This paper is aimed to report the development of a method for the determination of the binding rate of plasma protein with salvianolic acid B. In vitro, equilibrium dialysis method was used to imitate the binding process between salvianolic acid B and plasma protein, in vivo, ultrafiltration method was used and the binding rate with HPLC was determined. Plasma samples were treated with methanol to precipitate the protein, and the buffer solution was directly determined after filtering. The calibration curve of the buffer solution was linear in the range of 0.5-20 microg mL(-1). The calibration curve of the plasma was linear in the range of 2-200 microg mL(-1). The extract recovery was 68.6%-81.9%. RSDs of intra- and inter-day precisions were all less than 8.5%. The binding rates of plasma protein with salvianolic acid B in vitro was 75.2% and in vivo was 92.1%. This paper shows the high binding power of salvianolic acid B to plasma protein with high sensitivity, good reproduction, simple management and fulfilling the requirement.

Objective To develop a multivariate logistic regression model , and to predict the risk of ma-lignant partially cystic thyroid nodules .Methods 470 patients(662 nodules)treated with surgery and confirmed by pathological diagnosis were screened out .Their ultrasonographic morphology and vascularity of thyroid nod-ules, thyroid stimulating hormone(TSH)and clinical information were collected and analyzed retrospectively .The model was developed to calculate the individual risk and ROC curve was used to evaluate the predictive index . Results The regression model was:Z=-3.60+1.40X2+2.47X3+1.05X4+0.57X9+0.07X10+1.02X12 ( X2 represents eccentric acute-angle configuration , X3 represents microcalcification , X4 represents cystic-solid margin,X9 represents echogenecity of solid portion , X10 represents TSH,X12 represents gender );M=eZ/1+eZ (M represents probability of malignancy , e represents natural constant 2.72).When applied the model to the ver-ification group, the accuracy, sensitivity, specificity, misdiagnosis rate, missed diagnosis rate, negative likeli-hood ratio(LR-)and positive likelihood ratio(LR+)was 96.95%,100%, 96.68%, 3.32%, 0%, 0 and 30.12 respectively.The largest area under the receiver-operating characteristics curve (AUC)was 0.88,which proved the model has high diagnostic value .Conclusions The model has high accuracy to predict the risk of malignan-cy.M is closely related to malignant risk of partially cystic thyroid nodules .

A stable HMG-CoA reductase (HMGR) reaction in vitro was developed by a sensitive, selective and precise liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. The optimized enzyme reac-tion condition contained 1.5μg of HMGR, 20 nM of NADPH with 50 min of reaction time. The method was validated by several intra-and inter-day assays. The production transitions of m/z 147.0/59.1 and m/z 154.0/59.1 were used to detect and quantify mevalonolactone (MVAL) and MVAL-D7, respectively. The accuracy and precision of the method were evaluated over the concentration range of 0.005–1.000μg/mL for MVAL and 0.010–0.500μg/mL for lovastatin acid in three validation batch runs. The lower limit of quantitation was found to be 0.005μg/mL for MVAL and 0.010μg/mL for lovastatin acid. Intra-day and inter-day precision ranged from 0.95%to 2.39%and 2.26%to 3.38%for MVAL, 1.46%to 3.89%and 0.57% to 5.10% for lovastatin acid, respectively. The results showed that the active ingredients in Xuezhikang capsules were 12.2 and 14.5 mg/g, respectively. This assay method could be successfully applied to the quality control study of Xuezhikang capsule for the first time.

Objective A multivariate logistic regression model was built to estimate the risk of malignant partially cystic thyroid nodules.Methods Data of ultrasonographic morphology,microcalcifications,margin,colloid crystals and echogenicity of 766 thyroid nodules (of 506 patients) were analyzed retrospectively.All patients underwent surgery and the diagnosis were confirmed by pathology.The model was built to calculate the individual risk and evaluate the predictive index.Results The regression model was Z =-2.30 + 1.42X2 + 2.39X3 + 1.17X4-1.35 X7 + 0.62X9 ; P =eZ/1 + ez (P represents probability of malignancy,e represents natural constant 2.72).The largest area under the receiver-operating characteristics curve (AUC) was 0.86.When apply the model to the verification group(266 nodules),the accuracy,sensitivity,specificity,misdiagnosis rate,missed diagnosis rate,negative likelihood ratio and positive likelihood ratio were 93.61％,83.33％,94.63％,5.37％,16.67％、0.18 and 15.52 respectively.Conclusions This model based on the ultrasonographic variables increases the diagnostic accuracy of malignancy in patients with partially cystic thyroid nodules.

In order to develop a safer vaccine strain exploit Salmonella Pullorum vaccine strain as a live vaccine vector.Methods:AΔcrpΔasd mutant of S.pullorum C79-13 strain was constructed and it was developed E.coli donor strain mutant was generated through the two-step method introduced by χ7213 ( pREΔasd) was conjugated with the recipient of C 79-13Δcrp.The C79-13ΔcrpΔasd the transduction of recombinant suicide plasmid .Results:PCR and sequencing results showed that ΔsipBSL1344 was suc-cessfully constructed.The further study indicated that foreign DAP must be supplied for the ΔcrpΔasd mutant to grow,in addition,the asd gene was transmitted stably .Compared with C79-13 strain,the O antigens was identical to C79-13 strain,but the growth velocity was reduced significantly ,significantly reduced virulence .Conclusion: The ΔcrpΔasd mutant can accept asd+plasmid to construct host-vector balance lethal system , and this system can be used to express foreign gene efficiently and to develop potential oral multivalent vaccines.

OBJECTIVEThe color difference meter and electronic nose fingerprint analyzer were introduced to discriminate processed betel nut's different huohou and quantize empirical index during the processing of Chinese medicine.

METHODBy collecting the color's and the smell's characteristic parameters of betel nut and its processed products to set up huohou's math discrimination modules and 90% reference value scale.

RESULTWe set up a huohou's math discrimination module and 90% reference value range base on L a*b*, which discrimination coincidence was 100% and 90% reference value range respectively was Shengpin: L* (50.07-55.03), a* (4.136-6.906), b* (6.65-8.82); Chaopin: L* (43.874-47.998), a* (3.816-6.732), b* (3.786-6.558); Jiaopin: L* (38.744-40.616), a* (1.11-3.01), b* (-1.434-0.538). At the same time, we set up a huohou's math discrimination module base on smell's characteristic parameters too.

CONCLUSIONThe betel nut and its processed products' color and smell measurement based on the color difference meter and electronic nose fingerprint analyzer may be feasible, and provides a reference to better study on traditional Chinese medicine theory.

Objective: To develop an oral live vaccine vector which stably carries exogenous genes.Methods:SL1344ΔsipBΔasd host-vector balanced lethal system was constructed by the method of recombinant suicide plasmid-mediated allelic exchange on the basis of attenuated Salmonella typhinurium SL1344ΔsipB.Then,the biological characteristics of SL1344ΔsipBΔasd was analyzed.Results:The results showed that the mutant was stabile with the Δasd gene in vitro;the serotype and growth rate of SL1344ΔsipBΔasd strain was almost same as the parent SL1344ΔsipB and SL1344 strain.And the mutant strains remain swim ming zones.Virulence test in mice showed that the virulence of SL1344ΔsipBΔasd which carried complementary plasmid pYA3493 by electro-transformation decreased by 1.4%compared with SL1344.Conclusion: These results showed that the SL1344ΔsipBΔasd mutant was successfully constructed.It is likely that this mutant should be used as a live vector to express foreign genes.

Objective:In order to develop a safer vaccine strain exploit Salmonella typhimurium vaccine strain .A ΔsipB mutant of Salmonella typhimurium SL 1344 strain was constructed.Methods: Firstly, the recombinant suicide plasmid containing the missing 585 bp sipB ( PREΔsipB ) was built by homologous recombination , and screenned by two-step method.Results: PCR and sequencing results showed that SL 1344ΔsipB was successfully constructed.It was no significant changes compared with SL 1344.But compared with the parent strains SL 1344 , the mutant strain had obvious change in its virulence , oral challenge of bacteria in mice revealed that LD50 of the mutant strain was 1.70 ×108 CFU,the toxicity reduced about 1.4%.The protection rate induced by the sipB mutant was 50%,and the serum antibody peaked 14 d post-immunization.Conclusion:The SL1344ΔsipB mutant was constructed suc-cessfully,and genetic stability ,significantly reduced virulence.The study provides a new approach for further study of the relationship between the gene and pathogenicity of Salmonella typhimurium.It is likely that the ΔsipB mutant could be adapted to develope attenuated Salmonella vaccine.

In order to develop a recombinant attenuated Salmonella typhimurium as oral live vaccine vector, we constructed recombinant plasmid pYA-sopENt100 by replacing the trc promoter with the sopE promoter and secretion signal sequence sopENt100 of Salmonella typhimurium on the basis of plasmid pYA3493. Then, the complementary plasmid pYA-sopENt100 was transformed into ΔcrpΔasdSL1344 by electroporation to generate attenuated Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100). We further characterized ΔcrpΔasdSL1344 (pYA-sopENt100). We also constructed a recombinant strain ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) that harbored the reporter gene-enhanced green fluorescent protein (egfp) gene. Vero cells were infected with ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) and the ability of delivery foreign antigens was tested via Western blotting analysis. The results of PCR, enzyme digestion and sequencing showed that the ΔcrpΔasdSL1344 (pYA-sopENt100) type III secretion system was constructed successfully. The serotype of ΔcrpΔasdSL1344 (pYA-sopENt100) was identical to ΔcrpΔasdSL1344 and SL1344. Compared with wild strain SL1344, the biochemical characteristics of ΔcrpΔasdSL1344 (pYA-sopENt100) had obvious change, but it was basically the same with ΔcrpΔasdSL1344. The growth speed was much slower than that of the wild strain SL1344. The chicken virulence test (LD₅₀) showed that the virulence of ΔcrpΔasdSL1344 (pYA-sopENt100) was 7×10⁴ times lower than SL1344. In addition, we observed the 37 kDa SopENt100-egfp protein in the cultured supernatant of ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) strain by Western blotting analysis. However, both the 37 kDa SopENt100-egfp protein and 27 kDa EGFP protein were detected in ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)-infected Vero cells. These results demonstrated that the recombinant Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100) was successfully constructed, and it should be used as a live vaccine vector for expressing foreign genes.
Animals ; Bacterial Proteins ; genetics ; Cercopithecus aethiops ; Mice ; Plasmids ; Promoter Regions, Genetic ; Salmonella typhimurium ; genetics ; Type III Secretion Systems ; genetics ; Vaccines, Attenuated ; genetics ; Vero Cells ; Virulence