Performance dependence of a stick-slip piezoelectric actuator on the angle between the piezoelectric stack and mover.

The angle between the piezoelectric stack (PES) and the mover would significantly affect the output performances of stick-slip piezoelectric actuators in theory; however, this issue has not been explored. Accordingly, in this paper, to investigate the effects of the angle, a stick-slip piezoelectric actuator with a changeable angle between the PES and the mover was proposed, and its structure was briefly introduced first. Then, the relationship between the angle and the parasitic motion was theoretically calculated. After that, the output performances of the actuator were characterized under different angles between the PES and the mover. The results indicated that the angle between the PES and the mover significantly affected the one-step maximum displacement and the backward motion. After comprehensive analysis, the best output performances of the actuator were obtained under the angle of 0°.

The Active Constituent From Gynostemma Pentaphyllum Prevents Liver Fibrosis Through Regulation of the TGF-ß1/NDRG2/MAPK Axis.

Liver fibrosis resulting from chronic liver damage constitutes a major health care burden worldwide; however, no antifibrogenic agents are currently available. Our previous study reported that the small molecule NPLC0393 extracted from the herb Gynostemma pentaphyllum exerts efficient antifibrotic effects both in vivo and in vitro. In this study, a TMT-based quantitative proteomic study using a carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was performed to identify the potential target of NPLC0393. Combining this study with bioinformatic analysis of differentially expressed proteins between the CCl4 model and NPLC0393 treatment groups, we focused on the function of N-myc downstream-regulated gene 2 (NDRG2) involved in cell differentiation. In vitro studies showed that NPLC0393 prevented the TGF-ß1 stimulation-induced decrease in the NDRG2 level in hepatic stellate cells (HSCs). Functional studies indicated that NDRG2 can inhibit the activation of HSCs by preventing the phosphorylation of ERK and JNK. Furthermore, knockdown of NDRG2 abolished the ability of NPLC0393 to inhibit HSC activation. In conclusion, these results provide information on the mechanism underlying the antifibrotic effect of NPLC0393 and shed new light on the potential therapeutic function of the TGF-ß1/NDRG2/MAPK signaling axis in liver fibrosis.

A stick-slip piezoelectric actuator with high consistency in forward and reverse motions.

This paper presents a stick-slip piezoelectric actuator with high consistency in performances of forward and reverse motions. It is achieved by developing an integrated stator which bonds two lead zirconate titanate ceramic plates to a symmetrical flexible hinge mechanism. The working principle of the actuator was introduced, and the stator was optimized by finite-element analysis. Experimental results showed that the proposed actuator had an excellent consistency in output performances of forward and reverse motions with or without an external load. The positioning resolution, maximum speed, and maximum loading capacity of the actuator were 0.061 µm, 2195.29 µm/s, and 1.1 N, respectively. This study provides a solution for improving the forward and reverse motion consistency of stick-slip piezoelectric actuators.

TGF-ß1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular SAM.

BACKGROUND: Hepatic stellate cell (HSC) activation induced by transforming growth factor ß1 (TGF-ß1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-ß1 in such process are largely unknown. METHODS: We performed pharmacoproteomic profiling of the mice liver tissues from control, carbon tetrachloride (CCl4)-induced fibrosis and NPLC0393 administrated groups. The target gene MAT2A was overexpressed or knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional activity on MAT2A promoter via luciferase assay. Intracellular SAM contents were analyzed by LC-MS method. FINDINGS: We found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl4-induced fibrosis mice, and application of NPLC0393, a known small molecule inhibitor of TGF-ß1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-ß1 induces phosphorylation of p65, i.e., activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl4- and TGF-ß1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393. INTERPRETATION: This study identifies TGF-ß1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis. FUND: This work was supported by National Natural Science Foundation of China (No. 81500469, 81573873, 81774196 and 31800693), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502).

Integration of virtual screening and susceptibility test to discover active-site subpocket-specific biogenic inhibitors of Helicobacter pylori shikimate dehydrogenase

Shikimate dehydrogenase (HpSDH) (EC 1.1.1.25) is a key enzyme in the shikimate pathway of Helicobacter pylori (H. pylori), which catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. Targeting HpSDH has been recognized as an attractive therapeutic strategy against H. pylori infection. Here, the catalytic active site in the crystal structure of HpSDH in complex with its substrate NADPH and product shikimate was examined in detail; the site can be divided into three spatially separated subpockets that separately correspond to the binding regions of shikimate, NADPH dihydronicotinamide moiety, and NADPH adenine moiety. Subsequently, a cascading protocol that integrated virtual screening and antibacterial test was performed against a biogenic compound library to identify biologically active, subpocket-specific inhibitors. Consequently, five, eight, and six promising compounds for, respectively, subpockets 1, 2, and 3 were selected from the top-100 docking-ranked hits, from which 11 compounds were determined to have high or moderate antibacterial potencies against two reference H. pylori strains, with MIC range between 8 and 93 μg/mL. It is found that the HpSDH active site prefers to accommodate amphipathic and polar inhibitors that consist of an aromatic core as well as a number of oxygen-rich polar/charged substituents such as hydroxyl, carbonyl, and carboxyl groups. Subpockets 1- and 2-specific inhibitors exhibit a generally higher activity than subpocket 3-specific inhibitors. Molecular dynamics simulations revealed an intense nonbonded network of hydrogen bonds, π-π stacking, and van der Waals contacts at the tightly packed complex interfaces of active-site subpockets with their cognate inhibitors, conferring strong stability and specificity to these complex systems. Binding energetic analysis demonstrated that the identified potent inhibitors can target their cognate subpockets with an effective selectivity over noncognate ones

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Integration of virtual screening and susceptibility test to discover active-site subpocket-specific biogenic inhibitors of Helicobacter pylori shikimate dehydrogenase.

Shikimate dehydrogenase (HpSDH) (EC 1.1.1.25) is a key enzyme in the shikimate pathway of Helicobacter pylori (H. pylori), which catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. Targeting HpSDH has been recognized as an attractive therapeutic strategy against H. pylori infection. Here, the catalytic active site in the crystal structure of HpSDH in complex with its substrate NADPH and product shikimate was examined in detail; the site can be divided into three spatially separated subpockets that separately correspond to the binding regions of shikimate, NADPH dihydronicotinamide moiety, and NADPH adenine moiety. Subsequently, a cascading protocol that integrated virtual screening and antibacterial test was performed against a biogenic compound library to identify biologically active, subpocket-specific inhibitors. Consequently, five, eight, and six promising compounds for, respectively, subpockets 1, 2, and 3 were selected from the top-100 docking-ranked hits, from which 11 compounds were determined to have high or moderate antibacterial potencies against two reference H. pylori strains, with MIC range between 8 and 93 µg/mL. It is found that the HpSDH active site prefers to accommodate amphipathic and polar inhibitors that consist of an aromatic core as well as a number of oxygen-rich polar/charged substituents such as hydroxyl, carbonyl, and carboxyl groups. Subpockets 1- and 2-specific inhibitors exhibit a generally higher activity than subpocket 3-specific inhibitors. Molecular dynamics simulations revealed an intense nonbonded network of hydrogen bonds, π-π stacking, and van der Waals contacts at the tightly packed complex interfaces of active-site subpockets with their cognate inhibitors, conferring strong stability and specificity to these complex systems. Binding energetic analysis demonstrated that the identified potent inhibitors can target their cognate subpockets with an effective selectivity over noncognate ones.

MiR-542-3p controls hepatic stellate cell activation and fibrosis via targeting BMP-7.

There has been an increasing number of studies about microRNAs as key regulators in the development of hepatic fibrosis. Here, we demonstrate that miR-542-3p can promote hepatic fibrosis by downregulating the expression of bone morphogenetic protein 7 (BMP-7), which is known to antagonize transforming growth factor ß1 (TGFß1)-mediated fibrogenesis effect. The expression of miR-542-3p is increased in activated hepatic stellate cells (HSCs). Downregulation of MiR-542-3p by antisense inhibitors can inhibit HSCs activation markers, including α-smooth muscle actin (α-SMA) and collagen as well as TGFß signaling pathways. MiR-542-3p was significantly upregulated in carbon tetrachloride (CCl4 )-induced hepatic fibrosis in mice, and downregulation of miR-542-3p by lentivirus could prevent the development of hepatic fibrosis. In addition, miR-542-3p can directly bind to the 3'-untranslated region of BMP-7 mRNA, indicating that its profibrotic effect appears to be caused by its inhibition of BMP-7. Our results suggest that downregulation of miR-542-3p prevents liver fibrosis both in vitro and in vivo, highlighting its potential as a novel biomarker or therapeutic target for hepatic fibrosis.

Butein activates p53 in hepatocellular carcinoma cells via blocking MDM2-mediated ubiquitination.

INTRODUCTION: In this study, we aimed to investigate the effect of butein on p53 in hepatocellular carcinoma (HCC) cells and the related molecular mechanisms by which p53 was activated. METHODS: MTS assay and clonogenic survival assay were used to examine the antitumor activity of butein in vitro. Reporter gene assay was adopted to evaluate p53 transcriptional activity. Flow cytometry and western blotting were performed to study apoptosis induction and protein expression respectively. Xenograft model was applied to determine the in vivo efficacy and the expression of p53 in tumor tissue was detected by immunohistochemistry. RESULTS: HCC cell proliferation and clonogenic survival were significantly inhibited after butein treatment. With the activation of cleaved-PARP and capsase-3, butein induced apoptosis in HCC cells in a dose-dependent manner. The transcriptional activity of p53 was substantially promoted by butein, and the expression of p53-targeted gene was increased accordingly. Mechanism studies demonstrated that the interaction between MDM2 and p53 was blocked by butein and MDM2-mediated p53 ubiquitination was substantially decreased. Short-hairpin RNA experiment results showed that the sensitivity of HCC cells to butein was substantially impaired after p53 was knocked down and butein-induced apoptosis was dramatically decreased. In vivo experiments validated substantial antitumor efficacy of butein against HepG2 xenograft growth, and the expression of p53 in butein-treated tumor tissue was significantly increased. CONCLUSION: Butein demonstrated potent antitumor activities in HCC by activating p53, and butein or its analogs had therapeutic potential for HCC management.

Discovery of a Potential Plasma Protein Biomarker Panel for Acute-on-Chronic Liver Failure Induced by Hepatitis B Virus.

Hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF), characterized by an acute deterioration of liver function in the patients with chronic hepatitis B (CHB), is lack of predicting biomarkers for prognosis. Plasma is an ideal sample for biomarker discovery due to inexpensive and minimally invasive sampling and good reproducibility. In this study, immuno-depletion of high-abundance plasma proteins followed by iTRAQ-based quantitative proteomic approach was employed to analyze plasma samples from 20 healthy control people, 20 CHB patients and 20 HBV-ACLF patients, respectively. As a result, a total of 427 proteins were identified from these samples, and 42 proteins were differentially expressed in HBV-ACLF patients as compared to both CHB patients and healthy controls. According to bioinformatics analysis results, 6 proteins related to immune response (MMR), inflammatory response (OPN, HPX), blood coagulation (ATIII) and lipid metabolism (APO-CII, GP73) were selected as biomarker candidates. Further ELISA analysis confirmed the significant up-regulation of GP73, MMR, OPN and down-regulation of ATIII, HPX, APO-CII in HBV-ACLF plasma samples (p < 0.01). Moreover, receiver operating characteristic (ROC) curve analysis revealed high diagnostic value of these candidates in assessing HBV-ACLF. In conclusion, present quantitative proteomic study identified 6 novel HBV-ACLF biomarker candidates and might provide fundamental information for development of HBV-ACLF biomarker.

Rational Molecular Design of Potent PLK1 PBD Domain-binding Phosphopeptides Using Preferential Amino Acid Building Blocks.

Polo-like kinase 1 (PLK1) is an important regulator in diverse aspects of the cell cycle and proliferation. The protein has a highly conserved polo-box domain (PBD) present in C-terminal noncatalytic region, which exhibits a relatively broad sequence specificity in recognizing and binding phosphorylated substrates to control substrate phosphorylation by the kinase. In order to elucidate the structural basis, thermodynamic property, and biological implication underlying PBD-substrate recognition and association, a systematic amino acid preference profile of phosphopeptide interaction with PLK1 PBD domain was established via virtual mutagenesis analysis and mutation energy calculation, from which the contribution of different amino acids at each residue position of two reference phosphopeptides to domain-peptide binding was characterized comprehensively and quantitatively. With the profile, we are able to determine the favorable, neutral, and unfavorable amino acid types for each position of PBD-binding phosphopeptides, and we also explored the molecular origin of the broad sequence specificity in PBD-substrate recognition. To practice computational findings, the profile was further employed to guide rational design of potent PBD binders; three 6-mer phosphopeptides (i.e., IQSpSPC, LQSpTPF, and LNSpTPT) were successfully developed, which can efficiently target PBD domain with high affinity (Kd = 5.7 ± 1.1, 0.75 ± 0.18, and 7.2 ± 2.6 µm, resp.) as measured by a fluorescence anisotropy assay. The complex structure of PLK1 PBD domain with a newly designed, potent phosphopeptide LQSpTPF as well as diverse noncovalent chemical forces, such as H-bonds and hydrophobic interactions at the complex interface, were examined in detail to reveal the molecular mechanism of high affinity and stability of the complex system.

Aß peptide secretion is reduced by Radix Polygalae-induced autophagy via activation of the AMPK/mTOR pathway.

Radix Polygalae is a traditional Chinese medicine that has been used as a sedative and to improve memory for a number of years. The impact of Radix Polygalae in patients with Alzheimer's disease has been investigated. However the mechanisms underlying its effects remain unclear. In the current study, the toxicity of various doses (100, 40, 20, 10, 5 and 0 µg/ml) of Radix Polygalae was measured in the human neuroblastoma cell line (SH-SY5Y) using an MTT assay. Changes in amyloid ß (Aß) levels in the supernatant of Chinese hamster ovary (CHO) cells overexpressing ß-amyloid pro-protein (APP) and BACE1 (CHO-APP/BACE1), were detected using an ELISA assay. In order to confirm that the Aß reduction was associated with autophagy, the autophagy marker protein, light chain 3 (LC3), was measured by western blot analysis and autophagosomes were assessed using MDC staining. In addition, the mechanism underlying the autophagy induced by Radix Polygalae was analyzed using western blotting to measure the protein expression of mammalian target of rapamycin (mTOR), p70s6k, Raptor, protein kinase B and adenosine monophosphate-activated protein kinase (AMPK), in addition to the phosphorylated forms of these proteins. The results demonstrated no significant toxicity of Radix Polygalae in SH-SY5Y cells, at a dose of 100 µg/ml. The secretion of Aß was markedly reduced following treatment with Radix Polygalae, and this reduction occurred in a dose-dependent manner. The autophagy levels were shown to be enhanced in the drug treatment group, using fluorescence microscopy. In addition, levels of LC3II/LC3I, the marker protein of autophagy, were also increased. The results of the current study suggest that Radix Polygalae may aid in the elimination of the Aß peptide, via the induction of autophagy, by the AMPK/mTOR signaling pathway. These results may provide a basis for further kin vivo investigation.

Potent inhibition of TGF-ß signaling pathway regulator Abl: potential therapeutics for hepatic fibrosis.

Hepatic fibrosis is overly exuberant wound healing in which excessive connective tissue builds up in the liver. The treatment of hepatic fibrosis is still difficult and remains a challenge to the clinician. In recent years, the TGF-ß signaling pathway regulator tyrosine kinase Abl has been raised as a new and promising target of hepatic fibrosis therapy. Here, considering that there are numerous drugs and drug-like compounds being approved or under clinical development and experimental investigation, it is expected that some of the existing drugs can be re-exploited as new agents to target Abl with the capability of suppressing hepatic fibrosis. To achieve this, a synthetic protocol that integrated molecular docking, affinity scoring dynamics simulation and free energy analysis was described to systematically profile the inhibitory potency of various drugs and drug-like compounds against the kinase domain of Abl. Consequently, 4 out of 13 tested drug candidates were successfully identified to have high-Abl inhibitory activities. By visually examining the dynamics behavior, structural basis and energetic property of few typical Abl-drug complex cases, a significantly different pattern of non-bonded interactions between the binding of active and inactive drug ligands to Abl receptor was revealed; the former is defined by strong, specific chemical forces, while the latter can only form non-specific hydrophobic contacts with slight atomic collisions.

Genetic variants of interleukin-4 gene in autoimmune thyroid diseases: an updated meta-analysis.

Autoimmune thyroid diseases (AITDs) including Graves' disease (GD) and Hashimoto's thyroiditis (HT) are common autoimmune endocrine disorders. Interleukin-4 (IL-4), a cytokine secreted by T cells, plays a critical role in antigen-specific Th2 responses. The IL-4 gene is highly polymorphic and it has been reported that the polymorphism at -590 (T/C, rs2243250) in the promoter region of IL-4 may contribute to the development of AITDs. Recently, several case-control studies have examined the association of genetic variants of IL-4 with AITDs. However, the results of these studies remain conflicting. To systematically study the role of IL-4 in the pathogenesis of AITDs, we conducted a meta-analysis including 11 eligible studies (1847 cases and 2068 healthy controls). Fixed-effect or random-effect models were used to calculate pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs). Our results revealed a significant association between the IL-4 genetic variant (-590, T/C, rs2243250) and the risk of developing AITDs (TC + TT versus TT genotype: OR = 1.83, 95% CI = 1.083-3.091, p = 0.024). These findings demonstrate that the IL-4 rs2243250 genetic variant might play a key role in the development of AITDs.

Targeting human secretory phospholipase A2 with designed peptide inhibitors for inflammatory therapy.

Phospholipase A2 (PLA2) is potentially an important target for anti-inflammatory therapeutics. Here, we described a systematic scheme that integrated protein docking and peptide redocking, molecular dynamics simulation, and binding affinity analysis to rationally design PLA2 inhibitory peptides based on a solved PLA2 crystal structure. The scheme employed protein docking to sample the interaction modes of PLA2 with its natural inhibitor Clara cell protein, from which a number of peptide fragments, including a pentapeptide LLLGS, were cut off and redocked to serve as the lead entities of PLA2 inhibitory peptides. In addition, a systematic mutation energy map that characterized the binding free energy changes ΔG upon mutations of each position of the putative pentapeptide to 20 amino acids was also profiled, which was subsequently used to guide peptide structure optimization. In order to solidify the computational findings, we performed kinetic and inhibition studies of few designed peptides against human secretory PLA2. Consequently, eight peptides were successfully identified to have potent inhibition potency, in which the LLAYK and AVFRS were found to suppress enzymatic activity significantly (Ki = 0.75 ± 0.06 and 4.2 ± 0.3 µM, respectively). A further structure examination revealed that the designed peptides can form intensive nonpolar networks of van der Waals contacts and hydrophobic interactions at their complex interfaces with PLA2, conferring considerable stability and affinity for the formed complex systems.

Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1.

Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and K(m) values of the two substrates dUMP and CH(2) H(4) folate were determined to be 15.3 ± 1.25 µM and 0.35 ± 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery.

Two adjacent mutations on the dimer interface of SARS coronavirus 3C-like protease cause different conformational changes in crystal structure.

The 3C-like protease of SARS coronavirus (SARS-CoV 3CL(pro)) is vital for SARS-CoV replication and is a promising drug target. It has been extensively proved that only the dimeric enzyme is active. Here we discovered that two adjacent mutations (Ser139_Ala and Phe140_Ala) on the dimer interface resulted in completely different crystal structures of the enzyme, demonstrating the distinct roles of these two residues in maintaining the active conformation of SARS-CoV 3CL(pro). S139A is a monomer that is structurally similar to the two reported monomers G11A and R298A. However, this mutant still retains a small fraction of dimer in solution, which might account for its remaining activity. F140A is a dimer with the most collapsed active pocket discovered so far, well-reflecting the stabilizing role of this residue. Moreover, a plausible dimerization mechanism was also deduced from structural analysis. Our work is expected to provide insight on the dimerization-function relationship of SARS-CoV 3CL(pro).