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

Abstract: In order to investigate the feasibility of the extract from Cyclocarya paliurus as an additive in cigarettes, the volatile aromatic components were analyzed by gas chromatography-ion mobility spectrometry (GC-IMS), and C.paliurus extract was pyrolyzed to simulate cigarette smoking by TGA-GC/MS. The cracking products of C.paliurus were analyzed in a nitrogen environment, and the possible cracking mechanism of the products was reasonably speculated. The results showed that aldehydes, alcohols, and ketones were the primary volatile aroma components of the C.paliurus extract, comprising 62.28% of the total aroma components. The cracking products of C.paliurus extract varied greatly under different temperature conditions. A total of 79 compounds were identified at 200,360, and 440 ℃, among which 24 aromatic components were clearly identified as having significant effects on cigarette style, including aldehydes, ketones, alcohols, phenols, furans, benzene series, and other natural aromatic substances. Among them, phenols containing a high concentration are mainly formed by compounds containing the structure of guaiacol unit and eugenol unit by side chain cleavage, demethylation, demethoxylation, dehydration, etc. Furan and its derivatives are mainly formed by glycosides or glycoside compounds by breaking glucoside bonds and dehydration.

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 evaluate the clinical efficacy of the Governor Vessel-unblocking and mind-regulating acupuncture method plus repetitive transcranial magnetic stimulation (rTMS) in the treatment of post-stroke insomnia. Methods: A total of 72 patients with post-stroke insomnia were randomly divided into 2 groups, with 36 cases in each group. The control group received rTMS treatment with a frequency of 1 Hz and a motion threshold value of 90％. The observation group received acupuncture with Governor Vessel-unblocking and mind-regulating method based on the rTMS treatment of the control group. The points were Baihui (GV 20), Shenting (GV 24), Yintang (GV 29), Fengfu (GV 16), Sishencong (EX-HN 1), Shenmen (HT 7), Sanyinjiao (SP 6), Shenmai (BL 62) and Zhaohai (KI 6). The treatment was performed once a day for 5 d a week followed by 2 d of rest for 4 weeks. The improvements of Pittsburgh sleep quality index (PSQI), self-rating anxiety scale (SAS) and self-rating depression scale (SDS) scores were observed after treatment, and the clinical efficacy was compared between the two groups. Results: After 4 weeks of treatment, the PSQI, SAS, and SDS scores of the two groups were all reduced, and the intra-group differences were statistically significant (all P<0.001). After treatment, the three scores in the observation group were all lower than those in the control group, and the differences between the two groups were statistically significant (P<0.05, P<0.05, P<0.001). There was a statistically significant difference between the observation group and the control group comparing the clinical efficacy (P<0.05). Conclusion: The therapeutic effect of the Governor Vessel-unblocking and mind-regulating acupuncture method plus rTMS in treating post-stroke insomnia is better than rTMS alone, and it can better improve the anxiety and depression of patients.

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 explore the substantial resection limits of CO2 laser surgery for hypopharynx and the course of wound healing in animals, for the purpose of evaluating the clinic usefulness of transoral CO2 laser surgery in the treatment of selected hypopharyngeal carcinomas.

METHODSTwenty-three dogs were randomly assigned to two groups. Group one (11 dogs) received left piriform sinus resection, group two (12 dogs) received the resection of posterior wall of the hypopharynx. Six dogs in group one were killed immediately or 4, 8, 12, 16, 20 d post-operatively. Seven dogs in group two were killed immediately or 7, 14, 21, 28, 35, 42 d post-operatively. The whole larynx and hypopharynx were taken out and the specimens were examined by naked eyes and under microscope. The other 5 dogs in each group were fed until the wound healed, the duration were observed.

RESULTSAll the operations were successful and the results were satisfactory. In group one, the dogs could take food the day after operation; two dogs had slight cough during eating and recovered after five days. In group two, the dogs could take food the next day after operation, eight dogs had slight cough during eating and recovered after ten days. The excision dimension was satisfactory. In group one (resection of the lateral wall of piriform sinus), the size of raw surface was (7.5 +/- 0.8) cm2 (x +/- s) and the healing time was (18.4 +/- 1.5) d. In group two (resection of the posterior wall of the hypopharynx), the wound surface was (7.0 +/- 0.5) cm2 and the healing time was (39.8 +/- 1.9) d. The healing time in group two was significantly longer than that in group one (t = 19.535, P <0. 01). The post-operative healing process were observed, including cellulose membrane coverage, granulation filling and epithelization.

CONCLUSIONSTransoral CO2 laser was suitable for partial hypopharynx resection. Animals can recuperate well with little complications. Although the course of wound healing was delayed, wound surface can recover with good laryngeal and deglutition functions.

Animals ; Dogs ; Endoscopy ; Hypopharynx ; surgery ; Laser Therapy ; Lasers, Gas ; Pharyngectomy ; methods

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