【Objective】 To construct the eukaryotic expression vector carrying the human wild-type p27 and lacking nuclear localization signal p27△NLS coding sequences， and the express them in HEK293T cells， which may contribute to investigating the different locations and roles of p27 in the cytoplasm and nucleus. 【Methods】 Total RNA was prepared from human breast cancer MCF7 cells， and cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR）. After amplification of the p27 CDs and non-NLS fragments by PCR， full length p27WT （CDKN1B， NM_004064.5） and p27△NLS coding regions were obtained. PCR products were then subcloned into the eukaryotic expression vector pCMV-Blank. After identification with bacterial PCR， double restriction enzyme digestion and sequencing， they were defined officially as pCMV-p27WT and pCMV-p27△NLS， respectively. The recombinant plasmids were transfected into HEK293T cells by electroporation. After 48 h， the levels of p27 protein in the cytoplasm and nucleus were detected by Western blotting. 【Results】 The sequencing results showed that the sequences of p27WT and p27△NLS inserted into the plasmids were both correctly consistent with that of NM_004064.5. After transfection with pCMV-p27WT， total p27 protein expression was increased and distributed in both the cytoplasm and nucleus of HEK293T cells. After transfection with pCMV-p27△NLS， p27 protein was significantly increased and almost entirely localized in the cytoplasm of HEK293T cells. 【Conclusion】 The eukaryotic expression plasmids of human p27WT and p27△NLS coding sequences were successfully constructed and overexpressed in HEK293T cells. This research may lay a foundation for investigating the biological function of p27 in the cell cycle progression of tumor cells.

【Objective】 To construct the Rb luciferase reporter gene assay system and detect the activation ability of Rb gene for screening the targeted drugs. 【Methods】 The synthetic Rb gene sequence was annealed to form a double-stranded DNA structure and then inserted into the polyclonal site of pGL6-TA. The junction product was transformed into E.coli DH5α competent cells for expanded culture, and the constructed pGL6-Rb-Luc plasmid and pGL6-TA plasmid were transfected into HEK293 cells. The monoclonal cell line HEK293-Rb-Luc with stable expression was screened by G418, and the activation and inhibition of Rb in HEK293-Rb-Luc were tested by serum and CDK4/6 inhibitor Palbociclib. 【Results】 The sequence of Rb reaction elements in pGL6-Rb-Luc was completely correct. The recovery of serum culture significantly increased the luciferase activity in HEK293-Rb-Luc (P<0.001). Compared with 0 nmol/L, 25, 50, 75 and 100 nmol/L, CDK4/6 inhibitor Palbociclib made the inhibition rate of Rb activity rise to 6.90%, 40.23%, 50.57% and 52.07%, respectively (P<0.05). 【Conclusion】 The Rb luciferase reporter gene detection system HEK293-Rb-Luc was successfully constructed, which can effectively detect the activation level of Rb.

【Objective】 To clone the full-length of human kidney and brain protein (KIBRA) coding sequence in eukaryotic expression vector and provide a model for studying the biological function of KIBRA in breast cancer cells. 【Methods】 Total RNA of human breast cancer cell line MCF7 was extracted. After reverse transcription, the full length of KIBRA (NM_001161661.2) coding region was amplified by PCR, and cloned into eukaryotic expression vector pCMV-Blank. After identification, it was defined officially as pCMV-KIBRA. Then it was transfected into MCF7 cells, and the expression of KIBRA was detected by real-time PCR and Western blotting after 48 hours. The primary, secondary and tertiary structures and post-transcriptional modification sites of KIBRA were analyzed with bioinformatics software. 【Results】 Bacterial PCR, double enzyme digestion and DNA sequencing results showed that the correct sequence of KIBRA was inserted into the vector pCMV-KIBRA. The mRNA and protein expressions of KIBRA were significantly increased in MCF7 cells transfected with pCMV-KIBRA. Bioinformatics analysis showed that KIBRA was composed of 1119 amino acids. There were 52 phosphorylation sites, 1 acetylation site and 5 ubiquitination sites, and the protein structure was mainly α-helix and random coil. 【Conclusion】 The eukaryotic expression vector of full-length of human KIBRA coding sequence was successfully constructed and overexpressed in breast cancer cell line MCF7, which can lay a foundation for studying the biological function of KIBRA in breast cancer.

Objective:To understand the current practice of preoperative bowel preparation in elective colorectal surgery in China.Methods:A cross-sectional questionnaire survey was conducted through wechat. The content of the questionnaire survey included professional title of the participants, the hospital class, dietary preparation and protocol, oral laxatives and specific types, oral antibiotics, gastric intubation, and mechanical enema before elective colorectal surgery. A stratified analysis based on hospital class was conducted to understand their current practice of preoperative bowel preparation in elective colorectal surgery.Result:A total of 600 questionnaires were issued, and 516 (86.00%) questionnaires of participants from different hospitals, engaged in colorectal surgery or general surgeons were recovered, of which 366 were from tertiary hospitals (70.93%) and 150 from secondary hospitals (29.07%). For diet preparation, the proportions of right hemicolic, left hemicolic and rectal surgery were 81.59% (421/516), 84.88% (438/516) and 84.88% (438/516) respectively. The average time of preoperative dietary preparation was 2.03 days. The study showed that 85.85% (443/516) of surgeons chose oral laxatives for bowel preparation in all colorectal surgery, while only 4.26% (22/516) of surgeons did not choose oral laxatives. For mechanical enema, the proportions of right hemicolic, left hemicolic and rectal surgery were 19.19% (99/516), 30.04% (155/516) and 32.75% (169/516) respectively. Preoperative oral antibiotics was used by 34.69% (179/516) of the respondents. 94.38% (487/516) of participants were satisfied with bowel preparation, and 55.43% (286/516) of participants believed that preoperative bowel preparation was well tolerated. In terms of preoperative oral laxatives, there was no statistically significant difference between different levels of hospitals [secondary hospitals vs. tertiary hospitals: 90.00% (135/150) vs. 84.15% (308/366), χ 2=2.995, P=0.084]. Compared with the tertiary hospitals, the surgeons in the secondary hospitals accounted for higher proportions in diet preparation [87.33% (131/150) vs. 76.78% (281/366), χ 2=7.369, P=0.007], gastric intubation [54.00% (81/150) vs. 36.33% (133/366), χ 2=13.672, P<0.001], preoperative oral antibiotics [58.67% (88/150) vs. 24.86% (91/366), χ 2=12.259, P<0.001] and enema [28.67% (43/150) vs. 15.30% (56/366), χ 2=53.661, P<0.001]. Conclusion:Although the preoperative bowel preparation practice in elective colorectal surgery for most of surgeons in China is basically the same as the current international protocol, the proportions of mechanical enema and gastric intubation before surgery are still relatively high.

Objective:Surgical site infection (SSI) is the most common infectious complication after emergency abdominal surgery (EAS). To a large extent, most SSI can be prevented, but there are few relevant studies in China. This study mainly investigated the current situation of SSI occurrence after EAS in China, and further explored risk factors for SSI occurrence.Methods:Multi-center cross-sectional study was conducted. Clinical data of patients undergoing EAS in 33 hospitals across China between May 1, 2019 and June 7, 2019 were prospectively collected, including perioperative data and microbial culture results from infected incisions. The primary outcome was the incidence of SSI after EAS, while the secondary outcomes were postoperative hospital stay, ICU occupancy rate, length of ICU stay, hospitalization cost, and mortality within postoperative 30 days. Univariate and multivariate logistic regression models were used to analyze the risk factors of SSI after EAS.Results:A total of 660 EAS patients aged (47.9±18.3) years were enrolled in this study, including 56.5% of males (373/660). Forty-nine (7.4%) patients developed postoperative SSI. The main pathogen of SSI was Escherichia coli [culture positive rate was 32.7% (16/49)]. As compared to patients without SSI, those with SSI were more likely to be older (median 56 years vs. 46 years, U=19 973.5, P<0.001), male [71.4% (35/49) vs. 56.1% (343/611), χ 2=4.334, P=0.037] and diabetes [14.3% (7/49) vs. 5.1% (31/611), χ 2=5.498, P=0.015]; with-lower preoperative hemoglobin (median: 122.0 g/L vs. 143.5 g/L, U=11 471.5, P=0.006) and albumin (median: 35.5 g/L vs. 40.8 g/L, U=9452.0, P<0.001), with higher blood glucose (median: 6.9 mmol/L vs. 6.0 mmol/L, U=17 754.5, P<0.001); with intestinal obstruction [32.7% (16/49) vs. 9.2% (56/611), χ 2=25.749, P<0.001], with ASA score 3-4 [42.9% (21/49) vs. 13.9% (85/611), χ 2=25.563, P<0.001] and with high surgical risk [49.0% (24/49) vs. 7.0% (43/611), χ 2=105.301, P<0.001]. The main operative procedure resulting in SSI was laparotomy [81.6%(40/49) vs. 35.7%(218/611), χ 2=40.232, P<0.001]. Patients with SSI experienced significantly longer operation time (median: 150 minutes vs. 75 minutes, U=25 183.5, P<0.001). In terms of clinical outcome, higher ICU occupancy rate [51.0% (25/49) vs. 19.5% (119/611), χ 2=26.461, P<0.001], more hospitalization costs (median: 44 000 yuan vs. 15 000 yuan, U=24 660.0, P<0.001), longer postoperative hospital stay (median: 10 days vs. 5 days, U=23 100.0, P<0.001) and longer ICU occupancy time (median: 0 days vs. 0 days, U=19 541.5, P<0.001) were found in the SSI group. Multivariate logistic regression analysis showed that the elderly (OR=3.253, 95% CI: 1.178-8.985, P=0.023), colorectal surgery (OR=9.156, 95% CI: 3.655-22.937, P<0.001) and longer operation time (OR=15.912, 95% CI:6.858-36.916, P<0.001) were independent risk factors of SSI, while the laparoscopic surgery (OR=0.288, 95% CI: 0.119-0.694, P=0.006) was an independent protective factor for SSI. Conclusions:For patients undergoing EAS, attention should be paid to middle-aged and elderly patients and those of colorectal surgery. Laparoscopic surgery should be adopted when feasible and the operation time should be minimized, so as to reduce the incidence of SSI and to reduce the burden on patients and medical institutions.

Objective:To understand the current practice of preoperative bowel preparation in elective colorectal surgery in China.Methods:A cross-sectional questionnaire survey was conducted through wechat. The content of the questionnaire survey included professional title of the participants, the hospital class, dietary preparation and protocol, oral laxatives and specific types, oral antibiotics, gastric intubation, and mechanical enema before elective colorectal surgery. A stratified analysis based on hospital class was conducted to understand their current practice of preoperative bowel preparation in elective colorectal surgery.Result:A total of 600 questionnaires were issued, and 516 (86.00%) questionnaires of participants from different hospitals, engaged in colorectal surgery or general surgeons were recovered, of which 366 were from tertiary hospitals (70.93%) and 150 from secondary hospitals (29.07%). For diet preparation, the proportions of right hemicolic, left hemicolic and rectal surgery were 81.59% (421/516), 84.88% (438/516) and 84.88% (438/516) respectively. The average time of preoperative dietary preparation was 2.03 days. The study showed that 85.85% (443/516) of surgeons chose oral laxatives for bowel preparation in all colorectal surgery, while only 4.26% (22/516) of surgeons did not choose oral laxatives. For mechanical enema, the proportions of right hemicolic, left hemicolic and rectal surgery were 19.19% (99/516), 30.04% (155/516) and 32.75% (169/516) respectively. Preoperative oral antibiotics was used by 34.69% (179/516) of the respondents. 94.38% (487/516) of participants were satisfied with bowel preparation, and 55.43% (286/516) of participants believed that preoperative bowel preparation was well tolerated. In terms of preoperative oral laxatives, there was no statistically significant difference between different levels of hospitals [secondary hospitals vs. tertiary hospitals: 90.00% (135/150) vs. 84.15% (308/366), χ 2=2.995, P=0.084]. Compared with the tertiary hospitals, the surgeons in the secondary hospitals accounted for higher proportions in diet preparation [87.33% (131/150) vs. 76.78% (281/366), χ 2=7.369, P=0.007], gastric intubation [54.00% (81/150) vs. 36.33% (133/366), χ 2=13.672, P<0.001], preoperative oral antibiotics [58.67% (88/150) vs. 24.86% (91/366), χ 2=12.259, P<0.001] and enema [28.67% (43/150) vs. 15.30% (56/366), χ 2=53.661, P<0.001]. Conclusion:Although the preoperative bowel preparation practice in elective colorectal surgery for most of surgeons in China is basically the same as the current international protocol, the proportions of mechanical enema and gastric intubation before surgery are still relatively high.

Objective:Surgical site infection (SSI) is the most common infectious complication after emergency abdominal surgery (EAS). To a large extent, most SSI can be prevented, but there are few relevant studies in China. This study mainly investigated the current situation of SSI occurrence after EAS in China, and further explored risk factors for SSI occurrence.Methods:Multi-center cross-sectional study was conducted. Clinical data of patients undergoing EAS in 33 hospitals across China between May 1, 2019 and June 7, 2019 were prospectively collected, including perioperative data and microbial culture results from infected incisions. The primary outcome was the incidence of SSI after EAS, while the secondary outcomes were postoperative hospital stay, ICU occupancy rate, length of ICU stay, hospitalization cost, and mortality within postoperative 30 days. Univariate and multivariate logistic regression models were used to analyze the risk factors of SSI after EAS.Results:A total of 660 EAS patients aged (47.9±18.3) years were enrolled in this study, including 56.5% of males (373/660). Forty-nine (7.4%) patients developed postoperative SSI. The main pathogen of SSI was Escherichia coli [culture positive rate was 32.7% (16/49)]. As compared to patients without SSI, those with SSI were more likely to be older (median 56 years vs. 46 years, U=19 973.5, P<0.001), male [71.4% (35/49) vs. 56.1% (343/611), χ 2=4.334, P=0.037] and diabetes [14.3% (7/49) vs. 5.1% (31/611), χ 2=5.498, P=0.015]; with-lower preoperative hemoglobin (median: 122.0 g/L vs. 143.5 g/L, U=11 471.5, P=0.006) and albumin (median: 35.5 g/L vs. 40.8 g/L, U=9452.0, P<0.001), with higher blood glucose (median: 6.9 mmol/L vs. 6.0 mmol/L, U=17 754.5, P<0.001); with intestinal obstruction [32.7% (16/49) vs. 9.2% (56/611), χ 2=25.749, P<0.001], with ASA score 3-4 [42.9% (21/49) vs. 13.9% (85/611), χ 2=25.563, P<0.001] and with high surgical risk [49.0% (24/49) vs. 7.0% (43/611), χ 2=105.301, P<0.001]. The main operative procedure resulting in SSI was laparotomy [81.6%(40/49) vs. 35.7%(218/611), χ 2=40.232, P<0.001]. Patients with SSI experienced significantly longer operation time (median: 150 minutes vs. 75 minutes, U=25 183.5, P<0.001). In terms of clinical outcome, higher ICU occupancy rate [51.0% (25/49) vs. 19.5% (119/611), χ 2=26.461, P<0.001], more hospitalization costs (median: 44 000 yuan vs. 15 000 yuan, U=24 660.0, P<0.001), longer postoperative hospital stay (median: 10 days vs. 5 days, U=23 100.0, P<0.001) and longer ICU occupancy time (median: 0 days vs. 0 days, U=19 541.5, P<0.001) were found in the SSI group. Multivariate logistic regression analysis showed that the elderly (OR=3.253, 95% CI: 1.178-8.985, P=0.023), colorectal surgery (OR=9.156, 95% CI: 3.655-22.937, P<0.001) and longer operation time (OR=15.912, 95% CI:6.858-36.916, P<0.001) were independent risk factors of SSI, while the laparoscopic surgery (OR=0.288, 95% CI: 0.119-0.694, P=0.006) was an independent protective factor for SSI. Conclusions:For patients undergoing EAS, attention should be paid to middle-aged and elderly patients and those of colorectal surgery. Laparoscopic surgery should be adopted when feasible and the operation time should be minimized, so as to reduce the incidence of SSI and to reduce the burden on patients and medical institutions.

Objective To evaluate the effects of different depths of sedation on serum adiponectin (ADP) concentrations in elderly patients undergoing general anesthesia.Methods A total of 120 elderly patients of both sexes,aged 65-83 yr,weighing 45-75 kg,of American Society of Anesthesiologists physical status Ⅱ or Ⅲ,undergoing elective noncardiac surgery under general anesthesia,were divided into Ⅰ and Ⅱ groups (n =60 each) using a random number table.Propofol was given by closed-loop target-controlled infusion,and bispectral index value was maintained at 40-50 in group Ⅰ and at 50-60 in group Ⅱ.The cognitive function was assessed by the Montreal Cognitive Assessment at 1 day before operation ant 1 and 7 days after operation.Blood samples were collected from the internal jugular vein immediately before surgery,at 2 h after the beginning of surgery and at 1 and 7 days after surgery for determination of serum ADP and S-100β protein concentrations.Results Compared with group Ⅰ,Montreal Cognitive Assessment scores were significantly increased at 1 and 7 days after surgery,the serum concentrations of ADP were increased and S-100β protein concentrations in serum were decreased at 1 and 7 days after surgery,and the intraoperative requirement for ephedrine and atropine and incidence of postoperative cognitive dysfunction were decreased during surgery in group Ⅱ (P＜0.05).Conclusion Maintaining BIS value at 50-60 can reduce the development of postoperative cognitive dysfunction,which is related to the increased concentration of serum ADP in elderly patients undergoing general anesthesia.

Objective To compare health related quality of life (HRQOL) between modified and traditional cutaneous ureterostomy, and explore the reasons for these differences, in order to provide the basis of HRQOL for the choice of cutaneous ureterostomy. Methods A total of 53 patients underwent cutaneous ureterostomy were selected, and the patients were divided into traditional cutaneous ureterostomy group (traditional group, 21 cases) and modified cutaneous ureterostomy group (modified group, 32 cases) according to the surgery method. The patients were evaluated by functional assessment of cancer therapy-bladder (FACT-BL), and the HRQOL was compared between 2 groups. Results There were no statistical differences in HRQOL score at 1, 3, 6 and 9 months after surgery between 2 groups (P>0.05). The HRQOL score at 12 months after surgery was significantly higher in modified group than that in traditional group:(141.5 ± 10.4) scores vs. (123.1 ± 5.2) scores, and there was statistical difference (P<0.01). There were no statistical differences in the scores of physiology status, society/family status, emotional state and functional assessment of cancer therapy-general (FACT-G) at 12 months after surgery between 2 group (P>0.05). But the scores of functional status and bladder cancer special scale (BSS), total score of FACT-BL in modified group were significantly higher than those in traditional group:(26.0 ± 2.5) scores vs. (23.8 ± 3.5) scores, (46.7 ± 6.2) scores vs. (34.8 ± 5.5) scores, (143.9 ± 15.7) scores vs. (117.5 ± 8.1) scores, and there were statistical differences (P<0.01). Conclusions The HRQOL at 12 months after surgery in modified cutaneous ureterostomy is better than that in traditional cutaneous ureterostomy. Therefore, if the patient's physical condition permits, priority should be given to modified cutaneous ureterostomy to reduce the complications and improve the quality of life.

Objective To compare the clinical therapeutic effect of percutaneous nephrolithotomy (PCNL) and flexible ureteroscope lithotripsy (FUL) for unilateral lower-calyceal calculi with the diameter of 10-20 mm. Methods The clinical data of 65 patients with unilateral lower-calyceal calculi with the diameter of 10-20 mm were retrospectively analyzed. Thirty cases were treated with PCNL (PCNL group), and 35 cases were treated with FUL (FUL group). The operative time, success rate of lithotomy, haemoglobin decrease after operation, postoperative hospital stay, hospitalization expenses and complication were compared between 2 groups. Results Treatment was completed successfully in the patients of 2 groups, without ureteral perforation, avulsion and other serious complications intraoperatively and postoperatively. There were no statistical differences in success rate of lithotripsy, incidence of high fever after operation and postoperative analgesia rate between 2 groups (P>0.05). The operative time and hospitalization expenses in FUL group were significantly higher than those in PCNL group:(95.27 ± 22.69) min vs. (62.25 ± 20.73) min and (17 242 ± 2 679) yuan vs. (14 205 ± 1 654) yuan, and the haemoglobin decrease after operation and postoperative hospital stay time were significantly lower than those in PCNL group:(0.67 ± 0.33) g/L vs. (7.98 ± 4.33) g/L and (3.75 ± 0.78) d vs. (6.54 ± 1.68) d, and there were statistical differences (P<0.05). Conclusions For the treatment of lower-calyceal calculi with the diameter of 10-20 mm, the success rates of lithotripsy of PCNL and FUL are similar. FUL has less trauma, with shorter postoperative hospital stay time, but the cost is relatively high.