A method based on ultra performance liquid chromatography-tandem mass spectrometry ( UPLC-MS/MS) has been proposed for the determination of coccidiostat residues in chicken skin and fat. The sample was extracted with the combination of methanol, acetonitrile, and acetic acid, and cleaned-up by Sep-Pak tC18 solid phase extraction cartridge. Data acquisition under positive electrospray mode was performed by applying multiple reaction monitoring for both identification and quantification. The limits of detection and quantification for halofuginone and robenidine were 7 μg/kg and 20 μg/kg, respectively. The limit of detection of salinomycin, monensin, narasin, maduramicin, and lasalocid was 5 μg/kg, and limit of quantification was 15 μg/kg. The recovery was 75% to 110% in the spiked concentration range from 15 μg/kg to 200 μg/kg, with intra-day precision lower than 12. 8%, and inter-day precision lower than 13 . 4%.

In order to investigate the role of the PTEN expression in carcinogenesis and development of endometrial carcinoma and clarify whether and how PTEN and PI3K/Akt pathway relate to endometrial carcinoma, the expression of PTEN and phospho-Akt was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) methods and Western-blot from 24 cases of endometrial carcinoma, 10 cases of endometrial atypical hyperplasia, 10 cases of endometrial hyperplasia, and 10 cases of normal endometrium. SP immunohistochemical methods were used to measure levels of PTEN protein expression in following 5 study groups: 31 cases of endometrium in proliferative phase, 30 cases of endometrium in secretory phase, 71 cases of endometrial hyperplasia, 25 cases of atypical hyperplasia and 73 cases of endometrial carcinoma. Immunostaining score of PTEN was 3.39+/-0.15 in proliferative phase, 1.90+/-0.21 in secretory phase, 3.34+/-0.29 in endometrial hyperplasia, 0.62+/-0.11 in atypical hyperplasia, and 0.74+/-0.19 in endometrial carcinoma, respectively. PTEN mRNA relative value in normal endometrium, endometrial hyperplasia, endometrial atypical hyperplasia, and endometrial carcinoma was 2.45+/-0.51, 2.32+/-0.32, 0.46+/-0.11, and 0.35+/-0.13 respectively. The expression levels of PTEN mRNA and protein in patients with endometrial carcinoma and atypical hyperplasia were significantly lower than in those of proliferative phase and with endometrial hyperplasia. The level of PTEN expression in patients with endometrial carcinoma was significantly related to tissue type (P<0.005), differentiation (P<0.05) and clinical stage (P<0.05), but not to depth of myometrium invasion (P>0.05). Western blot analysis revealed that Phospho-Akt level in PTEN negative cases was significantly higher, and there was a negative correlation between PTEN and phospho-Akt (r=-0.8973, P<0.0001). It was suggested that loss of PTEN expression was an early event in endometrial tumorigenesis. The phosphorylation of Akt induced by the loss of PTEN took part in the tumorigenesis and development of endometrial carcinoma.

A strain of high-efficiency bioflocculant-producting bacteria,which was named TJ-3,was screened from sewage sludge.The strain was gram-negative and rod-shaped in physiological biochemical test and was identified as Pseudomonas aeruginosa by 16S rDNA Sequencing.According to the growth curve of the TJ-3 strain,the growth stabilization period is long so that Microbial Flocculants(MBF) production is stable.The MBF produced by the TJ-3 strain is able to flocculante kaolin suspension with 98.2%.The MBF activity distribution tests show that most of the active components of the bioflocculant exist in deposition after centrifufation.When pH is 8.5,1%(quality fraction) CaCl2 Solution dosing quantity is 3.5 mL,bioflocculant dosing quantity is 1.5 mL in 100 mL kaolin suspension,the bioflocculant has an optimal flocculation.

Purpose: This study aimed to investigate the mechanistic downregulation of mucin 6 (MUC6) and its influence on the progression of gastric cancer (GC). Materials and Methods: The expression of MUC6 was examined in 40 GC patients. The methylation status of the MUC6 promoter region was investigated using GC cell lines and GC tissue specimens by immunohistochemistry and/or quantitative polymerase chain reaction (qPCR). MUC6 was knocked down in the gastric epithelial cells (GES-1) cell and overexpressed in the SGC7901 cell.The effects of MUC6 knockdown and overexpression on cell migration and invasion were examined using Transwell assays. The effects of demethylation and methylation on MUC6 expression were examined by western blot, qPCR, or double luciferase reporter assays. Results: The expression of MUC6 in GC with lymph node metastasis and poor pathological stage was significantly lower than that in GC without lymph node metastasis and good pathological stage, respectively. While cell migration and invasion were significantly decreased after overexpression of MUC6, these abilities significantly increased after the knockdown of MUC6. The methylation levels of MUC6 in GC tissues and GC cell lines were significantly higher than those in para-cancerous tissues and normal GES.Promoter methylation could significantly reduce the binding of related transcription factors to the MUC6 promoter. The expression of MUC6 increased with the concentration and time of action of demethylation drugs. Conclusion Expression of MUC6 was regulated by promotor methylation. This methylation of the MUC6 promoter may lead to significant downregulation of MUC6 in GC and promote the progression of GC.

Purpose: This study aimed to investigate the mechanistic downregulation of mucin 6 (MUC6) and its influence on the progression of gastric cancer (GC). Materials and Methods: The expression of MUC6 was examined in 40 GC patients. The methylation status of the MUC6 promoter region was investigated using GC cell lines and GC tissue specimens by immunohistochemistry and/or quantitative polymerase chain reaction (qPCR). MUC6 was knocked down in the gastric epithelial cells (GES-1) cell and overexpressed in the SGC7901 cell.The effects of MUC6 knockdown and overexpression on cell migration and invasion were examined using Transwell assays. The effects of demethylation and methylation on MUC6 expression were examined by western blot, qPCR, or double luciferase reporter assays. Results: The expression of MUC6 in GC with lymph node metastasis and poor pathological stage was significantly lower than that in GC without lymph node metastasis and good pathological stage, respectively. While cell migration and invasion were significantly decreased after overexpression of MUC6, these abilities significantly increased after the knockdown of MUC6. The methylation levels of MUC6 in GC tissues and GC cell lines were significantly higher than those in para-cancerous tissues and normal GES.Promoter methylation could significantly reduce the binding of related transcription factors to the MUC6 promoter. The expression of MUC6 increased with the concentration and time of action of demethylation drugs. Conclusion Expression of MUC6 was regulated by promotor methylation. This methylation of the MUC6 promoter may lead to significant downregulation of MUC6 in GC and promote the progression of GC.

Objective: To explore the effect of pulsatile lavage on wound healing in diabetic foot ulcer patients. Methods: The random number table method was used to divide 86 patients of diabetic foot ulcers into two groups with 43 patients in each group. The control group disinfected and cleaned the wound by routine methods, while the experimental group received closed pulse irrigation with sewage collection unit. The two groups were debridement, dressing selection and wound dressing in a unified way. The frequency of dressing change, time of dressing change, efficacy, cost of dressing changes, score of wound pain and wound healing were observed. Results: The frequency of dressing change, dressing change time, wound healing time and total effective rate of the experimental group were (10.42±1.92) times, (12.19±2.37) min, (32.53±6.91) d and 86.04% (37/43), respectively, while those of the control group were (19.47±3.13) times, (21.65±3.99) min, (43.17±13.72) d and 51.16% (22/43), with statistically significant differences (t values were 4.545-16.127, χ² value was 13.214, all P < 0.01). However, the cost of dressing change in the experimental group was (3 278.78±220.92) yuan, and that in the control group was (3 195.75±206.54) yuan. There was no significant difference between the two groups (t value was -1.814, P > 0.05). The pain scores were (1.47±1.09), (0.57±0.72), (0.06±0.23), (0.003±0.01) points in the experimental group at the 2nd week,the 4th week,the 6th week and the 8th week after intervention, and they were (3.83±1.16), (2.73±1.41), (1.92±1.06), (1.43±0.70) points respectively in the control group, the differences were statistically significant (F_time=390.663, F_intergroup=76.011, F_interaction=4.210, all P < 0.01). The wound healing rates were (49.34±9.34)%, (86.26±13.33)%, (95.01±8.56)%, (97.28±3.62)% respectively in the experimental group in the 2nd week,the 4th week,the 6th week and the 8th week after intervention,while in the control group they were (26.64±5.19)%, (50.37±10.53)%, (64.84±12.27)% and (72.04±12.96)%. The differences between the two groups were statistically significant (F_time=354.487, F_intergroup= 921.230, F_interaction =23.154, all P < 0.01). Conclusions Pulsatile lavage can effectively clean the wound, reduce the frequency of dressing change, shorten the time of dressing change and wound healing, reduce the wound pain, improve the wound healing rate of diabetic foot ulcers, and did not increase the economic burden of patients, which was worthy of clinical application.

Objective To prepare the human papillomavirus (HPV) 16 peptide vaccine and explore the effect in vitro and in vivo. Methods (1) Prediction of the major histocompatibility complex (MHC) class I restricted T cell epitopes by bioinformatics target at transporter associated with antigen processing (TAP) and named by E7Pa, E7Pb, E7Pc separately. (2)In vivo, the C57BL/6 mice were divided into five groups with same amounts randomly after loading with TC-1 cells (HPV 16 positive tumor cells from C57BL/6 mouse), named as E7Pa + CpG,E7Pb + CpG,E7Pc + CpG (as experiment groups, and added 50 μg/ml E7Pa, E7Pb, E7Pc, respectively), CpG(as positive control group and added Con A with 12 mg/L final concentration) and blank control group (without any treatment). The T cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay at different time points;the lactate dehydrogenase (LDH) delivery method was used to test the cytolytie T lymphocyte (CTL) activity of mouse splenic lymphocyte in different ratio of effector cells and target cells (E:T);the related cytokines in tumor tissue and mouse peripheral blood were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The tumor volumes were measured to contrast the therapeutic effect in different groups. Results (1) Three peptide named E7Pa, E7Pb, E7Pc were successfully preparated which had high affinity and specificity. (2) After vaccination of 24, 48, 72,96 hours, MTT results shown that the proliferation rate in E7Pa + CpG group were(131±32)%, (302±15)%, (552±28)%, (731±24)% individually, which were much higher than those in blank control [(72± 15) %, (120 ± 57) %, (176 ±41)%, (288±29)% ;P<0.01], and the other groups i. e. E7Pb + CpG,E7Pc +CpG and CpG groups all proliferated much higher than those in blank control group with statistic signification (P<0. 05), but there was no significant difference between groups(P>0.05);the LDH delivery assay showed that when the ratio of E:T was 100:1, the activity of CTL in the E7Pa + CpG group was most powerful than the other groups with statistic signification (P<0. 01). Meanwhile, the ratio of E:T was concentration-dependent. Compared E7Pb + CpG, E7Pc + CpG or CpG groups with blank control group, there were significantly difference(P<0. 05) ,while there was no significant difference between groups(P >0. 05). The mRNA levels of interferon γ (IFN-γ), interleukin-2 (IL-2) in tumor tissue and peripheral blood in E7Pa + CpG group were significantly higher than those in blank control group (P<0. 01), which was the similar results when compared E7Pb + CpG, E7Pc + CpG or CpG groups with control group (P < 0. 05), and without significant difference between groups(P > 0. 05). The tumor volumes were suppressed obviously in all the experiment groups, especially at the 60th days, the volumes in ETPa + CpG group were much smaller than that in blank control group with statistic signification (P < 0. 01),which was the similar results that E7Pb + CpG, E7Pc + CpG or CpG groups had difference than blank control group with statistic signification (P < 0. 05), and without significant difference between groups(P >0. 05). Conclusion The HPV16 E7 peptide target at TAP combination with CpG as a vaccine could treat effectively the HPV16 E7 positive tumor in experiment.

Objective To construct eukaryotic expressing vector of mouse soluble CD160 and stably transfect into CHO cells for eukaryotic expression.Methods Recombinant soluble CD160(rsCD160) was constructed by gene recombination.Total RNA was extracted from the spleen of C57BL/6 mice.cDNA was amplified for the soluble form of CD160.Then,the PCR product was cloned tO pcDNA3.1 and pEGFP-N1.The recombinant plasmid was identified by restriction map and sequence analy-sis.The soluble CDl 60 expression in CHO cells transfected with recombinant psCDl 60 was verified by RT-PCR and Westernblot.The binding ability of psCD160 tO its ligand was detected by FACS.Results 520 bp mouse soluble CD160 gene was obtained.Recombinant mouse psCD160 was successfully constructed.After transfection,soluble CD160 expression in the culture supernatant of CHO cells was successfully detected.FACS analysis indicated that soluble CD160 could bind tO its ligand.Conclusion Recombinant mouse psCD160 is successfully constructed,which will benefit our further study on soluble CD160 for immune therapy against tumor in the future experiments.

Objective To explore the activated brain region of acute epilepsy in cat model induced by pentylenetetrazol(FFZ)with manganese enhanced-functional MR imaging(ME-fMRI),and evaluate the application of ME-fMRI on localization of the activated brain.Methods Forty cats were divided into 4 groups by random number table method as epileptic A and B groups as well as control A and B groups. Cats of epileptic groups were injected with PTZ(55 mg/kg)intramuscularly,and those of control groups were injected with the saline at same dose.The behavior change in the epileptic and control group A was observed and electroencephalogram(EEG)was also undertaken.Cats of epileptic and control group B were performed ME-fMRI,and the percentage of the enhanced signal intensity was then calculated.Results After injection with PTZ(55 mg/kg)intramuscularly,epileptic seizure was all evoked,and then EEG recording showed spike-wave and polyspike-wave complexes.The neocortex of cats of epileptic group B was diffusely phanero-enhanced on ME-fMRI.The percent enhancement of signal intensity in cortex of frontal lobe,parietal lobe and occipital lobe was(34.6?5.7)% and that in cortex of temporal lobe with(22.9? 6.5)%,whereas those of control group B with(14.9?4.5)% and(11.6?3.2)% respectively.And there was significant difference between the above different localization of the brain in the two groups (t=-10.43,-5.46 respectively,P

In order to investigate the role of the PTEN expression in carcinogenesis and develop-ment of endometrial carcinoma and clarify whether and how PTEN and PI3K/Akt pathway relate to endometrial carcinoma,the expression of PTEN and phospho-Akt was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) methods and Western-blot from 24 cases of endomctrial carcinoma,10 cases of endometrial atypical hyperplasia,10 cases of endometrial hy-perplasia,and 10 cases of normal endometrium.SP immunohistochemical methods were used to measure levels of PTEN protein expression in following 5 study groups:31 cases of endometrium in proliferative phase,30 cases of endometrium in secretory phase,71 cases of endometrial hyperplasia,25 cases of atypical hyperplasia and 73 cases of endometrial carcinoma.Immunostaining score of PTEN was 3.39±0.15 in proliferative phase,1.90±0.21 in secretory phase,3.34～0.29 in endometrial hyperplasia,0.624±0.11 in atypical hyperplasia,and 0.74±0.19 in endometrial carcinoma,respectively.PTEN mRNA relative value in normal endometrium,endometrial hyperplasia,endometrial atypical hyperplasia,and endometrial carcinoma was 2.45±0.51,2.32±0.32,0.46±0.11,and 0.35±0.13 respec-tively.The expression levels of PTEN mRNA and protein in patients with endometrial carcinoma and atypical hyperplasia were significantly lower than in those of proliferative phase and with endo-metrial hyperplasia.The level of PTEN expression in patients with endometrial carcinoma was sig-nificantly related to tissue type (P<0.005),differentiation (P<0.05) and clinical stage (P<0.05),but not to depth of myometrium invasion (P>0.05).Western blot analysis revealed that Phospho-Akt level in PTEN negative cases was significantly higher,and there was a negative correlation between PTEN and phospho-Akt (r=- 0.8973,P<0.0001).It was suggested that loss of PTEN expression was an early event in endometrial tumorigenesis.The phosphorylation of Akt induced by the loss of PTEN took part in the tumorigenesis and development of endometrial carcinoma.