Expression, purification and functional verification of recombinant human α-galactosidase A in suspension CHO-S / 中国药理学通报

Aim To express and purify rhα-Gal A with a 6 X His tag via using a serum-free expression system in high-density suspension culture of Chinese hamster ovary cells ( CHO-S) , and to verify the scavenging effect of rhα-Gal A on globular trisaccharide ceramide (Gb3 or GL3) . Methods The construction of recombinant protein expression vector, pcDNA4-GLA, was achieved by fusing the human α-galactosidase cDNA, gla, with 6 X His tag and artificial DNA synthesis. The expression plasmid was transfected into the suspended CHO-S to express rhα-Gal A and then purified. Following this procedure, we determined rhα-Gal A's expression, the enzymatic activity, and the glycosylation of the recombinant enzyme. Co-incubation with cultured cells was performed to examine whether rhα-Gal A could be taken up into the cells and effectively remove Gb3 substrates. Results rhα-Gal A was successfully expressed and purified after transiently transfecting pcDNA4-GLA into the suspended CHO-S, and the yield was up to (100 ±20. 6) mg • L

The PK bioanalysis method study of c-Met antibody-drug conjugate (RC108) in cynomolgus monkey / 药学学报

Antibody-drug conjugate (ADC) has the characteristics of low toxicity and high efficiency, and plays an important role in cancer treatment. However, due to the complexity of its structure, it brings difficulties in pharmacokinetic (PK) bioanalysis. This study established an analytical method for the detection of ADC (RC108) in cynomolgus monkey plasma by ligand-binding assay (LBA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which was used to analyze and quantify the total antibody, bound antibody and free drug in cynomolgus monkey plasma. Based on the LBA method, rabbit anti-RC108 Fab and mouse anti-MMAE (monomethyl auristatin E) mAb were pre-coated in 96-well plates as the total antibody and antibody binding reagents, respectively. The samples to be tested were added, and then the detection reagents were added in turn. Goat anti-human IgG (H+L)-HRP, chromogenic solution tetramethylbenzidine (TMB), H2SO4 terminate the reaction, read data at 450 nm/630 nm wavelength of microplate reader; LC-MS/MS analysis method quantifies MMAE concentration, and refer to relevant regulations for methodological validation. The analytical method for quantifying total antibody, bound antibody and free drug of RC108 drug obtained good accuracy and precision, and the selectivity, dilution linearity, hook effect, parallelism and stability were verified. Meet the requirements of biological analysis. Finally, a bioanalytical method for the determination of the concentration of the test substance RC108 (total antibody, conjugated antibody, free MMAE) in cynomolgus monkey plasma with high sensitivity and high throughput was established by LBA and LC-MS/MS method. Subsequent non-clinical research on PK research in cynomolgus monkeys will provide technical support.

Effects of Enriched Rehabilitation on Cognitive Function and Serum Arginine in Stroke Patients / 中国康复理论与实践

Objective:To observe the effects of enriched rehabilitation on cognitive function and serum arginine in stroke patients. Methods:From January, 2017 to June, 2018, 40 patients in our department were randomly divided into conventional group (n = 20) and enriched group (n = 20). The conventional group received cognitive rehabilitation, and the enriched group received enriched rehabilitation, once a day, six times a week for three months. Before treatment and three months after treatment, the cognitive function was assessed with Montreal Cognitive Assessment (MoCA), Digit Span Test and Stroop Color Word Test, and the levels of serum arginine and malondialdehyde were detected. Results:Finally, all the patients completed the experiment. There was no significant difference in the scores of MoCA, Digit Span Test and Stroop Color Word Test, and the level of serum arginine and malondialdehyde before treatment (t < 1.243, P > 0.05). Three months after treatment, all the above indexes improved (t > 2.170, P < 0.05), and were better in the enriched group than in the conventional group (t > 2.278, P < 0.05). Conclusion:Enriched rehabilitation could improve the cognitive function of patients after stroke, which might be related to the significant reduction of the elevated serum arginine and reduction of oxidative damage after stroke.

Investigation and analysis of health-related quality of life in myasthenia gravis patients with myasthenia gravis quality of life-15 Chinese version / 北京大学学报(医学版)

OBJECTIVE@#To investigate the level and influencing factors of health-related quality of life in myasthenia gravis (MG) patients with myasthenia gravis quality of life-15 (MGQOL-15) Chinese version and to provide corresponding measures in one tertiary hospital of Sichuan Province.@*METHODS@#We collected the general data (gender, age, body mass index BMI, marital status, educational level and employee status), clinical data [Osserman type, myasthenia gravis composite (MGC), other immunopathies, disease duration, frequency of outpatient visits per month, ratio of disease cost to income each month and frequency of symptoms during the past month] and the MGQOL-15 Chinese version from 168 myasthenia gravis patients in one tertiary hospital of Sichuan Province.@*RESULTS@#The mean score of MGQOL-15 was 17.67±12.78. The score of the item "My occupational skills and job status have been negatively affected." was the highest, followed by "I have trouble using my eyes." and "I am frustrated by my MG." Single factor analysis showed that MG patients' QOL were different with different disease severity MGC (F=19.353, P<0.001), ratio of disease cost to income each month (F=5.831, P<0.001) and the frequency of symptoms during the past month (F=9.128,P<0.001). Multiple regression analysis showed that disease severity MGC (β=0.743,P<0.001), ration of disease cost to income each month (β=3.347,P<0.001) and the frequency of symptoms during the past month (β=2.216,P<0.003) were the main predictors of HRQOL in the MG patients.@*CONCLUSION@#Our study showed that the MGQOL-15 is helpful for clinicians to evaluate MG patients' QOL regularly, investigate the influencing factors and implement corresponding interventions the so as to improve the patients' quality of life. Disease severity MGC, ratio of disease cost to income each month and the frequency of symptoms during the past month were the main predictors of MG patients' QOL. Clinicians should pay more attention to MG patients' disease severity MGC and the frequency of symptoms during the past month.

Current development of the second generation of mTOR inhibitors as anticancer agents / 癌症

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, acts as a "master switch" for cellular anabolic and catabolic processes, regulating the rate of cell growth and proliferation. Dysregulation of the mTOR signaling pathway occurs frequently in a variety of human tumors, and thus, mTOR has emerged as an important target for the design of anticancer agents. mTOR is found in two distinct multiprotein complexes within cells, mTORC1 and mTORC2. These two complexes consist of unique mTOR-interacting proteins and are regulated by different mechanisms. Enormous advances have been made in the development of drugs known as mTOR inhibitors. Rapamycin, the first defined inhibitor of mTOR, showed effectiveness as an anticancer agent in various preclinical models. Rapamycin analogues (rapalogs) with better pharmacologic properties have been developed. However, the clinical success of rapalogs has been limited to a few types of cancer. The discovery that mTORC2 directly phosphorylates Akt, an important survival kinase, adds new insight into the role of mTORC2 in cancer. This novel finding prompted efforts to develop the second generation of mTOR inhibitors that are able to target both mTORC1 and mTORC2. Here, we review the recent advances in the mTOR field and focus specifically on the current development of the second generation of mTOR inhibitors as anticancer agents.

Clinical analysis of rebleeding of ruptured aneurysms during the hospitalization / 中国脑血管病杂志

Objective: To investigate the time of rebleeding, the related risk factors and the prognosis of rebleeding in patients with ruptured intracranial aneurysms during the hospitalization. Methods: The clinical data of 253 hospitalized patients with ruptured intracranial aneurysms were analyzed retrospectively. They were divided into an early intervention (0 to 3 days) group (154 patients) and late intervention (4 to 28 days) groups (99 patients) according to the time of surgical or endovascular treatment. And then they were redivided into two subgroups according to whether they had rebleeding or not. Statistical methods were used to analyze the relationship between rebleeding and sex, age of the patients, site and size of the aneurysms, CT findings, Hunt-Hess grade, complicating hypertension, and the Glasgow Outcome Score (COS). Results: Circled digit one Of the 253 patients with ruptured intracranial aneurysms, 26 patients had rebleeding. The overall rebleeding rate was 10.3% (26/253) during hospitalization, 5. 2% (8/154) in the early intervention group and 18.2% (18/99) in the late intervention group; The median time between the first and to rebleeding was 6.5 days. Circled digit two There were significant differences in age and whether complicating hypertension or not between the patients with rebleeding and non-rebleeding in the early intervention group (P 0.05). Circled digit three There were no significant differences in patients sex and age, site and size of aneurysm, CT findings, and Hunt-Hess grade between the early intervention and late intervention groups in patients with rebleeding and non-rebleeding (P > 0.05). Circled digit four There were significant differences in GOS among the whole group of patients, early intervention and late intervention groups, in patients with or without rebleeding (P 0.05). Conclusions: Circled digit one The rebleeding rate of the ruptured aneurysms in the late intervention group was significantly higher than that in the early intervention group, and it is indicated that early surgery or endovascular treatment could reduce the incidence of rebleeding; Circled digit two the early rebleeding was associated with the age of patients and complicating hypertension; the advanced age and complicating hypertension were the risk factors for early rebleeding, while the late rebleeding was not significantly associated with the age of patients and complicating hypertension; Circled digit three There were no significant correlation between the rebleeding of ruptured aneurysms and sex, site and size of aneurysm, CT findings, and Hunt-Hess grade of the patients; Circled digit four the rebleeding of ruptured aneurysms markedly increased the mortality rate of patients.