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.

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.

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.

Cryo-EM structure and rational engineering of a superefficient ochratoxin A-detoxifying amidohydrolase.

Ochratoxin A (OTA) is among the most prevalent mycotoxins detected in agroproducts, posing serious threats to human and livestock health. Using enzymes to conduct OTA detoxification is an appealing potential strategy. The recently identified amidohydrolase from Stenotrophomonas acidaminiphila, termed ADH3, is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α (OTα) and L-ß-phenylalanine (Phe). To elucidate the catalytic mechanism of ADH3, we solved the single-particle cryo-electron microscopy (cryo-EM) structures of apo-form, Phe- and OTA-bound ADH3 to an overall resolution of 2.5-2.7 Å. The role of OTA-binding residues was investigated by structural, mutagenesis and biochemical analyses. We also rationally engineered ADH3 and obtained variant S88E, whose catalytic activity was elevated by 3.7-fold. Structural analysis of variant S88E indicates that the E88 side chain provides additional hydrogen bond interactions to the OTα moiety. Furthermore, the OTA-hydrolytic activity of variant S88E expressed in Pichia pastoris is comparable to that of Escherichia coli-expressed enzyme, revealing the feasibility of employing the industrial yeast strain to produce ADH3 and its variants for further applications. These results unveil a wealth of information about the catalytic mechanism of ADH3-mediated OTA degradation and provide a blueprint for rational engineering of high-efficiency OTA-detoxifying machineries.

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.

Mechanisms of action underlying the effect of Tongsaimai on wound healing based on experimental and network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongsaimai (TSM) is a traditional Chinese medicine that has several therapeutic qualities, including anti-inflammatory, anti-oxidative, and anti-vasculitis effects. However, its impacts and underlying mechanisms on wound healing remain unclear. AIM OF THE STUDY: The aim of our study was to evaluate TSM for its pro-healing effect and the relevant mechanisms using both experimental validation and network pharmacology analysis. MATERIALS AND METHODS: The components of TSM were detected by high-performance liquid chromatography combined with diode array detector (HPLC-DAD). Skin wounds with a diameter of 4 mm were created on the backs of mice, after which, topical treatments of 2.5-10% TSM were applied onto the lesions once daily for either 2 or 7 days. Then, the wound tissues were collected to determine the impacts of TSM on collagen deposition, epithelial cell proliferation, oxidative stress, inflammation, and angiogenesis. Moreover, the effects of TSM (0.5-2 mg/mL) on the cell viability of HUVECs and HaCaT cells were evaluated. RESULTS: A total of 11 components in TSM were identified by HPLC-DAD. TSM was found to enhance the rate of wound contraction and increase epithelial thickness and collagen deposition during the healing process. In addition, TSM increased SOD activity and downregulated MDA and IL-1ß levels in the wound tissues. Immunofluorescence analysis further indicated an increased expression of Ki67, CD31, and VEGF in wound tissues following TSM administration. Results of the network pharmacology analysis revealed that multiple pathways including VEGF, PI3K/Akt, and MAPK pathways were involved in the pharmacological actions of TSM on wound healing. Accordantly, in vitro experiments revealed that TSM promoted the proliferation of HUVECs and HaCaT cells while activating the PI3K/Akt pathway. CONCLUSIONS: Our results suggest that TSM may serve as a therapeutic medication to improve wound healing by employing multiple regulatory mechanisms that affect proliferation, angiogenesis, collagen deposition, oxidative stress, and inflammation.

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.

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.

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.

Nitrate availability controls translocation of the transcription factor NAC075 for cell-type-specific reprogramming of root growth.

Plant root architecture flexibly adapts to changing nitrate (NO3-) availability in the soil; however, the underlying molecular mechanism of this adaptive development remains under-studied. To explore the regulation of NO3--mediated root growth, we screened for low-nitrate-resistant mutant (lonr) and identified mutants that were defective in the NAC transcription factor NAC075 (lonr1) as being less sensitive to low NO3- in terms of primary root growth. We show that NAC075 is a mobile transcription factor relocating from the root stele tissues to the endodermis based on NO3- availability. Under low-NO3- availability, the kinase CBL-interacting protein kinase 1 (CIPK1) is activated, and it phosphorylates NAC075, restricting its movement from the stele, which leads to the transcriptional regulation of downstream target WRKY53, consequently leading to adapted root architecture. Our work thus identifies an adaptive mechanism involving translocation of transcription factor based on nutrient availability and leading to cell-specific reprogramming of plant root growth.

Structure-based rational design of a short-chain dehydrogenase/reductase for improving activity toward mycotoxin patulin.

Patulin is a fatal mycotoxin that is widely detected in drinking water and fruit-derived products contaminated by diverse filamentous fungi. CgSDR from Candida guilliermondii represents the first NADPH-dependent short-chain dehydrogenase/reductase that catalyzes the reduction of patulin to the nontoxic E-ascladiol. To elucidate the catalytic mechanism of CgSDR, we solved its crystal structure in complex with cofactor and substrate. Structural analyses indicate that patulin is situated in a hydrophobic pocket adjacent to the cofactor, with the hemiacetal ring orienting toward the nicotinamide moiety of NADPH. In addition, we conducted structure-guided engineering to modify substrate-binding residue V187 and obtained variant V187F, V187K and V187W, whose catalytic activity was elevated by 3.9-, 2.2- and 1.7-fold, respectively. The crystal structures of CgSDR variants suggest that introducing additional aromatic stacking or hydrogen-bonding interactions to bind the lactone ring of patulin might account for the observed enhanced activity. These results illustrate the catalytic mechanism of SDR-mediated patulin detoxification for the first time and provide the upgraded variants that exhibit tremendous potentials in industrial applications.

Structural analysis and engineering of aldo-keto reductase from glyphosate-resistant Echinochloa colona.

Glyphosate is a dominant organophosphate herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the shikimate pathway. Glyphosate is extensively applied since manufactured, which has led to the emergence of various glyphosate-resistant crops and weeds. However, the molecular mechanism of many glyphosate-resistance machineries remains unclear. Recently, the upregulated expression of two homologous aldo-keto reductases (AKRs), designated as AKR4C16 and AKR4C17, were found to contribute to the glyphosate resistance in Echinochloa colona. This represents the first naturally evolved glyphosate-degrading machinery reported in plants. Here, we report the three-dimensional structure of these two AKR enzymes in complex with cofactor by performing X-ray crystallography. Furthermore, the binding-mode of glyphosate were elucidated in a ternary complex of AKR4C17. Based on the structural information and the previous study, we proposed a possible mechanism of action of AKR-mediated glyphosate degradation. In addition, a variant F291D of AKR4C17 that was constructed based on structure-based engineering showed a 70% increase in glyphosate degradation. In conclusion, these results demonstrate the structural features and glyphosate-binding mode of AKR4C17, which increases our understanding of the enzymatic mechanism of glyphosate bio-degradation and provides an important basis for the designation of AKR-based glyphosate-resistance for further applications.

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.

Crystal structure and biochemical analysis of the specialized deoxynivalenol-detoxifying glyoxalase SPG from Gossypium hirsutum.

Deoxynivalenol (DON) and its acetylated derivatives such as 3-acetyldeoxynivalenol (3A-DON) and 15-acetyldeoxynivalenol (15A-DON) are notorious mycotoxins in Fusarium contaminated cereals, which pose a great threat to human and livestock health. The specialized glyoxalase I from Gossypium hirsutum (SPG) can lower the toxicity of 3A-DON by conducting isomerization to transfer C8 carbonyl to C7 and double bond from C9-C10 to C8-C9. Here we report that the substrate-flexible SPG can also recognize 15A-DON and DON, probably following the same isomerization mechanism as that for 3A-DON. The crystallographic, mutagenesis, and biochemical analyses revealed that SPG provides a hydrophobic pocket to accommodate the substrate and residue E167 might serve as the catalytic base. A variant SPGY62A that was constructed based on structure-based protein engineering exhibited elevated catalytic activity towards DON, 3A-DON, and 15A-DON by >70%. Furthermore, variant SPGY62A was successfully expressed in Pichia pastoris, whose catalytic activity was also compared to that produced in Escherichia coli. These results provide a blueprint for further protein engineering of SPG and reveal the potential applications of the enzyme in detoxifying DON, 3A-DON and 15A-DON.

Catalytically inactive lytic polysaccharide monooxygenase PcAA14A enhances the enzyme-mediated hydrolysis of polyethylene terephthalate.

The massive accumulation of polyethylene terephthalate (PET) in the global ecosystem is a growing environmental crisis. Development of environmental friendly strategies to achieve enzyme-catalyzed PET degradation has attracted tremendous attention. In this study, we demonstrated the synergistic effects of combining a specific PET-degrading enzyme IsPETaseEHA variant from PET-assimilating bacterium Ideonella sakaiensis and a lytic polysaccharide monooxygenase from a white-rot fungus Pycnoporus coccineus (PcAA14A) in PET degradation. We found that the presence of PcAA14A alone did not result in PET hydrolysis, but its presence could stimulate IsPETaseEHA-mediated hydrolytic efficiency by up to 1.3-fold. Notably, the stimulatory effects of PcAA14A on IsPETaseEHA-catalyzed PET hydrolysis were found to be independent of monooxygenase activity. Dose-effects of IsPETaseEHA and PcAA14A on PET hydrolysis were observed, with the optimal concentrations being determined to 25 µg/mL and 0.25 µg/mL, respectively. In the 5-day PET hydrolysis experiment, 1097 µM hydrolysis products were produced by adding the optimized concentrations of IsPETaseEHA and PcAA14A, which was 27.7% higher than those were produced by IsPETaseEHA alone. Our study reports the first time that PcAA14A could stimulate the IsPETaseEHA-mediated PET hydrolysis through a monooxygenase activity independent manner.

Enhancing PET hydrolytic enzyme activity by fusion of the cellulose-binding domain of cellobiohydrolase I from Trichoderma reesei.

The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (IsPETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETaseS121E/D186H/R280A (IsPETaseEHA) variant. We found that fusing a cellulose-binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETaseEHA showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETaseEHA_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 ℃ and 40 ℃, respectively. The catalytic activity of IsPETaseEHA_CBM was increased by 86 % when the protein concentration was increased from 2.5 µg/mL to 20 µg/mL. These findings suggest that the fusion of polymer-binding module to IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.

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.

Increased miR­187­3p expression after cerebral ischemia/reperfusion induces apoptosis via initiation of endoplasmic reticulum stress.

Ischemia/reperfusion (I/R) injury induces activation of the endoplasmic reticulum stress (ERS) pathway, accompanied by an increase in apoptosis. Multiple microRNAs (miRNAs/miRs) are dysregulated during I/R and contribute to I/R-induced injury. miRNAs act as suppressors of gene expression and negatively regulate gene expression by targeting the protein-coding sequence (CDS) of specific target mRNAs. Seipin is an endoplasmic reticulum protein that has recently been associated with ERS. We previously reported that seipin is the target gene of miR­187­3p. Therefore, we explored the involvement of miR-187-3p in I/R-induced ERS via the regulation of seipin. A rat MCAO/R model was established by 1 h of occlusion and 24 h reperfusion. Neurological deficits and infarction area were examined. PC12 cells were exposed to oxygen­glucose deprivation/reoxygenation (OGD/R) to model I/R. Expression levels of miR-187-3p and proteins related to ERS and apoptosis were measured using RT-PCR, western blotting, immunofluorescence, and immunohistochemistry, respectively. TUNEL staining was used to assay apoptosis. MCAO/R-induced morphological changes were analyzed with Nissl staining and Hematoxylin-eosin staining. I/R-induced ERS was closely associated with an increase in miR-1873p and a decrease in seipin expression. miR-187-3p agomir further activated the ERS pathway and promoted apoptosis but decreased seipin expression levels; these effects were reversed by miR-187-3p antagomir. Moreover, seipin knockdown aggravated ERS in PC12 cells after OGD/R, and this change was rescued by seipin overexpression. miR-187-3p antagomir did not suppress ERS and apoptosis in seipin knockdown PC12 cells after OGD/R. Our findings demonstrate that the inhibition of miR­187­3p attenuated I/R­induced cerebral injury by regulating seipin-mediated ERS.

[Spatial-temporal trend of grassland net primary production and their driving factors in the Loess Plateau, China]. / é»„åœŸé«˜åŽŸè‰åœ°å‡€åˆçº§ç”Ÿäº§åŠ›æ—¶ç©ºè¶‹åŠ¿åŠå ¶é©±åŠ¨å› ç´ .

Net primary productivity (NPP) of grassland is a key link and important part of the ecosystem's carbon cycle. We estimated the changes of NPP in grasslands of the Loess Plateau with unchanged land use types during 2000-2015 and analyzed its responses to the variation of main climate factors (annual precipitation, annual heavy rainfall, annual effective rainfall days, annual average temperature, annual maximum temperature, annual minimum temperature) using piecewise linear regression and Pearson correlation analysis. The driving factors of grassland NPP were further analyzed by pixel-by-pixel with boosted regression tree analysis. The results showed that annual mean grassland NPP in the Loess Plateau showed an increasing trend during the study period, with 51.3% of the total grassland area showing a significant increasing trend. The average increase rate of annual mean NPP declined from 15.23 g C·m-2·a-1 in 2000-2004 to 3.58 g C·m-2·a-1 in 2004-2015. There was a significant positive correlation between grassland NPP and precipitation, but negative correlation with temperature factors. Annual precipitation was the dominant climatic factor affecting NPP of the whole study area with the highest relative importance. Annual maximum temperature was the dominant driving force of grassland NPP of central Loess Plateau, while annual minimum temperature mainly affected the growth of grassland in high-altitude area of the western Loess Plateau.

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.