OTX1 gene is one of the pivotal transcriptional factors involved in the neurogenesis.In order to overexpress the OTX1 gene in distinct cell types and find out its contribution to the proliferation and differentiation of stem cells in vitro,OTX1 cDNA was subcloned into lentiviral vectors.The resulting constructions pDUETOTX1,pDUETGFPOTX1 and pDUETGFP were packaged in 293 cells producing viral particles to transduce 293T cells,SY5Y cells,mouse embryonic stem cells and E15 neural stem cells.It was proved that the transferred OTX1 gene was located in the nuclei of the transduced cells in stead of plasma.Lentivirus is an ideal vector delivering gene to different cells.The overexpression of OTX1 in transduced 293T cells were validated by Western blot and immunofluorescence.

Objective:To remove murine embryonic stem cells(mESC)from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting(MACS).Methods:Neural differentiation of mESC was induced by a 5-stage method.The specific cell surface marker,SSEA-1,was used to identify ES cells in the differentiating cells.The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test.The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate.After the optimization,stage 4 cells were dissociated into single cell suspension,incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM,and then sorted through the magnetic field.The rate of SSEA-1 positive cells in pre-and post-separation groups was assessed by flow cytometry,and the viability of cells was evaluated by trypan blue staining counting under light microscopy.Results:The proportion of SSEA-1 positive cells in the separated cells can be reduced from(7.19?1.36)% to(1.34?0.80)%.The survival rate of sorted cells was more than 92%,similar to that of pre-separation cells.Conclusions:The MACS system we used can effectively remove mESC from the differentiated cells.The sorted cells will be well provided for the subsequent studies about transplantation therapy.

Objective To investigate the effectiveness of problem-based learning in the biochemistry teaching in merging class of minority and Han students.Methods Totally 460 clinical medical students were divided into PBL group which contained 252 students and the traditional teaching group which involved 208 students,respectively.According to each team of seven to eight students,minority and Han students randomly arranged.Control group used classroom teaching mode,experimental group in addition to classroom lectures,had additional 12 hours of PBL teaching,but the theory classes for the two groups of students were taught by the same six teachers with rich teaching experience,and the teaching content and teaching material selection were also the same.At the end of the course,the learning outcomes were evaluated by using descriptive analysis and t test (α=0.05) based on the combination of theoretical examination,experimental practice and the questionnaire survey method.Results Compared with the traditional teaching group,the final scores were higher than those of PBL group (84.72 ± 6.99 and 80.34 ± 7.12,P＜0.05).There were also statistically significant between two groups according to different nationality(Minority:85.65 ± 5.27 and 79.70 ± 7.14;Han:83.91 ± 8.26 and 80.95 ± 7.08;P＜0.05),and interestingly the increased ratio of scores was higher in minority than that in Han.The questionnaire surveys indicated that the PBL teaching method could enhance professional and comprehensive qualities of students and more than 81.83％ students were satisfied with the new teaching mode.Conclusions The combination of tradition and PBL-based teaching methods improved the quality of biochemistry teaching of clinical medicine in merging class of minority and Han students in Xinjiang Medical University.

Inrecentyears,somecardiovascularguidelineshavegradualyintroducedambulatory blood pressure monitoring ( ABPM ) as an indispensable means for better diagnosis and management of hypertensive patients. ABPM can more comprehensively provide 3 types of information that has potential clinical value:the assessment of real or mean blood pressure level, the diurnal rhythm of blood pressure, and the blood pressure variability. This article review s the roles of ABPM in the outcome assessment in patients w ith acute ischemic stroke.

OBJECTIVETo discuss the epidemiological and clinical characteristics of the hospitalized children with hand foot and mouth disease (HFMD) in Yantai area.

METHODSEpidemiological and clinical data of HFMD children from 2009 to 2010 were summarized and analyzed retrospectively.

RESULTSMost of the infected (94.6%) were under 5 years old and the ratio between male and female was 1.5: 1. Oral mucosal pox or ulcer as well as hand and foot rashes were observed in all 931 patients. Fever and neurological disorders occurred in 840 (90.2%) and 121 (13.0%) patients respectively. The incidence was positively correlated with air temperature (r = 0.887, P < 0.001), with a peak in April to September (88.9%). The ratio of children from countryside, total duration of fever, serum concentration of c-reacting protein (CRP) and fasting blood glucose (FBG) were significantly higher in severe cases than in those mild ones. Multivariate analysis showed longer mean duration of fever( Odds ratio [OR], 1.491; 95% confidence interval [ CI] 1.170-1.901; P = 0.001) and hyperglycemia (OR, 1.124; 95% CI 1.016-1.245; P = 0.024) were independent risk factors of severity.

CONCLUSIONChildren younger than 5 years old are susceptible to HFMD and most cases occur in April to September. The monthly incidence is positively correlated with temperature of that month. Longer duration of fever and hyperglycemia are independent risk factors for severity. Most cases could have a favorable prognosis after timely diagnosis and proper intervention.

Adolescent ; Child ; Child, Preschool ; China ; epidemiology ; Epidemiologic Studies ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; Humans ; Incidence ; Infant ; Male ; Retrospective Studies ; Seasons ; Temperature

Nanoparticles have better applicability in the detection, treatment of cancer and various difficult diseases, but mononuclear phagocytosis system can seriously shorten the time of nanoparticles in vivo circulation, reduce the drug efficacy. The protein crown formed on the surface of the nanoparticle after entering the body can change its surface properties, interfere with the recognition of phagocytes, and thus affect its circulation time in vivo. This article outlines the general composition and formation process of protein crowns. It also summarizes the influence of the physical and chemical properties of nanoparticles, such as particle size, surface charge, hydrophilicity and surface materials on the formation of protein crowns. The protein crown affects the circulation of nanoparticles in vivo, mainly because the adsorbed opsonic protein promotes cell phagocytosis. Therefore, we also introduce the method of using protein crowns to promote the long circulation of nanoparticles in vivo. By designing appropriate physical and chemical properties, surface modification, and directed design of protein crowns, the adsorption of proteins on the surface of nanoparticles can be reduced. Therefore, it can reduce the clearance of nanoparticles in the mononuclear phagocytic system (mainly the phagocytes of the liver and spleen), and achieve the goal of long circulation of nanoparticles in the body.

Objective:To determine the prevalence and risk factors for age-related macular degeneration (AMD) in Tibetan of plateau area, and to establish a nomogram prediction model.Methods:A cross-sectional study was carried out.The 40-year-old and older Tibetan subjects were enrolled by cluster random sampling from Xinghai County and Tongde County with an average altitude of 3 000 meters in Qinghai Province.Questionnaire survey, routine ophthalmic examinations, direct fundus photography and health education were carried out among the subjects.The questionnaire items included age, gender, smoking, drinking, living duration in plateau region, daily sunshine duration, diabetes, hypertension and hyperlipidemia.A χ2 test was used to screen significantly factors, and risk factors for AMD were analyzed by logistic regression analysis.The nomogram plot was drawn by R software to establish a predicting model.The study protocol adhered to the Declaration of Helsinki and was approved by an Ethics Committee of Qinghai Provincial People's Hospital (No.2017-21). Written informed consent was obtained from each subject prior to any medical examination. Results:A total of 1 223 subjects were enrolled, and AMD were detected in 190 subjects with an prevalence of 15.54%.The risk factors for AMD were old age, living duration in plateau region, sunshine duration, smoking, hypertension and hyperlipidemia.Age was the first one of risk factors for AMD ( OR: 20.183, 95% CI: 9.536-29.805, P<0.001), followed by sunshine duration ( OR: 3.785, 95% CI: 1.581-8.721, P<0.001). The nomogram prediction model showed that when the questionnaire score was >150, the probability of suffering AMD was 50%.The decision curve showed that when the threshold probability was >25%, the predicted probability of AMD was the same as the actual probability. Conclusions:Pathogesis of AMD in Tibet is associated with old age, living duration in plateau area, sunshine duration, smoking, hypertension and hyperlipidemia.

Objective To discuss the epidemiological and clinical characteristics of the hospitalized children with hand foot and mouth disease (HFMD) in Yantai area.Methods Epidemiological and clinical data of HFMD children from 2009 to 2010 were summarized and analyzed retrospectively.Results Most of the infected ( 94.6％ ) were under 5 years old and the ratio between male and female was 1.5∶1.Oral mucosal pox or ulcer as well as hand and foot rashes were observed in all 931 patients.Fever and neurological disorders occurred in 840( 90.2％ )and 121 (13.0％) patients respectively.The incidence was positively correlated with air temperature ( r =0.887,P ＜ 0.001 ),with a peak in April to September (88.9％).The ratio of children from countryside,total duration of fever,serum concentration of c-reacting protein (CRP) and fasting blood glucose(FBG) were significantly higher in severe cases than in those mild ones.Multivariate analysis showed longer mean duration of fever( Odds ratio[ OR],1.491 ;95％ confidence interval[ CI] 1.170-1.901 ;P =0.001 ) and hyperglycemia( OR,1.124; 95％ CI 1.016-1.245 ; P =0.024 )were independent risk factors of severity.Conclusion Children younger than 5 years old are susceptible to HFMD and most cases occur in April to September.The monthly incidence is positively correlated with temperature of that month.Longer duration of fever and hyperglycemia are independent risk factors for severity.Most cases could have a favorable prognosis after timely diagnosis and proper intervention.

BACKGROUNDMycoplasma pneumoniae (M. pneumoniae) is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction (PCR) with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally.

METHODSIn this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results.

RESULTSAmong 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment.

CONCLUSIONSThe drug-resistant M. pneumoniae is very common in Beijing, China. Our modified allele-specific PCR analysis can identify erythromycin resistant mutations more rapidly from specimens than any other method currently available. Erythromycin is still effective for treating patients infected with the mutation negative M. pneumoniae, but this treatment fails to work on mutant organisms. This method can facilitate clinicians in selecting appropriate therapy within short timescales.

Alleles ; Anti-Bacterial Agents ; pharmacology ; China ; Drug Resistance, Bacterial ; genetics ; Erythromycin ; pharmacology ; Mycoplasma pneumoniae ; drug effects ; genetics ; Polymerase Chain Reaction ; methods

Tubulin is the major protein of microtubule. alpha- and beta- tubulins form heterodimers, while gamma-tubulin regulates microtubule organization. The present study aimed to observe the dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos. Immunofluorescence and laser confocal microscopy were used to detect the location of gamma-tubulin in preimplantation parthenogenetic embryos activated by SrCl2. The oocytes were collected at 13-14 h after hCG injection, and then activated with 10 mmol/L SrCl2 in Ca(2+)-free CZB medium with 5 mmol/L cytochalasin B (CB), fixed at 1 h intervals until 6 h after activation. The results showed that spindle was paralleled with the cell membrane all the time, when the meiosis of MII mouse oocytes resumed. The rotation of spindle was inhibited, but karyokinesis was not influenced. At 0 h after activation, i.e. at metaphase, gamma-tubulin was distributed mainly on the two poles of spindle. At 1 h after activation, i.e. at anaphase, following the separation of chromosomes, gamma-tubulin was transformed from dense to disperse. At 2 h after activation, gamma-tubulin was localized between the segregated sister chromatids at telophase. However, at 3-6 h after activation, gamma-tubulin concentrated around the two female pronuclei during their formation and juxtaposition. Moreover, another group of MII oocytes were activated for 6 h and cultured in droplets of KSOM medium under mineral oil in 5% CO2 in air at 37 degrees C to permit parthenogenetic development. The embryos were collected and fixed at 3 h, 14 h, 16 h, and 18 h of culture. At 3 h after culture, i.e. at mitotic interphase, it was shown that amorphous gamma-tubulin distributed around the nuclei of early parthenogenetic embryos. At 24 h after culture, i.e. at prometaphase, gamma-tubulin migrated along the spindle microtubule to the two poles. Our results showed that gamma-tubulin had similar location patterns at metaphase, anaphase and telophase in meiosis and mitosis. It was concluded that gamma-tubulin assembly in parthenogenetically activated oocytes facilitated the formation of negative pole cap and the stabilization of microtubule, thus promoting the spindle formation at meiosis and mitosis. The relocation of gamma-tubulin at anaphase and telophase might be induced by the event of segregation of homologous chromosome being pulled away by the spindle. gamma-tubulin might contribute to the migration and juxtaposition of the two female pronuclei as well.
Animals ; Embryo, Mammalian ; Embryonic Development ; Female ; Meiosis ; Mice ; Mitosis ; Oocytes ; cytology ; Parthenogenesis ; Spindle Apparatus ; physiology ; Tubulin ; physiology