Objective To compare the accuracy of stroke volume variation (SVV),central venous pressure (CVP) and puhnonary arterial wedge pressure (PAWP) in monitoring the changes in blood volume in the patients undergoing renal transplantation.Methods Sixteen patients with chronic renal failure,of American Society of Anesthesiologists physical status Ⅱ or Ⅲ,aged 18-55 yr,scheduled for elective allograft renal transplantation under general anesthesia,were enrolled in the study.SVV was continuously monitored with the FloTrac/Vigileo monitor,and CVP,PAWP and stroke volume index (SVI) were continuously monitored with the volumetric pulmonary artery catheter during surgery.The parameters of hemodynamics were recorded at 30 min after induction of anesthesia,5 min before renal artery opening,5 and 30 min after renal artery opening,and at the end of surgery.Hydroxyethyl starch 130/0.4 electrolyte solution 6 ml/kg was infused over 15 min via the central venous catheter to perform fluid responsiveness starting from 30 min after induction of anesthesia.Positive fluid responsiveness was defined as the change in SVI ≥ 15％.The relationship between SVV and CVP and between SVV and PAWP was analyzed using the Pearson correlation analysis.The receiver operating characteristic curve for CVP,SVV and PAWP in monitoring the changes in blood volume was drawn,and the area under the curve was calculated.Results Compared with the value at 5 min before renal artery opening,SVV was significantly increased after renal artery opening (P＜0.05),and no significant change was found in CVP and PAWP after renal artery opening (P＞0.05).SVV was negatively correlated with CVP,and r=-0.82 (P＜0.01);SVV was negatively correlated with PAWP,and r=-0.77 (P＜0.01).The area under the curve of SVV in monitoring the changes in blood volume was 0.87,and of CVP and PAWP was 0.69 and 0.66,respectively.Conclusion SVV provides better accuracy than CVP and PAWP in monitoring the changes in blood volume in the patients undergoing renal transplantation.

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.
Biosynthetic Pathways ; Ginsenosides ; biosynthesis ; Glycosyltransferases ; metabolism ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Synthetic Biology

Pichia pastoris is one of the most important systems used in the field of molecular biology for the expression of recombinant proteins. The system has advantages of high expression, high stability, high secretion, easy high-density fermentation and low cost. Many factors affect the expression of recombinant protein, such as gene copy number, codon usage preference, type of promoter, molecular chaperones, glycosylation, signal peptide and fermentation process. In this review, research advances of the above aspects are summarized, which lay a foundation for improving the expression of recombinant proteins in P. pastoris.
Fermentation ; Gene Dosage ; Glycosylation ; Molecular Chaperones ; Pichia ; metabolism ; Promoter Regions, Genetic ; Protein Sorting Signals ; Recombinant Proteins ; biosynthesis

Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.
Chromatography, High Pressure Liquid ; DNA Primers ; Down-Regulation ; Ergosterol ; metabolism ; Haploidy ; Intramolecular Transferases ; genetics ; Polymerase Chain Reaction ; Saccharomyces cerevisiae ; genetics ; Squalene ; analogs & derivatives ; metabolism

Objective To observe the effect of agmatine (AGM) on inflammatory factor in Kupffer cells of liver,and to investigate the protective effects of AGM on severe trauma-induced liver injury in mice and its possible mechanism.Methods Forty-two adult male BALB/c mice were randomly divided into sham group,model group,and AGM treatment group,with 14 mice in each group.The mice model of trauma-hemorrhage was reproduced by hindlimbs fracture combined with 35％ of orbital bleeding.The mice in the sham group were only anesthetized without other treatments.The mice in AGM treatment group were given intraperitoneal injection of 200 mg/kg AGM when limited recovery was performed,and the mice in model group were given the equal amount of normal saline.Seven mice in each group were sacrificed at 12 hours and 24 hours,respectively,after modeling,and blood samples and liver tissue were harvested,and liver Kupffer cells were isolated.Serum alanine aminotransferase (ALT),aspartate transaminase (AST)and lactic dehydrogenase (LDH) were determined with automatic biochemistry analyzer.Hepatic pathological changes were observed with light microscope using hematoxylin and eosin (HE) staining.The levels of tumor necrosis factor-α(TNF-o) and interleukin-6 (IL-6) in serum,hepatic homogenate and Kupffer cell supernatant were determined with enzyme linked immunosorbent assay (ELISA).The mRNA expressions of pro-inflammatory cytokines TNF-α and IL-6 in the Kupffer cell were determined by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR).Results ① The normal liver tissue structure was found in sham group.At 24 hours after modeling in the model group,the changes in pathobiology were found as following:neutrophil infiltration,hepatocytes swelling,hyperemia,and necrosis,as well as the abnormality of parameters reflecting liver function.AGM could significantly improve the pathological changes in liver tissue caused by severe trauma,and ameliorate the liver function.② There were no significant differences in the levels of TNF-α and IL-6 in serum and hepatic tissue at 12 hours after modeling,and the parameters at 24 hours in model group were higher than those at 12 hours,which were significantly higher than those of the sham group [serum TNF-α (ng/L):80.8±4.7 vs.34.7±4.7,IL-6 (ng/L):104.0±9.0 vs.55.4±3.3;liver TNF-α (ng/mg):405.2± 19.6 vs.57.2±10.0,IL-6 (ng/mg):58.4±7.7 vs.14.3±2.1,all P ＜ 0.01].AGM could effectively reduce the levels of TNF-o and IL-6 in serum and hepatic tissue [serum TNF-α (ng/L):58.2 ± 3.1 vs.80.8 ± 4.7,IL-6 (ng/L):74.1 ± 6.6 vs.104.0± 9.0;liver TNF-α (ng/mg):248.7 ± 22.5 vs.405.2 ± 19.6,IL-6 (ng/mg):22.5 ± 3.1 vs.58.4 ± 7.7,all P ＜ 0.01].③ The levels of TNF-o and IL-6 in Kupffer cells supernatant were significantly higher than those of the sham group,and they were further increased after lipopolysaccharide (LPS) stimulation for 24 hours.AGM could effectively reduce the levels of TNF-α and IL-6 in Kupffer cells [TNF-α (ng/L):256.6 ± 5.6 vs.465.5 ± 5.2,IL-6 (ng/L):1 185.5 ± 64.4 vs.2 018.8 ± 53.2,both P ＜ 0.01],and also decreased the mRNA expressions of TNF-α and IL-6 [TNF-α mRNA (2-△△Ct):7.2±0.4 vs.13.5±0.4,IL-6 mRNA (2-△△Ct):13.2±0.7 vs.21.3 ± 1.6,both P ＜ 0.01].Conclusion Agmatine can reduce trauma-induced acute hepatic injury via suppression of cytokines release in Kupffer cells,and can ameliorate the liver function.

BmK AngM1 is a long-chain scorpion toxin purified from the venom of Buthus martensii Karsch. It has been reported to exhibit evident analgesic effect and low toxicity, and has the potential to be a novel analgesic drug. The BmKAngM1 gene was transformed into Pichiapastoris GS115. Mut+ and Mut(s) recombinant strains were screened by phenotype and Mut+ recombinant strains were used to detect BmK AngMl gene copy number in the real-time PCR. Expression of BmK AngM1 in the Mut+ recombinant strain was compared with that of the Mut(s) recombinant strain with the same single copy of BmK AngM1 gene under the same condition. The results indicated that the transcription level of BmK AngM1 gene in the Mut(s) recombinant strain was 2.7 fold of that in the Mut recombinant strain in the real-time PCR, and the expression of BmK AngM 1 in the Mut(s) recombinant strain was 1.5 fold of that in the Mut+ recombinant strain. Therefore, Mut(s) recombinant strain showed better ability to express BmK AngM1 than Mut+ recombinant strain.

Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.

Pichia pastoris is one of the most important systems used in the field of molecular biology for the expression of recombinant proteins. The system has advantages of high expression, high stability, high secretion, easy high-density fermentation and low cost. Many factors affect the expression of recombinant protein, such as gene copy number, codon usage preference, type of promoter, molecular chaperones, glycosylation, signal peptide and fermentation process. In this review, research advances of the above aspects are summarized, which lay a foundation for improving the expression of recombinant proteins in P. pastoris.

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.