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.

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.

Objective To investigate the difference of effect between interventional treatments and intravenous therapy of lidamycin on VX2 rabbit liver cancer.Methods VX2 Carcinoma cells were surgically implanted into the left liver lobe of 12 New Zealand white rabbits to establish the VX2 rabbit liver tumor model.Tumor size was detected by type-B ultrasonic diagnostic instrument.The rabbits were randomly divided into two groups of six,respectively treated with the hepatic inter-ventional administration of lidamycin (LDM)(1 ml,0.05 mg/kg)under the guidance of digital subtraction angiography (DSA)(group A)and with the auricular intravenous administration of LDMat the same dose (group B).All the rabbits were sacrificed and anatomized on day 10 after treatment,whose liver tumor was fixed with 4% paraformaldehyde solution and embedded in paraffin.Proliferating cell nuclear antigen (PCNA)and CD34 expression in the sample sections of tumor tissue were assessed through immunohistochemical staining.The levels of alanine transaminase (ALT)and aspartate trans-aminase(AST)were detected by Cobas 8000.Finally,the inhibition of VX2 tumor was evaluated.Results The VX2 tumor volumes were all increased at 10 day after LDMtreatment.However,the tumors in group A were smaller than those of group B (P <0.05).The results of immunohistochemistry showed that the intervention therapy of LDM could further lower the expression of CD34 and PCNA compared to group B.Conclusion Hepatic interventional administration of LDM under the guidance of DSA produces a better effect on attenuating the tumor growth than the intravenous administration of LDM.

Objective: To observe the anti- arrhythmic effects of Lvfukang Capsules on the experimental arrhythmic models induced by aconitine in rats, and provid accordance for clinical medication. Methods: 50 rats were divided into model control group, positive control group, high, middle and low dosage groups of Lvfukang Capsules, respectively. All the dosage groups were treated with successive medication 3 days, arrhythmic models induced by aconitine for 30minutes after the last dosage. To observe and record the time of ventricular premature beat (VP) and ventricular fibrillation (VF). Results: All the dosage groups of Lvfukang Capsules significantly delayed the time of ventricular premature beat (VP) and ventricular tachycardia (VT) of arrhythmic models of rats (P

OBJECTIVETo establish the quality standard for carbonizing drug characteristic of ginger carbon.

METHODGingers and different carbonized gingers were compared by the absorption of pigment, tannin content, pH, mouth's coagulation time and bleeding time.

RESULTThe study resulted in the recommended carbonizing standard that the absorption capacity shall not be less than 7.50 mg x g(-1) for methylene blue, the tannin content shall not be less than 2.103 mg x g(-1), the pH shall be (5.50 +/- 0.10), and coagulation time and bleeding time shall be the shorter the better.

CONCLUSIONThe established assessment standard for carbonizing drug characteristic of ginger carbon is reasonable, easily operated and feasible.

Absorption ; Animals ; Blood Coagulation ; drug effects ; Carbon ; chemistry ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Ginger ; chemistry ; Hydrogen-Ion Concentration ; Mice ; Quality Control ; Tannins ; analysis

Objective:To explore the correlation analysis between psychological status and blood pressure of residents in medium-risk areas during the coronavirus disease 2019 (COVID-19) epidemic, and provide references for early psychological intervention and hypertension management during the COVID-19 epidemic.Methods:From January 4 to 8, 2021, the convenience sampling method was used to conduct psychological questionnaire surveys on residents and employees of enterprises in the medium-risk area and surrounding areas of Jiuxianqiao, Chaoyang District, Beijing. General data (gender, age, height, weight, history of smoking and drinking, previous medical history), and home blood pressure monitoring were collected. Pearson′s χ 2 test was used to compare different factors (anxiety, depression, age, gender, body mass index, smoking, drinking, and history of chronic diseases) in normal blood pressure or not. A multivariate logistic regression analysis model was established to explore independent risk factors of abnormal blood pressure. Results:No anxiety accounted for 75% (684/912), and anxiety accounted for 25% (228/912). No depression accounted for 67% (611/912), depression accounted for 33% (301/912). Both anxiety and depression accounted for 23% (210/912). The substandard blood pressure accounted for 10.3% (94/912). The incidence of poor blood pressure control in participants with anxiety was 30.7% (70/228), which was significantly higher than that of participants without anxiety 3.5% (24/684) (χ 2=136.779, P<0.001). The incidence of poor blood pressure control in participants with depression 27.2% (82/301) was significantly higher than that of participants without depression 2.0% (12/611) (χ 2=139.388, P<0.001). Multivariate logistic regression analysis showed anxiety ( OR=3.261, P<0.001), depression ( OR=7.928, P<0.001), and complications of chronic diseases ( OR=3.207, P<0.001) were closely related to whether blood pressure was under control. Conclusions:During the COVID-19 epidemic, the psychological status of residents and employees in medium-risk areas should be screened and interventions implemented accordingly to prevent unstable blood pressure caused by anxiety or depression.

Werner syndrome (WS) is a premature aging disorder that mainly affects tissues derived from mesoderm. We have recently developed a novel human WS model using WRN-deficient human mesenchymal stem cells (MSCs). This model recapitulates many phenotypic features of WS. Based on a screen of a number of chemicals, here we found that Vitamin C exerts most efficient rescue for many features in premature aging as shown in WRN-deficient MSCs, including cell growth arrest, increased reactive oxygen species levels, telomere attrition, excessive secretion of inflammatory factors, as well as disorganization of nuclear lamina and heterochromatin. Moreover, Vitamin C restores in vivo viability of MSCs in a mouse model. RNA sequencing analysis indicates that Vitamin C alters the expression of a series of genes involved in chromatin condensation, cell cycle regulation, DNA replication, and DNA damage repair pathways in WRN-deficient MSCs. Our results identify Vitamin C as a rejuvenating factor for WS MSCs, which holds the potential of being applied as a novel type of treatment of WS.
Animals ; Ascorbic Acid ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line ; Cellular Senescence ; drug effects ; DNA Damage ; DNA Repair ; drug effects ; DNA Replication ; drug effects ; Disease Models, Animal ; Heterochromatin ; metabolism ; pathology ; Humans ; Mesenchymal Stem Cells ; metabolism ; pathology ; Mice ; Nuclear Lamina ; metabolism ; pathology ; Reactive Oxygen Species ; metabolism ; Telomere Homeostasis ; drug effects ; Werner Syndrome ; drug therapy ; genetics ; metabolism