Objective To discuss blood collection tubes with different additives and their effects on the testing results of alcohol concentration in blood sam ples. Methods Blood sam ples from 10 volunteers were collected 2 hours after drinking with seven different types of disposable vacuum blood collection tubes, including ordinary tube without anticoagulant, coagulant tube, separating gel-coagulant tube, sodi-um citrate (1∶4) tube, sodium citrate (1∶9) tube, sodium citrate (9∶1) tube and EDTA-K2 tube. The al-cohol concentrations in these blood sam ples were analyzed by headspace gas chrom atography. Results The concentration testing results of the sam e blood sam ples in different types of tubes were different from one to another. The sequence was as follows:separating gel-coagulant tube>coagulant tube>ordi-nary tube without anticoagulant>EDTA-K2 tube>sodium citrate (1∶9) tube>sodium citrate (1∶4) tube, whereas the results of the sam e blood sam ple in sodium citrate (1∶9) tube and sodium citrate (9∶1) tube showed no obvious difference. Conclusion It is better to collect a suspicious drunk driver’s blood sam-ple using a disposable vacuum blood collection tube, with the EDTA-K2 tube being preferred.

Objective A method was developed for the determination diazepam and its main metabolites,nordiazepam, and oxazepam and then in urine by GC-ECD. Method The urine samples were hydrolyzed with ?-glucuronidase were extracted in organic solvent. The extractives were derived with BSTFA and the analytes as trimethylsilyl derivatives were determined by GC. Results Sensitive of the method is as low as snglme, the recovery is high then 70%. Conclusion This method is sensitive enough for the analysis of urine from the subjects over a 48 hour period after receiving a 10mg dose of diazepam orally.

China ; Female ; Hepatitis A Vaccines ; administration & dosage ; Hepatitis B ; prevention & control ; Hepatitis B Vaccines ; administration & dosage ; Humans ; Male ; Vaccination ; methods ; Vaccines, Combined ; administration & dosage

OBJECTIVE: To investigate the distribution of buprenorphione in the bodies of rabbits. METHODS: Buprenorphione was administrated to rabbits orally or by intravenous injection (0.04 mg/kg buprenorphione). Two hours after administration, rabbits were killed and their blood, urine, liver, kidney, lung, stomach, brain, heart, stomach content and feces were collected. The concentrations of buprenorphione in these body fluids and tissues were determined by liquid chromatography-mass spectrometry (LC-MS). RESULTS: The results show the distribution of buprenorphione in rabbit's body: urine>stomach content>brain >heart >stomach>lung> kidney > liver > blood> feces. CONCLUSION The method developed can be used for the detection of buprenorphione in biological fluids and tissues in forensic practice. Urine is the preferred sample for screening for buprenorphione abuse.
Analgesics, Opioid ; pharmacokinetics ; Animals ; Buprenorphine ; pharmacokinetics ; urine ; Female ; Male ; Rabbits ; Tissue Distribution

Objective To investigate the effect of phenformin combined with hexokinase inhibitor 2-deoxyglucose (2-DG) on the treatment of triple-negative breast cancer cell lines 4T1 and MDA-MB-231. Methods Following treatment with phenformin, 2-DG or phenformin combined with 2-DG on 4T1 and MDA-MB-231 cells for 48 h, the cell proliferation in each group was detected by SRB and the apoptosis of cells was detected by flow cytometry. The concentration of glucose and lactic acid in cell culture supernatant was detected by ELISA. The activity of mitochondrial respiratory chain complex Ⅰ was detected by FlexStation3 and the mitochondrial oxygen consumption (OCR) was assayed with the Seahorse X Fe Analyzer. Results The hexokinase expression (4.6±0.17,3.73±0.21), glucose consumption (356±31，397±42) μg/10⁵ cells , Lactic acid concentration (5.59±0.52, 7.83±0.78) μmol/L in the supernatant of 4T1 and MDA-MB-231 cells in Phenformin group were higher than that in control group ( 1±0.15，1±0.12 ) , ( 289±25，301±32) μg/10⁵cells , ( 2.37±0.18，4.01±0.45) μmol/L (P < 0.01). Even if the dose was reduced by 90%, the cell viability of phenformin combined with 2-DG group (64.63±2.28, 51.97±2.29) % was still higher than that of phenformin group (86.70±1.83, 85.53±1.46) % (P<0.001). The combination of the two drugs significantly promoted the apoptosis of 4T1 and MDA-MB-231. In addition, compared with the phenformin group (5.59±0.52, 7.83±0.78) μmol/L, the phenformin combined with 2-DG group (3.46±0.37, 5.18±0.62) μmol/L cell lactic acid production also greatly reduced (P<0.01). Compared with the phenformin or 2-DG single-drug group, the phenformin combined with 2-DG group can significantly inhibit the growth rate of tumors in tumor-bearing mice (P<0.01). The median survival time of tumor-bearing mice in the phenformin combined with 2-DG group was 72.5 d, which was higher than that in the phenformin group 57 d and 2-DG group 55.5 d (P<0.01). Conclusion Hexokinase inhibitor 2-DG significantly enhances the therapeutic effects of phenformin on triple-negative breast cancer cells.

A novel anaerobic fermentation process--multistage countercurrent fermentation was applied to improve the bioproduction of volatile fatty acids (VFAs) from excess municipal sludge. Results showed that the total VFAs concentration and the total VFAs yield reached (10.5 +/- 0.5) g/L and 0.20 gVFAs/gVS (Volatile solid) using this novel process. Comparing with the conventional anaerobic fermentation, the concentration and yield of total VFAs increased by 31% and by 54%, respectively. Moreover, removal ratio of organic solids also increased by 37% and it was 50% at the end of multistage countercurrent fermentation. We further investigated the mechanism of VFAs production. Results revealed that this novel process could reduce the inhibitory effect of VFAs on the acid-forming microorganisms, and the total VFAs yield and the removal ratio of organic solids respectively depended on the first stage and the third stage of this novel process. Therefore, the multistage countercurrent fermentation can efficiently improve the bioproduction of VFAs from excess municipal sludge, and relatively enhance the removal ratio of organic solids.
Anaerobiosis ; Bacteria, Anaerobic ; metabolism ; Bioreactors ; microbiology ; Fatty Acids, Volatile ; biosynthesis ; Fermentation ; Refuse Disposal ; instrumentation ; methods ; Sewage ; chemistry

To develop a magnetic nanoparticle chemiluminescence immunoassay (CLIA) for the determination of type Ⅰ procollagen N-terminal peptide (PINP) in human serum, we expressed a recombinant PINP-α1 protein in Corynebacterium glutamicum and used it as an immunogen to immunize BALB/c mice. We obtained three hybridoma cell lines that stably secret antibody against PINP-α1 protein. After further pairing and screening, we chose a monoclonal antibody 8C12 coupled with biotin as the capture antibody, and a monoclonal antibody 1F11 labeled horseradish peroxidase as the detection antibody. The antibodies combined with the serum samples, forming a sandwich complex which was used to detect the concentration of PINP in serum. After optimizing the conditions, we determined that the best working concentration of the capture antibody and the detection antibody were 3 μg/mL, and the incubation time was 30 minutes. The quantitative assay had a detection range of 5-1 100 ng/mL, with recovery rates between 93%-107% and the minimum detection limit of 1.22 ng/mL achieved. The intra-and inter-assay precisions were lower than 10%. The correlation coefficient of PINP results between this CLIA method and the Roche electrochemiluminescence immunoassay system was 0.906 2. Therefore, this CLIA method is specific and can be used to quantitatively detect the content of PINP in serum, which has the potential to become an auxiliary approach for bone disease examination.
Animals ; Humans ; Immunoassay ; Luminescence ; Mice ; Mice, Inbred BALB C ; Peptide Fragments/isolation & purification* ; Procollagen/isolation & purification*

Background Researches showed that elevatory blood glucose level results in long-term damage of cells and tissue,or metabolic memory phenomenon,and manipulation of hyperglycemic memory is a good approach in the prevention of diabetic complications.However,its mechanism is not clear.It is speculated that the pathogenesis of diabetic retinopathy (DR) in diabetic patients may be associated to related mechanisms.Uncoupling proteins (UCPs) can decrease the production of reactive oxygen species (ROS),which may be related to DR.Objective This study was to explore the association between DR and the single nucleotide polymorphisms (SNPs) of UCP genes in Chinese Han population with type 2 diabetes.Methods A cross-sectional study was performed.This study was approved by Ethic Committee of Affiliated First Hospital of Shanghai Jiao Tong University and complied with Declaration of Helsinki,and written informed consent was obtained from each subject prior to any medical examination.One thousand eight hundreds and seventy-five patients with type 2 diabetes mellitus were enrolled in Xinjing district of Shanghai city by cluster sampling from November 2014 to January 2015.The demographic and medical baseline characteristics,ocular examination and laboratory tests were obtained and periphery blood of 2 ml was collected for extraction of DNA.Eight tag SNPs of UCP1,three tag SNPs of UCP2,and seven tag SNPs of UCP3 were selected as marker locus for the detection of genotype by Sequenom Mass ARRAY.Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry platform were used for genotyping.Hardy-Weinberg equilibrium (HWE) analysis,allele and genotype frequencies,haplotype analysis,and association tests for DR and SNPs were performed by SAS and SHEsis software.Results A total of 530 DR patients were checked out from 1 875 subjects with type 2 diabetes mellitus,with the detection rate of 28.27％.rs660339 locn of UCP2 gene and rs1626521,rs668514 locus of UCP3 gene appeared to have low detectable rates,and the secondary allele base frequency of rs632862 in UCP2 gene was ＜0.01 and rs15763 of UCP3 gene was unmatched with HWE,therefore,these locus analysis was not included.In 13 SNPs locus included in the analysis,only 2 SNPs of UCP1 gene were related to DR.Compared with the non-diabetic retinopathy (NDR) patients,the G allele frequency of rs10011540 was increased (P =0.03,OR =1.31,95 ％ confidence interval[CI] =1.03-1.67,and T allele frequency of rs3811787 was decreased (P=0.04,OR=0.86,95％ CI=0.75-0.99) in DR patients.Genotyping detection showed that the C/C and A/A frequencies of rs3811790 in UCP1 gene were significantly more and C/A frequency was less in DR patients than those in NDR patients (all at P＜0.01).The logistic regression analysis indicated an association of SNPs of rs10011540 and rs3811787 with DR independent from glucose and disease duration.Conclusions The SNPs of rs10011540 and rs3811787 locus in UCP1 gene are associated with DR in Chinese type 2 diabetes patients.

We studied cutinase production from short-chain organic acids by Thermobifida fusca WSH03-11 to evaluate the possibility of converting municipal sludge to high value-added products. The optimum organic acid (8.0 g/L) and nitrogen source (1.5 g/L) concentrations were determined by the single factor experiments with butyric acid, propionic acid and acetic acid as the carbon sources. When lactic acid was used as the carbon source, the optimum organic acid (3.0 g/L) and nitrogen source (1.0 g/L) concentrations were obtained. Cutinase production by T. fusca WSH03-11 was further improved with butyric acid (by 31.0%), propionic acid (by 13.3%), acetic acid (by 43.8%) and lactic acid (by 73.2%) as carbon source, respectively, with the optimized cutin concentrations. Among these four short-chain organic acids, the average specific consumption rate of acetic acid was the highest, higher than that of propionic acid 1.3-folds, butyric acid 2.0-folds and lactic acid 2.2-folds. The highest cutinase activity reached 52.4 u/mL with butyric acid (8 g/L) as the sole carbon source, higher than that of lactic acid (3 g/L) 1.7-folds, acetic acid (8 g/L) 2.5-folds and propionic acid (8 g/L) 3.2-folds. The yield of cutinase activity on lactic acid (12.70 u/mg) higher than that of butyric acid 1.4-folds, propionic acid 3.0-folds and acetic acid 3.8-folds. T. fusca WSH03-11 consumed acetic acid firstly in mixed acids carbon sources, and the consumption of butyric acid was inhibited. Further studies indicated that the consumption rate of butyrate was decreased by 66.7% in the presence of 0.5 g/L acetic acid in the mixed acids. This was the first report concerning the production of cutinase by T. fusca with mixed organic acids as the carbon sources. The results presented here provided a novel and efficient approach to produce high value-add products from municipal sludge, and also established a foundation for the industrial production of cutinase by T. fusca WSH03-11 with cheap carbon sources from the processing of municipal sludge.
Acetates ; metabolism ; Actinomycetales ; growth & development ; metabolism ; Butyric Acid ; metabolism ; Carboxylic Ester Hydrolases ; biosynthesis ; Fermentation ; Organic Chemicals ; metabolism ; Propionates ; metabolism

Fermentation and induction conditions for recombinant Escherichia coli expressing Thermobifida fusca cutinase were optimized in flasks and 3L fermenter. Surface modification of poly (ethylene terephthalate) fibers with cutinase was also discussed. The results showed that, cutinase yield reached 128 U/mL by adding 2 g/L inducer lactose and 0.5% glycine. In the fed-batch culture in a 3 L fermenter, the maximum biomass cutinase activity was up to 506 U/mL, which is the highest bacterial cutinase activity reported by far. Recombinant cutinase was used to modify polyester fibers and terephthalic acid substance was detected by using UV analysis. The dyeing and wetting properties of cutinase treated fibers were higher than untreated fibers. Combined utilization of cutinase and Triton X-100 can significantly improve the hydrophilicity of polyester. This is the first report of surface modification on polyester fibers by bacterial cutinase.
Actinomycetales ; enzymology ; genetics ; Carboxylic Ester Hydrolases ; biosynthesis ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Polyethylene Glycols ; chemistry ; Polyethylene Terephthalates ; Recombinant Proteins ; biosynthesis ; genetics ; Surface Properties