[Meta-analysis of risk factors for all-cause mortality of pulmonary thromboembolism].

OBJECTIVE: To evaluate the causes of death and risk factors of pulmonary thromboembolism. METHODS: Pubmed, English Medical Current Contents, Chinese Conference Data and Chinese Biomedical Database were searched from January 1995 up to May 2011. And the references of these studies were also examined. Observational studies were assessed according to suggestion of quality assessment with references. Randomized control trials (RCT) were assessed with Jadad scale. Software RevMan 5.1 was used to examin the heterogeneity of trials. The fixed or random effect model was employed to pool the risk ratio and 95%CI. The results were expressed with risk ratio and 95%CI. RESULTS: Thirty-five studies with a total number of 19 613 cases of pulmonary thromboembolism (PTE) were included for final analysis. The average mortality rate was (10.7 ± 7.6)% (range 0.5%-30.0%). And the following factors increased the total mortality of pulmonary embolism: right ventricular hypokinesis or dysfunction (2.18(1.64-2.89), P = 0.000), elevated D-dimer (5.19(1.93-13.96), P = 0.001), elevated cardiac troponin (cTnI) (4.01(2.77-5.81), P = 0.000), hypotension (2.76(1.25-6.09), P = 0.010), malignancy (2.65(2.01-3.50), P = 0.000), congestive heart failure (1.90(1.62-2.22), P = 0.000), chronic lung disease (1.40(1.18-1.66), P = 0.000), tachycardia (1.65(1.23-2.20), P = 0.000), immobility (1.74(1.36-2.21), P = 0.000) and age > 65 years (1.24 (1.13-1.37), P = 0.000), etc. When multiple factors co-existed, the risk of death became more obvious. CONCLUSION: Elevated D-dimer, elevated cTnI, hypotension, malignancy, right ventricular hypokinesis or dysfunction, immobility, congestive heart failure, tachycardia, chronic lung disease, age > 65 years influence the mortality rate of pulmonary embolism.

[Meta-analysis of risk factors for recurrent pulmonary thromboembolism].

OBJECTIVE: To evaluate the risk factors of recurrent pulmonary thromboembolism (PTE) through Meta-analysis. METHODS: Chinese Journal Full-text Database, Chinese Biomedical Database, PubMed and Foreign Medical Journal Full-Text Service were searched for the paper relating to the risk factors of recurrent PTE from January 1995 to May 2011. And the references of these studies were also examined. Observational studies (cohort & case control) were assessed according to the method of quality assessment suggested within the references. Randomized control trials (RCTs) were assessed by the Jadad scale. Software RevMan 5.1 was used to examine the heterogeneity of trials. The fixed or random effect model was pooled to calculate the risk ratio (RR). And the results were expressed by RR (95%CI). RESULTS: Forty-two trials including 36 909 cases of PTE and/or deep vein thrombosis were analyzed. And the following factors were relative to recurrence: elevated D-dimer (1.77 (1.34 - 2.36), P = 0.000), idiopathic PTE (1.82 (1.61 - 2.05), P = 0.000), right ventricular dysfunction (RVD) (persistent RVD vs RVD regression (8.71 (2.38 - 31.91), P = 0.001); persistent RVD vs non-RVD (2.45 (1.26 - 4.76), P = 0.008), short anticoagulation duration (1.73 (1.32 - 2.28), P = 0.000), increased endogenous thrombin generation capacity (1.89 (1.39 - 2.56), P = 0.000), elevated factor VIII (1.96(1.40 - 2.74), P = 0.000), positive antiphospholipid antibodies (5.64 (4.09 - 7.77), P = 0.000), anti-thrombin defect (2.45 (1.26 - 4.76), P = 0.008) and males (1.47 (1.06 - 2.03), P = 0.020), etc. When multiple factors co-existed, the risk of recurrence became more obvious. CONCLUSIONS: Elevated D-dimer, idiopathic PTE and many other factors may influence the recurrence of pulmonary embolism. And most recurrent patients have two or more factors.

Efficacy of Glucocorticoids and Glucocorticoid-Induced Hyperglycaemia in Renal Disease: A Meta-Analysis of Randomized Controlled Trials.

Background: Glucocorticoids are the most effective anti-inflammatory and immunosuppressive drugs used to treat patients with renal disease. This study pooled the current evidence of the efficacy of Glucocorticoids and Glucocorticoid-induced hyperglycaemia in renal disease. Methods: We conducted a systematic literature search on PubMed, Cochrane Central, and Web of Science for relevant randomized controlled trials (RCTs) up to September 1, 2021. The meta-analysis, sensitivity analysis and bias analysis were performed using Review Manager 5. 3. Results: In this study, seven RCTs with 797 patients were included in our analysis. The analysis revealed that glucocorticoids had a certain alleviating effect on the reduction of renal function. (risk ratio [RR] 0.49 95% confidence interval [Cl] 0. 28 to 0.85, p =0.01) and reduction of proteinuria (weight mean difference [WMD] -0.43; 95% CI -0.57 to-0.28) when compared with the control group. Patients receiving glucocorticoids therapy did not have an increased risk of developing new-onset diabetes mellitus or impaired glucose tolerance. (RR 3.76 95% CI 0.54 to 26.10, p =0.18). For other safety outcomes, glucocorticoids therapy did not increase risk of respiratory infections (RR 1.63, 95% CI 0. 69to3. 89, p =0.27) and Gastrointestinal SAEs is relatively controversial (RR 1.10, 95% CI 0.32 to 3.79, p =0.88). Discussion. In conclusion, current clinical evidence indicates that glucocorticoids is efficacious and safe to renal disease compared with control. Further research comparing long-term glucocorticoids use is needed.

Loop-mediated isothermal amplification assay targeting the omp25 gene for rapid detection of Brucella spp.

A novel loop-mediated isothermal amplification (LAMP) assay was established to detect Brucella species DNA in milk and blood samples of animals and humans. This LAMP assay based on the sequence of highly repetitive omp25 gene was able to detect 9fg/µl Brucella spp. DNA with high sensitivity, which was 10 times higher than the nested PCR. The LAMP was evaluated for its specificity using 19 strains of six Brucella species and 28 related non-Brucella micro-organism strains as controls. The target 19 Brucella strains were all amplified, and no cross-reaction was found with all the non-Brucella micro-organism strains. Both nested PCR and LAMP assays were then used to detect Brucella spp. DNA in 78 milk samples and 113 blood samples from animals and 11 blood samples from humans, and the established LAMP assay yielded 99.0% concordance rate with the nested PCR. The LAMP assay should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of Brucellosis.

Functional Characterization of a New Bifunctional Terpene Synthase LpNES1 from a Medicinal Plant Laggera pter odonta.

Laggera pterodonta, known in China as 'Choulingdan' for its stimulous odor, has long been used as traditional herbal medicine. The essential oil of L. pterodonta, which exhibits various pharmacological activities, is a rich resource of monoterpenes and sesquiterpenes. To date, however, the terpene synthases responsible for their production remain unknown. In present study, a new terpene synthase gene (LpNES1) was identified from L. pterodonta, transcript level of which was significantly upregulated in response to methyl jasmonate treatment. Recombinant LpNES1 could synthesize (E)-nerolidol and minor ß-farnesene from farnesyl diphosphate and linalool from geranyl diphosphate in vitro. Whereas, only sesquiterpenes including (E)-nerolidol and minor ß-farnesene were released when LpNES1 was reconstituted in yeast, even coexpressed with a geranyl diphosphate synthase (ERG20WW). Combined with subcellular localization experiment, the result indicated that the cytosol-targeted LpNES1 was responsible for (E)-nerolidol biosynthesis exclusively in L. pterodonta. Additionally, the expression level of LpNES1 gene was more prominent in floral buds than that in other tissues. LpNES1 characterized in present study not only lays the molecular foundation for sesquiterpene biosynthesis of L. pterodonta, but provides a key element for further biosynthesis of bioactive compound in microbes.

Two new triterpenoids with antimicrobial activity from the leaves and twigs of Orophea yunnanensis.

Two new triterpenoids, 3-O-(4',5'-dihydroxybenzoyl)-lup-20(29)-en (1) and 3-O-(6'-desmethysyringyl)-13α-methyl-27-norolean-14-en-3ß-ol (2), were isolated from the leaves and twigs of Orophea yunnanensis. Their structures were identified by extensive spectroscopic experiments (NMR and MS) and comparison with literature data. Compounds 1 and 2 exhibited the moderate inhibitory activity against Sclerotinia sclerotiorum, Ceratocystis fimbriata and Verticillium dahliae Kleb with MIC values from 50 to 25 µg/mL, and also displayed the weak activity selectively against tested bacteria strains with MIC values from 100 to 50 µg/mL.

Three new hopane-type triterpenoids from the aerial part of Adiantum capillus-veneris and their antimicrobial activities.

Three new hopane-type triterpenoids (1-3), fern-7(8)-en-19α, 28-diol (1), pteron-14-ene-7α,19α,28-triol (2) and 3ß,4α,25-trihydroxyfilican (3), were isolated from the aerial parts of Adiantum capillus-veneris. Their structures were determined by NMR spectroscopic and mass spectrometric data. Compounds 2 and 3 exhibited remarkable antifungal activity against Helminthosporium maydis and Alternaria alternata with MIC values of 12.5-3.125 µg/mL, and compound 3 also against Verticillium dahliae Kleb with an MIC value of 3.125 µg/mL. In addition, compounds 1-3 also displayed weak antibacterial activity against Micrococcus lysodeikticus, Bacterium paratyphosum B and Pseudomonas aeruginosa with an MIC value of 100 µg/mL.

[Effect of p38 mitogen-activated protein kinase gene knockout on cell proliferation of embryonic fibroblasts in mice].

OBJECTIVE: To investigate the effect of p38 mitogen-activated protein kinase (MAPK) gene knockout on the proliferation of embryonic fibroblasts in mice (MEFs). METHODS: The expression of p38 in MEFs p38+/+ and p38(-/-) cells were detected by Western blotting. The growth curves of p38+/+ and p38(-/-) cells were plotted with the results of methylthiazoletetrazolium (MTT) colorimetric assay, and the ratios of different cell phases of p38+/+ and p38(-/-) cells were analyzed by flow cytometry. RESULTS: The growth curves showed that the growth rate was notably retarded and cell double time elongated in p38(-/-) cells, and there was 15.5% decrease of the number of p38(-/-) cells in comparison with that of p38+/+ cells in 96-hour culture. G2/M transition was inhibited in p38(-/-) cells. Meanwhile, G1/S transition was also inhibited in p38(-/-) cells, as shown by the results of flow cytometry. The ratios of G0/G1, G2/M, and S phases of p38+/+ cells were 34.47%, 10.81%, and 54.72%, respectively; while those of p38(-/-) cells were 48.49%, 4.06%, and 47.44%, respectively. There were 40.7% increase and 13.3% decrease in the cell numbers of G1 and S phases of p38(-/-) cells in comparison with those of p38+/+ cells, respectively. CONCLUSION: p38 gene knockout in MEFs leads to cell cycle arrest and decreased cell proliferation.

Three new phenanthrenes with antimicrobial activities from the aerial parts of Juncus effusus.

Three new phenanthrenes (1-3), designated as 2-methoxy-1,6-dimethyl-5-vinyl-9, 10-dihydrophenanthren-7-ol, 1,6-dimethyl-4,5-dihydropyrene-2,7-diol and 1-(3,7- dihydroxy-2,8-dimethyl-9,10-dihydrophenanthren-1-yl)ethanone, were isolated from the aerial parts of Juncus effusus. Their structures were determined by extensive spectroscopic experiments (NMR and MS) and comparing with those related known compounds. The antifungal and antibacterial activities of 1-3 were evaluated. Compound 1 showed remarkable antifungal activities against six agricultural pathogenic fungi (Rhizoctonia solani, Verticillium dahliae Kleb, Sclerotinia sclerotiorum, Gibberella saubinetii, Bipolaris zeicola, and Phytophthora parasitica) with minimum inhibitory concentration (MIC) values ranging from 3.125 to 12.5 µg/mL, and also displayed significant antibacterial activities against two human pathogenic bacteria (Bacterium paratyphosum B and Micrococcus lysodeikticus) with MIC values of 12.5 and 25 µg/mL, respectively.

[Construction and expression of green fluorescent protein fusion vector of p38 regulated/activated protein kinase].

OBJECTIVE: To construct the expression vector of p38 regulated/activated protein kinase (PRAK) fused to green fluorescent protein which is capable of expression in mammalian cells. METHODS: PRAK with His-tag in pET-14b expression vector was subcloned into the green fluorescent protein vector pEGFP-C2. The recombinant vector was then transfected into HeLa cells, followed by observation of the cells with fluorescent microscope. RESULTS: Identification by enzyme digestion, PCR and sequencing confirmed successful construction of the recombinant vector, which was highly expressed in HeLa cells. Green fluorescence of the fusion protein EGFP-PRAK was observed mainly in the cell nuclei. CONCLUSION: The expression vector of PRAK fused to green fluorescent protein is successfully constructed and expressed in mammalian cells, which may facilitate the study of intracellular localization and translocation of PRAK.

[Effect of Liangge San on lipopolysaccharide-induced nuclear factor kappa B activation in cultured mouse macrophages].

OBJECTIVE: To investigate the effect of Liangge San, a recipe of traditional Chinese herbal medicine, on lipopolysaccharide (LPS)-induced nuclear factor kB (NF-kB) activation in mouse macrophages cultured in vitro and explore the signal transduction mechanism of the detoxifying effect of Liangge San. METHODS: The mice were given oral administration of concentrated decoction of Liangge San to obtain the drug-containing serum. Macrophages from mouse abdominal cavity were collected, incubated and subsequently re-incubated with LPS and the prepared serum at different doses. Immunofluorescence method was adopted to examine the expression of NF-kB subunit p65 in the nuclei of the macrophages, and the fluorescence intensity of p65 expression was measured by laser scanning confocal microscope (LSCM). RESULTS: The fluorescence intensity of p65 expression in the nuclei of macrophages incubated with LPS for 1 h was significantly increased compared with that in the cells without LPS stimulation and Liangge San serum-treated cells. The fluorescence intensities were significantly decreased in cells treated with the inhibitor TLCK and different doses of Liangge San serum in comparison with those in LPS-stimulated cells. The fluorescence intensities were the lowest in cells treated with TLCK and high-dose Liangge San serum, and the cells treated with moderate and low doses of the serum both showed lower intensity compared with that of LPS-stimulated cells. p65 expression was similar between the macrophages incubated with LPS and those treated with serum that contained no Liangge San. CONCLUSIONS: Mouse serum containing Liangge San can inhibit LPS-induced p65 expression in mouse macrophages in a dose-dependent manner, which may be one of the signal transduction mechanisms of the detoxifying effect of Liangge San.

[Construction and expression of red fluorescent protein fusion vector incorporating p38 mitogen-activated protein kinase].

OBJECTIVE: To construct the vector that expresses the fusion protein of p38 mitogen-activated protein kinase (MAPK) and red fluorescent protein (RFP) in mammalian cells. METHODS: FLAG-tagged p38 MAPK in pcDNA3 vector was subcloned into RFP vector pDsRed1-N1, the construct of which was then transfected into HeLa cells and observed with fluorescence microscope. RESULTS: The recombinant plasmid was verified by enzyme digestion, PCR and sequence analysis, and p38 MAPK-RFP fusion protein was highly expressed in HeLa cells. Fluorescence microscope found the red fluorescence distributed all over the cytoplasm and in the nuclei as well. CONCLUSION: The expression vector for p38 MAPK-RFP fusion protein is successfully constructed and effective expression of this fusion protein is achieved, which might be instrumental in the study of intracellular localization of p38 MAPK.

Killing effect of suicide gene system under control by KDR promotor on human umbilical vein endothelial cells.

OBJECTIVE: To study the killing effect of adenovirus-mediated double suicide gene under the regulation of kinase domain-containing receptor (KDR) promoter on human umbilical vein endothelial cells (HUVECs). METHODS: The sequences of human KDR promoter gene, CD gene and TK gene were amplified by PCR, and the plasmid pKDR-CDglyTK was constructed. A two-step transformation protocol was employed for the construction of a recombinant adenoviral plasmid pAdKDR-CDglyTK that was transfected into 293 packaging cells to further multiply and purify the adenovirus. HUVECs were infected by the resultant recombinant adenovirus of different multiplicities of infection (MOI), and the infection rate was measured by observing the expression of green fluorescence protein (GFP). The infected cells were cultured in the culture media containing ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the killing effects were evaluated. RESULTS: Recombinant adenovirus AdKDR-CDglyTK were successfully constructed, which could efficiently infect HUVEC cells, with the infection rate associated with the MOI of the recombinant adenovirus. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of the recombinant adenovirus. The killing effect of the two produrgs used in combination was much stronger than that of exclusive use of GCV or 5-FC. CONCLUSIONS: Prodrug/KDR-CdglyTK system is effective in killing HUVEC cells, and its killing effect is correlated with the concentration of the prodrugs and the MOI of the recombinant adenovirus. Combination of the two prodrugs produces stronger killing effect on the cells.

Regulation of inducible nitricoxide synthase gene transcription by p38 mitogen-activated protein kinase in human embryonic kidney 293 cells.

OBJECTIVE: To study the transcriptional regulation of inducible nitric oxide synthase (iNOS) gene by p38 mitogen-activated protein kinase (MAPK). METHODS: With human embryonic kidney (HEK) 293 cells as the target and the assistance of lipofectamine-mediated co-transfection techniques and luciferase reporter gene systems, FLAG-tagged p38 isoforms (namely FLAG-p38 alpha, FLAG-p38 beta;, FLAG-p38 gamma and FLAG-p38 phi;) in pcDNA3, pcDNA3, piNOS-Luc and pCMV-beta; were transfected into HEK 293 cells, and the relative activity of luciferase was subsequently tested. RESULTS: Highest luciferase activity occurred only in p38 alpha group compared with the other three isoform groups under no stimulation. Under the stimulation by lipopolysaccharide (LPS), the luciferase activity of each group was obviously increased and the highest activity occurred in p38 beta group. CONCLUSION: LPS can induce transcription and activation of iNOS gene, and p38 MAPK is involved in the transcription regulation of iNOS gene in HEK 293 cells.

[Construction and expression of the fusion vector of His-tagged human ARPC2 gene].

OBJECTIVE: To construct the expression vector of His-ARPC2 fusion protein and obtain its expression and purification in E. coli. METHODS: ARPC2 cDNA codon region was amplified by PCR from human liver cDNA library and cloned into pET-14b vector following the routine procedures. After identification by enzyme digestion, PCR and sequencing, the positive clones were transformed into BL21 (DE3) competent cells, and the expression of His-ARPC2 fusion protein was induced with IPTG and further purified by Ni-NTA affinity chromatography. RESULTS: The constructed His-ARPC2 fusion protein vector was highly efficiently expressed in E. coli. With Ni-NTA affinity chromatography, a purified His fusion protein with relative molecular mass of approximately 36 000 was obtained. CONCLUSION: The expression vector of His-ARPC2 fusion protein is constructed, expressed and purified under non-denaturing conditions, which may significantly facilitate future study of the physiological functions of ARPC2 and characterization of its interaction proteins.

[Screening and identification of proteins interacting with p38 MAP kinase via T7 phage-display screening system].

OBJECTIVE: To screen and identify proteins that interact with p38 mitogen-activated protein (MAP) kinases by means of T7 phage-display screening system. METHOD: His-tagged fusion protein of p38 MAP kinase was used to coat a 96-well ELISA plate and Ni-NTA resin, which served as the media for screening human lung and liver T7 phage cDNA libraries. RESULTS: After 4 rounds of biopanning, 86 independent plaques were selected and processed by EDTA. The inserted gene fragments from these plaques were amplified by PCR, the products purified by a gel recovery method. The sequences of the insertions were identified and analyzed with BLAST program in GenBank. Forty-six clones were found to encode proteins. CONCLUSION: T7 phage-display screening system is convenient, rapid and effective for screening the P38 MAP kinase-binding proteins.

[Construction of vector of his-tagged cytoplasmic fragment of human toll like receptor 4 and its expression in E.coli].

OBJECTIVE: To construction of vector of his-tagged cytoplasmic fragment of human Toll like receptor 4 (hTLR4) and its expression in E.coli. METHODS: hTLR4 cytoplasmic cDNA codon domain was amplified by polymerase chain reaction (PCR) and cloned into pET-DsbA2.0 plasmid expressing His-DsbA fusion protein. After being identified by the assay of restrictional enzyme and sequencing, His-Dsb A fusion proteins were induced with isopropy-beta-D-thiogalactoside (IPTG) and further purified. RESULTS: A fusion protein with molecular weight of 42 kd was obtained. CONCLUSION: hTLR4 which was constructed and expressed successfully under nondenaturing conditions provides a tool for further studies.

Serological detection of bovine ephemeral fever virus using an indirect ELISA based on antigenic site G1 expressed in Pichia pastoris.

An indirect ELISA for the serological detection of bovine ephemeral fever virus (BEFV) infection in cattle is described in which a glycosylated protein of approximately 25 kDa (including the G1 antigenic site of the virus glycoprotein) expressed in Pichia pastoris GS115 was used as the coating antigen. The optimal concentration of coated antigen was 0.3 microg/well at a serum dilution of 1:40. The optimal positive threshold value of the assay was 1.88, as derived from receiver operating characteristic curve analysis. The test had 100% sensitivity and 96.7% specificity when compared with a micro-neutralisation test using 336 positive and 180 negative sera to BEFV, respectively. The inter-assay and intra-assay coefficients of variation for 15 sera were both <5.8% and there was no evidence of cross-reactivity between the tested coating antigen and antibodies to related rabies virus. The ELISA is an inexpensive and rapid serological detection method that would be suitable for screening for BEFV infection on a large scale.

[Construction of pNTAP-MK2 eukaryotic expression plasmid and establishment of a cell line for its stable expression].

OBJECTIVE: To construct pNTAP-MK2 eukaryotic expression plasmid and establish a HEK293 cell line stably expressing tandem affinity purification (TAP)-tagged MK2. METHODS: The MK2-encoding region was subcloned into the vector pNTAP to construct the recombinant plasmid pNTAP-MK2, which was subsequently transformed into DH5 alpha.E.coli. After identification by PCR, digestion with restriction endonuclease and sequencing, the recombinant expression plasmid was transfected into HEK293 cells via liposome, and the cell line with stable expression of exogenous TAP tag-MK2 gene was selected by antibiotic G418. The expression and localization of the fusion protein TAP tag-MK2 were detected by Western blotting and immunofluorescence assay. RESULTS: The results of PCR, restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pNTAP-MK2. Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection with G418 selection. Immunofluorescence assay identified the expression product TAP tag-MK2 mainly in the cell nuclei. CONCLUSION: The eukaryotic expression vector pNTAP-MK2 has been successfully constructed, and in the established cell line with stable expression of TAP tag-MK2, TAP tag does not influence the localization of exogenous MK2.

An indirect ELISA of classical swine fever virus based on quadruple antigenic epitope peptide expressed in E.coli.

In this study, a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV) E2 glycoprotein was expressed in E. coli to a obtain target protein. This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs. The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve (ROC) analysis based on 30 negative sera and 80 positive samples. The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination (IHA) test. The inter-assay and intra-assay coefficients of variation (CVs) for 16 sera were both ≤6.8%. No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus (BVDV) antibodies was observed.