Determination of insulin lispro in rat plasma by LC-MS/MS and its application in a pharmacokinetics study / 药学学报

Compared with human insulin, insulin lispro shows a faster hypoglycemic effect and a higher peak plasma concentration, which can better control postprandial hyperglycemia. In this study, we used a solid phase extraction pretreatment method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify insulin lispro in rat plasma. Bovine insulin was used as an internal standard. Plasma samples were separated on an ACQUITY UPLC Peptide CSH C18 column (2.1 mm × 50 mm, 1.7 μm) after solid phase extraction. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 1 162.5→217.2 for insulin lispro and m/z 1 157.5→136.0 for insulin bovine (internal standard). The method validation results showed that the linear range was 0.1 ng·mL-1 - 100 ng·mL-1; intra- and inter-day accuracy and precision met the acceptance criteria for biological sample analysis. The recovery of insulin lispro ranged from 63.1% to 68.1%. The method was applied in a pharmacokinetic study of insulin lispro following a single-dose subcutaneous administration to rats. Animal experiments were approved by the Experimental Animal Ethics Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Regulation of β-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells / 中国中药杂志

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in β-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the β-amyrin production. Compared with the control strain, the production of β-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the β-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of β-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

Study of heterologous efficient synthesis of β-amyrin and high-density fermentation / 中国中药杂志

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.

Investigation and optimization on ability of enzymatic hydrolysis of Mori Cortex residue / 中国中药杂志

Residue of Mori Cortex was studied to optimize its enzymatic hydrolysis process, and explore its potential as a carbon source for biochemistry and biofuel production. The cellulose content of diluted acid pretreated (DAP) and non-pretreated from Mori Cortex were measured in this study, and the results showed that the cellulose content of DAP and non-pretreated from Mori Cortex were 52.5% and 47%, respectively. This higher cellulose content indicated that residue of Mori Cortex had the potential to act as a carbon source for biochemistry and biofuel production. Enzymatic hydrolysis of pretreated and non-pretreated from Mori Cortex was conducted under different enzyme loading amount. 40 FPU·(g DW）⁻¹ enzyme loading was determined as the optimal amount by comparing the yield of sugar and the rate of enzymolysis. Under this condition, the concentrations of glucose, xylose, arabinose sugar were 23.82, 4.84, 3.6 g·L⁻¹, and the corresponding enzymatic hydrolysis rate was 45.33% which was 2.3 times higher than that of non-pretreated from Morus alba residues. Fed-batch enzymatic hydrolysis was conducted finally to get higher sugar yield, and the final glucose concentration reached up to 38 g·L⁻¹ with the enzymatic hydrolysis rate of 36.19%. The results indicated that Mori Cortex residue had higher cellulose and hemicellulose contents, so it had the potential to become a carbon source to produce the bio-chemicals and biofuels. Through enzymatic hydrolysis, it can be converted into microbial available monosaccharides; and through fermentation, it can be converted into high value-added chemicals, biofuels, etc., to solve the problem of residue pollution, and achieve the sustainable development and greening of Chinese pharmaceutical production process.

Clinical signiifcance of ICAM-1 expression and its correlation with prognosis of triple-negative breast cance r / 中国癌症杂志

Background and purpose:Triple negative breast cancer (TNBC) is with high invasion, poor prognosis and lack of usefull treatment. This study investigated expression status of ICAM-1 protein in TNBC in order to explore its relationship with clinicopathological features and outcome in patients.Methods:Fifty-nine tissue samples of TNBC were collected while 50 cases of para-carcinoma tissue samples were used as negative controls. Immunohistochemical staining was conducted to detect expression level of ICAM-1 protein. The relationship of ICAM-1 protein expression with clinicopathological features (age, tumor size, subtype, grade, status of lymph node metastasis, TNM stage, vascular tumor thrombus, nerve inifltration, Ki-67, p53 and E-cadherin expression) and outcome in patients were analyzed.Results:The ICAM-1 protein expression of TNBC was signiifcantly higher than that in adjacent tissues (P=0.000). ICAM-1 expression was related to status of lymph node metastasis, grade and TNM stage (with aP-value of 0.036, 0.027 and 0.048, respectively), while demonstrated an undeifned relationship with tumor size, subtype, vascular tumor thrombus and expression of Ki-67, p53 and E-cadherin. The disease-free survival (DFS) of ICAM-1 high expression set was shorter than that of the lower one but has nothing to do with overall survival (OS). In addition, Cox proportional hazards model showed that ICAM-1 expression and lymph node metastasis were independent risk factors of DFS in patients (HR=3.2, 95%CI: 1.6 to 6.4, HR=2.7, 95%CI: 1.28 to 5.9,P<0.05).Conclusion:ICAM-1 could serve as a predictive factor for differentiation status of TNBC. The high expression of ICAM-1 in TNBC may indicate poorer prognosis.

The inlfuence of diagnostic interval and other clinicopathological factors on the prognosis of triple-negative breast cancer / 中国癌症杂志

Background and purpose:The time from ifrst onset of symptoms or signs to a deifnitive diagnosis is deifned as diagnostic interval (DI). The relation of DI to other clinicopathological parameters andthe impact of DI on prognosis of patients with triple-negative breast cancer (TNBC) remain unclear.This article plans to make an intensive study of these questions.Methods:The clinical records of a series of 83 consecutively presenting unselected patients referred to the Shanghai Sixth People’s Hospital with diagnosed TNBC between September 2009 and September 2015 were retrospectively reviewed. Clinical and pathological factors included were investigated by univariate analysis using the Kaplan-Meier method, the factors associated with prognosis were further evaluated by multivariable analysis with Cox progression model.t-test and Kruskal-Wallis test were used to study the correlation between DI and other characters.Results:DI: stage T3>T1 (P=0.01), stageⅢ>Ⅱ (P=0.03) andⅠ (P=0.01). Compared with patients of DI≥3 months, the <3 months group had earlier age (P=0.028) and TNM stage (P=0.035). T stage, N stage, neoadjuvant chemotherapy, TNM stage and DI are inlfuencing factors of overall survival (OS). Age, T stage, N stage, TNM stage, menstrual status and neoadjuvant chemotherapy are inlfuencing factors of progression-free survival (PFS). TNM staging is an independent inlfuencing factor for OS and PFS.Conclusion:Patients with later disease stage were more likely to have a longer DI; The shorter DI, the earlier age and stage of disease; DI is the inlfuence factor of OS; TNM stage is an independent inlfuencing factor for OS and PFS.

Otogenic Brain Abscess Presenting with Gait Ataxia

Although the occurrence of otogenic brain abscess has been considerably reduced through improvements to antibiotics, brain abscesses remains one of the most significant life threatening complications of chronic otitis media. We report the case of a 67-year-old male patient who presented with gait ataxia and dizziness. Imaging studies revealed a left cerebellar abscess and extensive destructive changes to the labyrinth due to chronic otitis media. We conclude that otogenic brain abscess should be considered as differential diagnosis in patients with chronic otitis media who develop central vertigo.