NDV strain ZJ1 strain , a highly virulent NDV strain, has been prevalent among the waterfowls in China mainland in the past years. Multi-basic amino acid sequence distribute in the protease cleavage site of F protein of this strain. Recombinant expressing plasmid pCI-FT, was generated by converting multi-basic amino acid sequence of 112, 115, 117 of the protease cleavage site of F_ 0 protein, to the non-basic amino acid sequence characteristic of avirulent NDV strain. The result from co-expression of mutant or parental F protein with homologous HN protein in COS-1 cells revealed that both mutant and parental F protein had fusion activity. The result from co-expression of mutant or parental F protein with homologous HN protein in CEF cells showed that the cleavage activity of mutant F protein was significantly reduced. The study built a foundation for mutagenesis of amino acid sequence of the protease cleavage site of F_ 0 protein at the full-length cDNA clone level, study on factors contributing to virulence and construction of candidate vaccine strain, and so on.

Objective To prepare quercetin liposomes and establish a method for determination of its entrapment efficiency.Methods The film dispersion-homogenizing method was used to prepare quercetin liposomes.The formulation was optimized on the basis of orthogonal design and its entrapment efficiency was performed by the protamine sedimentation method.Results The optimal conditions were found to be cholesterol-egg phospholipid=1:3,quercetin-vehicle = 1:40,homogenization pressure 103.4 MPa for three times.The average entrapment efficiency of the optimized nano-liposomes was 92.1%.Conclusion The film dispersion-homogenizing method could be used to prepare quercetin liposomes.The protamine sedimentation method is convenient,accurate,and suitable for the determination of the entrapment effi- ciency of quercetin liposomes.

Objective To explore the iodine nutrition level of people, prevalence of iodine deficiency disorders and the thyroid function of women of childbearing age in pasturing areas of Tibet. Methods Thirty families were selected respectively in pastoral Dangxiong county and agricultural Qushui county of Lhasa in 2009,drinking water and edible salt samples were collected to test iodine content; at least 50 people from each crowd of the following populations including children aged 8 - 10, women of childbearing age of 18 - 49 old and male adults aged 18 - 60 were randomly sampled and to measure their urinary iodine content and for thyroid palpation. Direct titrimetric method was used to test salt iodine(GB/T 13025.7-1999); As3+-Ce4+ oxidation reduction process to test water iodine (GB/T 5750.1-2006); As3+-Ce4+ catalytic spectrophotometry using ammonium persulfate digestion to test urine iodine(WS/T 107-2006), and goiter examination was based on Diagnostic and Classificatory Criteria of Endemic Goiter (WS 276-2007). Results The median of water iodine was 1.3 μg/L in pasturing area and 0.7 μg/L in agricultural areas, there was no statistical significant difference between them(Z =- 1.809, P ＞ 0.05).There was no iodized salt used in pastoral people, but iodized salt coverage rate was 90.0％(27/30) in agricultural residents. The median of urinary iodine among people of pasturing areas was 50.2 μg/L, lower than that of agricultural areas( 193.2 μg/L, Z =- 10.48, P ＜ 0.01 ). However, the goiter rate in pasturing area[1.0％(1/100)]was significantly lower than that of agricultural areas[18.0％(18/100) , x2 =16.8, P ＜ 0.01]. Serum level of FT4 and TT4 in pastoral population[(14.0 ± 2.0)pmol/L, (85.6 ± 17.5)nmol/L] was significantly lower than that of agricultural areas[(16.2 ± 6.3)pmol/L, (95.4 ± 21.1)nmoL/L, t =- 2.06, - 2.20, all P ＜ 0.05]. The thyroid dysfunction rate[5.9％ (2/34)]and subclinical hypothyroidism rate[2.9％ (1/34)]in pastoral population was significantly lower than that of agricultural areas[25.5％(12/47), 21.3％(10/47), x2 =5.328, 5.651, all P ＜ 0.05]. Conclusions Pastoral areas of iodine intake is significantly lower than the agricultural areas, urinary iodine levels reflect a serious iodine deficiency in pastoral people, but the blood biochemical and urinary iodine and goiter rate does not match,and shows hidden iodine hunger, which does not constitute a goiter epidemic.

Objective To identify changes in the occurrence of endemic fluorosis in order to provide scientific basis for making countermeasures. Methods Five villages from 14 counties of mild, moderate and severe fluorosis affected areas were selected by stratified cluster sampling every year in the whole province during 2006 - 2010. Water and urinary fluorine were determined by ion selective electrode method(GB/T 8538-1995); dental fluorosis of children 8-12 years old was diagnosed with Dean method; skeletal fluorosis was diagnosed according to "clinical indexing standards of endemic skeletal fluorosis "(GB 16396-1996), between 2006 and 2008, and "clinical diagnosis standard of endemic skeletal fluorosis"(WS 192-2008) between 2009 and 2010. Results A total of 25 diseased villages were surveyed, 14 with water sources changed, covered a resident population of 8005 people, beneficiary population 7154, and accounting for 89.37% of the resident population; not changed villages 11. In accordance with the "State drinking water health standards", in the 14 changed villages the fluoride in drinking water was qualified (≤ 1.20 mg/L), there were 3 schools whose water fluorine content exceeded the standard; among the 11 villages that did not change water sources 7 drinking water samples fluorine content exceeded the standard. Of the 8 to 12 years old children in villages with changed water sources, 363 of them were checked and 142 dental fluorosis were found, the detection rate of dental fluorosis was 39.12% (142/363); in villages with water sources not changed, 303 children were checked, the detection rate of dental fluorosis was 43.89%(133/303). Of sixteen and elder adults in water source changed villages, 6424 people were checked and 403 skeletal fluorosis were found, skeletal fluorosis detection rate was 6.27% (403/6424); 3572 people were checked in not changed villages, the detection rate of skeletal fluorosis was 13.89%(496/3572). In water sources changed areas, geometric mean of urinary fluoride was in the normal reference value(WS/T 256-2005, 1.40 mg/L)or less. Conclusions Endemic fluorosis is decreased in water improved areas, but in unimproved areas the disease is still severe, and control of endemic fluorosis is still an arduous task.

Microsatellite instability(MSI)was defined according to the frequency of positive findings in a panel of MSI markers.High frequency MSI(MSI-H)was the phenotype in which repeat sequences were extraordinarily unstable, and was considered to be the bona fide phenotype of DNA mismatch repair defection. However base substitutions in some well studied oncogenes or tumor suppressors were reported to be uncommon in MSI-H tumors. To explore this obvious contradiction, the relationship between MSI and KRAS gene mutations were studied in a panel of 76 human colorectal carcinomas, the whole exon of MLH1 and MSH2 were sequenced for MSI-H tumors. KRAS gene mutation was confirmed by similar frequencies in tumors of different MSI status. Intriguingly, all of the KRAS mutant MSI-H tumors harbored sequence alterations in MLH1gene, which was a key player in DNA mismatch repair system. This implied that in MSI-H tumors carrying MMR mutations, KRAS mutation were frequently and almost exclusively occurred. Furthermore, these MMR mutants were uniformly carrying a unique modification + jumping type MSI, which was different to MSI-H tumors without MLH1 or MSH2 gene mutations. This study shaded lights on the heterogeneity of MSI-H tumors, and implied the connection between modification type MSI and DNA mismatch defection.

Eight fragments were amplified and cloned into pCR2.1 vector with the designed primers.The fragments,amplified with primer Ⅰ to Ⅶ,were subcloned into transcription vector to construct the plasmid pNDVZJI which contained the full-length cDNA of NDV ZJI strain.The eukaryotic expression vector pCI-L was constructed by subcloning the fragments,amplified with the primer Ⅴ,Ⅵ and Ⅷ,into the expression vector pCI-neo.The full-length cDNA clone,pNDVZJI,with three helper plasmids,pCI-NP、pCI-P and pCI-L,were cotranfected into BSR-T7/5 cell expressing T7 RNA polymerase.After inoculation of transfected cell culture into embryonated chicken eggs from specific pathogen free(SPF)flock,The NDV of ZJI strain was rescued successfully,which laid a good foundation for the further related research.

Objective:To observe the effect of Governor Vessel-unblocking and mind-refreshing acupuncture plus functional training on neural development in infants with brain damage and seek an effective method for early intervention of infantile brain damage.Methods:Eighty infants with brain injury were recruited and allocated to a treatment group and a control group by their visiting sequence,with 40 cases in each group.The control group received exercise training,40 min each session and 6 sessions a week,and tuina treatment,30 min each time and 6 times a week.Based on the treatment protocol for the control group,the treatment group additionally received Governor Vessel-unblocking and mind-refreshing acupuncture,3 times a week and 10 sessions as a course at a 2-week interval.Before the treatment and after 14-week treatment,the gross motor function measure (GMFM) and developmental quotient (DQ) of Bejing Gesell developmental scale were used to evaluate the development of the infants.Results:After the treatment,the GMFM score and DQs of Gesell scale all increased by different levels in the two groups,and the intra-group differences were statistically significant (all P＜0.05);the scores of the treatment group were superior to those of the control group,and the between-group differences were statistically significant (all P＜0.05).Conclusion:Governor Vessel-unblocking and mind-refreshing acupuncture plus functional training can significantly promote the development of gross motor and cognitive functions in infants with brain damage,and it is an early and effective intervention for infantile brain damage.

Objective To investigate the expression level and clinical significance of long non-coding RNA(LncRNA) growth arrest specific gene-antisense 1(GAS8-AS1) in papillary thyroid microcarcinoma(PTMC) patients. Methods We investigated the expression profile of GAS8-AS1 in tissue samples of patients with PTMC as well as nodular goiter(NG) by quantitative real-time polymerase chain reaction(RT-qPCR). Results GAS8-AS1 in cancer tissue was down-regulated in PTMC patients compared with adjacent thyroid tissue and NG samples(P<0.05). Lower level of GAS8-AS1 was also correlated with central cervical lymph node metastasis(CLNM, P<0.05). The area under the ROC curve for GAS8-AS1 was up to 0.717 3 in CLNM prediction(P<0.05). Conclusion GAS8-AS1 may act as a potential biomarker for PTC diagnosis and CLNM prediction.

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain,seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI,which contained the full-length cDNA of the NDV ZJI strain.The pNDV/ZJI,with three helper plasmids,pCIneoNP,pCIneoP and pCIneoL,were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase.After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock,an infectious NDV ZJI strain was successfully rescued.Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP.After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells,the recombinant NDV,NDV/ZJIGFP,was rescued.Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection,indicating that the GFP gene was expressed at a relatively high level.NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route.Four days post-infection,strong green fluorescence could be detected in the kidneys and tracheae,indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.

NP, P and L gene of Newcastle disease virus of goose origin were amplified and cloned into pGEM-T easy vector and then subcloned into pCI-neo expression vector respectively, the positive clones were identified by enzyme cutting, PCR and sequencing. GFP reporter gene was inserted into the downstream of recombinant expression plasmid of P gene, which of stop codon was deleted. The experiment of transfection of P and GFP recombinant plasmid on COS-1 cells and CEF showed that GFP gene expressed, and this demonstrated that P gene was also expressed. This research may be helpful for further study of reverse genetics and functional genome of NDV of goose origin.