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.

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.

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 .

Objective To explore the microsurgical treatment strategy of ruptured anterior communicating artery aneurysms.Methods From September 2009 to February 2013,thirty-three patients with ruptured anterior communicating artery aneurysms were treated with microsurgical clipping via modified pterion approach.The clinical and follow-up data were analyzed retrospectively.Results At 3 to 44 months period followed-up after microsurgical clipping of ruptured anterior communicating artery aneurysms were obtained in 32 patients.According to Glasgow Outcome Score,the curative effects score were 5 in 28 cases,four in 2 cases,three in 1 case and death in 1 case.Conclusion Microsurgical clipping of ruptured anterior communicating artery aneurysms via modified pterional approach was an effective method with sufficient exposure.It has less invasiveness to the brain tissue,reliable neck clipping and satisfactory results.Safety and effectiveness of this procedure are based on preoperative radiological evaluation of three-dimensional morphological specificity of aneurysms,identification of the parental arteries and its branches,as well as temporary proximal occlusion.

Objective To evaluate left intra-ventricular diastolic blood flow patterns and fluid dynamics in pre diabetics using vector flow mapping(VFM),and to explore the value of left intra-ventricular hydromechanics characteristics assessment of intracardiac flow by VFM with more sensitive indexes.Methods Forty four patients with type 2 diabetes,twenty-eight pre-diabetes patients and thirty-three normal volunteers underwent VFM to determine the left ventricular(LV) diastolic blood flow patterns and fluid dynamics.The two-dimensional color Doppler flow images data were acquired in standerd apical fourchamber view during 3 complete cardiac cycles.These images were analyzed using a dedicated off-line workstation.At different level (mitral annulus,papillary muscl and apex level),the diastole blood flow parameters of LV including flow velocity and volume,velocity gradients (△V),pressure gradients (△P),diastole flow volume,vortex intensity ( Ⅵ),vortex area ( VA),vortex radius (VA) were derived.Thedifferences of the above parameters were compared among the three groups.The correlationship between diastolic function parameters and LV hydromechanics indexes were analyzed.Results ① Compared to normal group and pre-diabetics group,the LV diastolic function parameters E/A and e/a in diabetics group were lower( P ＜0.01),with no differences for E/e( P ＞0.05).There were no significant diffrences of these indices between diabetics group and pre-diabetics group( P ＞0.05).②)Compared to normal group,the intraventricular peak flow velocity at basal level,△V and △P in diabetics group and pre-diabetics group were lower( P ＜0.01),but there were no significant differences of these indices between diabetics group and prediabetics group( P ＞ 0.05).③Compared to normal group,the diastolic positive volume at basal level in diabetics group and pre-diabetics group was lower ( P ＜0.01 ),but there was no significant differences of these indices between diabetics group and pre-diabetics group ( P ＞ 0.05).④ Compared to the normal group,the Ⅵ in diabetics and pre-diabetics group was decreased,but there were no significant differences ofthese indices between diabetics group and pre-diabetics group( P ＞0.05).The VA and VR in diabetics and pre-diabetics group were increased,but there were no significant differences of these indices between diabetics group and pre-diabetics group ( P ＞ 0.05).⑤ There were no correlationship between diastolic function parameters and LV hydromechanics indexes.Conclusions There is the same LV diastolic blood flow patterns and fluid dynamics existed in pre-diabetics as diabetics.VFM could be used as a new methods to demonstrate the early left intra-ventricular fluid dynamics damages in pre-diabetics patients.

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.

Animals ; Blood Glucose ; drug effects ; Body Weight ; drug effects ; Chronic Disease ; Female ; Hypoxia ; physiopathology ; KATP Channels ; drug effects ; Male ; Propylamines ; pharmacology ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley