Background and purpose:Previous studies have shown that Bubl was a critical component of the spindle checkpoint.Paclitaxel sensitivity was considered to be dependent on the functionality of this spindle checkpoint.This study investigated the effects of pEGFP-Bubl-shRNA plasmid stably transfected into the cell cycle and its sensitivity in human ovarian cancer cell line A2780.Methods:After the pEGFP-Bubl-shRNA plasmid and empty plasmids were constructed.they were transfected into A2780 cells by the Lipofectamine 2000~(TM).The nontransfected cells were the control.RT-PCR and Western blotting were used to determine the target gene and protein expression.The rate of proliferation inhibition was tested by an MTT assay,apoptosis and cell cycles were determined by flow cytometry,and the mitotic index was determined bv Hoechst33342 dye.Results:RT-PCR and Western blotting showed that pEGFP-Bubl-shRNA/A2780 group displayed a low expression of Bubl compared to the A2780 and pEGFP-Cl/A2780 group(P＜0.05).The sensitivity of the pEGFP-Bubl-shRNA/A2780 group was significantly lower than the non-transfccted and pEGFP-Cl/A2780 cells(P＜0.05).Flow cytometry revealed that the rates of G2/M phase and apoptosis were significantly lower in the pEGFP-Bubl-shRNA/A2780 group than those in the control group (P＜0.05).Conclusion:Bubl plays an important role in the paclitaxel treatment.A down-regulation of Bubl could reduce the drug sensitivity and rate of G_2/M cells in human ovarian cancer cell line A2780.

Objective: To analyze the expression of CMTM1-v17 in normal prostate tissue and prostate carcinoma originated cell lines, and study its impact on the transactivation of androgen receptor and the possible mechanism. Methods: The expression of CMTM1-v17 in normal prostate tissue was analyzed with immunohistochemistry method. In immounocytochemistry was used to analyze the expression of CMTM1-v17 in prostate carcinoma originated cell lines. Luciferase assay was used to study the impact of CMTM1-v17 on the transactivation of AR and its mechanism. Results: The results of immunohistochemistry showed that CMTM1-v17 was highly expressed in prostate. In prostate cancer originated cell lines, CMTM1-v17 could also be detected in prostate cancer originated cell lines PC3, Du145 and LNCaP. And the results of luciferase implied that the relative luciferase activity of the PC3 cells transfected with 1 ?g and 2 ?g pCDI-CMTM1-v17 plasmids separately were 70.8 and 34.7, compared with the control set as 100. When trichostatin A, the inhibitor for histone deacetylase, was used, the repression of androgen receptor could be recovered with trichostatin A treatment,for the relative luciferase activity of the PC3 cells transfected with 1 ?g and 2 ?g pCDI-CMTM1-v17 plasmids and treated with 100 nmol/L trichostatin A rebound to 90.9 and 86.4. Conclusion: CMTM1-v17 is highly expressed in both normal prostate and prostate carcinoma originated cell lines. It may recruit histone deacetylas to inhibit the function of androgen receptor.

In this article, the status of spindle assembly checkpoint and the alteration of its major component, Mad2 protein level were examined in A2780 and SKOV3 ovarian cancer cell lines. Recombinant eukaryotic expression plasmid pEGFP-Mad2 was transfected into paclitaxel-resistant SKOV3 cells and Mad2 protein was knocked down by Mad2-specific siRNA in paclitaxel-sensitive A2780 cells. Then the expression level of Mad2 gene was detected by Western blotting. Flow cytometry revealed that SKOV3 cells were not fully arrested in G(2)/M phase in contrast to A2780 cells in the presence of paclitaxel. However, paclitaxel sensitivity assay showed that sensitivity to paclitaxel was reversed after the transfection in both cell lines in terms of number of cells arrested at G(2)/M phase and the expression of Bcl-2 was significantly changed. These results suggest that weakened spindle checkpoint with reduced expression of Mad2 is associated with resistance to paclitaxel in ovarian cells and Bcl-2 may be involved in this process.

OBJECTIVETo validate the efficiency and accuracy of an improved Demons deformable registration algorithm and evaluate its application in contour recontouring in 4D-CT.

METHODSTo increase the additional Demons force and reallocate the bilateral forces to accelerate convergent speed, we propose a novel energy function as the similarity measure, and utilize a BFGS method for optimization to avoid specifying the numbers of iteration. Mathematical transformed deformable CT images and home-made deformable phantom were used to validate the accuracy of the improved algorithm, and its effectiveness for contour recontouring was tested.

RESULTSThe improved algorithm showed a relatively high registration accuracy and speed when compared with the classic Demons algorithm and optical flow based method. Visual inspection of the positions and shapes of the deformed contours agreed well with the physician-drawn contours.

CONCLUSIONDeformable registration is a key technique in 4D-CT, and this improved Demons algorithm for contour recontouring can significantly reduce the workload of the physicians. The registration accuracy of this method proves to be sufficient for clinical needs.

Objective To construct recombinant adenovirus expressing mouse ChREBP-α, and examine the effect of ChREBP-α overexpression on lipid synthesis in mouse primary hepatocytes. Methods The mouse ChREBP-α cDNA was subcloned into pShuttle-CMV vector, and the product was transformed into E. coli strain BJ5183 for homologous recombination with pAdEasy-1.The resultant recombinant vector was transfected into 293A cells for viral package. Mouse primary hepatocytes were infected with adenoviruses Ad-ChREBP-α, and gene expression was analyzed at mRNA and protein levels by real-time PCR and Western blotting analsysis. The expression levels of ChREBP target gene LPK were measured at mRNA level by real-time PCR, and the fatty acid synthesis rate was determined by [14C]-acetate incorporation. Results We successfully constructed recombinant adenoviruses expressing mouse ChREBP-α. Adenovirus-mediated overexpression of ChREBP-α markedly increased LPK mRNA expression and fatty acid synthesis in primary hepatocytes. Conclusion We have successfully constructed recombinant adenoviruses expressing mouse ChREBP-α, which is biologically active and can overexpress ChREBP-α in primary hepatocytes. Overexpression of ChREBP-α can enhance lipid synthesis.

A strain, Pseudomonas sp. X-2-45, with high and stable lipolytical activity was screened by continuously subculturing a lipase-producing bacterium P. sp. LP-1 in culture medium containing Jatropha oil as a sole carbon source. Its hydrolytic activity was 29.79 U/mL, which was increased by 288% as compared to that of parent strain. Furthermore, the growth and lipase synthesis of X-2-45, its catalytic ability to hydrolyze vegetable oils, as well as ester synthesis between fatty acids and organic alcohols were studied. Results showed that rates of bacterial growth and lipase synthesis were significantly raised. Bacterial biomass and lipase activity reached the highest level after 30 h of incubation. Moreover, growth stationary period was prolonged and lipase produced exhibited good stability in culture media during incubation period. Hydro- lytic activity of P. sp. X-2-45 lipase toward Jatropha oil was increased by 378% as compared to parent strain, suggesting that acclimation to Jatropha oil was an effective approach for improving substrate selectivity oflipase. Finally, results of ester synthesis catalyzed by P. sp. X-2-45 lipase indicated that this lipase could catalyze esterification reactions between lauric acid and n-butanol, n-octanol, 1-dodecanol or glycerol, palmitic acid or stearic acid and methanol, n-octanol, 1-dodecanol or glycerol, oleic acid and methanol, n-butanol, n-octanol, 1-dodecanol or glycerol.

Recent evidence has suggested that Akt2 plays an important role in the protection of cells from paclitaxel (PTX)-induced apoptosis and control of the cell cycle. In addition, some scholars suggested that the PTX sensitivity depends on a functional spindle assembly checkpoint. In the present study, we investigated the role of the Akt2/Bub1 cross-talking in apoptosis and cell cycle after exposure of the A2780 ovarian cancer cells to paclitaxel (PTX). Recombinant expression plasmid WT-Akt2 was transfected into A2780 cells by lipofectamine2000, and then the expression level of Akt2 gene was detected by using RT-PCR and Western blotting. Cell apoptosis and cell cycle were detected by flow cytometry and Hoechst 33342 staining after treatment with PTX. Moreover, we compared the expression level of Bub1 in different groups by Western blotting. Our study showed that up-regulation of Akt2 contributed to A2780 ovarian cancer cells overriding PTX-induced G(2)/M arrest, and inhibited Bub1 expression. Our findings might shed light on the molecular mechanism of PTX-induced resistance in ovarian cancer and help develop novel anti-neoplastic strategies.

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.

Objective To investigate the clinical efficacy of prucalopride in the treatment of severe chronic constipation.Methods The clinical data of 60 patients with severe chronic constipation [slow transit constipation (STC), functional defecation disorder (FDD) and constipation-predominant irritable bowel syndrome (IBS-C)] who were admitted to the Third Affiliated Hospital of Nanjing University of Chinese Medicine from February to August 2014 were prospectively analyzed.A prospective, clinical observational study was performed.Treatment plans included that patients withdrew the initial treatments of laxative and exema and took orally 2 mg prucalopride once daily for 2 weeks, and continued to be treated by oral prucalopride if frequency of the spontaneous complete bowel movement (SCBM) per week was satisfactory (or improvement of symptoms was more than 50％) till 4 weeks, and then were followed up after stopping prucalopride.If improvement of symptoms was less than 50％ after 2-week treatment, other treatment plans were performed according to symptoms of patients from week 3 to week 6 : (1) for patients with STC, prucalopride + two chain bacillus subtilis probiotic capsules were administered orally if patients were satisfied with frequency of SCBM per week and without improvement of abdominal distension;prucalopride + Chinese herb decoction were administered orally if patients had improvement of frequency of SCBM per week with abdominal distension or poor stool output;oral prucalopride + acupuncture were administered if patients were unsatisfied with frequency of SCBM per week or less bowel movements and without improvement of abdominal distension or poor stool output.(2) For patients with FDD, oral prucalopride + acupuncture + biofeed-back therapy were administered.(3) For patients with IBS-C, prucalopride + two chain bacillus subtilis probiotic capsules were administered orally if patients had abdominal distension;prucalopride Chinese herb decoction were administered orally if patients had improvement of frequency of SCBM per week and no improvement of abdominal distension or poor stool output.All patients used a diary for recording the frequency of SCBM per week, stool consistence, exertion in defecation and adverse reactions, which was submitted to doctors for inputting data at the return visit weekly.Results There was good overall medicine compliance in patients.Of 60 patients, 43 patients completed treatments (21 with STC, 11 with FDD and 11 with IBS-C).After 2-week treatment, there were 19 patients with satisfied therapeutic effects, 14 with improvement of constipation and 10 with poor therapeutic effects.After 4-week treatment, constipation in 17 patients was cured, constipation in 18 patients was improved,and constipation in 8 patients was not improved.Nineteen of 60 patients were complicated with adverse reactions within 1 week of the medication, including 6 patients dropping out of the trial due to medication withdrawal and others with improvement by symptomatic treatment or spontaneous remission.Conclusions Prucalopride is effective for the treatment of severe chronic constipation with a good toleration, and it can improve the overall satisfaction of patients combined with Chinese herb decoction and acupuncture.

Objective To analyze the characteristics of the genotype, phenotype, and follow-up of Gitelman's syndrome (GS) in the largest group of Chinese patients. Methods Sixty seven patients with GS underwent SLC12A3 gene analysis. Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. Additionally, the associations of genotypes and phenotypes were explored. Results Forty-one different SLC12A3 mutations were identified in 67 patients with GS, including 11 novel ones, and 5 recurrent ones. 3 families (5. 7% ) had triple SLC12A3 mutations. Typical hypocalciuria and hypomagnesemia were not found in 6(9% ) and 8 (11. 9% )patients, respectively. In addition, male patients had an earlier age of onset and a higher urinary fraction excretion of electrolytes. 2 patients presented with chronic kidney disease, 13 (19. 4% ) with type 2 diabetes, 14 (20. 9% )with impaired glucose tolerance, and 5(7. 5% ) with impaired fasting glucose. Conclusion This study revealed 41 mutations in 67 Chinese patients with GS, including 11 novel variants and 5 high-frequency ones. Fraction excretion of electrolyte in urine may be more sensitive in the evaluation of phenotype compared with those of blood. It is difficult to correct hypokalemia and hypomagnesemia in GS. Patients with GS are at higher risk of the development of diabetes than ordinary people.