Fusion PCR, which employs chimeric primers to generate PCR products with complementary ends in its amplifications, is a rapid and flexible method in joining different DNA fragments. This method can assemble DNA fragments without the treatment of restriction endonucleases and T4 DNA ligase. It offered a shortcut for the construction of homologous recombinant fragments. Through assembling three recombinant fragments successfully, fusion PCR procedure was improved and manipulation essentials of fusion PCR were described in detail. The results indicated that the improved procedure can assemble three or four fragments simultaneously, and the length of each fusion product is above 4.5 kb. The result recombinants were proposed to be use in further experiments, which structures had been confirmed by sequencing.

Based on a ?-tubulin gene EST sequence from Trichoderma harzianum mycelium cDNA library,a 1.74kb ?-tubulin gene coding region,together with a 1.5kb 5'-flanking region and a 1.0kb 3'-flanking region was cloned from T.harzianum by IPCR methods.The gene encodes a 446 amino acid polypeptide,which shows a high degree of homology with other fungal ?-tubulins.A three-dimensional model of the T.harzianum ?-tubulin was built based on the crystal structure of bovine tubulin using homology modeling method.Tertiary protein structure of the ?-tubulin should provide a basis for understanding a significant body of research on tubulin's properties and interactions with antimicrotubule agents.

Objective To observe the effect of cyanin on the expression of L-glutamate/ L-aspartate transporter (GLAST) in high glucose cultured retina Müller cells. Methods The retinal tissue of SpragueDawley (SD) rats was collected at postnatal 10 day, and Müller cells were isolated and cultured according to literature. The Müller ceils (2nd-4th generations) were treated with five different medium as normal group (group A), high glucose control group (group B), high glucose+30 μmol/L cyanin group (group C), high glucose+60 μmol/L cyanin group (group D) and high glucose+100 μmol/L cyanin group (group E). Cell relative survival rates (A value) were measured by MTT assay at 570 nm. The GLAST protein expression in M011er cells was observed by Western blot. Results MTT assay showed that the A value of the five group were 0. 450 8±0. 020 4, 0. 270 1±0. 031 4, 0. 332 0±0. 023 2, 0. 428 3±0. 017 2, 0. 361 9±0. 027 0,the cell relative survival rate were 100. 0%, 59. 9%, 73.6%, 95%, 80.3% respectively. The A value of group C, D, E were significantly higher than that of group B (F=32.25, P<0.05), the A value of group D were significantly higher than that of group C and E (F=21.07, P<0. 05). Western blot showed that the GLAST protein expression of group B was lower than that of group A (t=5.25, P<0. 05) ; there was no obvious changes of GLAST protein expression in group A, C, D and E (F= 2. 979, P>0.05).Conclusion Cyanin can rescue high glucose-induced GLAST reduction.

Objective·To discusses the high risk factors and therapy strategies for ovarian hyperstimulation syndrome (OHSS). Methods·Seventy-seven cases with moderate and severe OHSS were collected to be retrospectively analyzed in Shanghai Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine from January 2010 to December 2016. Results·Compared with non-pregnant patients, pregnant patients were more likely to have severe OHSS (P<0.05). Embryo transfer patients who underwent ovulation induction and oocyte retrieval became severe OHSS much more easily and had a much longer hospital stay than oocyte retrieval patients (P<0.05). Pumping ascites and pleural effusion or not has no effect on the length of hospital stay and the usage of low molecular dextran for patients with severe OHSS. Conclusion·The risk of OHSS should be evaluated to determine whether or not to carry out a fresh embryo transfer in patients who underwent ovulation induction and oocyte retrieval. For OHSS patients who have undergone embryo transfer or pregnancy, more attention should be paid to the progress of the disease, and individualized treatment is required.

Objective To explore the correlation of illness uncertainty and coping styles of patients with hepatitis B . Method A total of 254 patients with hepatitis B were assessed by Chinese version of Mishel′s uncertainty in illness scale ( MUIS ) and medical coping modes questionnaire ( MCMQ ) . Results The average score of illness uncertainty was at a higher level . The patients mainly used confrontation coping style. The illness uncertainty was positively correlated with the coping style of acceptance-resignation (P<0.05). Conclusion The illness uncertainty of patients with hepatitis B is at a higher level. Nursing intervention should be taken to reduce the degree of patients′uncertainty in illness and encourage them to take positive coping style.

AIM: To study the effect of heat shock protein (HSP) on the maturation of dendritic cells (DCs) and observe the morphological changes of DCs dynamically. METHODS: The HSP (gp96)-peptide complex was purified from the tissues of hepatocellular carcinoma. Hepatoma cells were treated by heat shock for preparation of the HSP expressed on cell surface and then marked with DiI fluorescence. Immature DCs from peripheral blood mononuclear cells were cocultured with two types of HSPs. The morphological changes of DCs were observed dynamically and the effects of HSPs on the maturation of DCs analyzed by Flowcytometer. RESULTS: The morphological changes and the processes of antigen capture of DCs cocultured with DiI marked tumor cells were well showed. Four ways to capture antigens of DCs were observed, including direct contact, besieging, forming bubbles and extending pseudopodia with bubbles on the terminals. Results also indicated that both types of HSP could promote the maturation of DCs. CONCLUSION: DiI is a good fluorescent dye suitable for the morphological studies of DCs. Four ways for DCs to capture antigens were indicated in this paper. Different types of HSP, purifed from tumor tissues or expressed on tumor cells surface, promote DC maturation, and the purified HSP is more effective. [

Immune thrombocytopenia (ITP) is a common bleeding disease in children.Recent advances has indicated that such factors as decreased platelet,fear of bleeding,prognosis,therapy associated side effect and restricted activities,usually decreased quality of life of patients and their parents.This review focuses on domestic and overseas researches on health-related quality of life (HRQoL) in children with ITP,in order to attract more scholars' attention.HRQoL should be recommended as an important indicator in treatment and management of ITP.

Coronary Vessel Anomalies ; Humans ; Myocardial Bridging

Objective To investigate the association between tumor necrosis factor(TNF) promot- er polymorphisms and susceptibility to ulcerative colitis(UC) in the Chinese Han population.Methods Blood samples from 110 unrelated UC patients and 292 healthy controls from Zhejiang Province,Eastern China were studied.Genotyping for 6 common TNF promoter polymorphisms (TNF—1031T/C,—863C/A,—857C/T,—380G/A,—308G/A,—238G/A)was carried out using polymerase chain se- quence-specific primer (PCR-SSP).Results TNF—308A was associated with susceptibility to UC (al- lele frequency patients 14.6% vs.controls 8.9%,P=0.02).TNF—857T was increased in patients but did not reach statistical significance (allele frequency 17.3% vs.12.2%,P=0.06).Haplotype analysis revealed 6 haplotypes including two (H5 and H3) which contained TNF-308A.H5 was associ- ated with disease (haplotype frequency patients—12.3% vs.controls 7.5%,P=0.03).Homozygosity for the haplotype H4 comprising the common alleles at each TNF promoter SNP was negatively associat- ed with disease (patients vs.controls 24.5% vs.34.9%,P＜0.05).Conclusions We report first the association of TNF-308A polymorphisms with UC in Chinese patients.The functional study of the poly- morphisms in Chinese Han population is required.

Chronic rhinosinusitis with nasal polyps (CRSwNP) and asthma is a common clinical refractory airway disease. Comprehensive treatment of nasal endoscopic surgery including nasal endoscopic surgery and medication, which can significantly improve nose-pulmonary symptoms and make sinusitis and asthma easier to be controlled by medication, has certain superiority. But the existence of disease heterogeneity of CRSwNP with asthma causes different reactions to the current treatment, which manifests as parts of polyps and asthma easy to recur and difficult to control. According to the research recently, the study of the heterogeneity of airway diseases, for example endotype, is a hot area of research. Endotype is a subtype of a condition, which is defined by a distinct functional or pathobiological mechanism. This is distinct from a phenotype, which is any observable characteristic or trait of a disease. Different Inflammatory subtypes often represent different pathophysiology and even different pathogenesis. The concept of inflammatory subtypes of airway diseases provides a new perspective for studies of airway diseases of endotype and the mechanism of combined airway diseases. This review summarizes recent advances in the clinical characterization and treatment of the CRSwNP with asthma. On this basis, we analyze and summarize the heterogeneity of CRSwNP and asthma separately from the perspective of inflammatory subtypes. Then according to the concept of the combined airway diseases and the common pathogenesis, we put forward the definition of inflammatory subtypes of the CRSwNP with asthma and preliminarily discuss the method of the definition.
Asthma ; complications ; Chronic Disease ; Endoscopy ; Humans ; Nasal Polyps ; complications ; Rhinitis ; complications ; diagnosis ; Sinusitis ; complications ; diagnosis