Central nervous system (CNS) damage caused by enterovirus 71 (EV71) infection is the main reason for its mortality and morbidity.In recent years,survival rate of EV71 infection-associated CNS damage in children is improved.However,the condition of survivors with sequelae is still unclear.It is important to understand its prognosis in order to improve the life quality of children with CNS damage after EV71 infection.In this paper,the prognosis and sequelae classifications of EV71 infection-associated CNS damage are described.

Interaction in classroom teaching is a bilateral activity that is difficult to control. Teachers and students are the two main aspects to realize the synchronization of teachers and students' thinking. For teachers, teachers should first make full use of multimedia resources, bring a colorful courseware into class-room teaching; secondly, teachers should stimulate students' interest in a reasonable way, so as to inject vitality into the classroom teaching; finally, teachers should mobilize the students' thinking and carefully regulate the changes in thinking between teachers and students through a proper teaching methods. In terms of students, students should not only give full play to their initiative, but also actively cooperate with teachers in order to realize the thought synchronization between teachers and students in classroom teaching and learning interaction and ultimately achieve the desired results.

BACKGROUND:Ghrelin is a newly discovered brain-gut peptide from the stomach of human and rats. As an endogenous ligand for growth hormone secretagogue receptor (GHSR), ghrelin can notably stimulate the release of growth hormone. Although GHSR is expressed in many peripheral tissues, little is known about the influence of ghrelin on bone metabolism and GHSR expression in bone tissue. OBJECTIVE:To review the research progress of ghrelin in bone metabolism.METHODS:The first author retrieved CNKI, WanFang, PubMed, and Springerlink databases with the keywords ofGhrelin, bone metabolismin Chinese and English, respectively. The studies regarding ghrelin and its involvement in bone metabolism were included, and repetitive ones were excluded. A total of 53 eligible literatures were selected through skimming abstracts. RESULTS AND CONCLUSION:Ghrelin is a peptide composed of 28 amino acids discovered in gastric endocrine cells and hypothalamic arcuate nucleus in mice and human, which makes a great effect on digestive, nervous, immune and endocrine systems, and also plays a role in hormone secretion, glucose metabolism, immunity, cell proliferation, and inflammation. Serum ghrelin makes a certain influence on bone growth and development, and promotes the differentiation and proliferation of osteoblasts, and inhibits its apoptosis. Additionally, ghrelin suppresses the early osteogenic differentiation of C3H10T1/2 cells by upregulating the expression of Runx2 protein, and attenuates adipogenic differentiation by downregulating PPARγ2 expression, thus inducing osteogenic differentiation. However, few studies have addressed the expression of GHSR in bone tissue.

Objective To analyze the whole genomic DNA methylation level and estrogen receptor α (ERα) gene promoter methylation status in systemic lupus erythematosus (SLE) with atherosclerosis (AS) in model mouse,and to explore its role in the pathogenesis of SLE with AS.Methods Eleven apoE-/-C57BL/6 mice were randomly divided into the model group (SLE+AS group) and the control group (AS group).Eleven wild C57BL/6 mice were also randomly divided into the model group (SLE group) and the control group (blank group).Single intraperitoneal injection of pristane 0.5 ml for the model group,single intraperitoneal injection of normal saline 0.5 ml for the control group.Eight months after injection,all mice were sacrificed,genomic DNA was extracted from spleen.The total genomic DNA methylation level was detected,and pyrosequencing was performed to determine the methylation status of ERα gene promoter.The differences between groups were compared.ANOVA,LSD-t test,Tamhane's T2 test were used for statistical analysis.Results The total genomic DNA methylation levels were (4.7±1.5)％,(5.1±0.5)％,(6.6±1.6)％,(7.5±1.6)％ respectively in the SLE+AS group,AS group,SLE group,blank group respectively,the average methylation indices of ERα gene promoter were (13.0±3.1)％,(26.7±7.2)％,(15.7±3.8)％ and (21.4±4.2)％ respectively.The total genomic DNA methylation level and the average methylation index of ERα gene promoter in the SLE+AS group and the SLE group was significantly lower than that of the blank group (P＜0.05).Compared with the AS group,the total genomic DNA methylation levels and the average methylation index of ERα gene promoter in the SLE+AS group were significantly decreased (P＜0.05).Conclusion The total genomic DNAs and the ERα gene promoters in SLE with AS model mouse are in low-methylation status.The results of this study suggest that epigenetics may play an important role in the pathogenesis of SLE with AS.

OBJECTIVE:To establish a method for the determination of 9 residual organic solvents in blonanserin as methanol, alcohol,isopropyl alcohol,acetonitrile,dichloromethane,hexane,ethyl acetate,tetrahydrofuran and methylbenzene. METHODS:Headspace gas chromatography was adopted. The determination was performed on DB-624 capillary column using temperature pro-gramming. The inlet temperature was 150 ℃,and flame ionization detector was used with temperature of 250 ℃. High purity nitro-gen was used as carrier gas with flow rate of 2.8 mL/min. The split ratio was 1:1,and headspace sample size was 1 mL. Head-space heating temperature was 90 ℃,and equilibration time was 35 min. RESULTS:The linear ranges of methanol,alcohol,iso-propyl alcohol,acetonitrile,dichloromethane,hexane,ethyl acetate,tetrahydrofuran and methylbenzene were 6-1500 μg/mL(r=0.9998),10-2500 μg/mL(r=0.9999),10-2500 μg/mL(r=0.9998),0.82-205 μg/mL(r=0.9994),1.2-300 μg/mL(r=0.9995), 0.58-145 μg/mL(r=0.9994),10-2500 μg/mL(r=0.9999),1.44-360 μg/mL(r=0.9996),1.78-445 μg/mL(r=0.9995),respec-tively. The limits of quantitation were 17.71,6.02,3.17,7.45,1.53,0.69,0.93,1.01,0.22 μg/mL;the limits of detection were 5.89,1.90,1.05,2.48,0.51,0.23,0.31,0.33,0.07 μg/mL,respectively. RSDs of precision and stability tests were all lower than 3.0%,and isopropanol was found in repeatability test (RSD=2.1%). The average recoveries ranged 96.67%-102.66%(RSD=1.9%,n=9),96.00%-101.83%(RSD=1.9%,n=9),97.17%-101.50%(RSD=1.4%,n=9),96.97%-102.44%(RSD=2.2%,n=9),95.83%-103.33%(RSD=2.5%,n=9),95.83%-100.28%(RSD=1.9%,n=9),98.17%-101.25%(RSD=1.0%,n=9),96.55%-102.30%(RSD=1.9%,n=9),96.30%-102.22%(RSD=1.8%,n=9),respectively. CONCLUSIONS:The method is simple,rapid,accurate and suitable for simultaneous determination of 9 residual organic solvents in blonanserin as methanol,alco-hol,isopropyl alcohol,acetonitrile,dichloromethane,hexane,ethyl acetate,tetrahydrofuran and methylbenzene.

Objective To investigate and summarize the clinical and high-resolution computed tomography(HRCT) characteristics of invasive pulmonary fungal infections(IPFIs)in children.Methods Clinical and HRCT data of 35 cases with IPFIs admitted in our hospital between March 2007 and July 2015 were retrospectively analyzed.The clinical and HRCT characteristics were summarized.Results Thirty-five patients consisted of 23 boys and 12 girls with mean age of(3.2±1.9) years.Host factors included acute leukemia (n=12),primary immunodeficiency disease (n=4),congenital heart disease (n=2),cerebral palsy (n=2),severe influenza A infection (H1N1) (n=2),ichthyosis (n=1),acquired immunodeficiency syndrome(n=1),systemic lupus erythematous (n=1),tubercular meningitis(n=1),mechanical ventilation(n=2).All patients were treated with broad-spectrum antibiotic,ranking by descending order:third-generation cephalosporins (28 cases),carbapenems(19 cases)and vancomycin (18 cases).Seventeen cases were treated with corticosteroids systemically and 12 cases with acute leukemia took antineoplastic medicine.The symptoms of IPFIs were intermittent or persistent fever,cough and rales.HRCT results:nodules (n=25,71.4％),subpleural patchy opacities (n=24,68.6％),mass (＞3cm) (n=4,11.4％),halo sign (n=27,77.1％),cavities (n=8,22.9％),air crescent sign (n=4,11.4％),miliary nodules (n=2,5.7％),pleural effusion (n=14,40％).Conclusion There are certain specific characteristics of IPFIs in children in clinical and HRCT aspects.The possible diagnosis of IPFIs can be made based on clinical and HRCT features.

Objective To establish a high-performance induction culture system for Raw264.7 cells to differentiate into osteoclasts(OC)invitro by improving the cell culture program.Methods The Raw264.7 cells were cultured withα-MEM medium containing 50 μg · L-1 M-CSF, 100 μg · L-1 RANKL, and 1 × 10-8 mol · L-1 1α,25-(OH)2 D3 in 5% CO2 for 12 d at 37℃. The cells were digested transiently every time before the medium was changed after every three days. The morphologic changes of the Raw264.7 cells were observed by inverted microscope.The maturation and phagotrophic function of OC were identified by HE,TRAP,FITC-phalloidin staining and immunofluorescence.Results The cells remained to grow in single layers all the time in most areas of the well during the whole induction by the improved culture program. The observation results of inverted microscope and HE staining showed that the growth area of the polykaryotic OC reached to 70% of the well on day 1 2. FITC-phalloidin staining showed that in the maturation of the OC, the cluster-shaped podosomes in the pseudopodia gradually transformed into rings,which finally fused to form a large belt surrounding the periphery of the cytoplasm. The calcitionin receptor (CTR) expressed by OC was markedly enhanced compared with the precursor cells by immunofluroescence staining,and a large number of red granules appeared in the cytoplasm of OC with TRAP staining on day 1 2. These results comfirmed that the obtained OC were maturated and owned phagotrophic function. Conclusion A high-performance induction culture system for Raw264. 7 cells to differentiate into OC in vitro induced by combination of 50μg · L-1 M-CSF, 100 μg · L-1 RANKL,and 1 × 10-8 mol·L-1 1α,25-(OH)2 D3 is established by improving the cell culture program.

Objective To observe the influence of Huiyang Shengji Ointment and its modified formulae on interleukin-1 (IL-1α) and thromboxane B2 (TXB2) in diabetic rats with chronic skin ulcers, and explore the mechanism for promoting the healing of ulcer.Methods Six out of 160 rats were randomly selected as a blank group, without any further processing. The remaining rats were made diabetic model and randomly divided into five groups after 2 weeks:1 d, 3 d, 5 d, 7 d and 14 d groups. Then, these groups were further divided into normal group (Vaseline ointments), model group (Vaseline ointments), Huiyang Shengji Ointment group (whole formula Ointment), Wenyang Yiqi group (Yiqi group, modified Wenyang Yiqi formula ointments) and Huoxue Shengji group (Huoxue group, modified Huoxue Shengji formula ointments). Normal group and model group were given Vaseline ointments;whole formula group, Yiqi group and Huoxue group were given corresponding ointment. Normal group used the method of skin excision, while other groups used STZ injection-hydrocortisone interference-skin excision-foreign body embedded preparation of composite factors for chronic skin ulcer model. After the appropriate treatment period, the rats were executed and tested for the contents of IL-1α and TXB2 in serum by enzyme-linked immunosorbent assay of five time points.Results In treatment 3 d, the contents of IL-1α in Yiqi group were significantly higher than the blank group, model group, whole formula group and Huoxue group (P<0.05). In treatment 5 d, the contents of IL-1α in whole formula group were significantly higher than the blank group and model group (P<0.05). In treatment 7 d, the contents of IL-1α in each treatment group were significantly higher than blank group and model group (P<0.05), and the whole formula group was higher than the Yiqi group and Huoxue group. In treatment 14 d, the contents of IL-1α in model group and Huoxue group were lower than the blank group (P<0.05). In treatment 3 d, the contents of TXB2 in normal group and the whole formula group were higher than the blank group (P<0.05). In treatment 5 d, the contents of TXB2 in whole formula group were higher than the blank group and the normal group (P<0.05). In treatment 7 d, the contents of TXB2 in Yiqi group were higher than the blank, the model, the whole formula and Huoxue groups (P<0.05). In treatment 14 d, the contents of TXB2 in Huoxue group were higher than the blank and model group (P<0.05), and the contents of TXB2 in the blank group and normal group was lower than those treatment groups (P<0.05).Conclusion Huiyang Shengji Ointment and its modified formulae could promote inflammation, stimulate secretion of inflammatory cytokines, while Yiqi Wenyang ointments played a more active role in promoting inflammation of the early phase of wound surface.

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2.METHODS:RT-PCR and Western blot was used to determine the expres-sion of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29.HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay.ELISPOT as-say was used to investigate the levels of IFN-γ.The cytotoxicity assay in vitro was also used to determine the ability of indu-cing T cell response by the peptides.RESULTS:The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule.ELISPOT assay showed P485 and P965 induced CTLs of IFN-γrelease form CTLs.The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION:The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.

Background The excessive growth of human Tenon fibroblasts (HTFs) is a primary cause of failure of anti-glaucomatous filtering surgery.To seek a drug of anti-fibrosis is of an important significance for improving the successful rate of anti-glaucomatous filtration surgery.Objective The goal of this study was to investigate the effect of paclitaxel on proliferation of HTFs in vitro.Methods Human Tenon tissue was obtained during the anti-glaucomatous filtering surgery.HTFs were cultivated using explant method and 3-6 generations of cells were used in the experiment.The cells were identified by immunochemistry using keratin,vimentin,fibronectin and S-100.Different concentrations (0,1 ×10-s,1 × 10-7,1 × 10-6 mol/L) of paclitaxel were added into the medium,and then the cell indexes (CI) in the various groups were detected by real-time cell electronic sensing (RT-CES) 24 hours after affection of paclitaxel.Apoptosis of the cells was examined using DAPI staining,and the proportion of the cells in different cycles were assayed by flow cytorneter 12 hours after addition of paclitaxel.The expressions of matrix metalloproteinase-1 (MMP-1) protein and mRNA were detected by ELISA and real-time PCR,respectively.Results The cells migrated from the cultivated tissue within 7 days with the fibrocyte-like shape.The cells showed the positive response for vimentin and absent response for keratin,fibronectin and S-100.The CI values were 1.093 ±0.191,0.665 ± 0.093 and 0.473 ± 0.117 in the 1 × 10-8,1 × 10-7 and 1 × 10-6 mol/L paclitaxel groups,showing significant rise in comparison with the 1.514 ±0.283 of the 0 mol/L paclitaxel group (all at P =0.000).The cell nuclei were normal in the 0 mol/L paclitaxel group,however,blue-fluorescent particles and apoptotic bodied were found in the cell nuclei after affection of paclitaxel.The proportion of G2/M phase of cells were (9.20±0.80) ％,(12.37±0.45)％ and (13.80±0.35)％ in the 1×10-8 mol/L,1×10-7 mol/L and 1×10-6 mol/L paclitaxel groups,which were higher than the (7.17±0.50) ％ in the 0 mol/L paclitaxel group (P=0.005,0.000,0.000).In addition,the relative expressing level of M MP-1 mRNA (MMP-1 mRNA/GAPDH mRNA) and the expression level of MMP-1 protein in the HTFs were significantly lower in the 1 ×10-8 mol/L,1 × 10-7 mol/L and 1 × 10-6 mol/L paclitaxel groups than those in the 0 mol/L group (all at P＜0.05).Conclusions Paclitaxel at the concentrations of 1 × 10-8 mol/L-1 × 10-6 mol/L inhibits the proliferation of HTFs in vitro by arresting the cellular mitosis and inducing cell apoptosis.These effects probably associated with down-regulation of MMP-1 expression in the HTFs.