Silver ions as EM marker of congo red ligation sites in amyloids and amyloid-like aggregates

Congo red (CR) is a known selective amyloid ligand. The focus of our work is identification (by EM imaging) of dye binding sites and their distribution in amyloids and amyloid-like aggregates formed in vitro. In order to produce the required contrast, CR has been indirectly combined with metal via including Titan yellow (TY) by intercalation which exhibits a relatively strong affinity for silver ions. The resulting combined ligand retains its ability to bind to proteins (which it owes to CR) and can easily be detected in EM studies thanks to TY. We have found, however, that in protein aggregates where unfolding is stabilized by aggregation and therefore is irreversible, TY alone may serve as both, the ligand and the metal carrier. The formation of ordered structures in amyloids was studied using IgG light chains with amyloidogenic properties, converted into amyloids by shaking. The resulting EM images were subjected to interpretation on the basis of the authors' earlier research on the CR/light chain complexation process. Our results indicate that dimeric light chains, which are the subject of our study, produce amyloids or amyloid-like complexes with chain-like properties and strong helicalization tendencies. Cursory analysis suggests that the edge polypeptide loops belonging to unstable light chains form intermolecular bridges which promote creation of loose gel deposits, or are otherwise engaged in the swapping processes leading to higher structural ordering

Self-Assembled Molecules – New Kind of Protein Ligands: Supramolecular Ligands

immunotargeting techniques; ligands; intramolecular immunological signals; congo red amyloid

Absorption and luminescence properties of beta-carotene with antioxidant and modified kaolinite and its application in OLED

Energy crises, especially fossil fuel-based energy, lead to increased use of new energy and renewable energy. In addition, research is also directed towards more efficient energy use through the development of energy storage materials such as batteries, and the use of energy-efficient materials. An OLED (organic light-emitting diode) is a light-emitting diode (LED) and is known for their high potential in a display, signage, and lighting applications. Carotenoids constitute an important class of linear -conjugated molecules that exhibit a high degree of electronic delocalization and ultrafast dynamic. The dye that is used as a photosensitizer plays an important role in the operation of DSSCs or other bifunctional material. In this research, we studied the photostability of the carotenoid compound (-carotene and fucoxanthin) using natural antioxidant and modified kaolinite. In this work, the carotenoid stability by the carotenoid compound/antioxidant binary is studied for the first time. Photostability of -carotene can be enhanced by antioxidant and modified kaolinite. Photoprotection efficiency of -carotene by curcumin was higher than fucoxanthin. Modified kaolinite decreased photodegradation of - carotene by shielded and protected from direct UV irradiation. Antioxidant fucoxanthin and curcumin decreased the electrochemical gap of the binary material. The electrochemical gap of carotene/curcumin is -1.61 eV, carotene/fucoxanthin is -1.75 eV compare to the only -carotene - 2.04 eV. Fucoxanthin can keep the first oxidation stage of -carotene. Therefore at the binary compound, electron was still reversible, but not for curcumin. Fucoxanthin in OLED devices NPD/Fx/ETL reduced significanly the EQE (%) almost 80% in Alq3 and 76% in BAlq. OLED device: NPD(50nm)/Fx(1nm)/Alq3(85nm) yielded an effeciency quantum external yield, EQE = 0.12% and CIE (0.4160, 0.5302). Fabrication OLED using -carotene as HTL and curcumin as EL layer resulted yellow color , EQE = 0.02%, with the composition MoO3(15nm)/NPD(40nm)/Car(10nm)/ Cur(15nm)/Alq3(70nm)/Ca(100nm). For application of - carotene in OLED device, its perfomance can be improved by using curcumin at the fabrication

The development of peptide-based inhibitors for Tau aggregation as a potential therapeutic for Alzheimer’s disease

There are currently approximately 50 million individuals worldwide with dementia resulting in predicted global societal costs of up to US $1 trillion. Approximately 60-70% of these individuals have Alzheimer’s disease, which results in a chronic and insidious decline in memory. One of the main proteins that misfolds in this disease is Tau protein, which aggregates into toxic oligomers and neurofibrillary tangles. It is these aggregates, which cause damage to the brain resulting in dementia. As a result, it is imperative to be able to prevent or suppress the pathogenic aggregation of this protein, so the onset of dementia is halted or delayed, improving quality of life. Certain amino acid sequences in Tau such as VQIINK and VQIVYK play important roles in aggregation. Targeting these sequences can potentially prevent aggregation. This project aims to produce effective peptide inhibitors based on the human Tau peptide sequences VQIINK and VQIVYK, to specifically target pathogenic Tau aggregation. Using molecular docking softrware ‘ICM-Pro’ the potential binding locations of a variety of peptide candidates were computationally investigated to determine which will be most successful in a laboratory setting. Recombinant TauΔ1-250 was incubated in the prescense of heparin and subsequently aggregated to display highly ordered parallel, in-register β-strand structures; including fibrils and paired helical filaments presenting the characteristic twist under transmission electron microscope. This aggregation was achieved using of 20μM Tau at pH 7.4 in the presence of 20mM Tris buffer, 1mM DTT, 5μM Heparin, and 15uM ThT and incubated at 37 °C for 48 hours. The first generation of peptides AG01, AG02, AG02, AG02R4, AG02R5, AGR502, AG02PR5, AG02R6, AG02R9, AG02TAT, AG02ΔI, AG02ΔV inhibited approximately 50% of Tau aggregation determined by Thioflavin-T (ThT) fluorescence assay. The next generation, AG03 was slightly more effective, however when retroinverted (RI-AG03) inhibited over 90% of Tau aggregation, confirmed by Thioflavin-T fluorescence assay, transmission electron microscopy, circular dichroism and Congo red birefringence. RI-AG03 was determined to be stable in cells at therapeutic concentrations. After determining stability of RI-AG03 using SDS-PAGE, thermal circular dichroism and mass spectrometry, it was tested in vivo in rough eye Drosophila model. Results suggested that RI-AG03 partially rescued the rough eye phenotype in this model. This research demonstrates that retro-inverted peptide RI-AG03 is a potent inhibitor of Tau aggregation and can be further developed as a novel therapeutic for Tauopathies like Alzhimer’s Disease

Microglial Activation by Amyloid-Beta

Paranjape, Geeta S.,Ph.D.,University of Missouri-Saint Louis, May 2012. Aβ(1-42) Protofibrils But Not Fibrils Activate Microglia. Major Professor: Michael R. Nichols. One of the hallmark features of the Alzheimer’s disease (AD) brain is the extracellular deposition of amyloid-β protein (Aβ) in both fibrillar (senile plaques) and diffuse forms. Significant proinflammatory markers including activated microglia and cytokines have been detected surrounding the plaques but are absent in diffuse areas suggesting that microglial activation is sensitive to Aβ structure. Since Aβ displays structural polymorphism in vitro, we sought to determine the relationship between Aβ aggregation state and microglial proinflammatory response. Size exclusion chromatography (SEC) purification of freshly reconstituted Aβ(1-42) in NaOH/F12 cell culture medium isolated classical 100 nm long curvilinear protofibrils which stimulated a robust production of microglial TNFα. The Aβ(1-42) protofibrils produced a concentration-dependent response in the low micromolar range. In contrast to Aβ(1-42), Aβ(1-40) required a pre-incubation of 24h at 25⁰C in order to produce protofibrils. Although both preparations were similar in morphology, Aβ(1-42) protofibrils were a much better stimulator of microglia than Aβ(1-40) protofibrils. Aβ(1-40) containing the Arctic mutation (E22G) formed protofibrils as soon as 3h after reconstitution , yet they were largely ineffective in stimulating microglia. None of the Aβ protofibril preparations were toxic to microglia suggesting that Aβ(1-42) protofibrils activate microglia in a manner independent of toxicity. As expected, freshly-purified Aβ(1-42) or Aβ(1-40) monomer were not effective in stimulating microglia, but surprisingly, neither were Aβ(1-42) fibrils even though they exhibited extensive Thioflavin-T fluorescence compared to protofibrils. These findings suggest that Aβ(1-42) protofibrils are the most effective inducers of a proinflammatory response in mouse microglia

Detection and thermal stabilization of virus based on surface properties

Viral diseases take the lives of millions of people each year. The most effective methods to prevent viral disease outbreak are viral detection to reduce contact with viral pathogens and vaccines to prevent disease. To reduce the costs of the detection of viruses and improve vaccine formulation, we explored viral surface properties. The properties we have focused on are viral hydrophobicity and surface charge using chemical force microscopy (CFM). CFM is a single-particle technique that measures the adhesion force of a functionalized atomic force microscopy (AFM) probe, and in this study, a virus covalently bound to a surface. The non-enveloped porcine parvovirus (PPV) and enveloped bovine viral diarrhea virus (BVDV) were used to demonstrate the use of CFM for viral particles with different surface properties. The high hydrophobicity of PPV and BVDV by CFM was used for a ligand-free, non-specific virus detection method that relies on the interaction of virus with osmolytes. It was previously found that the osmolyte mannitol can preferentially aggregate viruses while leaving proteins in solution. The virus was incubated with gold nanoparticles (AuNPs), and aggregation of the virus-AuNP complex with mannitol was detected by dynamic light scattering (DLS). The isoelectric point (pI) of PPV and BVDV by CFM was used for a vaccine formulation strategy of virus particle encapsulation by polymers that relies on electrostatic interactions of the virus with polypeptides. The random screen of different ratios of polyelectrolytes to encapsulate viruses could be reduced by knowing the virus pIs. An encapsulated non-enveloped PPV is thermally stabilized, demonstrating that this method is promising for formulating thermostable vaccines. We have developed a unique detection method and can improve vaccine formulation that would reduce the impact of viral diseases worldwide, based on the viral surface properties

Understanding Mechanistic Details of Neuroinflammatory Pathways Stimulated by the Alzheimer\u27s Disease Amyloid-Beta Protein

Alzheimer’s disease (AD) is characterized by neuroinflammation. Senile plaques composed of aggregated amyloid-β protein (Aβ) are found in AD patients’ brains. The Aβ is formed by the proteolytic cleavage of the amyloid precursor protein (APP) resulting in Aβ fragments that are 39-42 amino acids in length. The two most common peptides are Aβ(1-40) and Aβ(1-42), which differ by two amino acids, isoleucine and alanine. Within the brain of AD patients, Aβ monomer self-assembles to form several aggregate morphologies, including oligomers, protofibrils, and fibrils. Activated microglial cells and associated secreted proinflammatory cytokines surround these plaques producing a localized inflammatory environment in the brain. Several innate-immune pathways, including Toll-like receptors (TLRs) and the NLRP3 inflammasome, have been implicated in AD inflammation. Aβ plays a primary role in activating these pathways likely contributing to the progressive neurodegeneration in AD. In order to better understand the complexities of this interaction, I investigated the inflammatory response of microglia to Aβ(1-42) and Aβ(1-42)/Aβ(1-40) protofibrils, along with additional biophysical properties. Increased understanding of these pathological events will expand the current model of Aβ neuroinflammatory pathways and help identify new therapeutic targets for AD. My research has demonstrated that the Aβ(1-42) protofibrils triggered a time- and TLR/MyD88-dependent process that produced inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-1β (IL-1β) mRNA and intracellular pro and mature forms of IL-1β protein. Despite previous reports suggesting that NLRP3 activation requires two signals from two distinct molecules, my research indicated that Aβ(1-42) protofibrils alone could efficiently prime (TLR-dependent pro-IL-1β production) and activate (cleavage of pro-IL-1β to mature-IL-1β) the NLRP3 inflammasome. However, the increased intracellular mature-IL-1β did not translate into greater IL-1β secretion. Instead, we found that Aβ was able to elicit a very rapid, unsustainable, yet re-inducible quantized burst of secreted IL-1β, which occurred prior to Aβ priming of the microglia. These findings suggested a basal level of either pro- or mature-IL-1β in the cultured primary microglia yet revealed multiple sites of IL-1β regulation by Aβ(1-42) protofibrils. These sites, which are potential therapeutic targets, include TLR/MyD88-mediated priming, NLRP3 inflammasome activation, and modulation of the IL-1β secretory process