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.

Objective:To investigate the effects of ursolic acid (UA) on the proliferation and differentiation of osteoclasts (OC), and to explore its role in orthodontic force-induced root resorption and its relationship with OC.Methods:The mononuclear / macrophage cells RAW264.7 were induced to the OC.Tacrolimus acid phosphatase (TRAP) staining and bone resorption lacunae observation were used to identify the induction.CCK-8 method was used to select the appropriate concentration of UA for RAW264.7 cell-free biotoxicity and to observe its effect on the proliferation and differentiation of RAW264.7.In experimental groups, UA with gradient concentrations (1.0,2.5,5.0,10.0,20.0and 40.0 μmol·L-1)were added.UA was not added in control group.Results:The TRAP staining and bone resorption lacunae observation showed that after the RAW264.7 cells were induced for 5 d, the TRAP staining positive cells were found;the resorption lacunae were rounded,and oval, etc,the bottom wall was coarser,and the boundary was clear,which indicated that the RAW264.7 cells were successfully differentiated into the osteoclasts.The CCK-8 detection results showed that high concentration of UA (> 10.0 μmol·L-1) significantly inhibited the proliferation of OC;the appropriate concentration of UA (5.0 μmol·L-1) was in the biological safety concentration range and could inhibit the OC proliferation;low concentration of UA (<2.5 μmol·L-1) had no effect.Conclusion:RANKL can induce the differentiation and maturation of RAW264.7 cells.UA is correlated with the proliferation and differentiation of OC;UA has inhibitory effect on OC at the appropriate concentration (5.0 μmol·L-1) in a time-dependent manner.

Aged ; Cardiac Pacing, Artificial ; Cardiac Resynchronization Therapy ; Female ; Heart Ventricles ; Humans

Objective:To study the changes of oral health indexes in adolescent orthodontic patients under different brushing time, and to provide basis for the orthodontists to give effective dental health promotion in clinic.Methods:A total of 123 adolescent orthodontic patients were included, and they were divided into 1-3min, 3-5 min, 5-7 min and 7-10 min groups based on the brushing time recorded in the period of orthodontic treatment.The relationship between different brushing time and oral health indexes was evaluated 1,3,and 6 months after experiment, such as sulcus bleeding index(SBI),plaque index(PLI), debris index-simplified(DI-S) and enamel demineralization index(EDI).Results:Compared with 1-3 min and 3-5 min groups, the SBI, PLI, and DI-S of the patients in 5-7 min group were significantly reduced(P<0.05).There was no significant differences of SBI, PLI, DI-S , and EDI between 5-7 min group and 7-10 min group (P>0.05).Conclusion:Brushing teeth more than 5 min can improve the oral hygiene significantly and the best time is 5-7 min;the orthodontists should require the patients to brush teeth at least for 5 min.

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.

Objective To investigate the effect of silencing E6-associated protein(E6AP)on the level of p53 protein in human papilloma virus(HPV)negative cervical cancer cells(C33A cells).Methods The siRNA sequence silencing E6AP(siE6AP)and silencing control disordered siRNA sequence(siControl)were transfected into C33A cells with the mediation of LipofectamineTM2000 transfection reagent respectively.The silencing effect of siRNA on E6AP and the expression of p53and cleaved-caspase-3 proteins were detected by Western blot.Results The levels of E6AP protein in C33A cells of siE6AP group were significantly lower(t =-4.597,P<0.05),while the levels of p53 and cleaved-caspase-3 proteins were significantly higher than those of siControl group(t = 4.533 and 7.099 respectively,each P<0.05).Conclusion Silencing of E6AP significantly increased the expression of p53 protein in C33A cells,indicating that silencing of E6AP may restore the activity and function of p53 protein in C33A cells.

Objective To establish a network laboratory for blood cell analysis and better calibrate haematology analyzers in local lab.Methods According to GB/T 15481《General requirements for the competence testing and calibration laboratories》(idt ISO/IEC 17025),we established a network laboratory providing traceability for blood cell analysis.Complete blood count was traced to Calibration Laboratory in NCCL;The secondary standard haematology analyzer with the same model and calibrator with same lot number were used for verification for a long period.Fresh blood from healthy people was used to calibrate haematology analyzers.Results Gradually we have improved our laboratory quality management system, precision as well as accuracy,which was satisfactory.The unified blood sample was adopted to calibrate different equipments in our hospital and showed consistence when compared with calibration analyzer.The correlation coefficient of all tests is more than 0.99.The relative deviation of WBC,RBC,HCT,HGB and PLT are within?7%,?3.5%,?4%,?3% and?15%,respectively.Conclusions Secondary standard systems provides good comparable results with calibration laboratory.Its tracing mode and quality control scheme could ensure the traceability and accuracy of completed blood count.Furthermore,using elective fresh blood from healthy people,the comparable results from different analyzers were achievable.

Leptomeningeal carcinomatosis occurs in approximately 5% of patients with cancer. The most common cancers involving the leptomeninges are breast, lung cancer and melanoma. However, gastric adenocarcinoma has been rarely reported with leptomeningeal carcinomatosis. The presenting manifestations are usually headache, visual disturbances and seizures. We report a case of leptomeningeal metastasis that presented as a gastric cancer. A 49-year-old woman was admitted to our hospital with the symptoms of headache and melena for 10 days. The endoscopy showed a thickening of the folds of the stomach compatible with the diagnosis of a Borrman type IV gastric cancer. The biopsy revealed a signet ring cell carcinoma. The MRI of brain showed no abnormal findings; however, the patient complained of an intractable persistent headache, nausea and vomiting on admission day 6. The cytology examination of the cerebrospinal fluid supported the diagnosis of metastatic signet ring cell carcinoma.
Adrenal Cortex Hormones ; Carcinoma, Signet Ring Cell/*diagnosis/pathology/surgery ; Female ; Humans ; Mannitol ; Meningeal Neoplasms/*diagnosis/pathology/surgery ; Middle Aged ; Stomach Neoplasms/*diagnosis/pathology/surgery

Application of promoter trapping based on transformation in Magnaporthe grisea is reported in this paper. Two promoter-trapping vectors, designated as pCBGFP and pEGFPHPH, were constructed and transformed into protoplasts of M. grisea. A library of 1,077 transformants resistant to hygromycin B was generated. Of which, 448 transformants were found to express eGFP gene in different structures of M. grisea. Three transformants grew slowly, 5 transformants decreased in conidiation and 7 transformants reduced in pathogenicity greatly among these 448 transformants. Eleven transformants were checked by genomic southern blot randomly, and 9 of which were single-copy insertions. The promoter trapping technique has been applied successfully in M. grisea and can be used as a tool for functional genomic analysis.
Fungal Proteins ; biosynthesis ; genetics ; Gene Expression Regulation, Fungal ; genetics ; Genes, Reporter ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Magnaporthe ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics ; Protein Engineering ; methods ; Recombinant Proteins ; metabolism

PURPOSE: With recent advances of diagnostic methods and precise histopathologic examination, the incidence of synchronous multiple gastric cancer has increased. The purpose of this study was to evaluate the clinicopathologic features of patients with synchronous multiple gastric cancer. MATERIALS AND METHODS: We reviewed the clinicopathologic features of 189 patients with synchronous multiple gastric cancer out of 8,101 patients who underwent gastric resections for gastric cancers during 20 years from January 1977 to December 1996 at the Department of Surgery, Seoul National University Hospital, and compared them with single gastric cancer patients. The clinicopathologic features evaluated were age, sex, diagnostic method and accuracy, location of lesions, tumor size, histologic differentiation, Lauren classification, macroscopic classification, depth of invasion, lymph node metastasis, TNM stage, and type of operation and prognosis. RESULTS: The overall incidence of multiple synchronous gastric cancer was 2.33%. The mean age was 57.2 years old (27~84) and peak incidence was sixth decade. Male was predominant, that the sex ratio was 3.9: 1. Multiple gastric cancer was more frequent in old age, male and early gastric cancer patients. The number of lesions ranged from 2 to 5. In most cases, the lesions were located in lower two-thirds of the stomach. However, in 13 cases, lesions were located in both upper one-third and lower one-third. Only 33.3% of multiple cancer was diagnosed preoperatively, with the diagnostic accuracy of GFS was 30.0% and that of UGIS 26.1%. The most frequently missed lesions at preoperative examination were located in upper third of stomach, posterior wall of middle third and anterior wall of lower third of stomach. The most common macroscopic type was Borrmann type III (54.5%) in advanced lesions and type IIc (47.0%) in early lesions. Regarding the histologic differentiation, 58.7% of the cases were of the same differentiation and the cases composed of well differentiated adenocarcinomas were most common. According to the Laurens classification, 66.7% of lesions were intestinal type. As to the progression of the lesions, all lesions were early cancers in 75 cases, advanced cancers in 39 cases and advanced cancers were coexist with early cancers in 75 cases. Lymph node metastasis was less frequent than in single gastric cancer. Total gastrectomy was performed more frequently in multiple cancer patients than in single gastric cancer patients. The 5-year survival rate of patients with multiple gastric cancer was 70.2%, which was not significantly different from that of patients with single gastric cancer. CONCLUSIONS: Surgeons must keep in mind the possibility of multiple gastric lesions. More careful preoperative and intraoperative examination is mandatory to detect the possible accessory lesions, and postoperative periodic follow-up is necessary to detect any missed lesions, especially in the old age, male and early gastric cancer patients.
Adenocarcinoma ; Classification ; Follow-Up Studies ; Gastrectomy ; Humans ; Incidence ; Lymph Nodes ; Male ; Neoplasm Metastasis ; Postoperative Period ; Prognosis ; Seoul ; Sex Ratio ; Stomach ; Stomach Neoplasms* ; Survival Rate