Based on mass spectrometry(MS)-guided separation strategy, compound 1 was obtained from the roots of Rhus chinensis. By comprehensive analysis of high resolution-electrospray ionization-mass spectrometry(HR-ESI-MS), nuclear magnetic resonance(NMR) data, and quantum chemical calculation of NMR(qcc-NMR) parameters, compound 1 was elucidated as rhuslactone, a 17-epi-dammarane triterpenoid with a rare 17α-side chain. An HPLC-ELSD method for its quantification in R. chinensis was established and adopted for the quantification of rhuslactone in different batches of R. chinensis. Rhuslactone displayed a good linear relationship within the range of 0.021 3-1.07 μmol·mL~(-1 )(r=0.997 6), and the average recovery was 99.34% [relative standard deviation(RSD) 2.9%). Moreover, the results of the evaluation test of the preventive effects of rhusalctone on coronary heart disease(CHD) and thrombosis showed that rhuslactone(0.11 nmol·mL~(-1)) significantly alleviated heart enlargement and venous congestion and increased cardiac output(CO), blood flow velocity(BFV), and heart rate, thereby reducing thrombus formation in zebrafish with CHD. The effects of rhuslactone on CO and BFV were superior to that of digoxin(1.02 nmol·mL~(-1)), and its effect on improving heart rate was comparable to that of digoxin. This study provides experimental references for the isolation, identification, quality control, and application of rhuslactone from R. chinensis against CHD. It is worth mentioning that this study has discussed some omissions in the determination of the stereochemistry of C-17 in dammarane triterpenoids in the present coursebook Chemistry of Chinese Medicine and some research papers, that is, the compound may be 17-epi-dammarane triterpenoid. This paper has also proposed steps for the establishment of C-17 stereochemistry.
Animals ; Zebrafish ; Rhus/chemistry* ; Triterpenes/analysis* ; Coronary Disease ; Thrombosis

OBJECTIVE: To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. METHODS: The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. RESULTS: Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn't recognize mature VWF (without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. CONCLUSION Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease (VWD), and the prognostic monitoring of endothelial injury-related diseases.
Animals ; Mice ; Humans ; von Willebrand Factor ; Antibodies, Monoclonal ; Protein Precursors/metabolism* ; von Willebrand Diseases/diagnosis* ; Prognosis

OBJECTIVE: To investigate whether co-transfection of wild-type VWFpp with VWF mutant in D1 region is able to correct VWF defects in biosynthesis and secretion. METHODS: Four VWF mutant plasmids were single transfected into HEK 293 cells, or co-transfected into HEK 293 cells with the wild type VWFpp plasmids. The VWF in supernatant and lysate of transfected cells were analyzed by ELISA, vertical VWF multimer electrophoresis. The retention of VWF in endoplasmic reticulum of transfected cells were detected by immunofluorescence confocal microscope. RESULTS: In the vertical VWF multimer analysis, with co-expressing VWF mutant and VWFpp, the VWF multimer bands disappeared, and the VWF antigen in both supernatant and lysate of cells decreased, compared with the single expression of VWF mutant. Although the intracellular levels of VWF antigens decreased after co-expression, the retention rate of VWF mutant decreased in endoplasmic reticulum. CONCLUSION VWFpp can reduce the retention of VWF in endoplasmic reticulum, assists the transport of VWF between subcellular organelles. However, VWFpp inhibits the biosynthesis and secretion of VWF about the mutant in D1 domain.
HEK293 Cells ; Humans ; von Willebrand Diseases ; von Willebrand Factor/metabolism*

Objective: To investigate death and attrition in HIV-infected children under initial antiretroviral therapy (ART) and associated factors in Guangxi Zhuang autonomous region. Methods: This retrospective cohort study was conducted in HIV-infected children under initial ART in Guangxi from 2004 to 2019, data from ART information system of National comprehensive AIDS prevention and treatment information system. Cox proportional hazards models were used to assess factors associated with the death and attrition. Results: In 943 HIV-infected children, the overall mortality and attrition rates were 1.00/100 person-years and 0.77/100 person-years, respectively. The mortality and attrition rates within the first year of ART were 3.90/100 person-years and 1.67/100 person-years, respectively. The cumulative survival rate during the first, second, fifth and tenth year after ART was 96.14%, 95.80%, 93.68% and 91.54%, respectively. Multivariate Cox proportional hazards models results showed that being female (aHR=2.00, 95%CI: 1.17-3.40), CD4⁺T lymphocytes (CD4) counts before ART <200 cells/μl (aHR=2.79, 95%CI: 1.54-5.06), weight-for-age Z score before ART <-2 (aHR=2.38, 95%CI: 1.32-4.26), hemoglobin before ART <80 g/L (aHR=2.47, 95%CI: 1.24-4.92), initial ART with LPV/r (aHR=5.05, 95%CI: 1.15-22.12) were significantly associated with death; being female (aHR=2.23, 95%CI: 1.22-4.07) and initial ART with LPV/r (aHR=2.02, 95%CI: 1.07-3.79) were significantly associated with attrition. Conclusions: The effect of ART in HIV-infected children in Guangxi was better, but the mortality and attrition rates were high within the first year of treatment. It is necessary to strengthen the training in medical staff and health education in HIV-infected children and their parents in order to improve the treatment effect.
Anti-HIV Agents/therapeutic use* ; Child ; China/epidemiology* ; Female ; HIV Infections/drug therapy* ; Humans ; Male ; Proportional Hazards Models ; Retrospective Studies

Objective: To investigate the impact of pretreatment drug resistance (PDR) on virological effect among HIV-infected patients having received antiretroviral therapy (ART) after three years. Methods: The baseline survey of PDR among HIV-infected patients was conducted in 2018, with a three-year follow up study. The clinic data and virological laboratory test variables were statistically analyzed. Results: Of the 2 433 participants, 41.6% (1 012/2 433) were aged between 18 and 34, 82.8% (2 015/2 433) were males, 46.9% (1 142/2 433) had education of high school or above, 22.4% (544/2 433) were farmers, 33.8% (823/2 433) were unmarried, 48.1% (1 169/2 433) were infected heterosexually and 41.3% (1 004/2 433) were with CRF07_BC. The prevalence of PDR was 4.5% (109/2 433). The prevalence of virological suppression failure (viral load ≥50 copies/ml) and drug resistance at three years follow up after ART was 8.1%(196/2 433) and 2.5%(60/2 433) respectively. The prevalence of virological suppression failure and drug resistance at three years follow up after ART were 18.3% (20/109) and 7.6% (176/2 324), and 4.6% (5/109) and 2.4% (55/2 324) among participants with PDR and non-PDR, respectively. The results of multivariate logistic regression model showed that illiteracy (aOR=3.26, 95%CI: 1.82-5.86), primary and junior high school education (aOR=1.54, 95%CI: 1.09-2.18), CD4⁺T lymphocyte count <200/μl (aOR=2.77, 95%CI: 1.75-4.37) and CD4⁺T lymphocyte count 200-499/μl (aOR=1.55, 95%CI: 1.10-2.18) at a three year follow up visit after ART, missed drugs in the past month (aOR=4.24, 95%CI: 2.92-6.17), and PDR (aOR=2.84, 95%CI: 1.67-4.85) were statistically significant with virological suppression failure on treatment. Conclusions: The prevalence of PDR in China at a low level currently, and the virological suppression failure rate is low after three years of ART. It is necessary to strengthen drug resistance monitoring of HIV-infected patients and pay attention to the influence of PDR on treatment effect.
Male ; Humans ; Adolescent ; Female ; Follow-Up Studies ; Viral Load ; Treatment Failure ; Drug Resistance ; HIV Infections/drug therapy*

Objective:To apply real-time shear wave elastography to observe the effect of instrument-assisted soft tissue mobilization (IASTM) on Achilles tendons for healthy adults. Methods:From July to December, 2020, 52 healthy adults were assigned into control group (n = 15) and experimental group (n = 37) randomly. The experimental group received IASTM on left Achilles tendons, once another day for two weeks, while the control group received no treatment. The thickness and elastic modulus of the left Achilles tendons were measured with high-frequency ultrasound and shear wave ultrasound elastography on all the subjects, before treatment, immediately after the first treatment and three days after treatment, respectively. Results:Five cases dropped down in the experimental group. There was no significant difference in thickness and elastic Young's modulus of the left Achilles tendons between two groups before treatment (t < 0.630, P > 0.05). The thickness of the left Achilles tendons was less in the experimental group than in the control group immediately after the first treatment (t = 2.149, P < 0.05), while average and maximum elastic Young's modulus was less three days after treatment (t > 2.134, P < 0.05). Conclusion:Real-time shear wave elastography could quantify the thickness and elasticity of Achilles tendon, to evaluate the effect of IASTM.

OBJECTIVE: To obtain the recombinant protein of spacer domain in von Willebrand factor cleaving protease (ADAMTS13), and further study its biological function in ADAMTS13. METHODS: The prokaryotic expression vector was constructed by using the template of plasmid with full-length ADAMTS13, and then transfected into E coli., following the induction of IPTG with the low temperature (30 ℃). The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blot respectively. By Adding the recombinant protein to the plasma of immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, the activity of ADAMTS13 was tested. RESULTS: The prokaryotic expression vector was successfully constructed and the protein of spacer domain with the high purity was obtained. Western blot showed that the recombinant fragment could both react with monoclonal antibody against 6×His and polyclonal sheep IgG against ADAMTS13 (Gln34-Trp688). The protein formed a main lane at the position of 15 kDa with SDS-PAGE. It was demonstrated that the recombinant protein could efficiently elevate the ADAMTS13 activity in plasma of iTTP patients to reach normal level by functional experiment. CONCLUSION The recombinant protein has high purity and immune activity, which provides the experimental basis for further research on mechanism of iTTP involved in spacer domain.
ADAM Proteins ; ADAMTS13 Protein ; Animals ; Escherichia coli ; Humans ; Purpura, Thrombotic Thrombocytopenic ; Recombinant Proteins ; Sheep ; von Willebrand Factor

OBJECTIVE: To investigate the genetic carrier rate of thalassemia and its gene mutation types as well as the distribution characteristics among the people in Lingshui Li autonomous county of Hainan province, so as to provide the basis for making the prevention programs of thalassemia in administrative departments. METHODS: Samples were collected from couples undergoing premarital and pregestational screenings, in which the positive ones in preliminary screening were further tested by genetic diagnoses and the genotypes were analyzed. RESULTS: The rate of thalassemia gene carriers was 19.41% （274/1412） of the couples of childbearing age in Lingshui Li autonomous County of Hainan Province. In these carriers,α-thalassemia accounted for 83.21%（228/274）, β-thalassemia for 8.03%（22/274）, and both α-and β-thalassemia gene accounted for 8.76% （28/274）. CONCLUSION The carrying rate of thalassemia gene in population Lingshui Li autonomous county of Hainan province is high, and its distribution has geographical characteristics,the major type is α-thalassemia. Blood screening and genetic diagnosis of thalassemia should be strengthened, and corresponding measures should be taken to reduce its gene frequency.
China ; Genetic Testing ; Genotype ; Heterozygote ; Humans ; alpha-Thalassemia ; beta-Thalassemia

Objective To study the inhibitory effect of ursolic acid on the proliferation of human papillary thyroid carcinoma cell line TPC-1 in vitro. Method TPC-1 cells were treated with different concentrations of ursolic acid (control group:0μM, experimental group:3μM , 6μM, 12μM);MTT assay was used to observe the effect of the growth of TPC-1 cells on different concentrations of ursolic acid at the same time;Apoptosis and cell cycle distribution of TPC-1 cells were treated with ursolic acid by flow cytometry;The expression of Bcl-2, Bax and Caspase-9 mRNA in TPC-1 cells were treated with ursolic acid by QRT-PCR;The expression of Bcl-2, Bax and Caspase-9 protein in TPC-1 cells were treated with ursolic acid by Western blot. Results MTT assay showed that ursolic acid inhibited the proliferation of TPC-1 cells in a concentration and time-dependent manner, and the IC50 at 24 h, 48 h and 72 h was 14.21 μM, 10.56 μM, 10.39 μM; Flow cytometry showed that ursolic acid inhibited the apoptosis of TPC-1 cells in a concentration-dependent manner, and the growth of TPC-1 cells was arrested in S phase;QRT-PCR showed that Bcl-2, Bax and Caspase-9 mRNA were expressed in the control and experimental groups, ursolic acid inhibited the expression of Bcl-2 mRNA in a concentration-dependent manner and up-regulated the expression of Bax and Caspase-9 mRNA;Western blot results showed that Bcl-2, Bax and Caspase-9 were expressed in the control and experimental groups, ursolic acid inhibited the expression of Bcl-2 protein in a concentration-dependent manner and up-regulated the expression of Bax protein and Caspase-9 protein. Conclusion Ursolic acid can significantly inhibit the proliferation and induce apoptosis of human papillary thyroid TPC-1 cells, providing some ideas for the treatment of thyroid cancer.