Hsin-Yi Lin

The discovery of potential acetylcholinesterase inhibitors: A combination of pharmacophore modeling, virtual screening, and molecular docking studies

BackgroundAlzheimers disease (AD) is the most common cause of dementia characterized by progressive cognitive impairment in the elderly people. The most dramatic abnormalities are those of the cholinergic system. Acetylcholinesterase (AChE) plays a key role in the regulation of the cholinergic system, and hence, inhibition of AChE has emerged as one of the most promising strategies for the treatment of AD.MethodsIn this study, we suggest a workflow for the identification and prioritization of potential compounds targeted against AChE. In order to elucidate the essential structural features for AChE, three-dimensional pharmacophore models were constructed using Discovery Studio 2.5.5 (DS 2.5.5) program based on a set of known AChE inhibitors.ResultsThe best five-features pharmacophore model, which includes one hydrogen bond donor and four hydrophobic features, was generated from a training set of 62 compounds that yielded a correlation coefficient of R = 0.851 and a high prediction of fit values for a set of 26 test molecules with a correlation of R2 = 0.830. Our pharmacophore model also has a high Güner-Henry score and enrichment factor. Virtual screening performed on the NCI database obtained new inhibitors which have the potential to inhibit AChE and to protect neurons from Aβ toxicity. The hit compounds were subsequently subjected to molecular docking and evaluated by consensus scoring function, which resulted in 9 compounds with high pharmacophore fit values and predicted biological activity scores. These compounds showed interactions with important residues at the active site.ConclusionsThe information gained from this study may assist in the discovery of potential AChE inhibitors that are highly selective for its dual binding sites.

Molecular Dynamics Simulations to Investigate the Aggregation Behaviors of the Aß(17–42) Oligomers

Abstract The amyloid β-peptides (Aßs) are the main protein components of amyloid deposits in Alzheimers disease (AD). Detailed knowledge of the structure and assembly dynamics of Aß is important for the development of properly targeted AD therapeutics. So far, the process of the monomeric Aß assembling into oligomeric fibrils and the mechanism underlying the aggregation process remain unclear. In this study, several molecular dynamics simulations were conducted to investigate the aggregation behaviors of the Aß(17–42) oligomers associated with various numbers of monomers (dimer, trimer, tetramer, and pentamer). Our results showed that the structural stability of the Aß(17–42) oligomers increases with increasing the number of monomer. We further demonstrated that the native hydrophobic contacts are positive correlated with the ß-sheet contents, indicating that hydrophobic interaction plays an important role in maintaining the structural stability of the Aß(17–42) oligomers, particularly for those associated with more monomers. Our results also showed that the stability of the C-terminal hydrophobic segment 2 (residues 30–42) is higher than that of the N-terminal hydrophobic segment 1 (residues 17–21), suggesting that hydrophobic segment 2 may act as the nucleation site for aggregation. We further identified that Met35 residue initiates the hydrophobic interactions and that the intermolecular contact pairs, Gly33-Gly33 and Gly37-Gly37, form a stable “molecular notch”, which may mediate the packing of the ß-sheet involving many other hydrophobic residues during the early stage of amyloid-like fibril formation.

Controlled release of pentoxifylline from porous chitosan-pectin scaffolds

Measures to suppress inflammatory reactions are taken to prevent fibrous encapsulation of implants. It is proposed in this study that tissue engineered scaffolds that can slowly release anti-inflammatory drugs can help reduce inflammatory reactions around implants. Chitosan and chitosan cross-linked with different concentrations of pectin were made into films and porous scaffolds. Results seen from Fourier-transform infrared spectra and thermal gravimetric analysis showed that polyelectrolyte complexation took place between chitosan and pectin units. As the amounts of pectin added to chitosan increased (0%, 0.5%, 1%, and 2%) the scaffolds became more wettable (contact angle decreased from 81° to 76°), less swellable (swelling ratio decreased from 35% to 30%), and less capable of releasing pentoxifylline (PTX) (release efficacies decreased from 93% to 83%). Higher degrees of pectin cross-linking made the scaffolds more resistant to compression (Young’s modulus increased from 2.4 kPa to 3.7 kPa) and more favorable for initial cell attachment (percentage of attached cells increased from 55% to 67%). In vitro tests showed that, with the reduction of PTX release rates, PTX became more effective in inhibiting TNF-α and IL-6 production from activated macrophages. This investigation has demonstrated that the changes in the basic drug release properties of chitosan scaffolds were proportional to the amount of pectin added. The changes could help improve the effectiveness of PTX.

Molecular Dynamics Simulations to Gain Insights into the Stability and Morphologies of K3 Oligomers from β2-microglobulin

Abstract β2-Microglobulin (β2-m) forms amyloid fibrils in patients undergoing long-term hemodialysis. K3 peptide, a Ser20-Lys41 fragment of β2-m, has been known to form fibrils over a wide range of pH and solvent conditions. Recent solid-state NMR has revealed that K3 oligomer adopts a parallel U-shaped β-strand-turn-β-strand motif. In order to investigate the stability and morphologies of K3 oligomers with different sizes (dimer, trimer, and tetrameri and organizations (single and double layers), several all-atom molecular dynamics simulations were conducted at 310 K and pH 2 in water and 2,2,2-trifluoroethanol (TFE). For single-layered organizations, our results show that TFE destabilizes the stacking of K3 peptides due to the fact that TFE weakens the intermolecular hydrophobic interactions of K3 oligomers. In addition, we also identified that the loop region is stabilized by the hydrophobic cluster involving resides Y7, Fll, and I16. Our results further suggest that K3 tetramer is a potential minimal nucleus seed for the formation of K3 protofibrils. For dou-ble-layered organizations in water, our data demonstrate that K3 peptides can form various stable assemblies through different interfacial arrangements, such as NN, NC, and CC, by different driving forces. We further propose that the stacking of different interfaces between two facing β-sheets of K3 peptides could be related to different fibril morphologies, which is in good agreement with the previous experimental results, showing that K3 protofibrils associated to formed mature fibrils with a wide range of diameters from 4 to 15 nm when they were transferred from 20% (v/v) TFE to aqueous solution.

Chondrogenesis From Immortalized Human Mesenchymal Stem Cells: Comparison Between Collagen Gel and Pellet Culture Methods

Human mesenchymal stem cells (hMSCs) can differentiate into cells of connective tissue lineages, including cartilage. To overcome the limiting autogenous chondrocyte populations available in cartilage repair, various methods have been developed to maximize chondrogenesis of hMSCs in vitro, most of which use cells derived from primary culture. In this study, we compared chondrogenesis of immortalized hMSCs embedded in collagen gel to those grown in pellet culture. The hMSCs in collagen scaffolds expressed more glycosaminoglycan than those in pellet culture. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that the expression of genes encoding sox-9, aggrecan, and types I and II collagen increased in pellet culture over time. However, in the collagen cultures, only type II collagen and aggrecan expression increased over time, whereas sox-9 expression remained unchanged and type I collagen expression decreased. These results indicate that the immortalized hMSC line is a promising tool for further in vitro chondrogenic studies.

Pectin-chitosan-PVA nanofibrous scaffold made by electrospinning and its potential use as a skin tissue scaffold

Scaffolds made of chitosan nanofibers are often too mechanically weak for their application and often their manufacturing processes involve the use of harmful and flammable organic solvents. In the attempt to improve the mechanical properties of nanofibrous scaffolds made of chitosan without the use of harmful chemicals, pectin, an anionic polymer was blended with chitosan, a cationic polymer, to form a polyelectrolyte complex and electrospun into nanofibers for the first time. The electrospun chitosan-pectin scaffolds, when compared to electrospun chitosan scaffolds, had a 58% larger diameter, a 21% higher Young’s modulus, a 162% larger strain at break, and a 104% higher ultimate tensile strength. Compared to the chitosan scaffolds, the chitosan-pectin scaffolds’ swelling ratios decreased by 55% after 60 min in a saline solution and more quickly released the preloaded tetracycline HCl. The L929 fibroblast cells proliferated slightly slower on the chitosan-pectin scaffolds than on the chitosan scaffolds. Nonetheless, cells on both materials deposited similar levels of extracellular type I collagen on a per DNA basis. In conclusion, a novel chitosan-pectin nanofibrous scaffold with superior mechanical properties than a chitosan nanofibrous scaffold was successfully made without the use of harmful solvents.

Observation and Quantification of Gas Bubble Formation on a Mechanical Heart Valve

Clinical studies using transcranial Doppler ultrasonography in patients with mechanical heart valves (MHV) have detected gaseous emboli. The relationship of gaseous emboli release and cavitation on MHV has been a subject of debate in the literature. To study the influence of cavitation and gas content on the formation and growth of stable gas bubbles, a mock circulatory loop, which employed a Medtronic-Hall pyrolytic carbon disk valve in the mitral position, was used. A high-speed video camera allowed observation of cavitation and gas bubble release on the inflow valve surfaces as a function of cavitation intensity and carbon dioxide (CO2) concentration, while an ultrasonic monitoring system scanned the aortic outflow tract to quantify gas bubble production by calculating the gray scale levels of the images. In the absence of cavitation, no stable gas bubbles were formed. When gas bubbles were formed, they were first seen a few milliseconds after and in the vicinity of cavitation collapse. The volume of the gas bubbles detected in the aortic track increased with both increased CO2 and increased cavitation intensity. No correlation was observed between O2 concentration and bubble volume. We conclude that cavitation is an essential precursor to stable gas bubble formation, and CO2, the most soluble blood gas, is the major component of stable gas bubbles.

RING domains functioning as E3 ligases reveal distinct structural features: a molecular dynamics simulation study.

Abstract RING domain, a cysteine-rich motif that chelates two zinc ions, has been shown to regulate many biological processes such as mediating a crucial step in the ubiquitinylation pathway In order to investigate the distinct structural features for the RING domains functioning as E3 ligases, several molecular dynamics simulations involving the c-Cbl, CNOT4 (with E3 ligase function), and p44 (no E3 ligase function) RING domains were conducted in this study. Our results reveal that the structural stability of the recognition site is a basic requirement for the RING domains functioning as E3 ligases. The structural stability of the recognition site is maintained by the hydrophobic core and hydrogen bonding network. Another important structural feature of the RING domains functioning as E3 ligases is the stable distances between the recognition site and the zinc ion binding sites S1 and S2. Moreover, the RING domains functioning as E3 ligases seem to exhibit lower β stability due to the higher proportion of proline residues in their sequences. However, no significant difference of the other secondary (α and turn) and the tertiary structural stabilities can be observed among these three RING domains.

Characterization of electrospun nanofiber matrices made of collagen blends as potential skin substitutes

Collagen and its blends, collagen/polyvinyl alcohol (PVA) and collagen/chitosan/PVA, were made into nanofibers by electrospinning. The nanofibrous matrices were evaluated for their potential as skin substitutes. The addition of PVA to collagen increased the swelling ratio of the nanofibers, their Youngs modulus, strain at break and ultimate tensile strength. The addition of chitosan to collagen/PVA reduced its swelling ratio and its strain at break, but increased the Youngs modulus and ultimate tensile strength. Both PVA and chitosan stabilized the collagen fibers in an aqueous solution. The addition of PVA, but not chitosan, promoted initial fibroblast cell proliferation on the matrices. Compared to the skin substitute made of pure collagen, the substitutes with PVA and chitosan showed improved structural stability in aqueous solution, better tensile strength and similar or better biocompatibility in vitro.

Low-frequency Electromagnetic Field Exposure Accelerates Chondrocytic Phenotype Expression on Chitosan Substrate

To find whether low-frequency pulsed electromagnetic fields help repair larger cartilage defects with the assistance of tissue-engineered scaffolds, we tested their effect on the behavior of chondrocyte cells cultured on chitosan films. Primary porcine chondrocytes growing on chitosan films were exposed to low-frequency pulsed electromagnetic fields (frequency=75 Hz; impulse width=1.3 ms; strength=1.8-3 mT) 2 hours a day for 3 weeks. The cells that were not exposed to low-frequency pulsed electromagnetic fields served as controls. For 3 weeks, cell proliferation, viability, and expressions of type II collagen and glycosaminoglycan were measured weekly. Cell morphology and histological stains of glycosaminoglycan and type II collagen were performed at the end of the test. The cell proliferation and viability of the low-frequency pulsed electromagnetic fields group and the control were similar each week. By the end of the third week, cells in the low-frequency pulsed electromagnetic fields group deposited 28% more glycosaminoglycan than the control cells. The amounts of type II collagen deposited in the low-frequency pulsed electromagnetic fields group were 24% and 27% higher than those of the control group by week 2 and 3, respectively. Histological and immunohistochemical analyses confirmed the releases of glycosaminoglycan and type II collagen. Cells from both groups grew in aggregates and possessed a spherical shape after 3 weeks. These results suggest that low-frequency pulsed electromagnetic fields can enhance extracellular matrix production on chitosan substrate. Combining tissue engineering and low-frequency pulsed electromagnetic fields could further accelerate cartilage repair.