ObjectiveTo investigate the clinical significance of pyrosequencing assay for determining K-ras mutations in exon 2 codons 12 and 13 in clinical colorectal cancer tissues.Methods Genomic DNA,extracted from K-ras mutant cell lines SW480 (homozygous,c.35G ＞ T), DLD-1 (heterozygous,c.38G ＞ A) and wild-type HT-29,was first used as the sequencing template respectively to test the accuracy of pyrosequencing methodology.The SW480 and DLD-1 DNA was separately mixed with wild-type HT-29 DNA in proportions of 2％,3％,5％,10％,20％,30％ and 50％,the sensitivity for mutation detection was measured separately by pyrosequencing assay and directed Sanger DNA sequencing in the serial DNA mixture samples.The pyrosequencing assay results were compared with the corresponding Sanger sequencing and the datas were analysized by Fisher exact test.Pyrosequencing analysis was then performed for screening K-ras exon 2 mutations at codons 12 and 13 on DNA isolated from a panel of 30 colorectal cancer samples derived fromclinicalformalin-fixed andparaffinembedded(FFPE)tissues.ResultsCancer cell lines with known K-ras mutations ( SW480 and DLD-1 ) were readily detectable by pyrosequencing-based analysis.When the proportions of mutant colorectal cancer cell line DNA were 5％ and 10％ content,the mutation rates of K-ras gene detected by conventional Sanger DNA sequencing were 33.3％ (4/12) and 58.3％ (7/12) respectively,whereas the mutation rates detected by pyrosequencingbased assay were 91.7％ (11/12) and 100％(12/12) respectively,there were significant differences between those two sequencing methodology ( P ＜0.05).Furthermore,we found 10 patients with K-ras exon 2 point mutations at codons 12 and 13 by pyrosequencing-based assay from 30 colorectal cancer FFPE tissues,the point mutation rate was 33.3％ (10/30) and all of the mutations determined were heterozygous.The codon 12 was most frequently affected [30％ (9/30)].Mutations with the highest frequency were G ＞ A transitions [ 50％ ( 5/10 ) ],followed by G ＞ T transversions [ 30％ ( 3/10 ) ].Conclusion The pyrosequencing assay provides an accurate and sensitive method for mutation screening of K-ras exon 2 codons 12 and 13 in routine diagnostic specimens,thereby allowing the selection of the cancer treatment in clinical individualized practice.

AIM: To examine the expression and distribution of tumor necrosis factor-? (TNF-?), tumor necrosis factor receptor I (TNFR I) and apoptosis in oral lichen planus, and evaluate their roles and relation in the oral lichen. METHODS: Immunohistochemical technique and TUNEL were employed to study the expression of TNF-?, TNFR I and apoptosis in 50 cases of oral lichen planus and 10 normal oral mucosa specimens. RESULTS: Compared with the normal control group, TNF-? expression was upregulated in mononuclear cells in lamina propria and decreased in keratinocytes in oral lichen planus lesion ( P

Objective To investigate the common cause,inducement and treatment response in patients with status epileptic(SE) in children,in order to improve the level of prevention and treatment of the disease.Methods Valium and chloral hydrate be used in the patient untreated,followed with luminal.Deparkin injection be used in the patient uncontrolled,followed with syrup.Results Of 21 patients,3 cases with epilepsy hadn′t been treated;6 cases with epilepsy hadn′t been treated regularlly;3 cases with epilepsy place respiratory infection;4 cases were the first attack with no inducement;3 cases were suffering from viral encephalitis.Four cases with epilepsy were died who hadn′t been treated regularly.Deparkin might be helpful in treatment with SE,but poor control in patients with long time attacks.Conclusions The attack and prognosis of SE are closely relative to original sick.It is important to prevent the attack.Early diagnosis is the key factor to improve prognosis.

A novel stable blue agar plate which is convenient to preparing and more effective than the universal chrome azurol sulfonate (CAS) assay established by Schwyn and Neilands was designed by replacing MM9 growth medium and pipes with certain concentrate of phosphorus buffer solution which pH could be stabled at 6.8. It is more suitable for screening over- siderophores production bacteria. Since OD_ 630 of the sample is usually out of the range of spectrophotometer with CAS assay solution when quantifying the siderophores and the outcome is not steady,the measuring wavelength had been changed to 680 nm corresponding to the middle of max absorbance and the correlation between siderophores concentrations and OD was unchanged. But the detecting sensitivity is elevated by enlarged the absorbance differences among samples with different productivity of siderophores at 680 nm .

Atomic force microscopy is a powerful new tool to investigate the structure and measure the relationship between structure of surfaces and functions in microorganisms. Comparing with conventional electron microscope, atomic force microscope has higher resolution and can image real-time structures from atomic to molecular scale in three-dimensional mode under physiological condition. Therefore, atomic force microscope is being used in almost every aspect of microbiology and has gotten many exciting findings.

CAS (Chrome azurol S) assay was a universal method for detecting the bacterial siderophores, and Sugar-Asn liquid medium has been applied to the studies on siderophores from Pieudomonas. In this paper, Asp has been substituted for Asn, and MSA-CAS agar plate was developed by integrating the MSA (sugar-Asp) medium and CAS bright blue dye, which has been used in the universal CAS assay. On the aspect of siderophores detection , 8 strains of 7 species from Pseudomonas had been screened on MSA-CAS agar plates and universal CAS assay respectively. The results showed that MSA-CAS agar was higher-sensitive and lower basic fluorescent than universal CAS assay.

Aim To investigate the effect of ketamine plus fluoxetine on depressed behavior and the expres-sion of neuronal nitric oxide synthase (nNOS)and CA-PON in prefrontal lobe of mentally depressed rats at different time points,so as to study the possible mecha-nism of ketamine plus fluoxetine inducing antidepres-sant behavior.Methods Healthy adult male Sprague-Dawley rats,aged 2.5 ~3 months,weighing 220 ~270 g,were induced as the rodent model of depression by chronic unpredictable mild stress (CUMS).After the models of depression were established,96 of CUMS modeling successfully depressed rats were selected. Then they were randomly divided into four groups (n =24 each):the depressed group (group D,untreated group),ketamine group (group K,treated with intrap-eritoneal injection of ketamine 1 0 mg·kg -1 once a day for 3 days or 7 days),fluoxetine group (group F,trea-ted with gavage of fluoxetine 1 .8 mg·kg -1 once a day for 3 days or 7 days),or ketamine plus fluoxetine group (group KF,treated with intraperitoneal injection of ketamine 1 0 mg·kg -1 plus gavage of fluoxetine 1 .8 mg·kg -1 once a day for 3 days or 7 days).Open field test and sucrose preference test were performed 1 day before depression model was established,and 1 day before and after treatment.The rats were sacrificed 1 day after the last test for determination of the expres-sion of nNOS and CAPON protein (using immuno-his-tochemisity)and mRNA (by RT-PCR)in the prefron-tal lobe.Results After the models of depression were established,the total distance,rearing number and the sucrose preference percentage (SPP)were decreased significantly compared with those before (P <0.05). There was no significant difference among all groups in the total distance,rearing number and the SPP before treatment (P >0.05 ).Compared with groups D and F,the total distance was prolonged,the number of rea-ring and SPP were significantly increased,the expres-sion of nNOS protein and mRNA was down-regulated and the expression of CAPON protein and mRNA was up-regulated in groups K and KF,with 3 days’treat-ment (P <0.05).Compared with group D,the total distance was prolonged,the number of rearing and SPP were significantly increased,the expression of nNOS protein and mRNA was down-regulated and the expres-sion of CAPON protein and mRNA was up-regulated in groups K,F and KF with 7 days’treatment (P <0.05).Compared with group F,the total distance was prolonged,the number of rearing and SPP were signifi-cantly increased,the expression of nNOS protein and mRNA was down-regulated and the expression of CA-PON protein and mRNA was up-regulated in group KF with 7 days’treatment (P <0.05).Conclusion Co-administration of antidepressant fluoxetine with ket-amine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone and it can shorten the time to improve the depressive state through promoting the expression of CAPON and inhibiting nNOS activity in the prefrontal lobe of men-tally depressed rats.

Objective To compare the efficacy between the two-cuff swan neck catheter and the Tenckhoff catheter in continuous ambulatory peritoneal dialysis (CAPD) patients prospectively. Methods One hundred and ten patients with end-stage renal disease (ESRD) were selected as candidates, who received catheter implantation and CAPD therapy for the first time. Patients were divided into group A (swan neck catheter group) and group B (Tenckhoff catheter group), 55 patients for each group. Catheters of beth groups had a straight end and were implanted by routine surgical procedure. One-year follow-up was performed and information was recorded such as complications, survival time, quit of dialysis, death, etc. Survival analysis was carried out by Kaplan-Meier method and Log-Rank tests. Results At the end of follow-up, 17 patients died, 3 received renal transplantation, 8 were transferred to hemodialysis, 3 went to other hospitals, and 79 patients (71.8%) remained in our department for CAPD. Twenty-six patients of both groups had peritonitis with a total of 35 occurrences. The total incidence of peritonitis was 0.32 times/patient year, with the detailed figure of 0.35 times/patient year for group A and 0.29 times/patient year for group B respectively (P0.05). The time interval between the catheter implanting and the onset of peritonitis was (30±29) weeks and (29±24) weeks for group A and group B respectively (P0.05). The risk of developing peritonitis in both groups was 26.97% within 1 year. Tunnel infection occurred in 2 patients and exit-site infections in 9 patients of two groups. The incidence of tunnel plus exit-site infections was 0.1 times/patient year. Incidence of tunnel infection and the exit-site infection for group A was lower than that of group B (0 vs 0.036 times/patient year and 0.06 times/patient year vs 0.11 times/patient year respectively). However, the difference was not significant (P0.05). Mechanical complications of catheter (catheter migration, omcntum wrapping, leakage of peritoneal dialysates, slip out of outer cuff), incidence of inguinal hernia and bellyache between two groups were not significantly different (P0.05). There were 4 cases of catheter drawing in each group. Both two groups had the same 12-month technical survival rate as 92.73%. Of 17 dead cases, 7 were in group A and 10 in group B (P0.05). The main death causes were cardiocerebral events (47.1%) and infections (23.5%). The 12-month survival rate was 86.34% for group A and 80.68% for group B (P0.05). Conclusions There are no significant differences of infection, mechanical complications, technical survival rate and patients' survival rate between two groups. The efficacy of swan-neck catheter is similar to Tenckhoff catheter in CAPD patients.

Adult ; Chronic Disease ; Female ; Humans ; Placenta ; pathology ; Placenta Diseases ; diagnosis ; pathology ; Pregnancy

Objective To investigate the relationship between alteration of gene in cyclic adenosine monophosphate(cAMP) signal transduction system in rats after myocardial damage and changes of cardiac function and ventricular remodeling. Methods Twenty eight male Wistar rats weighing 220 g to 250 g were randomly divided into three groups: acute myocardial damage group(AMD, n = 10), chronic myocardial damage group (CMD,n = 9 ) and sham-operation group (control, n = 9). Animal model of acute myocardial damage was established by ligation of rats left coronary artery in the AMD and the CMD groups. Rats in control group were treated similarly, except that the coronary suture was not tied. Hemodynamics and echocardiography were measured before rats were sacrificed 24 hours after operation in control and AMD groups but those in CMD groups were sacrificed 8 weeks later. Cadiocyte apoptosis were estimated by TUNEL method, cAMP levels in heart were tested by radioimmunity and the mRNA expressions for inducible cAMP early repressor (ICER), cAMP response element binding protein (CREB), phosphodiesterase 3A (PDE3A) and bcl-2 were assayed by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results The difference of left ventricular end diastolic diameter (LVEDD), left ventricular end diastolic pressure(LVEDP), maximal rising and falling rate of ventricular pressure,left ventricular systolic pressure (LVSP), eject fraction (EF) and fraction shortening (FS) were statistically significant among the three groups(F = 285.9, 196.8, 83.2, 80.4, 54.9, 196.6, 95.2, all P ＜ 0.01). LVEDD[(7.03 ±0.28), (8.20 ± 0.27)mm] and LVEDP[(11.19 ± 2.89), (19.76 ± 3.34)mmHg] in AMD and CMD groups were significantly increased, compared with those in control group[ (5.05 ± 0.30)mm, (- 5.62 ± 3.01 )mmHg, all P ＜0.01 ]. While maximal rising rate[ (2964 ± 449), (2214 ± 434)mmHg/s] and falling rate[(- 2617 ± 441),(- 1891± 424)mmHg/s] of left ventricular pressure, LVSP[ (94.19 ± 4.03), (85.85 ± 6.39)mmHg], EF[ (41.6 ±5.9)%, (35.9 ± 4.1 )%] and FS[ (36.9 ± 4.6)%, (23.1 ± 4.9)%] of left ventricular in the two groups were lower than those in control[(4759 ± 406)mmHg/s, (- 4327 ± 388)mmHg/s, (116.29 ± 8.25)mmHg, (80.9 ± 5.6)%,(53.1 ± 4.3)%, all P ＜ 0.01 ]. These changes in CMD group were more significant than those in AMD groups(P ＜0.05 or P ＜ 0.01 ). The difference of apoptotic index, cAMP and expression of ICER, CREB, PDE3A mRNA and bcl-2 mRNA were statistically significant among the three groups(F= 172.5, 141.0, 540.8, 246.8, 165.1, 563.9,all P＜ 0.01 ). Apoptotic index[ (32.8 ± 4.2)‰, (18.4 ± 3.9)‰] and cAMP in heart[ (9.95 ± 0.30), (5.60 ± 0.25)nmol/kg] in AMD and CMD groups were increased compared to control group[ (3.9 ± 1.7)‰, (2.48 ± 0.29)nmol/kg,all P ＜ 0.01 ], and those in CMD group were lower than in AMD group(all P ＜ 0.01 ). Expression of ICER mRNA (1.434 ± 0.093, 0.942 ± 0.076) and CREB mRNA(5.70 ± 0.50, 2.64 ± 0.51) in AMD and CMD groups were higher, and expression of PDE3A mRNA(48.98 ± 8.14, 16.68 ± 8.46) were lower than those in control group (0.154 ± 0.063, 1.08 ± 0.35, 105.94 ± 12.61, all P ＜ 0.01 ). The three genes in CMD group were fewer than those in AMD group(all P ＜ 0.01 ). bcl-2 mRNA was up regulated in AMD group(4.55 ± 0.27) and was down regulated in CMD group(0.35 ± 0.15) compared to control(2.18 ± 0.30, all P＜ 0.01). Conclusions There is PDE3A-ICER positive-feedback loop leading to myocyte apoptosis and heart failure after myocardial damage. The downregulation of PDE3A mRNA observed in chronic myocardial damage may play a causative role in the progression of ventricular remodeling and heart failure, in part, by inducing ICER mRNA and promoting cardiac myocyte dysfunction.