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.

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.

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 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.

Based on analyzing image edge cloning theory and algorithms, a marginal clone algorithm is proposed with a combination of edge connectivity and noise removal algorithm. Simulation results show that the edge image detected by the algorithm has solved problems of traditional edge discontinuities and tsereve noise. Moreover, it has better edge recognition performance.
Algorithms ; Humans ; Image Enhancement ; methods ; Retinal Vessels

[Objects]To isolate and identify the pathogen of the large outbreak of dengue in Guangdong province in 2014. To understand the origin and the phylogenetic characteristics of the isolates ,and provide scientific foundation for the surveillance and prevention of dengue fever.[Methods]Collected the patient serum samples over all the Guangdong province during the 2014 outbreakperiod,isolated and identified the virus from these samples. Amplified complete E gene and complete genome with certain primers and sequenced all the products. Then the Phylogenetic ,Bayesian phylogeography and mutations analysis were carried.[Results]40 DENV-1 strains were isolated and identified. 40 complete E gene sequences and 6 complete genome sequences of DENV-1 were obtained. Phylogenetic analysis with E gene sequences revealed that the 40 isolates were classified into two genotypes including 16 genotypeⅠ(Asia)and 24 genotypeⅤ(America/Africa). 14 genotypeⅠisolates were clustered closest with isolates from Guangdong province(2013)and Sigapore(2013)which share the nucletide identities of 99.6% ~ 99.9%,other two genotypeⅠisolates were clustered with strains from Malaysia (2013) and both share the nucletide identities of 99.7%;24 genotypeⅤisolates were all classified in one clade with striains from Bangladesh(2009),China(2009)and Bhutan(2013)which share nucletide identities of 99.0%-99.9%. Further analysis with six complete genome sequences showed that five isolates were clustered closest with strains isolated from Guangdong province(2013)share the nucletide identities of 99.6%-99.8% while the sixth stains closest with strains isolated from Myanmar(2002)share the nucletide identities of 98.8%. The isolates have five amino acid mutations compared with strains epidemic in Guangdong province in 2013,three mutations(S88V,E203G,T275R)are in the EⅡdomain and one mutation (S305P)is in the EⅢdomain which associated with virulence.[Conclusions]During the outbreak in Guangdong province in 2014, DENV-1 is the predominant causative serotype,and there are at least two different kinds of genotypes of DENV-1 largely epidemiced in the whole province. Evolution analysis reveals the multiple origins of the isolates which may origin from Guangdong province , Sigapore,Malaysia,Myanmar so that we should enhance the study and surveillance of autochthonous and vectors in order to understand the epidemic way of dengue in Guangdong province. The isolates have had four mutations in the domain associated with virulence which remain further study to know their biological effects.

OBJECTIVETo study the effect of astragalosides (AST) on the anoxia/reoxygenation (A/R) injured neuron in rat.

METHODSPrimary cultured rat's hippocampal neurons were made into A/R model cells. The cell viability was detected by MTT assay and lactate dehydrogenase releasing methods; the activity of superoxide dismutase (SOD), and contents of malondialdehyde (MDA) and nitride oxide (NO) in culture supernate were detected; the apoptosis rate of hippocampal neurons after A/R was measured by flow cytometry with double-staining of Hoechst33258 and AnnexinV-PI; and intracellular calcium ion [Ca2+]i was observed with a cofocal laser-scanning microscope and determined by fluorescent probe Fluo-3/AM.

RESULTSAST enhanced the cell viability of neurons after A/R injury, increased SOD activity and decreased the MDA and NO contents in supernate, reduced the A/R-induced apoptosis and decreased the calcium overload in neurons.

CONCLUSIONAST has the protective effects on A/R injured neurons, the mechanism is possibly related with its anti-oxidation and calcium overload reducing actions.

Animals ; Calcium ; metabolism ; Cell Hypoxia ; drug effects ; Female ; Fetus ; Hippocampus ; cytology ; Malondialdehyde ; metabolism ; Neurons ; cytology ; Neuroprotective Agents ; pharmacology ; Nitric Oxide ; metabolism ; Pregnancy ; Primary Cell Culture ; methods ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; Saponins ; pharmacology ; Superoxide Dismutase ; metabolism ; Triterpenes ; pharmacology

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 Currently,pediatric 18F-FDG dose and acquisition durations are generally based on coarse extrapolation from adult guidelines.This study sought to determine whether shorter acquisition durations or a lower 18F-FDG injected activity could be used during pediatric 18F-FDG PET/CT examinations while maintaining diagnostic utility.Methods Thirty-six whole-body 18F-FDG PET/CT examinations were performed on 36 patients (weight,13-89 kg,(46.51±5.63) kg; age range,3-14 years old,(9.22±3.16) years old) with a weight-based injected activity (5.3 MBq/kg (0.144 mCi/kg)),fixed acquisition durations 180 S/FOV,VIP record acquisition mode using Discovery STE.For each examination,the Vip-mode data was truncated to form multiple datasets with shorter acquisition durations down to a minimum of 60 s/FOV (i.e.,60,80,100,120,140,160 s/FOV data were formed from single 180 s/FOV acquisition).168 image volumes were generated,randomized,and reviewed in a masked manner with corresponding CT image volumes by 6 radiologists.Overall,subjective adequacy and objective lesion detection accuracy by body region were evaluated.Results All examinations with maximum acquisition duration were graded as adequate and were used as the reference standard for detection accuracy.For patients more than 30 kg,when acquisition duration was more than 120 s/FOV,all PET/CT examinations were graded as adequate for clinical tasks,whereas,when acquisition duration was reduced to less than 120 s/FOV,lesion detection became less accurate.For patients less than 30 kg,lesion detection accuracy was perfect for acquisition times between 140 s/FOV and 180 s/FOV for all regions of the body.However,lesion detection became less accurate when imaging acquisition time was reduced less than 140 s/FOV.Conclusions When GE Discovery STE PET/CT was applied during pediatric PET/CT examination,using decreased acquisition times as a surrogate for 18F-FDG dose,18F-FDG dose can be reduced by approximately 33.33％ when patients weigh over 30 kg were scanned for 180 s/FOV.For patients less than 30 kg,18F-FDG dose can be reduced by approximately 22.22％ without losing diagnostic quality.Reduction of overall scan time potentially reduces motion artifacts,improves patient comfort,and decreases length of sedation.Alternatively,decreased 18F-FDG dose minimizes radiation risk.

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.