Objective To establish analysis methods of two-dimensional(2-DE)gel electrophoresis for human osteosarcoma.Methods A series of methods,including Immobilized pH gradient were used as ID.Some applications,such as sample preparation used as choice of IPG gel,were improved.Coomassie brilliant blue staining,ImageMaster 2D Elite 3.01 analysis software,MALDI-TOF/ TOF MS and SWISS-PROT database serching were used to separate and indentify the proteins of human osteosarcoma.Results The good use pattern including repetitive experiments showed that in three experiments,the amount of protein spots of the same team sample deviates from the relative standard as following,the average of variation coefficient(%) : 23.00?10.11 and 20.33?9.90;and the range of variation coefficient(%) were:3.80-6.89 and 2.706.89 from osteosarcoma and normal group respectively.The isoelectric point and molecular weigh of the same protein spots in three experiments deviated from relative standard as following:(8.93%?1.17)%,(10.16?2.02)%,(10.87?3.86)%,respectively.Therefore,better resolution and repetitive 2-DE atlas were obtained.The proteins from 11 pairs of sample were analysed by mass spectrometry,9 identified proteins(transthyretin precursor, Triosephosphate isomerase,slow skeletal muscle,cardiac muscle Troponin T,Cofilin-1,Myosin light chain 1,Calgranulin B,Heat-shock protein,Annexin A5, Fanconi anemia group D2 proteins) were more abundant in osteosarcoma tisstues and 2 proteins manganese SOD and carbonic dehydratase appeared down-regulation in osteosarcoma tisstues.Conclusion This optimized 2-DE map is an important tool for further study on osteosarcoma,and these identified proteins were related-proteins with osteosarcoma.It is suggested that the changes of the proteome are involved in the pathology processe of osteosarcoma.

OBJECTIVE:To prepare Puerarin polymeric micelles and establish a method to determine its entrapment efficiency. METHODS:Puerarin polymeric micelles were prepared by film dispersion method. The polymeric micelles and free drug were sepa-rated by centrifugal-millipore filter filtration method. The entrapment efficiency of puerarin polymeric micelles was determined by HPLC. Diamonsil C18(2)column was used with 1% citric acid solution-methanol(65∶35)at the flow rate of 1 ml/min. The detec-tion wavelength was set at 250 nm,and column temperature was room temperature. RESULTS:The prepared polymeric micelles were spherical and spherical-like in shape with a mean particle size of 54.12 nm,polydispersity index of 0.122,Zeta potential of -13.60 mV;the linear range of puerarin was 2-10μg/ml(R2=0.999 4)with average recovery rate of 99.2%(RSD=0.9%,n=3). The re-covery rate of free drug was 95.3%(RSD=1.7%,n=3). The mean entrapment efficiency and drug-loading amount of puerarin were(35.5±2.12)% and(0.3±0.07)%,respectively(n=3). CONCLUSIONS:Film dispersion method is suitable for the prepara-tion of Puerarin polymeric micelles. Established method is convenient,accurate and reliable for the content and entrapment efficien-cy determination of Puerarin polymeric micelles.

OBJECTIVE: To prepare Salinomycin nanostructured lipid carriers (Sal-NLCs) and optimize its formulation. METHODS: Sal-NLCs was prepared by emulsion evaporation-low temperature solidification method. Using particle size, Zeta potential, encapsulation efficiency and drug loading as evaluation indexes, central composite design-response surface methodology was used to optimize the amount of Sal, the ratio of solid lipid glyceryl bisstearate to liquid lipid glyceryl octanoate in oil phase, ratio of surface active agent polyoxyethylene 35 castor oil (EL) to polyethylene glycol-15-hydroxy stearate (HS 15), the amount of polyoxyethylene (40) stearate (P40). The morphology, particle size, polydispersity index (PDI), Zeta potential, encapsulation efficiency, drug loading and in vitro release mechanism of Sal-NLCs were investigated. RESULTS: The optimal prescription was as follows as Sal 0. 86 mg, glyceryl bisstearate 40.70 mg, glyceryl octanoate 11.30 mg, EL 44.05 mg, HS15 7.95 mg, P40 3.8 mg. Prepared Sal-NLCs was round-like and dispersed evenly. The particle size, PDI, Zeta potential, encapsulation efficiency and drug loading of prepared Sal-NLCs were(81.81 ± 2.60) nm, 0.183 ± 0.042, (-24.9 ± 3.4) mV,(94.35 ± 1.50)％ and (1.47 ±0.04)％ (n=5), respectively.24 h accumulative release rate was (99.81 ± 3.90)％ (n=3).Drug release behavior was in line with Higuchi model, and relative error of particle size, Zeta-potential, encapsulation efficiency and drug loading to predicted value of model were all lower than 4％. CONCLUSIONS: Sal-NLCs with sustained-release effect is prepared successfully according to optimized formulation, and its quality meets the expected standard.

Two strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in 2006 and 2016 and designated as FZ06A and FZ16A, respectively. Inoculation experiments showed that FZ06A caused 100% morbidity and 60% mortality, while FZ16A caused 100% morbidity without death. By using genomic sequence and phylogenetic analyses, close relationships between a Chinese highly pathogenic PRRSV strain and the FZ06A and FZ16A strains were observed. Based on the achieved results, multiple genomic variations in Nsp2, a unique N-glycosylation site (N³³→K³³), and a K151 amino acid (AA) substitution for virulence in the GP5 of FZ16A were detected; except the 30 AA deletion in the Nsp2-coding region. Inoculation experiments were conducted and weaker virulence of FZ16A than FZ06A was observed. Based on our results, a 30 AA deletion in the Nsp2-coding region is an unreliable genomic indicator of a high virulence PRRSV strain. The Nsp2 and GP5 differences, in addition to the virulence difference between these two highly pathogenic PRRSV strains, have the potential to be used to establish a basis for further study of PRRSV virulence determinants and to provide data useful in the development of vaccines against this economically devastating disease.
Asian Continental Ancestry Group ; China ; Genomics ; Humans ; Mortality ; Phylogeny ; Porcine Reproductive and Respiratory Syndrome ; Porcine respiratory and reproductive syndrome virus ; Vaccines ; Virulence

Objective To analyze the correlation between smoking and occurrence of intracranial artery stenosis.Methods From June 2015 to May 2016,a total of 10 711 inpatients with transient ischemic attack (TIA) or ischemic stroke from 20 basel hospitals of nationwide were enrolled using a cross-sectional study,76 patients with unknown smoking and smoking cessation years were excluded.Finally,a total of 10 635 patients were enrolled.Transcranial color coded sonography and/or transcranial Doppler were used evaluate the intracranial artery stenosis lesions.The basic risk factors for cerebrovascular disease (age,sex,smoking and smoking years,whether smoking cessation and years,hypertension,diabetes,hyperlipidemia,atrial fibrillation,and family history of stroke) were recorded.According to the different smoking years,the smoking years were divided into five groups:non-smoking,smoking time ≤10-year,11 to 20-year,21 to 30-year,and >30-year groups for trend chi square test.According to the different smoking cessation years in the smokers,the smoking cessation years were divided into four groups:non-cessation,cessation time 1 to 10-year,11 to 20-year,and >20-year groups for trend chi square test.The effects of different smoking years and different smoking cessation years on the occurrence of intracranial arterial stenosis were analyzed.Results The incidence of intracranial artery stenosis in the smokers (40.4%[1 433/3 547]) was significantly higher than that in the non-smoking patients (29.4%[2 085/7 088]).There was significant difference (χ2=128.850,P<0.01),and the incidence of cerebral infarction in the smokers (91.6%[3 250/3 547]) was significantly higher than the non-smokers (85.0%[6 027/7 088]).There was significant difference (χ2=92.328,P<0.01).Smoking was an independent risk factor for intracranial artery stenosis (OR,1.603;95%CI 1.456-1.765;P<0.01).With the increase of smoking years,the detection rate of intracranial arterial stenosis increased gradually (trend χ2=115.437,P<0.01).Whether giving up smoking had no significant effect on the incidence of intracranial artery stenosis in patients with ≥20 years of smoking (trend χ2=1.043,P=0.307).Conclusions Smoking is an independent risk factor for affecting intracranial artery stenosis;the risk of disease increases with the number of smoking years.Long-term smokers (≥20 years) cannot reduce the effect on intracranial artery stenosis,even if they give up smoking.

Objective To investigate the effect of serum lipid level on carotid artery stenosis in patients with ischemic cerebrovascular disease.Methods Using a multi-center cross-sectional study,10 711 consecutive inpatients with transient ischemic attack (TIA) or ischemic stroke diagnosed clearly in 20 stroke screening and prevention project base hospitals from June 2015 to May 2016 were enrolled.According to the results of carotid ultrasonography,1 560 patients with extracranial carotid artery stenosis rate≥50% screened were enrolled in the study.They were divided into a severe stenosis group (70%-99%) and a mild-moderate stenosis group (<70%).The distribution of total cholesterol (TC),triacylglycerol (TG),low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels in both groups of carotid stenosis patients were analyzed,and the quantitative classification was based on the normal range of serum lipids.The distributions of serum lipid levels in different grades in patients of both groups were compared with the non-parameter test.Results The incidence of dyslipidemia in the severe stenosis group was higher than that in the mild and moderate stenosis group.There was no significant difference between the two groups (54.4%[319/586]vs.48.3%[470/974],P<0.05).Dyslipidemia was an independent risk factor for severe carotid artery stenosis (OR,1.27,95% CI 1.24-1.30,P<0.01).The TC and LDL-C levels in patients of the severe stenosis group were significantly higher than those in the mild-moderate stenosis group (TC:3.98[3.31,4.82]mmol/L vs.3.91[3.31,4.53]mmol/L,LDL-C:2.48[1.86,3.14]vs.2.30[1.79,2.80];all P<0.01).With the increase of TC and LDL-C levels,there was significant differences between the severe stenosis group and the mild-moderate stenosis group (all P<0.05),and the proportions of TC >5.80 mmol/L (7.3%[43/586]vs.0.4%[4/974]) and LDL-C>3.12 mmol/L (26.3%[154/586]vs.10.0%[97/974]) in patients of the severe stenosis group were higher than those in the mild-moderate stenosis group (26.3%[154/586]vs.10.0%[97/974]).Conclusion The high LDL-C and TC levels may increase the incidence of severe carotid artery stenosis or occlusion.

Objective To investigate the differences of distribution characteristics and risk factors of large artery lesions in patients with ischemic cerebrovascular disease in different age groups in order to provide the basis for the prevention and treatment of stroke in different age groups.Methods From June 2015 to May 2016,a total of 10 711 consecutive inpatients with transient ischemic attack (TIA) and ischemic stroke from 20 centers nationwide were enrolled.Each 10 years was used as an age group from 40 years.All the patients were divided into 5 age groups.The differences of the different risk factors for cerebrovascular disease among the 5 groups were compared.All patients were separated by gender.The chi square test was used to compare the incidences of large artery stenosis of the intracranial and external and anterior and posterior circulation,and the number of vascular lesions in the same sex in different age groups.Results (1) The risk factors of elderly patients were mainly hypertension,diabetes mellitus,and atrial fibrillation (χ2=61.938,χ2=13.349,and χ2=55.940;all P<0.01).The smoking history,family history of cerebrovascular disease,and obesity were more frequent among the young and middle-aged people (χ2=131.505,χ2=7.298,and χ2=100.911,all P<0.01).(2) The linear trend chi square test results showed that the proportion of multivessel diseases in female and male extracranial arterial lesions increased gradually with the increase of age.(χ2=54.799,χ2=161.370,all P<0.01).The proportion of multivessel diseases in the intracranial artery in female decreased gradually (χ2=5.328,P=0.021),and that in male did not have obvious trend of change (χ2=0.289,P=0.591).(3) The linear trend chi square test results showed that the incidence of simple intracranial arterial stenosis in female and male intracranial arterial stenosis decreased gradually with the increase of age (χ2=20.090,χ2=42.351,all P<0.01),and the incidence of simple extracranial arterial stenosis increased gradually (χ2=40.311,χ2=90.698,all P<0.01).The incidence of both intracranial and extracranial artery stenoses increased gradually (χ2=12.077,χ2=45.887,all P<0.01).The incidence of simple posterior circulation vascular stenosis increased gradually in female (χ2=16.434,P<0.01),but that did not have obvious trend of change in male (χ2=1.701,P=0.192).The incidence of stenosis of both anterior and posterior arteries in female and male increased gradually (χ2=4.587,P=0.032;χ2=35.156,P<0.01).Conclusions The distribution of atherosclerotic lesions in ischemic cerebrovascular disease of the different age groups was different.No matter female or male patients,the majority of the young and middle-aged patients were intracranial artery lesions,and the elderly patients were mainly extracranial artery lesions.The majority of elderly women had posterior circulation artery lesions.Understanding the characteristics in patients with intracranial arterial lesion in different age groups will help to develop individualized stroke prevention and treatment strategies for the population of different age groups.

Objective To investigate the distribution characteristics and risk factors of intracranial and extracranial aterial lesions in Chinese patients with ischemic stroke . Methods In this multi‐center study ,2 310 continuously inpatients with ischemic stroke diagnosed in 20 stroke screening and prevention project base hospitals from June 2015 to M ay 2016 were enrolled . Carotid ultrasonography and transcranial color‐coded sonography or transcranial Doppler were performed in all patients to confirm the presence of cerebral artery stenosis or occlusion . According to the distribution of lesions ,the subjects were divided into 2 groups :the simple intracranial artery stenosis group and the simple extracranial artery stenosis group . T he difference of risk factors between the two groups was compared . Results Of the 2 310 patients with ischemic stroke ,1 516 ( 65 .6% ) had simple intracranial artery stenosis and 794 ( 34 .4% ) had simple extracranial artery stenosis . T he incidence of anterior circulation artery stenosis was higher in the group of intracranial artery stenosis than that in the extracranial artery stenosis group ( 68 .1% vs 48 .7% , P ＜0 .001) . Posterior circulation artery stenosis and combined anterior with posterior circulation artery stenosis were more common in patients with extracranial artery stenosis group than those in intracranial artery stenosis group ( 36 .4% vs 22 .1% ,14 .9% vs 9 .8% ;all P ＜0 .001) . Univariate analysis of risk factors for stroke showed that patients with intracranial arterial stenosis had a higher prevelence of hypertension , diabetes ,obesity ,and family history of stroke ,and their systolic blood pressure ,diastolic blood pressure , body mass index ( BM I) ,fasting blood‐glucose ,glycosylated hemoglobin ,triacylglycerol ,total cholesterol , and low‐density lipoprotein cholesterol were significantly higher than those in the extracranial arterial stenosis group ( all P ＜ 0 .05 ) . T he proportion of elderly ( ≥ 65 years old ) ,male and smokers in the extracranial arterial stenosis group was significantly higher than that in the intracranial arterial stenosis group ( all P ＜0 .05) . Multivariate logistic regression analysis showed that elderly ( ≥65 years old) ,male , and smoking history were independent risk factors for extracranial arterial stenosis ( OR＝ 2 .012 ,1 .637 , 1 .325 ,respectively ;all P ＜0 .05) . While hypertension ,diabetes ,less physical activity ,and high BM I levels were independent risk factors for simple intracranial arterial disease ( OR ＝ 1 .301 ,1 .252 ,1 .248 ,1 .030 , respectively ;all P ＜0 .05) . Conclusions There are significant differences in the distribution characteristics and risk factors of intracranial and extracranial aterial lesions in patients with ischemic stroke in China .