A strain Escherichia coli with high production of phytase was screened from pig excreta. Phytase gene appA, with 1,299 bp coding region in full length, was cloned from its genome by polymerase chain reaction (PCR) . The gene appA was then cloned into the prokaryotic expression vector pET-28a ( + ) . In the host BL21, the phytase appA was overexpressed by shaker-cultivation (up to 692 U/mL) . The enzymatic analysis of the prokaryotic derived appA phytase revealed that its optimal pH and temperature was 4.5 and 60℃, respectively.

Objective To evaluate the risks of vaccination operation in order to provide scientific basis for policy makings of vaccination operation.Methods The risk matrix and analytic hierarchy process approach were used to evaluate the risks of vaccination operation,including the possibility,severity,risk weight,and risk level.Results Seven items for the first level and thirty one items for the second level vaccination risk factors were determined.In the first level,E level risk (Extremely serious risk ) was vaccination implementation risk,and H level risk (high risk ) was the risks of pre -notification and health education and risks of vaccine and cold chain management.Vaccination implementation risk accounted the largest weight (weight coefficient=38.95%).In second level,E level risk was three inspection and seven verification before vaccination,and H level risk were personnel professional quality,personnel responsibility,pre -inspection,informed before vaccination, vaccination route, site and dose, vaccination operation, post vaccination notification and retention,and three inspection and seven verification before vaccination,accounted the largest weight (weight coefficient=10.14%).The weight of the logical consistency of the test results were satisfactory (consistency ratio<0.1 ).Conclusion The integrated application of risk matrix and analytic hierarchy process in risk assessment of vaccination operation helps to further regulate vaccination services and has application and promotion value.

Recent years, the development of medical devices kits is rapid. How to make the technical evaluation of medical devices kits more perfect bases on the two major principles of safe and effective, and to make kits in the market more normative and orderly, these issues for technical evaluation have to be considered. This article makes a study on current situation of production, classification of management and registration status, combined with existing regulations and related standards, and discusses technical evaluation related issues.
Equipment and Supplies ; standards

OBJECTIVETo evaluate the effect of surgical manipulation on the dissemination of cancer cells into blood circulation in patients with gastric cancer and to analyze its risk factors.

METHODSThis study included 45 consecutive patients with gastric cancer undergoing curative resection and 13 control cases (10 healthy persons and 3 patients with peptic ulcer receiving gastrectomy). Peripheral blood was obtained preoperatively and just after surgical manipulation. The mRNA levels of carcinoembryonic antigen (CEA) from the blood samples were assayed by reverse transcription-polymerase chain reaction(RT-PCR) and compared between the 2 groups.

RESULTSCEA mRNA was negative in all control cases. Of the 45 gastric cancer patients, the preoperative positive rate of CEA mRNA was 8.9%, while the postoperative positive rate was 48.9%, which was significantly higher than that of preoperation (P=0.000). Multivariable Logistic regression analysis showed that operative duration (P=0.014) and tumor depth (P=0.010) were independent risk factors for cancer cell dissemination. Furthermore, the operative duration in patients with positive postoperative CEA mRNA was markedly longer than that in patients with negative postoperative CEA mRNA (P=0.000), and positive rate of postoperative CEA mRNA in advanced gastric cancer was higher compared with that in early gastric cancer (P=0.034).

CONCLUSIONSSurgical manipulation of curative gastrectomy can provoke dissemination of cancer cells into blood circulation, and the operative duration and tumor invasion depth may be 2 of the risk factors for cancer cell dissemination.

Adult ; Aged ; Carcinoembryonic Antigen ; blood ; Case-Control Studies ; Female ; Gastrectomy ; adverse effects ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Neoplastic Cells, Circulating ; pathology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Risk Factors ; Stomach Neoplasms ; blood ; pathology ; surgery

Objective To explore the dosimetric effects of a self-developed planning mode of boundary range scattering dose(BRSD)on Cyberknife treatment of lung cancer brain metastases.Methods The positioning images of 15 patients with lung cancer brain metastases treated in the radiotherapy department of some institution from January 1,2021 to December 31,2021 were selected and introduced into Cyberknife Multiplan 4.0.3 treatment planning system.A fractionated stereotactic radiotherapy(FSRT)plan(as the FSRT planning group)and a BRSD plan(as the BRSD planning group)were developed for each patient.The FSRT planning group developed a plan for the planning target volume(PTV)in the conventional way,so that V100 covered more than 95％of the PTV;the BRSD planning group prepared a plan for the gross tumor volume(GTV)with the same parameter conditions as the FSRT planning group and the prescription dose was normalized to the PTV so that V100 covered more than 95％of the PTV.The dosimetric parameters of the target area and normal tissue of the 2 groups were compared by dose-volume histograms and isodose curves.Statistical analysis was performed using SPSS 24.0 software.Results The D98,Dmax and Dmean in the target area of the BRSD planning group were significantly higher than those of the FSRT planning group,and the differences were statistically significant(P<0.05);the differences in the conformity index,dose gradient index,and Dmean,V30,V24 and D3cc in normal tissue of the 2 groups were not statistically significant(P>0.05);the BRSD planning group gained a denser dose distribution when compared with the FSRT planning group.Conclusion The BRSD planning mode gains significant dosimetric advantage by enhancing the absorbed dose to the target area without increasing or decreasing the dose to normal tissue.

Abstract：Objective To prepare human monoclonal antibody against spike protein（S protein）of severe acute respiratory syndrome coronavirus 2（SARS⁃CoV⁃2）by using single B cell，and determine its neutralizing activity. Methods Venous blood with high antibody level was collected from people immunized with inactivated SARS⁃CoV⁃2 vaccine（Vero cells） twice，of which peripheral blood mononuclear cells（PBMCs）were isolated by lymphocyte stratified fluid and used to isolate single B cell expressing S protein antibody by magnetic beads coupled with S1 protein. Variable region genes of IgG heavy chain and light chain were amplified by nested PCR after reverse transcription of single B cell，which were connected with CMV promoter，IgG leader sequence，IgG constant region and polyA sequence by overlapping PCR to construct antibody linear expression cassette. Linear expression cassette of the heavy chain and light chain from the same B cell was transfected to HEK293T cells to express human monoclonal antibody of SARS⁃CoV⁃2 S protein. Immunoreactivity was detected by immuno⁃ fluorescence while neutralizing activity by pseudovirus neutralization test. Results A total of 26 monoclonal antibodies against SARS⁃CoV⁃2 S protein were expressed，which showed heavy chain and light chain protein bands of IgG antibody at

OBJECTIVETo study the alteration of protein Z (PZ) in patients with cardio-cerebral thrombotic diseases, its clinical significance and relations with FX.

METHODSPZ and FX:Ag were measured by ELISA, and plasma FX:C by first stage method. In 170 patients with acute ischemic stroke (AIS), 40 acute myocardial infarction (AMI) and 60 healthy adults as contrast, PZ, FX:C and FX:Ag were measured and compared between incipience and recurrence, different ages and genders.

RESULTSIn AIS and AMI groups, PZ levels decreased significantly to (940.02 +/- 229.82) microg/L and (1071.44 +/- 180.52) microg/L, respectively \[the contrast group was (2257.97 +/- 479.76) microg/L, P < 0.001\]. But FX:C and FX:Ag raised to (136.73 +/- 34.93)% and (135.54 +/- 54.39)% in AIS group; and to (139.53 +/- 29.18)%, (129.75 +/- 21.91)% in AMI group, respectively, while in the contrast group they were (94.33 +/- 22.00)% and (77.22 +/- 13.19)% (P < 0.001). In the comparative research between the AIS group, AMI group and the contrast group, PZ level was clearly found to negatively relate to the level of FX:C and FX:Ag (P < 0.001). Meanwhile, PZ level, FX:C and FX:Ag in recur-AIS group and recur-AMI group exhibited significant differences (P < 0.05) from those in the primary AIS and AMI groups, suggesting that the decrease of PZ levels reflected the pathological process of the disease. In addition, PZ level gradually decreased with the increase of age (P < 0.05), while FX:C and FX:Ag had no relations with age (P > 0.05). No correlation was found in sex with PZ level, FX:C, FX:Ag (P > 0.05).

CONCLUSIONPZ level was significantly decreased in AIS and AMI patients and was negatively related to FX:C and FX:Ag. The mechanism leading to FX increase may partially related with the decreased of PZ. PZ level was different in the primary and recurrent disease and was gradually decreased with the increase of age. Lack of PZ might be a etiological factor of cardio-cerebral thrombotic diseases.

Aged ; Aged, 80 and over ; Blood Proteins ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Factor X ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; blood ; Stroke ; blood

OBJECTIVETo evaluate the underlying mechanism of Jianpi Jiedu Recipe (, JJR) in the reversion of multidrug resistance concerning colorectal cancer in vitro and in vivo.

METHODSMice were treated orally with JJR at a daily 4.25 g/(kg·day) or injected with vinblastine (VCR) 2.5 mg/(kg·day) for 3 weeks after having been inoculated with HCT8/V cells; tumor tissues were assayed by hematoxylin and eosin staining. Firstly, the effects of JJR on the expression of cyclooxygenase-2 (COX-2) were tested by real-time polymerase chain reaction (PCR) technique and COX-2 gene silenced by siRNA. Secondly, the variation of intracellular concentration of oxaliplatin (L-OHP) was evaluated by the inductively coupled plasma mass spectroscopy (ICPMS) in HCT8/V and its COX-2 siRNA cells; the concentration of JJR combined with chemotherapeutic drugs and the reverse effect of multidrug resistance (MDR) in HCT8/V cells was evaluated by the MTT assay. Thirdly, real-time quantitative PCR and Western blot analysis were used to detect the multidrug resistance gene 1 (MDR1) mRNA and P-gp expression.

RESULTSJJR had an inhibitory effect on the growth of tumors in vivo, and it, in combination with chemotherapeutic drugs, could reverse the drug-resistance of HCT8/V cells and increase the sensitivity of HCT8/V cells to VCR, DDP, 5-Fu, and THP. ICP-MS results showed that JJR could increase the concentration of drugs in HCT8/V cells (P<0.01). Furthermore, it was shown that JJR could reverse drug resistance of colorectal cancer cells by decreasing MDR1 expression and P-gp level via downregulation of COX-2, which has been represented as one of the major mechanisms that contributes to the MDR phenotype (P<0.01).

CONCLUSIONJJR reversed multidrug resistance and enhanced the sensitivity to chemotherapy, which could be attributed to the down-regulation of COX-2 in MDR1/P-gp-mediated MDR colorectal cancer after chemotherapy.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Animals ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; drug therapy ; enzymology ; pathology ; Cyclooxygenase 2 ; genetics ; metabolism ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Green Fluorescent Proteins ; metabolism ; Humans ; Intracellular Space ; metabolism ; Mice, Inbred BALB C ; Organoplatinum Compounds ; metabolism ; RNA, Small Interfering ; metabolism ; Signal Transduction ; drug effects ; Vinblastine ; pharmacology ; therapeutic use ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the protective effect of high-pressure carbon monoxide for preservation of ex vivo rabbit heart graft in comparison with the conventional HTK cardioplegic solution preservation.

METHODSHeart grafts isolated from 85 New Zealand rabbits were randomly divided into Naive group (n=5), HTK group (n=40) and CO group (n=40). The grafts underwent no preservation procedures in Naive group, preserved at 4 degrees celsius; in HTK cardioplegic solution in HTK group, and preserved at 4 degrees celsius; in a high-pressure tank (PO2: PCO=3200 hPa: 800 hPa) in CO group with Krebs-Henseleit solution perfusion but without cardioplegic solution. After preservation for 2, 4, 6, 8, 10, 14, 18, and 24 h, 5 grafts from the two preservation groups were perfused for 30 min with a modified Langendorff apparatus and examined for left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), arrhythmia score (AS), myocardial ultrestructure, and cardiac enzyme profiles.

RESULTSAfter preservation for 6 to 24 h, the cardiac enzyme profiles and systolic and diastolic functions were significantly better in CO group than in HTK group, but these differences were not obvious between the two groups after graft preservation for 2 to 4 h. Significant changes in the myocardial ultrastructures occurred in the isolated hearts after a 24-h preservation in both CO and HTK groups, but the myocardial damages were milder in CO group.

CONCLUSIONPreservation using high-pressure carbon monoxide can better protect isolated rabbit heart graft than the conventional HTK preservation approach especially for prolonged graft preservation.

Animals ; Carbon Monoxide ; Cardioplegic Solutions ; Glucose ; Heart ; physiology ; Heart Transplantation ; Myocardium ; ultrastructure ; Rabbits ; Tissue Preservation ; methods ; Tromethamine

OBJECTIVETo construct an ERbeta expression vector and study its expression and function in different cancer cells.

METHODSStandard PCR was used to amplify the full-length coding sequence of ERbeta. The amplified ERbeta gene was cloned into the eukaryotic expression vector pCDNA3, generating pCDNA3-ERbeta. The ERbeta expression was detected by Western blot and in vitro translation. The biological activity of ERbeta was detected by transfecting the pCDNA3-ERbeta into SV40-transformed embryonic kidney cell line 293T,breast cancer cell lines MDA-MB-435, MDA-MB-436, SKBR3, and prostate cancer cell line PC-3, with reporters containing estrogen response elements.

RESULTSThe recombinant plasmid pCDNA3-ERbeta was confirmed by restriction analysis to contain the ERbeta gene. The 63 000 ERbeta expression was shown by Western blot and further confirmed by in vitro translation. The ERbeta expression in different cancer cells was demonstrated to stimulate the expression of the reporters containing estrogen response elements, ERE and C3.

CONCLUSIONERbeta protein is successfully expressed and has biological activity, laying solid foundation for further study on its role in cancer cells.

Breast Neoplasms ; metabolism ; pathology ; Cell Line ; Cell Line, Tumor ; Embryo, Mammalian ; Epithelial Cells ; Estrogen Receptor beta ; genetics ; metabolism ; Female ; Genes, Reporter ; genetics ; Genetic Vectors ; Humans ; Kidney ; cytology ; Male ; Plasmids ; Prostatic Neoplasms ; metabolism ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Response Elements ; genetics ; Transfection