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.

Anti-dplatelet has an important role in the treatment of coro-nary artery disease.The application of anti-dplatelet drug elopidogrel makes patients with coronary artery disease great benefit.However,re-current thrombosis still happened in some patients.In the review,we disccuss clopidogrel from the clinical application of evidence-dbased medicine to clinieal problems and countermeasures.

In this study, the buffering capacity of amphiphilic pH-sensitivity copolymer poly(2-ethyl-2-oxazoline)-cholesteryl methyl carbonate (PEOZ-CHMC) was evaluated. The ammonium sulfate gradient method was used to prepare doxorubicin hydrochloride (DOX x HCl)-loaded liposomes (DOX-L), and then the post-insertion method was used to prepare PEOZ-CHMC and polyethylene glycol-distearoyl phosphatidyl ethanolamine (PEG-DSPE) modified DOX x HCl-loaded liposomes (PEOZ-DOX-L and PEG-DOX-L). The physico-chemical properties, in vitro drugs release behavior, cellular toxicity and intracellular delivery of liposomes were evaluated, separately. The results showed that PEOZ-CHMC has a satisfactory buffering capacity. The sephadex G-50 column centrifugation method and dynamic light scattering were used to determine the encapsulation efficiency (EE) and particle size of liposomes. The EE and particle size of DOX-L were (97.3 ± 1.4) % and 120 nm, respectively, and the addition of PEOZ-CHMC or PEG-DSPE had no influence on EE and particle size. The zeta potentials of three kinds of liposomes were negative. The release behavior of various DOX liposomes in vitro was investigated by dialysis method. In phosphate buffer solution (PBS) at pH 7.4, DOX x HCl was released from PEOZ-DOX-L in a sustained manner. While in PBS at pH 5.0, the release rate of DOX x HCl from PEOZ-DOX-L increased significantly, which suggested DOX x HCl was released from PEOZ-DOX-L in a pH-dependent manner. The intracellular delivery of liposomes was investigated by confocal laser scanning microscopy (CLSM). The CLSM images indicated that PEOZ-DOX-L showed efficient intracellular trafficking including endosomal escape and release DOX x HCl into nucleus, as well as the DOX-L and PEG-DOX-L had no this effect. The cytotoxicity of liposomes against MCF-7 cells was detected by using MTT assay. The results showed that antiproliferative effects of PEOZ-DOX-L enhanced with pH value decreased, whereas DOX-L and PEG-DOX-L did not have any significant difference in inhibitions at different pH conditions. Therefore, the problems of the inhibition of cellular uptake of liposomes and the failed endosomal escape of pH-sensitive liposomes by PEG chain can be overcome by the pH-sensitive liposomes constructed by PEOZ-CHMC.
Cell Nucleus ; Doxorubicin ; analogs & derivatives ; chemistry ; Endosomes ; Formates ; chemistry ; Humans ; Liposomes ; chemistry ; MCF-7 Cells ; Microscopy, Confocal ; Particle Size ; Phosphatidylethanolamines ; Polyamines ; chemistry ; Polyethylene Glycols ; chemistry

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.

Animals ; Hepatitis B virus ; isolation & purification ; Mice ; Mice, Transgenic ; Microscopy, Immunoelectron ; Serum ; virology

Objective:To improve the genetic stability of HBV gene in transgenic mice.Methods:HBV transgenic mice were bred by backcross and double cross.The HBV gene expression and replication were studied with real-time PCR,ELISA and chemiluminescence.Results:The HBV transgenic mice have stably bred to 23rd generation.The serum HBsAg level is 4122.31?2044.74IU/ml;The rate of HBV transgenic mice whose serum HBV DNA reach 104~106copies/ml was 93.93%.The HBV replication and expression were improved markedly.There is no difference between male and female mice about serum HBsAg level.Conclusion:After breeding the HBV gene was expressed stably with high-level in transgenic mice.

OBJECTIVETo investigate the inhibitory effects of CaMKIIN on acute myeloid leukemia cell line HL-60 to explore a novel therapeutic target of leukemia.

METHODSHuman CaMK II N gene expression vector pcDNA3.1/hCaMKIIN or empty vector pcDNA3.1/myc-His (-) B was transfected into HL-60 cells by Lipofectamine 2000. Human CaMK II N proteins of transfected cells were detected by Western blot. Cell proliferation affected by human CaMKIIN was determined by MTT. Colony-forming assay was performed by soft agar growth system. The cells transfected with CaMKIIN were stained with Hoechst 33342 to detect the apoptotic proportion under fluorescence microscopy. Cell cycle was analyzed by flow cytometry.

RESULTSHuman CaMKIIN was stably transfected into HL-60 cells, and overexpression of human CaMKIIN inhibited the proliferation of HL-60/CaMKIIN cells compared to HL-60/mock cells and HL-60 cells [(0.44 ± 0.03) vs (0.94 ± 0.05) vs (0.94 ± 0.04), P<0.01]. The colony formation of HL-60/CaMKIIN was also markedly smaller[(21.00 ± 3.05)/500] than that of mock-transfected [(111.00±4.58)/500]] and control cells [(119.00±6.09)/500] (P<0.01). After 72 hrs-culture, the apoptotic proportion in cells transfected with CaMK II N was obviously higher than of cells transfected with mock DNA or control [(22.49 ± 2.15)% vs (7.17 ± 0.72)% vs (6.40 ± 0.55)%, P<0.01]. Up to (82.97 ± 2.90)% human CaMKIIN/HL-60 cells were arrested at G0/G1 phase, which was more than mock-transfected [(40.53 ± 2.38)%] and control cells [(41.63 ± 2.27)%] (P<0.05). Human CaMKIIN could down-regulate expression of Bcl-2 in transfected cells.

CONCLUSIONCaMK IIN up-regulation could inhibit proliferation and induce apoptosis of human acute myeloid leukemia cell HL-60.

Apoptosis ; Cell Proliferation ; Genetic Vectors ; HL-60 Cells ; Humans ; Proteins ; genetics ; metabolism ; Transfection ; Up-Regulation

The genome of CK/CH/SD09/005, an isolate of infectious bronchitis virus (IBV), was characterized to enable the further understanding of the epidemiology and evolution of IBV in China. Twenty-five pairs of primers were designed to amplify the full-length genome of CK/CH/SD09/005. The nucleotide sequence of CK/CH/SD09/005 was compared with reference IBV strains retrieved from GenBank. The phylogenic relationship between CK/CH/SD09/005 and the reference strains was analyzed based on S1 gene sequences. The complete genome of CK/CH/SD09/005 consisted of 27691 nucleotides (nt), excluding the 5' cap and 3' poly A tail. The whole-genome of CK/CH/SD09/005 shared 97 - 99% nucleotide sequence homology with the GX-NN09032 strain, which was the only complete genome that was closely related to CK/CH/SD09/005. When compared with all reference strains except GX-NN09032, CK/CH/SD09/005 showed the highest similarity to ck/CH/LDL/091022 and SDIB821/2012 (QX-like) in the replicase gene (Gene 1) and 3'UTR, with a sequence identity rate of 97% and 98%, respectively. However, CK/CH/SD09/005 exhibited lower levels of similarity with ck/CH/LDL/091022 and SDIB821/2012 in S-3a-3b-3c/ E-M-5a-5b-N with a sequence identity of 72% - 90%. CK/CH/SD09/005 showed the highest level of nucleotide identity with Korean strain 1011, and Chinese strains CK/CH/LXJ/02I, DK/CH/HN/ZZ2004 and YX10, in ORF 3c/E (97%), 5a (96%), 5b (99%) and N (96%), respectively. ORFs 3a, 3b and M of CK/CH/SD09/005 exhibited no more than 90% homology with the reference strains, excluding GX-NN09032. The phylogenic analysis based on the S1 gene revealed that CK/CH/SD09/005 and 39 published strains were classified into seven clades (genotypes). CK/CH/SD09/005 was distributed in clade IV with several isolates collected between 2007 and 2012. CK/CH/SD09/005 showed 66% - 69% and 72% - 81% nucleotide identities with the IBV strains of other six clades in the S1 and S2 subunits, respectively. More over, multiple substitutions were found throughout the entire S gene of CK/CH/SD09/005, while insertions and deletions were located within the S1 gene. These results indicated that CK/CH/SD09/005 is a novel variant that may be derived from the QX-like strains that are prevalent in China. Multiple genetic mechanisms, including recombinations, mutations, insertions and deletions, are likely to have contributed to the emergence of this IBV strain.
Animals ; Chickens ; China ; Coronavirus Infections ; veterinary ; virology ; Genome, Viral ; Genomics ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Sequence Homology, Amino Acid ; Viral Proteins ; chemistry ; genetics

OBJECTIVETo study the impact of maternal weight gain during pregnancy on the risk of infant obesity within 1 year old.

METHODSA total of 785 infants who were born in Hefei and participated children medical care in one district health center and their mothers were chosen as the research subjects from September 2010 to September 2011. Three groups were classified by weight gain during pregnancy according to the percentiles: excessive pregnancy weight gain group of 126 pairs, adequate pregnancy weight gain group of 542 pairs and inadequate pregnancy weight gain group of 117 pairs. Mother's general demographic information was collected. The height and weight were measured when the infant was 42 days, 3, 6, 9, and 12 months of physical examination. Z score was calculated. The differences of Z score in different groups were compared and the RR values of different weight gain during pregnancy on infant obesity were computed.

RESULTSThe weight-for-age Z score (WAZ) of infant at 42 days 3, 6, 9 and 12 months in excessive pregnancy weight gain group were 0.23 ± 0.93, 0.25 ± 1.03, 0.23 ± 0.99, 0.28 ± 1.09, 0.26 ± 1.14, respectively, all higher than that of the corresponding age in adequate pregnancy weight gain group (-0.04 ± 1.02, -0.07 ± 0.99, -0.05 ± 0.98, -0.06 ± 0.97, -0.07 ± 0.95, respectively). The differences were statistically significant (all P values < 0.05). In excessive pregnancy weight gain group, infant body mass index (BMI) at 9 months ((18.01 ± 0.15) kg/m(2)) and 12 months ((17.66 ± 0.15) kg/m(2)) were higher than that of adequate pregnancy weight gain group ((17.63 ± 0.13) and (17.22 ± 0.15) kg/m(2), respectively). The differences were statistically significant (all P values < 0.05). Differences of infant Height-for-age Z score (HAZ) among three groups were not statistically significant (all P values > 0.05). Compared to adequate pregnancy weight gain group, RR (95%CI) value of infant obesity in excessive pregnancy weight gain group was 1.86 (1.14 - 3.03).

CONCLUSIONExcessive maternal weight gain during pregnancy increased the risk of infant obesity within 1 year old.

Female ; Humans ; Infant ; Infant, Newborn ; Obesity ; epidemiology ; Pregnancy ; Pregnancy Trimesters ; Weight Gain

ObjectiveTo analyze Echinococcus infection in definitive and intermediate hosts in different zones of Qinghai plateau,Qinghai southern plateau,Qilian mountain-Hehuang valley and Chaidamu basin,and to provideascientificbasisfor developing controlstrategiesagainstEchinococcosisinfection. Methods Echinococcosis infection in definitive hosts,dogs and foxes,was identified by morphological observation; in domesticated and wild intermediate host animals was identified by anatomy and pathology; some of the suspected samples were further identified by molecular biological methods.ResultsStray dogs in different zones of Qinghai plateau were infected with Echinococcus granulosus,the infection rates were 38.71％(300/775),49.60％(124/250),and 9.76％(4/41 ) in Qinghai southem plateau,Qilian mountain-Hehuang valley and Chaidamu basin,respectively,and the difference was statistically significant(x2 =25.72,P ＜ 0.01 ).in addition,only Qinghai southern plateau dogs were infected with Echinococcus multiloularis,and the infection rate was 16.04％(98/611).The infection rates of fox with Echinococcus multilocularis were 22.89％(38/166) and 30.77％(12/39) in Qinghai southern plateau and Qilian mountain-Hehuang valley,respectively,and wolves were also found to be infected with Echinococcus granulosus in the same areas.The infection rates of domesticated sheep,yaks,goats and pigs with Echinococcosis were significantly different statistically in those different areas(x2 =82.70,41.82,212.63,194.58,all P ＜ 0.01 ).The infection rates of sheep and yaks were higher[43.43％(5664/13 042),49.47％(2917/5896),52.99％ (887/1674),42.18％ (779/1847),50.70％ (1049/2069),52.90％ (685/1295) ] in three areas.The infection rates of goats and pigs [3.26％ (7/215),0.00％ (0/108)] in Qinghai southern plateau were lower than that of other two areas[ 19.51％(119/610),26.91％(43/1598),47.91％(343/716),21.91％(71/324)].The infection rates of Ochotona curzoniae with Echinococcosis were 6.21％ (243/3910),1.80％ (3/167) and 0.00％ (0/199) in Qinghai southern plateau,Qilian mountain-Hehuang valley and Chaidamu basin,respectively,and the difference was statistically significant (x2 =18.50,P ＜ 0.01 ).Moreover,wild intermediate hosts of Echinococcosis,such as Microtus fuscus,Lepus oiostolus,Pseudois nayaur,Procapra picticaudata,and Prodorcas gutturosa were found to be infected only in Qinghai southern plateau.ConclusionsHuman is faced with a threat of Echinococcosis infection from various definitive hosts in different zones of Qinghai plateau.And stray dogs are the most crucial factor.The life-cycles of Echinococcus are very complicated in Qinghai plateau.Qinghai plateau is a key area in prevention and control of Echinococcosis infection in China.