Modulation of Amyloid and Tau Aggregation to Alleviate Cognitive Impairment in a Transgenic Mouse Model of Alzheimer's Disease.

Aggregation of misfolded amyloid-ß (Aß) and hyperphosphorylated tau proteins to plaques and tangles, respectively, is the major drug target of Alzheimer's disease (AD), as the former is an onset biomarker and the latter is associated with neurodegeneration. Thus, we report a small molecule drug candidate, DN5355, with a dual-targeting function toward aggregates of both Aß and tau. DN5355 was selected through a series of four screenings assessing 52 chemicals for their functions to inhibit and reverse the aggregation of Aß and tau by utilizing thioflavin T. When orally administered to AD transgenic mouse model 5XFAD, DN5355 significantly reduced cerebral Aß plaques and hyperphosphorylated tau tangles. In Y-maze spontaneous alteration and contextual fear conditioning tests, 5XFAD mice showed amelioration of cognitive deficits upon the oral administration of DN5355.

4-Acyl-3,4-dihydropyrrolo[1,2-a]pyrazine Derivative Rescued the Hippocampal-Dependent Cognitive Decline of 5XFAD Transgenic Mice by Dissociating Soluble and Insoluble Aß Aggregates.

Cerebral amyloid-ß (Aß) deposition is a representative hallmark of Alzheimer's disease (AD). Development of Aß-clearing small molecules could be an advantageous therapeutic strategy for Aß clearance considering the advantages in terms of side effects, cost-effectiveness, stability, and oral bioavailability. Here, we report an Aß-dissociating small molecule, YIAD-0121, a derivative of 4-acyl-3,4-dihydropyrrolo[1,2-a]pyrazine. Through a series of anti-Aß screening assays, YIAD-0121 was identified to inhibit Aß aggregation and dissociate preformed Aß fibrils in vitro. Furthermore, the administration of YIAD-0121 in 5XFAD transgenic AD mice inhibited the increase of cerebral Aß aggregation and progression of hippocampus-dependent cognitive decline, with ameliorated neuroinflammation.

Solid-phase synthesis and pathological evaluation of pyroglutamate amyloid-ß3-42 peptide.

Pyroglutamate amyloid-ß3-42 (AßpE3-42) is an N-terminally truncated and pyroglutamate-modified Aß peptide retaining highly hydrophobic, amyloidogenic, and neurotoxic properties. In Alzheimer's disease (AD) patients, AßpE3-42 peptides accumulate into oligomers and induce cellular toxicity and synaptic dysfunction. AßpE3-42 aggregates further seed the formation of amyloid plaques, which are the pathological hallmarks of AD. Given that AßpE3-42 peptides play critical roles in the development of neurodegeneration, a reliable and reproducible synthetic access to these peptides may support pathological and medicinal studies of AD. Here, we synthesized AßpE3-42 peptides through the microwave-assisted solid-phase peptide synthesis (SPPS). Utilizing thioflavin T fluorescence assay and dot blotting analysis with anti-amyloid oligomer antibody, the amyloidogenic activity of synthesized AßpE3-42 peptides was confirmed. We further observed the cytotoxicity of AßpE3-42 aggregates in cell viability test. To examine the cognitive deficits induced by synthetic AßpE3-42 peptides, AßpE3-42 oligomers were intracerebroventricularly injected into imprinting control region mice and Y-maze and Morris water maze tests were performed. We found that AßpE3-42 aggregates altered the expression level of postsynaptic density protein 95 in cortical lysates. Collectively, we produced AßpE3-42 peptides in the microwave-assisted SPPS and evaluated the amyloidogenic and pathological function of the synthesized peptides.

A Therapeutic Nanovaccine that Generates Anti-Amyloid Antibodies and Amyloid-specific Regulatory T Cells for Alzheimer's Disease.

Alzheimer's disease (AD), the most common cause of dementia, is a complex condition characterized by multiple pathophysiological mechanisms including amyloid-ß (Aß) plaque accumulation and neuroinflammation in the brain. The current immunotherapy approaches, such as anti-Aß monoclonal antibody (mAb) therapy, Aß vaccines, and adoptive regulatory T (Treg) cell transfer, target a single pathophysiological mechanism, which may lead to unsatisfactory therapeutic efficacy. Furthermore, Aß vaccines often induce T helper 1 (Th1) cell-mediated inflammatory responses. Here, a nanovaccine composed of lipid nanoparticles loaded with Aß peptides and rapamycin is developed, which targets multiple pathophysiological mechanisms, exhibits the combined effects of anti-Aß antibody therapy and adoptive Aß-specific Treg cell transfer, and can overcome the limitations of current immunotherapy approaches for AD. The Nanovaccine effectively delivers rapamycin and Aß peptides to dendritic cells, produces both anti-Aß antibodies and Aß-specific Treg cells, removes Aß plaques in the brain, alleviates neuroinflammation, prevents Th1 cell-mediated excessive immune responses, and inhibits cognitive impairment in mice. The nanovaccine shows higher efficacy in cognitive recovery than an Aß vaccine. Unlike anti-Aß mAb therapy and adoptive Treg cell transfer, both of which require complicated and costly manufacturing processes, the nanovaccine is easy-to-prepare and cost-effective. The nanovaccines can represent a novel treatment option for AD.

Quinacrine directly dissociates amyloid plaques in the brain of 5XFAD transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia characterized by the abnormal accumulation of amyloid-ß (Aß) in the brain. Aß misfolding is associated with neuroinflammation and synaptic dysfunction, leading to learning and memory deficits. Therefore, Aß production and aggregation have been one of the most popular drug targets for AD. Failures of drug candidates regulating the aforementioned Aß cascade stimulated development of immunotherapy agents for clearance of accumulated Aß in the brain. Here, we report that quinacrine, a blood-brain barrier penetrating antimalarial chemical drug, dissociates Aß plaques in the brain of AD transgenic mice. When co-incubated with pre-formed Aß fibrils, quinacrine decreased thioflavin T-positive ß-sheets in vitro, on top of its inhibitory function on the fibril formation. We confirmed that quinacrine induced dissociation of high-molecular-weight Aß aggregates into low-molecular-weight species by dot blots in association with size cut-off filtrations. Quinacrine was then administered to adult 5XFAD transgenic mice via weekly intravenous injections for 6 weeks, and we found a significant reduction of Aß plaques and astrocytosis in their cortex and hippocampus. In western blots of quinacrine-administered mouse brains, amelioration of AD-related biomarkers, glial fibrillary acidic protein, postsynaptic protein 95, phosphorylated cAMP response element-binding protein, phosphorylated c-Jun N-terminal kinase were observed. Lastly, quinacrine-stimulated dissociation of misfolded aggregates induced recovery of synaptic function associated with Aß in excitatory post-synaptic current recordings of primary rat cortical neurons treated with Aß aggregates and quinacrine. Collectively, quinacrine can directly dissociate Aß fibrils and alleviate decreased synaptic functions.

Azetidine-Bearing Non-Ribosomal Peptides, Bonnevillamides D and E, Isolated from a Carrion Beetle-Associated Actinomycete.

Two new nonribosomal peptides, bonnevillamides D and E (1 and 2), have been discovered in Streptomyces sp. UTZ13 isolated from the carrion beetle, Nicrophorus concolor. Combinational analysis of the UV, MS, and NMR spectroscopic data revealed that their planar structures were comprised of dichlorinated linear peptides containing nonproteinogenic amino acid residues, such as 4-methylazetidinecarboxylic acid and 4-O-acetyl-5-methylproline. The configurations of bonnevillamides D and E (1 and 2) were determined based on ROESY correlations, the advanced Marfey's method, phenylglycine methyl ester derivatization, molecular modeling, and circular dichroism spectroscopy. The nonribosomal peptide synthetase biosynthetic pathway of bonnevillamides D and E has been proposed using bioinformatic analysis of the whole-genome sequence data of Streptomyces sp. UTZ13. Their biological activity toward the aggregation of amyloid-ß, which is one of the key pathogenic proteins in Alzheimer's disease, was evaluated using a thioflavin T assay and gel electrophoresis. Bonnevillamides D and E reversed the fibril formation by inducing the monomerization of amyloid-ß aggregates.

Effect of Vanadium Addition on the High-Temperature Friction Behavior in Nanostructured Al-Cr-V-N Films Prepared by UBMS.

In recent year, vanadium-doped tribological films have become available as possible candidates for self-lubrication at high temperatures. In this work, quaternary Al-Cr-V-N films were deposited onto silicon wafer and WC-Co substrates by an unbalanced magnetron sputtering using high purity (99.99%) CrAl2 and V targets with argon-nitrogen reactive gases. EPMA results revealed that vanadium atoms can incorporated from 0 to 13 at.% into the films. The maximum hardness value was ~32 GPa at vanadium content of 7.1 at.% in the Al-Cr-V-N films. The high-temperature tribometer was used to analysis the friction characteristics of the films with elevated temperature. As a result of the high temperature friction test after heating up to 700 °C, the average friction coefficient decreased from 0.62 to 0.35 with increasing of vanadium contents in the Al-Cr-V-N films. It is concluded that the reduction of the friction coefficient is attributed to the formation of V2O5, which is a Magnéli phase that acts as a lubrication at high temperature.

Multi-Functional Cr-Al-Ti-Si-N Nanocomposite Films Deposited on WC-Co Substrate for Cutting Tools.

Multi-functional quinary Cr-Al-Ti-Si-N thin films were deposited onto WC-Co substrates using a cathodic arc evaporation system. In this study, the influence of silicon contents on the microstructure, mechanical, tribological, and oxidation properties of Cr-Al-Ti-Si-N thin films were systematically investigated and correlated for application of cutting tools. Based on results from various analyses, the Cr-Al-Ti-Si-N films showed excellent properties including mechanical, tribological, oxidation and adhesion values compared with those of the Cr-Al-Ti-N film. The Cr-Al-Ti-Si-N films with a Si content of around 4.21 at.% exhibited the highest hardness of 45 GPa, very low friction coefficient of 0.38 at room temperature against an Inconel alloy ball and superior adhesion property (105 N). The Cr-Al-Ti-Si-N films also showed excellent oxidation resistance after annealing in the ambient air at 1000 °C. Therefore, the Cr-Al-Ti-Si(4.21 at.%)-N films could be help to improve the performance of machining and cutting tools with application of the films.

High Temperature Oxidation Behavior of Cr-Al-Si-N Nanocomposite Films Deposited by Filtered Arc Ion Plating Technique.

High temperature oxidation behavior of nanocomposite films is very important characteristics for application of machining and cutting tools. Quaternary Cr-Al-Si-N nanocomposite films with various compositions were deposited onto WC-Co and Si wafer substrates using a filtered arc ion plating technique. The composition of the films were controlled by different combinations of CrAl2 and Cr4Si composite target power in a reactive gas mixture of high purity Ar and N2 during depositions. The instrumental analyses revealed that the synthesized Cr-Al-Si-N films with Si content of 2.78 at.% were nanocomposites consisting of nano-sized crystallites (3-7 nm in dia.) and a thin layer of amorphous Si3N4 phases. The nanohardness of the Cr-Al-Si-N films exhibited the maximum values of ~42 GPa at a Si content of ~2.78 at.% due to the microstructural change to nanocomposite as well as solid-solution hardening. The Cr-Al-Si-N film shows superior result of oxidation resistance at 1050 °C for 30 min in air. Based on the XRD and GDOES analyses on the oxidized films, it could be revealed that the enrichment of Al (17.94 at.%) and Cr (26.24 at.%) elements in the film leads to form an Al2O3 and Cr2O3 layer on the Cr-Al-Si-N film surface. Therefore, in this study, the microstructural changes on the mechanical properties and oxidation behavior with various compositions in the Cr-Al-Si-N nanocomposite films were discussed and correlated with the deposition parameters.

Oxidation Behavior of Ti-Al-Si-N-O Nanocomposite Films Deposited by Filtered Arc Ion Plating Technique.

Oxidation behavior of nanocomposite films is very important characteristics for application of machining and cutting tools. In this study, Ti-Al-Si-N-O nanocomposite films were fabricated onto WC-Co and Si wafer substrates. The composition of the Ti-Al-Si-N-O films was analyzed by X-ray photo-electron spectroscope (XPS). Also X-ray diffactometer (XRD) analysis was conducted to investigate the crystallinity and phase transformation of the films. As a result of XRD, Ti-Al(18 at.%)-Si-N-O films showed the great oxidation resistance of 950 °C for 30 min in air. Based on glow discharge optical emission spectroscopy (GDOES) depth profiles, Ti-Al(18 at.%)- Si-N-O film annealed at 950 °C for 30 min shows formation of aluminum oxide layer on the film surface. On the other hand, Ti-Al(7.56 at.%)-Si-N-O film had a titanium oxide layer on the surface after annealing at 950 °C for 30 min.

Microstructure and Mechanical Properties of Functional Graded Ti-Al-Si-N-O Nanocomposite Films Deposited by Filtered Arc Ion Plating Technique.

Functional graded Ti-Al-Si-N-O nanocomposite films were deposited onto WC-Co substrate by a filtered arc ion plating system using TiAl and TiSi composite targets under N2/Ar atmosphere. XRD and XPS analyses revealed that the synthesized Ti-Al-Si-N-O films were nanocomposite consisting of nanosized (Ti, Al, Si)N crystallites embedded in an amorphous Si3N4/SiO2 matrix. The hardness of the Ti-Al-Si-N-O films exhibited the maximum hardness values of ~47 GPa at a Si content of ~5.63 at.% due to the microstructural change to a nanocomposite as well as the solid-solution hardening. Besides, Ti-Al-Si-N-O film with Si content of around 5.63 at.% also showed perfect adhesive strength value of 105.3 N. These excellent mechanical properties of Ti-Al-Si-N-O films could be help to improve the performance of machining tools and cutting tools with application of the film. A comparative study on microstructural characteristics among Ti-Al-Si-N-O films with various Si contents is reported in this paper.

Oxidation and Tribological Behavior of Ti-B-C-N-Si Nanocomposite Films Deposited by Pulsed Unbalanced Magnetron Sputtering.

Quinary Ti-B-C-N-Si nanocomposite films were deposited onto AISI 304 substrates using a pulsed d.c. magnetron sputtering system. The quinary Ti-B-C-N-Si (5 at.%) film showed excellent tribological and wear properties compared with those of the Ti-B-C-N films. The steady friction coefficient of 0.151 and a wear rate of 2 × 10-6 mm3N-1m-1 were measured for the Ti-B-C-N-Si films. The oxidation behavior of Ti-B-C-N-Si nanocomposite films was systematically investigated using X-ray diffraction (XRD), and thermal analyzer with differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). It is concluded that the addition of Si into the Ti-B-C-N film improved the tribological properties and oxidation resistance of the Ti-B-C-N-Si films. The improvements are due to the formation of an amorphous SiOx phase, which plays a major role in the self-lubricant tribo-layers and oxidation barrier on the film surface or in the grain boundaries, respectively.

Processing, structure, and properties of nanostructured multifunctional tribological coatings.

Nanostructured, nanocomposite binary (TiC-a:C), ternary (Cr-Al-N), quaternary (Ti-B-C-N) and quinternary (Ti-Si-B-C-N) multicomponent films have been deposited using unbalanced magnetron sputtering (UBMS) and closed field unbalanced magnetron sputtering (CFUBMS) from both elemental and composite targets. Approaches to control the film chemistry, volume fraction and size of the multicomponent species, and pulsed ion energy (ion flux) bombardment to tailor the structure and properties of the films for specific tribological applications, e.g., low friction coefficient and low wear rate, are emphasized. The synthesized films are characterized using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), nanoindentation, and microtribometry. The relationships between processing parameters (pulsed ion energy and ion flux), thin film microstructure, mechanical and tribological properties are being investigated in terms of the nanocrystalline-nanocrystalline and nanocrystalline-amorphous composite thin film systems that are generated. In the Ti-Si-B-C-N films, nanocomposites of solid solutions, e.g., nanosized (Ti,C,N)B2 and Ti(C,N) crystallites are embedded in an amorphous TiSi2 and SiC matrix including some carbon, SiB4, BN, CN(x), TiO2 and B2O3 components. The Ti-Si-B-C-N coating with up to 150 W Si target power exhibited a hardness of about 35 GPa, a high H/E ratio of 0.095, and a low wear rate of from approximately 3 to approximately 10 x 10(-6) mm3/(Nm). In another aspect, using increased ion energy and ion flux, which are generated by pulsing the power of the target(s) in a closed field arrangement, to provide improved ion bombardment on tailoring the structure and properties of TiC-a:C and Cr-Al-N coatings are demonstrated. It was found that highly energetic species (up to hundreds eV) were found in the plasma by pulsing the power of the target(s) during magnetron sputtering. Applying higher pulse frequency and longer reverse time (lower duty cycle) will result in higher ion energy and ion flux in the plasma, which can be utilized to improve the film structure and properties. For example, optimum properties of the TiC-a:C coating were a hardness of 35 to 40 GPa and a COF of 0.2 to 0.22 for moderate maximum ion energies of 70 to 100 eV, and a super high hardness of 41 GPa and low wear rate of 3.41 x 10(-6) mm3N(-1) m(-1) was obtained for Cr-Al-N coatings deposited with a maximum ion energy of 122 eV. These conditions can be achieved by adjusting the pulsing parameters and target voltages. However, the pulsed ion energy together with the applied substrate bias are need to be carefully controlled in order to avoid excessive ion bombardment (e.g., the maximum ion energy is larger than 180 eV in the current study), which will responsible for an increase in point and line defects, and high residual stress in the crystalline structure.