Twenty-seven cases of simple type 2 diabetes mellitus,30 cases of coronary heart disease,and 32 cases of type 2 diabetes with coronary heart disease were enrolled in this study according to the results of coronary angiography.Meanwhile,32 healthy subjects were taken as a control group.The serum matrix metalloproteinase-9 (MMP-9) and other clinical and laboratory parameters were determined.The results showed that serum MMP-9 may play a minor role in the progression of coronary heart disease in type 2 diabetic patients.

Objective To evaluate the value of the neutrophil-lymphocyte ratio (NLR) in elderly type 2 diabetic patients (T2DM) with coronary heart disease (CHD).Methods We performed a retrospective observational study on 228 patients undergoing coronary angiography in Guizhou Provincial People's Hospital from April 2014 to July 2015.Patients were divided into three groups:the simple T2DM group (n=77),simple CHD group (n=72),and T2DM complicated with CHD group (n=79).Meanwhile,70 healthy elderly subjects served as the control group.The white blood cell count,high-sensitivity C-reactive protein (hs-CRP) and other clinical and laboratory parameters were collected,and NLR was calculated.Risk factors for CHD in T2DM patients were determined by logistic regression analysis.Multiple stepwise regression analysis was adopted to identify factors influencing NLR.Results The white blood cell count,neutrophil count,NLR and hs-CRP level in the simple T2DM,CHD,and T2DM+CHD groups were higher than in the control group [(7.48 1.81) 109/L,(7.72± 1.89) 109/L,(7.98±2.12) 109/L vs.(6.22± 1.61) 109/L;(4.49±1.38) 109/L,(4.88±1.56) 109/L,(5.35±1.40) 109/L vs.(3.52±0.84) 109/L;(2.84± 0.77),(3.07±0.79),(3.34±0.83) vs.(1.58±0.42);(2.92±0.65) mg/L,(3.20±0.86) mg/ L,(4.98±1.10) mg/L vs.(1.105±0.23) mg/L;respectively,P＜0.05 or P＜0.01].The lymphocyte count in the simple T2DM,CHD,and T2DM+CHD groups were lower than in the control group [(1.57±0.41) × 109/L,(1.58±0.40) × 109/L,(1.61±0.48) × 109/L vs.(2.22± 0.51) × 109/L,P＜0.05].NLR and hs-CRP levels in the T2DM+CHD group were higher than in the former two groups (all P＜0.05).Pearson correlation analysis showed that NLR was positively correlated with the Gensini score and hs-CRP level (r=0.7455 and 0.7792,both P＜0.01).Logistic regression analysis showed that NLR,hs-CRP levels and glycosylated hemoglobin A1c (HbA1c) were the risk factors for CHD in T2DM patients (OR=4.331,3.997 and 2.928,all P＜0.05).Multiple stepwise regression analysis showed that NLR was positively correlated with fasting plasma glucose,HbA1 c levels and systolic blood pressure (β' =0.3133,0.4720 and 0.3069,all P＜0.05).Conclusions NLR may be a valuable predictive factor for CHD in elderly T2DM patients.

Objective To explore the molecular mechanism of BRAFV600E inducing chromosome instability in Sbcl2 and SK-MEL31 melanoma cells.Methods The endogenous Mps1 in stable Sbcl2-and SK-MEL31-B-RafV600E expression cells were depleted by siRNA approach.To test the effect of B-RafV600E on the centrosome amplification and the formation of multipolar spindles,cells at S-phase with HU-treatment were arrested and then the centrosomes and mitotic spindles structure were detected through immunofluoresence.Results The percentage of B-RafV600E expressing Sbcl2 and SK-MEL31 cells (Sbcl2-B-RafV600E and SKMEL31-B-RafV600E) with centrosome amplification and multipolar spindle was reduced from 36 ％ to 6 ％ when Mps1 was absent.Conclusion B-RafV600E leads to centrosome amplification and multipolar spindle through Mps1,thus results in chromosome instability in Sbcl2 and SK-MEL31 melanoma cells.

Objective To understand the characteristics of minipigs infected withJapanese encephalitis virus(JEV).Methods After the brain tissues were treated, the pig brain tissue treatment solution was inoculated with BHK21 cells.Then, virus culture,indirect immunofluorescence assay, neutralization test, electron microscopic observation, and reverse transcription-polymerase chain reaction (RT-PCR) amplification of the new isolate E segment and PrM segment nucleotide sequence were performed and the genotype was identified.Results BHK21 cells were inoculated into 25 pigbrain tissues.Among them, three tissue-treated fluid couldinduce shrinkage and aggregation of BHK21 cells, and immunofluorescence staining showed strong green fluorescence response.The results of neutralization test showed that the neutralization titer of these three new isolates was 1:64, and the size of the virus particles was about 40nm under the electron microscope.The homology of both RT-PCR product sequencing results and E-segment of vaccine strain were 95%.Three new isolates were type GIII JEV.Conclusion The results ofthisstudydemonstrate that there is G III type Japanese encephalitis virus infection in the minipig farm.

Objective To develop RT-PCR for detection of TMEV and apply the method .Methods To design specific primers on the basis of GD VII ( GI:62039) genome sequences published in NCBI and establish RT-PCR.To verify the sensitivity and specificity of method after optimizing PCR .We infected 9 BALB/c mice intracerebrally and collected brain, heart, liver, spleen, lung, kidnet, cecal contents and serum samples the 6th day postinfection.The samples were tested by the TMEV RT-PCR.100 mouse cecal contents samples were also detected to apply the established method . Results The 371bp single band was amplified using GDVII as template .Sensitivity test showed that the RT-PCR method can detect as low as 0.69 pg/μL GDVII cDNA.There were no objective band amplified when encephalomyocarditis virus , lymphocytic choriomeningitis virus , Japanese B encephalitis virus , murine norovirus and normal mouse brain tissue were used as case-control .All infected mice showed symptom of different degrees such as depression and hind limb paralysis the 3th day postinoculation and two of infected mice died the 5th day postinoculation.Tissues such as heart, liver, spleen, lung, kidney, brain, cecal contents and serum were collected and tested for TMEV .All the brain samples were detected positive for GDVII and other tissues were all negative;The 100 cecal contents samples were tested and all were negative . Conclusions RT-PCR for TMEV GDVII strain can detect virus infection in mouse tissues efficiently and can be used as a powerful supplement for the national standard of lab animal .

Objective To develop a duplex PCR assay for detection of rat parvovirus H-1 and KRV and its application.Methods To design specific primers on the basis of H-1 ( NC_001358 ) and KRV ( U790330 ) genome sequences published in NCBI and establish a duplex PCR assay using H-1 and KRV DNA as templates.To verify the sensitivity and specificity of the method after optimizing PCR.The rats were infected by oral inoculation.The rats were divided into three groups:H-1 infection, KRV infection and mixed infection groups.To collect feces at the 4th, 6th, 8th and 10th days postinfection.Rats were euthanized on the 10th day and samples from heart, liver, spleen, lung, kidney and cecal contents were collected from each rat, then all the samples were screened with the duplex PCR.Results The 183 bp and 302 bp bands were amplified using H-1 and KRV as templates.The sensitivity test showed that the PCR method can detect as low as 3.8 pg/mL H-1 and 0.73 pg/mL KRV.There were no bands amplified when mouse minus virus, canine parvovirus and feline parvovirus were used as templates, showing that the specificity of the duplex PCR assay is very good. The nucleic acids of H-1 or KRV were detected in all rat feces on the 2th day postinfection and there was no obvious clinical symptoms in all the infected rats.The positive rates of H-1 were as follows:50%(4/8) heart tissues, 50%(4/8) liver tissues, 62.5%(5/8) spleen tissues, 50%(4/8) lung tissues, 37.5%(3/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of single infection group was higher than that of mixed infection group.The positive rates of KRV were as follows:0 (0/8) heart tissues, 25% (2/8) liver tissues, 87.5% (7/8) spleen tissues, 12.5% (1/8) lung tissues, 25%(2/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of mixed infection group was higher than that of single infection group.Conclusions The duplex PCR assay for H-1 and KRV established in this study can effectively detect H-1 or KRV infection in rat feces and other tissues, and can be used as an effective supplement to the national standard of lab animals.

Objective To establish a real-time fluorescent quantitative PCR ( Q-PCR) method for detection of feline herpesvirus 1 ( FHV-1 ) in experiment cats and clinical sick cats.Methods Primers and TaqMan probes were designed and synthesized according to the published FHV-1 specific sequences of TK gene.FHV DNA standards were prepared using molecular biological techniques.The linearity, specificity, sensitivity, stability of the established Q-PCR method were tested.The method was used to detect 48 samples of cats.Results The linear range was 102 copies/μL to 109 copies/μL.The developed Q-PCR method showed no cross reaction with herpes virus type 1 ( HSV-1 ) , canine herpesvirus (CHV), pig pseudo rabies virus (PRV) and cat parvovirus (FPV).The sensitivity was 10 copies/μL.The coefficient of variation ( CV ) was less than 5%.There were 33 positive cases detected in the 48 samples of cats. Conclusions The developed Q-PCR method is good in linearity, specificity, sensitivity, stability, and may be used for rapid quantitative detection of FHV-1 in cats.

Objective To establish and apply an effective PCR assay for detection of the Tupaia ( tree shrew) adenovirus ( TAV) .Methods According to NCBI Genbank, TAV genome DNA from 19418 to 19917 were synthetized and inserted into a plasmid as positive standards.One pair of primers was designed based on this sequence.Sixty blood samples and fifty-six stool samples from tree shrew were detected with this PCR assay.Results A PCR method for detection of TAV was successfully established, with a high specificity and the sensitivity was 13.5 ×10 -7μg/mL.The PCR results of testing sixty tree shrew blood DNA samples were negative.24 positive cases were tested among 56 stool DNA samples.Sequencing of the samples confirmed a 42.9%infection rate of TAV in tree shrew stool samples, well consistent with the PCR results.Conclusions The PCR method for detecting TAV established in this study has good specificity and high sensitivity, therefore, can be used in conventional detection of tree shrew adenovirus.

Objective To verify the detection ability of experimental animal quality detection laboratories in China for Staphylococcus aureus.Methods The testing samples for Staphylococcus aureus detection were prepared by bacterial culture, homogeneity test and stability test, according to the study plan approved by CNAS.Then the samples and operation instruction were sent to the participant laboratories.The detection reports from these laboratories should be submitted before the deadline expires, and the collected data were summarized and analyzed.Results There were 28 laboratories which joined to this test plan.Among them 22 laboratories ( 78.57%) achieved satisfactory test results, and six laboratories (21.43%) had unsatisfactory test results.27 Laboratories used the national standard detection assay, while only one labo-ratory used PCR assay.Conclusions Most of experimental animal quality testing laboratories in China have sufficient pro-ficiency in detection of Staphylococcus aureus.The obtained information are very helpful for the laboratory ability verification testing in future.

Objective To establish multiplex PCR assay for detection of chicken embryo lethal orphan virus (CELO)and egg drop syndrome virus (EDS).Methods According to GenBank gene sequence , two pairs of specific primers designed were amplified CELO long fiber protein and EDS hexon protein gene sequence .The specificity and sensitivity of multiplex PCR were tested .We also use the multiplex PCR to detect exogenous CELO and EDS in influenza virus.Results Two target bands have been successfully amplified and verified by sequencing .The specificity of the method is better , and the sensitivity is 10 -4μg/mL.The results of detecting exogenous CELO and EDS in 12 influenza virus were negative .Conclusion The multiplex PCR assay for detection of CELO and EDS was established successfully , which have good specificity and high sensitivity , and have high value and application prospect for detecting exogenous CELO and EDS in influenza virus .