Selective Inhibition of PAR4 (Protease-Activated Receptor 4)-Mediated Platelet Activation by a Synthetic Nonanticoagulant Heparin Analog.

Objective- PAR4 (protease-activated receptor 4), one of the thrombin receptors in human platelets, has emerged as a promising target for the treatment of arterial thrombotic disease. Previous studies implied that thrombin exosite II, known as a binding site for heparin, may be involved in thrombin-induced PAR4 activation. In the present study, a heparin octasaccharide analog containing the thrombin exosite II-binding domain of heparin was chemically synthesized and investigated for anti-PAR4 effect. Approach and Results- PAR4-mediated platelet aggregation was examined using either thrombin in the presence of a PAR1 antagonist or γ-thrombin, which selectively activates PAR4. SCH-28 specifically inhibits PAR4-mediated platelet aggregation, as well as the signaling events downstream of PAR4 in response to thrombin. Moreover, SCH-28 prevents thrombin-induced ß-arrestin recruitment to PAR4 but not PAR1 in Chinese Hamster Ovary-K1 cells using a commercial enzymatic complementation assay. Compared with heparin, SCH-28 is more potent in inhibiting PAR4-mediated platelet aggregation but has no significant anticoagulant activity. In an in vitro thrombosis model, SCH-28 reduces thrombus formation under whole blood arterial flow conditions. Conclusions- SCH-28, a synthetic small-molecular and nonanticoagulant heparin analog, inhibits thrombin-induced PAR4 activation by interfering with thrombin exosite II, a mechanism of action distinct from other PAR4 inhibitors that target the receptor. The characteristics of SCH-28 provide a new strategy for targeting PAR4 with the potential for the treatment of arterial thrombosis.

Dual-acting antibacterial porous chitosan film embedded with a photosensitizer.

This study proposes to develop a dual-acting antibacterial film of porous chitosan (Cs) embedded with small molecular compound, which possesses photosensitive characteristics with bactericidal efficacy, to promote the accelerated recovery of infectious wounds. The Cs/small molecular compound (Cs-cpd.2) dressing was prepared using the freeze-drying method. Characterization of the synthesized Cs-cpd.2 indicated that it has high porosity and moisture absorption effect, hence enhancing the absorption of wound exudate. Experimental results showed that Cs-cpd.2 dressing has good bactericidal and bacteriostatic effects on Staphylococcus aureus under visible-light irradiation and has antibacterial effect in the dark. It was also found that the small molecular compound does not have cytotoxicity at a dose of 0-5 µM. Furthermore, Cs-cpd.2 that contained small molecular compound with a concentration of 0.3-1 µM has positive effect on both the cell viability rate and cell proliferation rate of human fibroblast CG1639. Cs-cpd.2 can significantly promote cell proliferation when the small molecular compound and the basic fibroblast growth factor bFGF were added together. Therefore, the proposed Cs-cpd.2 dressing is feasible for photodynamic therapy (PDT) and clinical wound dressing applications.

Density Functional Theory Calculation May Confirm Arsenic-Thiol Adhesion as the Primary Mechanism of Arsenical Toxicity.

Previously, it was believed that methylation was the body's primary method to detoxify inorganic arsenic. However, recent research has shown that the metabolized intermediate known as MMAIII is more toxic than arsenite and arsenate, contradicting a previous understanding. Another important question arises: is arsenical toxicity truly caused by arsenic binding to proteins through arsenic thiol adhesion? Based on the toxicity order of the experiment, with MMAIII being the most toxic, followed by arsenite, arsenate, DMAV, and MMAV, density functional theory (DFT) calculations can provide a straightforward assessment of this issue. Our practice captures all the transition states associated with a specific imaginary-frequency vibration mode, including proton transfer and simultaneous departure of leaving group. We have obtained the energy barriers for five arsenicals reacting with thiol, alcohol, and amine separately. In addition to energetic favorability, the following are the energy barriers for arsenic's reaction with thiol ranked from low to high: MMAIII (25.4 kcal/mol), arsenite (27.7 kcal/mol), arsenate (32.8 kcal/mol), DMAV (36.2 kcal/mol), and MMAV (38.3 kcal/mol). Results show that the toxicity of arsenicals is mainly caused by their reaction with thiol rather than with alcohol or amine, as supported by the trend of decreasing toxicity and increasing energy barriers. Thus, this DFT calculation may confirm the paradigm that arsenic-thiol adhesion is the primary cause of arsenic toxicity in the body.

Synthesis, DNA-binding abilities and anticancer activities of triazole-pyrrolo[2,1-c][1,4]benzodiazepines hybrid scaffolds.

We synthesized a new series of PBD-hybrid derivatives having tethered triazoles and investigated for their cytotoxicity. The studies indicated that cis-olefin compounds induce higher cytotoxicity with increase in the G1 cell cycle phase compared with the trans-compounds. Quantitative RT-PCR assay indicated that compounds (16a-d) induced G1 phase arrest through down-regulation of cyclin D1 and up-regulation of p21, p27, and p53 mRNA expressions. Compounds 16a-d induced A375 early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide. Moreover, the Western blot analysis showed that A375 treated by compounds (16a-d) resulted in decreased levels of Bcl-2 and Bcl-xL, increased levels of Bax and Bad, and caspase/PARP degradation to identify apoptotic cells.

Novel anilinocoumarin derivatives as agents against hepatitis C virus by the induction of IFN-mediated antiviral responses.

The hepatitis C virus (HCV) is the main cause of progressive liver disease, leading to the development of liver cirrhosis and hepatocellular carcinoma (HCC). Novel anilinocoumarins were synthesized, and their efficacy against HCV replication was evaluated. We demonstrated that 3-(3',4',5'-trimethoxyanilin-1'-yl)methylaminocoumarin (6) exhibited strong anti-HCV activity at protein and RNA levels at non-toxic concentrations, with an EC(50) value of 12 ± 0.3 µM and a selective index (SI) value of 10. Combined treatment of compound 6 and interferon-α (IFN) or telaprevir induced a significant decrease in HCV RNA levels, respectively. We also found that the anti-HCV replication effect of compound 6 was due to the induction of IFN-mediated antiviral responses. This is the first report demonstrating that coumarins inhibit viral replication through an IFN-mediated anti-viral response. Collectively, compound 6 possessed potent activities against HCV replication and could be a new lead compound with higher selectivity and less toxicity.

Toll-like receptor and hepatitis B virus clearance in chronic infected patients: a long-term prospective cohort study in Taiwan.

BACKGROUND: We sought to elucidate the impacts of the Toll-like receptors (TLRs) on spontaneous hepatitis B virus (HBV) e antigen (HBeAg) and hepatitis B s antigen (HBsAg) seroconversion in chronic HBV-infected patients. METHODS: Human TLR2, TLR4, TLR5, and TLR9 gene polymorphisms were assessed in 278 HBeAg-positive, chronic HBV-infected patients. Additionally, HBV core antigen (HBcAg) in vitro stimulation using peripheral blood mononuclear cells (PBMCs) from 113 patients was done to assess interferon γ (IFN- γ) production. RESULTS: Of the study subjects, 204 (73.4%) developed spontaneous HBeAg seroconversion, 21 (7.6%) developed spontaneous HBsAg clearance, and 10 (3.6%) had spontaneous HBsAg seroconversion during the 19.1 ± 9.9 years of follow-up. The T allele at TLR5 rs5744174 (p.Phe616Leu) and the C allele at TLR9 rs5743836 promoter predicted earlier HBeAg seroconversion (hazard ratios [HRs], 2.45 and 3.65; P = .04, and .006, respectively). The TLR5 rs5744174 T allele carriers have higher PBMCs IFN-γ secretion to HBcAg stimulation (P= .0002). The G allele carriers at TLR4 rs4986790 (p.Asp299Gly) predicted spontaneous HBsAg seroclearance (HR, 18.73; P < .001) and seroconversion (HR, 43.45; P < .001). CONCLUSIONS: Toll-like receptor 5 rs5744174 (p.Phe616Leu) and TLR9 rs5743836 promoter area polymorphism were associated with earlier spontaneous HBeAg seroconversion. Toll-like receptor 4 rs4986790 (p.Asp299Gly) was associated with HBsAg seroclearance/seroconversion in chronic HBV patients. Toll-like receptor 5 rs5744174 (p.Phe616Leu) was associated with higher IFN-γ production in chronic HBV-infected patients.

Deep Learning-Based Automatic Duckweed Counting Using StarDist and Its Application on Measuring Growth Inhibition Potential of Rare Earth Elements as Contaminants of Emerging Concerns.

In recent years, there have been efforts to utilize surface water as a power source, material, and food. However, these efforts are impeded due to the vast amounts of contaminants and emerging contaminants introduced by anthropogenic activities. Herbicides such as Glyphosate and Glufosinate are commonly known to contaminate surface water through agricultural industries. In contrast, some emerging contaminants, such as rare earth elements, have started to enter the surface water from the production and waste of electronic products. Duckweeds are angiosperms from the Lemnaceae family and have been used for toxicity tests in aquatic environments, mainly those from the genus Lemna, and have been approved by OECD. In this study, we used duckweed from the genus Wolffia, which is smaller and considered a good indicator of metal pollutants in the aquatic environment. The growth rate of duckweed is the most common endpoint in observing pollutant toxicity. In order to observe and mark the fronds automatically, we used StarDist, a machine learning-based tool. StarDist is available as a plugin in ImageJ, simplifying and assisting the counting process. Python also helps arrange, manage, and calculate the inhibition percentage after duckweeds are exposed to contaminants. The toxicity test results showed Dysprosium to be the most toxic, with an IC50 value of 14.6 ppm, and Samarium as the least toxic, with an IC50 value of 279.4 ppm. In summary, we can provide a workflow for automatic frond counting using StarDist integrated with ImageJ and Python to simplify the detection, counting, data management, and calculation process.

A scaffold-independent subcellular event-based analysis: characterization of significant structural modifications.

General and singular subcellular events within the ligand-dependent receptor-mediated cellular response were separated by using the Jurs and the electrotopological state (ES) descriptors, allowing characterization of the significant structural modifications in a given set of collected peroxisome proliferator-activated receptor γ (PPARγ) agonists. The identified Jurs descriptor is the integrated function of all the general events but is scaffold-dependent. The top captured ES descriptors stand for significant structural modifications, i.e., singular events. To further elucidate the descriptor-event relationship, three biological data sets show that the Jurs descriptor can be further divided into three important descriptors, the log D, polar surface area, and shape-like descriptor. The identification of the essential descriptors for general events is the first regression, and the prioritization of all the possible structural modifications of the 46 collected thiazolidinedione PPARγ agonists is the second regression. As results, the top captured ES symbols can correspond to the singular ligand--receptor interactions as highlighted in the X-ray crystallographic image of rosiglitazone--PPARγ complex.

10-Shogaol, an antioxidant from Zingiber officinale for skin cell proliferation and migration enhancer.

In this work, one of Zingiber officinale components, 10-shogaol, was tested with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, metal chelating ability, and reducing power to show antioxidant activity. 10-Shogaol promoted human normal epidermal keratinocytes and dermal fibroblasts cell growths. 10-Shogaol enhanced growth factor production in transforming growth factor-ß (TGF-ß), platelet derived growth factor-αß (PDGF-αß) and vascular endothelial growth factors (VEGF) of both cells. In the in vitro wound healing assay for 12 or 24 h, with 10-shogaol, the fibroblasts and keratinocytes migrated more rapidly than the vehicle control group. Thus, this study substantiates the target compound, 10-shogaol, as an antioxidant for human skin cell growth and a migration enhancer with potential to be a novel wound repair agent.

Genetically Modified Soybean Detection Using a Biosensor Electrode with a Self-Assembled Monolayer of Gold Nanoparticles.

In this study, we proposed a genosensor that can qualitatively and quantitatively detect genetically modified soybeans using a simple electrode with evenly distributed single layer gold nanoparticles. The DNA sensing electrode is made by sputtering a gold film on the substrate, and then sequentially depositing 1,6-hexanedithiol and gold nanoparticles with sulfur groups on the substrate. Then, the complementary to the CaMV 35S promoter (P35S) was used as the capture probe. The target DNA directly extracted from the genetically modified soybeans rather than the synthesized DNA segments was used to construct the detection standard curve. The experimental results showed that our genosensor could directly detect genetically modified genes extracted from soybeans. We obtained two percentage calibration curves. The calibration curve corresponding to the lower percentage range (1-6%) exhibits a sensitivity of 2.36 Ω/% with R2 = 0.9983, while the calibration curve corresponding to the higher percentage range (6-40%) possesses a sensitivity of 0.1 Ω/% with R2 = 0.9928. The limit of detection would be 1%. The recovery rates for the 4% and 5.7% GMS DNA were measured to be 104.1% and 102.49% with RSD at 6.24% and 2.54%. The gold nanoparticle sensing electrode developed in this research is suitable for qualitative and quantitative detection of genetically modified soybeans and can be further applied to the detection of other genetically modified crops in the future.

Discovery of 7, 4'-dimethoxy-3-hydroxyflavone as a protease-activated receptor 4 antagonist with antithrombotic activity and less bleeding tendency in mice.

There is growing evidence of the importance of protease-activated receptor 4 (PAR4), one of thrombin receptors, as a therapeutic target in thrombotic cardiovascular diseases. In the present study, we utilized ligand-based virtual screening, bioassay, and structure-activity relationship study to discover PAR4 antagonists with new chemical scaffolds from natural origin, and examined their application as antiplatelet agents. By using these approaches, we have identified a flavonoid, 7, 4'-dimethoxy-3-hydroxyflavone, that exhibits anti-PAR4 activity. 7, 4'-Dimethoxy-3-hydroxyflavone inhibited PAR4-mediated human platelet aggregation, GPIIb/IIIa activation, and P-selectin secretion. Also, it inhibited PAR4 downstream signaling pathways, including Ca2+/protein kinase C, Akt, and MAP kinases ERK and p38, in human platelets, and suppressed PAR4-mediated ß-arrestin recruitment in CHO-K1 cells exogenously expressed human PAR4. In a microfluidic system, 7, 4'-dimethoxy-3-hydroxyflavone reduced thrombus formation on collagen-coated chambers at an arterial shear rate in recalcified whole blood. Furthermore, mice treated with 7, 4'-dimethoxy-3-hydroxyflavone were significantly protected from FeCl3-induced carotid arterial occlusions, without significantly affecting tail bleeding time. In conclusion, 7, 4'-dimethoxy-3-hydroxyflavone represents a new class of nature-based PAR4 antagonist, it shows effective in vivo antithrombotic properties with less bleeding tendency, and could be a potential candidate for developing new antiplatelet agents.

Lanthanides Toxicity in Zebrafish Embryos Are Correlated to Their Atomic Number.

Rare earth elements (REEs) are critical metallic materials with a broad application in industry and biomedicine. The exponential increase in REEs utilization might elevate the toxicity to aquatic animals if they are released into the water due to uncareful handling. The specific objective of our study is to explore comprehensively the critical factor of a model Lanthanide complex electronic structures for the acute toxicity of REEs based on utilizing zebrafish as a model animal. Based on the 96 h LC50 test, we found that the majority of light REEs display lower LC50 values (4.19-25.17 ppm) than heavy REEs (10.30-41.83 ppm); indicating that they are atomic number dependent. Later, linear regression analyses further show that the average carbon charge on the aromatic ring (aromatic Cavg charge) can be the most significant electronic structural factor responsible for the Lanthanides' toxicity in zebrafish embryos. Our results confirm a very strong correlation of LC50 to Lanthanide's atomic numbers (r = 0.72), Milliken charge (r = 0.70), and aromatic Cavg charge (r = -0.85). This most significant correlation suggests a possible toxicity mechanism that the Lanthanide cation's capability to stably bind to the aromatic ring on the residue of targeted proteins via a covalent chelating bond. Instead, the increasing ionic bond character can reduce REEs' toxicity. In addition, Lanthanide toxicity was also evaluated by observing the disruption of photo motor response (PMR) activity in zebrafish embryos. Our study provides the first in vivo evidence to demonstrate the correlation between an atomic number of Lanthanide ions and the Lanthanide toxicity to zebrafish embryos.

Comparison of the psychoactive activity of four primary Areca nut alkaloids in zebrafish by behavioral approach and molecular docking.

Areca palm nut (Areca catechu) has been listed as one of the most addictive substances, along with tobacco, alcohol, and caffeine. It belongs to the family Arecaceae and is widely used in Asia. Areca nut contains seven psychoactive alkaloids; however, the effects of these alkaloids on behaviors are rarely to be addressed in zebrafish. Therefore, this study aims to compare the psychoactive and potential adverse effects of four primary alkaloids (arecoline, arecaidine, guvacine, and guvacoline) isolated from areca nut on zebrafish. We found that four alkaloids induced hyperactivity-like behaviors in zebrafish larvae. Cooperating the results with the previous study, molecular docking scores suggested these alkaloids might bind to multiple muscarinic acetylcholine receptors (mAChRs), and various best binding modes were shown. According to the adult zebrafish behavioral test, arecoline was found to slightly increase the locomotor activity and caused tightening shoaling formations of adult zebrafish. Meanwhile, zebrafish exposed to arecaidine have reduced aggressiveness and conspecific social interaction. Similar to arecaidine, guvacoline treatment also caused abnormalities in zebrafish social behaviors. Furthermore, the fish displayed abnormal exploratory behaviors after being exposed to guvacoline. Interestingly, altered fear response behaviors were only displayed by guvacine-treated fish besides their lower locomotor activity. Based on the results of molecular docking, we hypothesize that the behavior alterations might be a consequence of the interaction between alkaloids and multiple mAChRs in the nervous system. In summary, our study found that each alkaloid specifically affects adult zebrafish behaviors.

Human interleukin-10 genotypes are associated with different precore/core gene mutation patterns in children with chronic hepatitis B virus infection.

OBJECTIVE: To elucidate the association between human interleukin-10 (IL-10) genotypes and hepatitis B virus (HBV) precore/core gene mutation in children with chronic HBV infection. STUDY DESIGN: The study group comprised of 21 children with chronic HBV infection with spontaneous hepatitis B e antigen (HBeAg) seroconversion who were followed for more than 10 years. Another nine children without HBeAg seroconversion served as the control subjects. Sera at the immune tolerance and inflammatory phase (alanine aminotransferase, >80 IU/L) were subjected to HBV precore/core sequence analysis. IL-10 -1082 polymorphism was also determined. RESULTS: HBV precore/core gene mutation increased significantly more in the inflammatory phase than in the tolerance phase (G1896A, 76.2% versus 4.8%; C1913A, 33.3% versus 0%; C2189A, 28.6% versus 4.8%; G2304A, 52.4% versus 14.3%) in study group (n = 21) but not the control group (n = 9). Subjects with the G/G genotype at the IL-10-1082 polymorphism site had higher C2189A mutation rate than the A allele carriers (P = .02). C2189A mutation carriers are associated with more viral load decrement from tolerance to inflammatory phase (P = .01) and earlier spontaneous HBeAg seroconversion (P = .01). CONCLUSIONS: The G/G genotype at the IL-10 -1082 polymorphism is associated with higher C2189A mutations, lower HBV viral load at immune inflammatory phase, and earlier spontaneous HBeAg seroconversion than A allele carriers.

Suppression of Heregulin-ß1/HER2-Modulated Invasive and Aggressive Phenotype of Breast Carcinoma by Pterostilbene via Inhibition of Matrix Metalloproteinase-9, p38 Kinase Cascade and Akt Activation.

Invasive breast cancer is the major cause of death among females and its incidence is closely linked to HER2 (human epidermal growth factor receptor 2) overexpression. Pterostilbene, a natural analog of resveratrol, exerts its cancer chemopreventive activity similar to resveratrol by inhibiting cancer cell proliferation and inducing apoptosis. However, the anti-invasive effect of pterostilbene on HER2-bearing breast cancer has not been evaluated. Here, we used heregulin-ß1 (HRG-ß1), a ligand for HER3, to transactivate HER2 signaling. We found that pterostilbene was able to suppress HRG-ß1-mediated cell invasion, motility and cell transformation of MCF-7 human breast carcinoma through down-regulation of matrix metalloproteinase-9 (MMP-9) activity and growth inhibition. In parallel, pterostilbene also inhibited protein and mRNA expression of MMP-9 driven by HRG-ß1, suggesting that pterostilbene decreased HRG-ß1-mediated MMP-9 induction via transcriptional regulation. Examining the signaling pathways responsible for HRG-ß1-associated MMP-9 induction and growth inhibition, we observed that pterostilbene, as well as SB203580 (p38 kinase inhibitor), can abolish the phosphorylation of p38 mitogen-activated protein kinase (p38 kinase), a downstream HRG-ß1-responsive kinase responsible for MMP-9 induction. In addition, HRG-ß1-driven Akt phosphorylation required for cell proliferation was also suppressed by pterostilbene. Taken together, our present results suggest that pterostilbene may serve as a chemopreventive agent to inhibit HRG-ß1/HER2-mediated aggressive and invasive phenotype of breast carcinoma through down-regulation of MMP-9, p38 kinase and Akt activation.

Evaluation of Effects of Ractopamine on Cardiovascular, Respiratory, and Locomotory Physiology in Animal Model Zebrafish Larvae.

Ractopamine (RAC) is a beta-adrenoceptor agonist that is used to promote lean and increased food conversion efficiency in livestock. This compound has been considered to be causing behavioral and physiological alterations in livestock like pig. Few studies have addressed the potential non-target effect of RAC in aquatic animals. In this study, we aimed to explore the potential physiological response after acute RAC exposure in zebrafish by evaluating multiple endpoints like locomotor activity, oxygen consumption, and cardiovascular performance. Zebrafish larvae were subjected to waterborne RAC exposure at 0.1, 1, 2, 4, or 8 ppm for 24 h, and the corresponding cardiovascular, respiratory, and locomotion activities were monitored and quantified. In addition, we also performed in silico molecular docking for RAC with 10 zebrafish endogenous ß-adrenergic receptors to elucidate the potential acting mechanism of RAC. Results show RAC administration can significantly boost locomotor activity, cardiac performance, oxygen consumption, and blood flow rate, but without affecting the cardiac rhythm regularity in zebrafish embryos. Based on structure-based flexible molecular docking, RAC display similar binding affinity to all ten subtypes of endogenous ß-adrenergic receptors, from adra1aa to adra2db, which are equivalent to the human one. This result suggests RAC might act as high potency and broad spectrum ß-adrenergic receptors agonist on boosting the locomotor activity, cardiac performance, and oxygen consumption in zebrafish. To validate our results, we co-incubated a well-known ß-blocker of propranolol (PROP) with RAC. PROP exposure tends to minimize the locomotor hyperactivity, high oxygen consumption, and cardiac rate in zebrafish larvae. In silico structure-based molecular simulation and binding affinity tests show PROP has an overall lower binding affinity than RAC. Taken together, our studies provide solid in vivo evidence to support that RAC plays crucial roles on modulating cardiovascular, respiratory, and locomotory physiology in zebrafish for the first time. In addition, the versatile functions of RAC as ß-agonist possibly mediated via receptor competition with PROP as ß-antagonist.

Pharmaceutical Assessment Suggests Locomotion Hyperactivity in Zebrafish Triggered by Arecoline Might Be Associated with Multiple Muscarinic Acetylcholine Receptors Activation.

Arecoline is one of the nicotinic acid-based alkaloids, which is found in the betel nut. In addition to its function as a muscarinic agonist, arecoline exhibits several adverse effects, such as inducing growth retardation and causing developmental defects in animal embryos, including zebrafish, chicken, and mice. In this study, we aimed to study the potential adverse effects of waterborne arecoline exposure on zebrafish larvae locomotor activity and investigate the possible mechanism of the arecoline effects in zebrafish behavior. The zebrafish behavior analysis, together with molecular docking and the antagonist co-exposure experiment using muscarinic acetylcholine receptor antagonists were conducted. Zebrafish larvae aged 96 h post-fertilization (hpf) were exposed to different concentrations (0.001, 0.01, 0.1, and 1 ppm) of arecoline for 30 min and 24 h, respectively, to find out the effect of arecoline in different time exposures. Locomotor activities were measured and quantified at 120 hpf. The results showed that arecoline caused zebrafish larvae locomotor hyperactivities, even at a very low concentration. For the mechanistic study, we conducted a structure-based molecular docking simulation and antagonist co-exposure experiment to explore the potential interactions between arecoline and eight subtypes, namely, M1a, M2a, M2b, M3a, M3b, M4a, M5a, and M5b, of zebrafish endogenous muscarinic acetylcholine receptors (mAChRs). Arecoline was predicted to show a strong binding affinity to most of the subtypes. We also discovered that the locomotion hyperactivity phenotypes triggered by arecoline could be rescued by co-incubating it with M1 to M4 mAChR antagonists. Taken together, by a pharmacological approach, we demonstrated that arecoline functions as a highly potent hyperactivity-stimulating compound in zebrafish that is mediated by multiple muscarinic acetylcholine receptors.

A cell-based reporter assay for inhibitor screening of hepatitis C virus RNA-dependent RNA polymerase.

The hepatitis C virus (HCV) NS5B, a RNA-dependent RNA polymerase (RdRp), is an attractive target for anti-HCV agents. The major disadvantages of the commonly used polymerase inhibitor screening involving the assessment of in vitro RNA synthesis are that it is incapable of demonstrating the cellular permeability and the cytotoxicity of compounds. To overcome these limitations, we created the BHK-NS5B-FRLuc reporter cell line that carries stably transfected NS5B and a bicistronic reporter gene, (+)FLuc-(-)UTR-RLuc, which can be used to simultaneously measure cellular toxicity and intracellular RdRp activity. The (+)FLuc-(-)UTR-RLuc construct comprises the firefly luciferase (FLuc) gene and the Renilla luciferase (RLuc) gene in reverse orientation flanked by both negative strands of the HCV 5'- and 3'-untranslated regions (UTRs), in which FLuc and RLuc reporter proteins are regulated by host polymerase and functional NS5B polymerase, respectively. The reporter system was validated with specific agents against NS5B polymerization. Additionally, this assay was placed in 96-well plates and had a Z'-factor value of approximately 0.75, which is amenable for facilitating high-throughput screening operations. Notably, in combination with the structured-based virtual screening, an imidazole derivative compound was evaluated as a candidate HCV RdRp inhibitor.

Tumour necrosis factor-α promoter region polymorphisms affect the course of spontaneous HBsAg clearance.

BACKGROUND: This study aimed to investigate the roles of tumour necrosis factor-α (TNF-α) gene polymorphisms in the spontaneous clearance of HBsAg after a hepatitis B virus (HBV) infection. METHODS: Polymorphisms in the TNF-α (-1031 T to C, -863 C to A, -857 C to T, -308 G to A and -238 G to A transition) gene were evaluated in 274 chronic HBV-infected patients and 194 patients with resolved HBV infection. The peripheral blood mononuclear cells (PBMC) isolated from 77 (28%) of the 274 chronic HBV-infected patients with negative HBeAg and positive antibody to HBeAg were stimulated with HBcAg. Data on TNF-α genotypes and phenotypes in subjects with/without the A allele at the TNF-α-863 promoter single nucleotide polymorphism (rs1800630) were compared. RESULTS: The A allele in the -863 promoter region of the TNF-α gene was present in 154 (56.2%) chronic HBV-infected patients and 87 (44.8%) patients who recovered from HBV infection (odds ratio 1.58; P<0.01). The TNF-α-863 A allele genotype predicted lower TNF-α production by PBMC after in vitro HBcAg stimulation (P<0.02). CONCLUSIONS: The A allele at the -863 locus of the promoter region of the TNF-α gene predicts lower HBcAg-inducible TNF-α secretion. It is also associated with chronicity of HBV infection.

Identifying new liver X receptor alpha modulators and distinguishing between agonists and antagonists by crystal ligand pocket screening.

Background: Modulators of LXRα are of high pharmacological interest as LXRα regulates fatty acid metabolism, inflammatory processes and cancer. We aim to identify new LXRα modulators and to recognize a distinguishable feature of agonists. Results&methodology: The ligand self-dock and largest-cavity-size searching purposely located two appropriate ligand-binding sites to reach the two aims. One is identifying the new modulators from Maybridge library. 20 new compounds are confirmed by the in vitro reporter gene assay. The other is denoting an agonist by at least one best docking pose having one hydrogen bond to LXRα Helix12 His421. Conclusion: Based on the quality x-ray binding pocket, we can identify new LXRα modulators and distinguish between agonists and antagonists by molecular docking.