphasize the importance of vaccine efficacy studies in providing data for models that can help to predict the efficacy of differing FMD control strategies.

Further evaluation of higher potency vaccines for early protection of cattle against FMDV direct contact challenge.

The effect of administering higher payload FMD vaccines 10 days prior to severe direct contact challenge on protection from clinical disease and sub-clinical infection was investigated in cattle using two antigen payloads (single strength and 10-fold). Regardless of antigen payload, vaccination was shown to significantly reduce the number of clinically infected animals, and significantly reduce virus excretion shortly after challenge, when compared with the unvaccinated group (P<0.05). Although FMDV transmission occurred from single strength vaccinated infected cattle to similarly vaccinated cattle held in indirect contact, no disease was induced in these animals. These studies further confirm that emergency vaccination does significantly reduce clinical disease and sub-clinical virus replication and excretion, particularly early post exposure, thereby reducing the possibility of transmission between animals and herds. To be most effective, however, the results also substantiate that time of vaccination prior to challenge significantly influences the number of animals becoming infected, so the decision to vaccinate should be made swiftly, to allow maximum opportunity for protective immunity to develop.

Secretory IgA as an indicator of oro-pharyngeal foot-and-mouth disease virus replication and as a tool for post vaccination surveillance.

A serotype-specific ELISA was developed to detect foot-and-mouth disease virus (FMDV) specific IgA antibody in the saliva of cattle, and the method was evaluated for its feasibility in detecting serotype O FMDV carrier animals, particularly amongst vaccinated cattle that had subsequently become sub-clinically infected. For this purpose, saliva samples were collected from naïve cattle (n = 173), FMDV challenged cattle (n = 10), FMDV vaccinated cattle (n = 40) and FMDV vaccinated-and-challenged cattle (n = 40). A subset of 29 cattle was sampled for 105-168 days after challenge. The FMDV infection status of each of the cattle was determined by virus isolation and RT-PCR tests on oesophago-pharyngeal fluids and the ability of the IgA test to detect viral infection and persistence was compared to an ELISA for the detection of serum antibodies against the 3ABC non-structural proteins of FMDV. Eleven out of twelve vaccinated cattle that were shown to be persistently infected with FMDV up to or beyond 28 days post challenge, were also detected by the IgA test on saliva. With some modification and further validation, this test could be useful in post-vaccination surveillance to help confirm the absence of sub-clinical infection in order to regain the FMD-free status of a region or country.