Objective To obtain highly expressing cell lines by inserting the glutamine synthetase (GS) screening system and replacing the promoter of the vector.Methods The mutation of the point BamHⅠwas induced to build a new vector pIRES2-EGFP.The marker gene GS was inserted by AseⅠ and NheⅠ, and the promoter hCMV was replaced by PacⅠand NheⅠ.The new vector pHGS1.0 and the vector pIRES2-enhanced screen fluorescein protein( EGFP)-B were inserted by the recombinant protein TEM8 ( 1-227 )-VEGFR1 domain2-IgG2 ( TV-IgG2 ) gene to analyze the advantages of the expression.Results The glutamine synsthetase is successfully inserted, the human cytomegalovirus replaced, and recombinant protein is increased 5-fold by human immunoglobulin quantification kit.Conclusion The GS system is a highly protein expressing system.

[ ABSTRACT] AIM:To investigate the genetic cause of 2 Chinese families with Marfan syndrome .METHODS:The clinical and laboratory investigations were performed in the 2 unrelated Chinese families .Family 1 had 1 patient with cardiac problem.Family 2 had 2 patients:one died, and the other with respiratory and cardiac problems .Next generation sequencing and Sanger sequencing in the Marfan syndrome causal gene FBN1 were performed in the patient , his unaffected sister and the parents of family 1.Sanger sequencing covering all the exons and intron-exon boundaries were performed in the patient and the parents in family 2.Bioinformatic analysis was engaged in the variations unravelled .Fifty healthy indi-viduals were also investigated in the same manner .RESULTS:Both patients were diagnosed with Marfan syndrome .A no-vel mutation c.4685G>A (p.Cys1562Tyr) was detected in the patient of family 1 but was absent in his parents and the unaffected sister .This is a previously unreported novel mutation .In the mutation a conserved Cys was substituted by a Tyr in amino acid 1562 affecting a TGF-βbinding domain and the secondary structure in the encoded protein .We also detected the mutation c.3706T>C (p.Cys1236Arg) in the patient of family 2.It was absent in the unaffected parents , and there-fore was a de novo mutation too.This mutation has been previously reported and known to be associated with neonatal Marfan syndrome .Both mutations were absent in the 50 healthy controls .We also compared the genotype and phenotypes of the 2 families.CONCLUSION:We report 2 de novo mutations in 2 Chinese families with Marfan syndrome .One of the 2 mutations is novel.The phenotype of the mutation c.4685G>A(p.Cys1562Tyr) in family 1 is associated with classical Marfan syndrome, while that of c.3706T>C (p.Cys1236Arg) in family 2 is with neonatal type of Marfan syndrome .De novo mutations may be a cause for a proportion of mutations underlying the disease .The novel mutation also expends the mutational spectrum of the FBN1 gene.

Objective To study the neuroprotective role of TFP5 in a MPTP-induced mouse model of Parkinson's disease (PD).Methods C57BL/6 mice were used as experimental animals.Briefly, 5 consecutive days of intraperitoneal injection of 25 mg/Kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was applied to induce mouse PD model.The mice were randomized into 5 groups including control group,model group, scrambled TFP5 peptide (Scb) group, TFP5 group and roscovitine group.On the 7th day after the first injection of MPTP,behavior tests were performed, and then western blot method was employed to detect the expression of p25 and phosphorylated MEF2D in substantia nigra.Tyrosine hydroxylase (TH) immunohistochemical staining was performed to observe the apoptosis of dopaminergic neurons in substantia nigra pars compacta (SNpc) 28 days after the first injection of MPTP.Results MPTP increased the expression of p25 (0.48±0.10 vs 0.26±0.02, P＜0.05) and phosphorylated MEF2D (0.81±0.10 vs 0.22±0.02, P＜0.05) in substantia nigra, but decreased the number of dopaminergic neurons in SNpc (348.67±24.40 vs 463.29± 19.61, P＜0.05),resulting in motor impairment in the model mice (P＜0.05).Intraperitoneal injection of 30mg/Kg of TFP5 for 3 days effectively reduced the excessive phosphorylation of MEF2D (0.25 ± 0.12 vs 0.81 ± 0.10, P＜ 0.05) in substantia nigra, rescued dopaminergic neuron reduction of SNpc (422.92±8.41 vs 348.67±24.40, P＜0.05), and improved the motor ability of the model mice (P ＜0.05).Roscovitine exerted almost same neuroprotective role as TFP5 ,while Scb had no protective effect.Conclusion TFP5 can rescue MPTP-induced damage of dopaminergic neurons in substantia nigra, and thus improve motor impairment of model mice,which may be mediated by the inhibition of Cdk5/p25 activity.

Objective To compare the effect of treating hyperglycemia on insulin pump therapy in newly diagnosed type 2 diabetes mellitus(T2DM) and the patients with failure to oral antidiabetic therapy. Methods Selecting 32 patients with newly diagnosed T2DM(NDM group)and 64 T2DM patients with failure to oral antidiabetic therapy(ODM group),which were treated by insulin pump,having no significant difference of the level of blood glucose,body mass index(BMI),age,proportion of sex between NDM group and ODM group. Results (1)The mean time and the maximal dosage of insulin for blood glucose to be targeted in NDM group were all lower than those in ODM group(P all

Objective: To investigate thestatus and control of blood lipid level among patients with type 2 diabetes mellitus（T2DM）in rural communities of Zhejiang Province，and to provide evidence for blood lipid control for T2DM. Methods: A sample of 10 343 patients with T2DM managed by communities from Jiashan，Suichang and Yongkang in 2016 were recruited. Through the diabetes registry system，physical examination and laboratory tests，data of demographic features，blood pressure，body mass index（BMI），waist circumstance（WC），glycated hemoglobin（HbA1c），total cholesterol（TC），triglyceride（TG），low-density lipoprotein cholesterol（LDL-C）and high-density lipoprotein cholesterol（HDL-C）were collected to learn the status of blood lipid control. Logistic regression analysis was conducted to explore the influencing factors for blood lipid control. Results: The control rate of TC，TG，LDL-C and HDL-C in patients with T2DM was 29.84%，58.72%，48.25% and 61.27%，respectively. About 11.76% of patients had all the four indicators in control，while 9.22% of patients failed in all. The higher control rates of all of the four indicators were seen in males than females，in older age，in lower BMI and in normal people than in central obese people（all P<0.05）. The results of multivariate logistic regression analysis showed that sex（OR=3.556，95%CI：3.070-4.119），age（OR=1.130，95%CI：1.060-1.204），WC（OR=0.989，95%CI：0.980-0.998）， BMI（OR=0.768，95%CI：0.688-0.857），systolic blood pressure（OR=0.991，95%CI：0.984-0.999），HbA1c level（OR=0.914，95%CI：0.876- 0.953），smoking（OR=0.768，95%CI：0.639-0.924）and drinking（OR=0.688，95%CI：0.536-0.884）were associated with the control of TC，TG，LDL-C and HDL-C in patients with T2DM. Conclusion The control rate of blood lipid is low in patients with T2DM in rural communities of Zhejiang Province，surveillance and interventions should be focused on sex，overweight/obesity，smoking，alcohol intake，blood glucose and blood pressure.

G mutation of SLC26A4, the parents and the second child were carriers of the same mutation, while the fetus had a wild-type form. Conclusion It is feasible to identify deafness related genes by screening for GJB2 and SLC26A4 mutation, thus providing correct prenatal diagnosis and avoiding deaf delivery of baby.

Objective: To learn the prevalence of dyslipidemia and its influencing factors among patients with type 2 diabetes mellitus (T2DM) in rural areas of Zhejiang Province, and to provide evidence for dyslipidemia control. Methods: A sample of 10 343 patients with T2DM managed by communities from three counties (Jiashan,Suichang and Yongkang) were recruited. Through the residents’ health record system and specific investigation, demographic features,physical examination results of blood pressure,height, weight, waist circumstance (WC), glycated hemoglobin (HbA1c),total cholesterol (TC),triglyceride (TG),low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were collected to analyze the spectrum of dyslipidemia. A logistic regression model was established to explore the associated factors of dyslipidemia. Results : The prevalence rate of dyslipidemia among the patients with T2DM in rural communities was 66.91%. The prevalence rates of higher level of TC, TG, LDL-C were 41.76%, 41.28%, 15.89%, respectively, while the prevalence rateof lower level of HDL-C was 16.92%. The prevalence rates of purely high TC,high TC with TG and purely high TG were 11.84%,11.75% and 11.47%,respectively. Higher level of TG with lower level of HDL-C was common in males (11.32%) and higher level of TC with TG was common in females (14.60%). The results from multivariate logistic regression analysis indicated that females(OR=1.499,95%CI:1.352-1.663),age(OR=0.992,95%CI:0.988-0.996),poor control of HbA1c(OR=1.241,95%CI:1.141-1.351), abdominal obesity(OR=1.169,95%CI:1.064-1.285),overweight(OR=1.384,95%CI:1.257-1.524)and obesity(OR=1.582,95%CI:1.352-1.852)were associated with dyslipidemia. Conclusions The prevalence of dyslipidemia is relatively higher among the patients with T2DM in rural communities of Zhejiang Province. Higher level of TC and TG contributed to most dyslipidemia. The patients with T2DM who are females,have poor control of plasma glucose and suffer from obesity should be under surveillance.

OBJECTIVETo observe blood uric acid levels and Goldstein grading, as well as their correlation in Wilson's disease (WD) patients with different Chinese medical syndrome types.

METHODSTotally 906 WD patients in line with inclusive criteria were assigned to 6 groups, i.e., the heart spirit confused by phlegm group (HSCP, 26 cases), the phlegm-fire disturbing heart group (PFDH, 90 cases), the retention of damp-heat group (RDH, 113 cases), deficiency of qi and blood group (DQB, 168 cases), the deficiency of Gan-yin and Shen-yin group (DGYSY, 327 cases), the deficiency of Gan and Shen group (DGS, 182 cases) due to different Chinese medical syndrome types. Recruited were another 160 healthy subjects having similar ages and diet structures, who came for medical examinations, as the healthy control group. Venous blood was collected from the medial cubital vein of each-patient on an empty stomach in early mornings to detect blood uric acid levels. Results Blood uric acid levels were lower in each syndrome type group than in the healthy control group (146.08 +/- 67.24 micromol/L in the HSCP group; 157.08 +/- 69.77 micromol/L in the PFDH group; 162.58 +/- 97.72 micromol/L in the RDH group; 156.20 +/- 62.63 micromol/L in the DQB group; 161.83 +/- 111.23 micromol/L in the DGYSY group; 194.41 +/- 90.01 micromol/L in the DGS group; 242.39 +/- 87.55 micromol/L in the healthy control group, P < 0.01). Blood uric acid levels were higher in the DGYSY group than in the other 5 syndrome groups (P < 0.01). Correlation analyses between Goldstein grading and blood uric acid showed that, along with increased Goldstein grade (that was aggravating disease conditions), WD patients' blood uric acid levels decreased (P < 0.01).

CONCLUSIONSWD patient's blood uric acid levels decreased more. Blood uric acid levels and Goldstein grading were different in various Chinese medical syndrome types. Blood uric acid levels had certain value in assessing the severity of WD.

Asian Continental Ancestry Group ; Heart ; Hepatolenticular Degeneration ; blood ; classification ; diagnosis ; Humans ; Medicine, Chinese Traditional ; Syndrome ; Uric Acid ; blood

OBJECTIVETo construct and express anti-human RBC and HIVgp160 fusion protein for rapid detection of antibody to HIV.

METHODSThe gene of the anti human RBC ScFv and HIV antigen were constructed together into expression vector. The fusion protein was expressed in E. coli.

RESULTSThe fusion protein was proved to be able to bind both anti-RBC and HIVgp160. It could cause agglutination of human RBC when HIVgp160 was present.

CONCLUSIONThe fusion protein has the potential in rapid detection of HIV.

Antibodies, Monoclonal ; immunology ; isolation & purification ; Autoantibodies ; immunology ; isolation & purification ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Erythrocytes ; immunology ; Gene Expression ; Genetic Vectors ; genetics ; HIV Antibodies ; blood ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; metabolism ; HIV Seropositivity ; blood ; Hemagglutination Tests ; methods ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

BACKGROUNDTo establish a molecular detection and typing assay for identification and typing of human enteroviruses (HEV) which is suitable for clinical detection and epidemiologic research.

METHODSUsing both primers specific for HEV genus and HEV typing primers and reverse transcription polymerase chain reaction (RT-PCR) the authors detected preliminarily HEV by agarose gel electrophoresis and then identified serotype through nucleotide sequence analysis of RT-PCR amplicons. The monospecific antisera neutralization was applied to validate the typing results.

RESULTSThe serotype of 18 suspicious HEV samples was identified: 4 Coxsackievirus type A24 (CVA24), 3 CVB3, 1 CVB2, 1 CVA9, 1 CVA15, 1 Echovirus type 3 (E3), 1 E6, 1 E9, 1 E11, 1 E14, 1 E33 and 1 Rhinovirus type 9. The result was validated by monospecitive antisera neutralization.

CONCLUSIONThis RT-PCR assay for HEV detection and typing may be suitable for clinical detection and epidemic research since this method is sensitive and specific for direct identification and typing of HEV.

DNA Primers ; Enterovirus ; classification ; genetics ; Enterovirus Infections ; diagnosis ; Humans ; RNA, Viral ; genetics ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Serotyping ; methods