Using multiple-fragment amplification combined with Gibson assembly to clone genes with site-directed mutations / 生物工程学报

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

Correlation between serum level of miRNA-106a expression with clinicopathological characteristics and prognosis of patients with renal cell carcinoma / 中华医学遗传学杂志

OBJECTIVE@#To analyze the expression of microRNA-106a(miR-106a) in renal cell carcinoma (RCC) and its correlation with clinicopathological characteristics and prognosis of patients.@*METHODS@#Serum samples of 64 patients with newly diagnosed RCC were collected as the study group, and serum samples of 40 healthy individuals were used as the control group. Real-time fluorescence quantitative PCR was used to determine the expression level of miR-106a in each group. The correlation between miR-106a expression and clinicopathological characteristics of the patients was studied with single factor analysis and multiple Logistic regression model. Kaplan-Meier survival curve was used to analyze its correlation with the prognosis of patients.@*RESULTS@#Before surgery, compared with the control group (1.17± 0.58), RCC patients with high- (9.15± 0.96) and low-expression(3.45± 0.37) had increased expression of miR-106a. Postoperatively, the expression level of miR-106a in both groups of patients decreased to 1.53± 0.18 and 1.75± 0.21, respectively. The area under the curve (AUC) of the diagnostic value of serum miR-106a for RCC was 0.782 (95% CI: 0.661-0.902). With an optimal cutoff value of 0.531, the sensitivity was 78.10% and the specificity was 75.00%. Serum miR-106a level of RCC patients with TNM stage T3 or T4, clinical stage II or III, lymph node metastasis, and recurrence were significantly increased. The high expression of serum miR-106a in RCC patients has an independent relationship with the tumor TNM stage and lymph node metastasis. Of the 64 follow-up patients, 4 were lost and 30 had died. Among them, the median survival time of patients in the miR-106a high expression group was 30 months, which was significantly shorter than that of the low expression group (52 months).@*CONCLUSION@#The serum level of miR-106a is elevated in RCC patients, and may be used as a molecular marker for the diagnosis of RCC. High serum expression of miR-106a is an independent predictor for tumor TNM stage and lymph node metastasis, as well as an independent predictor for poor prognosis of RCC patients.

Optimization of alkaline hydrolysis based on the side chain of diethyl ester 4-amino-N5-formyl-N8,N10-dideazatetrahydrofolic acid / 北京大学学报(医学版)

Objective: To optimize and establish the best hydrolysis method of diethyl ester 4-amino-N5-formyl-N8,N10-dideazatetrahydrofolate through the optimization of simple compound of diethyl N-(4-aminobenzoyl)-L-glutamate.Methods: To increase the low yield of hydrolysis reaction of diethyl ester 4-amino-N5-formyl-N8,N10-dideazatetrahydrofolate due to the by-products and difficult purification, we studied the effect of NaOH and KOH, two kinds of alkalis, three concentrations between 0.175-1 mol/L and five types of reaction time involved in 20, 30, 60, 120 and 180 min on the common side chain diethyl N-(4-aminobenzoyl)-L-glutamate.A high performance liquid chromatography was established for measuring the target product and the by-products in reaction liquid in different reaction conditions.Finally, on the basis of the best hydrolysis method of diethyl ester 4-amino-N5-formyl-N8,N10-dideazatetrahydrofolate, we completed the optimization of the hydrolysis reaction conditions of diethyl ester 4-amino-N5-formyl-N8,N10-dideazatetrahydrofolate.Results: We developed the best reaction condition for the hydrolysis of diethyl ester 4-amino-N5-formyl-N8,N10-dideazatetrahydrofolate, which could be carried out easily and efficiently.The results indicated that treated with the optimized condition of 0.3 mol/L KOH in 60 min at the room temperature, diethyl ester 4-amino-N5-formyl-N8,N10-dideazatetrahydrofolate was converted into its diacid derivative in 95.6 % yield, which turned to be a better reaction condition compared with the previous reaction condition.The structures of those compounds were identified to be correct by 1H nuclear magnetic resonance(1H NMR), 13C nuclear magnetic resonance(13C NMR) and electrospray ionization time of flight mass spectrometry (ESI-MS).The purity of the diacid derivative of the compound was determined to be 96% by high performance liquid chromatography(HPLC).The new hydrolysis reaction condition could not only avoid the formation of single ester hydrolysis product and amide bond hydrolysis product, but also improve the yield of the hydrolysis reaction.Conclusion: We have developed an efficient reaction for the hydrolysis of diethyl ester 4-amino-N5-formyl-N8,N10-dideazatetrahydro.Since the final step of the synthesis of classical folic acid antagonists is always the catalyzed hydrolysis of the side chain glutamate, the reaction also has great significance for anti-folic acid anti-tumor inhibitors synthesis.

Synthesis of 6-benzyl-1-[ (benzyloxy) methyl ]-3-hydroxy-5-(hydroxymethyl) pyrimi-dine-2,4(1H,3H)-dione / 北京大学学报(医学版)

Objective:To find the best synthesis method of 6-benzyl-1-[ ( benzyloxy ) methyl ]-3-hydro-xy-5-(hydroxymethyl)pyrimidine-2,4(1H,3H)-dione e for observing the change of its biological activity after N-3 hydroxylation .Methods:After trying some N-hydroxylation methods , the target compound was successfully synthesized via one-pot oxidizing process by sodium hydride ( NaH) and 3-chloroperbenzoic acid( m-CPBA);the anti-HIV reverse transcriptase ( RT) activity and integrase ( IN) activity of the tar-get compound was assayed via enzyme-linked immunesorbent assay ( ELISA) and phosphorylation of DNA package method .Results:The target compound could be obtained through the improved m-CPBA oxida-tive method by only one step , and the yield of the reaction could reach 60%-70%.And the structure of this compound was identified by 1 H NMR, 13 C NMR and MS;The activity result showed it added the an-ti-HIV IN activity after N-3 hydroxylation as well as retained the anti-HIV RT activity.Conclusion:The improved m-CPBA oxidative method is a convenient and efficient way to prepare the compound 6-benzyl-1-[(benzyloxy)methyl]-3-hydroxy-5-(hydroxymethyl)pyrimidine-2,4(1H,3H)-dione e which has both anti-HIV RT and IN activity .

Improved synthesis process of diethyl N-[4-[(2,4-diaminopyrido[3,2-d] pyrimidin-6-yl) methylamino] benzoyl]-L-glutamate / 北京大学学报(医学版)

Objective:To establish a new approach to synthesis of diethyl N-[4-[(2,4-diaminopyrido [3,2-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamate.Methods:Target compound (5) was syn-thesized by the use of (2,4-dioxo-tetrahydropyridopyrimidin-6-yl) methyl acetate (1) as starting material via hydrolysis, chlorination, condensation with diethyl (p-aminobenzoyl)glutamate and aminolysis.Re-sults:A new approach to synthesis of diethyl N-[4-[(2,4-diaminopyrido[3,2-d]pyrimidin-6-yl)methyl-amino]benzoyl]-L-glutamatewas established .This synthetic route has hydrolysis reaction , chlorination, diethyl N-( p-aminobenzoyl )-L-glutamate condensation reaction and ammonolysis reaction .The total yield is 36.7%.The structures of those compounds have identified by 1 H nuclear magnetic resonance , 13 C nu-clear magnetic resonance and mass spectrometry .This synthetic route avoid the unstable brominated re-action product and improves the harsh condition of ammonolysis reaction .Conclusion:The new synthetic route has improved the reaction condition and the stability of the intermediate , and increased the extent of the derivative compounds , which has great significance to anti-folic acid of anti-tumor inhibitor synthesis .

The Thinking of Promoting the Level of Pharmaceutical Universities New Drug Research and Development in Translational Medicine Platform / 中华医学科研管理杂志

As the main force of new drug research and development (R&D) in China,pharmaceutical universities are in charge of developing new drugs with our own intellectual property rights.The concept of translational medicine provides opportunity and challenge to improve the efficiency of new drug R&D.Several translational medicine centers have been set up in pharmaceutical universities,which provide practical conditions for new drug R&D in domestic.Administration management approaches to promote new drug R&D in translational medicine platform are analyzed in the paper.

Research advance in cGAS-STING pathway:a pathway unlock the anti-HIV function of immune system / 国际药学研究杂志

Stimulator of interferon genes(STING)are important adaptor proteins in innate immune-related signal pathway. The upstream protein,cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS),is cytosolic DNA sensor. Detection of cytosolic DNA,cGAS-STING pathway can be activated and subsequently activate the type Ⅰ interferon (IFN)antiviral response. Recent studies indicated that retroviruses especially HIV can activate the host innate immune system through cGAS-STING pathway and trigger robust immune response. In this review,we briefly describe the mechanism of innate immune activated by the newly discovered cGAS-STING pathway and highlight recent progress in STING agonist.

Purification and activity evaluation of methionine synthase / 药学学报

Methionine synthase (MS, EC2.1.1.13), a key enzyme in the folate metabolism area catalyzing methyl transfer from N5-methyltetrahydrofolate to homocysteine to give tetrahydrofolate and methionine, takes a core position in folate cycle, one-carbon-unit transfer and sculpture amino acid pathways. Cobalamin-dependent methionine synthase was purified from rat liver. The enzyme was purified 609-fold to near homogeneity by batch chromatography on DE-52, anion-exchange chromatography on Q Sepharose Fast Flow and CHT-I hydroxyapatite column and was identified by SDS-PAGE and Western blotting. The enzyme activity was determined by spectrophotometric assay. In addition, the influencing factor and optimal reaction condition were performed. The steady state kinetic of rat liver methionine synthase was similar to that of other mammalian cobalamin-dependent methionine synthase which employed a Ping-Pong mechanism. The result indicated that cobalamin-dependent methionine synthase purified from rat liver is suitable for screening and studying methionine synthase specific inhibitors.

Study on training scheme for adult with pharmacy the diploma of junior college trying to obtain the undergraduate diploma education / 中华医学科研管理杂志

To improve the pharmaceutical care ability of adult students,and ensure the safety in clinical medication. According to the requirement of the Ministry of Education of China, the new training scheme for adult Pharmaceutical undergraduates were drawn up by research and practice to meet the development needs of market economy and licensed pharmacist system in China.

Study on pharmacokinetics model for targeted drug delivery systems / 生物医学工程学杂志

Multi-compartment linear circulation mathematical model for targeted drug delivery systems was established on the bases of compartment theory and mass conservation theory. The function formulas of drug concentration-time in blood and target organ were established. According to this model, the drug concentration-time curve for the target organ can be plotted with reference to the data on blood. Based on the target organ drug concentration-time curve,the pharmacokinetics parameters of the target organ can also be calculated by the statistical moment. We further detected the practicability of the models by using the experimental data of drug concentration-time curve in blood and target organ of microspheres. The drug concentration-time curve in blood and in target organ predicted by mathematical model was agreed with that observed.

Quality standard for Compound Yiqi Granules / 中成药

AIM: To establish the quality standard for Compound Yiqi Granules (Radix Astragali, Radix Achyranthis Bidentatae, etc.). METHODS: Radix Achyranthis Bidentatae; Herba Epimedii were identified by TLC. The content of astragaloside IV in Compound Yiqi Granules was determined by TLCS. RESULTS: The methodological study showed a good linear correlation at the range of 1.12-5.60 ?g of astragaloside IV. The average recovery of astragaloside IV was 97.69% and RSD was 2.10%, respectively. CONCLUSION: The method is simple, accurate and with good repeatability. The method can be used for quality control of Compound Yiqi Granules.