Recombinant Fl-V (rFl-V) fusion protein is the main ingredient of the current candidate vaccine against Yersinia pestis infection, which has been under investigation in clinical trial in USA. We investigated the soluble expression conditions of rF1-V in Escherichia coli BL21 (DE3) that we constructed before. After scale-up and optimization of fermentation processes, we got the optimized fermentation process parameters: the culture was induced at the middle exponential phase with 50 µmol/L of IPTG at 25 °C for 5 h. Soluble rFl-V protein was isolated to 99% purity by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and gel filter chromatography. The protein recovery was above 20%. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing. Results of purity, quality and western blotting analysis indicated that the target protein is a consistent and properly folded product. Furthermore, the immunogenicity of various antigens formulated with aluminum hydroxide adjuvant was evaluated in mice. Serum antibody titers of 4 groups including 20 µg rFl, rV and rFl-V and 10 µg rFl+10 µg rV, were assayed by ELISA after 2 doses. The antibody titers of anti-Fl with 20 µg rFl-V were obviously higher than titers with other groups; meanwhile there were no significant difference of anti-V antibody titers among them. These findings confirm that rFl-V would be the active pharmaceutical ingredient of the plague subunit vaccine.
Adjuvants, Immunologic ; Animals ; Antibodies, Bacterial ; blood ; Antibody Formation ; Antigens, Bacterial ; immunology ; Blotting, Western ; Chromatography, Ion Exchange ; Enzyme-Linked Immunosorbent Assay ; Mice ; Plague ; prevention & control ; Plague Vaccine ; immunology ; Recombinant Fusion Proteins ; immunology ; Vaccines, Subunit ; immunology ; Yersinia pestis

OBJECTIVEHigh altitue pulmonary edema (HAPE) impacts seriously people's health at high altitude. Screening of susceptibility genes for HAPE will be used for the evaluation and protection of susceptible people.

METHODSWe performed a genome-wide association study (GWAS) using Affymetrix SNP array 6.0 in 23 HAPE patients and 17 healthy controls. GO and Pathway analysis softwares were used to analyze and draw gene network.

RESULTSThirty-nine SNPs were found to be significantly different between case and control groups (P < 10(-4)). GO and Pathway analysis of 27 genes around the 39 SNPs indicated that these genes mainly participate in the regulating of cell proliferation, regulation of nitrogen compound metabolic process and G-protein coupled receptor protein signaling pathway and so on.

CONCLUSIONIt suggests that these SNPs and genes found in this study may be associated with the susceptibility of HAPE.

Adult ; Altitude Sickness ; genetics ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Humans ; Hypertension, Pulmonary ; genetics ; Polymorphism, Single Nucleotide ; Young Adult

Objective To investigate the levels of literacy and numeracy in type 2 diabetes patients with poor glycemic status in communities of Shanghai,China,and to evaluate their associations with blood glucose level.Methods A total of 800 type 2 diabetes patients with recent HbA1c≥7.5％ or fasting plasma glucose level ≥10 mmol/L were recruited from 8 communities in Minhang district and Changning district of Shanghai,China,and were interviewed using a structured questionnaire during February 2015 and March 2016.Literacy and numeracy levels of all patients were evaluated using the validated Health Literacy Management Scale (HeLMS) and the 5-item version Diabetes Numeracy Test (DNT-5),respectively.Results The patients included in this study were observed to have higher levels of health literacy,with a median score of HeLMS being 116 [interquartile range (IQR),108-120] and a median correct rate of DNT-5 of 80％ (IQR,60％-100％).Age,educational level and occupation were significantly related with health literacy levels and numeracy.Sex and income were closely related with health literacy levels.HeLMS score was not significantly associated with HbA1c level (P =0.383),while the lower correct rate of DNT-5 was linked with a higher level of HbA1c.The median HbA1c level was 8.3％ (IQR 7.7％-9.4％) in the patients with the lowest tertile of DNT-5 correct rate,significantly higher than 8.2％ (IQR:7.5％-9.2％) in the medium and 8.0％ (IQR:7.5 ％-8.8 ％) in the highest tertile group (P =0.009).Conclusions Diabetes patients with poor glycemic status in communities of Shanghai have high levels of health literacy,which was significantly related with age,sex,educational level,occupation and income.Ability in numeracy may be a more important influence factor than health literacy for glycemic status of diabetes patients.

Adult ; Child ; Eosinophilia ; complications ; Female ; Hematologic Neoplasms ; complications ; Humans ; Male ; Middle Aged

Objective To investigate the mechanism of Toll-like receptor in intestinal mucosal injury induced by Cryptospo-ridium parvum infection in mice.Methods Totally 30 male BALB/c mice were randomly divided into a normal control group,1-week infection group and 2-week infection group.The mice of the 1-week and 2-week infection groups were sacrificed 7 days and 14 days after the infection respectively,and the mice of the normal control group were sacrificed 14 days after the infection.The model of intestinal infection of C.parvum in mice was built by using the immunosuppressive method and oocyst intragastric ad-ministration.The pathological changes of the intestinal mucosa of mice were observed with a light microscope and the villus height,crypt depth and ratio of villus height/crypt depth were measured.The ultrastructure of the intestinal mucosa of mice was observed by a transmission electron microscope(TEM).The expressions of TLR2 and TLR4 in the intestinal mucosa were tested by qPCR and Western blotting.Results Under the light microscope,the intestinal villi were dropsical,obviously atrophied and shortened,and the submucosal structure was dropsical.The height of chorionic villi and the ratio of villus height to crypt depth in the jejunum of the 1-week and 2-week infection groups were significantly lower than those in the normal control group(all P<0.05),while the depth of the recess of the former two was significantly increased(all P<0.05).With the extension of the infection time,the villus height and the ratio of villus height to crypt depth in the jejunum of mice decreased significantly(both P<0.05),and the crypt depth increased significantly(P<0.01).The TEM observation showed that the structure of the oocyst of C.parvum in the jejunum of the infected mouse was intact,the villi around the oocyst were abscission seriously,and the oocyst wall was fused with the epithelial cell membrane.The qPCR observation showed that compared with the normal control group,the expressions of TLR2 mRNA and TLR4 mRNA in the intestinal mucosa of the 1-week and 2-week infection groups were significantly higher(all P<0.05).In addition,the expressions of TLR2 and TLR4 mRNA in the 2-week infection group were significantly higher than those in the 1-week infection group(both P<0.05).The Western blotting showed that the expres-sions of TLR2 protein and TLR4 protein in the intestinal mucosa of the 1-week and 2-week infection groups were significantly higher than those of the normal control group(all P<0.05).Furthermore,the expressions of TLR2 and TLR4 protein in the 2-week infection group were significantly higher than those in the 1-week infection group(both P<0.05).Conclusions TLR2 and TLR4 are important receptors for intestinal mucosal recognition of C.parvum.The C.parvum infection may lead to intestinal mucosal damage possibly via the mechanisms associated with the up-regulation of TLR2 and TLR4 expressions.

Objective:To discuss the change of ferritin ( Fn) and ferroportin expression quantity and time-related feature in the alveolar macrophages of mice , infected with different virulence of Mycobacterium Tuberculosis infected .Methods:The prepared bacte-ria of H37Rv or BCG were injected intravenously into the mice tails .On the day 1, 3, 5, 7, 9, 11, 13 and 15, the lavage fluids were collected and the alveolar macrophages were obtained from each group of mice .The expression of FPN and Fn were detected with ELISA and /or Western blot analysis .Results:The expression of Fn in the group of either H 37Rv or BCG infected mice was decreased on the day 7, 9 and 11, and was lowest on the day 7, which showed significantly statistical difference compared to that on the other days (P<0.05).The expression of FNP in the infected mouse macrophage was decreased gradually , which was obvious on the day 5. The expression levels reached to the lowest on the day 7 and 9.The expression was much lower than that in the negative control group (P<0.05).Conclusion:The expression of Fn and FPN in macrophages isolated from lungs of mice infected with Mycobacterium tu -berculosis H37Rv or BCG become decreased , and there is no difference between these two infected mouse groups .

OBJECTIVESTo study the inhibitory effect of specially synthesized oligodeoxynucleotide (SODN) on malignant phenotype of human hepatocellular carcinoma Hep3B cells by complementary binding to the fourth promoter of IGF-2 gene.

METHODSA SODN was synthesized according to the sequence of the fourth promoter of IGF-2 gene, and was then transfected into Hep3B cells which overexpressed IGF-2. IGF-2 gene transcription activity and protein expression were assayed by RT-PCR and Western blot methods. The effect on cell growth by SODN was estimated by MTT method, and the effect on cell cycle distribution was measured by flow cytometry. Colony formation assay was performed on 6-well tissue culture dishes. Alteration on mobility and invasiveness were studied used transwell plates.

RESULTSIGF-2 mRNA and protein levels in Hep3B cells transfected with SODN were significantly lower in comparison with those in control groups (Hep3B cells and Hep3B cells transfected with a control oligodeoxynucleotide). Results also showed that SODN did not have inhibitory effects on cell growth and mobility of Hep3B cells, but did have an effect on its colony formation and invasiveness.

CONCLUSIONSODN has inhibitory effect on IGF-2 expression in Hep3B cells as a molecular switch, which partially alterates the malignant phenotype of this cell line.

Carcinoma, Hepatocellular ; pathology ; Humans ; Insulin-Like Growth Factor II ; biosynthesis ; genetics ; Liver Neoplasms ; pathology ; Neoplasm Proteins ; biosynthesis ; genetics ; pharmacology ; Oligonucleotides, Antisense ; biosynthesis ; genetics ; pharmacology ; Phenotype ; Tumor Cells, Cultured

OBJECTIVETo evaluate the possibility that using intracoronary delivery of autologus bone marrow-derived mesenchymal stem cells (MSCs) to improve the cardiac function after acute myocardial infarction (AMI) in miniature pig.

METHODSMSCs were cultured in Dulbecco's modified Eagle's medium-F12 (DMEM/F12) medium. AMI model was made by blocking the blood stream of the first diagonal branch in miniature pig, and released the branch after 90 minutes. After 10-14 days, (4-6) x 10(7) culture-expanded autologus 4', 6-diamidino-2-phenylindole (DAPI)-labelled MSCs were transplanted into each host heart's AMI area through intracoronary way. Ultrasonic cardiography (UCG) was performed to observe the left ventricular function at 3 months after transplantation. The cellular transplanted hearts were harvested and investigated by immunohistochemical analysis.

RESULTSLeft ventricular function of the MSCs group was improved significantly 3 months later compared with the control group [(54.65 +/- 3.39) vs (43.98 +/- 4.21)%, (P < 0.01)]. Exogenous MSCs survived and site-differentiated into cardiomyocytes in infracted hearts.

CONCLUSIONMSCs can play a benificial role to repair damaged heart. Heart function can be improved after MSCs transplantation in porcine myocardial infarction model.

Animals ; Female ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Myocardial Infarction ; pathology ; physiopathology ; therapy ; Swine ; Swine, Miniature ; Transplantation, Autologous ; Treatment Outcome

Objective To explore the correlation between Mycobacterium tuberculosis ( MTB ) PhoPR two-component system and drug resistance of MTB clinical isolates widespread in Xinjiang region by analyzing the expression of PhoP gene and PhoR gene among different isolates .Methods Total RNA of MTB was extracted from drug-susceptible strains , the strains only resistant to a single first-line anti-TB drugs (INH, RFP, SM and EB) and multidrug-resistant (MDR) strains, respectively.The purity of total RNA was checked by agarose gel electrophoresis .The expressions of PhoP gene and PhoR gene were quantified by using SYBR Green I qRT-PCR and the differences of their gene expression in different isolates were ana-lyzed.Results Compared with the drug-susceptible strains of MTB, the expression of PhoP gene was up-regulated for about 1.48 times in MTB strains resistant to RFP (RFP-MTB) and 2.74 times in MDR strain (P<0.05).Compared with MDR strain, the expressions of PhoP gene in the isolates resistant to INH (IN-HMTB), RFP (RFP-MTB), SM (SM-MTB) and EB (EB-MTB) were down-regulated for 0.70, 0.50, 0.25 and 0.21 times respectively.The expressions of PhoR gene were down-regulated for 0.36, 0.54, 0.35 and 0.19 times, respectively (P<0.05).The expressions of PhoR gene in the isolates of INH-MTB, RFP-MTB, SM-MTB and EB-MTB were up-regulated for 6.33, 4.56, 2.34, 1.85 and 9.06 times, respectively as compared with the drug-susceptible strains (P<0.05).Conclusion Significant differences of PhoR gene and PhoP gene expressions were observed among drug-susceptible strains , INH-MTB, RFP-MTB, SM-MTB, EB-MTB and MDR strains.Therefore, the Mycobacterium tuberculosis PhoPR two-component system is asso-ciated with the drug resistance of MTB strains prevalent in Xinjiang region .

Objective To establish an acute yunaconitine poisoning rat model with a single oral administration and to determine the contents of yunaconitine in rat tissues by UPLC-MS/MS method, then investigate the distribution of yunaconitine in rats. Method The rats were randomly divided into three groups and were intragastrically administered a single dose of 2.2mg/kg,1.1mg/kg,0.7mg/kg yunaconitine, respectively.. The rats were killed 2h later, the stomach tissue, intestine tissue, liver tissue, pancreas tissue, kidney tissue, lung tissue, spleen tissue, heart tissue, bladder tissue, testis tissue, brain tissue and heart blood samples were collected. The contents of yunaconitine in the biological materials were determined by UPLC-MS/MS method after the biological samples extracted by liquid-liquid extraction. Result A rat model of the yunaconitine poisoning was made with a single dose of 1.1mg/kg, the concentrations of yunaconitine displayed in the organs with the following order:stomach, small intestine, liver, pancreas, kidney, lung, spleen, heart, bladder, testis, heart blood and brain. Conclusion Yunaconitine was widely distributed in rats, especially the levels in the stomach, small intestine and liver were the highest. The conclusion provides a basis for the selection of test materials for the poisoning of Aconitum vilmorinianum Kom.