Abstract: This study is to improve the affinity of scFv-AK404R against VEGFR2. The secondary mutational library was constructed by hydrophilic shuffling in CDR3 region of the heavy chain. VEGFR2-specific screening was performed by phage display technology and the protein of mutants was expressed in periplasm of E.coli HB2151 and purified by affinity chromatography. The affinity constant of scFvs was measured by competitive ELISA, and the structure of scFvs was analyzed by bioinformatics. The result showed that a library with 6.4x10(5) scFv members was established by electro-transformation. Two mutated clones with high absorbance value were isolated after screening. After purification by affinity chromatography, electrophoretically pure scFv proteins were obtained. The competitive ELISA showed that the affinities of WZ01 and WZ02 were three times higher than that of the parental AK404R, and bioinformatics analysis showed that the enlarged contact surface and fitted closely with KDR3 surface may be the reasons for improved affinity. These results suggest that introducing hydrophilic amino acids to the heavy chain CDR3 region is an effective approach to improve the affinity of scFv.
Amino Acid Sequence ; Antibody Affinity ; Chromatography, Affinity ; Computational Biology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Hydrophobic and Hydrophilic Interactions ; Peptide Library ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology ; metabolism

OBJECTIVETo discuss the clinical results of hook plate internal fixation for the treatment of distal traumatic injuries of clavicle,and to analysis the mechanism of the complications.

METHODSFrom 2001.9 to 2008.2, 36 patients with fresh distal clavicle fractures of Neer type II and 14 patients with dislocation of Acromioclavicular joint of Tossy type III were treated with hook plate internal fixations. Among the patients, 36 patients were male and 14 patients were female, ranging in age from 19 to 77 years, with an average of 43 years. After the operation, the range of motion of shoulder joint was measured and the complications were studied. The Karlsson and Constant-Murley criteria were used to evaluate the therapeutic effects.

RESULTSAll the patients were followed up with a mean duration of 12 months (7 to 18 months). All the patients could move shoulder joint actively 1 week after operation, and recover to the normal ROM at the 6th week after operation. According to Karlsson criteria, 44 patients got an excellent result, 6 good. According to Constant-Murley criteria, the average score of wound shoulder was (73.8 +/- 8.3) before operation, and (59.6 +/- 6.7) after operation, and the postoperative score was higher than preoperative score. No incision infections were found and 5 patients with complications were cured. No re-dislocation of acromioclavicular joint occurred.

CONCLUSIONIt is advocated that hook plate for the treatment of distal clavicular fractures of Neer type II and acromioclavicular dislocation of Tossy type III could provide sufficient stability to allow early functional rehabilitation, and to decrease the rate of complications.

Adult ; Aged ; Bone Plates ; Clavicle ; injuries ; surgery ; Female ; Fracture Fixation, Internal ; instrumentation ; methods ; Fractures, Bone ; surgery ; Humans ; Male ; Middle Aged ; Treatment Outcome ; Young Adult

OBJECTIVETo investigate the biodegradation of tetrachloroethylene (PCE) by acclimated anaerobic sludge using different co-substrates, i.e., glucose, acetate, and lactate as electron donors.

METHODSHP-6890 gas chromatograph (GC) in combination with auto-sampler was used to analyze the concentration of PCE and its intermediates. Results PCE could be degraded by reductive dechlorination and the degradation reaction conformed to the first-order kinetic equation. The rate constants are k(lactate) > k(glucose) > k(acetate). The PCE degradation rate was the highest in the presence of lactate as an electron donor.

CONCLUSIONLactate is the most suitable electron donor for PCE degradation and the electron donors supplied by co-metabolic substrates are not the limiting factors for PCE degradation.

Acetates ; metabolism ; Anaerobiosis ; Bacteria, Anaerobic ; metabolism ; Biodegradation, Environmental ; Bioreactors ; Glucose ; metabolism ; Kinetics ; Lactic Acid ; metabolism ; Oxidation-Reduction ; Sewage ; microbiology ; Tetrachloroethylene ; metabolism

Objective To evaluate the commonly used consciousness scales according to longitudinal study on small-sample patients with minimally conscious state. Method Eleven patients with minimally consciousness, who were scanned using fMRI, were selected and scored by the commonly used consciousness scales in Wujing Hangzbeu Hospital. The 11 patients were classified into 2 groups according to the scores of CRS-R scale couple with the results of the fMRI study. The One-Way ANOVA method was used to analyze the inter-group difference of the commonly used consciousness scales and their subscales. Results Despite the communicative subscale of the CRS-R scales, other iterms lack of statistic significance in classification of the conscious state. Among the commonly used consciousness scales, the Wessex Head Injury Matrix (WHIM) scale presented the highest diagnostic value in consciousness state, whereas the Chinese Vegetative State Scale (CVSS) presented the lowest diagnostic value. Conclusions It indicated that the total scores of the consciousness scales and the scores of subscales of them presented poor diagnostic value in general, and big discrepancy of diagnostic value existed between the iterms of the scales by using objective tools.

OBJECTIVETo investigate the effect of 5-fluorouracil (5-FU) on the expression of ATP-binding cassette superfamily G member 2 (ABCG2) in human colon cancer cell SW480.

METHODSSW480 cells were treated with various concentrations of 5-FU. CCK8 assay was utilized to detect the 5-FU IC50 to SW480 cells. Positive expression of ABCG2 was detected by flow cytometry, and mRNA expression of ABCG2 was detected by real time polymerase chain reaction (RT-PCR).

RESULTSThe 5-FU IC50 to SW480 cells increased as the drug concentration increased (P<0.05). Flow cytometry revealed that positive expression rate of ABCG2 in normal SW480 cells (group A) was (6.26±0.86)%. Immediately after treatment with 5-FU for 48 hours, the positive expression rate of ABCG2 (group B) was (3.43±1.18)% (P<0.05). In the second passage of cells after treatment with 5-FU for 48 hours, the positive expression rate of ABCG2 (group C) was (12.91±3.42)% (P<0.05). The mRNA expression of ABCG2 detected by RT-PCR was in accordance with the results from flow cytometry.

CONCLUSIONExpression of ABCG2 in SW480 cells can be affected by various concentrations of 5-FU.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; metabolism ; Cell Line, Tumor ; Colonic Neoplasms ; metabolism ; Fluorouracil ; pharmacology ; Humans ; Neoplasm Proteins ; metabolism

OBJECTIVETo investigate the biodegradation of tetrachloroethylene (PCE) using methanol as electron donor by acclimated anaerobic sludge.

METHODSHP-6890 gas chromatograph (GC), together with HP-7694 autosampler, was used to analyze the concentration of PCE and intermediates.

RESULTSPCE could be decholrinated reductively to DCE via TCE, and probably further to VC and ethylene. The degradation of PCE and TCE conformed to first-order reaction kinetics. The reaction rate constants were 0.8991 d(-1) and 0.068 d(-1), respectively, and the corresponding half-life were 0.77 d and 10.19 d, respectively. TCE production rate constant was 0.1333 d(-1), showing that PCE was degraded more rapidly than TCE.

CONCLUSIONMethanol is an electron donor suitable for PCE degradation and the cometabolic electron donors are not limiting factors for PCE degradation.

Anaerobiosis ; Chromatography, Gas ; Kinetics ; Methanol ; metabolism ; Sewage ; microbiology ; Trichloroethylene ; metabolism

Objective: To observe the changes of PD-1 expression, mRNA level and cytotoxic activity of CD19 CAR-T cells during the culture process of CAR-T cells. Methods: The peripheral blood T cells of 6 lymphoma patients with high expression of PD-1 and 6 healthy volunteers were the source of CAR-T cells. The expression of PD-1 was analyzed by flow cytometry. The mRNA level of PD-1 was analyzed by PCR. The cell proliferation was analyzed by CCK-8 assay. The cytotoxicity was analyzed by LDH assay. Results: ①The transfection efficiency of high PD-1 expression T cells and healthy volunteer T cells were as the same (P>0.05) . ②The cell proliferation capacity of CD19 CAR-T cells from high PD-1 expression T cells or healthy volunteer T cells, with or without PD-1 inhibitor were as the same (P>0.05) . ③The cytotoxicity to lymphoma cells of high PD-1 expression T cells and CAR-T cells were lower than that of these two T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer T cells (P<0.001) . There was no difference of the cytotoxicity between the CAR-T cells from high PD-1 expression T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer (P>0.05) . ④There was no difference of the expression of PD-1 in all CAR-T cell groups during the culture process (P>0.05) . There was no difference of mRNA level of PD-1 in all groups during the culture process (P>0.05) . ⑤The PD-1 expression of CAR-T cells increased by the time of culture after contacting with lymphoma cells (P<0.001) . The PD-1 inhibitors could antagonize this effect. There was no difference of mRNA level of PD-1 in all groups after contacting with lymphoma cells (P>0.05) . Conclusion: The PD-1 expression of CAR-T cells from high PD-1 expression T cells increased by the time of culture after contacting with lymphoma cells. However, the mRNA level of PD-1 of all groups did not change, even if PD-1 inhibitor was applied.
Antigens, CD19 ; Humans ; Programmed Cell Death 1 Receptor/genetics* ; RNA, Messenger ; Receptors, Antigen, T-Cell ; T-Lymphocytes

Patients with late-stage cancer commonly have distant lymph node metastasis; however, poor health often contraindicates surgical treatment. Although the quality of life and overall survival for these patients are low, there is neither a consensus nor a guide for treatment. Ablation technique and surrounding tissue damage are two possible reasons for limited study of radiofrequency ablation in patients with superficial distant lymph node metastasis. Here, we report two patients treated successfully with ultrasound-guided radiofrequency ablation for superficial distant lymph node metastasis. In these patients, deionized water was injected to the surrounding tissues of the lymph node to decrease heat injury. Results from these patients suggest that radiofrequency ablation may play an important role in the treatment of patients with distant lymph node metastasis.
Catheter Ablation ; methods ; Esophageal Neoplasms ; diagnostic imaging ; pathology ; surgery ; Female ; Humans ; Lymph Nodes ; diagnostic imaging ; surgery ; Lymphatic Metastasis ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; diagnostic imaging ; pathology ; surgery ; Neoplasm Staging ; Ultrasonography, Interventional ; methods

To establish a fingerprint for Cimicifugae Rhizoma from different producing areas. Column kromasil (4.6 mm x 250 mm, 5 microm) was employed with acetonitrile-0.1% formic acid solution as the mobile phase for gradient elution. The flow rate was 1.0 mL x min(-1), the detection wavelength was 254 nm. Twenty chromatographic peaks were extracted as the common peaks of fingerprint, and 21 batches of samples were compared and classified with such methods as similarity evaluation, cluster analysis and principle component analysis. The results showed 12 common peaks and three categories of samples. The method was so highly reproducible, simple and reliable that it could provide basis for quality control and evaluation of Cimicifugae Rhizoma from different producing areas.
Chromatography, High Pressure Liquid ; methods ; Cimicifuga ; chemistry ; Drug Stability ; Quality Control

OBJECTIVEThe purposes of this thesis were to study the behavior about workers exposed to dust and provide scientific basis for health promotion.

METHODSWe designed a questionnaire and carry it on the 746 dust workers in the 3 representative corporations of Machinery, Ceramic, and Metallurgy Industry. All data were input into computer. And a database was established with Excel. SPSS11.5 statistical analysis software was used to analyze the influence on protecting behavioral between the application of qualifications, different jobs, training or protection, and other aspects etc.

RESULTSThe rates were 94.4% and 75.3% about the regular physical examination and requirements for protective equipment. The rate of choosing an effective way of protection was generally low (15.4%). There was significant difference for among different educational background workers (P < 0.01). The rates of choosing an effective way of protection (20.3%), the regular physical examination (98.3%) and requirements for protective equipment (86.4%) in the dust workers who participated in the training of dust protection were superior than those who did not participated in the training. There was the significant difference (P < 0.05, P < 0.01). There was the significant difference for the rate of effective way of protection, regular physical examination, and requirements for protective equipment among the different corporations (P < 0.05).

CONCLUSIONSDust workers' using rate about the choosing an effective way of protection was generally low in Machinery, Ceramic, and Metallurgy Industry. Those who were not educated had a lower using rate about the protection behavior, regular physical examination, and requirements for protective equipment than those educated.

Adolescent ; Adult ; Ceramics ; Choice Behavior ; Dust ; prevention & control ; Female ; Humans ; Industry ; Male ; Metallurgy ; Middle Aged ; Occupational Exposure ; prevention & control ; Respiratory Protective Devices ; utilization ; Young Adult