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This study engineered β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from β-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of β-carotene ketolase and β-carotene hydroxylase were improved for conversion of β-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.
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Objective:To study changes and clinical significance of serum interleukin-33(IL-33) of patients with adult measles complicated acute liver damage. Methods: Totally,88 adult measles patients were selected and divided into acute liver damage group (n=60) and no liver damage group(n=28). At the same time,20 healthy adults were selected as healthy controls. Serum IL-33 levels were measured by enzyme linked immunosorbent assays. Venous blood samples of IL-33 were drawn on the day of admission and were repeated at 7 and 14 days later. The correlation of IL-33 and biochemical indicators were analyzed. 60 patients with liver damage were divided into protecting liver and without protecting liver group in accordance with the method of random numbers. The level of serum IL-33 was compared at 7 and 14 days between the two groups of patients. Results: Patients with adult measles complicated acute liver damage and no liver damage had elevated serum IL-33 levels than that in the controls [( 205. 20 ± 25. 74 ) pg/ml, ( 168. 70 ± 18. 14)pg/ml,vs(132. 17±12. 41)pg/ml,P＜0. 05 for all],especially in acute liver damage group. The expression of serum IL-33 in no protecting liver group was significantly higher than protecting liver group at 7th day. IL-33 was no significant difference in patients between protecting liver and without protecting liver groups at 14th day(P＞0. 05). The level of IL-33 was a significant decrease in the treatment and there was no significant difference between healthy controls at 14th day(P＞0. 05). The level of IL-33 and liver damage were consistent. Serum IL-33 levels in patients with liver damage showed positive correlation with levels of ALT,GGT and IL-6 ( r=0. 392 1,0. 503 9,0. 724 9,P＜0. 001). Conclusion: IL-33 may have proinflammatory effects in the phase of adult measles complicated acute liver damage and its serum level reflects the severity of liver inflammation.
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An ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was established to identify the metabolites in rat plasma after oral administration of Zhikebao tablets. The high-resolution mass spectrometer was operated in positive and negative ion mode, respectively. First, full-scan was applied, which was dependent on a multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS). These were utilized to trigger the information dependent acquisition (IDA) function in the experiment. For the IDA criteria, the eight most intense candidate ions of per cycle were selected to do a product ion scan. Then Metabolite Pilot 2.0 software was utilized to load data to seek possible metabolites. The analytical models employed by Metabolite pilot 2.0 were established for representative compounds of the Papaveris Pericarpium and licorice in Zhikebao tablet. Finally, metabolites were identified according to accurate mass measurement and retention time. 38 components from the rat plasma after oral administration of the drug have been found, including 5 prototype opium alkaloids, liquiritin, glycyrrhizic acid and 31 relative metabolites. The metabolic transformation of Zhikebao tablet in rats was mainly induced by glucuronidation, sulfation, methylation, amine to carboxylic acid, hydrolysis and so on. In this paper, the metabolites of the main active components of Zhikebao tablet were tentatively identified, and the metabolic pathway was compared with that of single chemical drugs. Moreover, it laid the fundamental elucidation of further metabolism study of Zhikebao tablet or other compound traditional Chinese medicine preparations which containing Papaveris Pericarpium or licorice.
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The study was designed to develop the method for determination of 7 benzodiazepines concentration in human plasma. UHPLC-MS/MS was adopted to analyze plasma with protein precipitated by acetonitrile. Citalopram was used as an internal standard. Plasma samples were separated on CORTECS UHPLC C18 column with the mobile phase of aqueous solution (0.01% formic acid)-methanol (0.01% formic acid) at a flow rate of 0.3 mL·min-1. Multiple reaction monitoring (MRM) mode was performed in combiation with electrospray ionization source operating in the positive ionization mode. The liner calibration curve of midazolam, nitrazepam, estazolam, clonazepam, lorazepam, triazolam and diazepam were obtained in the concentration range of 1.05-840 (r=0.999 4), 2.06-824 (r=0.998 1), 2.02-1 616 (r=0.994 7), 6.18-2 472 (r=0.997 9), 6.12-2 448 (r=0.997 4), 3.02-2 416 (r=0.990 2), 1.02-816 (r=0.998 8) ng·mL-1, respectively. The lowest detection limit were 0.02, 0.52, 0.51, 1.55, 0.77, 0.76, 0.02 ng·mL-1, respectively. The RSD of inter-day and intra-day were less than 10.81%. The relative recovery was 81.46%-106.53%. The method was successfully applied to clinical analysis of blood samples from patients.
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Objective: To study the inhibition effect of siRNA on the expression of Wisp-1 gene in Hca-F of mouse hepatocellular carcinoma cells strain and also its effect on the proliferation, migration and adhesion of hepatocellular carcinoma cells. Methods: Three expression vectors of siRNA were constructed. Lipo2000 was employed to transfect Hca-F cells and Western blot was used to detect the inhibition effect of siRNA on the expression of Wisp-1 gene. Afterward, CCK8 was adopted to detect the effect of Wisp-1 siRNA on the proliferation of Hca-F cells; Annexin V-FITC/PI double staining flow cytometry was used to detect the effect of Wisp-1 siRNA on the apoptosis of Hca-F cells; Transwell was used to detect the effect of Wisp-1 siRNA on the migration of Hca-F cells. The in vitro cell adhesion kit was used to detect of Wisp-1 siRNA on the change in the components of extracellular matrix to which Hca-F cells adhered. Western blot was used to detect the activation of protein kinase B (AKT)/glycogen synthase kinase-3β pathway and the expression of downstream target protein p53 and matrix metalloproteinases-2. Results: The siRNA showed interference effect on the expression of Wisp-1 gene. Compared with the control group, after being transfected to cells, Wisp-1 siRNA could significantly inhibit the proliferation, migration and adhesion of Hca-F cells and also promote the cell apoptosis, which was related to the down-regulated phosphorylation of AKT and glycogen synthase kinase-3β and the expression of p53 and matrix metalloproteinases-2 (P < 0.05). Conclusions: The inhibition of Wisp-1 expression can reduce the proliferation, migration and adhesion of mouse hepatocellular carcinoma cells, which is related to the AKT/glycogen synthase kinase-3β pathway. Wisp-1 gene may be the potential target to cure the hepatocellular carcinoma.
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OBJECTIVE@#To study the inhibition effect of siRNA on the expression of Wisp-1 gene in Hca-F of mouse hepatocellular carcinoma cells strain and also its effect on the proliferation, migration and adhesion of hepatocellular carcinoma cells.@*METHODS@#Three expression vectors of siRNA were constructed. Lipo2000 was employed to transfect Hca-F cells and Western blot was used to detect the inhibition effect of siRNA on the expression of Wisp-1 gene. Afterward, CCK8 was adopted to detect the effect of Wisp-1 siRNA on the proliferation of Hca-F cells; Annexin V-FITC/PI double staining flow cytometry was used to detect the effect of Wisp-1 siRNA on the apoptosis of Hca-F cells; Transwell was used to detect the effect of Wisp-1 siRNA on the migration of Hca-F cells. The in vitro cell adhesion kit was used to detect of Wisp-1 siRNA on the change in the components of extracellular matrix to which Hca-F cells adhered. Western blot was used to detect the activation of protein kinase B (AKT)/glycogen synthase kinase-3β pathway and the expression of downstream target protein p53 and matrix metalloproteinases-2.@*RESULTS@#The siRNA showed interference effect on the expression of Wisp-1 gene. Compared with the control group, after being transfected to cells, Wisp-1 siRNA could significantly inhibit the proliferation, migration and adhesion of Hca-F cells and also promote the cell apoptosis, which was related to the down-regulated phosphorylation of AKT and glycogen synthase kinase-3β and the expression of p53 and matrix metalloproteinases-2 (P < 0.05).@*CONCLUSIONS@#The inhibition of Wisp-1 expression can reduce the proliferation, migration and adhesion of mouse hepatocellular carcinoma cells, which is related to the AKT/glycogen synthase kinase-3β pathway. Wisp-1 gene may be the potential target to cure the hepatocellular carcinoma.
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Objective To describe the distribution and related risk factors of lipodystrophy (LD) among AIDS patients treated with antiretroviral drugs.Methods A cross-sectional study was performed on 261 AIDS patients treated with antiretroviral drugs.All the subjects were followed in the Center for Disease Control and Prevention of two counties in northern Anhui province from May 25 to 30,2012.Data related to demography,physical examination,history of antiretroviral treatment,HIV plasma viral load,and CD4 +T cell count were collected.Clinical examination was based on an assessment of changes in face,legs,arms,buttocks (peripheral sites),back,chest,neck or abdomen or change in waist size (central sites) as quoted by the clinicians.Results LD was observed in 147 (56.3％) patients.The differences of age,gender,quality of sleep,weight and time of treatment between LD and non-lipodystrophy (NLD) groups were statistically significant (P＜0.05).Results from the Multivariate logistic regression analysis showed that the risk of women suffering from LD was 1.894 times ofthd males (95％CI:1.075-3.338).The risk of those with LD showed an 1.448-fold increase regarding the time of treatment for each additional year (95％CI:1.267-1.654).Patients with poor quality of sleep were prone to LD with 11.901 times more than those with good quality of sleep (95％CI:2.701-52.441).Conclusion LD was commonly observed in AIDS patients who were under antiretroviral therapy.Gender,tine of treatment and the quality of sleep appeared the main factors related to the results of observation.
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Momordica charantia L. is a vegetable widely cultivated around the world. Its fruits have been used in Asian countries as a folk medicine for the treatment of non-insulin-dependent diabetes mellitus. Here we report eight compounds isolated from the fruits of M. charantia. On the basis of NMR and MS spectroscopic analyses, these compounds were identified as momordicolide ((10E)-3-hydroxyl-dodeca-10-en-9-olide, 1), monordicophenoide A (4-hydroxyl-benzoic acid 4-O-beta-D-apiofuranosyl (1 --> 2)-O-beta-D-glucopyranoside, 2), dihydrophaseic acid 3-O-beta-D-glucopyranoside (3), 6,9-dihydroxy-megastigman-4,7-dien-3-one (blumenol, 4), guanosine (5), adenosine (6), uracil (7) and cytosine (8). Among them, 1 and 2 are new compounds. Compounds 3-5 were isolated from this plant for the first time.
Subject(s)
Fruit , Chemistry , Glycosides , Chemistry , Momordica charantia , Chemistry , Triterpenes , ChemistryABSTRACT
<p><b>OBJECTIVE</b>To study the effect of Supplemented Taoren Chengqi decoction (STCD) on the secretion of insulin and proliferation of NIT-1.</p><p><b>METHOD</b>The effect of STCD and the serum of rat after orally administrating of STCD on the secretion of insulin and proliferation of NIT-1 were studied. The proliferation of NIT-1 was measured by 3H-TdR incorporation and cell counting methods, while the secretion of insulin was measured from the cultured medium by the ultra sensitive rat insulin ELISIA kit.</p><p><b>RESULT</b>Both the STCD and the serum of rat after orally administrating of STCD significantly could increased the secretion of insulin and proliferation of NIT-1.</p><p><b>CONCLUSION</b>The treatment of the diabetic patients by STCD might be through with its improvement of secretion of insulin and proliferation on pancreatic beta-cell.</p>