A rapid, sensitive and convenient LC-MS/MS method was developed for the determination of γ-aminobutyric acid (GABA) in human plasma. d2-γ-Aminobutyric acid (d2-GABA) was synthesized as internal standard (IS). After extraction from human plasma by protein precipitation with acetonitrile, all analytes were separated on a Luna HILIC column (100 mm x 3.0 mm, 3 μm) using an isocratic mobile phase of water: acetonitrile: formic acid (20 : 80 : 0.12) with a flow rate of 0.5 mL x min(-1). Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode (MRM) in positive electrospray ionization using the transitions of m/z 104 --> 69 for GABA and m/z 106 --> 71 for d2-GABA. The method was linear in the concentration range of 5.00 to 1 000 ng x mL(-1). The intra- and inter-day precisions were within 9.9%, and accuracy ranged from 99.1% to 104%, within the acceptable limit across all concentrations. The method was successfully applied to a pharmacokinetic study of GABA tablets in healthy Chinese volunteers.
Chromatography, Liquid ; Humans ; Tandem Mass Spectrometry ; gamma-Aminobutyric Acid ; blood

Background and purpose: Because of the aggressive nature of hilar cholangiocarcinoma and the absence of effective adjuvant therapy, surgical radical resection offers hilar cholangiocarcinoma patients the only choice. Research focus include preoperative assessment, the use of preoperative biliary drainage, the range of hepatic resection, and the range of lymphadenectomy. To investigate the clinical experience and efifcacy of combined hepatectomy in the treatment of hilar cholangiocarcinoma. Methods: Two hundred and seven patients with hilar cholangiocarcinoma treated surgically in the First Afifliated Hospital of Kunming Medical University form Jan. 2007 to Oct. 2013 were retrospectively analyzed. Results:Of the 207 patients, 125 patients who received radical resection (R0 resection) and the curative resection rate was 60.4%. One hundred and iffty-six cases were treated in combined hepatectomy group, 51 cases in non-hepatectomy group, the rate of R0 resection was 70.5%in hepatectomy group and 29.4%in non-hepatectomy group, and the difference was signiifcant (P<0.01). Two patients died perioperatively, the main postoperative complications included hepatic function insufifciency and bile leakage. One hundred and seventy-two patients were followed up, the median survival time of the 102 patients who received R0 resection was 45 months, and the 1, 3, 5 year survival rates were 96.1%, 59.1%and 17.2%. The median survival time of the 70 patients who received R1-2 resection was 26 months, and the 1, 3 year survival rates were 81.3%and 19.2%, and none of the patient survived for over 5 years. The survival rate of patients who received R0 resection was signiifcantly higher than those who received R1-2 resection (χ2=39.121, P<0.01). In the hepatectomy group was awarded the R0 resection in patients with postoperative 1, 3, 5 year survival rate was 97.8%, 63.9% and 18.0%, in non-hepatectomy group received R0 resection in patients with postoperative 1, 3, 5 year survival rate was 83.3%, 20.8%and 8.3%. There were signiifcant differences in the postoperative survival rate between both group (χ2=5.988, P=0.014). Conclusion:Radical excision is the key to improve the long term survival. Combined hemihepatectomy and standardized lymph node resection has signiifcantly improved the radical resection rate and the efifcacy of treatment for hilar cholangiocarcinoma.

Background The construction of tissue-engineered corneal endothelium needs the functional seeding cells,so how to culture a large amount of functional corneal endothelial cells (CECs) is an urgent problem to be solved.Objective The aim of this study was to evaluate the role of aqueous humor on bovine CECs in vitro.Methods Aqueous humor of 1.2 ml was collected from the anterior chamber of bovine and sterilized,and the liquid supernatant was obtained.The bovine CECs were isolated from bovine cornea and then cultured in low glucose Dulbecco Modified Eagle Medium with 10％ fetal bovine serum (FBS) in vitro.Aqueous humor was added into the medium with the final concentration of 2.5％,5.0％,l0.0％,15.0％ and 20.0％,respectively,and no aqueous humor was added in the control group.Cell counting kit-8 (CCK-8) assay was used to detect the absorbency value of CECs for the evaluation of cell proliferation.Progression of the cell cycle was analyzed by flow cytometry (FCM).After confluence of the cells was reached,1 ml plastic spear tip was used to scratch the cell single layer,and the cells were incubated consequently in medium with 10％ FBS and with or without aqueous humor for 24 hours.Healing area of the cell single layer was measured.The cells were incubated at a density of 6 × 105 cells/ml and cultured using medium with or without 10.0％ aqueous human for 5 days,and the number of the cells was analyzed by DAPI fluorescence technique.Results Under the phase-contrast microscopy,the confluent CECs showed a slabstone-like and hexagonal appearance.CCK-8 assay revealed that the absorbance values of CECs was significantly different among the various culture groups (F=4.051,P =0.007),and the absorbance value in different concentrations of aqueous human culture groups was significantly higher than that in the control group (P ＜ 0.01).FCM showed that the percentage of the cells in S-G2 phases was (34.80-±3.13)％ in the 10.0％ aqueous humors group and (23.06±1.13)％ in the control group,showing a significant difference (t =-5.729,P=0.005).The scratch test showed that the healing area of the cell signal layer was (0.116±0.019) mm2 in the 10.0％ aqueous humors group and (0.358 ±0.049) mm2 in the control group,showing a significant difference (t =13.842,P =0.000).The density of cells in the 10.0％ aqueous humor group was (1439± 1 10)/field,which was more than (1162±45)/field in the control group (t =-11.020,P=0.000).Conclusions Aqueous humor at the concentration of 10.0％ promote the growth and proliferation of bovine CECs.The result suggests that 10.0％ aqueous humor can be used as a promoting agent during the culture of CECs.

The aim of this study was to detect the localization and level of tyrosine phosphorylated proteins during in vitro capacitation of guinea pig sperm. Sperm from mature guinea pigs were incubated in modified TALP under 5% CO_2 in air at 37 ℃. The capacitation effect was assessed by chlortetracycline (CTC) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum of sperm. Moreover, there were three proteins phosphorylated in this experiment. After 0 to 0.5h incubation, the protein of 40kDa was detected by anti-phosphotyrosine monoclonal antibody, and the intensity of this protein increased in the following incubation. Then, after 1h incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. Also, in 3h incubation, a protein of 45kDa was detected and the intensity of this protein increased in the following incubation.

The chemical constituents of Safflower injection were isolated and purified by polyamide, silica gel, Sephadex LH-20, ODS column chromatographies and preparative HPLC. As a result, sixteen compounds have been isolated. Based on the spectral data analysis, their structures were elucidated as scutellarin (1), kaempferol-3-O-β-rutinoside(2), hydroxysafflor yellow A(3), rutin (4), coumalic acid(5), adenosine(6), syringoside(7), (3E)-4-(4'-hydroxyphenyl)-3-buten-2-one(8), (8Z)-decaene-4, 6-diyne-1-Oβ-D-glucopyranoside(9), 4-hydroxybenzaldehyde (10), (2E, 8E) -tetradecadiene-4, 6-diyne-1, 12, 14-triol-1-O-β-D-glucopyranoside (11), kaem-pferol-3-O-β-sophorose (12), uridine (13), roseoside (14), cinnamic acid (15), and kaempferol (16). Compounds 1,2,7,9,11 and 12 were isolated from the Safflower injection for the first time. The anti-platelet aggregation activities of the isolated compounds were assayed. The results indicated all tested compounds exhibited potent activity except for 5, while 2, 3, 9 and 12 showed strong activity against platelet aggregation.
Animals ; Blood Platelets ; drug effects ; physiology ; Carthamus tinctorius ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Fibrinolytic Agents ; chemistry ; isolation & purification ; pharmacology ; Molecular Structure ; Platelet Aggregation ; drug effects ; Rabbits ; Spectrometry, Mass, Electrospray Ionization

A simple and rapid method was developed based on high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to determine sivelestat and its metabolite XW-IMP-A in human plasma. After a simple protein precipitation, the samples and internal standards were analyzed on a C18 column by a gradient elution program. The mobile phase consisted of 30% acetonitrile in methanol and 5 mmol · L(-1) ammonium acetate at a flow rate of 0.7 mL · min(-1). The mass spectrometric data was collected in multiple reaction monitoring mode (MRM) in the negative electrospray ionization. The standard curves were linear in the range of 10.0-15,000 ng · mL(-1) for sivelestat, and 2.50-1000 ng · mL(-1) for XW-IMP-A. The low limits of quantitation were identified at 10.0 and 2.50 ng · mL for sivelestat and XW-IMP-A, respectively. The intra- and inter-day precision were within 11.3% and 13.1% for sivelestat and XW-IMP-A, and accuracy was 0.3% and 0.6% for sivelestat and XW-IMP-A, within the acceptable limits across all concentrations. The method was successfully validated in the pharmacokinetic study of sivelestat in healthy Chinese volunteers.
Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Glycine ; analogs & derivatives ; blood ; Humans ; Inosine Monophosphate ; blood ; Reproducibility of Results ; Sulfonamides ; blood ; Tandem Mass Spectrometry

Background Induced pluripotent stem cells (iPSCs)can differentiate into various types of somatic cells without causing ethical controversy and immune rejection in clinical activity,which is similar to differentiation ability of embryonic stem cells.So,iPSCs may be used as seed cells for tissue engineering corneal endothelial reconstruction.Objective The present study was to survey the morphologic change of iPSCs after coculture with corneal endothelium cells(CECs) under the atomic force microscopy(AFM).Methods Rabbit CECs and human MMC-iPSCs were isolated and cultured respectively.The iPSCs were identified with the marker by immunochemistry.iPSCs passaged for 7 days were then cultured with 60％ confluent CECs to establish the co-culture model.The surface morphology and cellular membrane ultrastructure of differentiated iPSCs after induced by CECs were examined by AFM combination with inverted microscope,and compared with CECs and undifferentiated iPSCs.Results Thelengthand width were(66.93±10.48)μm and (44.85 ± 8.14) μm in CECs,(12.51±1.40)μm and (10.93 ±1.69) μm in uninduced iPSCs,and(36.12±10.29) μm and(31.53±9.65)μm in CECs-induced iPSCs.Both the length and width values of CECs-induced iPSCs were statistically bigger than those uninduced iPSCs,with significant differences between them (P＜0.05),but no significant difference was seen in the width valne of CECs-induced iPSCs in comparison with CECs(P＞0.05).The convex structure of CECs cytomembrane surface showed the digitation in shape with the size and height(2.11 ± 1.03) μm and (115.68±92.08) nm respectively,and the concave structure of cytomembrane surface of CECs was fenestrae-like depression and the size was (1.49 ± 0.65) μm.The numerical valuc of mean square root roughness (Rq)and average roughness (Ra)of cytomembrane surface of CECs were(39.20±7.82)nm and (30.37±5.32)nm respectively.The convex surface of cytomembrane of iPSCs was granular-like in shape with size and height(0.39±0.22)μm and(13.11±9.18)nm respectively.The concave surface of cytomembrane of iPSCs was worm-eaten-like concave with the size(0.34±0.18)μm.The numerical value of Rq and Ra of geometrical parameters of cytomembrane surface of iPSCs were (26.60 ± 4.93)nm and (9.97 ± 3.78) nm respectively.The convex surface of cytomembrane of induced iPSCs was digital-like in shape with the size and height (1.91±0.76) μm and(106.55±77.27) nm respectively.The concave surface of cytomembrane of induced iPSCs was fenestrae-like depression and the size of concave was(1.6l±1.25) μm.The numerical value of Rq and Ra on surface of cytomembrane of induced iPSCs was (57.33± 12.80) nm and (43.63± 11.17) nm respectively.The numerical values of the size and height of convex,the size of concave,Rq and Ra on surface of cytomembrane in induced iPSCs were statistically bigger than in iPSCs(P＜0.05)and were not significant differences in comparison with CECs (P＞0.05).Conclusions Morphology of iPSCs translate toward the CECs after induce for 7 days under the AFM.This outcome lays the foundation for further study on iPSCs.

Objective To design a new method to verify the position of multileaf collimator(MLC)leaf using a two-dimensional ion chamber array(2D-array).Methods 2D-array of PTW T10018 Seven29~(TM) was used to calibrate the accuracy of MLC leaf position of Elekta Precise accelerator.The edge function of the leaf position of MLC was measured and used as the reference value.The precision of MLC leaf was then evaluated through comparing the measured and reference values.Results The accuracy of MLC leaf position was found within?0.1 mm.Conclusion This method of verifying the accuracy of multileaf collimator leaf position is easy,simple and reliable

OBJECTIVETo explore the risk factors of esophageal cancer (EC) in the high-incidence regions, so as to provide scientific evidence for taking effective prevention measures.

METHODSA population based case-control family study was carried out. 1711 case family members in 505 families in which one of the couple or their first degree relatives suffered from EC were selected from high incidence in Henan province. Control families without neoplasm were selected from the same villages in matching conditions of age, sex, and family members. All information of case and control families was collected by Questionnaire of Life and Health of Inhabitant. The data were analyzed with logistic regression model.

RESULTSCompared with the control families,it was shown that hobby for smoked food [2.10% (36/1711), 0.82% (14/1711); chi2 = 9.82, P = 0.00; OR = 2.61, 95% CI: 1.40 - 4.85], hobby for fried food [7.17% (66/921), 3.91% (35/894) ; chi2 = 9.13, P = 0.00; OR = 1.90, 95% CI: 1.24 -2.89], hobby for raw and hard food [13.36% (123/921), 8.95% (80/894); chi2 = 8.87, P =0.03; OR =1.57, 95% CI: 1.16 - 2.11], and hobby for hot food [20.05% (343/1711), 15.20% (260/1711); chi2 = 13.87, P= 0.00; OR= 1.40, 95% CI: 1.17 - 1.67], the history with mental stimulated [6.72% (115/1711), 3.10% (53/1711); chi2 = 24.06, P = 0.00; OR = 2.25, 95% CI: 1.62 -3.14], upper digestive symptom history [19.40% (332/1711), 12.74% (218/ 1711); chi2 = 28.15, P = 0.00; OR= 1.65, 95% CI: 1.37 - 1.99] entered the last model, and were responsible for the higher risk of EC. Eating fast was shown to be a protective factor [20.85% (192/921), 25.14% (225/895); chi2 = 4.73, P =0.03; OR = 0.78, 95% CI: 0.63 - 0.98].

CONCLUSIONEC is a kind of malignant tumor caused by multiple factors. Prevention and control of EC should be initiated from environmental factors, life style, genetic factors and social-psychological factors comprehensively.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Causality ; Child ; Child, Preschool ; Esophageal Neoplasms ; epidemiology ; genetics ; Female ; Humans ; Incidence ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Risk Factors ; Surveys and Questionnaires ; Young Adult

BACKGROUNDBlood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) plays an important role in identifying functional cortical areas of the brain, especially in patients with gliomas. This study aimed to assess the value of fMRI in presurgical planning and functional outcome of patients with gliomas in the motor cortical areas.

METHODSTwenty-six patients with gliomas in the motor cortex were recruited in the study. Before operation, fMRI was performed in each patient to obtain the mapping of bilateral hands area on the primary sensorimotor cortex. This examination was performed on a 3.0T scanner with a bilateral hands movement paradigm. During microsurgery under awake anesthesia, the motor area was identified using direct electrical stimulation and compared with preoperative mapping. Finally the tumor was resected as much as possible with the motor cortex preserved in each patient. Karnofsky performance status (KPS) was evaluated in all patients before and after operation.

RESULTSTwenty-three patients showed a successful fMRI mapping. Among them, 19 were classified to be grade III; 4, grade II; 3, grade I. The operation time was about 7 hours in the 23 patients, 8.5 hours in the other 3. The pre- and postoperative KPS score was 82.3 +/- 8.6 and 94.2 +/- 8.1, respectively.

CONCLUSIONSPreoperative fMRI of the hand motor area shows a high consistency with intraoperative cortical electronic stimulation. Combined use of the two methods shows a maximum benefit in surgical treatment.

Adult ; Brain Neoplasms ; pathology ; surgery ; Female ; Glioma ; pathology ; surgery ; Humans ; Magnetic Resonance Imaging ; methods ; Male ; Middle Aged ; Motor Cortex ; pathology ; Oxygen ; blood