In high-risk patients,such as patients with hematologic malignancies,or patients after allogeneic stem-cell or solid-organ transplantation,early diagnosis of invasive aspergillosis(IA) is essential,as missing or delayed diagnosis of IA results in increasing rates of mortality.However,diagnosis of IA is difficult because classic tests have low sensitivity and specificity.The limited sensitivity and specificity of conventional assays for the detection of IA and the growing number of IA patients have led to the development of new assays.These methods include antigen detection systems,such as galactomannan(GM) assay,and different molecular methods(PCR assays).The GM assay is commercially available.However,it still need to be evaluated in large patient cohorts,especially children.A range of different PCR assays have been developed,targeting different gene regions,including a variety of amplicon detection methods.These molecular assays provide high potential in terms of sensitivity and specificity,but vary widely in their feasibility and up to now have not been standardised.Taken together,new non-culture-based diagnostic assays are noninvasive,simple,rapid and highly sensitive.Thus,they might be valuable tools to make early diagonosis,to reduce empirical antifungal therapy and to monitor the therapy.

Objective To preparing the reference material(RM) of lactate dehydrogenase(LD),we purification of LD from human erythrocyte(RBC)and studied its properties.Methods Using a modified procedure which including complete hemolysis of the RBC,hydroxyapatite treatment,(NH4)2 SO 4 Precipitation,CM- Sephadex C 50,Sephadex G100 and 5'- AMP- Sepharose 4B affinity chromatography.Results The purified LD has a sepecific activity of 163.0 kU/g protein.It is almost free of contaminating enzymes.Two corresponding bands were observed on both PAG plates stained for either protein or LD activity after electrophresis had been done.The apparent Michaelis constants were of 1.000 and 0.179 mmol/L for L-lactate and NAD and of 0.119 mmol/L 0.062 mmol/L for pyruvate and NADH respectively.Conclusion The final purified LD was found to be very similar to that in human serum in catalytic properties.It is intended to be a fundament to prepare a RM for the measurement of LD.

Objective: For preparing the reference material of alkaline phosphatase(ALP), the purification and properties of ALP from porcine kidney were studied. Methods: The ALP purification procedure included isolation microvillus of porcine kidney cortices by solubilization the membrance-binding enzymes with 1-butanol, precipitation with ammonium sulfate, chromatography on DEAE-Sephacel,ConA-Sepharose,Sephadex G-200 and a immuno-affinity column of anti-GGT antibodies. Results: The purified enzyme had a specific activity of 402 kU/g protein at 37℃and was almost free of contaminating enzymes. The apparent Michaelis constants was 1.35 mmol/L, and the optimum pH was 10.40. Conclusion: The kinetic properties of the preparation were very close to those of the enzyme present in the human serum, The product was suitable as enzyme preparation for producing the enzyme reference material.

Objective The positron generated at the dose deposition site by using high-energy carbon ions to hit the material annihilate with the negative electron in the material to release the gamma photon.The positron-emitting isotope (PEI) distributions in the target volume are activated significantly by carbon ions.Therefore, the mean values of positron emission tomography (PET) activity could be related to the delivered doses to the clinical target volume from carbon ion.This specialty can be used for the image registration fusion of the carbon ion treatment planning computed tomography (CT) and treatment verification PET-CT.After radiation in the almost same decay period, the relationship between the different target volume and the PET-CT SUV of different every single fraction dose can be found, then the range of SUV for the radiation target could be decided.So this PET-CT standardized uptake value (SUV) range can also provide a reference for the correlation and consistency in planning target dose verification and evaluation for the clinical trial.Methods The head phantom was used as a simulation of the real human body, the 1 cc, 4 cc, and 10 cc cube volume target contouring were done in the TPS, the 90 degree fixed carbon ion beams were delivered in different single fraction effective dose of 2.5 GyE, 5 GyE, and 8 GyE.After the beam delivery, later the PET-CT scanning was performed and parameters of scanning followed the trial regulation.The MIM Maestro software was used for the image processing and fusion to determine the maximum, minimum, average, and total values of SUV in the virtual clinical target volumes for the different single fraction dose.Results The results showed that for the same target volume, the SUV range of target had an approximate linear correlation with effective dose of target (P=0.000).The same effective dose for the different target volumes got the same SUV range (P>0.05).Conclusions For the carbon ion treatment plan, the SUV range from image registration and fusion of planning CT and PET-CT after treatment can be used to make an evaluation for accuracy of the dose distribution.And this method also could be used in the hyper-fraction treatment plan.In the SUV range research of different decay periods, the similar method can be performed for the exploration.

Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 micromol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylation status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1 kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

Objective Invasive candidiasis is associated with a significant mortality clinically.The purpose of this study was to observe the T cells immune response to fructose bisphosphate aldolase (Fba), an immunodominant antigen of Candida albicans, and determine whether the antigen has the possibility of priming cellular immune protection in invasive candiasis.Methods Using ELISPOT assay, we determined the frequencies of positive spot-forming cells (SFCs) of Fba antigen-specific T cells secreting IFN-γ, IL-4 and IL-17A in the peripheral blood mononuclear cells (PBMCs) of 26 healthy individuals.Results After Fba stimulation, the frequencies of positive SFCs of IFN-γ, IL-4 and IL-17A in the 26 healthy subjects were 23 (9.75, 42.50), 0 (0, 0.25) and 1.5 (0.75, 8.25), respectively, with statistically significant differences among the three (P<0.01).The response rates of IFN-γ (100% [26/26]) and IL-17A (76.92% [20/26]) were significantly higher than that of IL-4 (15.38% [4/26]) (P<0.01).Fba-induced strong response (SCFs ≥20) for IFN-γ was observed in 57.69% (15/26) of the healthy individuals, that for IL-17A in only 1, while that for IL-4 in none.Responses of both Th1 and Th17 cells to Fba were found in 65.38% (17/26) of the subjects, that of Th1 cells in 19.23% (5/26), but that of Th2 cells in none.Conclusion Fba of Candida albicans can induce immunodominant responses of Th1 and Th17 cells and is a potential vaccine against invasive candiasis.

Objective The effects of candesartan,an angiotensin Ⅱ type 1 receptor blocker (ARB) were investigated on advanced glycation end-products accumulation and the receptor for AGE (RAGE) expression in type 2 diabetic KK/Ta mouse kidneys.MethodsKK/Ta mice(n=72)were random divided into three groups(n=24) and it was treated with candesartan [4 mg/(kg·d)] or vehicle from 6 or 12 to 28 weeks of age.BALB/c mice(n=24) treated with vehicle were used as controls.Body weight,blood pressure,blood glucose,urinary microalbumin,urinary creatinine and serum creatinine were measured every four weeks.At 28 weeks,renal expressions of carboxymethyllysine and RAGE were evaluated by immunohistochemistry and/or competitive RT-PCR.Results KK/Ta mice developed high body weight,high blood glucose,and high urinary microalbumin/creatinine ratio in KK/Ta mice at 28 weeks of age,and it was significantly higher than that of BALB/c mice [(427.49±89.37)mg/g vs (9.54±3.25)mg/g,P＜0.01 ].Protein and mRNA expressions of RAGE were upregulated in KK/Ta kidneys with increased immunostaining intensities of carboxymethyllysine.Candesartan treatment has markedly reduced urinary microalbumin/creatinine ratio [Early treatment group (32.18±9.41)mg/g,Late treatment group (53.20±7.26)mg/g,P＜0.01 ].Treatment with candesartan down-regulated the protein and mRNA expressions of RAGE and reduced the accumulation of carboxymethyllysine.There were no significant differences between the two treatment groups (from 6 or 12 weeks).ConclusionsThe results suggest that candesartan,an ARB,reduces advanced glycation end-products accumulation and subsequent albuminuria by down-regulating RAGE expression in type 2 diabetic KK/Ta mouse kidneys.

Objective To study molecular epidemiology and carbapenem-resistance mechanism of four Escherichia coli strains isolated from general surgery wards. Methods Antibiotic susceptibility was carried out by K-B gar diffusion and agar dilution methods. Carbapenemases were screened by three dimensional test and EDTA-Na_2-disk synergy test. Pulsed-field gel electropboresis (PFGE) was performed to analyze molecular epidemiology of isolates. Plasmid was extracted by using an alkalinelysis technique. Conjunction experiment, transformation assay, specific PCR and DNA sequencing were performed to confirm carbapenemase genotype and its transmission mechanism Results Four Escherichia coli isolates were resistant to most antimicrobials including carbapenem. PFGE showed that the four isolates belong to four different clonal strains. Specific PCR and DNA sequence analysis identified that carbapenem resistance in four clinical isolates was mediated by KPC-2 encoded on an approximately 56 000 bp plasmid, and this plasmid did not harbor aminoglycosides and fluorquinolones resistant genes. Conclusion Four Escherichia coli isolates with carbapenem resistance are obtained from our hospital, and KPC-2 plasmid is main cause of carbapenem resistance in these isolates.

Hypermethylation in the promoter region is an important epigenetic mechanism for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferases are considered to be the main cause of promoter hypermethylation. In order to explore the roles of two methyltransferase members (DNMT1 and DNMT3b) in the cholangiocarcinoma tumorigenesis, antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene was constructed respectively, and were co-transfected into the human cholangiocarcinoma cell line QBC-939 to observe their biological effects on the cell growth and proliferation ability, apoptosis, cell cycle alteration, and the tumorigenesis ability in the subcutaneous tissue of nude mouse. The results demonstrated that co-transfection with antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene and single transfection with antisense eukaryotic expression plasmid of DNMT1 gene can suppress the growth and proliferation of QBC-939, block the cell cycle at G1 phase, increase the apoptosis rate, minimize the tumor size in the subcutaneous tissue of nude mouse. The suppressing biological effect of co-transfection is stronger than single transfection with antisense DNMT1. Meanwhile, single transfection with antisense eukaryotic expression plasmid of DNMT3b gene has no effects on the biological characteristics of QBC-939. This study suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function in inactivation of tumor suppressor genes. Combination DNMT1 and DNMT3b will increase their biological effects and have the synergistic effect on suppressing the growth of human cholangiocarcinoma cell line QBC-939.
Apoptosis ; Biliary Tract Neoplasms/*metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma/*metabolism ; DNA (Cytosine-5-)-Methyltransferase/*biosynthesis ; DNA (Cytosine-5-)-Methyltransferase/genetics ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Mice, Nude ; Neoplasm Transplantation

Objective 30 Pseudomonasaeruginosa mechanism of resistance to quinolones.Methods For the determination of ciprofloxacin MIC by agar dilution method.Used PCR on DNA gyrase and topoisomerase Ⅳ,resistance genes gyrA,gyrB, parC and parE were amplified,and BLAST,to determine whether there was resistance to bits mutation point;using pulsed-field gel electrophoresis (PFGE)of these 30 strains homology analysis.Results The 28 bacterial strains gyrA gene ampli-fied fragment of 137 points were C→T mutation causes T83I;17 strains gyrB gene amplified fragment of 351 G→C lead to G466A;parC gene amplification 21 bacteria fragment 277 point increase with C→U mutation causes S87L change two differ-ent strains parE gene locus C→U mutation A425V and A473V cause change.PFGE results:30 Pseudomonas aeruginosa could be divided into six clones,Aclone 4,B clone 7,C clone 3,D clone 14,and two other single clones.Conclusion The tar-get mutant strains closely related to the epidemic clone type,the same changes in the same pop-type strains of drug targets, and proportional to the level of ciprofloxacin MICs value,the more the number of mutated genes,MICs value higher.GyrA gene most prone to mutation,the mutation was also the first to be discovered,more than any other target of the mutation mutations on binding of drugs and targets that would be the focus of concern.