A Single Active-Site Mutagenesis Confers Enhanced Activity and/or Changed Product Distribution to a Pentalenene Synthase from Streptomyces sp. PSKA01.

Pentalenene is a ternary cyclic sesquiterpene formed via the ionization and cyclization of farnesyl pyrophosphate (FPP), which is catalyzed by pentalenene synthase (PentS). To better understand the cyclization reactions, it is necessary to identify more key sites and elucidate their roles in terms of catalytic activity and product specificity control. Previous studies primarily relied on the crystal structure of PentS to analyze and verify critical active sites in the active cavity, while this study started with the function of PentS and screened a novel key site through random mutagenesis. In this study, we constructed a pentalenene synthetic pathway in E. coli BL21(DE3) and generated PentS variants with random mutations to construct a mutant library. A mutant, PentS-13, with a varied product diversity, was obtained through shake-flask fermentation and product identification. After sequencing and the functional verification of the mutation sites, it was found that T182A, located in the G2 helix, was responsible for the phenotype of PentS-13. The site-saturation mutagenesis of T182 demonstrated that mutations at this site not only affected the solubility and activity of the enzyme but also affected the specificity of the product. The other products were generated through different routes and via different carbocation intermediates, indicating that the 182 active site is crucial for PentS to stabilize and guide the regioselectivity of carbocations. Molecular docking and molecular dynamics simulations suggested that these mutations may induce changes in the shape and volume of the active cavity and disturb hydrophobic/polar interactions that were sufficient to reposition reactive intermediates for alternative reaction pathways. This article provides rational explanations for these findings, which may generally allow for the protein engineering of other terpene synthases to improve their catalytic efficiency or modify their specificities.

[Sagittaria sagittifolia polysaccharides regulates Nrf2/HO-1 to relieve liver injury caused by multiple heavy metals in vivo and in vitro].

This study explored whether Sagittaria sagittifolia polysaccharides(SSP) activates the nuclear factor erythroid-2-related factor2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway to protect against liver damage jointly induced by multiple heavy metals. First, based on the proportion of dietary intake of six heavy metals in rice available in Beijing market, a heavy metal mixture was prepared for inducing mouse liver injury and HepG2 cell injury. Forty male Kunming mice were divided into five groups: control group, model group, glutathione positive control group, and low-and high-dose SSP groups, with eight mice in each group. After 30 days of intragastric administration, the liver injury in mice was observed by HE staining. In the in vitro experiment, MTT assay was conducted to detect the effects of SSP at 0.25, 0.5, 1, and 2 mg·mL~(-1) on HepG2 cell survival at different time points. The content of alanine transaminase(ALT) and aspartate aminotransferase(AST) in the 48-h cell culture fluid was measured using micro-plate cultivation method, followed by the detection of the change in reactive oxygen species(ROS) content by flow cytometry. The mRNA expression levels of Nrf2 and HO-1 in cells were determined by RT-PCR, and their protein expression by Western blot. HE staining results showed that compared with the model group, the SSP administration groups exhibited significantly alleviated inflammatory cell infiltration and fatty infiltration in the liver, with better outcomes observed in the high-dose SSP group. In the in vitro MTT assay, compared with the model group, SSP at four concentrations all significantly increased the cell survival rate, decreased the ALT, AST, and ROS content(P<0.05), and down-regulated Nrf2 and HO-1 mRNA and protein expression(P<0.05). SSP significantly improves inflammatory infiltration in the liver tissue of mice exposed to a variety of heavy metals and corrects the liver fat degeneration, which may be related to its regulation of the Nrf2/HO-1 signaling pathway, reduction of ROS, and alleviation of oxidative damage.

Sagittaria sagittifolia polysaccharide protects against six-heavy-metal-induced hepatic injury associated with the activation of Nrf2 pathway to regulate oxidative stress and apoptosis.

The hepatic protective role of Sagittaria sagittifolia polysaccharide (SSP) and its possible mechanism were discussed in mice and L02 hepatocytes injured by heavy metals mixture of Cd + Cr (VI) + Pb + Mn + Zn + Cu. After 30-day intervention, blood and liver samples were collected for the relevant assessments. Methyl thiazolyl tetrazolium (MTT) assay showed 24 h was the best protecting point and the SSP protection at 1 mg/mL was strongest in L02 hepatocytes. SSP can alleviated hepatic injury, as evidenced by significantly decreased the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and the malondialdehyde (MDA) content, also increased the superoxide dismutase (SOD) activity and glutathione (GSH), total sulphydryl (T-SH) contents. SSP effectively reduced pathological damage of mice and accumulation of heavy metals in liver, as well as decreased the level of reactive oxygen species (ROS) in L02 hepatocytes. After SSP treatment, the protein expressions or gene transcription of nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H dehydrogenase, quinone 1 (NQO1) and heme oxygenase1 (HO-1) decreased in L02. The protein expression of Nrf2 and NQO1 were increased while HO-1 was decreased in liver. Besides, SSP can attenuates apoptosis through reducing the protein expression of Bcl-2-associated X protein (Bax) and caspase-3, and increasing B-cell lymphoma gene 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl). SSP protects against six-heavy-metal-induced hepatic injury in mice and L02 hepatocytes. Supported by Nrf2 gene silencing, the mechanisms may correlate with activating Nrf2 pathway to mitigate oxidative stress and apoptosis.

Theoretical scheme of nonvolatile strain-switchable high/low resistance based on novel strain-tunable magnetic anisotropy in the Mn2.25Co0.75Ga0.5Sn0.5/MgO superlattice.

It is highly desirable to effectively tune electronic and magnetic properties using simple means such as applying an external electrical field or strain in spintronics research. Here, we investigate the strain manipulation of the electronic structure and magnetic anisotropy in (Mn2.25Co0.75Ga0.5Sn0.5)2/(MgO)1 superlattices using first-principles calculations, where Mn2.25Co0.75Ga0.5Sn0.5 (MCGS) is a newly proposed spin gapless semiconductor with 100% spin polarization. We report that -3 to -5% compressive strain and 1 to 2% tensile strain induce the (MCGS)2/(MgO)1 superlattice to present half-metallic character with 100% spin-polarization. More importantly, biaxial strain has the ability to modify the magnetic anisotropy of the (MCGS)2/(MgO)1 superlattice effectively. The easy magnetization axis can automatically reverse between the out-of-plane and in-plane directions by applying compressive and tensile strain to the MCGS layer. Remarkably, the perpendicular magnetic anisotropy (PMA) of the (MCGS)2/(MgO)1 superlattice increases almost linearly with the increase in compressive strain, while the in-plane magnetic anisotropy (IMA) under tensile strain shows a trend of initially increasing and then decreasing. Actually, strain can be generated by depositing (MCGS)2/(MgO)1 films on ferroelectric substrates. Thus, we propose a new scheme of nonvolatile solely strain-switchable (usually electrically induced) high/low resistance in magnetic tunnel junctions without the aid of a magnetic field. Our results will be important for exploring and realizing stain involved high-performance spintronic devices.

Sagittaria sagittifolia polysaccharide interferes with arachidonic acid metabolism in non-alcoholic fatty liver disease mice via Nrf2/HO-1 signaling pathway.

BACKGROUNDS: Non-alcoholic fatty liver disease (NAFLD) is currently one of the most common chronic liver diseases especially in developed countries. Modern research shows an obvious protective effect of Sagittaria sagittifolia L. (Alismataceae) on glucose and lipid metabolism disorders. Previous studies had reported that Sagittaria sagittifolia polysaccharide (SSP) has potent protective effects on drug-induced liver injury. Based on this, we speculated that Sagittaria sagittifolia polysaccharide also has protective effects on NAFLD and performed experiments to explore this more. METHODS: Outstanding protective effects of SSP against NAFLD in mice was observed with Hematoxylin and Eosin (H&E) and uranium acetate-citrate stain in our prophase research. By performing bioinformatics analysis on plasma metabolic data which is obtained from ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS), we found the regulatory mechanisms and key nodes behind the beneficial effect with IPA (Ingenuity Pathway Analysis) software. Immunohistochemical staining and Western blot were performed for further validation on expression variations of key proteins. RESULTS: Regulatory pathways were enriched with 33 significant differential metabolites that responded to SSP treatment in plasma, and specifically, the ones related to arachidonic acid metabolism showed high participation. Moreover, the expression patterns of upstream regulators, Nrf2 and HO-1, were found to be significantly regulated upon SSP treatment. CONCLUSIONS: In conclusion, our findings illustrated a novel perspective that SSP exerts preventive protection against high-fat diet-induced NAFLD by interfering with arachidonic acid metabolism via Nrf2/HO-1 signaling pathway in liver oxidative stress, providing an attractive point for the breakthrough of related natural medicine development.

Atomic switches of metallic point contacts by plasmonic heating.

Electronic switches with nanoscale dimensions satisfy an urgent demand for further device miniaturization. A recent heavily investigated approach for nanoswitches is the use of molecular junctions that employ photochromic molecules that toggle between two distinct isoforms. In contrast to the reports on this approach, we demonstrate that the conductance switch behavior can be realized with only a bare metallic contact without any molecules under light illumination. We demonstrate that the conductance of bare metallic quantum contacts can be reversibly switched over eight orders of magnitude, which substantially exceeds the performance of molecular switches. After the switch process, the gap size between two electrodes can be precisely adjusted with subangstrom accuracy by controlling the light intensity or polarization. Supported by simulations, we reveal a more general and straightforward mechanism for nanoswitching behavior, i.e., atomic switches can be realized by the expansion of nanoelectrodes due to plasmonic heating.

Metabolomic Study to Determine the Mechanism Underlying the Effects of Sagittaria sagittifolia Polysaccharide on Isoniazid- and Rifampicin-Induced Hepatotoxicity in Mice.

In this study, a non-targeted metabolic profiling method based on ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was used to characterize the plasma metabolic profile associated with the protective effects of the Sagittaria sagittifolia polysaccharide (SSP) on isoniazid (INH)-and rifampicin (RFP)-induced hepatotoxicity in mice. Fourteen potential biomarkers were identified from the plasma of SSP-treated mice. The protective effects of SSP on hepatotoxicity caused by the combination of INH and RFP (INH/RFP) were further elucidated by investigating the related metabolic pathways. INH/RFP was found to disrupt fatty acid metabolism, the tricarboxylic acid cycle, amino acid metabolism, taurine metabolism, and the ornithine cycle. The results of the metabolomics study showed that SSP provided protective effects against INH/RFP-induced liver injury by partially regulating perturbed metabolic pathways.

Determination of estradiol valerate in pharmaceutical preparations and human serum by flow injection chemiluminescence.

A novel method for the detection of trace estradiol valerate (EV) in pharmaceutical preparations and human serum was developed by inhibition of luminol chemiluminescence (CL) by estradiol valerate on the zinc deuteroporphyrin (ZnDP)-enhanced luminol-K3 Fe(CN)6 chemiluminescence system. Under optimized experimental conditions, CL intensity and concentration of estradiol valerate had a good linear relationship in the ranges of 8.0 × 10(-8) to 1.0 × 10(-5) g/mL. Detection limit (3σ) was estimated to be 3.5 × 10(-8) g/mL. The proposed method was applied successfully for the determination of estradiol valerate in pharmaceutical preparations and human serum and recoveries were 97.0-105.0% and 95.5-106.0%, respectively. The possible mechanism of the CL system is discussed.