The leakage of glutamic-pyruvic transaminase ( GPT ) in the supernatant of primary cultures of rat hepatocytes has been used to evaluate the toxicity of the classical hepatotoxin carbon tetrachloride ( CC14 ) . The response of the isolated hepatocyte's preparation to incubate with CCl4 showed a clear dose dependency from 5 to 20 ml/ L in this system and the toxicity increased -with time of incubation. Glycyrrhizin ( 100mg/L ) and sodium caffeic acid 10 to 100 mg/L remarkably reduced GPT release in the medium of hepatocytes with CC14 while Bupleurum kunmingense polysaccharides had no significant effect at the same condition. Glycyrrhizin and caffeic acid might have protective effects against CC14. Further study is necessary. These findings also indicated that the primary cultures of rat hepatocytes is a suitable system for evaluating liver protective drugs as well as studying the function of cell metabolism.

AIM:To express recombinant hCD154-GST fusion protein, to prepare anti-hCD154 monoclonal antibody, and to investigate the effect of anti-hCD154 monoclonal antibody on graft rejection. METHODS AND RESULTS: Total RNA was prepared from human peripheral blood mononuclear cell (PBMC) activated with 10ng/mL PMA and 1 ?g/mL PHA for 8h, the total RNA was reversetranscribed to cDNA. The entire coding region and a part of the 3'non-coding regions were amplified by PCR using a pair of primers designed and synthesized according to the sequence of human CD154 gene from gene bank. The amplified product, a 820bp DNA fragment was cloned into pGEX-4T-1 plasmid expressing glutathione S-transferase(GST). The cloned insert was identified by double digestion of the cloned pGEX-4T-1 plasmid with retriction enzymes BamHⅠand EcoRⅠ.The fusion protein expression plasmid of PGEX-4T-1/hCD154 was constructed, then transformed to E coli BL21. The human CD154-GST fusion protein expression was induced by IPTG in BL21. The expression of recombinant 26kD GST and 55kD human CD154-GST fusion protein were confirmed by SDS-PAGE. CONCLUSION: We have express the recombinant human CD154-GST fusion protein. The expressed hCD154-GST fusion protein will be used to prepare anti-hCD154 monoclonal antibody, to investigate the role of anti-CD154 monoclonal antibody on graft rejection.

Objective: To clone and express Treponema pallidum (TP) specific antigens P15 and P47 and use them in clinical examination. Methods: P15 and p47 genes synthesized were inserted into a GST fusion expression vector. The recombinant antigens were purified by affinity chromatography and then coated on microplates to establish an ELISA kit. Using this kit, national TP standards, sera of normal persons and patients with TP and other diseases were tested. Results: The synthesized p15 and p47 genes sequenced were identical with those of GenBank. National TP standards were tested by ELISA kit which coated with these recombinant antigens, and the results accorded with the standards. High sensitivity and specificity was showed when 2 recombinant antigens were used in ELISA to detect the sera of normal persons and patients with TP and other diseases. Conclusion: The recombinant TP specific antigen P15 and P47 are suitable for establishment of ELISA kit in clinical examination.

Objective　To compare the endocytic routes of exogenous antigen between murine dendritic cells (DCs) and macrophages (Mφs). Methods　Murine bone marrow-derived DCs and peritoneal Mφs were pulsed with horseradish peroxidase (HRP)-5 nm colloidal gold for 10 minutes, then grouped and chased for 0-120 minutes in culture medium. Intracellular distribution of 5 nm colloidal gold was explored by means of the cellular enzymatic-chemistry of acid phosphatase and MHC Ⅱ immuno-cytochemistry under electron microscope. Results　After 10 minutes of pulse with HRP-5 nm colloidal gold and 30 minutes of chase, most HRP-5 nm gold particles internalized by DCs entered into MHC class Ⅱ compartments (MⅡCs), and a small portion entered into acid phosphatase-positive lysosomes. In contrast to DCs, most Mφs lysosomes were accessed by HRP-5 nm gold particles, and a small portion of HRP-5 nm gold particles entered into MⅡCs. After 60 minutes of chase, 5 nm gold particles could hardly be seen within Mφs, whereas most 5 nm gold particles were still retained in DCs. Conclusions　The endocytic route of exogenous antigen in DCs seems to be different from that in Mφs. Antigens taken by Mφs mainly enter into lysosomes within 30 minutes. In the case of DCs, most internalized antigens enter into MⅡCs, which may be related to their unique antigen-presenting function . In addition, Mφs seem to have more powerful capacity to scavenge exogenous antigen than DCs.

Human kallikrein-1 (hK1) gene was cloned from kidney tissues cDNA, it was inserted into the plasmid pPICZalphaA, then the yeast expression vector pPICZalpha-hK1 was constructed. After transformed into Pichia pastoris host X33, high-level expression transformants were screened by escalating the concentration of Zeocin (from 500 to 700 microg/mL) of YPD plate and medium. When temperature was 30 degrees C, pH 6.0 with induction duration of 64 hours in the 30 L fermenter, the highest yield can reach about 6500 u/L (1.25 g/L). The variation of glycosylation resulted in two kinds of molecules, i.e. rhK1-H with a heavy molecular weight and rhK1-L with a light one. rhK1 was purified from the supernatant through Phenyl hydrophobic interaction, Cu(2+)-charged Chelating and Anion-exchange chromatography. 0.28 g rhK1-H and 0.62 g rhK1-L can be purified from one liter supernatant. The yield recovery was 72% with a purity of > 96%. So far our yield of rhK1 is superior than known recombinant expression method reported by other researchers.
Amino Acid Sequence ; Base Sequence ; Chromatography, Ion Exchange ; methods ; Genetic Vectors ; genetics ; Humans ; Kidney ; metabolism ; Molecular Sequence Data ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Tissue Kallikreins ; biosynthesis ; genetics

Objective To summarize the experience of patient positing,port placements setting and robot cart docking in pediatric robot-assisted laparoscopic upper urinary tract operations.Methods From March 2017 to December 2017,140 robot-assisted laparoscopic upper urinary tract procedures were performed in our institution,including 110 cases of pyeloplasty,15 upper pole heminephroureterectomy,12 simple nephrectomy and 3 adrenalectomy.There were 103 males and 37 females with a range age from 1 month to 18 years.The assistant surgeon was adjacent to the instrument nurse,and patients were placed in a supine position with 60°-80° inclination and keep the legs low to the body.Room setup and patient positioning were similar to the traditional laparoscopic surgery.Semi-hidden incision technique was used in 140 patients:the camera port was placed umbilicus,two additional arm ports (one 5 mm and one 8 mm) were placed under direct vision,the 8 mm arm port was placed on the line of a Pfannenstiel incision and the 5 mm arm port was placed below the Xiphoid along the midline.Finally,a 3 or 5 mm assistant port was placed approximately 3 cm lateral to the inferior arm port,the line of a Pfannenstiel incision.Results The average time was (11.5 ± 3.2) min (10.5-16.5 min) from skin incision to robot cart docking completed.All surgeries were successfully completed without open conversion.One patient required an additional assist port for severe adhesion after the previously open surgery,there was no injury to other viscera.Average operative time was (146.9 ± 48.7)min (78-259 min) and average post-operative hospitalization time was (5.7 ± 1.4) d(4-10 d),respectively.There was no visual scar on abdominal 6 weeks postoperatively,and all parents made comments about their satisfaction with the cosmetic appearance.All operations got complete success at a mean follow up of 6 (1-9) months.Conclusions A good room setup,patient positioning and the semi-hidden incision technique port placements are maintaining the safety of the patient,avoiding compression injuries,allowing maximum mobility of the robotic arms,and facilitating a smooth and efficient surgery,and improving post-operative recovery.

Objective:To establish a disk (CD) microfluidic chip detection platform for the rapid detection of CALR-1 and CALR-2 mutations in patients with cerebral infarction, and summarize its clinical application value.Methods:Based on microfluidic technology and loop mediated isothermal amplification technology, a CD microfluidic chip detection platform for simultaneous detection of CALR-1 and CALR-2 gene mutations were established, and the sensitivity, specificity, repeatability and accuracy of the platform were verified. A total of 124 patients with cerebral infarction treated in Huashan Hospital, Shanghai Medical College, Fudan University from November 2019 to March 2021 were prospectively selected into the experimental group; and 80 healthy subjects were included in the control group. The CALR-1 and CALR-2 gene mutations in anticoagulant peripheral blood samples were detected by the CD microfluidic chip. Each chip could detect 4 samples at the same time and synchronously detect 3 indexes of each sample. The detection results could be obtained after isothermal amplification for 40 min. At the same time, sequencing method was used to verify the test results, and the consistency of the results of the two detection methods was compared.Results:Using this CD microfluidic chip platform, the synchronous amplification of 3 indexes in the sample could be completed within 40 min without the need of thermal circulation, and the whole detection process of the sample could be completed within 60 min. For samples with a high concentration of target nucleic acid, typical positive signals could be visualized after amplification for 10 min, and the test results would be available within 30 minutes after receiving the samples. The detection sensitivity of CD microfluidic chip method for CALR-1 and CALR-2 mutation load concentration was 1.0% and 0.5% respectively. Nonspecific amplification was not observed for the non-target nucleic acid samples, indicating the high specificity of this method. The coincidence rates of intra and inter batch repeatability were 100% (20/20) respectively. Two samples with CALR gene mutation were found in the cerebral infarction group, both of which were CALR-1 mutations (L367fs*46). There was no CALR-1 or CALR-2 mutation in the control group. The detection results of CD microfluidic chip method were completely consistent with the sequencing verification results (100% [204/204]).Conclusions:The CD microfluidic chip method could be used for the detection of CALR-1 and CALR-2 gene mutations in clinical samples of patients with cerebral infarction. This method has the advantages of high detection sensitivity, good detection specificity, fast detection speed and high detection flux, which is helpful to clarify the etiology of patients with cerebral infarction.

Objective: To analyze the association of eating dining locations and their association with nutritional status among Chinese children aged 6-17 years,so as to provide reference for guiding children s reasonable diet. Methods: Stratified random cluster sampling was used to select children aged 6 to 17 years from 28 cities and rural areas of 14 provinces in East, North, Central, South, Southwest, Northwest, Northeast of China, and a total of 52 535 children were included in the study from 2019 to 2021. Information including dining locations, demographic characteristics, dietary intakes and physical activity were collected through a questionnaire survey. Fasting body height and weight were measured in the morning. Unordered multiclass Logistic regression analysis was conducted to assess the relationship between dining locations and nutritional status in children. Results: Regarding children s dining locations, 66.3% ate breakfast at home,25.8% ate breakfast at school,7.9% ate breakfast outside (small dining tables, restaurants, stalls, etc.); 67.7% ate dinner at home,29.0% ate dinner at school,3.3% ate dinner outside; and 63.6% ate lunch at school,30.8% ate lunch at home,5.7% ate lunch outside. The prevalence rates of overweight/obesity and undernutrition were 28.6% and 9.3%, respectively. The adjusted multiclass Logistic regression analysis (controlling for age, region, parental education, household income, total energy intake, and moderate-to-vigorous physical activity) demonstrated that, compared to eating at home, school based breakfast and dinner consumption was associated with significantly lower overweight/obesity risks for both genders (boys: breakfast OR =0.70, 95% CI =0.65-0.75; dinner OR =0.80, 95% CI = 0.74- 0.86; girls: breakfast OR = 0.89 , 95% CI = 0.82-0.96; dinner OR =0.88, 95% CI =0.81-0.95), whereas eating lunch away from home significantly increased overweight/obesity risks (boys: OR =1.32, 95% CI =1.17-1.48; girls: OR =1.43, 95% CI =1.26- 1.62 ), with all associations being statistically significant ( P <0.05). After adjusting for confounding factors, boys who ate breakfast away from home showed a significantly reduced risk of undernutrition ( OR =0.80,95% CI =0.66-0.97), while those consuming lunch away from home had an increased risk ( OR =1.26, 95% CI =1.01-1.57) ( P <0.05). Conclusions The choice of dining locations for children is becoming more diverse, and a relatively high proportion of children eat meals outside the home and at school. Eating out have a higher risk of malnutrition for children. School feeding may be beneficial to children s physical health.