Objective To establish a multiplex real-time quantitative polymerase chain reaction (MRQPCR) assay for fast and simultaneous detection of Escherichia coli (E.coli) and Candida albicans (C.albicans) genes in human whole blood,in order to facilitate differentiation of the types of microorganism and evaluation of the severity of bacterial or fungi translocation due to impaired gut barrier,hence providing help to select specific antimicrobial agents.Methods The β-D-galactosidase gene of E.coli and ITS2 gene of C.albicans were selected as the target genes for designing primers and probes.E.coli and C.albicans genomes were extracted with QIAamp(R) DNA Blood Mini Kit,and the 25 μl TaqMan MRQ-PCR amplification reaction system was established.18 simulated human whole blood samples and 10 whole blood samples from febrile surgical patients were detected for E.coli and C.albicans genes using MRQ-PCR.Results The specificity of the primers and probes were excellent.The correlation coefficients of the standard curves of E.coli and C.albicans were 0.994-0.999 and 0.994-0.998,respectively;and the efficiency of amplification were 0.894-1.022 and 0.905-1.028,respectively.In the standard samples,the lowest detection limits of E.coli and C.albicans were 13.9 copies/μl and 0.8 cfu/μl,respectively;the sensitivity was 100％ and 99.69％,the specificity was 100％ and 94.73％,respectively;the average recovery rates were (101.89 ± 5.69)％ and (103.74 ± 4.64)％ respectively;the intra-batch coefficients of variance (CV) in detecting the genes were (13.14 ± 10.27)％ and (19.18 ± 8.54)％,respectively,and the inter-batch CV were (14.35 ± 9.34)％ and (18.31 ± 10.25) ％,respectively.In human whole blood,the lowest detection limits of E.coli and C.albicans were 12 455.2 copies/ml and 800.3 cfu/ml,respectively;the average recovery rates were (111.60 ± 11.06) ％ and (99.96 ± 6.16) ％,respectively;the intra-batch CV in detecting the genes were (11.02 ± 5.65) ％ and (8.14 ± 7.29)％,respectively,and the average inter-batch CV were (12.88 ± 7.59)％ and (18.62 ± 9.14)％.Conclusions MRQ-PCR is a rapid,sensitive,specific,accurate,and reproducible method for simultaneous detection of E.coli and C.albicans genes in human whole blood,with sample-,cost-,and time-saving advantages.It is a promising technique for rapid differentiation between fungi and bacteria,which could help targeted administration and evaluation of antimicrobial agents,and help to assess the consequence of gut barrier damage and the efficacy of treatment.

Objectives To monitor the residents prevalence of endemic arsenism in the disease affected areas in Inner Mongolia, so as to provide feasible suggestions for control of arsenism in the future. Methods Monitoring data were obtained from the Project of Endemic Disease Prevention Granted by Central Government in 2010 - 2012, and the conditions of arsenism patients from 38 endemic arsenic villages were analyzed among different year, age and gender. Results The detection rate of arsenism of the 38 surveillance villages was 7.38%(517/7 004) in 2010, 7.10%(482/6 784) in 2011 and 6.62% (431/6 514) in 2012. The arsenism patients of mild;moderate and severe cases from 2010 to 2012, accounted for 74.47% (385/517), 74.27% (358/482), 75.17% (324/431); 16.83% (87/517), 16.60% (80/482), 15.78% (68/431) and 8.7% (45/517), 9.13% (44/482), 9.05% (39/431), respectively. For skin lesions, the detection rates of keratosis, pigmentation and depigmentation from 2010 to 2012, were 8.08%(566/7 004), 7.90%(536/6 784), 7.77%(506/6 514);3.27%(229/7 004), 3.29%(223/6 784), 2.87%(187/6 514) and 6.68% (468/7 004), 6.63% (450/6 784), 5.82% (379/6 514), respectively, showed a declining trend. It also showed a declining trend with age, and the patients were mainly 40 years old people and older, and the highest detection rate was in the 60- 70 years old group[15.54%(143/920)、14.72%(135/917)、13.36%(136/1 018)]. For gender distribution, the detection rate of the three years was higher in male than female [male 8.24%(300/3 639), 7.99%(283/3 542), 7.71%(260/3 372);female 6.45%(217/3 365), 6.14%(199/3 242), 5.44%(171/3 142),χ2=8.24, 8.77, 13.54, all P〈0.01]. Conclusion There is no big change of arsenism conditions in 2010-2012, with a slight declining trend.

Objective To investigate the changes of follicular regulatory T cells ( Tfr cells) and follicular T helper cells ( Tfh cells) in peripheral blood of children with myasthenia gravis ( MG) . Methods We recruited 28 MG patients and 20 healthy subjects in this study. The percentages of Tfh and Tfr cells in peripheral blood samples were measured by flow cytometry. Real-time PCR was performed to detect the ex-pression of transcription factors and regulatory factors of Bcl-6, c-MAF, Blimp-1 and PD-1 at mRNA level. ELISA was used to detect the levels of IL-2, IL-6, IL-10 and IL-21 in plasma samples and the titers of Ach-Rab and PsMab. Results Compared with the healthy subjects, the MG patients showed higher percentages of Tfh cells and lower percentages of Tfr cells before receiving treatment. The expression of Bcl-6 and c-MAF on CD4+T lymphocytes cells at transcriptional level were significantly enhanced, while the expression of Blimp-1 on CD4+T cells and the expression of PD-1 on Treg cells at transcriptional level were inhibited in the MG patients in comparison with those in healthy subjects. Moreover, decreased levels of IL-2 and increased levels of IL-21 were found in plasma samples collected from the MG patients. Conclusion The decreased percentages of Tfr cells and increased percentages of Tfh cells in patients with MG resulted in abnormal ratios of Tfr/Tfh cells, which might be involved in the immunological pathogenesis of MG. Several changes in the patients with MG might be responsible for the imbalanced ratio of Tfr/Tfh cells, which included changes of IL-2 and IL-21 in microenvironment, enhanced expression of Bcl-6 and c-MAF at mRNA level and inhibited expression of Blimp-1 at mRNA level on CD4+T cells as well as over-expression of PD-1 at mRNA level on Treg cells.

Objective To explore the influence of human chorionic gonadotropin(HCG)on testicular germ cell apoptosis of experimental unilateral cryptorchidism in rats.Methods Forty immature male Sprague-Dawley rats were randomly divided into unilateral cryptorchi-dism group(n=20) and sham operation group(n=20).The 2 groups were divided into group treated with HCG and group without HCG.At age 21 days,unilateral cryptorchidism was produced.Half of the rats were injected with 20 U HCG from day 22 to 34 every other day.At age of 35 d and 60 d,rats were sacrificed for detection germ cell apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL).Results Apoptosis index(AI) of cryptorchidism testis were higher compared with the scrotal testis in sham operation group(P0.05).AI of the scrotal testis of sham operation group and unilateral cryptorchidism group with HCG was higher compared with the correspond groups without HCG,and there were significant difference of AI between sham operation group and the correspond group without HCG at age 35 days(P0.05).Conclusions AI of testicular is increased both in cryptorchidism testis and scrotal testis in experimental unilateral cryptorchidism;HCG adds the number of apoptotic germ cells,and histology damage of testis is not completely recovery after stop using HCG.So clinical application of HCG must be cautious and operation ought to be done as early as possible in cryptorchidism.

Objective To explore metabolic characteristics of and risk factors for newly diagnosed type 2 diabetes mellitus(T2DM) combined with non-alcoholic fatty liver disease (NAFLD).Methods One hundred and forty-two cases of newly diagnosed T2DM were divided into two groups according to whether they have comorbid NAFLD:group A (without NAFLD,n =79) and group B (combined with NAFLD,n =63).Data collected included body height,body weight,blood pressure,fasting plasma glucose (FPG),blood lipid,serum uric acid (UA),HbA1c and fasting insulin,body mass index and insulin resistance index with homeostasis model(HOMA-IR) were calculated to compare the clinical and biochemical parameters between groups A and B.Results (1) The difference of age and blood pressure between groups A and B were not statistical different (P ＞ 0.05).Compared with group A,BMI ((26.79 ± 1.93) kg/m2 vs (24.61 ± 2.46) kg/m2,t =5.76),FINS((15.49±2.44) mU/L vs (13.20±2.17) mU/L),t =5.91),HOMA-IR((6.74± 1.32) vs (5.65 ±1.10),t =5.37),glycerin trimyristate (TG) ((2.94 ± 0.65) mmol/L vs (1.74 ± 0.46) mmol/L),t =12.86),low density lipoprotein cholesterin (LDL-C) ((3.46 ±0.73) mmol/L vs (2.78 ±0.86) mmol/L,t =5.07) and UA((342.41 ±71.49) mmol/L vs (312.98 ±66.24) mmol/L,t =2.54) were significantly higherand hight density lipoprotein cholesterin (HDL-C) ((0.99 ± 0.17) mmol/L vs (1.21 ± 0.29) mmol/L,t =5.33) was significantly lower in group B (P ＜ 0.05).(2) Using whether to combined with NAFLD as dependent variable,and BMI,FINS,HOMA-IR,TG,LDL-C,HDL-C and UA as independent variable,logistics regression analysis showed that BMI,HOMA-IR and TG were risk factors for NAFLD(OR =2.838,19.241,and 2.019 respectively,P ＜ 0.05).Conclusion Newly diagnosed type 2 diabetes mellitus combined with NAFLD have more obvious dyslipidemia and insulin resistance.Obesity,insulin resistance,hyper-triglyceridemia are risk factors for newly diagnosed type 2 diabetes mellitus combined with NAFLD.

Objective To study the hemodynamic changes and pathologic foundation of rabbit models of radiation-induced lung injury (RILI) via 64-slice CT pulmonary perfusion imaging ( CTPI),in order to seek the correlation between the alterations of the hemodynamic parameters and pathophysiology.Methods Seventy-two healthy New Zealand rabbits were randomly classified into two groups:test group ( n =36),received 25 Gy with single fraction irradiation in a whole unilateral lung; control group ( n =36),received sham-irradiation.Each group was divided into 12 subgroups respectively according to post- and pseudo-irradiation time points (1,6,12,24,48,72 h and 1,2,4,8,16,24 w).Each rabbit underwent HRCT and CTPI at every pre- and post-radiation time point.All rabbits were sacrificed,and morphology of specimens was observed using light- and electron microscope. The changing regularity of HRCT,CTPI parameters and pathology were analyzed and compared with each other in order to find the correlation among them.The CTPI parameters of the test and control groups were compared using t test.The CTPI parameters and pathological values were analyzed using linear correlation with two variables,the detection rates of RILI by CTPI and HRCT was compared using Chi-square test.Results ( 1 ) The changes of CTPI parameters from control group after irradiation was relatively stable,but in test group those parameters including rBF,rBV and rPS,at pre- and post-irradiation time points (0,72 h and 2 w),were respectively 1.01 ± 0.09,1.86 ± 0.20,1.43 ±0.12,1.03 ±0.08,1.63 ±0.19,1.56±0.14,0.96±0.12,1.54 ±0.17 and 1.83 ±0.24.The corresponding parameters before and after irradiation were significantly different ( t =2.90-6.37,P ＞ 0.05).(2)In test group,capillary endothelial cells,basement membrane and alveolar epithelial cells,as the main injury targets,showed certain alterations in pathology.There was a significant correlation between the changes of CTPI parameters ( rBF and rBV) and pathophysiology in control group ( r =0.74,0.83,P ＜0.05 ),with the dependent relationship between rPS and the amounts of RBC outside the capillary and the destruction of basement membrane( r =0.87,0.88,P ＜ 0.01 ).(3)The detection rate of RILI with CTPI (72.2％,26/36) was obviously higher than that with HRCT( 16.7％,6/36,x2 =4.37,P =0.036).Conclusions CTPI parameters is capable of revealing the rule of hemodynamic process and reflecting the pathophysiologic state of different stages of RILI.By the time of detecting RILI,the detection rate of CTPI is clearly superior to that of HRCT,which yields potential value in predicting RILL

Objective To investigate the relationship between C734A and G-2964A polymorphism of cytochrome P450 1A2 gene and clinical efficacy of duloxetine.Methods 223 patients with depression were treated with duloxetine for six weeks.The clinical efficacy was evaluated with the Hamilton rating scale for depression (HAMD) ;single nucleotide polymorphism (SNP) at position C734A and G-2964A of CYP1A2 gene were identified with restriction fragment length polymorphism(RFLPs) ;then one-way ANOVA was adopted to analyze the relationship between SNP and clinical efficacy.Results (1) In 223 patients,the frequency of allele A at locus 734 was 63.64％,while that of allele A at locus-2964 was 26.82％.(2) 220 patient underwent the whole treating course.The conjoint analysis of two locuses indicated that the decreasing ratio of HAMD score of high-activity group,middle-activity group and low-activity after treatment was (56.05± 10.13) ％,(66.36± 8.66) ％ and (73.82± 7.10) ％ respectively,the differences obtained by pairwise comparison of the three groups were of statistical significance(P＜0.01).Conclusion There is close relationship between C734A and G-2964A polymorphism of CYP1A2 gene and clinical efficacy of duloxetine in the treatment for depression.

18 fungal strains including N.coenophialum,N.lolii, N.huerfanum、N.chisosum、N.aotearoae、N.sp.and 8 varieties of grass seeds belonging to Festuca arundinacea and Lolium perenne have been studied.With amplification of IS1～IS3 and F1～R1 of genomic DNA, the primers Tub-2-F～Tub-2-R from Tubulin-2 gene and F3～R3 from NC25 gene have been designed.A PCR method to detect N.coenophialum and N.lolii was established, and also a nested-PCR method to detect N.coenophialum and N.lolii in single seed was established.These PCR detection methods are strongly special and much credible and rapid-speeded.

Objective To establish the real-time quantitative PCR (RQ-PCR) assay for detecting Candida albicans (C.albicans) in whole blood and its clinical application in the febrile surgical patients who may develop gut barrier damage and gut microorganism translocation.Methods The NAG1 gene,which is a single copy in C.albicans genome,was selected as the target gene for designing the primers and probe.The plasmid was fabricated and produced as standard samples.C.albicans genomes were extracted with QIAamp(R) DNA Blood Mini Kit,and the total 20 μl TaqMan RQ-PCR amplification reaction system was established.The 74 venous blood samples from the surgical febrile patients were detected for C.albicans load.Results The specificities of the primers and probe were excellent,the correlation coefficients of the standard curves were between 0.9918 and 0.9985,and the efficiency of amplification was 0.88-1.027 for the samples above the lowest detection limit (100 copies/μl examine fluid,or nearly 1.1 × 103 cfu/ml whole blood).The average accuracy of the RQ-PCR equipment was (99.64±2.08) %,the sensitivity was 97.46%,the specificity was 100%,and the average coefficients of variation (CV) of the intra-and inter-assay were (14.76±2.64)% and (17.85±3.53)%,respectively.The average recovery rate of C.albicans DNA in whole blood samples was (88.60±5.73) %,and the average CV of recovery rate was (11.70 ±5.36) %.The number of copies of C.albicans genes per unit blood was not significantly different among the same original blood samples stored separately under-20℃ for 3 or 6 months when compared with its freshly collected blood (P = 0.267).In the 74 whole blood samples obtained from the febrile surgical patients,the positive rate of C.albicans genes was 2.7% and the highest load was 4.42×103 cfu/ml.Conclusions RQ-PCR is a rapid,sensitive,highly specific,and reproducible method in detecting C.albicans NAG1 gene.Clinically it can be used to quantitatively evaluate the numbers of C.albicans in the whole blood.A small percentage of the febrile surgical patients may develop blood infection of C.albicans.

Objective To establish the rabbit model of radiation-induced lung injury (RILI) for the study of CT perfusion. Methods Forty-eight New Zealand rabbits were randomly divided into two groups, 36 rabbits in test group were administered with 25 Gy of single fractionated irradiation in the whole unilateral lung, and the other 12 rabbits in control group were sham-irradiated. All rabbits were sacrificed at 1, 6, 12, 24, 48, 72 h, and 1,2, 4, 8, 16, 24 week after irradiation respectively, then six specimens were extracted from upper, middle and lower fields of bilateral lungs, respectively. The pathological changes were observed with light and electron microscopies. The expression of TNF-α and TGF-β1 in local lung tissue was detected by immunohistochemistry. Results In test group, RILI occurred at early stage,characterized by acute inflammatory reaction, and featured by the progressing fibrosis at later stage. The expression of TNF-α and TGF-β1 1 and 72 h post-irradiation were statistically different between test and control groups (t = 3.04-14. 95,P ＜ 0. 05 ). Thickness of alveolar wall, density of pulmonary interstitium 12 h of post-irradiation, amount of fibroblast and fibrocyte from interstitium 24 h post-irradiation were statistically different between two groups ( t = 4.44-39. 78, P＜ 0.05 ), and correlated with the time postirradiation (r = 0. 821, 0. 872, 0. 682). There was statistical differences among the relative amount of collagen fibers at time points post-irradiation in test group ( F = 100.31, P ＜0.05), while no difference in control group ( F= 1.00, P ＜ 0.05 ). The relative amount of collagen fibers was statistically different between two groups 72 h post-irradiation (t = 3.07-45.18, P＜0.05 ), and correlated with the time postirradiation (r = 0.993 ). Conclusions Stable and reliable rabbit model of RILI could be established through single fractionated irradiation in whole unilateral lung with 25 Gy of high-energy X-rays, which may simulate the occurrence and development of evolution of RILI.