[摘 要] 目的：探讨在靶向HER2的CAR的CD3ζ链胞内区引入YRHQ基序对CAR-T细胞的特异性杀伤活性及免疫记忆形成的影响。方法：通过DNA合成获得包含靶向HER2的编码抗原受体H28ζ或H28ζ(YRHQ)的DNA片段，通过慢病毒载体将不同CAR的DNA片段分别转导健康人外周血T细胞，制备靶向HER2的H28ζ-CAR-T及H28ζ(YRHQ)-CAR-T细胞。扩增过程中对不同CAR-T细胞进行计数，FCM检测CAR的表达率。将CAR-T细胞分别与HER2阳性的SKOV3、MDA-MB-453或HER2阴性的MCF-7细胞共培养，LDH释放法检测其杀伤活性，ELISA法检测IL-2、IFN-γ和颗粒酶B的水平，WB法检测STAT3磷酸化水平及免疫检查点分子TIM-3和PD-1的表达，通过FCM检测CCR7、CD45RO的表达，分析CAR-T细胞的表型。结果：H28ζ-CAR-T和H28ζ(YRHQ)-CAR-T细胞扩增能力较好，体外培养7 d时扩增4~5倍。H28ζ-CAR和H28ζ(YRHQ)-CAR表达率分别为（33.3±2.85）%和（28.30±3.2）%。H28ξ(YRHQ)-CAR-T细胞的杀伤活性较H28ζ-CAR-T细胞更高（P<0.05）。经HER2抗原刺激后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞的STAT3磷酸化水平较H28ξ-CAR-T细胞明显升高（P<0.01）；而两者间PD-1和TIM-3的表达无明显差异。未经抗原刺激的CAR-T细胞CCR7和CD45RO表达与正常T细胞比较差异无统计学意义（均P>0.05），与SKOV3细胞共培养后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞中TEM细胞比例明显增加、TCM细胞比例明显减少（均P<0.05）。结论：在CD3胞内区引入YRHQ基序可在一定程度上提高CAR-T细胞的杀伤潜力。

Objective To investigate the early diagnostic and the prognostic value of amplitude integrated e-lectroencephalography(aEEG)in neonates with hypoxic - ischemic encephalopathy( HIE). Methods The medical data subjects were admitted to the Department of Neonatology,Children's Hospital of Hebei Province from January 2012 to December 2013. Ninety term infants with HIE were divided into 3 groups(mild,moderate and severe),and they were investigated respectively by aEEG monitoring within 12 hours after birth,and all of the infants accepted cranial magnetic resonance imaging(MRI)on 3 to 7 days after birth. The outcomes by MRI were divided into 3 groups(mildly abnor-mal,moderately abnormal and severely abnormal). The findings of aEEG monitoring were divided into 3 groups(nor-mal,mildly abnormal and severely abnormal),the correlation between the findings of aEEG and the severity of HIE was analyzed. The correlation between the results of aEEG and severity of MRI were analyzed. Behavior evaluation of infants with HIE were applied by Neonatal Behavioral Neurological Assessment(NBNA)score on 7 d,14 d,28 d after birth and prognostic evaluation of children with HIE was conducted based on Children's Development Center of China infants intelligence development test at 12 months of age. Results (1)Among 90 term infants with HIE,44 cases(48. 9% ) had mild HIE,29 cases(32. 2% )moderate and 17 cases(18. 9% )severe HIE;49 cases(54. 4% )had mildly ab-normal MRI,23 cases(25. 6% )moderately abnormal MRI and 18 cases(20. 0% )severely abnormal MRI;43 cases (47. 8% )had normal aEEG,25 cases(27. 8% )mildly abnormal and 22 cases(24. 4% )severely abnormal aEEG. (2)The findings of aEEG classification were correlated with the severity of HIE(r = 0. 970 7,P ﹤ 0. 001). The findings of aEEG classification were correlated with the severity of MRI(r = 0. 933 5,P ﹤ 0. 001).(3)NBNA score with severe-ly abnormal aEEG was obviously lower than that with the mildly abnormal aEEG. NBNA score on 7 d after birth:(14. 1 ± 4. 2)scores vs(32. 2 ± 2. 3)scores,on 14 d after birth:(17. 8 ± 5. 6)scores vs(33. 4 ± 2. 1)scores,on 28 d after birth:(18. 9 ± 8. 4)scores vs(34. 6 ± 2. 6)scores,and the difference was statistically significant(all P ﹤0. 05).(4)The infants with HIE were followed at 12 months of age. The development quotient mental development in-dex(MDI)and psychomotor development index(PDI)with severely abnormal aEEG were obviously lower than that with the mildly abnormal aEEG[MDI(65. 1 ± 4. 1)scores vs(89. 1 ± 6. 7)scores,PDI(67. 5 ± 10. 1)scores vs(90. 7 ± 8. 3)scores],the difference was statistically significant(all P ﹤ 0. 05). Conclusion It is indicated that aEEG can early evaluate the severity of HIE and help predict its neurological outcome.

Objective To analyze the changes of WBC classification,sugar and protein in cerebro-spinal fluid(CSF)and pathogenic bacteria of neonatal purulent meningitis.Methods Thirty-one neonates with bacterial meningitis in our department of neonatology from June 201 1 to June 2013 were enrolled,and the clinical features,pathogenic bacteria,laboratory examination of CSF were analyzed.Results Fever (90.3%),convulsions(67.7%)and changed consciousness(58.1 %)were common clinical symptoms.The incidences of other nervous system abnormal signs such as gastrointestinal dysfunction(25.8%),abnormal breathing(16.1 %),cervical resistance(16.1 %),bulging fontanel(9.7%)were lower.The Gram -negative bacteria was more than Gram -positive in both blood and CSF culture.The escherichia coli was the most common bacteria,with positive rate of 38.1 %(8 /21 )in blood culture and 55.5%(5 /9)in CSF culture.The germiculture positive rate in CSF was lower than in blood culture (29.0% vs.67.7%).Polymorphonuclear leukocyte(PMN)[(79.61 ±12.06)%]was the most predominant cell of the leukocyte classification in CSF within 1 week in all cases,PMN was still predominant in 1 to 2 weeks in 7 cases,while only 2 cases in 2 to 3 weeks still dominated by PMN,PMN was not the predominant cell 3 weeks later.Conclusion In the typi-cal neonatal purulent meningitis,PMN was the predominant cell in CSF within the first week,but the propor-tion of monocyte gradually increased and was dominant later.Escherichia coli was a common bacteria caused by this disease.

Objective To evaluate the clinical efficacy and safety of flexible fiberoptic bronchoscopy (FFB) and bronchoalveolar lavage (BAL) in treatment of neonatal atelectasis.Methods Eligible patients, who were diagnosed as neonatal pulmonary atelectasis and admitted consecutively to the neonatal intensive care unit of the Children's Hospital of Hebei Province from January 2013 to January 2015, were included in the study.They were randomly assigned to FFB group (n=30) and control group (n=28).Newborns in the FFB group received BAL under FFB, while those controls received tracheal irrigation after intubation.The duration of lung recruitment, oxygen exposure and antibiotic administration, hospital stay, culture results of respiratory secretions, prognosis and total expenses during hospitalization were compared between the two groups.Complications of FFB were also recorded.Chi-square test and t-test were performed for statistical analysis.Results (1) In the FFB group, atelectasis occurred in the upper fight lobe (n=26), upper lobes of both sides (n=1), lower right lobe (n=2) and lower left lobe (n=1), while in the control group, atelectasis occurred in the upper right lobe (n=26), lower left lobe (n=1) and middle right lobe (n=2) (x2=0.094, P ＞ 0.05).(2) The positive rate of bacteria culture results showed no difference between bronchoalveolar lavage fluid in FFB group and tracheal secretions in the controls [43％(13/30) vs 32％(9/28), x2=0.770, P ＞ 0.05].(3) The duration of lung recruitment, antibiotic administration and hospital stay of the FFB group were all shorter than those of the control group [(4.7±3.4) vs (7.4±6.6) d, (14.0±4.5) vs (20.3±10.9) d, (15.1±4.7) vs (21.8±12.3) d, t=-5.718, 8.604 and 7.733, all P ＜ 0.05].(4) Among babies in the FFB group, nine experienced fever and returned to normal after physical cooling;three showed more shadow in chest X-ray with aggravated dyspnea during a short period, and relieved 12 hours later;two had minimal hemorrhage from tracheal mucous membrane;one showed crying hoarse.Serious complications, such as pneumothorax, massive bleeding or cardiac arrest, did not occurred.No death or refuse of treatment was reported.Conclusion FFB and BAL is much more effective than tracheal irrigation after intubation in treatment of neonatal atelectasis without any severe complications.

Fermentative production of lactic acid, an important bio-based chemicals, has made considerable progress. In addition to the food industry and production of polylactic acid, lactic acid also can be used as an important platform chemical for the production of acrylic acid, pyruvic acid, 1,2-propanediol, and lactic acid esters. This article summarizes the recent progress in biocatalytic production of lactic acid derivatives by dehydration, dehydrogenation, reduction, and esterification. Trends in the biotransformation of lactic acid are also discussed.
Acrylates ; metabolism ; Bacteria ; genetics ; metabolism ; Biotechnology ; methods ; Biotransformation ; Fermentation ; Industrial Microbiology ; methods ; Lactic Acid ; metabolism ; Propylene Glycol ; metabolism ; Pyruvic Acid ; metabolism ; Yeasts ; genetics ; metabolism

Objective To detect the immune effect of FbaAmAb2 against the surface protein of group A Straptococcus (GAS),and explore the pathogenesis and therapy of GAS infections.Methods By subclonal and bacterial ELISA,the positive hybridoma cells were screened that can produce better titers of FbaAmAb2 against GAS-surface FbaA protein,and were injected into the peritoneal cavities of BALB/c mice to produce ascites.The collected ascites were performed to dilute,as follows,original ascite,1:2,1:4,1:8,and 1:16 to test tube agglutination.Based on the results,we selected appropriate dilution to passively immunize mice,and then challenged the mice with GAS,evaluating FbaAmAb2 neutralizing ability with GAS in mice by the survival rate of the immunized mice.Whether FbaAmAb2 could inhibit the binding of factor H to GAS was confirmed by the invasive inhibition assay.Results The IgG titer of bacteria solution ELISA is 1:160 and the titer of tube agglutination is 1∶8.The protect rates of FbaAmAb2 on preventing mice with GAS infections are as follows:66.67％ in original ascite and 1:2 diluted groups,and 50％ in 1:4 diluted group.Mice in each experimental group were evoked significantly protective immune responses compared with the PBS control by SPSS analysis.FbaAmAb2 can competitively inhibit factor H binding to the surface proteins FbaA of GAS,which decreased the entry of GAS into the cytoplasm of human epithelial cells through the binding of factor H.Conclusion FbaAmAb2 is promising to be used in emergent prevention or the clinical therapy for GAS infection and it is promising starting points for pharmacologic targeting and further development of new therapeutic agents for GAS.

Objective The truncated fibronectin-binding proteins A (Fba protein) were cloned and expressed, then animals were immunized with Fba protein and subsequently challenged with group A streptococcus (GAS) to further investigate protective antibodies induced by each domain and determine the most immunogenic domain. Methods Fba proteins, which were divided into four overlaps based on the structural domains, were truncated and expressed. The fba truncated genes were amplified by polymerase chain reaction (PCR) with SSI-9 of GAS as template, and cloned into prokaryotic expression plasmid pGEX-2T and expressed in E. coli BL-21. The products were confirmed by Western blot and purified by affinity chromatography column. Female BALB/c mice were immunized with the four truncated proteins respectively, with phosphate buffered solution (PBS) as control. The serum IgG of mice was detected by enzyme-linked immunosorbent assay (ELISA). After the third immunization, mice were challenged with fatal dose of GAS (M+ Fba+ ) to evaluate the protective rates in each group. The data were compared by analysis of variance and Fisher's exact test.Results Prokaryotic expression plasmids of pGEX-2T/FbaAl, pGEX-2T/FbaA2, pGEX-2T/FbaA3 and pGEX-2T/FbaA4 were successfully constructed and the four truncated proteins were expressed and purified successfully. Serum levels of IgG in each experimental group gradually increased with immunization with Fba protein more times. After the third immunization, the IgG titer against FbaA2 1290.2, P<0. 01). After GAS challenge, four out of eight mice were protected in FbaA2 protein group, while two out of eight mice in FbaAl protein, FbaA3 protein and FbaA4 protein groups,respectively (P<0. 05). Conclusions Four truncated Fba proteins are constructed and expressed successfully. Truncated FbaA2 protein could be able to induce strongest protective immune response.

Objective To Discuss the impacts of different dosage of atorvastatirs on serum hsCRP,IL-10 and MCP-1 levels on post-intervention patients with coronary stenting. Methods 93 post-intervention patients with coronary stenting were selected and randomly divided into 3 groups.Each group took different dosage of oral atorvastatins after the operation for more than one week.The dosage for each group was 10 mg,20 mg and 40 mg,respectively.Each patient was phlebotomized for three times,which are 24 hours before the operation,24 hours after the operation and one week after the operation.Serum MCP-1,IL-10 and hs-CRP levels were measured by enzyme linked immunosorbent assay(ELISA)and immunoturbidimetry(ITM). Results Serum hs-CRP and MCP-1 levels of post-intervention patients were significantly higher than those of pre-intervention.This illustrated that the serum hsCRP and MCP-1 levels were closely related to PCI.Serum hs-CRP and MCP-1 levels decreased in those patients one week after operation which proves they are negatively correlated with the dosage of atorvastatins.There was no statistic evidence to prove the correlation between different dosage of atorvastatins and the level of serum IL-10.The ratio of MCP-1/IL-10 at 24h post-intervention patient was significantly higher than pre-intervention,which proves the ratio was negatively correlated with the dosage of atorvastatins. Conclusion Atorvastatins decreases serum MCP-1 and hs-CRP levels after PCI.Serum MCP-1 and hs-CRP levels were negatively correlated with the dosage of atorvastatins.

Microbial metabolic engineering and synthetic biology are important disciplines of microbial technology nowadays. Microbial cells are fast growing, easy to be cultivated in large scale, clear in genetic background and convenient in genetic modification. They play an important role in many domains. Microbial cell factory means an artificial microbial metabolic system that can be used in chemical production. The construction of a microbial cell factory needs transferring of multiple genes or a whole metabolic pathway, which may cause some problems such as metabolism imbalance and accumulation of mesostates. This review focuses on the regulation strategies of different levels involving simultaneous engagement of multiple genes. Future perspectives on the development of this domain were also discussed.
Bacteria ; genetics ; Fungi ; genetics ; Gene Expression Regulation ; Gene Regulatory Networks ; genetics ; Genetic Engineering ; Industrial Microbiology ; Metabolic Engineering ; methods ; Metabolic Networks and Pathways ; genetics

Spore surface display is one of attractive microorganism surface display systems. With the advantage of resistance attribute and specific assembly pattern, the technology of spore surface display now is attracting more and more attention. According to the current reports and main achievements of spore surface display, the structure and assembly of spores, the principle for construction and some existing spore surface display systems were elaborated in this paper. Now with the unique property of spores, the technique is not only widely used in production of vaccines but also has great applied potential in the field of biocatalysis and cell-factory.
Bacillus subtilis ; genetics ; metabolism ; Biocatalysis ; Biotechnology ; methods ; Gene Expression Regulation, Bacterial ; Genetic Engineering ; methods ; Recombinant Proteins ; genetics ; metabolism ; Spores, Bacterial ; genetics ; metabolism ; Tetanus Toxoid ; genetics ; immunology