A sensitive and selective method was developed for the separation and characterization of related substances (RSs) in EVT-401 by hyphenated LC–MS techniques. Complete separation of the RSs was achieved with an Inertsil ODS-SP column (250 mm×4.6 mm, 5 μm) by linear gradient elution using a mobile phase consisting of 0.2% formic acid solution, methanol and acetonitrile. EVT-401 was found to be susceptible to acid, alkaline and oxidative stresses, while relatively stable under photolytic and thermal dry stress conditions. Fourteen RSs including six process-related substances and eight degradation products were detected and identified in EVT-401 with positive ESI high-resolution TOF-MS analysis of their parent ions and the corresponding product mass spectra elucidation, and some of them were further verified by chemical synthesis and NMR spectroscopy. The specific LC–MS method developed for separation, identification and characterization of RSs is valuable for EVT-401 manufacturing process optimization and quality control.

ObjectiveThe aim of the present study was to investigate the incorporation of plasmid DNA (pDNA) onto a coronary stent by chemo-immuno-conjugation for achieving site-specific gene delivery.MethodsA gene eluting stent was fabricated by reacting with polyallylamine bisphosphonate (PAA-BP) to introduce amine reactive groups on the surface.Then an anti-DNA antibody was chemically coupled and pDNA was immunologically tethered on the stent surface.Radioactive-labeled antibody was used to evaluate binding capacity and stability.ResultsThe presence of amine groups on the modified stent surface was confirmed by XPS and AFM analysis.The isotope label assay indicated that the amount of antibody chemically linked on the stents was 15-fold higher than that of the control stent and its retention time was also significantly longer.ConclusionThe results suggested that a large amount of reactive amine groups were introduced on the PAA-BP modified 316L coronary stent surface.This study provide a potential metal surface modification method that could facilitate coupling and tethering of biological molecules such as anti-DNA antibody and plasmid DNA (pDNA) to achieve sustained and highly localized gene delivery for substrate-mediated gene transfection.

This study aimed to identify the related substances of phloroglucinol injection by two-dimensional liquid chromatography quadrupole time-of-flight mass spectrometry (2D-LC-Q-TOF/MS). The first-dimensional separation was carried out on an HSS T3 (250 mm × 4.6 mm, 5 μm) column by gradient elution using 1.36 g·L^-1 potassium dihydrogen phosphate buffer solution (pH adjusted to 3.0 with diluted phosphoric acid) and acetonitrile as the mobile phases. The separated components were then trapped in switch valve tube lines respectively and delivered to the second-dimensional desalting gradient elution which was performed with a BDS C18 (100 mm × 4.6 mm, 2.4 μm) column using 0.1% formic acid and methanol as the mobile phases. After rapid desalting, electrospray-ionization quadrupole time-of-flight high resolution mass spectrometry was used for determining the accurate masses and elemental compositions of the parents and their product ions for both phloroglucinol and its related substance. Structures of the related substances were then figured out by mass spectrometry elucidation, organic reaction mechanism analysis, and/or comparison with reference substances. Under the established analytical conditions, phloroglucinol and its related substances were adequately separated, 17 main related substances were detected and identified in the injection and its stressed samples for the first time. The identification results can provide reference for the quality control of phloroglucinol injection.

OBJECTIVETo investigate the effect of human serum albumin (HSA) on cell attachment of human gingival epithelial cells (HGE).

METHODSHGE were primary cultured with keratinocyte serum-free medium (KSFM) and dispase. The cultured cells were immunohistochemically stained by monoclonal anti-pan cytokeratin. MTT test was employed to investigate the influence of HSA on the cell attachment on polystyrene surface. The cell growth curve of HGE which were cultured in KSFM with 50 g/L HSA was observed.

RESULTSThe results showed significant decrease in cell numbers within 8 hours after HGE were inoculated, in which the polystyrene surface was preincubated with 50 g/L HSA. But it did not prove to be the case from 10 hours to 24 hours after HGE were inoculated. There were no significant difference within 24 hours in cell numbers between cultured in KSFM with 50 g/L HSA and control. The cell numbers in cell growth curve of HGE in KSFM with and without 50 g/L HSA did not show significant difference.

CONCLUSIONSHSA preincubation on polystyrene were produce inhibitory effect of HGE attachment in early stage.

Cell Adhesion ; drug effects ; Cell Count ; Cell Division ; drug effects ; Cells, Cultured ; Epithelial Cells ; cytology ; drug effects ; Gingiva ; cytology ; drug effects ; Humans ; Polystyrenes ; Serum Albumin ; pharmacology

OBJECTIVETo observe the therapeutic effect of Yiqi Yangyin principle (YQYY, the treating principle in TCM to supplement Qi and nourish Yin) on corticosteroid withdrawal in patients with systemic lupus erythematosus (SLE) in remission stage and its influence on some immune parameters.

METHODSThe SLE patients were divided into two groups, 30 in the treated group and 10 in the control group, who were treated by conventional method with corticosteroids and/or immunosuppressant in acute progressive stage, and YQYY was added to the treated group in remission stage.

RESULTSThe total effective rate was 93.3% in the treated group and 90.0% in the control group, comparison between the two groups showed significant difference by Ridit test (P < 0.05). The immune parameters, IgG and C3 were significantly improved after treatment in the treated group (P < 0.01), but changed insignificantly in the control group. The maintaining dose of prednisone used in the two groups was 7.08 +/- 5.26 mg/d and 11.72 +/- 6.48 mg/d respectively, the amount used in the treated group was significantly lower than that in the control.

CONCLUSIONUsing mainly YQYY Principle to treat SLE in remission stage could withdraw the corticosteroid smoothly, relieve symptoms and improve immune function.

Adolescent ; Adult ; Child ; Complement C3 ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Immunoglobulin G ; blood ; Lupus Erythematosus, Systemic ; drug therapy ; Male ; Middle Aged ; Phytotherapy ; Prednisone ; adverse effects ; therapeutic use ; Substance Withdrawal Syndrome ; drug therapy

OBJECTIVETo prepare a rapid and sensitive diagnostic kit for detection of CA50 based on time-resolved fluoroimmunoassay.

METHODSA sandwich-TRFIA diagmostic kit was developed using anti-CA50 monoclonal antibody and all parameters of the kit were evaluated.

RESULTSThe linear measurement range of the kit was (5 - 300) U/ml. The sensitivity was 0.2 U/ml. The intra- and inter-assay coefficients of variation were 4.3% - 8.2% and 7.7% - 11.2%, respectively. There was no cross-reaction with CEA, CA12-5, CA15-3 and AFP. The cross reactivity with CA19-9 was 0.7 U/ml. The correlation coefficient of detection results of 107 blood samples between this newly developed kit and commercially available CA50 RIA kit was 0.901.

CONCLUSIONThis newly developed CA50-TRFIA kit is a valuable test tool for clinical application with even better sensitivity, specificity and accuracy.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, Tumor-Associated, Carbohydrate ; blood ; Female ; Fluoroimmunoassay ; instrumentation ; methods ; Humans ; Male ; Middle Aged ; Radioimmunoassay ; Reagent Kits, Diagnostic ; standards ; Reference Standards ; Reproducibility of Results

BACKGROUNDType 2 diabetes mellitus (T2DM) results from the complex association of insulin resistance and pancreatic β-cell failure. Recent studies have shown that patients diagnosed with T2DM present with a significant decrease in β-cell function, which can be further compromised during the progression of the disease. Several mechanisms have been shown to play a role in this process such as glucotoxicity and lipotoxicity, which contribute to accelerating insulin secretion. In this regard, Chinese medicine has a certain advantage. This experiment was performed to observe the effect of a Chinese medicine named Kaiyuqingre formula (KYQRF) on β-cell function and its mechanisms of action therein.

METHODSHigh glucose was used to set up a model of β-cell function failure. At the same time, medicated serum of KYQRF with different doses were administered to the cells. Rosiglitazone was taken as a control to observe the changes in insulin secretion, ATP-sensitive K(+) channels (K(ATP) channel) and uncoupling protein-2 (UCP-2) in each group.

RESULTSKYQRF had some effects on the insulin secretion. In a low glucose environment, no effective change in insulin secretion was observed (P > 0.05). However, insulin levels increased significantly when INS-1 cells were exposed to a high glucose environment (P < 0.05). KYQRF could also enhance cell viability (P < 0.05) in an effect similar to rosiglitazone. Although KYQRF had no effect on inwardly rectifying potassium channels (Kir6.2) (P > 0.05), it could decrease the overexpression of both UCP-2 and sulfonylurea receptor 1 (P < 0.05).

CONCLUSIONKYQRF can protect islet function by decreasing UCP-2 and sulfonylurea receptor 1.

ATP-Binding Cassette Transporters ; genetics ; Animals ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Glucose ; pharmacology ; Insulin ; secretion ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Ion Channels ; genetics ; Male ; Mitochondrial Proteins ; genetics ; Potassium Channels, Inwardly Rectifying ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Drug ; genetics ; Sulfonylurea Receptors ; Thiazolidinediones ; pharmacology ; Uncoupling Protein 2

Objective To study the genotypes of representative Mycobacterium tuberculosis (M.tuberculosis) strains from China with spacer oligonucleotide typing (spoligotyping),and to investigate the prevalence of different genotypes TB in China,and analyse the relationship between genotype and drug resistance.Methods 4017 clinical isolates were collected by Chinese Center for Disease Control and Prevention from 2007 to 2008 in 31 provinces in China according to sampling principle of epidemiology.Drug susceptibility testing was performed using proportion method,and spoligotyping was chosen to carry out genotyping of these M.tuberculosis.In addition,chi-square test was used to compare the differences among the detection rate of different genotypes.Results Among the 4017 M.tuberculosis isolates,2500 ( 62.2％ ) isolates belonged to Beijing genotype.The percentage of Beijing genotypes in the northern of China was higher than that in the southern of China ( 76.5％ vs.53.2％,x2 =219.69,P ＜ 0.05 ),while T1 genotypes were more common in the southern China,compared with that in northern China ( 13.3％ vs.4.3％,x2 =219.69,P ＜ 0.05 ).The differences were statistically significant.The proportions of Rifampinresistant (21.7％ vs.21.7％ ),Ofloxacin-resistant (4.9％ vs.2.4％ ) and Multidrug-resistant ( 11.3％vs.7.4％ ) isolates among Beijing genotype strains were significantly higher than those among non-Beijing strains (x2 =22.10,14.42 and 14.83,respectively,P ＜ 0.05 ).Conclusions Beijing genotype was still predominant epidemic genotypes.The percentage of Beijing genotype showed difference between distinct areas,and the percentage of Beijing genotypes in northern China was higher than that in southern China.Beijing genotype strains reveal correlation with Rifampin-resistance,Ofloxacin-resistance and Multidrug-resistance.

Objective To explore the expression of eukaryocyte initiation factors-4E (eIF-4E) in normal esophageal epithelium and different esophageal lesions tissue and its relationship with vascular endothelial growth factor (VEGF).Methods Thirty normal esophageal incisal margin tissues,32 atypical hyperplasia tissues,20 carcinoma in situ tissues and 117 esophageal invasive carcinoma tissues were selected from the biopsy specimens of the Department of Pathology,the First Affiliated Hospital of Xinxiang Medical University from March 2014 to March 2016.The expression of eIF-4E and VEGF were detected by immunohistochemistry method and the correlation between them was analyzed.Results The positive expression rate of eIF-4E in normal esophageal epithelium,atypical hyperplasia,carcinoma in situ and invasive carcinoma tissues was 0.0％ (0/ 30),9.4％ (3/32),45.0％ (9/20) and 80.3％ (94/117) respectively.There was no statistic difference in the positive expression rate of eIF-4E in normal esophageal epithelium and atypical hyperplasia tissues (P ＞ 0.05);there was statistic difference in the positive expression rate of eIF-4E in the other esophageal tissues(P ＜ 0.05).In 117 invasive carcinoma tissues,the positive expression rate of eIF-4E in metastatic carcinoma tissues (93.2％,55/59) was significantly higher than that in the non metastatic carcinoma tissues (67.2％,39/58) (P ＜ 0.05).The positive expression rate of eIF-4E in esophageal carcinoma tissues which invaded into shallow muscle layer,deep muscular layer and adventitia was 70.0％ (28/40),73.8％ (31/42) and 100.0％ (35/ 35) respectively;there was no statistic difference in positive expression rate of eIF-4E in the carcinoma tissues which invaded into shallow muscle layer and deep muscular layer(P ＞ 0.05);the positive expression rate of eIF-4E in the carcinoma tissues which invaded into shallow muscle layer and deep muscular layer was significantly lower than that in the carcinoma tissues which invaded into adventitia(P ＜0.01).The expression of eIF-4E in the esophageal invasive carcinoma tissues was positive VEGF(x2 =51.460,P ＜ O.05).Conclusion eIF-4E play an important role in the canceration of normal esophageal and the invasion,metastasis of esophageal carcinoma.The expression of eIF-4E is correlate with VEGF in esophageal carcinoma tissues.

This study was aimed to investigate the clinical value of multiplex nested reverse transcription PCR (RT-PCR) in detecting MLL-related fusion genes in myelodysplastic syndrome (MDS). Ten MLL-related genes (dupMLL, MLL-ELL, MLL-ENL, MLL-AF6, MLL-AF9, MLL-AF10, MLL-AF17, MLL-CBP, MLL-AF1P, MLL-AF1Q) in 221 MDS cases were detected by multiplex nested RT-PCR. The results indicated that 20 patients were detected with positive result among 221 patients and the positive rate was 9.05%. The number of the positive cases and positive rates of the above mentioned 10 fusion genes were in order: 7 (3.16%), 2 (0.9%), 1 (0.45%), 1 (0.45%), 2 (0.9%), 2 (0.9%), 1 (0.45%), 2 (0.9%), 1 (0.45%), 1 (10.45%). It is concluded that the multiplex nested RT-PCR has been confirmed to be able to detect 10 fusion genes in MDS patients, which can provide important evidences for assessing diagnosis and treatment, and give related necessary information about minimal residual disease and its prognosis.
Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Karyotyping ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; Oncogene Proteins, Fusion ; genetics ; Polymerase Chain Reaction ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Translocation, Genetic ; Young Adult