Objective To construct a luciferase reporter plasmid containing interferon regulatory factor 3(IRF-3)human gene promoter and to evaluate promoter activity in human embryonic kidney(HEK)-293 cells.Methods The 1 000 bp fragment was amplified by PCR with human genomic DNA as a template and was directionally cloned into pGL3-basic multiple cloning sites to construct the luciferase repor-ter plasmid pGL3-pIRF-3.Transfection of HEK-293 cells with the promoter-driven lucife-rase construct was performed to induce lucife-rase gene expression and calculate the relative luciferase activity unit(RLU).Promoter sequence of 1 000 bp upstream of transcription initiation site of IRF-3 was analyzed by using Promoter 2.0 Prediction software.Results DNA sequencing and restriction endonuclease analysis verified the successful construction of the plasmid pGL3-pIRF-3.This IRF-3 promoter exhibited a strong promoter activity with an increase of 42.2-fold of RLU in HEK-293 cells when compared with pGL-3 basic vector.The transfection experiment confirmed that the levels of its activation were significantly higher than that in controls in HEK-293 cells.Function analysis of IRF-3 promoter disclosed seve-ral GATA-1 and specific protein 1(Sp1) sites and E2F in minimal promoter region.Conclusion The plasmid pGL3-pIRF-3 promoter is successfully constructed and has a strong basal promoter activity in HEK-293 cells.

Objective To explore the relationship between X - linked spondyloepiphyseal dysplasia tarda (SEDL) gene escaping X chromosome inactivation( XCI) and SEDL phenotype. Methods RT - PCR was performed on total RNA which was isolated from blood samples of patients, female carriers and controls. Patients and female carriers were selected from the pedigree with SEDL caused by the mutation (IVS2 - 2A→C) of the gene. cDNA was analyzed by polyacrylamide gelelectrophoresis(PAGE). Results PAGE data indicateed that female carriers expressed both normal and mutant SEDL mRNA,meaning the SEDL gene escaping XCI. Family investigation showed carrier females in the SEDL pedigree presented no symptoms. Conclusions The SEDL gene escaping X chromosome in-activation is firstly identified from human body. This may explain that carrier females present no symptoms.

Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.
Chromatography, High Pressure Liquid ; DNA Primers ; Down-Regulation ; Ergosterol ; metabolism ; Haploidy ; Intramolecular Transferases ; genetics ; Polymerase Chain Reaction ; Saccharomyces cerevisiae ; genetics ; Squalene ; analogs & derivatives ; metabolism

Pichia pastoris is one of the most important systems used in the field of molecular biology for the expression of recombinant proteins. The system has advantages of high expression, high stability, high secretion, easy high-density fermentation and low cost. Many factors affect the expression of recombinant protein, such as gene copy number, codon usage preference, type of promoter, molecular chaperones, glycosylation, signal peptide and fermentation process. In this review, research advances of the above aspects are summarized, which lay a foundation for improving the expression of recombinant proteins in P. pastoris.
Fermentation ; Gene Dosage ; Glycosylation ; Molecular Chaperones ; Pichia ; metabolism ; Promoter Regions, Genetic ; Protein Sorting Signals ; Recombinant Proteins ; biosynthesis

Objective To evaluate the feasibility of application of PBL method on otolaryngology education for medical undergraduates.Methods Totally 61 medical undergraduates of Class 2009 at Peking Union Medical College were enrolled into this study.There are 22 males and 39 females and aged 23-26 years old.They were grouped randomly into two groups: the PBL group (n=29) and the traditional teaching method group (n=32).Questionnaires were collected and statistical analyses were conducted.Results The scores of the evaluations for education of otolaryngology after teaching were much better than those before teaching in the PBL group in most aspects according to the questionnaires (P<0.05).In traditional teaching method group, there are only few aspects` scores much better than those before teaching.Comparing the two teaching methods shows, in most aspects, the scores of PBL group are much higher than those of the traditional teaching method group.ConclusionsCombination of PBL with other teaching method will be an ideal way for medical education in otolaryngology.

Objective To probe the relationship between the connectivity of premotor area detected by resting state functional MRI and stereotyped behavior of ASD.Methods 39 children patients with ASD and 42 healthy controls with matched age,sex and IQ were recruited.All the 81 subjects performed scaling with behavior scale index and underwent resting-state fMRI scans.After the fMRI data precession,the premotors in left and right sides were selected as ROI seeds to perform functional connectivity.Results Compared with healthy controls,children patients showed significant increased connectivity between the left premotor area and posterior cingulate cortex,as well as decreased functional connectivity between the left premotor area and the left insula.In addition, a negative linear correlation was showed between the connectivity of the left premotor and the insula and the behavioral scores.Con-clusion Imbalanced premotor functional connectivity may be one possible mechanism of stereotyped behavior of ASD,representing an attention deficit and/or impaired sensory perception to the external salient stimuli.

Objective To observe the relationship between computed tomography-gross tumor volume (GTV) and non-surgical T stage in patients with esophageal squamous cell carcinoma (ESCC) and the survival rates of patients with different GTVs,and to investigate the impact of GTV on the prognosis of ESCC after three-dimensional radiotherapy.Methods A retrospective analysis was performed on 223 ESCC patients without lymph node metastasis and distant metastasis who were hospitalized from July 2003 to January 2009.The prescribed doses of three-dimensional radiotherapy ranged from 50-70 Gy.These patients were divided into 3 or 4 groups according to different percentile intervals of GTVs.The Spearman rank correlation analysis was used for investigating the relationship between non-surgical T stage and GTV.The Kaplan-Meier method was used for calculating survival rates,and the log-rank test was used for survival difference analysis.Results The follow-up rate was 98.2％.A total of 163 patients were followed up for at least 3 years.The median GTVs of patients with T1 +2 ESCC,T3 ESCC,and T4 ESCC were 19.31 cm3,33.69 cm3,and 41.25 cm3,respectively,exhibiting a positive correlation between non-surgical T stage and GTV (P =0.000).The 5-year survival rates were 59％,43％,and 24％ (P =0.000) in 3 GTV-based groups and were 55％,51％,31％,and 24％ (P =0.004) in 4 GTV-based groups.The primary cause of death for the patients with GTVs of ≤35 cm3 and ＞35 cm3 was failure of local control (57.9％ vs 52.1％) ; 21.9％ and 13.8％ of the patients with a GTV of ＞ 35 cm3 died of uncontrol and excessive bleeding,versus 9.4％ and 3.1％ of the patients with a GTV of ≤ 35 cm3 (P =0.046 ; P =0.029) ;2 of the patients with a GTV of ＞ 35 cm3 died of grade 5 radiation pneumonitis.Conclusions GTV is positively correlated with nonsurgical T stage in esophageal cancer patients who receive radiotherapy.The 3-level and 4-level grading of GTV can be used for prognostic evaluation,and the 3-level grading of GTV is more closely related to prognosis.The patients with a larger GTV have higher incidence of uncontrol and bleeding and risk of treatment-related death than those with a smaller GTV after radiotherapy.

ObjectiveTo explore the application of diffusion-weighted magnetic resonance imaging (DWMRI) in precise radiotherapy of esophageal carcinoma.MethodsThirty-seven patients with biopsy proven esophageal cancer from March 2010 to January 2011 were included.To delineate the gross tumor volume (GTV) using CT and DWMRI images,each patient was examined by DWMRI and CT scan using the same position before radiotherapy.To compare the maximum diameters and volumes of tumor between CT and DWMRI. The tumor lengths measured by esophagogram,esophagoscope,CT and DWMRI were compared.ResultsTumor lengths under esophagogram,esophagoscope,CT and DWMRI were 5.70 cm,6.06 cm,7.97 cm and 5.79 cm respectively. The lengths between CT and esophagogram,CT and esophagoscope,CT and DWMRI had statistical significance respectively (F=4.88,P=0.003).The maximum diameters of tumor shown on CT and DWMRI were 3.79 cm and 3.81 cm respectively ( t =-0.32,P=0.751 ).The GTV were 45.75 cm3 and 38.05 cm3 in CT and DWMRI respectively (t=5.30,P =0.001 ).53 lymph nodes were assessed positive on both CT and DWMRI.DWMRI excluded 25 positive lymph nodes assesed by CT; also confirmed 15 negative lymph nodes excluded by CT,6 of which were paraesophageal lymph nodes.The addition of DWMRI information altered the clinical stage in 6 patients.ConclusionsTumor lengths measured on DWMRI and esophagogram had the optimal approximation.It was easy to find paraesophageal lymph nodes via DWMRI.With the addition of DWMRI information,the target range and clinical stage were alerted in some patients.

ObjectiveTo analyze the prognosis of 784 patients according with clinical staging of esophageal carcinoma treated with non-surgical methods,investigate the predictive value and deficiency of the clinical staging.MethodsFrom July 2003 to January 2009,784 patients with esophageal carcinoma received 3DCRT treatment.The prescribed doses ranged from 50 Gy-70 Gy with median dose of 60 Gy,1.8-2.0 Gy/fraction,1 fraction/day,5 fractions/week.65 patients received prescription dose of＜60 Gy and all the others'≥60 Gy.All the patients were divided into subgroups according to different T,N and TNM stages.Therapeutic effect was evaluated.ResultsThe follow up rate was 97.1％,503 patients were followed up for more than 3 years and 122 were followed up for more than 5 years.The 1-,3-,5-year local control rates and overall survival rates were 77.2％,54.2％,46.5％ and 69.5％,34.9％,23.9％,respectively,with median survival time of 21 months.There were significant differences of survival curves for different T stages,N stages and TNM stages.For the groups of stage Ⅰ,Ⅱ and Ⅲ,the 1-,3-,5-year survival rates were 86.4％,47.6％,45.1％ ;84.7％,46.3％,36.4％ and 64.0％,30.9％,19.1％,respectively ( x2 =29.34,P =0.000).There were 752 patients with squamous cell carcinoma ( 95.9％ )and 32 patients with non-squamous cell carcinoma (4.1％ ),the median survival time were 21 and 16 months,respectively ( x2 =4.44,P =0.035 ).There were significant difference of survival rates for the subgroups whose GTV volume ≤20 cm3,20 -40 cm3,40 -60 cm3 and ＞60 cm3 (54 months,29 months,21months and 14months,x2 =68.71,P =0.000).ConclusionsThe clinical staging of esophageal carcinoma treated with non-surgical methods could predict the prognosis accurately,for patients with different pathology and GTV volumes,there were variance in the prognosis,so we advised the complement of the two factors in the draft of clinical stages.

Objective To investigate the effects of dosimetric differences in gross tumor volume ( GTV ) on local control and survival rates in patients with esophageal carcinoma undergoing three?dimensional ( 3D) radiotherapy,and to provide a basis for clinical treatment. Methods From January 2004 to December 2010, 548 patients with esophageal carcinoma received conventional fractionated 3D radiotherapy with a prescribed dose of 60 Gy. All patients were divided into low?dose group and high?dose group according to the dosimetric differences in GTV. The survival and local control rates were compared between the two groups. The survival rates were calculated using the Kaplan?Meier method and analyzed using the logrank test. The Cox regression model was used for the multivariate prognostic analysis. Results The number of sample were 456 and 216 patients at 5 and 7 years followed time. The 1?,3?,5?,and 7?year local control rates were significantly higher in the high?dose group than in the low?dose group ( 83?5% vs. 71?3%, 62?6% vs. 44?8%,57?5% vs. 41?7%,52?9% vs. 38?8%,P=0?000).The 1?,3?,5?,and 7?year survival rates were also significantly higher in the high?dose group than in the low?dose group ( 79?6% vs. 66?3%, 44?3% vs. 29?7%, 34?0% vs. 21?8%, 26?1% vs. 17?0%, P=0?000 ) . The univariate prognostic analysis using the Cox regression model showed that Dmin , Dmean , and D100 for GTV were prognostic factors ( P=0?000,0?001,0?000).In all the 548 patients,201 were assigned to the high?dose group and the others to the low?dose group. Compared with the high?dose group, the low?dose group showed significantly larger GTV (38?2 vs. 48?1 cm3,P=0?002) and more advanced T stages (P=0?035).The stratified analysis showed that the 1?,3?,5?,and 7?year local control and survival rates were significantly higher in the high?dose group than in the low?dose group,regardless of tumor location,GTV,TNM stage,or chemotherapy. The multivariate analysis using the Cox regression model indicated that tumor location and grouping based on the radiation dose to GTV were independent prognostic factors. Conclusions In 3D radiotherapy for treating esophageal carcinoma,a high?quality treatment plan and GTV dose assurance improve the survival rates in patients. The patients with lower Dmin ,Dmean ,and D100 for GTV than the prescribed dose have a poor prognosis.