[Quality control of digestive tract reconstruction after proximal gastrectomy].

With the increasing incidence of esophagogastric junction carcinoma, the application rate of proximal gastrectomy has been rising annually. There is a wide variety of methods for digestive tract reconstruction after proximal gastrectomy, and some of these reconstruction methods have been introduced relatively recently, with limited clinical experience, which led to a lack of standardization. Such a situation will inevitably result in inconsistent clinical outcomes of proximal gastrectomy with digestive tract reconstruction. To promote the standardization of digestive tract reconstruction after proximal gastrectomy, improve the clinical efficacy of proximal gastrectomy, and reduce the occurrence of postoperative complications, this article elaborates on the indications, surgical steps and technical points of the four methods after proximal gastrectomy recommended by the "Chinese consensus on digestive tract reconstruction after proximal gastrectomy (2020 edition)", such as double tract, side overlap, double flaps and gastric tube reconstruction, providing guidance for the application of digestive tract reconstruction after proximal gastrectomy.

[Research progress in anti-reflux reconstructions and mechanism after proximal gastrectomy].

The electrophysiological activity of the gastrointestinal tract and the mechanical anti-reflux structure of the gastroesophageal junction are the basis of the anti-reflux function of the stomach. Proximal gastrectomy destroys the mechanical structure and normal electrophysiological channels of the anti-reflux. Therefore, the residual gastric function is disordered. Moreover, gastroesophageal reflux is one of the most serious complications. The emergence of various types of anti-reflux surgery through the mechanism of reconstructing mechanical anti-reflux barrier and establishing buffer zone, and the preservation of, the pacing area and vagus nerve of the stomach, the continuity of the jejunal bowel, the original gastroenteric electrophysiological activity of the gastrointestinal tract, and the physiological function of the pyloric sphincter, are all important measures for gastric conservative operations. There are many types of reconstructive approaches after proximal gastrectomy. The design based on the anti-reflux mechanism and the functional reconstruction of mechanical barrier, and the protection of gastrointestinal electrophysiological activities are important considerations for the selected of reconstructive approaches after proximal gastrectomy. In clinical practice, we should consider the principle of individualization and the safety of radical resection of tumor to select a rational reconstructive approaches after proximal gastrectomy.

[Diagnosis, prevention and treatment of post-operative rare complications after radical gastrectomy for gastric cancer].

Radical gastrectomy for gastric cancer results in various post-operative complications, and the influencing factors are complicated. The diagnosis, treatment and prevention of common complications have been reported in many literatures. However, there are few reports on the prevention and treatment of rare complications. Rare complications after radical gastrectomy are often overlooked due to their low incidence. In addition, there are few guidelines and expert consensus regarding to the rare complications. Therefore, clinicians may lack experience in the diagnosis, treatment and prevention of rare complications after radical gastrectomy. Based on the literature review and the author's experience, this article systematically reviews seven rare complications after radical gastrectomy (duodenal stump fistula, pancreatic fistula, chyle leakage, esophagomediastinal fistula, internal hernia, gastroparesis, and intussusception). This article aims to provide a comprehensive reference for the diagnosis, treatment and prevention of rare complications after radical gastrectomy for gastric cancer patients.

[Real-world data analysis of 3012 patients undergoing laparoscopic radical gastrectomy in a single center over the past 12 years].

Objective: To Summarize the safety, clinical outcome and technical evolution of laparoscopic gastric cancer surgery. Methods: A retrospective cohort study was carried out. Clinical data of 3012 patients who underwent laparoscopic radical gastrectomy for gastric cancer from January 2010 to March 2022 at Department of General Surgery, the First Affiliated Hospital of Nanjing Medical University were retrospectively collected and analyzed. Case inclusion criteria were gastric malignancies confirmed by pathology, without distant metastasis by examination before operation and exploration during operation, patients undergoing laparoscopic radical gastrectomy, intact function of important organs and with complete data. Exclusion criteria were patients who underwent emergency gastric cancer resection due to gastric bleeding, perforation or obstruction, etc., tumor found to invade adjacent organs such as pancreas or transverse colon during the operation, conversion to open surgery during the operation, those who had other malignant tumors (except thyroid cancer) within 5 years, and those had severe cardiopulmonary, liver, or kidney insufficiency before surgery. Outcomes included: (1) baseline information of patients; (2) trend of the quantity of laparoscopic radical gastrectomy year by year; (3) evolution of the mode of digestive tract reconstruction; (4) periopertive outcome short-term complication was defined as complication occurring within 30 days after operation and classified accordiny to the clavien-Dindo criteria; and (5) 5-year overall survival. SPSS software was used for statistical analysis. Continuous variables that obeyed the normal distribution were expressed in the form of Mean±SD. Days of hospital stay that did not follow a normal distribution were expressed as median (Q1,Q3), and the Mann-Whiney U test was used for comparison. Discrete variables were expressed as cases (%), and chi-square test or rank sum test was used for comparison between groups. Linear regression analysis was used to analyze the relationship between the amount of surgery and the year of surgery. Kaplan-Meier method and log-rank test were used for survival analysis. Two-tailed P<0.05 was considered as statistically significant. Results: Among the 3012 cases, 2114 were male and 898 were female. The patients' average age at surgery was (61.1±10.7) years old. According to the number of cumulative cases, the patients were divided into three groups: early, intermediate and late, with 1004 patients in each group. The early group consisted of patients undergoing operation from January 2010 to October 2018, the intermediate group consisted of patients undergoing operation from October 2018 to January 2021, and the late group consisted of patients undergoing operation from January 2021 to March 2022. (1) General information: There were 691 (68.8%), 699 (69.6%) and 724 (72.1%) male patients in early, intermediate and late groups respectively; the average age increased from 56.6 years in 2010 to 62.8 years in March 2022. As for the tumor stage T1, T2, T3, T4, there were 49.0%, 14.4%, 23.9% and 12.6% in the early group; 47.5%, 12.9%, 26.9% and 12.6% in the intermediate group; 39.7%, 14.6%, 30.0%, and 15.6% in the late group, respectively. Patients with N0, N1, N2, N3a, N3b stage were 56.8%, 13.7%, 13.4%, 11.0%, and 5.0% in the early group; 55.7%, 12.9%, 12.8%, 11.6%, and 6.9% in the intermediate group; 51.0%, 16.1%, 12.8%, 12.5%, and 7.5% in the late group, respectively. (2) Year-by-year change in the number of gastric cancer operations: From 19 cases per year in 2010 to 786 per year in 2021, the annual number of gastric cancer operations was proportional to the year of operation (y=47.505x, R2=0.67). The proportion of patients with stage I disease showed a fluctuating downward trend over time, while the proportion of patients with stage III disease increased slightly, accounting for 34% until March 2022. (3) Evolution of digestive tract reconstruction methods: Except in 2010, the digestive tract reconstruction method of distal gastrectomy focused on Billroth-II+Braun anastomosis among patients undergoing laparoscopic gastric cancer surgery in other years, whose proportion had gradually increased from less than 20% in 2016 to about 70% after 2021; the gastrointestinal reconstruction methods after total gastrectomy had gradually increased in π anastomosis and overlap anastomosis since 2016, of which π anastomosis reached about 65% in 2019, and overlap anastomosis reached almost 30% in 2020; the anastomosis methods after proximal gastrectomy had been mainly double-channel anastomosis (54%) and esophagogastric anastomosis (30%) since 2016, and double-channel anastomosis accounted for up to 70% in 2019. (4) Operation time: The operation time of early, intermediate and late group was (193.3±49.8) min, (186.9±44.3) min and (206.7±51.4) min respectively. Intermediate group was significantly shorter than early group (t=3.005, P=0.003), while late group was significantly longer than early group (t=5.875, P<0.001) and intermediate group (t=9.180, P<0.001). (5) Postoperative hospital stay: The median length of hospital stay for gastric cancer patients in early, intermediate and late groups was 9 (8, 11) d, 8 (7, 10) d, and 8 (7.5, 10) d respectively. The postoperative hospital stay of intermediate group and late group was significantly shorter than that of early group (Z=-12.467, Z=-5.981, both P<0.001), but there was no significant difference between intermediate group and late group (Z=0.415,P=0.678). (6) Postoperative complication: The morbidity of short-term complication in early, intermediate and late group was 20.4% (205/1004), 16.2% (163/1004), and 16.2% (162/1004) respectively, and above morbidity of intermediate group and late group was significantly lower than that of early group (χ2=5.869, P=0.015; χ2=6.165, P=0.013), while there was no significant difference between intermediate group and late group (χ2=0.004,P=0.952). The morbidity of short-term complication of grade IIIor higher was 8.0% (80/1004), 7.6% (76/1004), and 4.9% (49/1004) in early, intermediate and late group respectively, and above morbidity of late group was significantly lower than that of early and intermediate group (χ2=7.965, P=0.005; χ2=6.219,P=0.013), while there was no significant difference between intermediate group and early group (χ2=0.111,P=0.739). (7) Survival analysis: The follow-up deadline for survival data was December 31, 2021, and the median follow-up time was 29.5 months. The overall 5-year survival rate of all the patients was 74.7%. The 5-year survival rates of stage I, II and III patients were 92.0%, 77.2%, and 40.3% respectively and 5-year survival rates of patients with stage IA, IB, IIA, IIB, IIIA, IIIB and IIIC were 93.2%, 87.8%, 81.1%, 72.7%, 46.2%, 37.1%, and 34.0% respectively. Conclusions: The number of laparoscopic gastric cancer operation in our center is increasing year by year. With the maturity of laparoscopic technology, the morbidity of complication in laparoscopic gastric cancer surgery is decreasing.

[Anti-reflux gastrointestinal reconstruction techniques after laparoscopic proximal gastrectomy].

Laparoscopic techniques are more and more poplular in proximal gastrectomy. The traditional esophagogastric anastomosis may lead to severe reflux esophagitis after surgery, affecting patient's quality of life. In recent years, multiple methods of digestive tract reconstruction after laparoscopic proximal gastrectomy capable of resisting reflux have been applied to the clinic. Combining the results of the latest clinical studies and our clinical experience, we elaborate the views on digestive tract reconstruction after laparoscopic proximal gastrectomy. Esophagogastric anastomosis (posterior esophagogastric anastomosis, anterior esophagogastric anastomosis, gastric tube reconstruction, lateral esophagogastric anastomosis, Kamikawa anastomosis and modified Kamikawa anastomosis, etc.) and esophagojejunal anastomosis (interposition jejunum, interposition jejunum with pouch, and double-channel anastomosis, etc.) are mainly discussed. Of course, the anti-reflux mechanisms of different surgical procedures are not the same, the anti-reflux effects are variable, and the surgical difficulties under laparoscopy are also different. Therefore, how to choose a rational reconstruction method after proximal gastrectomy needs to be comprehensively considered based on patient's own situation and technical level of the surgeons.

[Role of crenel lateral lumbar interbody fusion in the precise selection of fusion level in posterior correction for adult degenerative scoliosis].

Objective: To investigate the role of minimally invasive crenel lateral lumbar interbody fusion (CLIF) in the decision of fusion level in posterior correction for severe adult degenerative scoliosis. Methods: This is a prospective study.Patients with level Ⅴ and Ⅵ of Lenke-Silva classification who were treated at Department of Orthopedics,the Second Affiliated Hospital, School of Medicine, Zhejiang University from June 2016 to March 2019 were included.First,the enrolled patients completed the preoperative clinical and imaging examination,the Lenke-Silva classification was evaluated,the surgical segments in first-stage CLIF was determined and the fusion segments required for single-stage posterior correction was predicted.After the first-stage CLIF,patients received reassessment of Lenke-Silva classification and global coronal and sagittal balance.Patients were divided into two groups:the effective group (level of Lenke-Silva classification decreased) and the ineffective group (level of Lenke-Silva classification unchanged).Second-stage posterior surgery was performed based on the results of reassessments.The fusion segment,Cobb angle,parameters of global coronal and sagittal plane,visual analogue pain score (VAS) and Oswestry disability index (ODI) were compared between the two groups preoperatively,after first-stage CLIF,second-stage posterior fixation and at the final follow-up.The potential factors associated with the decrease of the level of Lenke-Silva classification were recorded and compared between the two groups.Independent sample t test,repeated measure analysis of variance,rank sum test,χ2 test or Fisher exact method were used to compare the difference among groups. Results: Fifty-four patients were enrolled,including 8 males and 46 females,aged (68.8±5.8) years (range:56 to 77 years).Preoperatively,26 patients were classified as level Ⅴ by Lenke-Silva classification,28 cases were grade Ⅵ.CLIF was performed in 194 segments,with 114 segments(58.8%) receiving anterior column realignment (ACR) and 15 segments(7.7%) using hyperlordotic cages.After first-stage CLIF,22 patients with level Ⅴ and 10 patients with Ⅵ of Lenke-Silva classification decreased and were classified into effective group.The level of the remaining 4 patients with level Ⅴ and 18 patients with grade Ⅵ unchanged and were classified into ineffective group.Preoperatively,the apical vertebrae was below L1 in all 32 patients of effective group and 18 (81.8%,18/22) patients of ineffective group.The difference was statistically significant (P=0.023).There were 7 (31.8%,7/22) patients had continuous osteophyte in front of the intervertebral space in ineffective group,while none patient had continuous osteophyte in front of the intervertebral space in effective group,and the difference was statistically significant (P=0.001).In first-stage CLIF,more intraoperative ACR(71.2% vs.39.5%,χ²=20.660,P<0.01)and hyperlordotic cage (12.7% vs.0,P=0.001) were used in the effective group,while there was less severe cage subsidence after the operation (5.9% vs.15.8%,χ²=4.793,P=0.029) in effective group.After first-stage CLIF,there was no difference in the Cobb angle between the two groups.While,lumbar lordosis (LL) in effective group (34.0±8.3)° was greater than that of the ineffective group (25.5±9.7)° (t=3.478,P=0.001),and the difference between the pelvic incidence (PI) and LL in effective group (15.7±4.6)°was significantly smaller than ineffective group(20.0±10.8)° (t=-2.129,P=0.038).The posterior fusion levels was less,the rate of fusion to thoracic spine region and the actual fusion segment was less than that of single-stage posterior correction in effective group (all P<0.01).All patients were follow-up for 24 to 45 months.There was no significant difference in radiological and clinical results between the two groups after first-,second-stage surgery and at the final follow-up (all P>0.05). Conclusions: First-stage CLIF decreased the Lenke-Silva classification of some patients with severe degenerative scoliosis.Combined with the reassessment of Lenke-Silva classification and global coronal and sagittal plane,it helps to accurately determine the fusion segment.Decrease of Lenke-Silva classification is associated with the preoperative level of apical vertebrae,continuous osteophytes in front of the intervertebral space,intraoperative use of ACR and hyperlordotic cage and the degree of cage subsidence postoperatively.

Adjuvant chemotherapy for patients with gastric neuroendocrine carcinomas or mixed adenoneuroendocrine carcinomas.

BACKGROUND: The aim of this study was to evaluate whether adjuvant chemotherapy is associated with improved survival in patients with resectable gastric neuroendocrine carcinomas (G-NECs) or mixed adenoneuroendocrine carcinomas (G-MANECs). METHODS: The study included patients with G-NECs or G-MANECs who underwent surgery in one of 21 centres in China between 2004 and 2016. Propensity score matching analysis was used to reduce selection bias, and overall survival (OS) in different treatment groups was estimated by the Kaplan-Meier method. RESULTS: In total, 804 patients with resectable G-NECs or G-MANECs were included, of whom 490 (60·9 per cent) received adjuvant chemotherapy. After propensity score matching, OS in the chemotherapy group was similar to that in the no-chemotherapy group. Among patients with G-NECs, survival in the fluorouracil (5-FU)-based chemotherapy group and the non-5-FU-based chemotherapy group was similar to that in the no-chemotherapy group. Similarly, etoposide plus cisplatin or irinotecan plus cisplatin was not associated with better OS in patients with G-NECs. Among patients with G-MANECs, OS in the non-5-FU-based chemotherapy group was worse than that in the no-chemotherapy group. Patients with G-MANECs did not have better OS when platinum-based chemotherapy was used. CONCLUSION: There was no survival benefit in patients who received adjuvant chemotherapy for G-NECs or G-MANECs.

ANTECEDENTES: El objetivo de este estudio fue evaluar si la quimioterapia adyuvante mejoraba la supervivencia en pacientes con carcinomas gástricos resecables neuroendocrinos (gastric neuroendocrine carcinomas, G-NECs) y carcinomas adenoneuroendocrinos mixtos (mixed adenoneuroendocrine carcinomas, G-MANECs). MÉTODOS: Se incluyeron pacientes con G-NECs y G-MANECs tratados quirúrgicamente en 21 centros en China entre 2004 y 2016. Se utilizó un análisis de emparejamiento por puntaje de propensión para reducir el sesgo de selección y el método de Kaplan-Meier para estimar la supervivencia global (overall survival, OS) de los pacientes en los diferentes grupos de tratamiento. RESULTADOS: En total, se incluyeron en el estudio 804 pacientes con G-NECs y G-MANECs resecables y 490 pacientes (60,9%) recibieron quimioterapia adyuvante. Después del emparejamiento por puntaje de propensión, la OS del grupo con quimioterapia fue similar a la del grupo sin quimioterapia. En los pacientes con G-NECs, la supervivencia en los grupos con quimioterapia basada en 5-FU (fluorouracilo) y de quimioterapia sin 5-FU fue similar a la del grupo sin quimioterapia. Asimismo, la combinación de etopósido y cisplatino o de irinotecán y cisplatino no se asoció con una mejor OS en pacientes con G-NECs. En pacientes con G-MANECs, la OS del grupo con quimioterapia sin 5-FU fue peor que la del grupo sin quimioterapia. Los pacientes con G-MANECs no presentaron una mejor OS cuando se administró quimioterapia basada en platinos. CONCLUSIÓN: La administración de quimioterapia adyuvante en pacientes con G-NECs y G-MANECs no mejoró la supervivencia.

[Minimally invasive surgery for the severe degenerative lumbar scoliosis: two stage protocol].

Objective: To investigate the surgical outcome of minimally invasive surgery(MIS) for severe degenerative lumbar scoliosis(DLS) and put forward a two-stage MIS surgical strategy. Methods: Prospective study of MISDEF Ⅲ DLS patients from June 2016 to August 2017 in the Second Affiliated Hospital of Zhejiang University was carried out, excluding the patients whose apex vertebrae of scoliosis was above L(1/2) level or whose facet joint got spontaneous fusion. Fifty-three patients were included in this study for staging evaluation and MIS surgical treatment. Information was recorded, including gender, age, body mass index, follow-up period, pelvic incidence (PI), blood loss, operation time, visual analogue pain score (VAS), Oswestry disability index (ODI), complications in the perioperative period and follow-up period, and also the radiographic parameters such as scoliosis Cobb angle, the mismatch between pelvic incidence and lumbar lordosis (PI-LL), sagittalvertical axis (SVA), coronal balance (CB) before and after each stage of surgery or latest follow-up. The paired-samples t test was used to analyze the effectiveness of staging surgery. Results: Fifty-three patients (18 males and 35 females) were included in this study. All patients had completed clinical and the follow-up records, with an average follow-up period of 11.52 months (6-20 months). A total of 168 segments fusions were performed with CLIF, 113 segments were performed with anterior column realignment (ACR), the average correct angle was 15.6°±6.3°(7°-28°) in sagittal plane each level. After the stage Ⅰ surgery, lumbar scoliosis cobb had been corrected for 55.35%, after the stage Ⅱ surgery, rate of correction was 75.6%. PI-LL had been matched (-32.8°±14.9° to -2.5°±9.4°), SVA was changed from 5.7 cm to 0.6 cm, the stage Ⅰ rate of correction was 80.3 and stage Ⅱ was 88.8%, pelvic tilt (PT), lumbar lordosis (LL) and CB had been restored; 13 (24.5%) patients were performed paraspinal approach transforaminal decompression. The posterior minimally invasive fixation indexes: 11(20.8%) patients were performed paraspinal approach transforaminal multi-segment transforaminal osteotomy (TFO) and internal fixation; 36(67.92%) cases were performed paraspinal approach transforaminal multi-segment fixation; 6(11.33%) patients were treated with percutaneous pedicle screw fixation. The average fixed segments was 7.4±1.4 in each patient. The blood loss of stage Ⅰ and stage Ⅱ operation was (157±71) ml, (343±224)ml, respectively. The operation time was (214±60) min, (190±54)min respectively in the two stage operations. The low back pain and leg pain VAS score and ODI improved after the stage Ⅰ and Ⅱ surgery (t=17.948, 10.099, 14.619, all P<0.001). Conclusions: MIS for the severe degenerative lumbar scoliosis can achieve good clinical outcome and deformity correction. The two-stage protocol has the advantages of less complications and is well-tolerated.

Correlations of MGMT genetic polymorphisms with temozolomide resistance and prognosis of patients with malignant gliomas: a population-based study in China.

This study aims to investigate the associations of O6-methylguanine-DNA methyltransferase (MGMT) genetic polymorphisms (Leu84Phe and Ile143Val) with temozolomide (TMZ) resistance and prognosis of patients with malignant gliomas. A total of 212 patients diagnosed with malignant gliomas were enrolled in this study as the case group. All of these patients took oral TMZ and were assigned into the TMZ-sensitive (complete response+partial response) and the TMZ-resistant (stable disease+progressive disease) groups based on the clinical response after chemotherapy. The polymerase chain reaction-restriction fragment length polymorphism was used to identify the gene polymorphism of Leu84Phe and Ile143Val. The survival time and survival outcomes of all the patients were obtained by follow-up. There were significant differences in the genotype and allele of Leu84Phe between the TMZ-sensitive and the TMZ-resistant groups. The CT, TT and CT+TT genotypes and the T allele of MGMT gene Leu84Phe may be associated with increasing TMZ resistance in patients with malignant gliomas. Logistic regression analysis showed that Leu84Phe of MGMT gene and pathological grade were independent risk factors for the increase of TMZ resistance in patients with malignant gliomas. Kaplan-Meier survival curve revealed that the average survival time of patients with the CT+TT and CC genotypes of Leu84Phe in the two groups was statistically significant. COX regression analysis showed that Leu84Phe, degree of resection and pathological grade were independent prognostic factors for patients with malignant gliomas. Our study demonstrates that Leu84Phe of MGMT gene might be a risk factor of TMZ resistance and poor prognosis of patients with malignant gliomas.

Establishment of a novel triple-transgenic mouse: conditionally and liver-specifically expressing hepatitis C virus NS3/4A protease.

It is well known that NS3/4A protein plays crucial roles in the hepatitis C virus (HCV) replication. NS3/4A protein also results to virus-mediated immune evasion and persistence of infection through the interaction with host proteins. However, the lack of a suitable animal model hampers studies of HCV NS3/4A protein interaction with host proteins, which impacts immunopathology due to infection. Here, transgenic vector containing transcriptional regulation and Fluc reporter gene was constructed to conditionally express NS3/4A protein under the dual control of Tet-On regulatory system and Cre/LoxP gene-knockout system. NS3/4A transgenic founder mice were continuously crossed with Lap transgenic mice expressing reverse tetracycline-controlled transcriptional activator (rtTA), the NS3/4A/Lap double transgenic mouse lines with liver-specifically and conditionally expressing reporter (luciferase Fluc) under control of Tet-On system were established. The NS3/4A/Lap double transgenic mouse are mated with Lap/LC-1 double transgenic mouse with liver-specifically and conditionally expressing Cre recombinase under control of Tet-On system, NS3/4A/Lap/LC-1 triple transgenic mouse were generated. In vivo bioluminescent imaging, western blotting and immunohistochemical staining (IHS) was used to confirm that NS3/4A protein was strictly expressed in the liver of Doxycycline-induced triple transgenic mice. The results show that we established a triple-transgenic mouse model conditionally expressing the HCV NS3/4A protein under strict control of the Tet-On regulatory system and Cre/loxP system. This novel transgenic mouse model expressing NS3/4A in a temporally and spatially-specific manner will be useful for studying interactions between HCV NS3/4A protein and the host, also for evaluating NS3/4A protease inhibitors.

Construction and immunogenicity of the DNA vaccine of Mycobacterium Tuberculosis dormancy antigen rv1733c.

We aimed to construct the DNA vaccine encoding Mycobacterium Tuberculosis (Mtb) dormancy antigen Rv1733c and investigate its immunogenicity in mice. The recombinant plasmid pcDNA-Rv1733c was transfected into P815 cells and its product was detected by indirect immunofluorescence. The mice were immunized once every 2 weeks by intramuscular injection of pcDNA-Rv1733c plasmid for a total of three times. The specific antibodies in the serum of the immunized mice were detected by enzyme-linked immunosorbent assay at the indicted time. Enzyme-linked immunosorbent spot was applied to determine the levels of IFN-γ, IL-2 and IL-4 secreted by splenic lymphocytes. Total cytotoxicity T lymphocyte (CTL) active of the splenic lymphocytes was detected by lactate dehydrogenase assay. Additionally, we analysed the percentages of CD4⁺ and CD8⁺ T cells in splenic lymphocytes using flow cytometry. The specific antibody was detected at 2 weeks after the first immunization, and the antibody titre was increased with time which was reached to 1:1600 at 8 weeks. The stimulation index of spleen lymphocytes and the levels of IFN-γ, IL-2 and IL-4 of pcDNA-Rv1733c-immunized mice were both higher than those of saline-immunized mice (P < 0.05). However, no difference was found in the percentages of CD4⁺ and CD8⁺ T cells and the activity of CTL between the pcDNA-Rv1733c- and saline-immunized mice (P > 0.05). So we got the conclusion that the plasmid pcDNA-Rv1733c DNA could induce specific humoral and cellular immunity in mice. Improving the immune effect of Rv1733c by several strategies, such as choosing appropriate immunization route and adjuvant, would be significant for Rv1733c as new tuberculosis vaccine.

High segregation distortion in maize B73 x teosinte crosses.

Two genetic linkage maps of cultivated maize inbred lines and teosinte species were constructed. One population comprised 81 F(2) individuals derived from a cross between maize inbred line B73 and Zea mays ssp parviglumis, while the second consisted of 63 backcross individuals from a cross of maize inbred line B73 with Z. mays ssp diploperennis. In the B73 x Z. mays ssp parviglumis F(2) population, 172 simple sequence repeat (SSR) markers were mapped to 10 chromosomes, which covered 2210.8 cM. In the B73 x Z. mays ssp diploperennis backcross population, 258 SSR markers were mapped to 10 chromosomes, covering 1357.7 cM. Comparison of the two maps revealed that the total map length of Z. mays ssp diploperennis covers 1357.7 cM, which is about 61.4% of that of Z. mays ssp parviglumis (2210.8 cM). Extensive segregation distortion regions were found on chromosomes 1, 2, 3, 5, 6, 7, and 10 in the B73 x Z. mays ssp parviglumis F(2) population and on chromosomes 1-5 and 8-10 in the B73 x Z. mays ssp parviglumis backcross population. Segregation distortion analysis confirmed that the segregation distortion ratio in the interspecific population B73 x Z. mays ssp diploperennis was higher than in B73 x Z. mays ssp parviglumis. We found that the recombination distances are highly variable in these genetic crosses between cultivated and wild species of maize.

Properties of hepatitis C virus minigenome containing mutated 5'UTR region and luciferase transgene.

UNLABELLED: Sequences at the 3'UTR of Hepatitis C virus (HCV) negative-strand (-)RNA play an important role in the initiation of positive-strand (+)RNA synthesis. However, the underlying mechanism in cellular context is still unclear. In this report, we designed several cDNA-based HCV-like minigenomes containing different mutations at the 5'UTR of (+)RNA. These (+)RNAs transcribed from the minigenomes in vitro were transfected into HCV replicon cells for producing (-)RNAs with deletions of different stem loops (SL) at the 3'-end. The results showed that expression of the antisense transgene from minigenome increased, when the minigenome containing deletion of SL-C1+D1+E1 at the 3'-end of (-)RNA was transfected into the HCV replicon cells compared to that of the full minigenome. The expression of the transgene from minigenome decreased using other mutant minigenomes containing deletions SL-A1, SL-A1+B1, and SL-A1+B1+C1 at the 3'-end of (-)RNA. Finally, the transgene from SL-C1+D1+E1 of (-)RNA using CMV promoter-driven minigenome was expressed at higher level than full minigenome in HCV replicon cell lines. These results indicated that the region of (-)RNA interacting with HCV replicase may locate in the SL-C1+D1+E1 region of (-)RNA. KEYWORDS: Hepatitis C virus; minigenome; RNA dependent RNA polymerase; replication.

Surface hydrophilisation and antibacterial functionalisation for microporous polypropylene membranes.

The surface properties of polymer membranes are crucial to their separation performances. For the microporous polypropylene membranes, the high hydrophobicity and lack of functionality easily cause protein adsorption and subsequent microorganism attachment and biofilm formation, i.e. biofouling. Thus, their applications in water treatment, bioseparation and biomedical fields are largely limited. Surface hydrophilisation and antibacterial functionalisation are, therefore, reasonably necessary. This review provides a concise summarisation of related studies according to the surface modification strategies. Especially, the interfacial crosslinking approach developed in our previous studies is presented in detail.

Development of a cell-based assay for monitoring hepatitis C virus ns3/4a protease activity.

Hepatitis C virus (HCV) NS3/4A (non-structural 3 and 4 B) protease plays a key role in the processing of polyprotein precursor and it becomes an attractive target for antiviral drug discovery. We developed a cell-based assay for monitoring of the NS3/4A protease activity in mammalian cells that is an important step in screening of specific drugs against the protease. The recombinant caspase 3 (rCasp3) was used as the specific substrate for NS3/4A protease. The endogenous cleavage sites in the procaspase 3 molecule were substituted by decapeptides specific for NS3/4A protease. The activation of rCasp3 depended on its specific cleavage by NS3/4A protease and resulted in an apoptosis of stable cells expressing the protease. The difference in cell viability between the cells expressing NS3/4A protease transfected with rCasp3 and the counterparts pretreated with NS3/4A protease inhibitors could be estimated by a spectrophotometry based on 3-(4,5-dimethylthioazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) staining of cells in microplates. Thus, we developed a simple and cost-effective colorimetric assay for evaluating NS3/4A protease activity enabling the screening of candidate NS3/4A protease inhibitors.

A novel hepatitis C virus vaccine approach using recombinant Bacillus Calmette-Guerin expressing multi-epitope antigen.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. HCV infection is associated with high morbidity and has become a major problem in public health. Until now, there has been no effective prophylactic or therapeutic vaccine. BCG, a live vaccine typically used for tuberculosis prevention, has been increasingly utilized as a vector for the expression of recombinant proteins that will induce specific humoral and cellular immune responses. In this study, recombinant BCG (rBCG) was engineered to express a HCV multi-epitope antigen CtEm, and HLA-A2.1 transgenic mice were immunized with rBCG-CtEm. High levels of specific anti-HCV antibodies targeted to mimotopes of HVR1 were detected in the serum. HCV-specific lymphocyte proliferation assay, cytokine determination and cytotoxicity assay indicated that HCV epitope-specific cellular immune responses were elicited in vitro. The rBCG-CtEm immunization conferred protection against infection with the recombinant vaccinia virus (rVV-HCV-CNS) in vivo. These results suggest that rBCG expressing multi-epitope antigen may serve as an effective vaccine against HCV infection.

[Preliminary studies on tissue culture and agrobacterium-medicated transformation of Brassica campestris ssp. chinensis].

The hypocotyls and cotyledons of the asepetic seedling of Brassica campestris ssp. chinensis L cv. Pudongaijiecai) were used as explants for tissue culture. Adventitious buds were differentiated on modified MS medium supplemented with TDZ 1-2 mg/L, NAA 0.2-1 mg/L and AgNO3 7.5 mg/L. The percentage of explants which formed buds of cotyledons was about 56%, and that of hypocotyls was about 37%. When the regenerated explants were transferred onto MS medium with 2 i.p. 5 mg/L and NAA 0.1 mg/L for two weeks, whole plantlets were obtained by culturing the regenerated shoots on 1/2 MS medium with NAA 0.1 mg/L. Agrobacterium tumefaciens strain (LBA 4404/PBI 121) carrying the GUS gene and Npt II gene was used for transformation. After 2 days of coculture, the hypocotyls and cotyledons were transferred onto regenerated medium containing CP 300 mg/L for bud formation. After 4-5 weeks, the differentiated buds were transferred onto selection medium with CP 200 mg/L and Km 10 mg/L for 1 month, then the green shoots were transferred onto the rooting medium containing Cef 100 mg/L and Km 20 mg/L. 4-5 weeks later, plantlets with Km resistance were obtained and some of them showed higher enzymatic activities of beta-glucuronidase than control ones.

[Cleavage activity of ribozymes on a target RNA of tobacco mosaic virus in E. coli].

A target cDNA fragment from TMV RNA was inserted to a reporter gene CAT immediately to the 3' end of the translation initiation codon ATG resulting in the formation of a chimeric CAT gene in an in vivo transcription and expression vector. The in vivo activities of various ribozymes on the target sequence were observed by determining the changes of the CAT activities of the chimeric CAT gene expressed in E. coli. The CAT activity was reduced by up to 30% when a specific ribozyme RZ1, RZ1A or RZ1B was transcribed, no change in CAT activity was observed when a non-specific ribozyme RZ3 was expressed. The protein electrophoresis and primer extension experiments indicated that this reduction in CAT activity was due to the specific cleavage of the ribozymes to the chimeric CAT mRNA at the target region hence the decrease in CAT protein synthesis.

[Cloning and expression of an antifreeze peptide gene of winter flounder in E. coli].

Antifreeze peptide (AFP) produced in some animals is able to lower freezing temperature to prevent serum from freezing. There is a great potential to apply AFP in various circumstances wherever tolerance to cold environment is required. cDNA copies of AFP and proAFP coding sequence were synthesized according to the published sequence from a winter flounder (Pseudopleuronectes americanus). Expression of AFP or proAFP cDNA was initially tested in E. coli using a procaryotic expression vector. Level of the expression was essentially low indicated by SDS-PAGE and protein staining procedures. In order to detect the low level expression, the AFP (or proAFP) cDNA was fused, in frame, to the 5'-end of the CAT reporter gene resulting in a chimeric AFP/CAT (or proAFP/CAT) cDNA. The product translated from such a chimeric mRNA must contain a N'-terminal AFP (or proAFP) portion and C'-terminal CAT portion. Thus, low level of expression of AFP (or proAFP) in the fused form may be detected indirectly by sensitive CAT assay as long as the chimeric CAT still remains the activity. As expected, CAT activity has been clearly detected in protein extract from induced E. coli indicating that AFP (or proAFP) cDNA clones be expressed in E. coli.

Structure-specific binding of wound tumor virus transcripts by a host factor: involvement of both terminal nucleotide domains.

A gel retardation assay was used to demonstrate binding of wound tumor virus transcripts by a protein component of leafhopper vector cell extracts. Comparative binding studies employing terminally modified and internally deleted transcripts established that the segment-specific inverted repeats present in the terminal domains of the viral transcripts were necessary but not sufficient for optimal binding. An additional involvement of internal sequences in either the formation or the stabilization of the binding complex was indicated. Results of competitive binding experiments confirmed the sequence- and structure-specificity of the protein-RNA interaction and revealed apparent differences in the ability of individual viral transcripts to form a stable binding complex. Possible implications of structure-specific interactions between wound tumor virus transcripts and a host component and the role of the terminal inverted repeats are discussed.