Objective To improve EP9-A2 for Bias estimation among multiple quantitative detection systems within full range of AMR.Methods 40 patients specimens were determined twice for serum total cholesterol by four detection systems(A,B,C and D).With system A served as comparative method,Bias between A and the other three was evaluated according to CLSI EP9-A2 separately.Furthermore,DD(distance from deviation to tolerable error)and its average confidence intervals between every two sys-tem were calculated and compared with zero.The confidence interval of greater than zero was served as criteria for accepting bias between every two system.Results Bias between A and the other three meet the analytical quality specification according to EP9-A2,although that of D system was positive,and those of B and C system were negative.DD between every two system obeyed nor-mality distribution.All biases between every two system wes acceptable except that between B and D,causing of their interval con-taining zero.After correcting of results from system D,Biases between every two system were all acceptable.Plots of confidence in-terval could provide a full range bias assessment within AMR.Conclusion Comparability and Bias estimation in full range of AMR for results between every two system among 3 or more systems could be evaluated by confidence intervals.

Objective Role of melanocortin receptor 4 (MCAR) in excitatory amino acid release from rat astrocytes in spinal cord. Methods Astrocytes were isolated from the spinal cord of newborn pathogen-free Wistar rats ( 1-3 days after birth) and cultured in serum-free Neurobasal/B27 liquid culture medium. After 4 passages the primary cultured astrocytes were randomly divided into 3 groups (6 wells each): group Ⅰ control (group C); group Ⅱ the astrocytes were exposed to TNF-α 10 μg/L (group T) and group Ⅲ the astrocytes were exposed to TNF-α 10 μg/L and HS014 (selective MC4R antagonist) 1 μmol/L (group TH). The astrocytes were incubated at 37 ℃ for 3 h. The supernatant was collected for determination of glutamic acid (Glu) and aspartic acid (Asp)concentrations by HPLC-MS/MS. Results TNF-α significantly increased Glu and Asp release from astrocytes in group T as compared with group C. The Glu and Asp concentrations were significantly lower in group TH than in group T. Conclusion MG4R is involved in the excitatory amino acid release from astrocytes in the spinal cord.

Poor and monotonous work could easily lead to a decrease of arousal level of the monitoring work personnel. In order to improve the performance of monitoring work, low arousal level needs to be recognized and awakened. We proposed a recognition method of low arousal by the electroencephalogram (EEG) as the object of study to recognize the low arousal level in the vigilance. We used wavelet packet transform to decompose the EEG signal so the EEG rhythms of each component were obtained, and then we calculated the parameters of relative energy and energy ratio of high-low frequency, and constructed the feature vector to monitor low arousal state in the operation. We finally used support vector machine (SVM) to recognize the low arousal state in the simulate operation. The experimental results showed that the method introduced in this article could well distinguish low arousal level from arousal level in the vigilance and it could also get a high recognition rate. Have been compared with other analysis methods, the present method could more effectively recognize low arousal level and provide better technical support for wake-up mechanism of low arousal state.
Arousal ; Brain ; physiology ; Electroencephalography ; Humans ; Signal Processing, Computer-Assisted ; Support Vector Machine ; Wavelet Analysis

Objective To determine the influencing factors of post-stroke depression by machine learning.Methods Stroke patients' medical records (688 cases eligible) were extracted from record system,including age,gender,pulse manifestation,complexion,tongue quality,fur,Chinese medicine intervention,body mass index (BMI),blood pressure,blood glucose,blood triglyceride,blood total cholesterol,smoking history,drinking history,depression family history,stroke lesion site in imaging,as well as the final depression judgment.Single rule algorithm (1R) was adopted to learn.The risk factors influencing post-stroke patients' depression in extracted information were determined.Then the cases collected were divided into the training dataset (500 cases) and the test dataset (188cases).Optimal discriminant results were obtained by random forest model.Results Single rule algorithm showed that the most important influencing factor of post-stroke depression was stroke lesion site.By computer speculation,stroke lesions in the frontal and temporal lobes were most prone to post-stroke depression.Basal ganglia,brain stem,cerebellum,medulla oblongata and occipital lobe lesions were less likely to cause depression.The accurate classification rate could amount to 88.95％ (612/688 cases).Random forest model determination was made in the former 500cases in the training dataset.The total correct rate of determining depression was 98.2％.The total correct rate of determination in 188 cases of the test dataset was 99.47％.Six hundred and eighty-eight patients' data were learnt by random forest model.The total correct rate was 98.84％.The importance measure results showed that top 3 important indexes of post-stroke depression were lesion site,Chinese medicine intervention and depression family history.Conclusion Patients with lesions in the frontal & temporal lobes and depression family history were most prone to post-stroke depression.

Objective To further research the biological functions of PES1,the ovarian SKOV3 cell line with inducible stable PES1 expression is established by using Tet-on system.Methods PES1 was cloned into pTRE-Tight vector via PCR and its expression was identified. After transfected the regulating plasmid pTet-on,SKOV3 cells were screened with G418 and re-transfected pTRE-Tight-PES1.The positive cell clones were screened out with hygromycin and were induced by doxycycline (Dox) to definite the best induction concentration.Growth velocity of SKOV3 cells stably expressing PES1 induced by Dox was detected with viola crystallina.Results The SKOV3 cells with inducible PES1 expression were screened out after the cells were transfected pTRE-Tight-PES1 constructed.Dox could dose-dependently induce the PES1 expression with the concentration under 2 mg/L,and 2 mg/L of Dox induced the highest PES1 expression.Growth velocity of SKOV3 cells transfected pTRE-Tight has no significant difference between the SKOV3 cells transfected nothing induced with Dox.However,the SKOV3 cells transfected pTRE-Tight-PES1 grew faster than the cells transfected pTRE-Tight or without transfection in the fourth day (P =0.001 ).Conclusion The inducible stable PES1 expression SKOV3 cells are successfully established and could be used to be an effective cell model to research the biological functions of PES1.The expression of PES1 could promote the growth of SKOV3 cells.

Objective To isolate and clone novel O-superfamily conotoxin genes of Conus capitaneus Linnaeus collected from Hainan,which would provide the initial drug leads to investigate and develop Conus capitaneus conotoxins.Methods 3'-RACE(Rapid Amplification of cDNA Ends) was used for cloning the novel O-superfamily conotoxins.The specific amplified cDNA fragments were sequenced and analyzed,as well as the genetic diversity of the O-superfamily conotoxins.Results The full-length cDNA(CaHr91N) of a new O-superfamily conotoxin(CaHr91) was cloned and sequenced from Conus capitaneus Linnaeus.The novel conotoxin precursor CaHr91P with 77 amino acids(aa) encoded by the cDNA consists of three typical regions of signal with 21aa, pro-peptide with 22aa and mature peptide with 34aa.Predicted sequence of the toxin region CaHr91M is "ECREQSQGC TNTSPPCC SGLRC SGQSQGGVC ISN" with a common O-superfamily cysteine pattern C-C-CC-C-C.Percent identities between CaHr91P and other published homologue O-superfamily sequences were compared systematically,as well as research status on conopeptides from C.capitaneus.Conlusion The elucidated cDNA of the novel CaHr91P conotoxin will be the basis for a better understanding of its bioactivity and application,as well as finding more novel conotoxins from C.capitaneus.

Objective To analyze the characteristics and significance of matrix metalloproteinase-9 (MMP-9) expression in the pathophysiological processes of tuberculous meningitis in mice.Methods Sixteen mice were intracerebroventricularly injected with H37RV suspension as the model group.Meanwhile,the other 16 mice were injected with 0.9％ sodium chloride solution as the control group.Thirty days later,all mice were decapitated and the brain tissue were respectively used to for Mycobacterium tuberculosis (M.tuberculosis) incubation,pathological changes observation,MMP-9 activity detection by zymography,blood-brain-barrier permeability and moisture content detection,and immunofluorescence stain of MMP-9,glial fibrillary acidic protein (GFAP) and integrin αM (OX-42).The t test was used to compare the differences between the two groups.Results Every experimental mouse was injected with (1.271±0.111) × 106 colony-forming units (cfu) M.tuberculosis.Thirty days later,the amount of M.tuberculosis in brain tissue homogenates was (4.900± 1.407) × 104 cfu/mL,and the hematoxylin and eosin staining showed dilatation of subarachnoid and ventricular and infiltration of a large number of inflammatory cells.The cumulative absorbance (A) of MMP-9 bands of brain tissue was 47 821 ± 19 932 in the model group and 10 082 ± 3 544 in the control group.The difference was statistically significant (t =3.728,P=0.010).The evans blue (EB) content of brain tissue was (11.8 ± 3.6) μg/g in model group and (4.7 ±3.4) μg/g in control group.The difference was statistically significant (t=2.887,P=0.028).The moisture of brain tissue was 0.849±0.035 in model group and 0.775±0.037 in control group.The difference was statistically significant (t=2.925,P=0.026).The immunofluorescence staining showed that the infected brain tissue expressed high degrees of MMP-9,GFAP and OX-42.And MMP-9 was overlapped with both GFAP and OX-42 obviously.Conclusions The activity of MMP-9 is significantly enhanced in brain tissue of mice suffering from tuberculous meningitis and participates in blood-brain barrier damage,tissue edema and inflammatory cells exudation.Microglia cells-astrocytes network is involved in the secretion of MMP-9.

Objective:To explore the effect of heparanase (HPSE) on the cell adhesion and invasion ability of hepatoma carcino-ma (HC) cell. Methods:HPSE expressions in human HC cell lines (BEL-7402, HepG2, and HCCLM3) were measured by real-time re-verse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Four recombinant miRNA vectors, pcD-NATM6.2-GW/EmGFP-miR-HPSE (pcDNA-miR-HPSE), were constructed and transfected into HCCLM3 cells. Full-length cDNA of HPSE gene was cloned into pIRES2-EGFP vector and transfected into HepG2 cells. Transfection efficiency was observed with fluores-cence microscope. HPSE expressions in transfected cells were measured by real-time RT-PCR and Western blot analysis. Adher-ence ability was determined with microplate reader, and invasion and migration abilities were detected with Transwell chambers. Re-sults:Both HPSE mRNA and protein relative expression levels were higher in the three types of HC cells than those in normal hepato-cyte (P<0.05). HPSE had the highest expression level in HCCLM3 cells and the lowest expression level in HepG2 cells (P<0.05). All five recombinant vectors met the experimental requirements. The transfection efficiencies were 75%-85%. The four miRNA vectors, pcDNA-miR-HPSE, significantly decreased HPSE expression in transfected HCCLM3 cells (P<0.05), and pcDNA-miR-HPSE-1 showed the best interference effect (P<0.05). Plasmid pIRES2-EGFP-HPSE increased HPSE expression in transfected HepG2 cells (P<0.05). After pcDNA-miR-HPSE-1 was transfected, the HCCLM3 cell adherence rate and the cell invasion and migration numbers dropped by almost 50%(P<0.01). After transfection of pIRES2-EGFP-HPSE, the HepG2 cell adherence rate and the cell invasion and migration numbers increased by nearly 40%(P<0.05). Conclusion:Different HPSE vectors could regulate bi-directionally the adher-ence, invasion, and migration abilities of transfected HC cells. HPSE may be related with adherence aside from invasion of HC cell.

Objective To improve patient postoperative comfort of vitrectomy with tamponnade in the prone position, design a new prone position headrest to reduce complications caused by improper body position and observe its clinical effect. Methods According to the postoperative position of the patients, 360 cases were collected. The patients were divided into the control group and the observation group with 180 cases of each group. Observation group was treated with the new prone position headrest nursing, control group were treated with routine prone position. The comfort of patients, postoperative adverse reactions, success rate of retina reattachment were observed. Results According to simplified comfortable situation scale, physiological, psychological, social culture and environment of each individual score respectively was (2.74±0.21), (3.54±0.29) , (3.25±0.23), (3.36±0.27) points in observation group and (2.30± 0.19), (2.92±0.31), (2.93±0.26), (2.79±0.30) points in control group, and there were significant differences (t=12.368-20.845, all P<0.05). The daily posture duration in postoperative first time and 5 days was respectively (220.00±25.08), (1008.00 ± 20.32) min in observation group and (85.00±28.07), (650.00± 30.12) min in control group, and there were significant differences(t=48.117, 133.194, all P<0.01). The incidence of corneal edema, conjunctival congestion, water turbidity in observation group were lower than those in control group at 4 weeks after surgery, and there were statistically significant difference (U=6.308,8.130, 6.875, P < 0.01). The incidence of high intraocular pressure, recurrent retinal detachment rate and reduction rate in observation group were lower than those in control group at 4 weeks after surgery, and there were statistically significant difference (χ2=9.000, 10.540, 11.770, P < 0.01). Conclusions The new prone headrest can effectively improve the resection of vitreous body with tamponade patients in comfort, ensure the operation effect.

Objective To investigate the effect of inhibition of glycogen synthase kinase?3 beta ( GSK?3β) activity on sevoflurane postconditioning?induced cardioprotection in diabetic rats. Methods Healthy adult male Sprague?Dawley rats, weighing 250-300 g, in which diabetes mellitus was induced by intraperitoneal 1% streptozotocin 60 mg∕kg combined with high?fat and high?sucrose diet and confirmed by blood glucose level >16. 7 mmol∕L. Forty rats with diabetes mellitus were divided into 5 groups ( n=8 each) using a random number table: sham operation group ( S group ) , ischemia?reperfusion ( I∕R ) group, sevoflurane postconditioning group ( SP group) , GSK?3β inhibitor SB216763 group ( SB group) , and sevoflurane postconditioning plus SB216763 group ( SS group ) . Myocardial ischemia was induced by 30 min occlusion of the left anterior descending branch of the coronary artery followed by 120 min reperfu?
sion. The rats inhaled sevoflurane with the end?tidal concentration of 2.5% for 5 min starting from 1 min be?fore reperfusion in group SP. SB216763 0.2 mg∕kg was injected via the caudal vein at 1 min before reperfu?sion in group SB. In group SS, the rats inhaled sevoflurane with the end?tidal concentration of 2.5% for 5 min starting from 1 min before reperfusion, and SB216763 0.2 mg∕kg was injected via the caudal vein at 1 min before reperfusion. At 120 min of reperfusion, blood samples were collected from the carotid artery for determination of serum creatine kinase?MB (CK?MB) activity and cardiac troponin I (cTnI) concentra?tions. Myocardial specimens were collected at 120 min of reperfusion for microscopic examination of the pathological changes and for determination of myocardial infarct size ( by 2,3,5?triphenyltetrazolium chlo?ride staining) and phosphorylated GSK?3β (p?GSK?3β) expression (by Western blot). Results Com?pared with group S, the myocardial infarct size and serum CK?MB activity and cTnI concentration were sig?nificantly increased, and the expression of p?GSK?3βwas significantly down?regulated in I∕R, SP, SB and SS groups (P<0.05). Compared with group I∕R, the myocardial infarct size and serum CK?MB activity and cTnI concentration were significantly decreased, and the expression of p?GSK?3β was significantly up?regulated in SB and SS groups (P<0.05), and no significant change was found in the parameters men?tioned above in group SP ( P>0.05) . Compared with group SB, the myocardial infarct size and serum CK?MB activity and cTnI concentration were significantly decreased, and the expression of p?GSK?3β was sig?nificantly up?regulated in group SS (P<0.05). The pathological changes of myocardium were significantly attenuated in SB and SS groups as compared with group I∕R and group SP . Conclusion Inhibition of GSK?3β activity can improve sevoflurane postconditioning?induced cardioprotection in diabetic rats.