Objective To provide the experimental basis for the further research of the interacting proteins with Stathmin ,the Stathmin gene Pichia pastoris expression system was constructed ,the expressed Stathmin product was purified and identified .Meth‐ods Stathmin gene was amplified from tumor cell line of SKBR3 by PCR method and cloned into the yeast expression vector pPIC3 .5K .The recombinant vector pPIC3 .5K‐Stathmin was constructed and transformed into Pichia pastoris GS115 .The positive clones were screened by YPD medium containing Geneticin 600 μg/mL .Expression was induced with 0 .5% methanol and expres‐sion products were identified by SDS‐PAGE and Western Blotting .Results DNA sequencing result showed that the gene fragment was consistent with Stathmin gene sequence .pPIC3 .5K‐Stathmin was selected from YPD culture medium containing Geneticin ,and the positive clones were identified by PCR .SDS‐PAGE showed that a 37 × 103 protein band could be seen on the PAGE gel after Coomassie Blue staining ,which was further confirmed and identified as Stathmin protein by Western Blotting .Conclusion Stathmin yeast expression vector is successfully constructed and expressed in Pichia pastoris ,which laid the foundation for the study of inter‐acting proteins with Stathmin ,and for the preparation of the biological treatment drugs of Stahtmin target .

Background Uveal effusion syndrome is uncommon in clinic.To understand the clinical characteristics of uveal effusion syndrome is helpful for rescuing visual acuity of patient.Objective This study was to discuss the diagnosis,classification and surgical outcome of uveal effusion syndrome.Methods This was a descriptive study.The clinical data of 14 eys from 10 patients with uveal effusion syndrome,ineluding ophthalmologic examination,B-scan sonography,ultrasound biomicroscopy (UBM),fundus fluorescence angiography (FFA),indocyanine green angiography (ICGA),surgical treatment and prognosis,were retrospectively analyzed.The follow-up period was 6 months.Results The fundus findings of all impacted eyes showed bullous-shape retinal detachment (RD).B-scan sonography revealed retinal and choroidal detachment.A annular peripheral ciliochoroidal detachment was observed in the cases under the UBM.FFA exhibited leopard spots without any leakage from choroid into the subretinal space.ICGA demonstrated diffusely choroidal granular hyperfluorescence in the very early phase,which presented with an increasing intensity as time lapse until the late phase.Full-thickness sclerectomy was performed on 4 eyes of 2 patients and subscleral sclerectomy was performed in 1 eye of 1 patient,achieving a retinal anatomic reattachment after surgery.All of the patients finished the fellow-up.No recurrence of RD was seen during the followup duration.Conclusions Comprehensive preoperative evaluation,including ophthalmologic ultrasonography,MRI and CT,is crucial for accurate classification of uveal effusion syndrome and determine of proper management strategy.

Objective To explore the relationship between the Stathmin gene expression in breast cancer cells MDA-MB-231, MCF-7 and the biological behaviours such as cell growth,adhesion and invasion,and provide experimental basis of breast cancer metastasis for further study.Methods Used RT-PCR and Western Blot methods to detect the Stathmin gene expres-sion levels in MDA-MB-231 and MCF-7 cells,and in the mean while to test the MDA-MB-231 and MCF-7 cell growth,adhe-sion,invasion ability by CCK-8 cell proliferation experiments,cell adhesion experiments,cell invasion experiments,then, analyed the relationship of Stathmin gene expression and cell growth,adhesion,invasion ability.Results Over-expression levels of Stathmin gene were observed both in the MDA-MB-231 and MCF-7 cells (F=10.173,P<0.05),and furthermore, the expression levels of Stathmin gene in MDA-MB-231 cells was higher than in MCF-7 cells (t=4.562,P<0.05).While, the growth,adhesion and invasion ability of the MDA-MB-231 cells was higher than that of MCF-7 cells(P<0.05).Conclu-sion The higher level of Stathmin gene expression,the stronger breast cancer cells had ability of growth,invasion,and ad-hesive.The Stathmin gene expression levels was closely correlated with breast cancer cell invasive.

?AIM:To evaluate the objective visual quality of patients who underwent corneal cross-linking for the keratoconus using double-pass analysis system.? METHODS: Advanced keratoconus patients who underwent UV - riboflavin corneal cross - linking from January to July 2015 were included. The outcomes of their objective scattering index ( OSI ) , predicted visual acuity ( VA ) , the cut - off frequency of modulation transfer function ( MTF cut- off ) , the Strehl ratio ( SR ) were compared before and 6mo after corneal cross-linking.?RESULTS: A total of 13 patients ( 16 eyes ) were included. There was no statistically significant difference between pre- and 6mo postoperative data in uncorrected visual acuity, best corrected visual acuity, refractions and mean value of Sim-k (P>0. 05). Non-invasive average tear film break up time ( NIAvg-BUT ) detected by the Sirius system decreased after corneal cross-linking ( P<0. 05 ) . Using double - pass analysis system, no statistically significant change was found in MTF cut off, Strehl Ratio, OSI before and after treatment(P>0. 05). Tear Film Analysis Mean OSI increased at 6mo postoperatively (P<0. 05).? CONCLUSION: The subjective visual quality isn’t effected by corneal cross-linking. The tear stabilities of patients are influenced by these operations at 6mo postoperatively. More observations on long-term effect are needed to be taken in the future.

Objective To retrospectivel y analyze the abdominal CT findings and pathological results of the chronic schist osomiasis so as to improve the diagnostic accuracy of the disease. M ethods The plain abdominal CT scanning was performed in 103 cases an d enhanced CT scanning in 81 cases. The pathological specimen which was consist ent with the section of CT scan was obtained in each cases. Results On CT scanning, liver cirrhosis was seen in 84 cases, various calci fication in liver in 71 cases, liver cancer in 12 cases, enlargement of sple en in 78 cases, calcification in spleen in 13 cases, wall-thickening in colon i n 27 cases, calcification in colon in 31 cases, and colon cancer in 9 cases. Pa thological examination revealed various fibrosis and formation of pseudolobule. The eggs and calcification could be seen in pseudolobule and septa, colonic sub mucosa, and regional lymph nodes. Fibrous hyperplasia in colonic wall and hyper plasia in mucous membrane were obvious. Fibrous hyperplasia and calcification w ere seen in spleen, but the eggs were not found. Conclusion The liver and colon are the major organs affected by chronic schistosomias is in abdomen, and the CT findings are obvious too. The pathological features o f spleen are accompanied with liver cirrhosis. CT is the important imaging meth od in diagnosing chronic schistosomiasis and pathological changes.

OBJECTIVETo study the effect of NADPH oxidase on hypoxia-inducible factor (HIF)-1alpha and endothelin (ET)-1 expression in human umbilical endothelia cells (HUVECs) and its possible mechanism.

METHODSTwenty-five bottles of HUVECs culture fluid were randomly assigned into five groups: group A (normoxic control), group B (hypoxic), group C (NADPH oxidase inhibitor apocynin + normoxic), group D (H2O2 which can degrade HIF-1alpha rapidly+hypoxic) and group E (H2O2+apocynin+normoxic), with five bottles in each group. The culture supernates were collected and the total protein was extracted 3 hrs after treatment. Western Blot and ELISA were used to detect the HIF-1alpha protein expression in HUVECs and the ET-1 level in the culture supernates respectively.

RESULTSThere was a lower expression of HIF-1alpha protein (0.336 +/- 0.012) and lower ET-1 levels (5.87 +/- 2.22 pg/mL) in group A. The HIF-1alpha protein expression in groups B and C (0.773 +/- 0.018 and 0.888 +/- 0.022) and ET-1 levels (95.38 +/- 8.06 and 33.67 +/- 4.21 pg/mL) were noticeably higher than in group A (P < 0.05). The groups D and E had the HIF-1alpha protein expression levels similar to group A, but the ET-1 levels in group D (108.43 +/- 8.38 pg/mL) and group E (109.66 +/- 5.80 pg/mL) were significantly higher than in group A (P < 0.05).

CONCLUSIONSHypoxia or apocynin can increase the HIF-1alpha and ET-1 expression in HUVECs. H2O2 can inhibit the HIF-1alpha expression but increase the ET-1levels. It is speculated that NADPH oxidase as an oxygen sensor regulates the HIF-1alpha expression by changing the intracellular redox reaction and that except HIF-1, H2O2 might contribute to ET-1 synthesis and release.

Blotting, Western ; Cell Hypoxia ; Cells, Cultured ; Endothelial Cells ; metabolism ; Endothelin-1 ; analysis ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; metabolism ; NADPH Oxidases ; physiology

Objective:To construct the recombinant prokaryotic plasmid to express HCV HLA-A2 restricted multi-CTL epitopes and to purify the fused protein for antigenic analysis.Methods:The human ubiquitin gene and multi-CTL epitopes gene was synthesized respectively,and digested by restrict enzyme before being cloned into pRSET-A.Then it was transformed into E.coli DH5α and the positive recombinant plasmid named pRSET-Ub-Mep was sequenced.Target protein was distinctly expressed after transformed into E.coli BL21 and induced with IPTG.Thus the protein was scanned and purified on Ni~(2+)-NTA column as well as Western blot performed after solubility analysis.Results:The recombinant plasmid pRSET-Ub-Mep was successfully constructed and it could efficiently express the target gene.Protein production was mainly in inclusion body and could be purified through Ni~(2+)-NTA column.The purified protein kept the antigen activity.Conclusion:The gene encoding for HCV HLA-A2-restricted multi-CTL epitopes is efficiently expressed and the target protein is purified,which establishes a foundation of further research to evaluate the cellular immune response induced by the target gene.

Objective: To understand sleep behavior among primary and middle school students and its impact on overweight and obesity changes, to provide evidence for developing obesity prevention and controlling strategies in children and adolescents. Methods: Primary and middle school students from three cities in Zhejiang Province who participated in questionnaire surveys and physical measurements in both 2017 and 2019 were selected. A follow up dataset of 605 students was developed and the relationship between sleep duration and body mass index was analyzed. Results: From 2017 to 2019, BMI Z scores for male and female participants increased by 0.24 and 0.13, respectively. BMI Z scores increased by 0.29 in students of 9-12 years old and increased by 0.11 and 0.25 in urban and rural students, respectively ( P <0.05). The prevalence of insufficient sleep duration increased from 37.0 % to 41.8% simultaneously ( χ 2=3.68, P =0.06). After adjusting for confounding factors, the BMI Z score of students with insufficient sleep was 0.20 higher than those with sufficient sleep duration ( P <0.01). Compared with participants who had sufficient sleep duration from 2017 to 2019, participants whose sleep duration changed from sufficient to insufficient, and those who always had insufficient sleep duration increased by 0.23, respectively ( P <0.05). Conclusion Insufficient sleep duration is a risk factor for obesity. Shortened sleep duration is related to weight gain, and maintaining sufficient sleep duration may reduce the risk of obesity in children and adolescents.

OBJECTIVETo investigate the effect of Evn-50 extracted from Vitex negundo on human breast cancer cell line MCF-7 and MCF-7/TAM-R cells in vitro.

METHODSMCF-7 and tamoxifen-resistant MCF-7/TAM-R cells were treated with Evn-50,tamoxifen or combination of Evn-50 and tamoxifen. Cell proliferation inhibition rates were determined by MTT assay. The apoptosis rate and the change of cell cycle were detected by PI staining flow cytometry. Protein expression of phospho-MAPK 44/42 (Thr202/Tyr204),MAPK P44/42, phospho-AKT (Ser473) and AKT were detected with Western blotting.

RESULTSThe viability of MCF-7 cells was decreased in combination group [(28.65 ±11.43)%] and Evn-50 group [(53.02 ±15.14)%] compared with TAM group (P<0.01). The cell viability of MCF-7/TAM-R in combination group [(42.11 ±14.30)%] was significantly lower than that in TAM group [(92.18 ±13.16)%] (P<0.01). The cell apoptosis rate was dependent on the time of treatment in all groups,the effects on apoptosis and G2/M phase cells were most prominent at 72 h (P<0.01). Western blotting revealed that protein levels of phosphorylated AKT and p-MAPK44/42 decreased,while the expression of total AKT and MAPK44/42 was stable. In MCF-7/TAM-R cells,the expression of phosphorylation of AKT and MAPK44/42 protein was not changed in Evn-50 or TAM alone group,but significantly inhibited in the combination group at 72 h.

CONCLUSIONEvn-50 can inhibit cell growth and induce apoptosis in MCF-7 and MCF-7/TAM-R cells,it can reverse tamoxifen-resistance of MCF-7/TAM-R cells.The mechanisms may be related to the down-regulation of phosphorylated ERK1/2 in MAPK signal pathway and phosphorylated AKT in AKT signal pathway.

Apoptosis ; drug effects ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; MCF-7 Cells ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects ; Tamoxifen ; therapeutic use ; Vitex ; chemistry

OBJECTIVETo develop a bacteria expression system to produce the fusion protein of humanized anti-HBsAg scFV and interferon-alpha.

METHODSThe expression vector was constructed after cleaving the plasmids harboring the humanized anti-HBsAg scFv and interferon alpha respectively and ligating to linearized pET22b subsequence. The expression of fusion protein in E.coli was analyzed by SDS-PAGE. The binding activity and antiviral activity of the fusion protein was characterized by competing inhibition test and cytopathic effect reduction.

RESULTSThe plasmid harboring the in frame arranged fusion gene was constructed and identified. After induction for 12h, a new band close to 4.5 10(4) was observed using SDS-PAGE. Results of competing ELISA and cytopathic effect reduction showed the fusion protein retained its specific binding activity and antiviral activities.

CONCLUSIONSThe construction and expression of the fusion gene of humanized anti-HBsAg scFv and interferon in E.coli are successful.

Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Hepatitis B Antibodies ; biosynthesis ; genetics ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B virus ; drug effects ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Interferon-alpha ; biosynthesis ; genetics ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology