Taxa-4(5),11(12)-diene is the precursor for paclitaxel biosynthesis. The diterpenoid paclitaxel (marketed as Taxol), a plant secondary metabolite isolated from yew, is an effective drug widely used in the treatment of numerous cancers. However, further application of taxol has been restricted due to its low yield in plants and the difficulties in extraction. To increase the intact isoprene flux, we constructed the fusion gene plasmid pBgGGTS and individual cassette plasmid pBgGGgTS to enhance the expression levels of geranylgeranyl diphosphate synthase gene (ggpps) and a taxadiene synthase gene (ts) in Coprinopsis cinerea. These two plasmids were separately transformed into C. cinerea LT2 strain, resulting in several putative transformants. Putative transformants were determined by PCR technique, indicating that 5 out of 13 putative transformants transformed by pBgGGTS and 6 out of 13 putative transformants transformed by pBgGGgTS, respectively. Additionally, the Southern blotting analysis of these 10 transformants confirmed that both ggpps and ts gene were stably integrated into the genome of C. cinerea. Crude extracts from each of the transformants were analyzed. There is no difference in the mycelium extracts among the wild-type LT2 and two types of transformants. However, analysis of culture filtrates indicated that an additional GC peak was found at the retention time of 16.762 min which was absent in the wild type control. The mass fragmentation pattern of this peak had the same diagnostic ions with taxa-4(5),11(12)-diene. According to peak area, the amounts of taxa-4(5),11(12)-diene in each fermented broth were 44 ng/L (transformed with pBgGGgTS) and 30 ng/L (transformed with pBgGGTS), respectively. In conclusion, co-expression of the ggpps and ts gene could increase the taxadiene production in C. cinerea.
Agaricales ; metabolism ; Alkenes ; metabolism ; Diterpenes ; metabolism ; Farnesyltranstransferase ; genetics ; metabolism ; Genetic Engineering ; Isomerases ; genetics ; metabolism ; Paclitaxel ; Plasmids

BACKGROUND:The preemptive analgesia is stil a controversial issue. Existing studies have not paid much attention to effects of preoperative factors on the hypersensitivity of peripheral and central mechanisms. Visual analog scale scores cannot subjectively and repeatedly reveal patient’s pain.
OBJECTIVE:To investigate the validity of the preventive analgesia effect of Celebrex in patients with total knee arthroplasty.
METHODS:Patients with osteoarthritis of the knee who received total knee arthroplasty were accessed by Pittsburgh sleep quality index, self-rating depression scale and self-rating anxiety scale. In al , thirty patients were enrol ed in the study. They were randomized into Celebrex group and vitamin C group, and each group had 15 patients. The patients in the Celebrex group and vitamin C group took 200 mg Celebrex and vitamin C, respectively, twice a day from day 2 to day 4. Both of their knees were evaluated by resting visual analogue scale and moving visual analogue scale in the evening of day 1 before treatment and day 3 after treatment. Meanwhile, the pain threshold and pain tolerance were accessed by a pain-threshold machine.
RESULTS AND CONCLUSION:No statistical significance of the changes of resting and moving visual analogue scale scores was found in both knees in the Celebrex group (P>0.05). The pain threshold of both knees were significantly increased (P<0.05), and the severe knee, which had less visual analogue scale scores than the minor one, turned out to increase more obviously than the minor knee (P>0.05). There were no significant changes in the pain tolerance in both knees (P>0.05). The changing values of resting or moving visual analogue scale were not significantly correlated with the pain threshold and pain tolerance (P>0.05). There were no significant changes in visual analogue scale scores, pain threshold and pain tolerance in both knees of the vitamin C group (P>0.05). Celebrex could increase the pain threshold of patients receiving total knee arthroplasty, especial y the severe knee, which indicates that the Celebrex is good for the preventive analgesia. Comparatively speaking, the pain threshold might be more sensitive than visual analogue scale in revealing the change of pain after analgesia. There is no significant correlation between visual analogue scale score and the hypersensitivity of pain.

ObjectiveTo observe the effects of human IL-10 gene transfection on the mRNA and protein expressions of IL-1β and TNF-α in the penumbra area following focal cerebral ischemia-reperfusion injury in rats and to investigate its neuroprotective mechanism. MethodsRats were divided into four groups: normal control group, ischemic control group, empty plasmid group and human IL-10 gene transfected group. The mRNA and protein expressions of IL-1β and TNF-α in the penumbra area were detected by fluorescence real-time quantitative PCR and ELISA respectively. ResultsIn normal control group, ischemic control group, empty plasmid group and human IL-10 gene transfected group, the levels of protein expression of TNF-α in penumbra area were(0.66±0. 04) ,(1.16±0.26),(1. 155±0. 26)ng/g and(0. 84±0. 05)ng/g, and the levels of protein expression of IL-1βin penumbra area were(0.37±0.05), (1.25±0.39), (1.21±0.57) ng/g and(0.62+0.05)ng/g, respectively. Compared with normal control group, the levels of protein expression of TNF-α and 1L-1β were significantly higher in other three groups(all P＜0. 01), and lower in human IL-10 gene transfected group than in ischemic control group and empty plasmid group(all P＜0. 01). In normal control group, ischemic control group, empty plasmid group and human IL-10 gene transfectedgroup, the levels of mRNA expression of TNF-α in penumbra area were 1.00 ±0.53,9.42±1.83,9.69±1.96 and 3.53±1.09, and the levels of mRNA expression of IL-1β in penumbra area were 1.00 ±0.51,27. 81±4.84,23.96 ± 4.90 and 13.55± 4.45, respectively. Compared with normal control group, the levels of mRNA expression of TNF-α and IL-1β were significantly higher in other three groups(all P＜0. 01), and lower in human IL-10 gene transfected group than in ischemic control group and empty plasmid group(all P＜0. 01). ConclusionsThe human IL-10 gene transfection may play an protective effect on cerebral ischemia through inhibiting mRNA and protein expression of IL-1β and TNF-α in the penumbra area following focal cerebral ischemia-reperfusion in rats.

Objective To evaluate measurement of the submucosal thickness with endoscopic ultrasonography (EUS) for activity of Crohn disease (CD).Methods Ten patients with active stage of CD and 10 healthy controls (HC) underwent EUS.Simple endoscopic score for Crohn disease(SES-CD)and submucosal thickness at the most severe lesions were measured and recorded.Submucosal thickness of the same region in CD patients were measured at remissive stage.In order to analyze the relationship between submucosal thickness and the stage of CD, submucosal thickness were compared among patients at active stage of CD, remissive stage of CD and HC.And the cut-off value of submucosal thickness was calculated to diagnose the stage of CD.Results The mean submucosal thicknesses of active stage and remissive stage of CD were 6.48±1.95 mm and 2.47±1.08 mm,respectively (P<0.01).The correlation analysis showed that submucosal thickness had a positive correlation with Crohn disease activity index(CDAI)(r=0.708,P<0.01) and SES-CD(r=0.807,P<0.01).Receiver operating characteristic curve analysis was used for 10 cases of CD patients and the area under the curve was 0.985(P<0.01).The cut-off value of submucosal thickness to diagnose active stage of CD was 3.85 mm, and the sensitivity and specificity reached 100% and 90% respectively.The Youden index was 0.9.Conclusion Measurement of gastrointestinal submucosal thickness by EUS could contribute to evaluate the stage of CD and to guide clinical treatment.

Objective To evaluate the safety and efficacy of endoscopic ultrasonography(EUS) guided ethanol ablation in patients with insulinoma. Methods The data of 10 patients with insulinoma trea-ted at the First Affiliated Hospital of Guangxi Medical University from December 2013 to January 2015 were prospectively analyzed. Results The patients were given EUS-guided ethanol ablation with dose of 0. 10 to 2. 00 ml(average 0. 70 ± 0. 62 ml)in pancreatic lesions for 15 times. No complications were observed dur-ing and after the procedure. The blood glucose improved after the procedure[4. 8(3. 9-5. 5)mmol/ L VS 2. 4 (1. 9-2. 5)mmol/ L,P < 0. 05]and the serum insulin level significantly decreased[83. 7(40. 1-143. 5) pmol/ L VS 177. 3(66. 5-200. 6)pmol/ L,P<0. 05]. The average hospital stay was(4. 3±1. 5)days. The patients were followed up for 6-12 months. EUS indicated that the echo of pancreatic lesions changed from high to low. CE-EUS revealed low enhancement and lack of blood supply. Conclusion EUS-guided ethanol ablation may become a promising minimally invasive treatment for insulinoma because of its safety,efficacy and low price. Trail registration Clinical Trial.gov,NCT02121366.

Objective To study the detection of Na+/H+exchanger regulatory factor 3(NHERF3) protein expression in renal carcinoma and its correlation with malignant biological behavior. Methods Renal clear cell carcinoma and their adjacent tissues of forty- eight patients were collected. Immunohistochemical method was used to detect the positive expression of NHERF3 protein,and specific expression was detected by Western-blot.Patients were further divided into high NHERF3 group and low NHERF3 group according to median expression of NHERF3 protein,and each group had 24 cases.The expressions of proliferation,invasion and autophagy genes in tumor tissues were detected by fluorescent quantitative PCR.Results The positive rate of NHERF3 protein and the expression of NHERF3 protein in renal carcinoma tissue was significantly lower than that in paracancerous tissue(P<0.05).Expressions of proliferation genes such as k-Ras,c-Myc,TRPC1 mRNA in low NHERF3 group were higher than those in high NHERF3 group:141.74 ± 18.95 vs. 100.00 ± 0.00, 135.88 ± 16.32 vs. 100.00 ± 0.00, 137.21 ± 16.98 vs.100.00 ± 0.00;MIIP,FOXO1 mRNA levels were lower than those in high NHERF3 group: 43.19 ± 5.88 vs. 100.00 ± 0.00, 38.76 ± 4.51 vs. 100.00 ± 0.00; expressions of invasion genes such as CD74, Fascin, MACC1, TRPM8 mRNA were significantly higher than those in high NHERF3 group:152.18 ± 17.64 vs. 100.00 ± 0.00, 146.29 ± 17.63 vs. 100.00 ± 0.00, 139.76 ± 15.82 vs. 100.00 ± 0.00,150.47 ± 17.95 vs.100.00 ± 0.00;expressions of autophagy genes such as Beclin-1,LC3 mRNA were significantly lower than those in high NHERF3 group: 63.21 ± 7.09 vs. 100.00 ± 0.00, 56.28 ± 7.15 vs. 100.00 ± 0.00; EZH2 mRNA level was higher than that in high NHERF3 group:159.47 ± 17.82 vs.100.00 ± 0.00,and there were significant differences(P<0.05).Conclusions The positive rate of NHERF3 protein expression and the amount of protein expression in renal carcinoma tissue is increased, and the specific expression is closely related to tumor proliferation, invasion and activity of autophagy.

Objective To study the diagnostic value of narrow-band imaging ( NBI) combined with endoscopic ultrasonography ( EUS) for ampullary tumors. Methods A total of 21 patients suspected with ampullary lesions by imaging or endoscopic examination from December 2015 to March 2017 were enrolled in this prospective study. All patients underwent NBI and EUS, and 20 patients underwent biopsy. The type of ampullary tumor was predicted by preoperative examination, and appropriate treatment methods were chosen. The final diagnosis was confirmed by biopsy, surgical pathology, and clinical follow-up for more than 6 months. The accuracy of NBI combined with EUS and biopsy in diagnosis of ampullary malignant tumors was calculated according to the gold standard. The Chi-square test was used to compare diagnostic accuracies. Results The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of NBI combined with EUS in diagnosis of ampullary malignancies were 94. 1％ (16/17), 100. 0％ (4/4), 95. 2％ (20/21), 100. 0％ (16/16), and 80. 0％ (4/5), respectively. The corresponding indicators of preoperative biopsy were 41. 2％ ( 7/17) , 100. 0％ ( 3/3) , 50. 0％ ( 10/20) , 100. 0％ ( 7/7) , and 23. 1％( 3/13) , respectively. The accuracy of NBI combined with EUS in diagnosing ampullary malignant tumor was significantly higher compared with preoperative biopsy ( P=0. 004) . Conclusion NBI combined with EUS can more accurately predict benign or malignant ampullary tumor, and better guide the choice of surgical methods compared with preoperative biopsy.