A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.
Artificial Gene Fusion ; Cholera Toxin ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; G(M1) Ganglioside ; metabolism ; Proinsulin ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Chinese acupuncture and moxibustion has been widely accepted as a useful therapeutics all over the world, but its mechanism has not been fully defined. For this purpose, a reticular framework of whole-body fascia and connective tissues has been established by means of digitized virtual human technique. The virtual acupoints represented three-dimensionally were compared with the sites for stimulation in practice of traditional Chinese medicine (TCM) acupuncture therapy. The results showed that the fascial network constituted by the connective tissues may be the anatomical basis for acupuncture therapy. We found that the acupoints were mainly located where thick connective tissues were present. In this fascial network, sensitive nerve endings, active cells and lymphatic vessels abounded in the sites with thick connective tissue, and needling at these sites induced definite biological effects. In light of biological phylogeny and embryo development, we believe that the connective tissue network may constitute a new functional system in the human body, the Self-supervision and control system (Fasciology), which provides a theoretical base for acupuncture therapy.
Acupuncture ; methods ; Acupuncture Points ; Connective Tissue ; anatomy & histology ; Fascia ; anatomy & histology ; Female ; Humans ; Medicine, Chinese Traditional ; methods ; Meridians ; Models, Anatomic ; Models, Neurological

OBJECTIVEThrough comparison of HER2/neu oncogene detected by chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) in breast cancer, to explore the effect of CISH on detecting gene amplification of HER2.

METHODSSelected formalin-fixed paraffin-embedded breast samples whose pathological types were infiltrating ductal carcinomas (255 retrospective samples, 271 prospective samples), and these samples were detected by IHC and CISH.

RESULTS(1) In the retrospective study, CISH identified gene amplification in 91.6% of IHC score 3+ tumors (120/131) and in 56.5% of IHC score 2+ tumors (39/69), thus the concordant ratio between IHC and CISH was 81.2% (207/255). The two results showed significant correlation (P<0.01). (2) In the prospective study, the ratio of HER2 protein over expression detected by IHC was 31.7%, the ratio of HER2 gene amplification detected by CISH was 27.3%. CISH identified gene amplification in 91.4% of IHC score 3+ tumors (53/58) and in 46.4% of IHC score 2+ tumors (13/28), Concordant ratio between IHC and CISH was 89.7% (243/271). Two results showed significant correlation (P<0.01). (3) Paired CISH/FISH results were concordant in 14 of 15 cases. The remaining case was detected by FISH, but showed no HER2 gene amplification by CISH. (4) The gene amplification by CISH had a significantly reverse correlation with ER and PR expression (P<0.01).

CONCLUSIONSThe results of HER2 gene amplification detected by CISH have high concordance with the results detectd by IHC and FISH. CISH is a novel technique for detecting HER2 gene amplification.

Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Female ; Gene Amplification ; Humans ; Immunohistochemistry ; methods ; In Situ Hybridization ; methods ; Prospective Studies ; Receptor, ErbB-2 ; genetics ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism ; Retrospective Studies

BACKGROUND: Chinese medicine is effective for preventing and treating glucocorticoid-induced osteoporosis, however, the underlying mechanism remains unclear. DKK1, an inhibitor of Wnt/β-catenin signaling pathway, can be up-regulated by glucocorticoid. Thereafter, DKK1 is an important target in the prevention and treatment of osteoporosis. OBJECTIVE: To explore the regulatory effect of Zuogui pill on DKK1 in the prevention and treatment of glucocorticoid-induced osteoporosis. METHODS: Eighteen three-month-old female Sprague-Dawley rats were randomly divided into three groups: control group, model group and Zuogui pill group. Rats in the model and Zuogui pill groups received the subcutaneous injection of dexamethasone to establish the model of glucocorticoid-induced osteoporosis. The Zuogui pill group rats were administrated Zuogui pill extracts, and the control rats were given the same volume of normal saline. At 1 month after modeling, the lumbar vertebrae were removed to test the bone mass and microstructures by micro-CT scanning. The biomechanical properties were detected by compression test. The mRNA expression levels of DKK1, Runx2 and CTSK were determined by Qpcr. The serum alkaline phosphatase activity was tested. RESULTS AND CONCLUSION: Compared with the control group, in the model group, the volumetric bone mineral density, trabecular bone volume fraction, trabecular number, and trabecular thickness were significantly decreased (P ＜ 0.05), the trabecular separation and structure model index were significantly increased (P ＜ 0.05). The serum alkaline phosphatase activity was on a decline. The mRNA expression level of DKK1 showed a significant up-regulation (P ＜ 0.05). The mRNA expression level of Runx2 showed a down-regulated trend while mRNA expression level of CTSK showed an up-regulated trend. Compared with the model group, the Zuogui pill group showed significantly enhanced volumetric bone mineral density, trabecular bone volume fraction, and trabecular number (P ＜ 0.05); the structure model index was significantly decreased (P ＜ 0.05); the trabecular separation was reduced; the serum alkaline phosphatase activity was enhanced; the mRNA expression level of DKK1 showed a significant down-regulation (P ＜ 0.05); the mRNA expression level of Runx2 showed an up-regulated trend while mRNA expression level of CTSK showed a down-regulated trend. The vertebral compressive strength in the Zuogui pill group was significantly higher than that in the model group (P＜0.05). In summary, Zuogui pill prevents and treats glucocorticoid-induced osteoporosis possibly through the down-regulation of mRNA expression of DKK1.

In black tea,theaflavins (TFs) are one important class of substances that determine sensory quality and have significant medicinal activities. In addition to the four kinds of common TFs,there may be many other theaflavin analogues (TFAs) with similar chemical structures in tea,but the study on them is very limited. Based on the characteristic sub-structure,mass spectrometry (MS) and MS/MS information,a method for screening and annotation of TFAs from the complex ultra high performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) data was proposed in this work. By analyzing the oxidation and polymerization process of a few TFs,the theoretical reaction model of TFs were summarized,which was used to calculate the precursor ion values of potential TFAs. Meanwhile,the diagnostic fragmentation ions and neutral loss of TFAs according to the fragmentation pathways obtained from chemical standards or documented in literatures were summarized. As a result,36 kinds of compounds were successfully annotated based on the calculated precursor ion values and the MS fragmentation patterns,among which 6 kinds of compounds were reported for the first time in tea. In vitro synthesis experiments were carried out to verified the annotation results. Based on the results of quantitation of 36 kinds of TFAs,a partial least squares-discriminant analysis model was used to investigate the changes of these components during black tea manufacturing. The results indicated that these novel TFAs could be used to effectively distinguish the black tea samples before and after fermentation.

OBJECTIVETo study the HIV incidence and risk factors among sero-negative spouses of HIV patients in Dehong prefecture of Yunnan province.

METHODSA cohort of sero-negative spouses of the HIV patients had been developed and followed up since November, 2005. HIV new infections and related behaviors had been investigated every six months.

RESULTSBy the end of June, 2008, 790 sero-negative spouses of HIV patients had been recruited, of whom 702 were followed-up for at least one time. During the total 1202.35 person-years, 31 new HIV infections were identified, with an overall incidence of 2.58/100 person-years. The HIV incidence rates were 2.22/100 person-years in 2006, 2.95/100 person-years in 2007 and 2.74/100 person-years in 2008. Data from the Cox proportional hazard regression model indicated that those who resided in Yingjiang county [hazard ratio (HR) = 4.37, 95%CI: 1.48 - 12.90, P = 0.008], ever using drugs (HR = 3.49, 95%CI: 1.09 - 11.18, P = 0.035), or having an HIV-infected spouse who never exposed to antiretroviral treatment (HR = 3.60, 95%CI: 1.41 - 9.16, P = 0.007) were at higher risk for HIV infection.

CONCLUSIONSero-negative spouses of HIV patients in Dehong prefecture of Yunnan province had a relatively high incidence of HIV new infection during 2006-2008. More efforts should put on those people living in these areas, having a history of drug use or having an HIV-infected spouse who had never been exposed to antiretroviral treatment.

Adolescent ; Adult ; China ; epidemiology ; Female ; HIV Infections ; epidemiology ; HIV Seronegativity ; Humans ; Incidence ; Male ; Middle Aged ; Prospective Studies ; Risk Factors ; Sexual Behavior ; Spouses ; Young Adult

OBJECTIVETo identify risk factors associated with patients suffered multiple level osteoporosis vertebral compression fractures(OVCFs).

METHODSFrom March 2011 to March 2015, 199 patients suffered osteoporotic were classified into multiple level OVCFs group and single level OVCF group. Multivariate logistic regression analysis were performed to identify risks factors associated with multiple level OVCFs.

RESULTSAll the patients underwent OVCF, including 71 multiple level OVCFs and 128 single level OVCF. There were no differences in the age, gender, BMI, hypertension and diabetes between two groups. While multiple level OVCFs were associated with spinal deformity index SDI[(2<=SDI<4, OR=2.587, 95% CI(1.148, 5.828);SDI>=-4, OR=7.775, 95% CI(3.272, 18.478)], BMD[(T<-4.5SD, OR=2.608, 95% CI(1.038, 6.551)].

CONCLUSIONSSDI and BMD might be the risk factors for multiple level OVCFs.

Objective To study the HIV incidence and risk factors among sero-negative spouses of HIV patients in Dehong prefecture of Yunnan province. Methods A cohort of sero-negative spouses of the HIV patients had been developed and followed up since November, 2005.HIV new infections and related behaviors had been investigated every six months. Results By the end of June, 2008, 790 sero-negative spouses of HIV patients had been recruited, of whom 702 were followed-up for at least one time. During the total 1202.35 person-years, 31 new HIV infections were identified, with an overall incidence of 2.58/100 preson-years. The HIV incidence rates were 2.22/100person-years in 2006, 2.95/100 person-years in 2007 and 2.74/100 person-years in 2008. Data from the Cox proportional hazard regression model indicated that those who resided in Yingjiang county [hazard ratio (HR) =4.37, 95% CI: 1.48-12.90, P=0.008] , ever using drugs (HR=3.49, 95% CI:1.09-11.18, P=0.035) , or having an HIV-infected spouse who never exposed to antiretroviraltreatment (HR=3.60, 95% CI: 1.41-9.16, P=0.007) were at higher risk for HIV infection.Conclusion Sero-negative spouses of HIV patients in Dehong prefecture of Yunnan province had a relatively high incidence of HIV new infection during 2006-2008. More efforts should put on those people living in these areas, having a history of drug use or having an HIV-infected spouse who had never been exposed to antiretroviral treatment.

Aged ; Diskectomy, Percutaneous ; Endoscopy ; Humans ; Intervertebral Disc Displacement/surgery* ; Lumbar Vertebrae/surgery* ; Spinal Stenosis/surgery* ; Treatment Outcome

OBJECTIVE: To explore the influencing factors of low back pain and the relationship of the influence of bad working posture, weight load and frequency of load and the dose-response relationship among the occupational workers of key industries in China. METHODS: A total of 57 501 employees from 15 key industries in China were selected as research subjects using stratified cluster sampling method. The occurrence of low back pain in the past one year, as well as occupational factors such as job type, labor organization and work posture were investigated by using the Chinese version Musculoskeletal Disorders Questionnaire. RESULTS: The prevalence of low back pain in the occupational population of key industries in China was 16.4%(9 448/57 501). Multivariate Logistic regression analysis showed that the risk of low back pain in females was higher than that in males(P<0.01). Married, obese, occasional and frequent smokers, and a history of lower back disease were associated with increased risk of low back pain(all P<0.05). The risk of low back pain was associated with older age, higher education level, and lower frequency of physical exercise(all P<0.01). The risk of low back pain was higher with longer working time, greater back curvature, and the high frequency of long standing and sitting position work, uncomfortable working posture, repeated operation per minute, and lifting>5 kg weight(all P<0.01). CONCLUSION: The influencing factors of low back pain in the occupational population of key industries in China include bad working posture, high frequency load, weight load and other individual factors. There is a dose-response relationship with low back posture load and frequency of load.