Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.

Objective:To evaluate the effect of continuing intervention on prophylactic application of antibiotics in sterile operation in urology department by clinical pharmacist to provide reference for the clinical prophylactic application of antibiotics. Methods:All cases of discharged patients underwent sterile operation in urology department of our hospital from July 2010 to June 2014 were divided into three groups according to the intervention time and methods: non-intervention group(n=141), stage Ⅰ intervention group(n=139), stage Ⅱ intervention group (n=162) and stage Ⅲ intervention group (n=137). The prophylactic application of antibiotics was statistically analyzed. Results:After the continuing intervention, the prophylactic application rate of antibiotics in the three inter-vention groups was decreased significantly from 100% before the intervention respectively to 34. 5%,18. 5% and 14. 6% after the in-tervention (P<0. 01). The rationality rate of prophylactic application was improved significantly from 36. 9% before the intervention respectively to 58. 3%, 63. 3% and 85. 0% after the intervention (P<0. 01). The course of prophylactic application was decreased significantly from (138.2 ±31.6)h respectively to (89.9 ±48.0)h,(72.8 ±32.5)h and(45.1 ±29.5)h (P<0.01) and the post-operative infection rate was decreased from 2. 8% respectively to 2. 1%,1. 8% and 1. 4%. Conclusion:The pharmaceutical interven-tion is feasible and valid to improve the rational prophylactic use of antibiotics in urological surgery.

ObjectiveTo investigate the effects of different amounts of iodine intake on the cellular and humoral immune in Grave's disease (GD) patients.MethodsThe clinical GD cases were diagnosed by thyroid fine needle Cytology examination.Patients in GD group are divided into GD group Ⅰ and GD group Ⅱ based on the median of urine iodine.The blood levels of FT4,FT3,TSH,TPOAb,TGAb,TRAb and TNF-t were detected.The difference and association of these parameters between these groups were analyzed.ResultsThe TNF-αt level in GD Ⅰ group was higher than that of GD Ⅱ group( P ＞ 0.05 ) ;The average level of TRAb of GD Ⅰgroup ( [ 1.4 ±0.2 ] U/L) were higher than that of GD Ⅱ group ( [ 1.2 ± 0.1 ] U/L) ( P ＜ 0.05 ) ;The positive rates of TGAb and TPOAb of GD Ⅰ group were higher than that of GD Ⅱ group ( P ＜ 0.05 ).The percentages of patients with high level of TGAb and TPOAb in GD Ⅰ group ( 78.9％ 、84.2％ ) were higher than that in GD Ⅱ group (50.0％,62.5％ ) ( x2 =6.79,10.70,P ＜0.05 ) ; Analysis showed a linear positive correlation of TNF-αwith TRAb and TPOAb ( r is 0.489 and 0.563,P ＜ 0.01 ).ConclusionIodine is an important factor to the development of Graves disease.Excessive iodine intake will exaggerate the GD condition and patients with GD should be controlled for iodine intake.

Objective To study the relationship between cellular immunity in vivo,humoral immunity and different iodine intakes in patients with hashimoto thyroiditis(HT).Methods Seventy-six HT patients were divided into two groups acconding to the median of urine iodine (MUI =491.20 μ g/L):HT I group (urine iodine≥MUI) with 37 cases and HT Ⅱ group (urine iodine ＜ MUI) with 39 cases.And 49healthy persons were selected as control group.The level of free three triiodothyronine (FT3),free thyroxine (FT4),thyroid stimulating hormone (TSH),thyroglobulin antibody (TGAb),thyroid peroxidase antibody (TPOAb),thyroid hormone receptor antibody ( TRAb ),tumor necrosis factor-alpha ( TNF- α )of all groups were detected.Results The levels of FT3 and FT4 in HT I group [ (2.67 ± 1.93 ),( 4.22 ± 3.77) pmol/L ]and HT Ⅱ group [ ( 3.19 ± 1.63 ),( 5.99 ± 3.97 ) pmol/L ] were significantly lower than those in control group [(5.30± 1.10),(16.50 ±2.70) pmol/L] (P ＜ 0.01).The levels of TNF-α in HT I group [(6.14 ± 1.83)ng/L] and HT Ⅱ group [ (6.09 ± 1.50) ng/L] were both obviously higher than that in control group [ ( 1.90 ±0.60) ng/L] (P ＜ 0.01 ).The levels of FT3 and FT4 were lower and TNF α was higher in HT I group than those in HT Ⅱ group,but there was no statistically significance (P ＞ 0.05 ).The positive rate of TPOAb,TGAb in HT I group [97.3％(36/37),81.1％(30/37)] and HT Ⅱ group [89.7％(35/39),74.4％(29/39)]were significantly higher than those in contnol group [ 18.4％(9/49),12.2％(6/49 ) ] (P ＜ 0.01 ).There was no statistically difference of the positive rate of TPOAb,TGAb and TRAb between HT I group and HT Ⅱ group (P ＞ 0.05).While the percentage of patients with high titer of TPOAb and TGAb in HT I group was higher than that in HT [Ⅱ group,and there was statistical difference(P ＜ 0.05 ).The level of TRAb in HT I group was higher than that in HT Ⅱ group [ ( 1.25 ± 0.14) mU/L vs.( 1.16 ± 0.21 ) mU/L ],but there was no significant difference (P ＞ 0.05).Correlated anlysis showed that FT3 was negatively correlated with TGAb and TPOAb (r =0.342,-0.397,P ＜0.05),and TNF-αwas positively correhted with TGAb and TPOAb (r =0.405,0.561,P ＜ 0.05).Conclusions High iodine intake influences the autoimmune mechanism of HT patients.The iodine intake should be limited in HT patients.

Objective To improve the diagnosis and treatment ot adrenocorticotropin-independent macornodular adrenal hyperplasia (AIMAH).Methods The clinical data of 17 cases with AIMAH from 2000 to 2011 were analyzed retrospectively,including 3 subclinical AIMAH,10 clinical AIMAH and 4 highrisk AIMAH patient,with common radiological characteristic of bilaterally enlarged adrenal glands with multiple nodules like ginger.The 3 cases of subclinical AIMAH patients presented with decreased serum ACTH,normal or slightly elevated plasma cortisol and urinary free cortisol level,no suppression following 1 mg overnight dexamethasone suppression test and absence of clinical signs of Cushing syndrome (CS).While clinical AIMAH and high-risk AIMAH presented with clinical signs of CS,elevated plasma cortisol and urinary free cortisol level,suppressed serum ACTH,loss of normal circadian rhythm in cortisol secretion and no suppression following the low-dose and high-dose overnight dexamethasone suppression test.Among the 4 cases of high-risk AIMAH,2 cases presented with osteoporosis,2 cases with hepatic dysfunction,3 cases with cardiopulmonary dysfunction,and 4 cases with severe hypertension.Three cases of subclinical AIMAH were treated with symptomatic treatment,10 cases of clinical AIMAH patients with surgical operation,4 cases of high-risk AIMAH patients with ketoconazole and surgical operation.Results Three subclinical AIMAH patients received symptomatic treatment and discharged from hospital with normal blood pressure and blood glucose.During the period of follow-up from 3 months to 3 years,endocrine results were normal.Seven clinical AIM AH patients underwent unilateral adrenal tumor resection plus ipsilateral partial adrenalectomy or total adrenalectomy.CS disappeared completely after 6 to 9 months.Two clinical AIMAH patients underwent simultaneous bilateral adrenalectomy.One case died of adrenal crisis after operation,and the other case presented with adrenal insufficiency but returned to normal after glucocorticoid replacement therapy,no Nelson's syndrome happened during the follow-up for 5 years.One clinical AIMAH patient undertook unilateral adrenalectomy twice by interval,followed by routine corticosteroid replacement therapy.Followed up for 10 years,no Nelson's syndrome happened.Four high-risk AIMAH patients received ketoconazole and then underwent right total adrenalectomy.Cortisol levels returned to normal after 1 to 2 months and during the follow-up for 1 to 3 years,the laboratory examinations maintained normal.Conclusions Different treatment methods should be adapted to different subtypes of AIMAH.For subclinical AIMAH,the principal treatment is symptomatic,and close follow-up with regular adrenal imaging and endocrine examination is required.Surgical operation should be performed when clinical symptoms of AIMAH appear.Medical management is essential for high-risk AIMAH to inhibit the production of cortisol at first.Once these patients could stand the stimulation caused by operation,the adrenal glands should be resected as soon as possible.The unilateral adrenalectomy is an effective treatment for clinical AIMAH.

Objective To investigate the relationship between the serum SRC-3 levels and the bone loss severity in postmenopausal women. Methods Fifty-eight PMW with osteopenia or osteoporosis and nineteen healthy PMW were enrolled in this study from June 2012 to September 2013. BMD at the lumbar spine and femoral neck were observed by DXA Lunar Prodigy Vision. The levels of serum SRC-3 were detected by ELISA. The diagnosis value was evaluated by the ROC curves analysis. Results The levels of serum SRC-3 were significant higher in the normal group than those in the osteopenia or the osteoporosis groups (P<0.001 for both), no statistical significance was found between the osteopenia and the osteoporosis group(P=0.056). The levels of serum SRC-3 were negatively correlated with the BMD diagnosis grading (r=-0.543, P < 0.001). By using the ROC curve analysis, the serum level of SRC-3 for PMW with osteoporosis and osteopenia were found to be 0.297 ng/mL and 0.347 ng/mL, respectively. The levels of serum SRC-3 were positively associated with BMI (r=0.395, P<0.001) and LS-BMD (r=0.503,P<0.001) in the postmenopausal women. Conclusion SRC-3 might be an useful index to reflect the severity of lumbar spine bone loss.

Objective To develop a sensitive,specific, simple and rapid quantitative real-time PCR (Q-PCR) assay for detection of Staphylococcus aureus with SmartCycler.Methods According to the nuc gene sequences specific to S.aureus, a pair of primers and one TaqMan probe were designed. An internal amplification control (IAC) which is a chimeric double-stranded DNA constructed from a fragment of the Listeria monocytogenes hly gene flanked by the nuc-specific target sequences was added to the reaction system. This IAC was detected using a second TaqMan probe labeled with a different fluorophore. The performance of the nuc-IAC Q-PCR was evaluated using artificially contaminated drinking water and commercial UTH whole milk samples spiked with ATCC 6538.Results The nuc-IAC assay could be used reliably for detection with a sensitivity of 5 copies of linear plasmid DNA per reaction, 10 fg of genomic DNA in 62.5% of the reactions or 50 cfu/ml S.aureus cells with 50% probability. The quantification was linear (r~2≥0.998) over a 6-log dynamic range, with a PCR efficiency over 0.967. The 5×10~2 CFU per 25 ml mimic sample of drinking water or milk could be detected by this assay consistently and quantifiably.Conclusion The nuc-IAC Q-PCR assay for S.aureus is developed. It could not only be applied for the quantitative detection of S.aureus, but also prevent the false negatives and underestimations of contamination loads due to PCR failure.

Objective To investigate the effect of dexmedetomidine (DEX) on inflammatory mediators in different periods of sepsis and its impact on survival rate of rats with sepsis.Methods Ninety male Wistar rats were randomly divided into three groups:control group (group C),sepsis model group (group S),and DEX treatment group (group D).Lipopolysaccharide (LPS) was injected to the vein to reproduce a classic sepsis model,while in control group,equivalent volume of NS was used instead.Different treatments were given to the corresponding groups,and then venous blood was taken at given intervals.Behavioral changes and mortality rate of rats within 48 hours after experiment were recorded.The levels of the early inflammatory mediators such as tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6) and the late inflammatory mediators such as high-mobility group boxl protein (HMGB1) in blood serum were detected by ELISA.Results Among three groups,there were significant differences in mortality rate of rats,the serum level of TNF-αt,IL-6 and HMGB1 (all P ＜ 0.05).Conclusion DEX has a certain anti-inflammatory effects on early and late stages of sepsis in rats with sepsis,and can also reduce the mortality rate.

Objective To evaluate the diagnostic performance of enhancement value and morphological features by using mul-tiphasic MDCT on differentiating untypical T4a from T3 gastric cancer.Methods Fifty-one histopathologically proven T3 and T4a gastric cancer patients with smooth serosa were collected retrospectively.Three radiologists read all images regarding morphological features,while the CT value and enhancement value of regions of interest (ROIs)located in the outer of lesion were calculated.Cut-off analysis was performed to determine optimal threshold levels of enhancement value to discriminate T4a and T3 gastric cancer.Di-agnostic performance of enhancement value and combination of enhancement value and morphologic assessment were compared with morphologic assessment by means of receiver operating characteristic (ROC)curve analysis.Results The sensitivity and specificity of morphological features was 66.67% and 33.33% respectively;the area under the ROC of enhancement value (between venous phase and plain scan)for differentiating T4a from T3 gastric cancer was 0.82,with a cut-off at 43.6 HU,sensitivity of 74.07% and specificity of 70.83%.Combined conventional standard and enhancement value,sensitivity of 100.00% and specificity 26.31%. Conclusion For differentiation of T4a and T3 gastric cancer by means of MDCT,enhancement value is found to be superior to con-ventional standard.