Objective:According to the GB/T 15000.3-2008， to develop a fucosterol certified reference material based on the project approved by Standardization Administration. Method:Fucosterol was isolated from Laminaria japonica dried thallus via 95% ethanol extraction， vacuum concentration， repeated column chromatography separation， recrystallization in petroleum ether-ethyl acetate， and residual solvent removal. Its chemical structure was identified by elemental analysis （EA）， infrared spectrum （IR）， mass spectrometry （MS）， nuclear magnetic resonance （NMR） and X-ray diffraction （XRD）. Its homogeneity， stability， and cooperative certification conducted by 8 laboratories were carried out by high performance liquid chromatography with evaporative light scattering detector. Result:For the fucosterol reference material， the certified value of purity was 99.54% with expanded uncertainty of 0.16% in confidence interval of 95%， the stability was good within 24 months storage period at 2-4 ℃， which met the technical requirements of reference material and passed the acceptance organized by Standardization Administration. Conclusion:The national standard materials of fucosterol has been successfully developed， which can be used for the determination of this component， the evaluation of detection methods， and the detection and quality control of related products.

Objective To evaluate the effects of a peer support education program in patients with type 1 diabetes mellitus.Methods A total of 68 patients with type 1 diabetes mellitus were enrolled and randomized into the experimental group and the control group,with 34 cases in each group.Both groups received diabetes health education for six months;the experimental group was given peer support education,and the control group was given routine health education.Fasting blood glucose,2-hour postprandial blood glucose,HbA1c,scores of China Diabetes Stress Scale,and perceived number of times for attending education programs were compared between two groups before intervention,and 3,6 months after intervention.Results The significant differences were found in HbA1c and 2-hour postprandial blood glucose between two groups and among time.Intervention and time interactively affected fasting blood glucose,2-hour postprandial blood glucose and HbA1c of two groups significantly.There were significant differences in total score,the scores of regular life,emotion and human relationship dimensions of Diabetes Stress Scale between two groups (P＜0.05).Among time the differences can be found in the total score and the score of each dimension (P＜0.01).Intervention and time interactively affected the total score and the scores of each dimension of Diabetes Stress Scale in two groups.Conclusion Peer support education program can promote blood glucose control in type 1 diabetes mellitus,improve their psychological status and increase their enthusiasm for being involved in disease management.

OBJECTIVEThis paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumoniae) antigens in humans with the variable domains (VD) 2 and 3 of the major outer membrane protein (MOMPVD2-VD3) and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae.

METHODSMOMPVD2-VD3 were overexpressed in Escherichia coli and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I (156 patients with typical respiratory illness clinically confirmed before) and Group II (57 healthy donors).

RESULTSIn Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II "healthy donors", 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively.

CONCLUSIONThe novel MOMPVD2-VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.

Animals ; Antibodies, Monoclonal ; Bacterial Outer Membrane Proteins ; immunology ; Chlamydophila Infections ; diagnosis ; microbiology ; Chlamydophila pneumoniae ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Mice ; Protein Structure, Tertiary

To analyze hematopoietic kinetics of mouse embryonic aorta-gonad-mesonephros (AGM) region, an in vitro tissue culture method was developed in this study, partly based on previous reports. After 2 days of tissue culture, a significant number of erythro myeloid progenitors, as quantitated by colony forming assay was detected in the AGM region. Moreover, the cells from cultured E10.5 AGM region could generate 10.8 ± 3.5 colony-forming unit in spleen (CFU-S) per tissue on average. Transplantation of cultured E10.5-E11.0 AGM cells resulted in efficient (85.7% repopulated) and long-term (> 4 months) reconstitution of lethally irradiated adult recipients with remarkable chimerism [(51.12 ± 21.17)%]. The multilineage contribution of donor cells was validated by significant engraftment of myeloid and/or lymphoid cells in peripheral blood, bone marrow, spleen and thymus of recipients. Taken together, the tissue culture method can enable us to manipulate the AGM region in vitro, fulfilling a systematic evaluation of developmental kinetics of various hematopoietic precursor cells, particularly HSC, in normal and mutant mid-gestation mouse embryos.
Animals ; Female ; Gonads ; cytology ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; Hematopoietic System ; embryology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Tissue Culture Techniques ; methods

The anatomical location of lymphocyte ontogeny in the developing mouse embryo remains controversial. To define the site that can generate lymphocytes de novo, the intraembryonic splanchnopleura (SP) and extraembryonic yolk sac (YS) at 8.5 days postcoitum, when systemic circulation is not established, were investigated. The results indicated that in standard colony forming assay, the cells from both splanchnopleura and yolk sac formed typical myeloerythroid colonies, but their types were distinct. When cocultured with the OP9, the splanchnopleura produced B cells expressing B220, CD19 and surface IgM. Using a three-step culture protocols with the OP9 expressing Delta-like 1 as feeders, the splanchnopleura produced immature T precursor cells (CD44-/CD25+) and more mature single positive T cells (CD4+/CD8-) after 16 days of incubation. However, the yolk sac failed to generate B and T lymphocytes under identical conditions. It is concluded that prior to linked embryonic circulation, the splanchnopleura other than the yolk sac had robust lymphoid potential in vitro. In the future, more reliable evidence from novel model animals will ultimately delineate the embryonic origin of lymphocytes in vivo.
Animals ; B-Lymphocytes ; cytology ; Cell Differentiation ; Coculture Techniques ; Female ; Hematopoietic Stem Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Pregnancy ; T-Lymphocytes ; cytology ; Yolk Sac ; cytology

Objective To evaluate the shaping ability of hand-used ProTaper on curved canals using Endodontic Cube.Methods Fifty-four curved root canals in vitro were selected and dirided into three groups according to the Curved degree(α),group A:0°≤α＜25°,group B:25°≤α＜40°,group C:40°≤α＜55°.Endodontic Cube was assembled.and each sample was sectioned perpendicular to the axis of the tooth into four sections with Isomet-Buhler in low speed.Then the root canals were prepared with hand-used ProTaper.Before and after shaping,photograph of all the sections were taken under a stereomicroscope.Statistical analyses were performed.Results The dentin cutting quantity of the whole canal prepared with ProTaper in group B and C Was larger than that of group A.The deviation distance of the whole canal prepared by ProTaper in group C was significantly larger than that in group A,and the deviation distance in middle portion larger than that in group B.The maintaining ability in the middle portion ofgroup C by ProTaper wag worse than that of group A and B.Condusious The curvature of root canal mayincreasethe cutting quantity of the dentin and reduce the ability of remaining original canal shape prepared by ProTaper.

Objective To delimit the natural infectious focus, including the distribution of wildlife,species, ecology of intermediate hosts and final host of Angiostrongylus cantonensis, as well as the routes of transmission and epidemiological characteristics and wildlife of human Angiostrongylus cantonensis, based on human diverging cases identified in Shenzhen, southern area of China. Methods Data including rate of infection and density of Angiostrongylus cantonensis among different hosts in 12 different areas in Shenzhen was collected, using microscope to inspect homogenate liquids of snails. Wild mice were captured with mouse cage to examine the adult Angiostrongylus cantonensis. Using larva isolated from wild-snails-infected rats to observe the life cycle of Angiostrongylus cantonensis. Results Wild life of Angiostrongylus cantonensis existed in the southwest part of Shenzhen with its majority intermediate hosts as Achatina fulica. The overall rate of infection was 31% in wildlife and final host was found to be Rattus andersoni, Achatina fulica which were extensively distributed in the shrub region of Shenzhen because of suitable climate,humidity and vegetation for generating the life cycle of Achatina fulica. Human infected Angiostrongylus cantonensis was mainly due to eating raw snails or vegetables contaminated by larva of Angiostrongylus cantonensis.The peak of infection was seen from April to November in Shenzhen area.Conclusion Wildlife of Angiostrongylus cantonensis existed in the southwest part of Shenzhen with major wildlife reservoir including fresh water snail and wild mouse. The existence of natural focus Angiostrongylus cantonensis was now recognized as an important source of human angiostrongliasis in Shenzhen area.

AIMTo investigate the pharmacokinetics of mycophenolic acid (MPA), an active metabolite of mycophenolate mofetil (MMF) in Chinese adult liver transplant patients.

METHODSThirty-eight liver transplant patients (male 30, female 8) receiving MMF 1.0 g, twice daily in accordance with the recommended regimen were included in this study. Plasma MPA concentrations were measured by high performance liquid chromatography at 0.5, 1, 1.5, 2, 4, 6, 8, 10 and 12 h after the administration of a single dose. Pharmacokinetic parameters were calculated with 3P97 software.

RESULTSThe plasma MPA concentration-time curve was characterized with an early sharp peak reached at 0.5 - 6.0 h after oral administration. And in some patients there was a small second peak due to enterohepatic circulation of mycophenolic acid glucuronide (MPAG), which underwent deglucuronidation and re-absorption as MPA at 4 to 12 h postdose. The mean peak plasma concentration (C(max)) and area under concentration-time curve (AUC(0-12 h)) were (12 +/- 7) microg x mL(-1) and (44 +/- 16) microg x h x mL(-1), respectively. However, a large variability of pharmacokinetic parameters existed in these patients.

CONCLUSIONIn view of the inter-individual variability of MMF pharmacokinetics, plasma MPA concentration should be monitored routinely after MMF administration for individual patient.

Adult ; Aged ; Area Under Curve ; Female ; Humans ; Immunosuppressive Agents ; pharmacokinetics ; Liver Transplantation ; Male ; Middle Aged ; Mycophenolic Acid ; analogs & derivatives ; pharmacokinetics

OBJECTIVETo determine the physicochemical properties of the mutanase of Trichoderma harzianum isolated from China and to study the influence of mutanase on the adherence of oral Streptococci and the structure of oral biofilms.

METHODSSix fungal strains belonging to Trichoderma were tested for mutanase production in the same cultural condition, the strain producing the highest mutanase activity was studied further and the pH and temperature optimum of the enzyme was determined. The RT-PCR method was used to obtain the gene coding for mutanase and the product was cloned to pMD18-T simple vector for sequencing. Inhibition effects of mutanase on the adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 were studied by adherence test. The optical sectioning of biofilms with or without mutanase supplementation were analyzed by confocal laser scanning microscopy (CLSM).

RESULTSThe highest enzymatic activity was achieved by Trichoderma harzianum Th1, the maximum activity was at pH 5.5 and at 40 degrees C. The nucleotide sequence was 92% homology with that of a known gene coding a mutanase (GenBank accession No. AJ243799). The adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 was significantly inhibited by mutanase. Compared with control, the biofilms with mutanase supplementation had lower height and sparser structure.

CONCLUSIONSThe mutanase from Trichoderma harzianum Th1 can inhibit the adherence of oral Streptococci and had an influence on the structure of oral biofilms.

Bacterial Adhesion ; drug effects ; Biofilms ; Glycoside Hydrolases ; chemistry ; physiology ; Streptococcus mutans ; drug effects ; Streptococcus sobrinus ; drug effects ; Trichoderma ; enzymology ; pathogenicity

OBJECTIVETo investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells.

METHODSThe A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants.

RESULTSSpecific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX.

CONCLUSIONSCTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.

Animals ; Antigens, CD ; genetics ; immunology ; metabolism ; CTLA-4 Antigen ; Cricetinae ; Dental Caries ; prevention & control ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, DNA ; genetics ; immunology