【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 investigate the clinical efficacy of proximal gastrectomy and total gastrectomy in the treatment of Siewert type Ⅱ and Ⅲ adenocarcinoma of esophagogastric junction (AEG).Methods:The retrospective cohort study was conducted. The clinicopathological data of 170 patients with Siewert type Ⅱ and Ⅲ AEG who were admitted to Guangdong Provincial People′s Hospital from January 2010 to December 2018 were collected. There were 125 males and 45 females, aged from 30 to 85 years, with a median age of 64 years. Of the 170 patients, 82 cases undergoing proximal gastrectomy were allocated into the proximal gastrectomy group and 88 cases undergoing total gastrectomy were allocated into the total gastrectomy group. Observation indica-tors: (1) surgical and postoperative situations; (2) follow-up and survival; (3) analysis of prognostic factors. Follow-up was conducted using telephone interview and outpatient examination to detect survival of patients up to December 2021. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was analyzed using the t test. Measure-ment data with skewed distribution were represented as M( Q1, Q3) or M(range), and comparison between groups was analyzed using the Mann-Whitney U test. Count data were described as absolute numbers or percentages, and comparison between groups was analyzed using the chi-square test or Fisher exact probability. Comparison of ordinal data was analyzed using the rank sum test. Kaplan-Meier method was used to draw survival curves, and Log-Rank test was used for survival analysis. COX proportional hazard model was used for univariate and multivariate analyses. Variables with P<0.1 in univariate analysis were included for multivariate analysis. Results:(1) Surgical and postoperative situations. Cases with surgical approach as transthoracic or thoraco-abdominal approach, transabdominal approach, the operation time, cases with volume of intra-operative blood loss ≤100 mL or >100 mL, cases with length of proximal margin ≤1.5 cm or >1.5 cm, cases with radical surgery outcome as R 0, R 1, R 2, the number of lymph nodes harvest, cases with anastomotic leakage, cases with anastomotic stricture, cases with incision infection, cases with pleural infection or effusion, cases with abdominal infection or ascites were 61, 21, (211±18)minutes, 46, 36, 44, 38, 73, 6, 3, 15(9,22), 5, 2, 2, 4, 2 in the proximal gastrectomy group, respec-tively. The above indicators were 12, 76, (263±15)minutes, 27, 61, 45, 43, 82, 4, 2, 23(18,32), 4, 1, 3, 1, 4 in the total gastrectomy group, respectively. There were significant differences in the surgical approach, operation time, volume of intraoperative blood loss and the number of lymph nodes harvest between the two groups ( χ2=63.94, t=-25.50, χ2=11.19, Z=-5.62, P<0.05). There was no significant difference in the length of proximal margin or radical surgery outcome between the two groups ( χ2=0.11, Z=-0.95, P>0.05) and there was no significant difference in the anastomotic leakage, anastomotic stricture, incision infection, pleural infection or effusion, abdominal infection or ascites between the two groups ( P>0.05). (2) Follow-up and survival. All the 170 patients were followed up for 89(64,106)months. Of the 170 patients, the 5-year overall survival rates were 43.8% and 35.5% of the Siewert type Ⅱ and Ⅲ AEG patients, respectively, showing no significant difference between them ( χ2=0.87, P>0.05). Of the patients with Siewert type Ⅱ AEG, the 5-year overall survival rates were 41.7% and 54.3% in the patients with proximal gastrectomy and the total gastrectomy, respectively, showing no significant difference between them ( χ2=1.05, P>0.05). Of the patients with Siewert type Ⅲ AEG, the 5-year overall survival rates were 31.3% and 37.5% in the patients with proximal gastrectomy and the total gastrectomy, respectively, showing no significant difference between them ( χ2=0.33, P>0.05). The 5-year overall survival rates were 39.0% and 44.2% in the proximal gastrectomy group and the total gastrectomy group, respectively, showing no significant difference between the two groups ( χ2=0.63, P>0.05). Of the patients in TNM stage Ⅰ, stage Ⅱ, stage Ⅲ, the 5-year overall survival rates were 65.3%, 36.3%, 27.1% in the proximal gastrectomy group, versus 83.3%, 48.0%, 39.7% in the total gastrectomy group, showing no signifi-cant difference between the two groups ( χ2=0.02, 1.50, 1.21, P>0.05). (3) Analysis of prognostic factors. Results of univariate analysis showed that pathological N staging, degree of tumor differen-tiation and radical surgery outcome were related factors influencing prognosis of AEG patients ( hazard ratio=1.71, 1.70, 2.85, 95% confidence interval as 1.16-2.60, 1.15-2.50, 1.58-5.14, P<0.05). Results of multivariate analysis showed that pathological N staging and radical surgery outcome were independent factors influencing prognosis of AEG patients ( hazard ratio=1.55, 2.18, 95% confidence interval as 1.05-2.31, 1.18-4.02, P<0.05). Conclusions:There is no significant difference in the prognosis of Siewert type Ⅱ and Ⅲ AEG patients undergoing proximal gastrectomy or total gastrectomy. Proximal gastrectomy can be used for the treatment of advanced Siewert type Ⅱ and Ⅲ AEG.

【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: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: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 investigate the correlation between the expression of (epidermal growth factor receptor-2,HER-2) and clinicopathological features and survival of colorectal cancer.Methods From Jan 2005 to Dec 2015,all colorectal cancer cases were enrolled that the expression levels of HER-2 were detected in Guangdong Provincial People's Hospital.Clinicopathological features of the tumors and survival of the patients were analyzed.Results A total of 1 463 cases were collected in 10 years,including 711 cases (48.6％) of HER-2 (-),470 cases (32.1％) of HER-2 (+),249 cases (17％) of HER-2 (+ +),and 33 cases (2.3％) of HER-2 (+ + +).Correlation analysis shows that the expression levels of HER-2 were significantly correlated with tumor differentiation and the depth of tumor invasion(T stage),but not correlated with gender,age,tumor location,N stage,M stage,TNM stage and overall survival,and disease-free survival.Conclusion The expression of HER-2 may be related to tumor differentiation and growth infiltration,but it cannot be used as a predictor of prognosis in patients with colorectal cancer.

Objective: To investigate the value and feasibility of C reactive protein (CRP) in predicting postoperative anastomotic leakage in rectal cancer patients with enhanced recovery after surgery (ERAS) for safer implementation of this ERAS. Methods: A cohort study on serum CRP of 455 rectal cancer patients undergoing laparoscopic radical resection according to the ERAS procedure at Gastrointestinal Unit of General Surgery Department, Guangdong General Hospital from August 2014 to June 2017 was retrospectively carried out. The serum CRP level was measured before operation and at postoperative days 1-7, and the serum CRP level of the groups with and without anastomotic leakage was compared to analyze its prediction for anastomotic leakage. Diagnostic standard of anastomotic leakage was based on the definition of postoperative anastomotic leakage in rectal cancer from International Study Group of Rectal Cancer (ISREC) : (1) Postoperative localized or diffuse peritonitis occurred, or fecal liquid was found from the abdominal drainage tube; (2) When anastomotic leakage was uncertain, peritoneal or pelvic computed tomography scan should be used to confirm. Results: All the 455 patients underwent surgery successfully, and 41 patients (9.0%) had anastomotic leakage postoperatively. Patients with anastomotic leakage were diagnosed (4.0 ± 2.0) days postoperatively, of whom 8 cases (19.5%) were diagnosed more than 5 days postoperatively. Serum CRP levels in patients with anastomotic leakage continued to increase within 1-4 days postoperatively[ (50.04 ± 27.98) mg/L to (122.75 ± 52.98) mg/L]and decreased 5 days postoperatively[ (92.02 ± 58.26) mg/L], both were higher than those of non-anastomotic leakage group, and the difference was statistically significant (all P < 0.05, except postoperative day 2) . The serum CRP level of non-anastomotic leakage group reached the peak on the second postoperative day[ (83.10 ± 37.45) mg/L] and decreased 3 days postoperatively[ (48.01 ± 27.59) mg/L]. The ROC curve was drawn with the anastomotic leakage as the state variable, and the CRP level as the detection variable. The area under the curve (AUC) at postoperative 1, 2, 4, 5, 6 and 7 days was 0.74, 0.58, 0.83, 0.82, 0.65, and 0.70, respectively. The maximum was at postoperative day 3[0.93 (95%CI: 0.86-0.99) ]. The Youden index was 0.72, and the threshold of CRP was 80.09 mg/L, as the cut-off point to predict anastomotic leakage, with sensitivity, specificity, and positive predictive value of 79.3%, 92.3%, and 74.2%, respectively. Conclusions Monitoring the postoperative serum CRP level can help predict the occurrence of anastomotic leakage after laparoscopic surgery for rectal cancer. When the serum CRP level is > 80.09 mg/L on the third postoperative day, the CRP level has the largest value in predicting postoperative anastomotic leakage, and the safety of ERAS has a certain clinical significance as well.

A rapid and accurate determination method of lipids in microalgae plays a significant role in an efficient breeding process for high-lipid production of microalgae. Using low field nuclear magnetic resonance (LF-NMR), we developed a direct quantitative method for cellular lipids in Chlorella protothecoides cells. The LF-NMR signal had a linear relationship with the lipid content in the microalgae cells for both dry cell samples and algal broth samples (R2 > 0.99). These results indicated that we could use this method for accurate determination of microalgal lipids. Although LF-NMR is a rapid and easy lipid determination method in comparison to conventional methods, low efficiency would limit its application in high throughput screening. Therefore, we developed a novel combined high throughput screening method for high-lipid content mutants of C. protothecoides. Namely, we initially applied Nile red staining method for semi-quantification of lipid in the pre-screening process, and following with LF-NMR method for accurate lipid determination in re-screening process. Finally, we adopted this novel screening method in the breeding process of high-lipid content heterotrophic cells of C. protothecoides. From 3 098 mutated strains 108 high-lipid content strains were selected through pre-screening process, and then 9 mutants with high-lipid production were obtained in the re-screening process. In a consequence, with heterotrophical cultivation of 168 h, the lipid concentration could reach 5 g/L, and the highest lipid content exceeded 20% (W/W), which was almost two-fold to that of the wild strain. All these results demonstrated that the novel breeding process was reliable and feasible for improving the screening efficiency.
Chlorophyta ; chemistry ; Heterotrophic Processes ; High-Throughput Screening Assays ; Lipids ; analysis ; Magnetic Resonance Spectroscopy ; Microalgae ; chemistry ; Staining and Labeling

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.