A fast and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of prodrug of paclitaxel (Pro-PTX) and paclitaxel (PTX) in rat plasma was developed. The plasma samples were subjected to protein precipitation with acetonitrile (0.1% formic acid), and then separated by LC with an Ultimate AQ-C18 column (50 mm × 3.0 mm, 3 μm) and acetonitrile-1 mmol·L^-1 ammonium formate (containing 0.1% formic acid) as the mobile phase. Multiple reaction monitoring (MRM) scanning mode was used to detect the ion responses m/z 983.4→415.2 (Pro-PTX), m/z 854.4→286.1 (PTX) and m/z 808.3→527.2 (Docetaxel, internal standard) by using a triple quadrupole tandem mass spectrometer with electrospray ionization source and positive ion mode. The method validation results show that the linear ranges of Pro-PTX and PTX in plasma were 2.00-500 ng·mL^-1 (r > 0.99) and 4.00-1 000 ng·mL^-1 (r > 0.99), the lower limit of quantification was 2.00 ng·mL^-1 and 4.00 ng·mL^-1, respectively; the quality control samples with low, medium and high concentrations of Pro-PTX and PTX were within the batch, the relative standard deviation (RSD) between batches were all less than 9%; the relative deviation (RE) was within the range of ± 9%; In the stability test, both Pro-PTX and PTX in plasma were stable under various storage conditions. The method was sensitive, specific, and reproducible, and was suitable for the pharmacokinetic study of Pro-PTX in rats. Animal experiments were approved by the Ethics Committee of Laboratory Animal Management and Animal Welfare, Institute of Materia Medica, Chinese Academy of Medical Sciences (No.: 00003552).

Objective To study the characteristic of inhibition on platelet P2Y12 and short-term change after clopidogrel intake in patients with cardiovascular disease. Methods Thirty-two patients with cardiovascular disease were enrolled. Samples at baseline, 10 h and 36 h after 300 mg loading dose and 75 mg/d maintenance dose of clopidogrel with 100 mg/d Aspirin intake were measured respectively. Platelet aggregation (PAgT) was measured on thromboelastograph(TEG) induced by ADP/AA. INH was detected and calculated activated by Kaolin, AA, ADP and Activator((R)) in the TEG reagent. CD62p and VASP phosphorylation (PRI), platelet activation markers were tested with FACSCalibur Flow Cytometry, and platelet secretion activity and suppression of P2Y12 receptor were detected respectively. The changes of indicators were compared before and after clopidogrel intake, and evaluate their function in platelet receptor activation. Results INHADP at baseline was (11.5 ±9.3)%, and increased to (42.5 ±29.1)% statistically (t =3.155, P<0.05) after taking the P2Y12 at 10 h, but decreased to (20.4±13.1)% at 36 h, non-statistical to baseline (t = 2.078, P > 0. 05) , INHAA increases from baseline level (56. 6 ± 36. 6) % to (83.0 ±27. 3)% at 10h(t=2.086,P>0.05) and (85. 4 ±20. 8)% at 36 h (t= 1. 888, P>0.05), no statistical defferences were found. Inhibition on platelet activativation induced by ADP function well till 36 h after 300 mg loading dose. PAgTADP decrease from (53. 7 ± 14. 1)% at baseline to (49. 2 ±22. 8)% at 10 h non-statistically (t=0.656, P>0.05), and (40.7±12.8)% at 36 h statistically (t=2.418, P<0.05), however PAgTAA decrease at both 10 h and 36 h statiscally, from (34. 3 ± 18. 1) % to (17.4 ± 13. 1) % , (t=3.134, P<0.05) and (14.6±5.1)%, (t=2.532, P<0.05), respectively. Data of PAgT was not corresponding to that from TEG for the difference in sample type partly. PRI in VASP assay was (78. 6 ± 22.3)% before loading dose, and decreased to (70.7 ±9.4)% at 10 h without significance (t = l. 194, P>0.05) and (59.6 ±28.0)% at 36 h (t=1.930,P<0.05) statistically, similarly to INHADP,indicating that within 36 h clopidogrel did not have strong inhibitory effect on the ADP receptor. On the contrary, CD62p changed from (7. 5 ± 1. 4) % at baseline to (4. 2 ± 1. 1) % statistically (10 h, t = 18. 027, P < 0. 05) and ( 4. 3 ± 0. 2 ) % non-statistically (36 h, t = 2. 908, P > 0. 05 ). Inhibition of secretion activity reflected by CD62p was significant. In contrast, it was more obvious inhibition in COX-1 passway, while the inhibition of P2Y12 receptor varied due to assay difference. Conclusions AA-induced platelet activation is significantly decreased in the inhibition of clopidogrel and aspirin, while ADP receptor is significantly inhibited until 36 h after the loading dose of clopidogrel. Platelet function in whole blood reflects total activity of platelet interaction with other components, in which no significant inhibition could be witnessed within 10 h.

Objective To evaluate performance of PlateletMapping(R) and RapidTEGTM based on thrombelastography by blocking platelet function with Reopro.Methods PlateletMapping was carried out with whole blood from healthy volunteers mixed with Reopro in vitro in a serial of titration.TEG(R) ACT of heparinized blood was tested with Rapid TEG kits.Linearity, repeatability and validity were calculated for two methods.Results MA activated by Kaolin, AA and ADP decreased with the increase of the concentration of Reopro. Inhibition rates (%)for AA and ADP induced aggregation were repeatable in channels and systems.At the Reopro levels of (1-4) × 10-2 mg, inhibition rate increased statistically( AA:27.99% ± 2.8% vs 63.37% ± 0.0% ,t = 21.9, P ＜ 0.01;ADP: 35.9% ± 0.56% vs 91.42% ± 1.14%,t=58.9,P ＜ 0.01 ) after addition of Reopro.Dose-dependent effect relationship could be seen between TEG(R) ACT and heparin;In Rapid TEG assay, measurement repeatability of K, α angle, MA and TEG(R)ACT were all good ( CV ＜ 5% ) except for R.Conclusions PlateletMapping(R) is sensitive to the inhibition of platelet function with good precision with dose-dependent effect.Moreover, Rapid TEG provides analysis of the overall coagulation function besides monitoring heparin therapy.

4-Hydroxycoumarins ; urine ; Animals ; Chromatography, High Pressure Liquid ; methods ; Sensitivity and Specificity

Bacillus subtilis ZC-7 was obtained by implantation with N+ ions beam to B.subtilis AS1.398,and compared with the AS1.398 neutral protease,the enzyme activity of ZC-7 neutral protease was about 1 timeshigher in previous research.A neutral protease was purified from the culture of B.Subtilis ZC-7 by the procedures including amoninium sulfate precipitation,ultrafiltration,DEAE-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography.By multi-step purification,the ZC-7 neutral protease was purified to 78.5 folds and its yield was 27.7%,at last,the specific activity of ZC-7 neutral protease was up to 4.1?105U/mg.Analysed by SDS-PAGE,the purified protease has shown a molecular mass of about 42kDa.The Km for casein hydrolysis was 3.67?10-3?g/ml and the Vmax was 12.21?g/min.The optimum pH and temperature forhydrolysis of casein were 7.0 and 55℃,respectively.This protease was stable up to 40℃ within the pH range of 6.5 and 8.0.EDTA,isopropanol and alcohol nearly inhibited its activity while some ions such as Ca2+,Mg2+,Fe3+ can improve its activity.In addition,it could resist 1 mol/L H2O2.

Objective To explore the mastery of relevant knowledge and skills for autism rehabilitation teachers in Beijing private rehabilitation agencies. Methods 51 autism rehabilitation teachers were selected from 10 Beijing private rehabilitation agencies and tested with self-prepared questionnaire. Results Rehabilitation teachers believed that professional knowledge, rehabilitation skills and special education basics ranked the former three positions as for knowledge importance; teachers' basic skills, professional knowledge and special education basics ranked the former three positions as for knowledge mastery. There were difference in academic background and business life. 95% teachers believed that the training form was knowledge plus case plus guidance. The best training time was weekends or summer and winter vacations. The curriculum expected mainly focused on behavior modification, class management, and training system that generally used for children with autism. Conclusion Autism rehabilitation teachers in Beijing private rehabilitation agencies urgently need training in the aspects of expertise, rehabilitation skills and so on.

OBJECTIVETo investigate clinical efficacy and feasibility of double-plating fixation via anteriormiddle approach in treating type C3 distal femoral fractures.

METHODSFrom August 2008 to August 2011, 12 cases with type C3 distal femoral fractures were treated, including 5 open fractures and 7 closed fractures. Among them, there were 8 males, 4 females with an average of 40 years (ranged, 25 to 55 years). There were 7 in left side, 5 in right side. Nine cases were caused by car accident, 3 cases by falling down. The duration from injury to hospital was form 20 minutes to 5 days (mean 135 min). After tibia bone traction for 5 to 8 days, the operation were performed by double-plating fixation via anteriormiddle approach, and autograft of iliac bone or allograft bone grafting were given to bone defect. Knee joint function was evaluated according to Merchanetal criteria.

RESULTSThe operation time was from 110 to 160 min, with an average of 135 min, the blood loss was from 300 ml to 500 ml,with an average of 400 ml. Post-operative wound were stage I healing. All patients were followed up from 16 to 36 months (mean 24 months). No infection, reduction loss, nonunion, deep vein thrombosis occurred. Bone healing time was for 18 to 24 weeks with an average of 21 weeks. According to the Merchanetal criteria, 4 cases got excellent results, 6 good, 1 fair and 1 poor.

CONCLUSIONDouble-plating fixation via anteriormiddle approach for type C3 distal femoral fractures is an effective way, which has advantages of obvious exposure, simple manipulation, anatomical reduction, stable fixation. However,operation indications and operating instructions should be strictly followed.

Adult ; Bone Plates ; Female ; Femoral Fractures ; diagnostic imaging ; surgery ; Fracture Fixation, Internal ; instrumentation ; Humans ; Male ; Middle Aged ; Tomography, X-Ray Computed ; Treatment Outcome

OBJECTIVEIn this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB) in modulating adhesion molecules expressions.

METHODSNeutrophils and BEAS-2B cells (one human bronchial epithelial cell line) were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction (real-time qPCR). Phosphorylated p38 MAPK and inhibitor κB were analyzed by Western blot.

RESULTSIn co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly (P<0.05). Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor κB in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin (P<0.05).

CONCLUSIONSCoculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF-κB pathways, and exhibiting its anti-inflflammation activity.

Animals ; Base Sequence ; Bronchi ; cytology ; enzymology ; metabolism ; Cattle ; Cell Adhesion Molecules ; metabolism ; Cell Line ; Coculture Techniques ; DNA Primers ; Down-Regulation ; drug effects ; Epithelial Cells ; enzymology ; metabolism ; Isoflavones ; pharmacology ; NF-kappa B ; metabolism ; Neutrophils ; enzymology ; metabolism ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVEThis review aimed to update the progress of microRNA (miRNA) in early detection of ovarian cancer. We discussed the current clinical diagnosis methods and biomarkers of ovarian cancer, especially the methods of miRNA in early detection of ovarian cancer.

DATA SOURCESWe collected all relevant studies about miRNA and ovarian cancer in PubMed and CNKI from 1995 to 2015.

STUDY SELECTIONWe included all relevant studies concerning miRNA in early detection of ovarian cancer, and excluded the duplicated articles.

RESULTSmiRNAs play a key role in various biological processes of ovarian cancer, such as development, proliferation, differentiation, apoptosis and metastasis, and these phenomena appear in the early-stage. Therefore, miRNA can be used as a new biomarker for early diagnosis of ovarian cancer, intervention on miRNA expression of known target genes, and potential target genes can achieve the effect of early prevention. With the development of nanoscience and technology, analysis methods of miRNA are also quickly developed, which may provide better characterization of early detection of ovarian cancer.

CONCLUSIONSIn the near future, miRNA therapy could be a powerful tool for ovarian cancer prevention and treatment, and combining with the new analysis technology and new nanomaterials, point-of-care tests for miRNA with high throughput, high sensitivity, and strong specificity are developed to achieve the application of diagnostic kits in screening of early ovarian cancer.

Early Detection of Cancer ; methods ; Female ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; MicroRNAs ; genetics ; Ovarian Neoplasms ; genetics

Based on the analysis of the management of Director 's target responsibility system in China Academy of Chinese Medical Sciences these years, the methods and experience were introduced in this paper. How to further improve the management was also discussed, providing a certain reference for the target responsibility system management of scientific research institutes.