Using the character of natural aggregation of CHO cells, and an ultrasonic and sedimentation column combined perfusion system to promote cells aggregation and retention into bioreactor,recombinant CHO cell strain MK3-A2 was cultured,which could secrete rhTNK-tPA, by a serum-free perfusion culture system. The culture periods in this two experiments were as long as 77 and 110 days respectively. The cells density reached 2?107 cells /ml. The average volumetric productivity of rhTNK-tPA was 89 mg/L?d, and the highest one was 216mg/L?d.The cells aggregation rate was approximately 90%, and the diameters of most of them were 285～570?m. During the perfusion culture the cells retention rate almost kept in 95% and the viability of cells was more than 85%.Thus, it means that aggregation culture with such perfusion system could be used to scale up produce biopharmaceuticals instead of microcarrier culture system.

One pair of degenerate primer was designed according to conserved motifs of the psaB (A2 subunit of photosystem I) of Chlamydomonas reinhardtii, Chlamydomonas moewusii, Chlorella vulgaris and Mesostigma viride, and a total RNA of Dunaliella salina (D. salina) was extracted with TRIzol reagent. A cDNA fragment, about 1.8kb in length, from green algal D. salina was obtained through RT-PCR method. The resulting PCR product was cloned into T-vector and screened to determine its sequence. Homologous analysis of the deduced amino acid sequence was performed by BLAST and subsequeqtly compared with GenBank data. The obtained cDNA sequence was 1815 bp long, which encodes 605 amino acids (GenBank accession number: AY820754). The sequence shared high homologue with the following psaB: Chlamydomonas reinhardtii 92%, Chlamydomonas moewusii 91%, Chlorella vulgaris 86%, Mesostigma viride 85%, Physcomitrella patens subsp. Patens 85% and Nephroselmis olivacea 84%. It can be concluded that the cloned sequence is psaB cDNA fragment from D. salina.
Algal Proteins ; genetics ; Amino Acid Sequence ; Animals ; Chlamydomonas reinhardtii ; genetics ; Chlorophyta ; genetics ; metabolism ; Cloning, Molecular ; DNA, Complementary ; genetics ; Molecular Sequence Data ; Photosystem I Protein Complex ; genetics ; Sequence Analysis, Protein ; Sequence Homology, Amino Acid

BACKGROUNDImpaired exercise capacity is one of the most common clinical manifestations in patients with chronic heart failure (CHF). The severity of reduced exercise capacity is an indicator of disease prognosis. The aim of the current study was to investigate the association between left heart size and mass with exercise capacity.

METHODSA total of 74 patients were enrolled in the study, with 37 having congestive heart failure (left ventricular ejection fraction (LVEF) < 0.45) and the other 37 with coronary heart disease (by coronary angiography) serving as the control group (LVEF > 0.55). Echocardiography and cardiopulmonary exercise test were performed. The multiply linear regression model was used to evaluate the association between echocardiogrphic indices and exercise capacities.

RESULTSThe study showed that left ventricular end diastolic/systolic diameter (LVEDD/LVESD), left atrial diameter (LAD) and left ventricular mass index (LVMI) were significantly enlarged in patients with chronic heart failure compared with controls (P < 0.01). The VO(2)AT, Peak VO(2), Load AT, and Load Peak in chronic heart failure patients were also significantly reduced compared with controls (P < 0.05), VE/VCO(2) slope was increased in patients with chronic heart failure (P < 0.01). Multivariate linear regression analysis indicated that the patients' exercise capacity was significantly associated with the left heart size and mass, however, the direction and/or strength of the associations sometimes varied in chronic heart failure patients and controls. Load AT correlated negatively with LVEDD in chronic heart failure patients (P = 0.012), while Load AT correlated positively with LVEDD in control patients (P = 0.006). VE/VCO(2) slope correlated positively with LAD (B = 0.477, P < 0.0001) in chronic heart failure patients, while the VE/VCO(2) slope correlated negatively with LAD in control patients (P = 0.009).

CONCLUSIONThe study indicates that the size of LVEDD and LAD are important determinants of exercise capacity in patients with CHF, which may be helpful to identify exercise tolerance for routine monitoring of systolic heart failure.

Aged ; Cardiac Volume ; physiology ; Echocardiography ; Exercise Test ; Exercise Tolerance ; physiology ; Female ; Heart Failure ; physiopathology ; Heart Ventricles ; diagnostic imaging ; pathology ; physiopathology ; Humans ; Male ; Middle Aged ; Radiography

Adult ; Female ; Humans ; Hyperlipidemias ; Pancreatitis ; Pregnancy ; Pregnancy Complications ; Pregnancy Trimester, Third

To measure particle size distribution of phenols in mainstream cigarette smoke aerosol,the particles of cigarette smoke aerosol were divided into 12 stages using single channel smoking machine coupled electrical low-pressure impactor (ELPI) and collected by 12 polyester films.The collected particles were weighted and then analyzed by ultra-high performance liquid chromatography-fluorescence detection (UPLC-FLD) to determine the 14 phenols in the different size particles.The results showed that the aerosols collecting method had good stability with relative standard deviation (RSD) of collected particles mass less than 10%.The analyzing results of 14 phenols by UPLC-FLD showed that the linear correlation coefficients(R2) were greater than 0.9959,with detection limits were less than 1.2 ng/cig and recoveries were 80.1%-115.0%.The distributions of 14 phenols with respect to smoke aerosol particle size were investigated.The results indicated that except 4-ethyl-2-methoxy-phenol not detected,the other 13 phenols were detected in mainstream cigarette smoke aerosol.The content of 13 phenols appeared increasing at first and then decreasing with increase of the particle size which distributed in a pattern similar to that of particle mass.All of 13 phenols were present in higher amounts in the medium size particles (0.261-0.722 μm) with peak content in particles 0.431 μm.The distribution of concentrations (ratio of content to particle mass) of 13 phenols in different size particles was different.The concentrations of phenol and mono-substituted phenol appeared to first increase and then decrease with increasing smoke aerosol particle size and were higher in medium size particles (0.261-0.772 μm).The concentrations of benzenediol and mono-substituted benzenediol were uniformly distributed in medium size particles (0.144-1.166 μm),and the concentration of disubstituted phenol was uniform throughout the particles of varying sizes.

OBJECTIVETo determine the differentially expressed serum proteins in patients with hepatoma carcinoma and identify a putative diagnostic marker.

METHODThe isobaric tags for relative and absolute quantitation (iTRAQ) labeling method and LC-MALDI-TOF/TOF MS detection method were used to quantify serum proteins in hepatocellular carcinoma patients (n =20) and healthy individuals (n =20). Real-time reverse transcription-polymerase chain reaction was used to verify the differentially expressed proteins by analyzing the corresponding mRNA expression levels in the hepatic carcinoma and healthy hepatocyte samples, as well as in 30 pairs of patient-matched hepatic carcinoma and adjacent normal tissue samples. Western blot analysis was used to verify the protein expression in hepatic carcinoma cells.

RESULTFifty-one proteins were significantly differentially expressed between the hepatic carcinoma group and healthy controls. The iTRAQ protein profile showed that the serum level of clusterin was significantly lower in hepatoma carcinoma patients. The mRNA level of clusterin was 20-fold lower in hepatic carcinoma cells than in healthy hepatocytes, and was 2.38-fold lower in hepatoma tissues than that in adjacent normal tissues. The clusterin protein levels were significantly lower in hepatic carcinoma cells (8.06 vs normal hepatocytes: 27.81; P less than 0.01).

CONCLUSIONThe serum expression of clusterin is significantly decreased in both serum and tissues of hepatic carcinoma patients. The relationship between hepatic carcinoma and clusterin should be evaluated in future studies.

Biomarkers ; Carcinoma, Hepatocellular ; metabolism ; Case-Control Studies ; Clusterin ; blood ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; Mass Spectrometry ; RNA, Messenger ; genetics ; Tumor Cells, Cultured

BACKGROUNDThere is no research, either at home or abroad, focusing on assessing the cardiopulmonary functional reserve and exercise tolerance in patients with pulmonary embolism (PE), but the benefits of early exercise are well recognized. The goals of this study were to assess cardiopulmonary functional reserve in treated PE patients using the inert gas rebreathing method of the cardiopulmonary exercise test (CPET), and to compare it with traditional methods.

METHODSCPET on the bicycle ergometer were performed in 40 patients with age, gender, body mass index, systolic blood pressure, and pulmonary function matched. The first group was the PE group composed of 16 PE patients (5 male, 11 female) who were given the standard antithrombotic therapy for two weeks. The second group was composed of 24 normal individuals (10 male, 14 female). Both groups were evaluated by cardiac ultrasound examination, 6-minute walking test (6MWT), and CPET.

RESULTS(1) Right ventricular systolic pressure (RVSP) in the PE group increased significantly compared to the control group, (34.81 ± 8.15) mmHg to (19.75 ± 3.47) mmHg (P < 0.01). But neither right atrial end-systolic diameter (RASD) nor right ventricular end-diastolic diameter (RVDD) in the PE patients had changed when compared with the controls. The 6-minute walk distance was significantly reduced in the PE patients compared with normal subjects, (447.81 ± 79.20) m vs. (513.75 ± 31.45) m (P < 0.01). Both anaerobic threshold oxygen consumption (VO(2)AT) and peak oxygen consumption (VO(2)peak) were significantly lower in patients with PE, while CO(2) equivalent ventilation (VE/VCO(2) slope) was higher; VO(2)AT (9.44 ± 3.82) ml×kg(-1)×min(-1) vs. (14.62 ± 2.93) ml×kg(-1)×min(-1) (P < 0.01) and VO2peak (12.26 ± 4.06) ml×kg(-1)×min(-1) vs. (23.46 ± 6.15) ml×kg(-1)×min(-1) (P < 0.01) and VE/VCO(2) slope 35.47 ± 6.66 vs. 26.94 ± 3.16 (P < 0.01). There was no significant difference in resting cardiac output (CO) between the PE and normal groups, whereas peak cardiac output (peak CO) and the difference between exercise and resting cardiac output (ΔCO) were both significantly reduced in the PE group; peak CO (5.97 ± 2.25) L/min to (8.50 ± 3.13) L/min (P < 0.01), ΔCO (1.29 ± 1.59) L/min to (3.97 ± 2.02) L/min (P < 0.01). (2) The 6-minute walk distance did not correlated with CPET except for the VO2 peak in patients with PE, r = 0.675 (P < 0.01).

CONCLUSIONSThe cardiopulmonary functional reserve was reduced in patients with PE. CPET is an accurate, quantitative evaluation of cardiopulmonary functional reserve for PE patients.

Aged ; Exercise Test ; methods ; Exercise Tolerance ; physiology ; Female ; Humans ; Male ; Middle Aged ; Oxygen Consumption ; physiology ; Pulmonary Embolism ; physiopathology ; therapy

OBJECTIVE: To explore the guiding significance of artificial intelligence for clinical practice of breast cancer after operation,and to evaluate the concordance of the Watson for oncology(WFO)with the breast cancer treatment guidelines of NCCN and Chinese Anti-Cancer Association.METHODS: A total of 100 patients who were diagnosed as breast cancer after accepting surgical operations from January 2016 to December 2017 in China-Japan Union Hospital of Jilin University were enrolled.The expressions of ER,PR,Her-2 and Ki-67 protein in cancer tissues after operation were detected by using immunohistochemitry,and inputed the clinical cases information of these 100 patients into the WFO system.Finally,the treatment recommendations were compared with the breast cancer treatment guidelinesof NCCN and the China Anticancer Association.The Kappa test was used to determine the concordance.RESULTS: There were 64 cases(64%)of all 100 patients with the same treatment plans,the clinical stage Ⅱ(42/44 cases,95.5%)to Ⅲ(15/16 cases,93.6%)and triple negative breast cancer(7/8 cases,87.5%)treatment showed a higher concordance(Kappa value ≥ 0.75);The age≥ 41 years old,Luminal A of the immunophenotype and pathological grade Ⅲ also had a certain concordance,and the concordance of 41-55 years old were average,but it is not ideal of the cases that older than 55 years old or the pathological grade Ⅲ;On the contrary,the treatment recommendations of clinical stage Ⅰ(7/40 cases,17.5%),especially for both the lymph node metastasis negative were very inconsistency(Kappa value <0).But there was no statistical significance in the cases of the age ≤ 41 years old,pathological gradeⅠto Ⅱ,Luminal B and HER-2 type of immunophenotype.CONCLUSION: WFO in the patients of clinical stageⅡ to Ⅲ and triple negative breast cancer(P<0.05)has a certain reference value in the afore-mentioned situations;And WFO could be considering in the cases of age ≥ 41 years of age or older,Luminal A of immunophenotype and pathological grade Ⅲ;In contrast,for clinical stage Ⅰ of early breast cancer patients,WFO is not recommended.WFO could offer some reasonable treatment recommendations in partial patients with artificial intelligent computer system,and it may provide clinical decisionsupport for clinicians,and it has some guiding significance in breast cancer postoperative clinical practice,clinical learning and research.

Objective To understand the health lifestyles and its influencing factors of scientific and technical workers in Zhejiang Province and to provide the basis for developing intervention measures.Methods A total of 800 non -medical scientific and technical workers were selected randomly by multi -stage stratified sampling method,and investigation of health lifestyles was carried out with questionnaire.Results A total of 748 valid questionnaires were collected with response rate of 93.50%,among which male accounted for 60.24%,smokers 26.29%,excessive drinkers 48.64% and physical activity lack 52.49%.There were statistical differences in the dietary intake of vegetables,fruits,beans and milk products between genders (all P <0.05 ).The respondents considered that pressure and entertainment were the main causes of smoking and excessive drinking.Multiple logistic regression analysis showed that the pressure of life was an independent risk factor (OR =1 .82),female and those with higher education level were the protective factors (OR =0.35 and 0.52 respectively).Conclusion Smoking,excessive drinking and physical activity lack are the main problems of lifestyles in non -medical scientific and technical workers of Zhejiang Province.Life stress may be one of the important influencing factors.Men in dietary needs to be strengthened.

Purpose To explore the β-catenin role in the process of invasion and metastasis of esophageal cancer.Methods Transfection-effective β-catenin gene segments of siRNA interference in human esophageal Eca-109 cells was used to downregulate β-catenin expression:CCK-8 multiplication experiment was carried out to observe the esophageal cancer cell proliferation.Transwell chambers experiment was used to observe its invasion,migration ability.Western blot was used to detect the expression of WISP2 and TCF4,E-cadherin protein.Results CCK-8 multiplication experiment showed that in the interference group (the efficient transfection of β-catenin down-regulation group by siRNA) cell proliferation ability significantly decreased as compared with the blank control group (the untreated group)and the negative control group (the transfection group meaningless fragments) (P ＜ 0.05),and there was no statistical significance between the blank and negative control groups (P ＞0.05).The invasion and migration ability of the interference group was lower than that in the blank control group and the negative control group (P ＜ 0.05) by the transwell chambers experiment.Western blot showed that the protein lever of WISP2 and E-cadherin in interference group was higher than those in the blank control group and the negative control group (P ＜ 0.05).TCF4 protein expression in the interference group was lower than that of the blank control group and the negative control group (P ＜ 0.05).Conclusions After the β-catenin expression is down-regulated,Wnt signaling pathway-related factors are significantly changed.It can be speculated that the silencing of β-catenin in Wnt signaling pathway may hinder the esophageal cancer cell proliferation by up-regulating E-cadherin expression to obstruct epithelial mesenchymal transition (EMT) and to inhibit tumor cell proliferation.Invasion and metastasis of the tumor are also inhibited by reducing TCF4 expression and promoting WISP2 downstream target genes expression.Therefore,β-catenin gene is expected to be a target for the treatment of esophageal cancer.