Objective To study the species and amount of bacteria in sputum of patients with ventilator associated pneumonia (VAP) by using 16S rDNA sequencing analysis,and to explore the new method for etiologic diagnosis of VAP.Methods Bronchoalveolar lavage sputum samples were collected from 31 patients with VAP.Bacterial DNA of the samples were extracted and identified by polymerase chain reaction (PCR).At the same time,sputum specimens were processed for routine bacterial culture.The high flux sequencing experiment was conducted on PCR positive samples with 16S rDNA macro genome sequencing technology,and sequencing results were analyzed using bioinformatics,then the results between the sequencing and bacteria culture were compared.Results ① 550 bp of specific DNA sequences were amplified in sputum specimens from 27 cases of the 31 patients with VAP,and they were used for sequencing analysis.103 856 sequences were obtained from those sputum specimens using 16S rDNA sequencing,yielding approximately 39 Mb of raw data.Tag sequencing was able to inform genus level in all 27 samples.② Alpha-diversity analysis showed that sputum samples of patients with VAP had significantly higher variability and richness in bacterial species (Shannon index values 1.20,Simpson index values 0.48).Rarefaction curve analysis showed that there were more species that were not detected by sequencing from some VAP sputum samples.③ Analysis of 27 sputum samples with VAP by using 16S rDNA sequences yielded four phyla:namely Acitinobacteria,Bacteroidetes,Firmicutes,Proteobacteda.With genus as a classification,it was found that the dominant species included Streptococcus 88.9％ (24/27),Limnohabitans 77.8％ (21/27),Acinetobacter 70.4％ (19/27),Sphingomonas 63.0％ (17/27),Prevotella 63.0％ (17/27),Klebsiella 55.6％ (15/27),Pseudomonas 55.6％ (15/27),Aquabacterium 55.6％ (15/27),and Corynebacterium 55.6％ (15/27).④ Pyrophosphate sequencing discovered that Prevotella,Limnohabitans,Aquabacterium,Sphingomonas might not be detected by routine bacteria culture.Among seven species which were identified by both methods,pyrophosphate sequencing yielded higher positive rate than that of ordinary bacteria culture [Streptococcus:88.9％ (24/27) vs.18.5％ (5/27),KlebsieHa:55.6％ (15/27) vs.18.5％ (5/2 7),Acinetobacter:70.4％ (19/27) vs.37.0％ (10/27),Corynebacterium:55.6％ (15/27) vs.7.4％ (2/27),P＜0.05 or P＜0.01].Sequencing positive rate was found to increase positive rate for culture of Pseudomonas [55.6％ (15/27) vs.25.9％ (7/27),P=0.050].No significant differences were observed between sequencing and ordinary bacteria culture for detection Staphylococcus [7.4％ (2/27) vs.11.1％ (3/27)] and Neisseria bacteria genera [18.5％ (5/27) vs.3.7％(1/27),both P＞0.05].Conclusions 16S rDNA sequencing analysis confirmed that pathogenic bacteria in sputum of VAP were complicated with multiple drug resistant strains.Compared with routine bacterial culture,pyrophosphate sequencing had higher positive rate in detecting pathogens.16S rDNA gene sequencing technology may become a new method for etiological diagnosis of VAP.

OBJECTIVETo establish a HPLC method for the analysis of Danshensu (DSS), protocatechuic acid (PA) and protocatechuic aldehyde (PAL) of Xiangdan injection in rat's plasma, and to study pharmacokinetic characteristics of Xiangdan injection components in rats with m-hydroxybenzoic acid as internal standard.

METHODprotein was precipitated by 10% trichloroacetic acid and extracted by ethyl acetate. The plasma concentration was detected by HPLC, The pharmacokinetics parameters of DSS, PA and PAL were calculated by DAS2.0 software after iv injection.

RESULTDSS, PA and PAL have a good linear relationship in 0.68-44.0 mg x L(-1), 0.43-14.0 mg x L(-1) and 0.38-12.0 mg x L(-1), respectively. The average recoveries were more than 92% and the RSD of precision and stability of the test were between 0.4%-4.8%. DSS, PA and PAL showed a two-compartment open model, the half-life of absorption (t(1/2alpha)) were (6.26 +/- 4.6), (5.93 +/- 4.9), (18.44 +/- 2.4) min, the half-life of elimination (t(1/2beta)) were (64.11 +/- 8.8), (63.28 +/- 0.13), (69.315 +/- 0) min, the area under curve(AUC(0-infinity)) were (852.98 +/- 175.6), (83.84 +/- 58.8), (147.79 +/- 12.3) mg x min(-1) x L(-1).

CONCLUSIONA method with high recovery rate and good stability was established to determine the blood concentration of DSS, PA, PAL in Xiangdan injection and applied in its pharmacokinetics successfully.

Animals ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Female ; Hydroxybenzoates ; blood ; pharmacokinetics ; Injections ; Limit of Detection ; Linear Models ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo detect potential mutation in a pedigree affected with congenital nephrogenic diabetes insipidus (NDI).

METHODSClinical data of a male patient affected with NDI was collected. Genomic DNA was extracted from peripheral blood samples from the patient and five family members. The whole coding region of the arginine vasopressin receptor 2 (AVPR2) gene was amplified by PCR and directly sequenced.

RESULTSThe patient presented polyuria and polydipsia postnatally. Computerized tomography revealed bilateral hydronephrosis and hydroureter. The patient was responsive to hydrochlorothiazide but not to desmopressin. DNA analysis identified a hemizygous missence mutation c.295 T>C in exon 2 of the AVPR2 gene in the proband. His mother and grandmother were both heterozygous for the same mutation.

CONCLUSIONThe congenital NDI in the patient was probably due to mutation of the AVPR2 gene.

Adolescent ; Base Sequence ; DNA Mutational Analysis ; Diabetes Insipidus, Nephrogenic ; congenital ; genetics ; Exons ; genetics ; Family Health ; Female ; Genetic Predisposition to Disease ; genetics ; Humans ; Male ; Mutation ; Pedigree ; Receptors, Vasopressin ; genetics

Objective: To study serum zinc level in pregnancy and umbilical cord blood and their association with newborn birth weight. Methods: Pregnant women accepting obstetric examination in Ma'anshan Maternal and Child Care Center were recruited from May 2013 to September 2014. The follow up was conducted during their first, second and third trimesters of pregnancy and the self-designed questionnaire was used to collect information of social and demographic characteristics. Blood samples in the first, second pregnancy period and umbilical cord blood samples were collected and serum concentrations of zinc were assayed. 3 239 mother-infant entered the final analysis. We divided serum zinc level into low (<P₂₅), medium (P₂₅-P₇₅) and high (>P₇₅) groups according to their exposure concentrations at each trimesters. Non-conditional multivariate logistic regression model was conducted to evaluate the association between serum zinc level in first, second trimesters of pregnancy and umbilical cord blood with small for gestational age (SGA) and large for gestational age (LGA). Results: Serum zinc level in P₅₀ (P₂₅-P₇₅) during the first, second trimesters and cord blood were 1 016.18 (907.09-1 145.60), 813.36 (732.47-897.89) and 903.44 (808.71-1 015.64) μg/L, respectively. The prevalence of zinc deficiency during the first, second trimesters and cord blood were 1.5% (44/2 957), 15.9% (492/3 087) and 6.5% (176/2 707), respectively. The prevalence of total SGA and LGA were 9.7% (313/3 239) and 16.5% (536/3 239), respectively. Compared to high-level serum zinc group, the risk of SGA (OR (95%CI) in low-level serum zinc group during first trimesters was 1.51 (1.05-2.19)). Serum zinc level among second pregnancy period and umbilical cord blood had no statistically significant effect on SGA and LGA (both P values >0.05). Conclusion Zinc nutritional status of pregnant women in Ma'anshan city was at a good level. The low serum zinc level in first trimester increased the risk of SGA.

OBJECTIVETo investigate the expression level and analyze the clinical significance of NT5C2, which is an nucleoside analogues metabolism related gene, in children with acute leukemia (AL).

METHODSReal-time PCR and immunohistochemistry were presented to detect the level of NT5C2 mRNA and its protein product cN- Ⅱ in bone marrow samples of 63 patients initially diagnosed with AL, 15 patients who achieved complete remission, 7 patients who relapsed and 16 non- hematologic malignancie controls. The expression of NT5C2 mRNA in different groups of AL and its relevance with clinical indicators were analyzed.

RESULTS①The expression of NT5C2 mRNA in newly diagnosed B-ALL, TALL, AML and controls were 1.16 (0.89-2.25, 0.96 (0.74-1.25, 1.66 (0.84-3.15) and 0.88 (0.61-1.21), respectively. NT5C2 mRNA expression in AML (P<0.01) and B-ALL (P<0.05) cases were higher than that in controls; NT5C2 mRNA expression in T- ALL and in controls showed no significant difference (P>0.05). Changes of NT5C2 mRNA level were observed between preliminary diagnosis and complete remission in 15 patients. NT5C2 mRNA levels were significantly decreased in complete remission stage than that in newly diagnosis AL (P<0.01). NT5C2 mRNA levels of relapsed-refractory group were higher than that of complete remission group and controls (P<0.01). ② Immunohistochemical staining results revealed that NT5C2 protein levels were consistent with the trend of mRNA levels. ③NT5C2 mRNA levels in AML (r=0.434) and T-ALL (r=0.389) were positively correlated with risk classification (P<0.05). ④ During chemotherapy of patients with AML, the NR rate of bone marrow in NT5C2 high expression group was higher than that of low expression group after 9 days induction chemotherapy (35.2% vs 0) and before consolidation therapy (25.0% vs 0); The positive rate of minimal-residual disease (36.4% vs 14.3%) and relapse rate of AL (38.5% vs 28.6%) were increased in NT5C2 high expressed patients than that in low expressed patients, but all the differences were insignificant (P>0.05).

CONCLUSIONHigh expression of NT5C2 was found to be a related risk factor of AL children with unfavourable prognosis. NT5C2 promises a new target for guiding individualized chemotherapy and evaluating the prognosis of childhood acute leukemia and monitoring recurrence.

5'-Nucleotidase ; metabolism ; Bone Marrow ; metabolism ; Child ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; Neoplasm, Residual ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; Prognosis ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Recurrence ; Remission Induction