Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroAR.aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroAE.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroAR.aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroAE.coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5  substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.The 5-enolpyruvylshikimate-3-phosphate synthase  is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition.  Zea mays L.), canola (Brassica napa L.), cotton (Gossypium hirsutum L.), alfalfa (Medicago sativa L.), and soybeans account for about 82% of biotech crops.The enzyme 5-enolpyruvylshikimate-3-phosphate synthase  is a carboxyvinyl transferase that catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P) and forms enolpyruvylshikimate-3-phosphate. It is the sixth key enzyme in the shikimate pathway, which is essential for the production of aromatic amino acids Phe, Tyr, and Trp, as well as chorismate-derived secondary metabolites in plants, fungi, and microorganisms Escherichia coli and Salmonella typhimurium, the catalytic activity of which was inhibited at low micromolar concentrations of glyphosate E. coli and Pro101Ser in S. typhimurium have been shown to present resistance to glyphosate. Class II EPSPS, identified from some bacteria, such as Pseudomonas strain PG2982, Agrobacterium tumefaciens strain CP4, Streptococcus pneumoniae, and Staphylococcus aureus, was distinguished by its capability to sustain efficient catalysis in the presence of high glyphosate concentrations In the past years, a number of glyphosate-resistant bacteria and glyphosate-tolerant plants have been identified. EPSPS from different organisms has been divided into two classes according to intrinsic glyphosate sensitivity. Class I EPSPS was generally identified from plants and bacteria, such as Rahnella aquatilis strain from the rhizosphere of grape in glyphosate-contaminated vineyards. The strain showed a significant inhibition to the pathogen causing crown galls, a worldwide plant disease in grape-growing regions R. aquatilis strain EPSPS, AroAR.aquatilis, are described. Although this EPSPS shares 88% amino acid identity with the EPSPS (P07638) from E. coli, it has more excellent kinetic qualities as it possesses similar binding affinity for the natural substrate PEP but lower affinity sensitivity to glyphosate than the E. coli EPSPS. The protein also confers glyphosate tolerance on transgenic plants in greenhouse. Using site-directed mutagenesis, two regions at subdomains 3 and 5 were identified to contribute to increased tolerance to glyphosate.In vineyards, glyphosate is widely used to control weeds directly beneath the vines. Soil and rhizosphere microorganisms from vineyards can harbor highly glyphosate-tolerant EPSPS. In this study, we have isolated a glyphosate-tolerant R. aquatilis by the biolog identification system. It was small, rod shaped, and Gram negative, and the biochemical test results showed that it was negative for cytochrome oxidase and positive for catalase. The 16S rDNA gene sequence  confirmed that GR20 was strongly related to R. aquatilis (99.8%).After selection on low-concentration glyphosate and further verification on high-concentration glyphosate, one strain has been isolated from the rhizosphere of grape. This strain, named GR20, grew well on M9 plates in the presence of 200 mM glyphosate. GR20 was identified as R. aquatilis GR20, a genomic library was constructed from this strain. There were about 2×105 ampicillin-resistant clones on the LB plates. Selection on M9 plates containing 60 mM glyphosate led to the identification of five clones. The plasmid of all these clones was extracted and digested with EcoRI and HindIII. The clone harboring a plasmid pGR20-5 with about 2.1 kb insert fragment was selected to be sequenced subsequently.To isolate the gene involved in glyphosate tolerance from R. aquatilis (GenBank sequence accession number GQ499276). To determine whether ORF427 was translated, AroAR. aquatilis was expressed and purified. The translated protein of ORF427 was determined to be 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), which agreed well with the molecular size of the predicted product , the most different amino acids between AroAR. aquatilis and AroAE.coli were lined on four-stranded β-sheet structures  in subdomain 5 R. aquatilis belonged to class I EPSPS . Cells expressing the regions mutant m35 AroAR.aquatilis grew much worse than the cells expressing the wild type. The growth potential of the cells expressing m35 AroAR.aquatilis was similar to that of cells expressing AroAE.coli in the presence of 100–150 mM glyphosate  and two AroAE.coli transgenic lines (Ec3 and Ec7) were analyzed for gene expression using RT-PCR analysis. The results of RT-PCR showed that the inserted genes were actively and stably transcribed in transgenic plants  and the f-strand of subdomain 5 are key domains for glyphosate resistance activity.Sequence analysis showed that it was significantly different between AroAE. coli, then extracted and purified for activity assay. The Km [PEP] and Km [S3P] value of AroAR.aquatilis was similar with that of AroAE.coli. However, the Ki [glyphosate] and IC50 [glyphosate] of AroAR.aquatilis values are approximately 35-fold and 5-fold higher than the values of AroAE.coli respectively  have no significant change. But the values of Ki [glyphosate] and IC50 [glyphosate] decreased when compared with that of the wild type. The ratios of the wild type enzyme over the mutant m3AroAR.aquatilis were approximately 2.8 and 1.5 for Ki [glyphosate] and IC50 [glyphosate] respectively, while the ratios over the mutant m5AroAR.aquatilis were 3.7 and 2.0. It indicated that the f-strand of the subdomain 5 was more important for glyphosate resistance than the c-strand of subdomain 3  could complement E. coli AroA mutations. Sequence analysis revealed that AroAR.aquatilis was most closely related to the EPSPS of E. coli AroAE.coli and belonged to class I AroA enzymes  of this domain are changed greatly  had particularly favorable characteristics. The TIPS mutations have been introduced into the endogenous EPSPS enzyme of Z. mays to produce the first commercial varieties of glyphosate-resistant maize . In this research, we found that AroAR.aquatilis, which showed 88% amino acid identity with the AroA from E. coli, had greater glyphosate tolerance without decreasing its affinity for PEP and S3P. AroAR.aquatilis may help to obtain a new type of class I AroA proteins that not only show high tolerance to glyphosate but also have high affinity for PEP. This can be done through domain replacement or multi-sites mutation but not single-site mutation. Through site-directed mutation and optimal codon usage, the new aroA gene is likely to improve its efficiency for glyphosate tolerance in engineering crops.Although a large number of AroA enzymes have been identified and tested as glyphosate resistant, only AroA variants derived from the E. coli strain ER2799 (with the EPSPS gene deleted from its genome) was a kind gift from Professor Thomas C. Evans, Jr. . Agrobacterium strains were bought from the Agricultural Culture Collection of China.S3P, glyphosate (free acid form), and PEP were purchased from Sigma-Aldrich. All other chemicals were of analytical grade. The field studies were conducted in a vineyard which located at Hongqi village, Minheng district, Shanghai, China. Glyphosate has been used extensively as a broad-spectrum control agent to kills weed plants over 10 years in this vineyard.This study involved collection of 10 samples of soil from the rhizosphere of grape in glyphosate used extensively vineyards, without human participants and animal work, therefore the local ethics committee considered that informed verbal consent was appropriate and this was approved by district officials, village, sub-village leaders and the owner of this vineyards. No specific permits were required for the field studies. Also the field studies did not involve endangered or protected species and no specific permissions were required for these locations. And local ethics committee waived the need for consent.Isolation of the glyphosate-tolerant strains was carried out with soil samples from the rhizospheres of grape in glyphosate-contaminated vineyard. A pool of soils from the rhizospheres of grape in 10 different sites in this vineyard were washed with 0.9% (w/v) NaCl solution and then spread onto M9 plates containing 60 mM glyphosate. Dozens of colonies were isolated after 2 days of incubation at 28°C and further screened on M9 plates in the presence of 200 mM glyphosate. One well-grown strain, GR20, was chosen for further studies.5′-AGAGTTTGATCCTGGCTCAG-3′) and 16SF (5′-ACGGCTACCTTGTTACGACTTC-3′). The PCR fragment was cloned and sequenced.The morphological, cultural, biochemical, and physiological characteristics of GR20 were examined according to the methods previously reported by Farmer Sau3A, and DNA fragments of 2 to 4 kb were purified and inserted into the BamHI site of dephosphorylated pUC18 E. coli DH5α-competent cells. Then the genomic library plasmid was transformed to the E. coli aroA mutant strain ER2799 and screened on M9 agar plates supplemented with 60 mM glyphosate. One colony harboring a plasmid pAroA-Ra5 with about 2.0 kb insert fragment was selected to be sequenced subsequently.Chromosomal DNA isolated from strain GR20 was partially digested with 5′-GAGAGAGGATCCATGGAATCCCTGACATTACAAC-3′) and GR20F2 (5′-GAGAGAGAGCTCTTATGCCAGTGTGCTGATTGC-3′) were used to amplify the EPSPS gene R.aquatilisAroA from pAroA-Ra5. Multiple-site mutagenesis was carried out in order to induce mutations in the hinge region  and the subdomain 5  of R.aquatilisAroA by PAGE-mediated overlap extension PCR 5′-GCTTTTGCACAGGGTAAAACCGTCCTGACTAACCT GCT CGA-3′) and M1F (5′-TCGAGCAGGTTAGTCAGGACGGTTTTACCCTGTGCAAAAGC-3′) for mutations in the hinge region and M2Z (5′-ACCACGAGTACCAGCAATTTGTCGTCAAAGGA CGTCAGCATTA-3′) and M2F (5′-TAATGCTGACGTCCTTTGACGACAAATTGCTGGTACTCGT GGT-3′) for mutations in subdomain 5. The first PCRs were performed to amplify three mutant fragments using GR20Z and M1F, M1Z and M2F, and M2Z and GR20F. PCR products were resolved using 8% polyacrylamide gels, recovered and purified by electroelution into dialysis bag, and concentrated by ethanol precipitation. The full-length mutant gene R.aquatilism35AroA was obtained by PCR with a pair of primer GR20Z and GR20F and the mixed templates combined with the above three purified DNA fragments. Single-site mutagenesis was carried out to obtain the mutant gene R.aquatilism5AroA or R.aquatilism3AroA through direct amplification from pAroA-Ra5 with the prime M1Z and M1F or M2Z and M2F. In order to further testify that the f-strand of the subdomain 5 was important for glyphosate binding, we have generated m5AroAE.coli with the subdomain 5 of AroAR.aquatili. Also, the G96AAroAE.coliG96AAroAR.aquatilis were created using a PCR-based staggered extension process.Primers GR20Z2 (5′-CGGGATCCGTTAATGCCGAAATTTTGCTTAATC-3′) and EF1 (5′-CGGAGCT CAGGTCCGAAAAAAAACGCCGAC-3′) were used to amplify the EPSPS gene E.coliAroA from the chromosomal DNA of E.coli. The amplified fragments of the wild type gene R.aquatilisAroA, mutant gene R.aquatilism35AroA and E.coliAroA were digested with BamHI and SacI and ligated into the pYPX251 vector (GenBank accession number AY178046) to construct recombinant plasmids p251-AroA-Ra, p251-m35AroA-Ra and p251-AroA-Ec, respectively. These EPSPS genes would be driven by the moderate promoter of aacC1.Primers EZ1  and TSPF (5′-CAGAGGATCCTCTGTGC AGTGACCACTGAT-3′). The PCR fragment was digested with SalI and BamHI, cloned into the corresponding restriction sites of pGEM3Z , and confirmed by DNA sequencing. The PstI-SalI fragment of double CaMV35S promoter was inserted upstream of the TSP sequence in the sequence confirmed plasmid. The fused fragment and aroA gene were cloned into the Agrobacterium binary vector pYF7716 PstI-BamHI and BamHI-SacI restriction sites, respectively. The final constructs D35S:TSP:aroA:Nos  by electroporation. The tobacco (Nicotiana tabacum cv Xanthi) was transformed by the leaf discs method via the Agrobacterium-mediated transformation procedure described by Wang et al. A DNA fragment encoding the chloroplast transit peptide of tobacco  was PCR amplified from the genomic DNA of tobacco using primers TSPZ , and first-strand cDNA was synthesized using 5 µg total RNA with the Reverse Transcription System . The fragments of the tobacco Actin gene were amplified from the same cDNAs as a control to normalize the amount of cDNA using primers TactZ (5′-CAATGAACTTCGTGTGGCTCC-3′) and TactF (5′-CGGAATCTCTCAGCACCAATG-3′). The specific fragment of about 200 bp from R.aquatilisAroA, R.aquatilism35AroA and E.coliAroA gene was amplified from transgenic plants using the same amount of cDNA. The PCR products were separated on 2% agarose gel and quantified using a Model Gel Doc 1000 . The expression pattern of those aroA genes was evaluated with a Shine Tech Gel Analyser . The same results were obtained for three independent experiments.The transgenic plants were confirmed by PCR with the specific primers GR20Z2 and GR20F2 for 1 generations were transplanted into soil and grown to five- to six-leaf stage. After spraying with the 5 mM glyphosate for 2 weeks, injury of the transgenic plants was observed visually.The leaf discs were cut from individual transgenic tobacco and then moved to Murashige and Skoog (MS) medium containing 0 to 0.8 mM glyphosate and grown for 3 weeks with 16 h of light and 8 h of dark. Seedlings of TR.aquatilisAroA and the mutants R.aquatilism35AroA, R.aquatilism3AroA, and R.aquatilism5AroA was obtained by PCR from the positive colony with the following two primers: GR20Z (5′-GAACCATGGAATCCCTGACATTACAACCCGT-3′) and GR20F (5′-GTTCTCGAGTGCCAGTGTGCTGATTGCCGCCA-3′). Primer GR20Z contains a mutation where the initiation start codon ATG was replaced with GUG. The fragment was digested with NcoI and XhoI, cloned into the corresponding restriction sites of pET-28a , and confirmed by DNA sequencing. Then the plasmid was transformed to E. coli BL21 . The expressed protein was purified using a HisTrap HP kit (Amersham Biosciences) according to the manufacturer's instructions. The AroAE.coli from E. coli was overexpressed and purified according to the method used above. Also the protein of R.aquatilisAroA was further blotted onto a polyvinylidene difluoride membrane for N-terminal protein sequencing using an Applied Biosystems Procise sequencer.DNA fragments containing the entire coding region of EPSPS activity was measured by determining the amount of inorganic phosphate produced in the reaction using the malachite green dye assay method Km values for PEP, Ki values for glyphosate, and IC50 values (the concentration of glyphosate inhibiting enzyme activity by 50%) were measured as described by Tian et al. Rahnella aquatilis was deposited as GenBank Accession number GQ499276.The novel 1284 bp DNA sequence encoding the 5-enolpyruvylshikimate-3-phosphate synthase from Figure S1Overexpression and purification of AroAR.aquatilis. Lane 1, molecular mass marker; lane 2, protein of E.coli ER2799; lane 3, overexpression of AroAR.aquatilis induced with IPTG; lane 4, purified protein with HisTrap HP kit.(TIF)Click here for additional data file.Figure S2Kinetic properties of AroAE.coli., m35AroAR.aquatilis, m3AroAR.aquatilis, m5AroAR.aquatilis, wild type AroAR.aquatilis, m5AroAE.coli, G96AAroAE.coli and G96AroAR.aquatilis. A. Steady-state kinetics of AroAE.coli., m35AroAR.aquatilis, m3AroAR.aquatilis, m5AroAR.aquatilis, wild type AroAR.aquatilis, m5AroAE.coli, G96AAroAE.coli and G96AroAR.aquatilis Activities were assayed in Hepes buffer at 28°C in the presence of 1 mM S3P and various concentrations of PEP. B: Steady-state kinetics of AroAE.coli., m35AroAR.aquatilis, m3AroAR.aquatilis, m5AroAR.aquatilis, wild type AroAR.aquatilis, m5AroAE.coli, G96AAroAE.coli and G96AroAR.aquatilis. Activities were assayed in Hepes buffer at 28°C in the presence of 1 mM PEP and various concentrations of S3P. C. IC50 values of AroAE.coli., m35AroAR.aquatilis, m3AroAR.aquatilis, m5AroAR.aquatilis, wild type AroAR.aquatilis, m5AroAE.coli, G96AAroAE.coli and G96AroAR.aquatilis. The IC50 values of glyphosate inhibition were determined by fitting the data to the equation v = minV+ (maxV−minV)/ [1+ ([I]/IC50)s], and v was determined at 1 mM PEP and 1 mM S3P, with glyphosate concentrations ranging from 0.0001 to 100 mM.(TIF)Click here for additional data file.


Radix Salvia miltiorrhiza (Danshen) has been used as a principal herb in treating cardiovascular diseases in Chinese medicine. Salvianolic acid B (SA-B), a water-soluble active component of Danshen, was found to have anti-myocardial ischemia (anti-MI) effect. This study aims to investigate mechanisms of SA-B on MI.Five conventional Western medicines  with different mechanisms for treating cardiovascular diseases were selected as positive references to compare with SA-B in changing of the metabolomic profiles in MI rats under treatment. Potential mechanisms of SA-B were further investigated in H9C2 cell line.2+ and inhibiting protein kinase A (PKA).The metabolomic profiles between SA-B- and propranolol-treated MI rats were similar, since there was a big overlap between the two groups in the PLS-DA score plot. Finally, it was demonstrated that SA-B exhibited a protective effect on MI mainly by decreasing the concentration of cyclic adenosine monophosphate (cAMP) and CaSA-B and propanolol exhibited similar metabolomic profiles, indicating that the two drugs might have a similar mechanism. The phosphorylation of L-type Ca2+ channel causes the Ca2+ influx, leading to stronger muscle contraction. The phosphorylation of phospholamban accelerates Ca2+ uptake into the sarcoplasmic reticulum, increasing the rate and extent of muscle relaxation [Myocardial ischemia (MI) is characterized by ischemia in the heart muscle. It is the most common cause of death, and a major reason for hospital admissions . In MI, laxation .2+, leading to slow heart rate, decreased myocardial contractility, reduced cardiac output, and decreased myocardial oxygen consumption. Besides, Western medicines with other mechanisms of action, such as isosorbide dinitrate (a vasodilator), verapamil , captopril (an angiotensin converting enzyme inhibitor) and trimethazine (a fatty acid oxidation inhibitor), are also commonly used in the prevention and treatment of MI.β-adrenergic blockers such as propranolol  could inradix salvia miltiorrhiza (Danshen) is also widely used in Chinese medicine for the treatment of cardiovascular diseases [Danshen preparations in China medicine, including Compound Danshen Tablet, Compound Danshen Dripping Pill and Compound Danshen Granule [In recent years, diseases . Danshen Granule -8 have bDanshen on rat femoral artery was primarily produced by the inhibition of Ca2+ influx in the vascular smooth muscle cells. Kim et al. [Danshen could activate the endothelial nitric oxide synthase to induce vasodilation and reduce blood pressure. However, there is no final conclusion about mechanisms of Danshen. Thus, most scientists hope to increase understanding of mechanisms of Danshen by analyzing active components of Danshen. Presently, many active components have been isolated and identified in Danshen, such as tanshinone IIA, and salvianolic acid B (SA-B) [Lam et al. ,10 foundm et al.  found thB (SA-B) -14.Danshen, is effective for the protection of heart from ischemia [SA-B, a water-soluble active component of ischemia . Severalischemia -20.2-agonist but later specifically introduced as a growth-promoting agent, showed a pattern of mRNA and lipid expression almost identical to that showed by clenbuterol and salbutamol, β2-agonists. In the same way, anti-MI drugs may be categorized into classes based on their mechanisms of action.Metabolomics is an emerging technique in the field of "omics" research and it is the systematic study of metabolites and its profile in a biological matrix, such as a cell, organ or organism ,22. It aIn this study, metabolomic approach was used to explore potential mechanisms of SA-B by comparing with five Western medicines  conventionally used for the treatment of cardiovascular diseases.® Non-Radio cAMP-Dependent Protein Kinase Assay were purchased from Promega .HPLC grade acetonitrile was purchased from JT Baker . Spectroscopic grade formic acid, leucine enkephalin, dimethyl sulfoxide (DMSO) and 3--2, 5-diphenyltetrazolium bromide were purchased from Sigma/Aldrich . Distilled water was purified "in-house" using a Milli-Q20 system Millipore . SA-B was purchased from the National Institute for the Control of Pharmaceutical and Biological Products . Isosorbide dinitrate was purchased from Forward Co., Ltd . Verapamil was purchased from Shanghai Pharmaceutical (Group) Co., Ltd . Propranolol was purchased from Shanghai Xinpashi Pharmaceutical Co., Ltd . Captopril was purchased from Shanghai Hengshan Pharmaceutical Factory . Trimethazine was purchased from Servier Pharmaceutical Factory . Fluo3/AM was purchased from Dojindo Laboratory . cAMP-Glo™ Assay and PepTagFifty male Sprague-Dawley rats (185-215 g) were purchased from the Slac Laboratory Animal Co., Ltd . The animals were housed in stainless steel metabolic cages under controlled conditions of humidity (40-60%), temperature (23-27°C), and a 12 h light-dark cycle. They were allowed free access to food and tap water. MI model was induced by left anterior descending coronary artery ligation . The sha2 incubator  at 37°C with 5% CO2. The cells were seeded at a density of 1 × 104/well in a 96-well plate, and cultured for 12 h for adherence. Cells were pre-treated with propranolol (0.01 mg/mL) or SA-B at concentrations of 0.001, 0.01 and 0.1 mg/mL for 2 h, and the cells were incubated in a Tri-Gas/CO2 incubator  at 37°C with 5% CO2 and 95% N2 which was anoxic circumstance for 24 h. The cells were incubated in a humidified CO2 incubator  at 37°C with 5% CO2 as a control group. Ca2+, cAMP and PKA in the cells were measured. SA-B and propranolol were dissolved in DMSO. Appropriate amount of DMSO was also added to control media so that the same final concentration of DMSO (less than 0.1%) can be kept in all groups.The H9C2 cell line  was obtained from the Cell Bank of the Chinese Academy of Sciences and maintained in Dulbecco's Modified Eagle's Medium  containing 10% fetal bovine serum (FBS), penicillin-G (100 U/mL) and streptomycin (100 U/mL), and incubated in a humidified COg for 10 min at 4°C, and the supernatants were collected. Acetonitrile (300 μL) was added to the supernatant (100 μL). The mixture was shaken vigorously for 30 s and centrifuged at 9562.5 × g for 10 min at 4°C. After centrifugation, the supernatant was analyzed on an ACQUITY™ UPLC system coupled to a Micromass Q-Tof Micro™  equipped with an electrospray ionization source. A 2.1 mm i.d. × 100 mm ACQUITY™ 1.7 μm column  was used. The column was maintained at 45°C. Mobile phase A was formic acid (0.1%) in water; mobile phase B was formic acid (0.1%) in acetonitrile; injection volume was 5 μL; flow rate was 400 μL/min. The gradient duration program was: 0-1.5 min, 5% of B; 1.5-9 min, 5-100% of B; 9-14 min wash with 100% of B; and a 3 min recycle time. The parameters of mass detection were: desolvation gas at 400 L/h; cone gas at 20 L/h; desolvation temperature at 250°C; source temperature at 100°C; capillary voltage at 3000 V; cone voltage at 30 V; collision energy at 5 eV, while it was set at 20 eV in MS/MS mode to identify potential biomarkers. The data acquisition rate was set as 0.4 s, with 0.1 s inter-scan delay. Lock spray was used to calibrate accuracy of mass. Leucine enkephalin was used as the lock mass . Data were collected in the continuum mode and averaged over 10 scans. The lock spray frequency was set at 10 s. Full scan mass range of 50-1000 m/z was acquired.A blood sample (1 mL) from each rat was collected and kept in 2.5 mL heparin-coated tubes. These samples were centrifuged at 2789.1 × Cell viability was assessed by the MTT assay . The cel2+-sensitive was detected by dyeing with fluo-3/AM [2+ in cells were examined by flow cytometry .The Caluo-3/AM . The chaThe cAMP-Glo™ assay was used to monitor changes in cAMP concentrations . The lumThe activity of PKA was detected by non-radioactive PepTag PKA assay kit  with dye-labeled Kemptide as substrate according to the manufacturer's protocol . The celpost hoc test. P values less than 0.05 were considered significant.The spectral data were exported by Micromass MarkerLynx™ applications manager version 4.1 software . The raw data of each sample was normalized to total area to correct for the MS response shift from the first injection to the last injection due to the long duration, overnight or longer, of an LC-MS analysis. The sum of the ion peak area within each sample was set at 10,000. After processing, partial least squares discriminant analysis (PLS-DA) was used for analysis of metabolite profiles, which was performed by the SIMCA-P software version 11 . In cell culture, each assay was performed in triplicate and repeated three times. The data were expressed as mean ± standard deviation (SD). The significance was tested by one-way analyses of variance (ANOVA) of the SPSS 13.0 for Windows , followed by the Duncan This study aims to investigate mechanisms of SA-B on MI. Firstly, metabolomics in combination with multivariate statistical analysis was used to display potential mechanisms of SA-B by comparing with five Western medicines  conventionally used for the treatment of cardiovascular diseases. Secondly, the potential mechanisms of SA-B suggested by the metabolomic study were further investigated in H9C2 cell line.In our previous study , a total2-intercept in permutation test should be less than 0.05. The intercept value of Q2 for this model was -0.315, which meant no overfitting.PLS-DA model  was builP < 0.05, SA-B-treated groups versus MI group) [The mean levels of 22 identified MI biomarkers in SA-B-treated rats were calculated to further evaluate the regulations of SA-B. As shown in Table I group) . The res2 = -0.301, which suggested there was no overfitting. The result indicated that SA-B and propanolol might have a similar mechanism in treating MI, since the drugs with the same mechanism showed similar effects on the metabolomic profile [In order to investigate the mechanisms of SA-B, we selected five Western medicines  with different mechanisms for treating MI as positive references. PLS-DA model  was esta profile .P < 0.05, SA-B-treated group versus MI group)  . In this study, we focused on the influences of SA-B and propranolol on the downstream regulation in β-adrenoceptor signaling pathway. The effects of SA-B on the levels of on cAMP, PKA and Ca2+ in cells were investigated by H9C2 cell line, which is a commercially available myogenic cell line derived from embryonic rat heart ventricle [As aforementioned, propranolol  could inentricle  and is wentricle .2+, cAMP and PKA assays showed that SA-B significantly decreased Ca2+ concentration in anoxic H9C2 cells . We also found that both SA-B and proprannolol significantly inhibited the increased levels of cAMP induced by anoxia , but required a high dosage (0.1 mg/mL) to decrease the concentration of cAMP. The cell experiments demonstrated that SA-B had a similar role in regulation of concentrations of Ca2+, cAMP and activity of PKA as proprannolol.The viability of H9C2 cells was not affected significantly by pretreatment with SA-B up to 0.1 mg/mL for 24 h Figure , indicata Figure . SA-B de2+, cAMP and activity of PKA as proprannolol. While the drug action of the metabolites of SA-B is still under investigation, our results suggest that metabolomics is a good analytical method for understanding mechanisms of SA-B.Our previous studies suggested that the metabolomic profile was changed in MI rats , and SA-Danshen, and is potentially effective for the protection of heart from ischemia-reperfusion. Metabolomic results showed that SA-B and propranolol had similar effects on metabolic profiles of MI rats. The cell experiments demonstrated that SA-B had protective effects on MI mainly by decreasing Ca2+ and cAMP and inhibiting the activation of PKA. This study indicates that SA-B and propanolol might have a similar mechanism in treating MI.SA-B is a water-soluble active component of MI: myocardial ischemia; AC: Adenylate cyclase; ATP: adenosine triphosphate; cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; SA-B: salvianolic acid B; VEGF: vascular endothelial growth factor; DMSO: dimethyl sulfoxide; ECG: electrocardiograms; DMEM: Dulbecco's Modified Eagle's Medium; FBS: fetal bovine serum; OD: optical density value; PLS-DA: partial least squares discriminant analysis; SD: standard deviation; ANOVA: one-way analyses of variance.The authors declare that they have no competing interests.YHL, YZ, TJL and WDZ designed the study. YHL conducted the metabolomic study and wrote the manuscript. YZ assisted in the cell experiments and in writing the manuscript. XRL and XL assisted in writing the manuscript. SMN, TJL and WDZ amended the manuscript. All authors read and approved the final manuscript.


During the development of a therapeutic antibody, large numbers of monoclonal antibodies are required to screen for those that are best suited for the desired activity. Although the single cell-based immunoglobulin variable gene cloning technique is a powerful tool, the current methods remain an obstacle to the rapid production of large numbers of recombinant antibodies.We have developed a novel overlap extension polymerase chain reaction, the target-selective joint polymerase chain reaction (TS-jPCR), and applied it to the generation of linear immunoglobulin gene expression constructs. TS-jPCR is conducted using a PCR-amplified immunoglobulin variable gene and an immunoglobulin gene-selective cassette (Ig-cassette) that contains all essential elements for antibody expression and overlapping areas of immunoglobulin gene-specific homology. The TS-jPCR technique is simple and specific; the 3'-random nucleotide-tailed immunoglobulin variable gene fragment and the Ig-cassette are assembled into a linear immunoglobulin expression construct, even in the presence of nonspecifically amplified DNA. We also developed a robotic magnetic beads handling instrument for single cell-based cDNA synthesis to amplify immunoglobulin variable genes by rapid amplification of 5' cDNA ends PCR. Using these methods, we were able to produce recombinant monoclonal antibodies from large numbers of single plasma cells within four days.Our system reduces the burden of antibody discovery and engineering by rapidly producing large numbers of recombinant monoclonal antibodies in a short period of time. Recombinant monoclonal antibody technology comprises a series of molecular approaches that allows for the production of therapeutic antibodies ,2. MolecBecause of the need for a more convenient method for the generation of recombinant antibodies, the overlap extension polymerase chain reaction method (overlap PCR) has been developed. In this method, a PCR-amplified V gene fragment is joined to DNA cassettes by PCR to build a linear immunoglobulin gene expression (Ig-expression) construct -17. WhilThis study describes a novel overlap PCR method termed target-selective joint PCR (TS-jPCR). With this method, a PCR-amplified V gene fragment can be selectively assembled into a linear Ig-expression construct, even in the presence of nonspecifically amplified DNA fragments. TS-jPCR is accomplished by joining the 3'-random nucleotide-tailed V gene fragment and an immunoglobulin-selective cassette (Ig-cassette). The Ig-cassette contains all the essential elements for antibody expression and V-gene-specific long homology overlaps within a single DNA molecule.We also developed a robotic magnetic head handling instrument (MAGrahder) that allows for automated single cell-based cDNA synthesis and 3' end homopolymer tailing using the MAGrahd method . The MAGTo evaluate the performance of TS-jPCR, we conducted a pilot experiment using an artificially amplified mouse V gene and a mock DNA fragment Figure . The mocThe scheme in Figure When TS-jPCR is conducted with the Ig-cassette and the tailed mock DNA, the melted strands of the Ig-cassette and mock DNA partially hybridize with each other via their short homology overlaps (P1 to VP1 and P2 to VP2) to generate unstable hybrid duplexes. It is important to note that these structures render the polymerase unable to extend any strands because of the 3'-end mismatches. Therefore, nonspecifically amplified DNA that lacks the V gene-specific sequence (T1 and T2) internal to the primer-derived sequence (P1 and P2) is unable to be joined to the Ig-cassette Figure .Single cell-based cDNA synthesis is a costly and time-consuming process for the production of recombinant monoclonal antibodies from large numbers of single cells. To speed up this process, we developed an automatic non-contact magnetic power transmission instrument, MAGrahder. MAGrahder is composed of a MAGrahd reactor tray and a desktop robot. The MAGrahd reactor tray can be assembled from a reusable tray and a single-use super-hydrophobic film upon which a 24 × 8 grid of cup-shaped structures to hold drops are embossed Figure . The robWe next attempted to demonstrate the utility of TS-jPCR by producing Ig-expression constructs from amplified V genes. After 3'-end random nucleotide tailing of the 5'-RACE PCR products, they were joined to the respective Ig-cassette using TS-jPCR. As shown in Figure We have established TS-jPCR and used it for the rapid production of recombinant monoclonal antibodies from mouse plasma cells. While the principle of this procedure is essentially the same as other recently reported PCR-based methods, TS-jPCR has several useful features that overcome the limitations of the current methods. First, purification of the amplified V genes represents a significant, major bottleneck for the high-throughput production of recombinant antibodies. In contrast, TS-jPCR enables the production of Ig-expression constructs, even in cases where the nonspecific amplification product happens to be the same size as the V genes. Second, current PCR-based methods sometimes suffer low Ig-expression construct yields. This result is likely due to inefficient hybrid duplex formation between the V gene and the promoter or terminator cassette, which is mediated through their short homology overlaps (~25 bp). TS-jPCR can produce a sufficient amount of linear expression construct for DNA transfection and direct sequencing by using a single cassette that has long and specific homology overlaps (over 50 bp) at both ends.Several methods for single cell-based cDNA synthesis have been reported to amplify V genes by 5'-RACE PCR, including the use of multi-well methods . HoweverWe recently developed a target-selective homologous recombination cloning method (TS-HR) for the single-step insertion of PCR-amplified V gene fragments into expression vectors . BecauseStarting from fifty-three isolated mouse plasma cells, we achieved amplification of the VH and VL genes within a single day. On the next day, each of the amplified V genes was joined to an Ig-cassette by TS-jPCR to generate Ig-expression constructs that can be used for DNA transfection and direct sequencing. Our methods are directly applicable to rapid and scalable automation for the generation of large numbers of recombinant monoclonal antibodies.® HD Transfection Reagent and TdT were obtained from Roche Applied Science. PrimeSTAR™ HS DNA Polymerase was obtained from Takara Bio. Restriction enzymes were obtained from New England Biolabs. Titermax Gold Adjuvant was obtained from CytRx Corporation. A SHOTMASTER 300 desktop robot was purchased from Musashi Engineering. The animal experiment was approved by the Committee on Animal Experimentation at Toyama University.Dynabeads oligo(dT)25 magnetic beads, Dulbecco's Modified Eagle's Medium, SuperScript III Reverse Transcriptase and One-shot TOP10 competent cells were obtained from Invitrogen. Super-hydrophobic film was obtained from Alcan Packaging. The FuGENEGATATCACGTGTGCCTGGTCAAGGGCTATTTCCCTGAG-3' and 5'-- CTCCGCGGCCGCTGGGATCATTTACCAGGAGAGT -3' (restriction sites underlined). A DNA fragment encoding 309 bp of the mouse immunoglobulin kappa (IgK) constant region (441-750 of nucleotide accession number: AF466770) was amplified with the primers 5'--GATATCACGTGCTGTATCCATCTTCCCACCATCC -3' and 5'--TCTCGCGGCCGCTGTCTCTAACACTCATTCCTG-3'. A DNA fragment containing the pUC replication origin, an ampicillin resistance gene and a SacB gene was amplified from pDNR (Takara-Clontech) by PCR with the primers 5'-AGAGAGACCCGGGCCAGGAACCGTAAAAAGGCCG-3'and 5'-AGAGAGACCCGGGACGTCCACATATACCTGCC-3'. The dC13/dG13 linker DNA was made by annealing the oligonucleotides 5'--GATCCCCCCCCCCCCCGATATC-3' and 5'--GATCGATATCGGGGGGGGGGGG -3'. The dC13/dG13 linker, the pDNR-derived DNA fragment and the mouse IgG constant gene fragment were inserted into the Bam HI/Not I site of pGFP N1 plasmid (Clontech). The pJON-mIgG plasmid was generated from the resulting plasmid by removing the pEGFP N1-derived sequence spanning from the kanamycin gene to the pUC replication origin by PCR. The pJON-mIgK plasmid was constructed with the dC13/dG13 linker, the pDNR-derived DNA fragment and the mouse IgK constant gene fragment in the same manner. A map of the plasmids is shown in Figure A DNA fragment encoding 901 bp of the mouse immunoglobulin gamma (IgG) constant region (559-1460 of nucleotide accession number: AB097849) was amplified with the primers 5'-- The MAGrahd reactor tray consists of three components: drops, magnetic beads and a super-hydrophobic film-layered tray. The upper tray plate of an Omni tray (Nunc) was cut in a rectangular shape and was fixed to a thin glass cover plate  instead of a plastic board. Super-hydrophobic film was fabricated by pressing the film with an aluminum master stamp that contained the positive relief features against a silicone plate. The master stamp used here was made using a standard end mill process. The fabricated film has a 24 × 8 grid of positive relief features, which capture drops on a planer surface. The grid consists of two arcs that are arranged to form a cup-shaped enclosure with diameter of 3 mm. The arcs in each body project 0.15 mm upwards from a planar surface and are separated from one another by gaps of approximately 2.5 mm in width on the upper side to allow magnetic beads to move into the cup and 1 mm in width on the bottom side to allow magnetic beads to move through the grids. Twenty-four grids are arranged in rows, whose pitch matches the pitch of standard 384-well plates. The fabricated film was bound to the inner surface of the thin glass plate. On this film, droplets were spontaneously self-centered on the generated pattern, which enabled parallel sample processing by MAGrahder.A SHOTMASTER 300 desktop robot was converted into MAGrahder by mounting a magnetic head. Twenty-four neodymium magnet rods  were installed on the head, whose pitch matches the pitch of standard 96-well plates. With a specially designed software program, the non-contact magnetic power transmission can be adapted to achieve optimal output.Plasma cells were isolated from popliteal lymph nodes of egg albumin-immunized mice using a CD138+ plasma cell isolation kit as previously described . Single VH and VL chain genes were amplified by single cell-based 5'-RACE PCR with 3'-end homopolymer-tailed cDNAs as templates. Briefly, the first round of PCR was performed with a dC13 forward primer (5'-CGGTACCGCGGGCCCGGGATCCCCCCCCCCCCCDN-3') and a mixture of reverse primers (P0) specific for the respective IgG (5'-ACCYTGCATTTGAACTCCTTGCC-3') and IgK (5'-ACTGCCATCAATCTTCCACTTGACA-3') constant regions. PCR was performed using PrimeStar DNA polymerase in 1× PrimeStar GC buffer with the BIO-RAD MyCycler . The resulting PCR mixtures were next diluted at a 1:10 ratio with water, and 1 μl of each was used for the second round of PCR. The second round of PCR was performed with a forward primer (P1) that annealed to the dC13 forward primer used in the first PCR and the respective nested reverse primer (P2) specific for the IgG (5'-CTGGACAGGGATCCAGAGTTCCA-3') or IgK (5'-ACTGAGGCACCTCCAGATGTTAACT-3') constant regions.GTGGTGCCCATCCTGG -3' and 5'-CTGGACAGGGATCCAGAGTTCCATGCCGAGAGTGATCCCG -3' (underlined sequences are homologous to the GFP gene). The mock DNA sequence (727 bp) consisted of the upper primer-derived sequence (P1 region), a portion of the GFP gene sequence (712-1372 of nucleotide accession number: U55762), and the lower primer-derived sequence (P2 region). The mock DNA was used as a nonspecifically amplified DNA sequence for the pilot experiment. The mouse V gene fragment consisted of the upper primer-derived sequence (P1 region), the poly dC/dG sequence (T1 region), the mouse IgG V region, part of IgG constant region (T2 region) (560-600 of nucleotide accession number: AB097849) and the lower primer-derived sequence (P2 region) (601-623 of nucleotide accession number: AB097849). The amplified DNA fragments were purified using a spin column. A schematic illustration of the DNA fragment and Ig-cassettes is found in Figure A mock DNA sequence was amplified by PCR from pEGFP-N1 (Takara-Clontech) with the primers 5'-CTTCGAATTCTGCAGTCGACGGTACCGCGGGCCGGGAfter the second round of PCR, 1 μl of the PCR solution was mixed with 1 μl of TdT solution (1 × PrimeStar GC buffer containing 0.2 mM of each dNTP and 2 units of TdT) and incubated at 37°C for 30 min for 3'-end random nucleotide tailing. TdT was heat inactivated at 94°C for 5 min.The random nucleotide-tailed PCR products (50-100 ng) were mixed with 20 μl of PCR mixture containing 10 ng of the IgH- or IgK-cassette, 0.2 mM of each dNTP, 1.25 U of PrimeStar DNA polymerase and 400 nM of the P3 and P4 PCR primers (5'-AGAGAAACCGTCTATCAGGGCGATGGC-3' and 5'-AGAGACCCTTTGACGTTGGAGTCCACG-3') in 1× PrimeStar GC buffer. TS-jPCR was performed for 5 cycles with denaturation at 95°C for 30 s and annealing and strand elongation at 70°C for 240 s, followed by 25-30 cycles with denaturation at 95°C for 30 s, primer annealing at 60°C for 30 s and primer extension at 72°C for 1 min.The unpurified TS-jPCR products (1 μl each) of paired IgH- and IgL-expression constructs were co-transfected with FuGENE HD Transfection Reagent (Roche) into 293FT cells grown in 96-well culture dishes without the risk of endotoxin contamination derived from the plasmid purification step out of bacteria. Two days after transfection, the cell culture supernatants were analyzed for the secretion of recombinant antibodies. Antibody concentration and antibody reactivity were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) as described previously .http://imgt.cines.fr/IMGT_vquest/share/textes/[The nucleotide sequence was determined using an Applied Biosystems 373 DNA sequencer with a BigDye Terminator v3.1 Cycle Sequence Kit (Applied Biosystems). The alignment of the V-(D)-J sequence was performed using IMGT/V-QUEST e/textes/.PCR: polymerase chain reaction; TS-jPCR: target-selective joint PCR; GFP: green fluorescent protein; V: immunoglobulin variable; VL: immunoglobulin light chain variable; VH: immunoglobulin heavy chain variable; 5'-RACE PCR: rapid amplification of 5'cDNA ends polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin gamma; IgK: immunoglobulin kappa; TdT: terminal deoxynucleotidyl transferase, RLU: relative light unit.MY performed the experiments and data analysis described in this study. NK designed the experiments and drafted the manuscript. MI organized the experiments. All authors read and approved the final manuscript.


Artemether is the derivative extracted from Chinese traditional herb and originally used for malaria. Artemether also has potential therapeutic effects against tumors. Vascular cell adhesion molecule-1 (VCAM-1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we investigated the role and mechanism of artemether combined with shRNA interference of VCAM-1 (shRNA-VCAM-1) on the migration, invasion and apoptosis of glioma cells. U87 human glioma cells were treated with artemether at various concentrations and shRNA interfering technology was employed to silence the expression of VCAM-1. Cell viability, migration, invasiveness and apoptosis were assessed with MTT, wound healing, Transwell and Annexin V-FITC/PI staining. The expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and phosphorylated Akt (p-Akt) was checked by Western blot assay. Results showed that artemether and shRNA-VCAM-1 not only significantly inhibited the migration, invasiveness and expression of MMP-2/9 and p-Akt, but also promoted the apoptosis of U87 cells. Combined treatment of both displayed the maximum inhibitory effects on the malignant biological behavior of glioma cells. Our work revealed the potential therapeutic effects of artemether and antiVCAM-1 in the treatments of gliomas. Gliomas, the most common primary malignant tumors in the central nervous system, are refractory to the surgical therapy of extensive resection and are easy to be recurrent due to their highly invasive and infiltrative growth pattern In recent years, studies have demonstrated that artemisinin and its derivatives had a significant cytotoxic effects toward cancer cells and could reverse multiple drug resistance of tumors Interactions between extracellular matrix (ECM) and adhesion molecules are essential for the development and angiogenesis of gliomas In the present study, we first investigated the effects of artemether on the proliferation, migration, invasion and apoptosis of human glioma U87 cells. Based on this, RNA interference technique was employed to silence the expression of VCAM-1. Afterwards, we examined the effects of artemether combining with shRNA interference of VCAM-1 (shRNA-VCAM-1) on the cell migration, invasion, the expressions of MMP-2/9, phosphorylated Akt (p-Akt) and cell apoptosis, aiming at providing novel experimental evidence for the treatment of human brain glioma.2.The human U87 MG cell line was purchased from Nanjing KGI Biotechnology Co., Ltd. U87 MG cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12)  supplemented with 10% fetal bovine serum (FBS)  in an incubator at 37°C in humidified atmosphere and 5% CO5′-CACCGGAAGCCGATCACAGTCAAGTTTCAAGAGAACTTGACTGTGATCGGCTTCCTTTTTTG-3′; Antisense: 5′-GATCCAAAAAAGGAAGCCGATCACAGTCAAGTTCTCTTGAAACTTGACTGTGATCGGCTTCC-3′. The sequence of sh negative control (shNC) was Sense: 5′-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTG-3′; Antisense: 5′-GATCCAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGACGTGACACGTTCGGAGAAC-3′. U87 cells were transfected by Lipofectamine LTX and plus reagent (Invitrogen) according to the manufacturer’s manual. The medium containing transfection reagents was replaced with DMEM/F-12 supplemented with 10% FBS 18 h after transfection. The cells were collected 48 h after transfection, processed in the following experiments and prepared for protein extraction. The silence efficiency of VCAM-1 was tested by Western blot (described below).Cells were seeded into 24 well plates (Corning) until they reached 50–60% confluence prior to transfection. Afterwards, the stable transfection was performed. Four short hairpin RNAs (shRNA) targeting on VCAM-1 gene were designed and synthesized by Shanghai Jima pharmaceutical technology, China. The most applicable shRNA (shRNA-VCAM-1) was identified by G418 concentration gradient screening and applied in the following experiments. The sequence of shRNA-VCAM-1 was Sense: P<0.05).Cell viability and proliferation activity was checked with MTT assay as described previously 5/well until they reached 80% confluence. The scratching wounds were created in the monolayer of confluent U87 cells with a pipette tip. The width of wounds was assessed to be the same at the beginning of the experiments. The wells were rinsed with PBS three times to remove floating cells and debris. To test the effects of artemether on the migration of U87 cells, parental U87 cells were seeded and serum-free DMEM/F12 with or without artemether  was added. Then these cells were incubated for 48 h. We also compared the effects of artemether with shRNA-VCAM-1. Parental U87 cells and shRNA-VCAM-1-U87 cells were seeded and either blank serum-free DMEM/F12 or serum-free DMEM/F12 containing artemether (300 µmol/L) was added in each cell line. The culture plates were incubated at 37°C and in 5% CO2. Wound healing was measured and recorded photographically over time using phase- contrast microscopy at 0, 24 and 48 h.Scratch wound healing assay was adapted to evaluate the migration ability of U87 cells according to the previous protocols 4/ml and 200 µl of U87 cell suspension was added into the upper chambers. 800 µl of DMEM/F12 supplemented with 10% FBS was placed in the lower chambers. Serum free DMEM/F12 served as a negative control. Artemether was added in the suspension of parental U87 cells at various concentrations  and artemether at the concentration of 300 µmol/L was also added into the suspension of shRNA-VCAM-1-U87 cells. After incubated for 48 h, the inserts were taken out and cells remained on the upper surface of the filters were removed carefully with a cotton wool swab. The cells migrating to the underside surface were washed with PBS gently and fixed with methanol and glacial acetic acid (mixed at 3∶1) for 30 min at room temperature and stained in Giemsa stain for 15 min. The average number of invasive cells was counted in six random high-power fields (×400).The effects of artemether and shRNA-VCAM-1 on the invasion were checked using Transwell invasion assay with inserts of 8-µm pore size (Corning Costar) as described previously U87 cells were stimulated as previously described. The cells were washed three times with ice-cold PBS to stop the stimulation. Then, the cells were collected and lysed in ice-cold radio immunoprecipitation assay lysis buffer containing 50 mM Tris (PH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride and EDTA  for 30 min. Then the pellet was disrupted with an ultrasonic crusher and samples were centrifuged at 17,000 rpm for 60 min at 4°C. The supernatant was collected as the soluble fraction and transferred to a new tube. The protein concentration of the soluble material was determined with BCA protein assay kit , with bovine serum albumin used as a standard. Equal amounts of proteins (20–25 µg) were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and processed for immunoblotting with a rabbit multiclonal antibody for VCAM-1 , a goat multiple clonal antibody for Akt1/2, a rabbit multiple antibody for p-Akt 1/2/3 (Ser473) and rabbit multiple clonal antibodies for MMP-2/9. A mouse polyclonal anti-β-actin antibody  was used as an internal control. All the protein bands were scanned using ChemiImager 5500 V2.03 software, and the integrated density values (IDV) were calculated by computerized image analysis system (Fluor Chen 2.0) and normalized with that of β-actin.2 and Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 seperately. The hydrolysis of these fluorescent substrates by protein supernatant  exacted from parental U87 cells or U87 cells treated with artemether , shRNA-VCAM-1 and artemether (300 µmol/L)+shRNA-VCAM-1 was measured at 37°C. The rate of product formation in each well was recorded at 420 nm using a microplate luminometer and plot data was analyzed according to the manufacturer’s instruction. The activity of the MMPs was expressed as picomoles substrate hydrolyzed per second.Kinetic analyses were performed to check the enzymatic activity of MMP-2/9 with fluorescently quenched gelatin as described previously 6/ml, stained with Annexin V-FITC and PI in darkness for 15 min at room temperature and then analyzed by flow cytometry .Apoptosis of U87 cells was analyzed with Annexin V-fluorescein isothiocyanate (FITC)/Propidium iodide (PI) apoptosis detection kit . Briefly, cells were cultured in 6-cm dishes and treated with or without artemether (300 µmol/L) for 48 h. Then they were trypsinized with 0.25% Typsin, washed with PBS twice and collected by centrifugation . The collection of cells was suspended with binding buffer at the density of 1×10t-test and differences between multiple groups were assessed with Bonferroni test. P<0.05 was considered to be statistically significant.All results were described as mean ± S.D. Statistical analysis was performed with SPSS 13 software. Differences between groups were assessed using a Student’s P<0.01). Moreover, significant proliferation inhibition was observed in 48-hour and 72-hour groups compared with the 24-hour groups.The effects of artemether on the proliferation of U87 cells were assessed with MTT method. U87 cells were treated with artemether at various concentration levels  for 24–72 h. As shown in Nevertheless, the cell viability did not vary significantly in different concentration levels above 400 µmol/L , nor did it change obviously in 48 or 72 h groups. These results indicated that artemether inhibited the proliferation of U87 cells significantly at 400 µmol/L for 48 h. Therefore 48 h was selected as the optimal administration time in the following studies. The IC50 for 24 h, 48 h and 72 h was 948.47±10.2 µmol/L, 354.81±13.8 µmol/L and 275.42±9.5 µmol/L respectively. Based on IC50 measured in this work, concentrations of 150, 300 and 600 µmol/L were selected to be used in the following experiments according to the literature The effects of artemether on the migration and invasiveness of U87 cells were checked by the means of wound healing and Transwell assays. U87 cells were treated with artemether at various concentrations of 0, 150, 300 and 600 µmol/L for 0, 24 and 48 h. The results of wound healing assay displayed that healing over the scratch decreased gradually with increased concentrations of artemether . ArtemetP<0.05, P<0.01, P<0.01 respectively).Then the effects of artemether on the invasiveness of U87 cells were assessed with Transwell assay. The results were shown in P<0.01). 600 µmol/L displayed the maximum inhibition on both protein expressions. The kinetic analysis of catalyzed hydrolysis using a synthetic fluorogenic substrate was performed to investigate the effects of artemether on the catalytic behavior of MMP-2 and MMP-9 according to the literature The family of MMPs has been identified as the invasion related proteins. MMP-2 and MMP-9 are two important members, which play important roles in the invasion and malignancy of glioma cells It has been reported that VCAM-1 is highly expressed in the glioma tissues. VCAM-1 mediates the migration of leukocytes and messenchymal stromal cells. Nevertheless, the role of VCAM-1 in the migration and invasion of glioma cells has not been clearly identified. In the present study, we also tried to investigate the role of VCAM-1 in the malignancy of U87 cells and whether the inhibitory effects would be enhanced if combined with shRNA-VCAM-1. The expression of VCAM-1 was inhibited with shRNA stable transfection technology. The silence of VCAM-1 protein was assessed with Western Blot and the results were displayed in Next, the role of VCAM-1 in the migration and invasiveness of human glioma cells was investigated with shRNA-VCAM-1 U87 cells. The results of wound healing assay revealed that migration of shRNA-VCAM-1 U87 cells was inhibited significantly compared with control group . SimilarP<0.01). Moreover, artemether+shRNA-VCAM-1 displayed the greatest inhibition on the expression of MMPs over single artemether or shRNA-VCAM-1 groups (P<0.05). The enzymatic activity was reduced significantly with the treatments of arthemeter or shRNA-VCAM-1 compared with control group and artemether+shRNA-VCAM-1 group displayed the maximum inhibition on the MMP activity  and artemether+shRNA-VCAM-1 displayed the maximum promoting effect on the apoptosis over artemether or shRNA-VCAM-1 alone (P<0.01).The effects of artemether (300 µmol/L) and shRNA-VCAM-1 alone or in combination on the apoptosis of U87 cells were also investigated. The apoptosis was evaluated by Annexin V-FITC/PI staining. The results were shown in P<0.01). However, no significant inhibition was obtained in the shRNA-VCAM-1 group (P>0.05). Interestingly, artemether+shRNA-VCAM-1 produced further inhibition on p-Akt expression than artemether alone (P<0.01).Phosphoinositide-3-kinase (PI3K)/Akt pathway is involved in many biotic activities. It has also been clarified that PI3K/Akt pathway also plays a major role in the malignancy of glioma cells such proliferation and invasiveness In the present study, we discovered that artemether inhibited the proliferation, migration and invasion of human malignant glioma U87 cell line in a dose-dependent manner. We also showed that artemether significantly promoted the apoptosis of U87 cells and inhibited the expressions of MMP-2 and MMP-9, which are the two major proteolylic enzymes involved in the invasion and metastasis of gliomas Artemisinin is the product isolated from the plant of Artemisia annua, a Chinese traditional herb. Artemisinin and its derivatives are originally used in the therapy of malaria, especially antidrug malaria As an important adhesion molecule, VCAM-1 plays an important role in the migration and proliferation of endothelial cells to induce angiogenesis The mechanisms relating the inhibitory effects of artemether and shRNA-VCAM-1 on the malignancy of glioma cells remain poorly understood. Previous study has revealed that VCAM-1 was involved in the regulation of MMP-2 and MMP-9 Artemether reduces the migration and invasion capability of glioma cells and promotes the apoptosis of U87 human glioma cells by inhibiting MMP-2, MMP-9 and p-Akt expressions. The combination of artemether with shRNA-VCAM-1 shows a synergistic inhibitory effect on the malignancy of glioma cells.


RNase H is an endonuclease that hydrolyzes the RNA strand in RNA/DNA hybrids. Retroviral reverse transcriptases harbor a C-terminal RNase H domain whose activity is essential for viral replication. The RNase H degrades the viral genomic RNA after the first DNA strand is synthesized. Here, we report the biophysical and enzymatic properties of the RNase H domain of prototype foamy virus (PFV) as an independently purified protein. Sequence comparisons with other retroviral RNases H indicated that PFV RNase H harbors a basic protrusion, including a basic loop and the so-called C-helix, which was suggested to be important for activity and substrate binding and is absent in the RNase H domain of human immunodeficiency virus. So far, no structure of a retroviral RNase H containing a C-helix is available.D for the RNase H domain in the low micromolar range which is about 4000-fold higher than that of the full-length protease-reverse transcriptase enzyme. Analysis of the RNase H cleavage pattern using a [32P]-labeled substrate indicates that the independent RNase H domain cleaves the substrate non-specifically. The purified RNase H domain exhibits a well defined three-dimensional structure in solution which is stabilized in the presence of Mg2+ ions.RNase H activity assays demonstrate that the PFV RNase H domain is active, although its activity is about 200-fold reduced as compared to the full length protease-reverse transcriptase enzyme. Fluorescence equilibrium titrations with an RNA/DNA substrate revealed a KOur data demonstrate that the independent PFV RNase H domain is structured and active. The presence of the C-helix in PFV RNase H could be confirmed by assigning the protein backbone and calculating the chemical shift index using NMR spectroscopy. Retroviral reverse transcription describes the formation of a double-stranded DNA using the single-stranded viral RNA genome as a template. The process is catalyzed by the viral reverse transcriptase which harbors a polymerase and an RNase H domain.retroviridae and follow a distinct replication pattern unique among retroviruses: (a) reverse transcription occurs predominantly in the virus producing cell (b) the pol-gene coding for the viral enzymes is expressed from an independently spliced mRNA and (c) the viral protease is not cleaved off from the Pol precursor protein, leading to a mature protease-reverse transcriptase (PR-RT)  at the 5' end to visualize the cleavage products. To obtain cleavage of the substrate, we had to use almost 700 fold more RNase H-(Q591-N751) than PR-RT   determined (see below) for the RNase H domain  Figure . This isn Figure . The cleD) for substrate binding. In the full length PR-RT enzyme, binding of the RNase H domain is amplified by the polymerase domain which also binds to the substrate and, due to its larger size, covers a larger portion of it. To analyze whether the higher RNase H activity of the full length PR-RT can,at least in part, be explained by a higher binding affinity of PR-RT, we performed fluorescence equilibrium titrations with a fluorescently labeled DNA/RNA primer/template substrate. The KD value of 9.1 nM (± 1.7) we obtained for the full length PFV PR-RT  + (k2/k1), or if k2 can be neglected KM = k-1/k1 = KD, indicating that the KM for the RNase H domain is greater than 40 μM.To get more information on the kinetic parameters, we set out to determine the dissociation constant  allows the individual detection of peptide backbone amide signals. Thus, each signal represents a single amino acid of the peptide chain, and the spectrum serves as a fingerprint of the folding state of a protein. Adoption of a defined structure increases the dispersion of signals of amide protons in the HSQC spectrum to 6.9 - 9.7 ppm, relative to a random coil structure, which only exhibits an amide proton signal range from 7.8 - 8.7 ppm.Proper folding of the RNase H domain was analyzed by NMR spectroscopy using uniformly 2+ well-dispersed signals  typical for a protein with a defined tertiary structure are obtained  allowed the assignment of the backbone resonances for all residues except, D607, N639, N692-K695, Q725-N728, S730, I731, E734. The assigned  HSQC spectrum is depicted in Figure 680 - S686), as determined by the chemical shift index (CSI) method (see below), are marked by boxes. The proline at position 682 is not visible in the HSQC spectrum due to the lack of a backbone N-H bond.NMR data analysis of triple resonance experiments of uniformly To determine the secondary structure elements and to prove the existence of the C-helix experimentally, we compared the observed NMR resonance frequencies with tabulated values found in random coil peptides by the CSI method Figure . The sys594-I603, N612-Y623, N631-I635, V664-T668, I716-E721) show upfield chemical shifts of α- and carbonyl carbons and downfield chemical shifts of their α-protons, relative to random coil values and thus can be identified as β-strands  could only be solved with a mutant protein lacking the C-helix -ATP  and 20 U T4 polynucleotide kinase  as described 0 = total concentration of substrate DNA/RNA; KD = dissociation constant; R = ratio of fluorescence intensities of the bound and free substrate.where A = anisotropy measured; A15N labeled RNase H-(Q591-N751) was analyzed in 5 mM Na-phosphate pH 6.8 or 7.0, 100 mM NaCl, 0.5 mM DTT, 10% D2O (v/v) in the absence or presence of 6 mM MgCl2. For NMR resonance assignment standard double and triple resonance through-bond correlation experiments [15N,13C labeled RNase H-(Q591-N751), in 5 mM Na-phosphate pH 7.0, 100 mM NaCl, 6 mM MgCl2, 0.5 mM DTT, 10% D2O (v/v) at 25°C on a Bruker Avance 700 MHz spectrometer equipped with a cryogenically cooled probe. In-house protocols were used to process the NMR data and the program NMRview were utilized for analysis .1 mM uniformly eriments ,47 were 591-N751) assignments have been deposited in the BioMagResBank, accession number: 17745The PFV RNase H-(QEscherichia coli; FV: foamy virus; GB1: immunoglobulin binding domain B1 of streptococcal protein G; HIV-1: human immunodeficiency virus type 1; IN: integrase; IPTG: isopropyl-thiogalactoside; PFV: prototype foamy virus; MoMLV: Moloney murine leukemia virus; PPT: polypurine tract; PR: protease; RHBD: RNA-hybrid binding domain; RNase H: ribonuclease H; RT: reverse transcriptase; TEV: tobacco etch virus;E. coli: The authors declare that they have no competing interests.BMW conceived and coordinated the study. BL and FM performed the experiments. BL, BMW, MJH and KS analyzed the data. BMW, BL and KS wrote the paper. All authors read and approved the manuscript.


The NiII atom, lying on an inversion center, is four-coordinated by the phenolate O atoms and imine N atoms of two Schiff base ligands, forming a square-planar geometry. The O- and N-donor atoms are mutually trans. In the crystal structure, the nitrate anions are linked to the complex cations by intermolecular N—H⋯O hydrogen bonds.The asymmetric unit of the title compound, [Ni(C DOI: 10.1107/S1600536810025663/sj5031Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Pica pica) in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2, PA, and NP genes, and H5N1-like  PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46) discovered in our recent study. Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants. However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice. Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies ( H9N2 subtype influenza viruses, derived from Eurasian and North American major gene pools Furthermore, G1-like H9N2 viruses (e.g. HK/1073/99 or HK/1074/99) have been detected in humans since 1999 which caused mild disease symptoms It is noteworthy that H9N2 influenza viruses have been isolated from a variety of birds, including pheasant, chukkar, partridge, guinea fowl, and pigeon in the past two decades e.g. A/magpie/Korea/YJD174/2007) and H5N1  can be isolated from magpies indicate that magpies may play an important role in the evolution and ecology of influenza A virus. Black-billed magpies (BbM) are very common birds in southern China and have lived in close proximity to human settlements and poultry farms for centuries. In order to evaluate the role of black-billed magpies in the evolution of influenza A viruses, we characterized three H9N2 influenza viruses isolated from wild black-billed magpies during two epidemic surveys of influenza viruses in Guangxi, China in 2005. Eight complete gene segments of isolated BbM viruses were amplified, sequenced and analyzed phylogenetically; and their molecular properties and evolutionary characteristics were determined. In addition, their antigenicity, replication and pathogenicity in chickens and mice were also investigated.Outbreaks of H9N2 influenza viruses in domestic poultry have resulted in huge economic losses. And human infections by H9N2 viruses reported in 1999 A total of 32 paired tracheal and cloacal swabs were collected from black-billed magpies living in the wild bird reservoir in Guangxi of China in 2005. Collected samples were placed into viral transport media and maintained at −70°C until they were inoculated into 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs. Three H9N2 subtype in fluenza viruses were isolated from three separate birds among 32 black-billed magpies (isolation rate 9.4%), and were named A/Black-billed Magpie/Guangxi/29/2005 (BbM/GX/29/05) to A/Black-billed Magpie/Guangxi/31/2005 (BbM/GX/31/05). Of these BbM viruses, BbM/GX/29/05 was isolated from the cloacal swab, while BbM/GX/30/05 and BbM/GX/31/05 were isolated from the tracheal samples.Phylogenetic analysis of HA genes showed that H9N2 viruses were divided into two distinctive clusters with similar branching patterns as previously reported Phylogenetic analysis of NA genes revealed a similar evolutionary pattern to that of HA genes . NA geneThe phylogenetic tree of PB1 genes was separated into eight distinct evolutionary lineages, including G1-like, H5N1-like, KR323-like, SH/F/98-like, DE113-like, Y439-like, WI/1/66-like, and BJ94-like . Most ofIn the phylogenetic tree of PB2 genes, there were eight different lineages: BJ94-like, Y439-like, H5N1-like, SH/F/98-like, DE113-like, KR323-like, G1-like, and WI/1/66-like . The BbMPhylogenetic analysis of PA genes showed that at least eight different lineages were recognized  . The BbMThe phylogenetic tree of NP genes had seven distinct lineages comprised of SH/F/98-like, DE113-like, G1-like, Y439-like, KR323-like, BJ94-like and WI/1/66-like . The NP In the phylogenetic tree of M genes, the BbM viruses grouped with the BJ94-like lineage containing Ck/SH/F/98 and Ck/HLJ/35/00 , and werFor the NS genes, BbM viruses were almost identical in comparison with 2005-2006 Guangxi isolates (99.7% to 100% homology). All these isolates belonged to the BJ94-like lineage and shared a common ancestor with Ck/SH/F/98 . These rTo further determine the genetic characterization and probable origin, the cladogram of BbM viruses and H9N2 representative strains from various lineages was genetically analyzed . The resPanorama genotypical analysis from our recent study revealed that H9N2 influenza viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series, designated as A–G representing HK/G1/97, BJ/1/94, KR/96323/96, DE/113/95, HK/289/78, HK/AF157/92, and WI/1/66 lineage viruses, respectively The deduced amino acid sequences of BbM virus proteins were also compared with those of H9N2 reference viruses. Results indicated that the HAs of BbM viruses shared the same R-A-S-R amino acid motif at the connecting peptide between HA1 and HA2 with those of 2005-2006 H9N2 Guangxi isolates from different hosts . AlternaAccording to NetNGlyc 1.0 Server predicted results, eight N-linked potential glycosylation sites (PGS) were conserved in HA proteins of BbM viruses  A1, of w10EID50/ml. Results of mouse experiments demonstrated that infection by BbM viruses induced 5% to 10% weight loss of the inoculated mice and BbM viruses could be isolated from lung tissues of infected mice on day 3 p.i. with titers of 4.5 to 5.2 log10EID50/ml. However, no virus was recovered from spleens and brains and no deaths were observed in inoculated mice during the experiment period. These findings showed that BbM viruses replicated efficiently in chickens and mice but were not lethal to them.To assess the pathogenicity of BbM viruses in chickens and mice, 6-week-old, SPF chickens and 8-week-old, SPF BALB/c mice were inoculated intranasally (i.n.). Virus replication, morbidity, and mortality were assessed . ResultsThe antigenic properties of H9N2 in fluenza viruses from black-billed magpies were investigated with post-infection chicken antisera from three different viruses by HI assay . NumericGiven the capability for H9N2 influenza viruses to infect wild birds, poultry, and humans in Asia, further evaluation of potential reservoir species such as black-billed magpies in the evolution of influenza viruses is necessary. Characterization of three H9N2 influenza viruses in this study, isolated firstly from wild black-billed magpies , revealed that these BbM viruses were circulating in Guangxi of China in 2005. They represent novel genotypical reassortants, containing BJ94-like, SH/F/98-like, and H5N1-like gene segments and substituting for previous SH/F/98-like influenza viruses . AnalysiResults of phylogenetic analysis demonstrated that HA, NA, M and NS genes of BbM viruses were closely related to those of BJ94-like viruses. Three internal genes, PB2, PA and NP, were descended from SH/F/98-like viruses which may have been the donor of H5N1-like inner genes Results of genotypical analysis showed that BbM viruses represent genotype B46 viruses, which documented the existence of H5N1-like PB1 genes, resulting in a single cluster in the PB1 phylogenetic tree and a unique genotype. Genotype B46 virus was first recognized in chickens in 2004 and became predominant in poultry, pigs, and birds during 2005-2006 Molecular analysis demonstrated that all BbM viruses maintained the similar HA connecting peptide motifs with terrestrial poultry viruses of low pathogenicity in chicken , and alsIt was reported recently that Qa/HK/G1/97 replicated efficiently in the human alveolar epithelial A549 cells whereas other H9N2 viruses, Dk/HK/Y280/97 and Ck/HK/G9/97, replicated poorly The aquatic birds including ducks, shorebirds, and gulls have been regarded as the natural reservoir for in fluenza A virus, with sixteen HA and nine NA subtypes reported to date In summary, our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. These BbM viruses were highly related to H5N1-like viruses in ribonucleoprotein complex genes, and are capable of replicating efficiently in chickens and mice without previous adaptation, suggesting that they may require relatively less genetic changes such as exchange of gene segments or accumulation of critical mutations to become more pathogenic to hosts or make the avian-to-mammalian transmission. Therefore, conducting long-term molecular epidemiological investigations of H9N2 influenza viruses in black-billed magpies is necessary to better understand the genetic evolution of BbM viruses and elucidate their potential role in future influenza outbreaks in poultry and epidemics in humans.This study was approved by the Ethical Committee of Animal Experiments of the Institute of Zoology, Chinese Academy of Sciences, which does not issue a number to any animal study. All animal care and use were conducted in strict accordance with the Animal Research Committee guidelines of the Institute of Zoology, Chinese Academy of Sciences. All surgery was performed under anesthesia, and all efforts were made to minimize suffering.Two epidemic surveys on avian influenza viruses in wild black-billed magpies were conducted at the mountain forest near Zhongzhou village in Guangxi, China in March and May of 2005. Briefly, tracheal and cloacal swabs were collected from trapped black-billed magpies, and were eluted with PBS containing 2000U/ml penicillin and 1000ug/ml streptomycin at 4°C for 12 h. Samples were centrifuged at 5000 g for 10 min at 4°C and the supernatant was inoculated into the allantoic cavity of 10-day-old SPF embryonated chicken eggs. Inoculated embryos were incubated at 37°C and allantoic fluid was harvested 48–72 h p.i. TM DNA polymerase with specific primers (primer sequences available on request). The PCR products were purified with an agarose gel DNA extraction kit (Tiangen) and directly sequenced by the Invitrogen Biotechnology Company. Obtained sequences were assembled and compiled with the SeqMan program of the Lasergene 6.0 , and were deposited in GenBank.Viral RNA was extracted from infected allantoic fluid with Trizol LS Reagent (Invitrogen) and was reverse-transcribed into cDNA by using universal influenza primer with the M-MLV reverse transcriptase (Promega). Eight full-length influenza virus genes were amplified by using the TaKaRa Ex Taqhttp://www.mbio.ncsu.edu/BioEdit/bioedit.html). To determine sequence similarities and evolutionary relationships, pairwise sequence alignments were performed and neighbor-joining trees were constructed using the MEGA version 4.0.2 program (http://www.megasoftware.net/). Amino acid sequences of HA, NA, PB2, PB1, M and NS proteins were also aligned and analyzed by Clustal W in the BioEdit program. Potential N-linked glycosylation sites in HA and NA proteins were predicted with the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/).To better understand the evolution of H9N2 viruses in black-billed magpies, the complete genomes of BbM viruses were sequenced and analyzed phylogenetically, and neighbor-joining phylogenetic trees were constructed for eight gene segments based on nucleotide sequences. The reference sequences were available from GenBank . NucleotAccording to available genomic information of different H9N2 genotypes from our recent study 6.0 EID50 of virus-allantoic fluid To determine the pathogenicity of isolated viruses in chickens, 6-week-old, SPF White Leghorn chickens  were i.n. inoculated with 0.1 ml 106.0 EID50 of the virus-allantoic fluid in a volume of 0.05 ml, and were observed daily for 14 days for signs of disease. Control mice were inoculated with PBS. On day 3, three mice from each group were sacrificed for collection of lungs, spleens, and brains under sterile conditions for virus titration in eggs 10EID50 per 1 ml ± standard deviation (SD). The remaining inoculated mice were monitored daily for weight loss and mortality.To determine the pathogenicity of isolated viruses in mice, groups of eight 8-week-old SPF female BALB/c mice  were i.n. inoculated under anesthesia with 10Antigenic analysis was performed using antisera to three different H9N2 viruses generated in 6-week-old SPF White Leghorn chickens. The antigenicity of three BbM H9N2 influenza viruses were determined by HI assays as described previously The nucleotide sequences for influenza virus genes obtained from this study have been deposited to GenBank under accession numbers GU121379 to GU121386 and HM590759 to HM590774.Figure S1Potential N-linked glycosylation sites in HA and NA proteins of the BbM viruses (BbM/GX/29/05 as a template) predicted by the NetNGlyc 1.0 Server analysis.(TIF)Click here for additional data file.Figure S2Potential N-linked glycosylation sites in HA1 proteins of the BbM viruses based on the 1jsdA HA1 template. In blue HA1 protein, red arrow-head indicates mutable potential glycosylation sites at positions 82 and 218, while black arrow-head indicates conserved potential glycosylation sites at positions 29, 141, 298 and 305.(TIF)Click here for additional data file.Table S1Accession numbers of nucleic acid sequences used in this study.(DOC)Click here for additional data file.Table S2Amino acid differences at the whole PB1 protein between the BbM viruses (BbM/GX/29/05 as a template) and analyzed reference viruses from different lineages.(DOC)Click here for additional data file.


Geminiviruses mainly infect terminally differentiated tissues and cells in plants. They need to reprogramme host cellular machinery for DNA replication. This process is thought to be mediated by inactivation of cell-cycle repressor proteins and by induction of host DNA synthesis protein expression through actions of the geminviral replication initiator protein (Rep).Nicotiana benthamiana pOri2 line, which is transformed with a transgene consisting of a direct repeat of the African cassava mosaic virus (ACMV)-replication origin (Ori) flanking a non-viral DNA region, and virus-induced RNA silencing (VIGS), the impact of host gene expression on replication of the ACMV-derived replicon was investigated. The ACMV Rep trans-replicated the viral episomal replicon in leaves of young but not older pOri2 plants. Upon VIGS-mediated down-regulation of N. benthamiana NbRBR1, the retinoblastoma-related protein gene coding for a negative cell-cycle suppressor, recovered the ability of ACMV Rep for trans DNA replication, whereas the silencing of NbPCNA coding for the sliding clamp of DNA polymerase had no effect.Exploiting a These results suggest that the cellular machinery for DNA replication in differentiated tissues of older leaves cannot be reprogrammed by Rep alone but may need other uncharacterised viral and plant factors. Begomovirus, family Geminiviridae. ACMV possesses two circular DNAs, designated DNA-A and DNA-B of approximately 2.7 kb [cis-acting elements required for replication and transcriptional modulation of viral gene expression [African cassava mosaic virus (ACMV) is a single-stranded (ss) DNA virus in the genus y 2.7 kb , both arpression ,4. The bpression ,6. Rep apression ,6,8,9. Apression , affect pression , disturbpression ,13.in planta trans-replication system [Nicotiana benthamiana retinoblastoma-related (NbRBR1) and proliferating cell nuclear antigen (NbPCNA) genes, , on viral DNA replication. The potato virus X (PVX)-based vector for virus induced RNA silencing (VIGS) was employed to knock-down NbRBR1 and NbPCNA expression. To achieve this, a 420-bp fragment corresponding to the 5' end of NbRBR1 (Genbank accession number: AY699399) was obtained through reverse-transcription (RT)-PCR with total RNAs template from healthy N. benthamiana leaves using Qiagen RNeasy Minikit (Qiagen) for extraction and the primer pair PP369 (5' ACGACATCGATATGGGTGGAGCTGAATAATTGTTC 3') and PP449 (5' CTCTTCCGGCCGTCTGAACCATACAGATTG 3'). The RT-PCR product was digested with ClaI/EagI and cloned into the same sites of the modified PVX/GFP vector to produce PVX/NbRBR1-GFP  was obtained through RT-PCR using a pair of primers PP398 (5' TTTTCAATCGATATGTTGGGAATTACGGCTTGT 3') and PP450 (5' AATTACCGGCCGGTCACCGATTACTGCTAAGGT 3') to generate PVX/NbPCNA-GFP  were inoculated with RNA transcripts for PVX/GFP, PVX/NbRBR1-GFP or PVX/NbPCNA-GFP as previously described [NbRBR1 resulted in growth retardation, abnormal leaf development, and newly emerged leaves were irregularly shaped and had a definite curl downwards running the whole circumference of the leaf  RT-PCR. Total RNAs (100 ng) were extracted at 31 dpi from leaves at the age of 56-days old plants, and used as templates together with one of the two sets of primers PP369 and PP370 (5' TCTTGCGGCCGTCGCTTGTAGTACTTGCTTAAAAG 3') or PP398 and PP439 (5' TTGAGACGGCCGCACCTTCCTTTGTCGCAGAAATTA 3') for sqRT-PCR assays. These primers allowed a specific detection of the 500-nt portion corresponding to the endogenous mRNA transcripts of the two target genes. Reduction in NbRBR1 . These data suggest that the expressed ACMV Rep is only able to initiate DNA replication in older leaves if expression of the cell cycle block NbRBR1 is reduced.The effect of silencing of m Figure . Trans-D4 Figure . Express4 Figure  and temp4 Figure  detected4 Figure . HoweverS Figure , circulaS Figure . SimilarTomato golden mosaic virus, Rep-RBR interaction and PCNA accumulation are important for virus replication and infectivity in N. benthamiana [Geminiviral Rep is not a DNA polymerase. However, during viral DNA replication the oligomeric Rep protein cleaves the viral replication origin TAATATT↓AC and acts as an ATP-dependent ligase to re-circularise progeny ssDNA ,16,17. Ethamiana ,24,26. Othamiana -29. Indethamiana . HoweverRBR1. For instance, there may be low expression of NbRBR1 in S-phase cells of growing leaves of young plants whereas in older leaves high NbRBR1 expression could effectively maintain cells in G1 phase with minimal replication activity. Indeed, in older tissues NbRBR1 mRNA was readily detectable (Figure NbPCNA, an auxiliary protein of DNA polymerase, could not restore the deficiency of DNA replication (Figure NbRBR1 expression by VIGS may allow G1 cells of older leaves progressing into the S phase. Consequently, DNA synthesis functionality is re-activated and then exploited by ACMV Rep (Figure ACMV Rep was unable to initiate RCR in cells of mature leaves of older pOri2 plants although it was effective in triggering RCR in cells of growing leaves of young plants. It is possible that young growing leaves possess many S-phase cells with an active DNA replication machinery, which ceases to function in cells of older mature leaves. One factor affecting the cellular DNA replication capacity can be different expression levels of genes that encode cell cycle modulators such as e Figure . Howevern Figure . Howeverp Figure . Thus, oThe authors declare that they have no competing interests.GB designed and performed experiments; MG, NS and YL contributed through discussion and revised paper. YH initiated the project, designed experiments and wrote paper. All authors read and approved the final manuscript.


JE from Jeotgalicoccus sp. ATCC 8456, a new member of the CYP152 peroxygenase family, was recently found to catalyze the unusual decarboxylation of long-chain fatty acids to form α-alkenes using H2O2 as the sole electron and oxygen donor. Because aliphatic α-alkenes are important chemicals that can be used as biofuels to replace fossil fuels, or for making lubricants, polymers and detergents, studies on OleTJE fatty acid decarboxylase are significant and may lead to commercial production of biogenic α-alkenes in the future, which are renewable and more environmentally friendly than petroleum-derived equivalents.Cytochrome P450 OleT2O2-independent activity of OleTJE for the first time. In the presence of NADPH and O2, this P450 enzyme efficiently decarboxylates long-chain fatty acids (C12 to C20) in vitro when partnering with either the fused P450 reductase domain RhFRED from Rhodococcus sp. or the separate flavodoxin/flavodoxin reductase from Escherichia coli. In vivo, expression of OleTJE or OleTJE-RhFRED in different E. coli strains overproducing free fatty acids resulted in production of variant levels of multiple α-alkenes, with a highest total hydrocarbon titer of 97.6 mg·l-1.We report the H2O2-independent activity of OleTJE not only raises a number of fundamental questions on the monooxygenase-like mechanism of this peroxygenase, but also will direct the future metabolic engineering work toward improvement of O2/redox partner(s)/NADPH for overproduction of α-alkenes by OleTJE.The discovery of the H The urgency to develop sustainable fossil fuel alternatives is driven by rapidly increasing global consumption, irreversibly diminishing reserves, unpredictable geopolitical factors, fluctuating price of crude oil, growing concerns about national energy security, and serious environmental concerns surrounding immense greenhouse gas emissions mainly resulting from combustion of fossil fuels -3. BiofuMicrococcus luteus/.The GC-MS analytical method for hydrocarbon and fatty acid samples was adapted from Guan et al.. The anaGC inlet  were notde et al. except feviously . Quantifβ-D-thiogalactopyranoside; LB: lysogeny broth; OD: optical density; PCR: polymerase chain reaction; RhFRED: Rhodococcus fusion reductase.ACP: acyl carrier protein; CoA: coenzyme A; CYP: cytochrome P450 enzyme; DTT: dithiothreitol; FdR: flavodoxin reductase; Fld: flavodoxin; GC-MS: gas chromatography-mass spectrometry; IPTG: Isopropyl-The authors declare that they have no competing interests.SL conceived of the study. YL, CW, XL and SL designed the experiments. YL, JY and WZ performed the experiments including plasmid construction, protein overexpression, purification, characterization and enzymatic assays. YL, CW and WG carried out GC-MS analysis. YL, CW and SL drafted the manuscript. JY, WZ, WG and XL helped to revise the manuscript. All authors read and approved the final manuscript.(A) OleTJE and (B) OleTJE-RhFRED. M, protein marker.SDS-PAGE analysis of purified Click here for file(A) OleTJE and (B) OleTJE-RhFRED.CO-bound reduced spectra of purified Click here for fileBSβ from Bacillus subtilis); CYP152A2 (P450CLA from Clostridium acetobutylicum); CYP152B1 (P450SPα from Sphingomonas paucimobilis); CYP152B2 (from Azotobacter vinelandii); CYP152C1 (from Rhodobacter sphaeroides); CYP152C2 (from Rhodobacter sphaeroides); CYP152D1 (from Streptomyces scabies); CYP152E1 (from Cyanothece sp. CCY0110); CYP107L1 (P450PikC from Streptomyces venezuelae); CYP102A1 (P450BM3 from Bacillus megaterium); CYP101A1 (P450cam from Pseudomonas putida). Conserved amino acid residues are shaded. The Arg and Pro absolutely conserved in CYP152 family are marked by asterisks.Protein sequence alignment of CYP152 family members and three selected P450 monooxygenases. Protein sequences were obtained from NCBI protein databases. CYP152A1  and internal standard (IS), respectively.Click here for fileThe dry cell weight of the YL5-8 cultures using LB broth.Click here for fileProduction profile of free fatty acids by the strain XL100/(pMSD8 + pMSD15).Click here for filePrimers used in this study.Click here for file(A) pET28b-oleTJE and (B) pET28b-oleTJE-RhFRED expression vectors.Plasmid maps for Click here for fileα- and β-hydroxy myristic acid authentic standards on HP-INNOWAX capillary column. (A) Without derivatization, α-hydroxy myristic acid was unseen due to its high boiling point. Importantly, no thermally degraded terminal olefin (1-tridecene) was observed in the dashed box. (B) Without derivatization, β-hydroxy myristic acid was unseen due to its high boiling point. Again, no thermally degraded terminal olefin (1-tridecene) was observed in the dashed box. (C) The decarboxylation reaction of myristic acid (2 h) catalyzed by OleTJE. The peak shown in the dashed box corresponds to 1-tridecene.GC-MS analysis of the Click here for file