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Amyloid fibrillogenesis is a pathogenic protein aggregation process that occurs through a highly ordered process of protein-protein interactions. To better understand the protein-protein interactions involved in amyloid fibril formation, we formed nanogold colloid aggregates by stepwise additions of ∼2 nmol of amyloid ß 1-40 peptide (Aß1-40) at pH ∼3.7 and ∼25 °C. The processes of protein corona formation and building of gold colloid [diameters (d) of 20 and 80 nm] aggregates were confirmed by a red-shift of the surface plasmon resonance (SPR) band, λpeak, as the number of Aß1-40 peptides [N(Aß1-40)] increased. The normalized red-shift of λpeak, Δλ, was correlated with the degree of protein aggregation, and this process was approximated as the adsorption isotherm explained by the Langmuir-Freundlich model. As the coverage fraction (θ) was analyzed as a function of ϕ, which is the N(Aß1-40) per total surface area of nanogold colloids available for adsorption, the parameters for explaining the Langmuir-Freundlich model were in good agreement for both 20 and 80 nm gold, indicating that ϕ could define the stage of the aggregation process. Surface-enhanced Raman scattering (SERS) imaging was conducted at designated values of ϕ and suggested that a protein-gold surface interaction during the initial adsorption stage may be dependent on the nanosize. The 20 nm gold case seems to prefer a relatively smaller contacting section, such as a -C-N or C═C bond, but a plane of the benzene ring may play a significant role for 80 nm gold. Regardless of the size of the particles, the ß-sheet and random coil conformations were considered to be used to form gold colloid aggregates. The methodology developed in this study allows for new insights into protein-protein interactions at distinct stages of aggregation.

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BACKGROUND: Amyloid beta (Aß) peptides, such as Aß1-40 or Aß1-42 are regarded as hallmark neuropathological biomarkers associated with Alzheimer's disease (AD). The formation of an aggregates by Aß1-40 or Aß1-42-coated gold nano-particles are hypothesized to contain conformation of Aß oligomers, which could exist only at an initial stage of fibrillogenesis. NEW METHOD: The attempt of in-situ detection of externally initiated gold colloid (ca. 80 nm diameter) aggregates in the middle section of the hippocampus of the Long Evans Cohen's Alzheimer's disease rat model was conducted through the Surface Enhanced Raman Scattering (SERS) method. RESULTS: The SERS spectral features contained modes associated with ß-sheet interactions and a significant number of modes that were previously reported in SERS shifts for Alzheimer diseased rodent and human brain tissues; thereby, strongly implying a containment of amyloid fibrils. The spectral patterns were further examined and compared with those collected from in-vitro gold colloid aggregates which were formed from Aß1-40 - or Aß1-42 -coated 80 nm gold colloid under pH ∼4, pH ∼7, and pH ∼10, and the best matched datasets were found with that of the aggregates of Aß1-42 -coated 80 nm gold colloid at ∼pH 4.0. The morphology and physical size of this specific gold colloid aggregate was clearly different from those found in-vitro. COMPARISON WITH EXISTING METHOD(S): The amyloid fibril with a ß-sheet conformation identified in previously reported in AD mouse/human brain tissues was involved in a formation of the gold colloid aggregates. However, to our surprise, best explanation for the observed SERS spectral features was possible with those in vitro Aß1-42 -coated 80 nm gold colloid under pH ∼4. CONCLUSIONS: A formation of gold colloid aggregates was confirmed in the AD rat hippocampal brain section with unique physical morphology compared to those observed in in-vitro Aß1-42 or Aß1-40 mediated gold colloid aggregates. It was concluded that a ß-sheet conformation identified in previously reported in AD mouse/human brain tissues was in volved in a formation of the gold colloid aggregates.

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The adsorption process of SARS-CoV-2 Omicron spike protein to the nano-gold colloid surfaces was examined by monitoring the surface plasmon resonance (SPR) band shift of gold-nano particles ranging between diameters of d = 10-100 nm. The externally changed pH between 3 and 11 at 24.5 ± 0.4 °C initiated a reversible formation of the gold colloid aggregates, where formation/deformation of the aggregates were monitored by red/blue shift of the peak of the SPR band. There was no sign of reversible aggregation for d = 10, 15, and 20 nm gold colloids. A clear undulation of the peak shift corresponding to pH hopping between pH ~3 and ~11 was confirmed for colloidal d > 30 nm. This degree of the reversibility was compared to previously reported SARS-CoV-2 Alpha spike protein coated gold colloids. It was concluded that Omicron spike protein possesses a similar low affinity for gold nano particle d < 20 nm and possesses the higher affinity to the gold nanoparticles of d > 30 nm. However, the Omicron spike protein conformation was presumed to be more denatured compared to the SARS-CoV-2 Alpha spike protein. Our finding suggested Omicron spike protein was more acid labile/flexible.

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The affinity of the SARS-CoV-2 spike protein (S protein) to gold nano-particles was examined through spectral shifts of SPR (Surface Plasmon Resonance) band. Gold nano-colloidal particles are sensitive to the conformational change of the protein adsorbed over the particles' surface. As the pH value was gradually lowered from approximately neutral pH to an acidic pH (ca. pH 2), all mixtures of S protein with the gold colloids ≥30 nm in diameter exhibited a drastic red-shift of the average SPR band peak at one pH value more than that observed for bare gold colloids. The surface coverage fraction (Θ) of S protein over the nano-particle's surface was extracted and all showed relatively small coverage values (i.e., Θ ~ 0.30). The SPR band peak shift was also examined as the pH values were hopped between pH ~ 3 and pH ~ 10 (pH hopping). As the pH values hopped, an alternation of the average SPR band peaks were observed. A significant amplitude of an alternation was especially observed for the mixture of S protein with gold ≥30 nm of gold size implying the reproduction of pH induced reversible protein folding. We hypothesize that the pH hopping scheme captured a reversible transition between folded or Down conformation (pH ≥ ~7) and unfolded or Up (pH ~ 3) conformation of RBD (receptor binding domain). The acidic condition may also dimerize the S protein through RBD. The Up conformation or dimerization of S protein are considered to be connected to the other gold nano particles forming gold nano-particle aggregates.

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Gold nano-particles were coated with the spike protein (S protein) of SARS-CoV-2 and exposed to increasingly acidic conditions. Their responses were investigated by monitoring the surface plasmon resonance (SPR) band shift. As the external pH was gradually changed from neutral pH to pH ∼2 the peak of the SPR band showed a significant red-shift, with a sigmoidal feature implying the formation of the gold-protein aggregates. The coating of S protein changed the surface property of the gold enough to extract the coverage fraction of protein over nano particles, Θ, which did not exhibit clear nano-size dependence. The geometrical simulation to explain Θ showed the average axial length to be a = 7. 25 nm and b =8.00 nm when the S-protein was hypothesized as a prolate shape with spiking-out orientation. As the pH value externally hopped between pH∼3 and pH∼10, a behavior of reversible protein folding was observed for particles with diameters >30 nm. It was concluded that S protein adsorption conformation was impacted by the size (diameter, d) of a core nano-gold, where head-to-head dimerized S protein was estimated for d ≤ 80 nm and a parallel in opposite directions formation for d = 100 nm.

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The adsorption of amyloidogenic peptides, amyloid beta 1-40 (Aß1-40), alpha-synuclein (α-syn), and beta 2 microglobulin (ß2m), was attempted over the surface of nano-gold colloidal particles, ranging from d = 10 to 100 nm in diameter (d). The spectroscopic inspection between pH 2 and pH 12 successfully extracted the critical pH point (pHo) at which the color change of the amyloidogenic peptide-coated nano-gold colloids occurred due to aggregation of the nano-gold colloids. The change in surface property caused by the degree of peptide coverage was hypothesized to reflect the ΔpHo, which is the difference in pHo between bare gold colloids and peptide coated gold colloids. The coverage ratio (Θ) for all amyloidogenic peptides over gold colloid of different sizes was extracted by assuming Θ = 0 at ΔpHo = 0. Remarkably, Θ was found to have a nano-gold colloidal size dependence, however, this nano-size dependence was not simply correlated with d. The geometric analysis and simulation of reproducing Θ was conducted by assuming a prolate shape of all amyloidogenic peptides. The simulation concluded that a spiking-out orientation of a prolate was required in order to reproduce the extracted Θ. The involvement of a secondary layer was suggested; this secondary layer was considered to be due to the networking of the peptides. An extracted average distance of networking between adjacent gold colloids supports the binding of peptides as if they are "entangled" and enclosed in an interfacial distance that was found to be approximately 2 nm. The complex nano-size dependence of Θ was explained by available spacing between adjacent prolates. When the secondary layer was formed, Aß1-40 and α-syn possessed a higher affinity to a partially negative nano-gold colloidal surface. However, ß2m peptides tend to interact with each other. This difference was explained by the difference in partial charge distribution over a monomer. Both Aß1-40 and α-syn are considered to have a partial charge (especially δ+) distribution centering around the prolate axis. The ß2m, however, possesses a distorted charge distribution. For a lower Θ (i.e., Θ <0.5), a prolate was assumed to conduct a gyration motion, maintaining the spiking-out orientation to fill in the unoccupied space with a tilting angle ranging between 5° and 58° depending on the nano-scale and peptide coated to the gold colloid.

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Because Congo red (CR) can bind to critical intermediate structural forms of amyloid beta (Aß), it has been suggested as a potential therapeutic agent against neurodegenerative disorders such as Alzheimer's disease. In this study, the interaction of CR with Aß(12-28) was investigated by use of isothermal titration calorimetry (ITC). Studies conducted between 15 and 35 °C show that binding of CR to Aß(12-28) was strongly dependent on temperature, with a decrease in CR-Aß(12-28) complexation as temperature increases, presumably because of conformational changes within Aß(12-28) at the highest temperatures, that conceal the CR binding sites. In fact, no CR binding was observed at 35 °C. The binding of CR to Aß(12-28) was associated with favorable changes in both enthalpy and entropy that resulted in binding constants (K) of between 10(5) and 10(6) M (-1). An early (and more intense) entropy-driven CR disaggregation phase (K ~10(7)-10(8) M (-1)) was observed before the onset of CR-Aß(12-28) complexation. Only CR disaggregation was observed at 35 °C. These results may provide further insights into the ability of CR to inhibit Aß toxicity in neurodegenerative diseases.

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To investigate the molecular mechanism underlying heme oxygenase-1 (HO-1)-modulated infiltration of neutrophils, the sepsis model of cecal ligation and puncture in Sprague-Dawley rats was used. In vivo induction and suppression of HO-1 were performed by pretreatment with cobalt protoporphyrin IX (CoPP) and zinc protoporphyrin IX, respectively. Tricarbonyldichlororuthenium(II) dimer, [Ru(CO)3Cl2]2 (a carbon monoxide [CO] releaser), and hemoglobin (a CO scavenger) were used to examine the participation of HO-1/CO in the effect of CoPP pretreatment on formylated peptide (fMLP)-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation. Anisomycin (a p38 MAPK activator) and SB203580 (a p38 MAPK inhibitor) were used to examine p38 MAPK mediation in the attenuation of fMLP-attracted migration by HO-1. The results demonstrated that zinc protoporphyrin IX and CoPP pretreatment conferred enhancing and inhibitory effects, respectively, on hepatic neutrophil infiltration. Pretreatment with CoPP inhibited fMLP-induced migration and p38 MAPK phosphorylation in neutrophils ex vivo. The [Ru(CO)3Cl2]2 stimulated whereas hemoglobin diminished the suppression of fMLP-induced p38 MAPK phosphorylation by CoPP. Moreover, anisomycin diminished the suppressive effects of CoPP pretreatment on fMLP-induced migration, actin polymerization, polarization, and migration speed of neutrophils. These results suggest that HO-1 in neutrophil attenuates its infiltration during sepsis via the inactivation of p38 MAPK. Understanding the mechanism that diminishes neutrophil infiltration by HO-1 may help prevent hepatic failure during sepsis.

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The absorption spectrum of the amyloid beta 1-40 peptide (Abeta(1-40)) conjugated to gold colloidal suspension of 15, 20, 30, and 40 nm size were examined under temperature ranging from 5 to 50 degrees C. As the pH was externally altered repetitively between pH 4 and 10, Abeta(1-40)-coated 20 nm gold colloid nanoparticles exhibited a reversible color change at the entire temperature range tested in this study except for 5 +/- 0.2 degrees C. This reversible change may be due to the fact that hydrophilic Abeta(1-40) evolves between a three-dimensional network containing mainly beta-sheet and alpha-helices, and an intermediate of this process implies a reversible step reported as initiation of the fibrillogenesis in Alzheimer's disease. When other nanosize particles were investigated, Abeta(1-40)-coated 30 and 40 nm colloids exhibited the reversible color change when temperature was lowered to 18 +/- 0.2 and 6 +/- 0.2 degrees C, respectively. This specific and unique size and temperature dependence in reversible color change strongly suggests that the noncovalent intrinsic intermolecular potential formed between the nanocolloidal surface and each Abeta(1-40) monomer conjugated at the surface drives the process.

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Aß(1-40) coated 20 nm gold colloidal nanoparticles exhibit a reversible color change as pH is externally altered between pH 4 and 10. This reversible process may contain important information on the initial reversible step reported for the fibrillogenesis of Aß (a hallmark of Alzheimer's disease). We examined this reversible color change by microscopic investigations. AFM images on graphite surfaces revealed the morphology of Aß aggregates with gold colloids. TEM images clearly demonstrate the correspondence between spectroscopic features and conformational changes of the gold colloid.

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We investigated the effect of charge-charge interactions on the mobilities of rhodamine 6G (R6G), Nile Red, sulforhodamine B, and Oregon Green 514 (ORG) guest molecules within a silica sol-gel host as the guest charge progressed from positive to neutral to negative. Through classification of the mobility as fixed, tumbling, or intermediate behavior, we were able to distinguish differences in surface attraction as the guest charge was varied. On the basis of our results, an attractive charge (as tested by cationic R6G) does not contribute significantly to mobility within dry films. However, an increase in the cationic influence is observed in water-equilibrated environments. A comparison of ORG in dry and water- and phosphate-buffer-equilibrated films indicates that charge repulsion does significantly increase dye rotational mobility (to a maximum of 24 +/- 3% tumbling molecules). However, in view of the percentage of tumbling molecules found, charge-charge interactions do not appear to be the dominant force controlling guest mobility.

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