[Cloning and expression analysis of U6 promoters in Panax quinquefolius].

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.

Seven new 2-(2-phenylethyl) chromone derivatives from agarwood of Aquilaria agallocha with inhibitory effects on nitric oxide production.

Seven new 2-(2-Phenethyl) chromone derivatives (1-7), including four 2-(2-Phenethyl) chromones (1-4), one 6, 7, 8 trihydroxy-2-(2-Phenethyl) chromone (5), one acetylated 5, 6, 7, 8-tetrahydroxy-2-(2-Phenethyl) chromone (6), and one chlorine-containing 5, 6, 7, 8-tetrahydro-2-(2-Phenethyl) chromone (7), along with eight known compounds (8-15), were isolated from agarwood originating from Aquilaria agallocha Roxb.. Their structures were determined mainly by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR) analysis. The absolute configurations of 3-7 were resolved by electronic circular dichroism (ECD) calculations. Nearly all compounds were evaluated for their anti-inflammatory activities in RAW264.7 cells. Compounds 1 and 7-11 displayed significant anti-inflammatory activities with IC50 values ranging from 3.71 to 32.04 µM.

Eudesmane-type and agarospirane-type sesquiterpenes from agarwood of Aquilaria agallocha.

Eleven previously unreported sesquiterpenes, including nine eudesmane-type (agalleudesmanol A-I) and two agarospirane-type sesquiterpenes (agarospiranic aldehyde A-B), together with eight known sesquiterpenes, were isolated from the agarwood of Aquilaria agallocha Roxb. The structures were established based on extensive spectroscopic analyses, including infrared (IR), high-resolution electrospray ionisation mass spectrometry (HRESIMS), nuclear magnetic resonance (NMR), X-ray diffraction, quantum chemical calculations based on empirical electronic circular dichroism (ECD) data, and DP4+ probability analysis. The bioactivity of these undescribed compounds against lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells was evaluated. Compounds 1 and 2 exhibited significant anti-inflammatory activities, with IC50 values of 5.46-14.07 µM (aminoguanidine as positive control, IC50 20.33 ± 1.08 µM).

Dissolved inorganic carbon determines the abundance of microbial primary producers and primary production in Tibetan Plateau lakes.

Climate change globally accelerates the shrinkage of inland lakes, resulting in increases in both water salinity and dissolved inorganic carbon (DIC). The increases of salinity and DIC generate contrasting effects on microbial primary producers and primary production, however, their combined effects remain unclear in aquatic ecosystems. We hypothesized that increased DIC mitigates the constraints of enhanced salinity on microbial primary producers and primary production. To test this, we employed isotope labeling and molecular methods to explore primary production and four dominant types of microbial primary producers (form IA, IB, IC and ID) in lakes on the Tibetan Plateau. Results showed that DIC was positively correlated with the abundance of the form IAB and ID microbial primary producers and primary production (all P < 0.001) and offset salinity constraints. Structural equation models elucidated that DIC substantially enhanced primary production by stimulating the abundance of form ID microbial primary producers. The abundance of form ID primary producers explained more variations (14.6%) of primary production than form IAB (6%) and physicochemical factors (6.8%). Diatoms (form ID) played a determinant role in primary production in the lakes by adapting to high DIC and high salinity. Our findings suggest that inland lakes may support higher primary productivity in future climate change scenarios.

Antioxidative 2-(2-phenylethyl)chromones in Chinese eaglewood from Aquilaria sinensis.

Two new compounds 6,7-dimethoxy-2-[2-(2'-hydroxyphenyl)ethyl]chromone (1) and 6,7-dimethoxy-2-[2-(4'-hydroxyphenyl)ethenyl]chromone (2), together with ten known 2-(2-phenylethyl)chromones (3-12) were isolated from the resinous wood of Aquilaria sinensis (Lour.) Gilg. Their structures were elucidated by detailed IR, MS, NMR spectroscopic analyses, and comparison with reported. The absolute configuration of 3 was confirmed by Cu Kα X-ray crystallographic experiment, and the X-ray crystallographic data of 3 were firstly reported. Compounds 2, 8, 10, and 11 exhibited strong ABTS•+ radical scavenging activity, with IC50 values of 12.3, 16.5, 12.1, and 34.7 µM, respectively.[Formula: see text].

Degradation in vitro and in vivo of ß-TCP/MCPM-based premixed calcium phosphate cement.

Premixed calcium phosphate cements (CPCs) have been developed to shorten the surgical time of conventional CPCs. However, there is lack of investigation on degradation behavior of premixed CPCs in vitro and in vivo. In this study, the premixed CPCs are prepared by mixing glycerol or polyethylene glycol (PEG) with the CPC power (ß-tricalcium phosphate (ß-TCP) and monocalcium phosphate monohydrate (MCPM)), and their degradation performances including the microstructure, chemical composition and mechanical properties are systematically evaluated both in vitro and in vivo (subcutaneous implantations in rabbits). When the premixed CPCs aged in PBS or FBS, results show weight loss of the specimens, decreased pH value and increased calcium ion concentration of aging media. Meanwhile, the setting products convert from dicalcium phosphate dihydrate (DCPD) to dicalcium phosphate anhydrous (DCPA), and no hydroxyapatite deposit. The specimen size and the molecular weight of non-aqueous solvent can modulate the setting product of premixed CPCs. For the larger specimens, DCPA is the main setting product, for the smaller ones, the composite contained DCPD and DCPA. With the decrease of the molecular weight of the non-aqueous solvent PEG, the setting product change from both DCPD and DCPA to DCPA due to the quicker exchange rate of PEG with water. After a period of subcutaneous implantation, the surface of the grafts obviously disintegrated with the formation of porous structures, but their internal morphology do not obviously change.

Three new C-glycosyflavones with acetyl substitutions from Swertia mileensis.

Three new acetylated C-glycosylflavones, 3″,6″-di-O-acetylswertiajaponin (1), 4″,6″-di-O-acetylswertiajaponin (2), and 6″-O-acetylswertiajaponin (3), together with six known compounds were isolated from the whole herb of Swertia mileensis. Their structures were elucidated on extensive NMR experiments and mass spectrometry studies. 1H and 13C NMR data exhibited doublet signals at room temperature. Variable temperature 1H NMR experiments were carried out to investigate the presence of rotational isomerism of C-glycosylflavones. All compounds showed potential antioxidant activities against apoptosis of H2O2-induced human embryo liver L02 cells.

High mechanical strength chitosan-based hydrogels cross-linked with poly(ethylene glycol)/polycaprolactone micelles for the controlled release of drugs/growth factors.

High mechanical strength hydrogels without burst release are known to be beneficial to deliver bioactive molecules including drugs and growth factors. Herein, chitosan-based hydrogels are fabricated by the covalent attachment of poly(ethylene glycol) diacrylate (PEGDA) to thiol groups of thiolated natural polymers via Michael addition reaction under physiological conditions. Poly(ethylene glycol-b-caprolactone-b-ethylene glycol) (PECL) micelles bearing double bonds act as both fillers and chemical cross-linkers to mechanically reinforce chitosan-based hydrogels, which is confirmed by the results of rheological behavior and compressive strength measurements. Indomethacin (IMC) and/or basic fibroblast growth factor (bFGF) are/is entrapped into the PECL micelle cross-linked hydrogel network primarily through hydrophobic interaction and specific affinity to thiolated heparin, respectively. After a relatively quick initial release, rather than an initial burst commonly occurring in conventional hydrogels based on drug delivery systems, IMC and/or bFGF are/is released from these PECL micelle cross-linked hydrogels at a slower rate until a steady state is reached. The release rate of IMC and/or bFGF could be readily tuned by varying the micelle amount and the thiolated heparin content in a polymer matrix.

Alginate/PEG based microcarriers with cleavable crosslinkage for expansion and non-invasive harvest of human umbilical cord blood mesenchymal stem cells.

Porous microcarriers are increasingly used to expand and harvest stem cells. Generally, the cells are harvested via proteolytic enzyme treatment, which always leads to damages to stem cells. To address this disadvantage, a series of alginate/PEG (AL/PEG) semi-interpenetrating network microcarriers are prepared in this study. In this AL/PEG system, the chemically cross-linked alginate networks are formed via the reaction between carboxylic acid group of alginate and di-terminated amine groups of cystamine. PEG is introduced to modulate the degradation of microcarriers, which does not participate in this cross-linked reaction, while it interpenetrates in alginate network via physical interactions. In addition, chitosan are coated on the surface of AL/PEG to improve the mechanical strength via the electrostatic interactions. Biocompatible fibronectin are also coated on these microcarriers to modulate the biological behaviors of cells seeded in microcarriers. Results suggest that the size of AL/PEG microcarriers can be modulated via adjusting the contents and molecular weight of PEG. Moreover, the microcarriers are designed to be degraded with cleavage of disulfide crosslinkage. By changing the type and concentration of reductant, the ratio of AL to PEG, and the magnitude of chitosan coating, the degradation ability of AL/PEG microcarriers can be well controlled. In addition, AL/PEG microcarriers can support the attachment and proliferation of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs). More importantly, the expanded hUCB-MSCs can be detached from microcarriers after addition of reductant, which indeed reduce the cell damage caused by proteolytic enzyme treatment. Therefore, it is convinced that AL/PEG based microcarriers will be a promising candidate for large-scale expansion of hUCB-MSCs.

Three new sulphur glycosides from the seeds of Descurainia sophia.

Three new sulphur glycosides, raphanuside B-D (1-3), together with a known sulphur glycoside, raphanuside (4) were isolated from the decoction of the seeds of Descurainia sophia (L.) Webb ex Prantl, and the compound 4 was reported for the first time from this plant. Their structures were identified by means of UV, IR, 1D, 2D NMR (HSQC, HMBC and NOESY) and HR-ESI-MS spectroscopic data.

Extractions of oil from Descurainia sophia seed using supercritical CO2, chemical compositions by GC-MS and evaluation of the anti-tussive, expectorant and anti-asthmatic activities.

Descurainia sophia is widely distributed in China and is one of the most troublesome annual weeds. It has diverse medicinal usage. D. sophia has abundant oil, making it an important oil plant in China. The main goal of this study was to obtain the maximum yield of the oil by an optimal selection of supercritical fluid extraction parameters. According to the central composite design and response surface methodology for supercritical fluid extraction method, a quadratic polynomial model was used to predict the yield of D. sophia seed oil. A series of runs was performed to assess the optimal extraction conditions. The results indicated that the extraction pressure had the greatest impact on oil yield within the range of the operating conditions studied. A total of approximately 67 compounds were separated in D. sophia seed oil by GC-MS, of which 51 compounds represented 98.21% of the total oils, for the first time. This study was also aimed at evaluating the anti-asthmatic, anti-tussive and expectorant activities in vivo of D. sophia seed oil which supplied for further research on bioactive constituents and pharmacological mechanisms.

Contributions of Fusarium virguliforme and Heterodera glycines to the disease complex of sudden death syndrome of soybean.

BACKGROUND: Sudden death syndrome (SDS) of soybean caused by Fusarium virguliforme spreads and reduces soybean yields through the North Central region of the U.S. The fungal pathogen and Heterodera glycines are difficult to manage. METHODOLOGY/PRINCIPAL FINDINGS: The objective was to determine the contributions of H. glycines and F. virguliforme to SDS severity and effects on soybean yield. To quantify DNA of F. virguliforme in soybean roots and soil, a specific real time qPCR assay was developed. The assay was used on materials from soybean field microplots that contained in a four-factor factorial-design: (i) untreated or methyl bromide-fumigated; (ii) non-infested or infested with F. virguliforme; (iii) non-infested or infested with H. glycines; (iv) natural precipitation or additional weekly watering. In years 2 and 3 of the trial, soil and watering treatments were maintained. Roots of soybean 'Williams 82' were collected for necrosis ratings at the full seed growth stage R6. Foliar symptoms of SDS (area under the disease progress curve, AUDPC), root necrosis, and seed yield parameters were related to population densities of H. glycines and the relative DNA concentrations of F. virguliforme in the roots and soil. The specific and sensitive real time qPCR was used. Data from microplots were introduced into models of AUDPC, root necrosis, and seed yield parameters with the frequency of H. glycines and F. virguliforme, and among each other. The models confirmed the close interrelationship of H. glycines with the development of SDS, and allowed for predictions of disease risk based on populations of these two pathogens in soil. CONCLUSIONS/SIGNIFICANCE: The results modeled the synergistic interaction between H. glycines and F. virguliforme quantitatively in previously infested field plots and explained previous findings of their interaction. Under these conditions, F. virguliforme was mildly aggressive and depended on infection of H. glycines to cause highly severe SDS.

Chemically crosslinked alginate porous microcarriers modified with bioactive molecule for expansion of human hepatocellular carcinoma cells.

Microcarrier is an essential matrix for the large-scale culture of anchorage-dependent cells. In this study, chemical cross-linked alginate porous microcarriers (AMC) were prepared using microemulsion and freeze-drying technology. Moreover, chitosan was coated on the surface of microcarriers (AMC-CS) via electrostatic interactions to improve the mechanical strength. The size of AMC can be modulated through adjusting the concentration of alginate, amount of dispersant and stirring rate. The surface chemical characteristics and morphology of AMC-CS were evaluated by Fourier transformed infrared, X-ray photoelectron spectroscopy, and scanning electron microscope. Fibronectin (Fn) or heparin/basic fibroblast growth factor (bFGF) was then immobilized on the surface of microcarriers via layer-by-layer technology to improve the cytocompatibility. Our data suggested that the size of AMC can be accurately modulated from 90 µm to 900 µm with a narrow size distribution. Micropore structures of AMC-CS were relatively disordered and the pore size ranged between 20 µm and 100 µm. Using AMC after modified with Fn or bFGF as the cell expansion microcarriers, we showed that the proliferation rates of HepG2 cells increased significantly, reaching to more than 30-fold of cell expansion after 10 days of culture, with minor cellular damage caused by the microcarriers. Moreover, the AMC microcarriers modified with Fn or bFGF can increase albumin secretion of HepG2. We suggest that our new modified AMC-based microcarriers will be an attractive candidate for the large-scale cell culture of therapeutic cells.

A new kaempferol trioside from Silphium perfoliatum.

A new apiose-containing kaempferol trioside, kaempferol-3-O-α-L-rhamnosyl-(1‴ → 6″)-O-ß-D-galactopyranosyl-7-O-ß-D-apiofuranoside, along with 16 known compounds, were isolated from 50% acetone extract of Silphium perfoliatum L. Their structures were elucidated by acid hydrolysis and spectroscopic techniques including UV, IR, MS, ¹H, ¹³C, and 2D-NMR. In addition, the pharmacological activity of compound 1 was tested with HepG2 and Balb/c mice (splenic lymphocytes and thymic lymphocytes) in vitro, and it exhibited inhibitory effect on the proliferation of HepG2 cells and showed the immunosuppressive activity.

Characteristics of oxytetracycline sorption and potential bioavailability in soils with various physical-chemical properties.

Veterinary antibiotics are widely used for disease treatment, prevention and animal growth promoting. Frequent detection of veterinary antibiotics in environments, caused by land application of untreated or even treated antibiotics-containing animal wastes, has posed the growing concern of their adverse effect on natural ecosystems. Oxytetracycline (OTC) is one of the most widely-used veterinary antibiotics in livestock industry. OTC present as a cation, zwitterions, or net negatively charged ion in soils complicates predicting its sorption characteristics and potential bioavailability and toxicity. This study was to identify soil properties influencing OTC sorption and its subsequent bioavailability in five soils with various physical-chemical properties. A solution used to determine bioavailable analytes in soils and sediments, 1 M MgCl(2) (pH 8.5), was chosen to desorb the potentially bioavailable fraction of OTC sorbed onto soils. Our results demonstrated that soils with higher illite content and permanent cation exchange capacity have higher OTC sorption capacity, but increase the availability of sorbed OTC indicated by higher release of sorbed OTC from soils into aqueous phase in 1 M MgCl(2) (pH 8.5). Reversely, soil organic matter (SOM), clay, kaolinite, variable cation exchange capacity, DCB-Fe and -Al have lower OTC sorption capacity, but decrease the release of sorbed OTC from soils into 1 M MgCl(2). These findings indicate that SOM and clay greatly influence OTC adsorption and potential availability. This study contributes significantly to our understanding of the potential bioavailability of sorbed OTC and the effects of soil properties on OTC sorption behaviors in soils.