MicroRNA408 negatively regulates salt tolerance by affecting secondary cell wall development in maize.

Although microRNA408 (miR408) is a highly conserved miRNA, the miR408 response to salt stress differs among plant species. Here, we show that miR408 transcripts are strongly repressed by salt stress and methyl viologen treatment in maize (Zea mays). Application of N, N1-dimethylthiourea partly relieved the NaCl-induced down-regulation of miR408. Transgenic maize overexpressing MIR408b is hypersensitive to salt stress. Overexpression of MIR408b enhanced the rate of net Na+ efflux, caused Na+ to locate in the inter-cellular space, reduced lignin accumulation, and reduced the number of cells in vascular bundles under salt stress. We further demonstrated that miR408 targets ZmLACCASE9 (ZmLAC9). Knockout of MIR408a or MIR408b or overexpression of ZmLAC9 increased the accumulation of lignin, thickened the walls of pavement cells, and improved salt tolerance of maize. Transcriptome profiles of the wild-type and MIR408b-overexpressing transgenic maize with or without salt stress indicated that miR408 negatively regulates the expression of cell wall biogenesis genes under salt conditions. These results indicate that miR408 negatively regulates salt tolerance by regulating secondary cell wall development in maize.

Variational Bayesian Algorithms for Maneuvering Target Tracking with Nonlinear Measurements in Sensor Networks.

The variational Bayesian method solves nonlinear estimation problems by iteratively computing the integral of the marginal density. Many researchers have demonstrated the fact its performance depends on the linear approximation in the computation of the variational density in the iteration and the degree of nonlinearity of the underlying scenario. In this paper, two methods for computing the variational density, namely, the natural gradient method and the simultaneous perturbation stochastic method, are used to implement a variational Bayesian Kalman filter for maneuvering target tracking using Doppler measurements. The latter are collected from a set of sensors subject to single-hop network constraints. We propose a distributed fusion variational Bayesian Kalman filter for a networked maneuvering target tracking scenario and both of the evidence lower bound and the posterior Cramér-Rao lower bound of the proposed methods are presented. The simulation results are compared with centralized fusion in terms of posterior Cramér-Rao lower bounds, root-mean-squared errors and the 3σ bound.

Spatiotemporal optical properties of dissolved organic matter in a sluice-controlled coastal plain river with both salinity and trophic gradients.

Due to the combined effect of sluices and sea tide, the sluice-controlled coastal plain river would be characterized by both trophic state and salinity gradients, affecting the spatiotemporal optical properties of dissolved organic matter (DOM). In this study, we investigated the spatiotemporal variation of water quality parameters and optical properties of DOM in the Haihe River, a representative sluice-controlled coastal plain river in Tianjin, China. A significant salinity gradient and four trophic states were observed in the water body of the Haihe River. Two humic- and one protein-like substances were identified from the DOM by the three-dimensional fluorescence spectra combined with the parallel factor (PARAFAC) analysis. Pearson's correlation analysis and redundancy analysis (RDA) showed that the salinity significantly affected the abundance of chromophoric DOM (CDOM) but did not cause significant changes in the fluorescence optical characteristics. In addition, the effect of Trophic state index (TSI) on the CDOM abundance was greater than that on the fluorescence intensity of fluorescent dissolved organic matter (FDOM). In the water body with both salinity and trophic state gradients, TSI posed a greater influence than salinity on the CDOM abundance. Our results fill the research gap in spatiotemporal DOM characteristics and water quality variation in water bodies with both salinity and trophic state gradients. These results are beneficial for clarifying the joint influence of saline intrusion and sluices on the DOM characteristics and water quality in sluice-controlled coastal plain rivers.

Structural and Functional Insights into a Nonheme Iron- and α-Ketoglutarate-Dependent Halogenase That Catalyzes Chlorination of Nucleotide Substrates.

Nonheme iron- and α-ketoglutarate (αKG)-dependent halogenases (NHFeHals), which catalyze the regio- and stereoselective halogenation of the unactivated C(sp3)-H bonds, exhibit tremendous potential in the challenging asymmetric halogenation. AdeV from Actinomadura sp. ATCC 39365 is the first identified carrier protein-free NHFeHal that catalyzes the chlorination of nucleotide 2'-deoxyadenosine-5'-monophosphate (2'-dAMP) to afford 2'-chloro-2'-deoxyadenosine-5'-monophosphate. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG at resolutions of 1.76 and 1.74 Å, respectively. AdeV possesses a typical ß-sandwich topology with H194, H252, αKG, chloride, and one water molecule coordinating FeII in the active site. Molecular docking, mutagenesis, and biochemical analyses reveal that the hydrophobic interactions and hydrogen bond network between the substrate-binding pocket and the adenine, deoxyribose, and phosphate moieties of 2'-dAMP are essential for substrate recognition. Residues H111, R177, and H192 might play important roles in the second-sphere interactions that control reaction partitioning. This study provides valuable insights into the catalytic selectivity of AdeV and will facilitate the rational engineering of AdeV and other NHFeHals for synthesis of halogenated nucleotides. IMPORTANCE Halogenated nucleotides are a group of important antibiotics and are clinically used as antiviral and anticancer drugs. AdeV is the first carrier protein-independent nonheme iron- and α-ketoglutarate (αKG)-dependent halogenase (NHFeHal) that can selectively halogenate nucleotides and exhibits restricted substrate specificity toward several 2'-dAMP analogues. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG. Molecular docking, mutagenesis, and biochemical analyses provide important insights into the catalytic selectivity of AdeV. This study will facilitate the rational engineering of AdeV and other carrier protein-independent NHFeHals for synthesis of halogenated nucleotides.

Structural dissection of unnatural ginsenoside-biosynthetic UDP-glycosyltransferase Bs-YjiC from Bacillus subtilis for substrate promiscuity.

Glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferases (UGT) contributes to the chemical and functional diversity of a number of natural products. Bacillus subtilis Bs-YjiC is a robust and versatile UGT that holds potentials in the biosynthesis of unnatural bioactive ginsenosides. To understand the molecular mechanism underlying the substrate promiscuity of Bs-YjiC, we solved crystal structures of Bs-YjiC and its binary complex with uridine diphosphate (UDP) at resolution of 2.18 Å and 2.44 Å, respectively. Bs-YjiC adopts the classical GT-B fold containing the N-terminal and C-terminal domains that accommodate the sugar acceptor and UDP-glucose, respectively. Molecular docking indicates that the spacious sugar-acceptor binding pocket of Bs-YjiC might be responsible for its broad substrate spectrum and unique glycosylation patterns toward protopanaxadiol-(PPD) and PPD-type ginsenosides. Our study reveals the structural basis for the aglycone promiscuity of Bs-YjiC and will facilitate the protein engineering of Bs-YjiC to synthesize novel bioactive glycosylated compounds.

Water quality characteristics and assessment of Yongding New River by improved comprehensive water quality identification index based on game theory.

The Yongding New River is essential for the water supplies of Tianjin. To date, there is no comprehensive report that assesses the year-round water quality of the Yongding New River Main stream. Moreover, little attention has been given to determining a combined weight for improving the traditional comprehensive water quality identification index (ICWQII) by the game theory. Seven water quality parameters were investigated monthly along the main stream of the Yongding New River from May 2018 to April 2019. Organic contaminants and nitrogen pollution were mainly caused by point sources pollution, and the total phosphorus mainly by non-point source pollution. Dramatic spatio-temporal variations of water quality parameters were jointly caused by different pollutant sources and hydrometeorological factors. In terms of this study, an improved comprehensive water quality identification index (ICWQII) based on entropy weight or variation coefficient and traditional CWQII underestimated the water qualities, and an ICWQII based on the superstandard multiple method overvalued the assessments. By contrast, water qualities assessments done with an ICWQII based on the game theory matched perfectly with the practical situation. The ICWQII based on game theory proposed in this study takes into account not only the degree of disorder and variation of water quality data, but also the influence of standard-exceeded pollution indicators, whose results are relatively reasonable. All findings and the ICWQII based on game theory can provide scientific support for decisions related to the water environment management of the Yongding New River and other waters.

[Identification of chemical constituents in Guizhi Fuling Capsules by UPLC-Q-TOF-MS/MS].

To qualitatively characterize the chemical composition of Guizhi Fuling Capsules using UPLC-ESI-Q-TOF-MS/MS. The analysis was performed on Agilent ZORBAX RRHD Eclipes Plus C_(18)(2.1 mm×100 mm, 1.8 µm) column,that was eluted with mobile phase consisting of acetonitrile and 0.1% formic acid in a gradient mode. The flow rate was 0.4 mL·min~(-1), and column temperature was 30 ℃. Tandem mass spectrometry was acquired in both negative and positive ESI modes. These components were further analyzed based on high-resolution mass-to-charge ratios, fragment ion species, reference substances and literature data. In conclusion, a total of 200 compounds were identified, in which 40 were verified with reference substances. The current study laid a foundation for in-depth studies of its mass balance and pharmacodynamics.

Crystal structure and proposed mechanism of an enantioselective hydroalkoxylation enzyme from Penicillium herquei.

Hydroalkoxylation is a useful and efficient reaction which generates C-O bond and produces cyclic ethers, the common structural elements of natural products. The dedicative enzyme which can catalyze enantioselective hydroalkoxylation named PhnH was recently identified in the herqueinone biosynthetic gene from Penicillium herquei. It catalyzes addition of a phenol to the terminal olefin on substrate to produce a dihydrobenzofuran. Here, the crystal structure of PhnH is reported and the putative substrate-binding pocket is illustrated. Through docking experiment, possible substrate-binding poses are displayed and the catalytic mechanism is therefore proposed. Our findings form the basis for further studies of enantioselective hydroalkoxylation enzymes.

An Iterative Nonlinear Filter Using Variational Bayesian Optimization.

We propose an iterative nonlinear estimator based on the technique of variational Bayesian optimization. The posterior distribution of the underlying system state is approximated by a solvable variational distribution approached iteratively using evidence lower bound optimization subject to a minimal weighted Kullback-Leibler divergence, where a penalty factor is considered to adjust the step size of the iteration. Based on linearization, the iterative nonlinear filter is derived in a closed-form. The performance of the proposed algorithm is compared with several nonlinear filters in the literature using simulated target tracking examples.

[A new aurone with anti-inflammatory activity from Cleistocalyx operculatus flower buds].

A new compound(Z)-6-hydroxy-4-methoxy-5,7-dimethylaurone was isolated from Cleistocalyx operculatus flower buds. Its structure was identified by spectroscopic data including MS, ¹H-NMR, ¹³C-NMR HSQC and HMBC. A known compound, 2',4'-dihydroxy-6'-methoxy-3'5'-dimethylchalcone (DMC), was also isolated and identified,and used as material to synthesize (Z)-6-hydroxy-4-methoxy-5,7-dimethylaurone.Anti-inflammatory activities of the two compounds were tested in vitro. The results showed that (Z)-6-hydroxy-4-methoxy-5,7-dimethylaurone possesses much stronger PGE2 inhibitory activity (IC50 6.12 nmol·L⁻¹) than the positive control ibuprofen （68.66 nmol·L⁻¹ï¼‰.

[Effect of rat intestinal flora in vitro on metabolites of acteoside].

Acteoside was used for anaerobic incubation with rat intestinal flora in vitro. HPLC was used to detect the changes of acteoside at different incubation time points and HPLC-Q-TOF-MS was used to identify the metabolites of acteoside. The results showed that acteoside could be metabolized by rat intestinal flora in vitro and the metabolites were 3,4-dihydroxyphenyl acid, caffeic acid and 3-(3'-hydroxyphenyl) propionic acid.

Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD(+) , NADPH, or NAD(+) /10-Oxogeranial: Reaction Mechanisms.

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD(+) , NADPH or NAD(+) /10-oxogeranial were solved. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form the byproduct S-10-oxo-citronellal. Tyr178 Oζ is positioned 2.5 Šfrom the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1-C2 to form the cisoid isomer, leading to formation of the cis-enolate, together with rotation about C4-C5, which enables cyclization and lactol production. The structure is similar to that of progesterone-5ß-reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10-oxogeranial structure also serves as a model for ß-face hydride attack in progesterone 5ß-reductases and is of general interest in the context of asymmetric synthesis.

Structure and inhibition of tuberculosinol synthase and decaprenyl diphosphate synthase from Mycobacterium tuberculosis.

We have obtained the structure of the bacterial diterpene synthase, tuberculosinol/iso-tuberculosinol synthase (Rv3378c) from Mycobacterium tuberculosis , a target for anti-infective therapies that block virulence factor formation. This phosphatase adopts the same fold as found in the Z- or cis-prenyltransferases. We also obtained structures containing the tuberculosinyl diphosphate substrate together with one bisphosphonate inhibitor-bound structure. These structures together with the results of site-directed mutagenesis suggest an unusual mechanism of action involving two Tyr residues. Given the similarity in local and global structure between Rv3378c and the M. tuberculosis cis-decaprenyl diphosphate synthase (DPPS; Rv2361c), the possibility exists for the development of inhibitors that target not only virulence but also cell wall biosynthesis, based in part on the structures reported here.

Structural and functional insights into the self-sufficient flavin-dependent halogenase.

Flavin-dependent halogenases (FDHs) have tremendous applications in synthetic chemistry. A single-component FDH, AetF, exhibits both halogenase and reductase activities in a continuous polypeptide chain. AetF exhibits broad substrate promiscuity and catalyzes the two-step bromination of l-tryptophan (l-Trp) to produce 5-bromotryptophan (5-Br-Trp) and 5,7-dibromo-l-tryptophan (5,7-di-Br-Trp). To elucidate the mechanism of action of AetF, we solved its crystal structure in complex with FAD, FAD/NADP+, FAD/l-Trp, and FAD/5-Br-Trp at resolutions of 1.92-2.23 Å. The obtained crystal structures depict the unprecedented topology of single-component FDH. Structural analysis revealed that the substrate flexibility and dibromination capability of AetF could be attributed to its spacious substrate-binding pocket. In addition, highly-regulated interaction networks between the substrate-recognizing residues and 5-Br-Trp are crucial for the dibromination activity of AetF. Several Ala variants underwent monobromination with >98 % C5-regioselectivity toward l-Trp. These results reveal the catalytic mechanism of single-component FDH for the first time and contribute to efficient FDH protein engineering for biocatalytic halogenation.

Chemical Profiling of Shen-Wu-Yi-Shen Tablets Using UPLC-Q-TOF-MS/MS and Its Quality Evaluation Based on UPLC-DAD Combined with Multivariate Statistical Analysis.

Shen-Wu-Yi-Shen tablets (SWYST) is a traditional Chinese medicine prescription used for treating chronic kidney disease (CKD). This study aims to characterize the constituents in SWYST and evaluate the quality based on the quantification of multiple bioactive components. SWYST samples were analyzed with ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and a data-processing strategy. As a result, 215 compounds in SWYST were unambiguously identified or tentatively characterized, including 14 potential new compounds. Meanwhile, strategies based on characteristic fragments for rapid identification were summarized, indicating that the qualitative method is accurate and feasible. Notably, the glucose esters of laccaic acid D-type anthraquinone were first found and their fragmentation patterns were described by comparing that of O-glycoside isomers. Besides, based on comparisons of the cleavage ways of mono-acyl glucose with different acyl groups or acylation sites, differences in fragmentation pathways between 1,2-di-O-acyl glucose and 1,6-di-O-acyl glucose were proposed for the first time and verified by reference substances. In addition, a validated UPLC-DAD was established for the determination of 11 major bioactive components related to treatment of CKD (albiflorin, paeoniflorin, 2,3,5,4'-tetrahydroxy-stilbene-2-O-ß-d-glucoside (TSG), 1-O-galloyl-2-O-cinnamoyl-ß-d-glucose, emodin-8-O-ß-d-glucoside, chrysophanol-O-ß-d-glucoside, aloe-emodin, rhein, emodin, chrysophanol and physcion). Moreover, TSG and 1-O-galloyl-2-O-cinnamoyl-ß-d-glucose were found as the quality markers related to the origins of SWYST based on multivariate statistical analysis. Conclusively, the findings in this work provide a feasible reference for further studies on quality research and mechanisms of action in treating CKD.

Functional characterization and structural basis of an efficient ochratoxin A-degrading amidohydrolase.

Ochratoxin A (OTA) contamination in various agro-products poses a serious threat to the global food safety and human health, leading to enormous economic losses. Enzyme-mediated OTA degradation is an appealing strategy, and the search for more efficient enzymes is a prerequisite for achieving this goal. Here, a novel amidohydrolase, termed PwADH, was demonstrated to exhibit 7.3-fold higher activity than that of the most efficient OTA-degrading ADH3 previously reported. Cryo-electron microscopy structure analysis indicated that additional hydrogen-bond interactions among OTA and the adjacent residue H163, the more compact substrate-binding pocket, and the wider entry to the substrate-access cavity might account for the more efficient OTA-degrading activity of PwADH compared with that of ADH3. We conducted a structure-guided rational design of PwADH and obtained an upgraded variant, G88D, whose OTA-degrading activity was elevated by 1.2-fold. In addition, PwADH and the upgraded G88D were successfully expressed in the industrial yeast Pichia pastoris, and their catalytic activities were compared to those of their counterparts produced in E. coli, revealing the feasibility of producing PwADH and its variants in industrial yeast strains. These results illustrate the structural basis of a novel, efficient OTA-degrading amidohydrolase and will be beneficial for the development of high-efficiency OTA-degrading approaches.

Insights into TIM-barrel prenyl transferase mechanisms: crystal structures of PcrB from Bacillus subtilis and Staphylococcus aureus.

Well structured: As a new triose phosphate isomerase (TIM) barrel-fold prenyl transferase, PcrB catalyzes the production of heptaprenylglyceryl phosphate from heptaprenyl diphosphate and glycerol-1-phosphate. Crystal structures of PcrB from Bacillus subtilis and Staphylococcus aureus in complex with ligands were solved, and together with site-directed mutagenesis and bioinformatics analyses, clearly reveal the catalytic mechanism of the enzyme.

Crystallization and preliminary X-ray diffraction analysis of YisP protein from Bacillus subtilis subsp. subtilis strain 168.

YisP is an enzyme involved in the pathway of biofilm formation in bacteria and is predicted to possess squalene synthase activity. A BlastP search using the YisP protein sequence from Bacillus subtilis subsp. subtilis strain 168 shows that it shares 23% identity with the dehydrosqualene synthase from Staphylococcus aureus. The YisP from B. subtilis 168 was expressed in Escherichia coli and the recombinant protein was purified and crystallized. The crystals, which belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 43.966, b = 77.576, c = 91.378 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.92 Šresolution. Structure determination using MAD and MIR methods is in progress.

Screening and characterization of xenobiotics in rat bio-samples after oral administration of Shen-Wu-Yi-Shen tablet using UPLC-Q-TOF-MS/MS combined with a targeted and non-targeted strategy.

Shen-Wu-Yi-Shen tablet (SWYST), a well-known traditional Chinese medicine prescription (TCMP), has been effectively used for treating chronic kidney disease (CKD) in clinically. However, an in-depth study of in vivo metabolism of SWYST is lacking. In this study, a targeted and non-targeted strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) was developed to screen and characterize SWYST-related xenobiotics in rats. Based on the in-house library, a chemical database of SWYST including 215 constituents was constructed through "find by formula" and further verified by characteristic fragmentations or the literatures. Then the constructed chemical database was applied for the targeted screening of prototypes. As for metabolites, the non-targeted screening was achieved combined the peak picking using the function "find by auto-MS/MS" and peak filtration of the prototypes and endogenous components, while the targeted screening was performed using Metabolite ID according to the possible metabolic reactions. Furthermore, the potential metabolites were preliminarily identified by comparison of the parent compounds or references to the literatures. As a result, 201 exogenous components (87 prototypes and 121 metabolites) were characterized in rats after administration of SWYST, including 55 (17 prototypes and 38 metabolites) in plasma, 151 (52 prototypes and 99 metabolites) in urine, and 121 (74 prototypes and 47 metabolites) in feces. Finally, their possible metabolic pathways were summarized, and the metabolic reactions mainly involved phase I reactions (hydroxylation, deoxygenation, hydrogenation, methylation, oxidation, hydrolysis and esterification) and phase II reactions (glucuronidation and sulfation). The findings of this research reveal the potential active ingredients of SWYST, providing an important material basis for the pharmacokinetics and pharmacodynamics of SWYST.

Comparative pharmacokinetics of six bioactive components of Shen-Wu-Yi-Shen tablets in normal and chronic renal failure rats based on UPLC-TSQ-MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Shen-Wu-Yi-Shen tablets (SWYST), a Chinese patent medicine consisting of 12 herbal medicines, was formulated by a famous TCM nephrologist, Zou Yunxiang. It is clinically used to improve the symptoms of nausea, vomiting, poor appetite, dry mouth and throat, and dry stool in patients with chronic renal failure (CRF) accompanied by qi and yin deficiency, dampness, and turbidity. SWYST can reduce urea nitrogen, blood creatinine, and urinary protein loss, and increase the endogenous creatinine clearance rate. However, little is known about its pharmacokinetics. AIM OF STUDY: To compare the pharmacokinetics of six bioactive components after oral administration of SWYST in normal and adenine-induced CRF rats. MATERIALS AND METHODS: A method based on ultra-performance liquid chromatography coupled with a triple-stage quadrupole mass spectrometer (UPLC-TSQ-MS/MS) was developed and validated to determine the six bioactive compounds (albiflorin, paeoniflorin, plantagoguanidinic acid, rhein, aloe-emodin, and emodin) in rat plasma. Rat plasma samples were prepared using protein precipitation. Chromatography was performed on an Agilent Eclipse Plus C18 column (3.0 × 50 mm, 1.8 µm) using gradient elution with a mobile phase composed of acetonitrile and water containing 0.1% (v/v) formic acid, while detection was achieved by electrospray ionization MS under the multiple selective reaction monitoring modes. After SWYST administration, rat plasma was collected at different time points, and the pharmacokinetic parameters of six analytes were calculated and analyzed based on the measured plasma concentrations. RESULTS: The UPLC-TSQ-MS/MS method was fully validated for its satisfactory linearity (r ≥ 0.9913), good precisions (RSD <11.5%), and accuracy (RE: -13.4∼13.1%), as well as acceptable limits in the extraction recoveries, matrix effects, and stability (RSD <15%). In normal rats, the six analytes were rapidly absorbed (Tmax ≤ 2 h), and approximately 80% of their total exposure was eliminated within 10 h. Moreover, in normal rats, the AUC0-t and Cmax of albiflorin, plantagoguanidinic acid, and rhein exhibited linear pharmacokinetics within the dose ranges, while that of paeoniflorin is non-linear. However, in CRF rats, the six analytes exhibited reduced elimination and significantly different AUC or Cmax values. These changes may reflect a decreased renal clearance rate or inhibition of drug-metabolizing enzymes and transporters in the liver and gastrointestinal tract caused by CRF. CONCLUSIONS: A sensitive UPLC-TSQ-MS/MS method was validated and used to investigate the pharmacokinetics of SWYST in normal and CRF rats. This is the first study to investigate the pharmacokinetics of SWYST, and our findings elucidate the causes of their different pharmacokinetic behaviors in CRF rats. Furthermore, the results provide useful information to guide further research on the pharmacokinetic-pharmacodynamic correlation and clinical application of SWYST.