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The water quality in the drinking water reservoir directly affects people's quality of life and health. When external pollution input is effectively controlled, endogenous release is considered the main cause of water quality deterioration. As the major nitrogen (N) and phosphorus (P) sources in reservoirs, sediment plays a vital role in affecting the water quality. To understand the spatial and temporal variation of N and P in the sediment, this study analyzed the current characteristics and cumulative effects of a semi-humid reservoir, Yuqiao Reservoir, in North China. The N and P concentrations in the reservoir sediment were decreased along the flow direction, while the minimum values were recorded at the central sediment profile. External input and algal deposition were the main factors leading to higher sediment concentrations in the east (Re-E) and west (Re-W) areas of reservoir sediment profiles. According to the long-term datasets, the peaks of both sediment total nitrogen content and deposition rate were observed in the 2010s, which has increased about three times and six times than in the1990s, respectively. Therefore, the increase in phosphorus concentration may be the main reason for eutrophication in water in recent years. The mineralization of organic matter has a significant promoting effect on releasing N and P from sediments, which will intensify eutrophication in water dominated by P and bring huge challenges to water environment management. This study highlights that the current imbalance in N and P inputs into reservoirs and the endogenous P release from sediment will have a significant impact on water quality., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A 28-year-old woman was found to have coagulation factor Ⅷ activity (FⅧ∶C) <1% and von Willebrand factor antigen (VWF∶Ag) <1% during routine prenatal examinations. No pathogenic variation was found in the exon region of the VWF gene using next-generation sequencing. The clinical presentation of this patient does not match the clinical characteristics of type Ⅲ hemophilia [von Willebrand disease (VWD) ]; therefore, third-generation sequencing technology was used to perform whole-genome sequencing on the patient and her family members. Multiple members of the patient's paternal family carried a heterozygous variant of VPS33B, c.869G>C. The family members carrying this variant all had varying degrees of reduced VWF levels (39% -56% ). Moreover, the proband was detected with the heterozygous variant c.1474dupA in GP1BA. The ACMG and Clinvar databases determined that this variation was associated with platelet-type pseudo VWD. The decrease in VWF levels caused by heterozygous variations in VPS33B in families is the first international report, and no previous studies have reported cases of severe decrease in plasma VWF levels caused by double heterozygous variations in VPS33B and GP1BA.

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.

We developed a copper-catalyzed enantio- and diastereoselective boron conjugate addition to α-alkyl α,β-unsaturated esters under base-free conditions. The approach showed excellent enantioselectivities (87-99% ee) and moderate to good conversions (51-99%), albeit with moderate diastereoselectivities (1 : 1-17 : 1 dr). The synthetic utility of this protocol was demonstrated.

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.

Objective: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbβ3 on the surface of platelet membrane., Methods: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbβ3 on the surface of platelet., Results: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbβ3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05)., Conclusion: A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.

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.

Objectives: Based on previous reports that ginsenosides have been shown to exert better preventive effects on cisplatin-induced kidney injury, the present work aims to evaluate the protective effects of ginsenoside Rb3 (G-Rb3) on cisplatin-induced renal damage and underlying mechanisms in vivo and in vitro., Materials and Methods: The protective effect of G-Rb3 on cisplatin-induced acute renal failure in ICR mouse model and HEK293 cell model was investigated, and the underlying possible mechanisms were also explored. For animal experiment, renal function, kidney histology, inflammation, oxidative stress, relative protein molecules involved in apoptosis and autophagy signalling pathways were assessed. In addition, rapamycin (a specific inhibitor of mTOR), compound C (a specific inhibitor of AMPK) and acetylcysteine (NAC, a specific ROS scavenger) were employed to testify the effects of AMPK/mTOR signal pathway on the protective effects of G-Rb3 in HEK293 cells., Results: Pre-treatment with G-Rb3 at doses of 10 and 20 mg/kg for ten days significantly reversed the increases in serum creatinine (CRE), blood urea nitrogen (BUN) and malondialdehyde (MDA), and decrease in glutathione (GSH) content and superoxide dismutase (SOD) activity. Histopathological examination further revealed that G-Rb3 inhibited cisplatin-induced nephrotoxicity. G-Rb3 diminished cisplatin-induced increase in protein expression levels of p62, Atg3, Atg5 and Atg7, and decrease in protein expression level of p-mTOR and the ratio of LC3-I/LC3-II, indicating that G-Rb3 suppressed cisplatin-induced activation of autophagy. Inhibition of autophagy induced inactivation of apoptosis, which suggested that autophagy played an adverse effect on cisplatin-evoked renal damage. Further, we found that G-Rb3 might potentially modulate the expressions of AMPK-related signal pathways., Conclusions: These findings clearly suggested that G-Rb3-mediated alleviation of cisplatin-induced nephrotoxicity was in part due to regulation of AMPK-/mTOR-mediated autophagy and inhibition of apoptosis in vitro and in vivo., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Objective: To assess the significance of DDAVP use in the diagnosis and treatment of VWD. Methods: An analysis of 15 VWD cases who referred to Hematology Division of First affiliated Hospital of Soochow University and treated with DDAVP from March 2016 to August 2018 was conducted. Efficacy and treatment response of DDAVP were monitored by observations of changes in factor Ⅷ procoagulant (FⅧ∶C) and von Willebrand Factor (VWF) related indicators before and 2 h after DDAVP injection. Results: Of 15 cases with VWD, 7 males and 8 females with a median age of 23 (6-46) years, 7 of 9 type I VWD patients achieved complete response (CR) , 1 type 2A VWD case CR, 5 type 3 VWD ones no response (NR) . The VWF multimer analysis in 5 patients combined with other plasma VWF values were in accordance with the known diagnosis. Conclusions: DDAVP was effective in most type 1 patients, and ineffective in some type 2 and almost all type 3 cases. It was helpful for diagnosis and subsequent treatment planning.

The present study was designed to evaluate protective activity of an ethanol extract of the stems of Schisandra chinensis (SCE) and explore its possible molecular mechanisms on acetaminophen (APAP) induced hepatotoxicity in a mouse model. The results of HPLC analysis showed that the main components of SCE included schisandrol A, schisandrol B, deoxyschisandrin, schisandrin B, and schisandrin C and their contents were 5.83, 7.11, 2.13, 4.86, 0.42 mg·g -1 , respectively. SCE extract was given for 7 consecutive days before a single hepatotoxic dose of APAP (250 mg·kg -1 ) was injected to mice. Our results showed that SCE pretreatment ameliorated liver dysfunction and oxidative stress, which was evidenced by significant decreases in aspartate transaminase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) contents and elevations in reduced glutathione (GSH) and superoxide dismutase (SOD) levels. These findings were associated with the result that the SCE pretreatment significantly decreased expression levels of 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT). SCE also significantly decreased the expression levels of Bax, mitogen- activated protein kinase (MAPK), and cleaved caspase-3 by APAP exposure. Furthermore, supplementation with SCE suppressed the expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), suggesting alleviation of inflammatory response. In summary, these findings from the present study clearly demonstrated that SCE exerted significant alleviation in APAP-induced oxidative stress, inflammation and apoptosis mainly via regulating MAPK and caspase-3 signaling pathways., (Copyright © 2018 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Nephrotoxicity induced by cisplatin in 30% of all cisplatin treated patients seriously limits its clinical implication as a widely used anticancer agent, and may even cause patients to alter or give up cisplatin therapy. The purpose of this study is to test a protective effect of American ginseng berry extract (AGBE) on cisplatin-induced nephrotoxicity in mice. In this study, the histopathological changes and elevated levels of serum creatinine (CRE) and urea nitrogen (BUN) caused by cisplatin were significantly diminished by AGBE treatment. Oxidative stress caused by cisplatin, evidenced by increases in kidney tissues malondialdehyde (MDA) content, cytochrome P450 E1 (CYP2E1), renal 4-hydroxynonenal (4-HNE) levels and decreases of glutathione (GSH) and superoxide dismutase (SOD) contents, was significantly ameliorated by AGBE pretreatment. The expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were inhibited by AGBE treatment, suggesting a suppression of inflammatory response. Additionally, AGBE clearly inhibited cisplatin-induced activations of nuclear factor-kappa B (NF-κB) and mitogen activated protein kinase (MAPK) signal pathways. Supplementation of cisplatin-intoxicated mice with AGBE also significantly reduced apoptotic protein levels of Bax, cleaved caspase-3, cytochrome c and increased anti-apoptotic protein Bcl-2. These findings highlight nephroprotective effect of AGBE against cisplatin-evoked nephrotoxicity through ROS-mediated MAPK and NF-κB signaling pathways., (Copyright © 2017. Published by Elsevier Ltd.)

Objective: To establish the hybridoma cell lines secreting anti-ADAMTS13-T7 monoclonal antibodies (MA6) and to construct the MAb directed against different domains of ADAMTS13-T7., Methods: The trucated type protein ADAMTS13 of eukaryote-expressed recombinant ADAMTS13-T7 was constructed and was purified by using Ni-NTA agarose. Then the purified ADAMTS13 was used to immunize the BALB/c mice; the antiserum titer of ADAMTS13 protein of immunized mice was deteceted by ELISA. The spleen was taken from mice for construcing the single cell suspension, then the single cell suspension was mixed with myeloma cells SP2/0 at 10:1 ratio for cell fusion, and the elution culture of hybridoma cells was carried out; after 2 weeks, the wells in which clones were well grown were selected for detection, then the positive clonal wells were expansively cultured and the detection again was performed., Results: The purified ADAMTS13-T7 protein was gained. The ELISA showed that the antiserum titer in mice immumized by ADAMTS13-T7 protein was 1:20000. The results of fusion culture by hybridoma techmique showed that 30 hyridoma cell lines secreting MAb against recombinant ADAMTS13 were established., Conclusion: The hybridoma cell lines secreting MAb against recombinant ADAMTS13 have been successfully established by fusion culture, which provide more powerful tools for further screening the MAb with certain functional activity and studying the structure and function of ADAMTS13.

Although cisplatin is an anticancer drug that has activity against malignant tumor, it often causes nephrotoxicity. Previous reports have confirmed that the saponins from the leaves of P. quinquefolium (PQS) exerted many pharmacological activities. However, the renoprotective effects of PQS were still unknown. The purpose of the present research was to discuss renoprotective effect of PQS in a mouse model of cisplatin-induced acute kidney injury (AKI). The levels of blood urea nitrogen (BUN) and serum creatinine (CRE) were evidently increased in cisplatin-intoxicated mice, which were reversed by PQS. Renal oxidative stress, evidenced by increased malondialdehyde (MDA) level and decline of glutathione (GSH) and superoxide dismutase (SOD) activities, was significantly alleviated by PQS pretreatment. The suppression of inflammatory response by PQS was realized through the decrease the mRNA expression levels of tumor necrosis factor-α ( TNF-α ) and interleukin-1β ( IL-1β ) in kidney tissues, which were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Simultaneously, the overexpression of cytochrome P450 E1 (CYP2E1) and heme oxygenase-1 (HO-1) were attenuated by PQS. Furthermore, the effects of Western blotting demonstrated that PQS administration significantly suppressed the protein expression levels of nicotinamide adenine dinucleotide phosphate oxidase type 4 (Nox4), cleaved Caspase-3, cleaved Caspase-9, Bax, nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS), suggesting the inhibition of apoptosis and inflammation response. Overall, PQS may possess protective effects in cisplatin-induced AKI through suppression of oxidative stress, inflammation and apoptosis., Competing Interests: The authors declare no conflict of interest.

Objective: To deepen the understanding of acquired von Willebrand syndrome (AVWS)., Methods: The clinical data of 3 patients were analyzed and related literature were reviewed., Results: ① Case 1, a 70- year- old male, diagnosed as Waldenstrom macroglobulinemia and AVWS, was presented with spontaneous epitaxis and bruising. The VWF∶Ag level was 16%. Treatment was initiated with VWF concentrates. Two cycles of chemotherapy with Bortezomib, thalidomide and Dexamethasone were followed. Partial remission was achieved. Half- year' follow- up showed no sign of spontaneous hemorrhage. ② Case 2, a 48- year- old female, diagnosed as monoclonal gammopathy of undetermined significance and AVWS, was presented with repeated epitaxis. The VWF∶Ag level was 7%. Because the bleeding was slight and self-relieved, no specific treatment was addressed. She was followed up for one and a half year. ③ Case 3, a 50- year- old man, diagnosed as monoclonal gammopathy of undetermined significance and AVWS, was referred to our hospital for presentation with significant hematomas. VWF∶ Ag was reduced to 12%. VWF- containing cryoprecipitate, plasma, intravenous immunoglobulin and rituximab were used to control his bleeding symptom. During the follow-up, spontaneous hemorrhage still occurred occasionally., Conclusions: Acquired von Willebrand syndrome presented with heterogeneous symptoms. The level of VWF∶Ag and VWF∶Rco for patients with bleeding disorder should be performed. Abnormal bleeding symptoms in elderly patients without personal or family history of bleeding should prompt consideration of the underlying disorders. Treatment included controlling acute bleeding, curing the underlying diseases and preventing bleeding in high- risk situations. The prognosis of acquired von Willebrand syndrome is mainly related to the underlying diseases.

Objective: To construct and identify the monoclonal antibodies against von willebrand factor cleaving protease(ADAMTS13), and to study their biological function., Methods: BALB/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein (ADAMTS13-T7). Murine anti-human ADAMTS13 monoclonal antibodies (McAbs) were constructed by standard hybridoma technology and identified by ELISA. The recognition of McAbs with full-length recombinant ADAMTS13 protein was identified by Western blot. In function assay, the influence of McAbs on the proteolysis of vWF by ADAMTS13 was observed., Results: A group of 6 murine anti-ADAMTS13 McAbs was obtained with the clone number 1G11, 2F11, 6G3, 9E1, 10A8 and 10B4. In ELISA, the highest titers of 1G11 and 2F11 were observed, both of which showed a higher affinity to ADAMTS13-T7 than full-length ADAMTS13. The Western blot demonstrated that the 6 McAbs all could recognize ADAMTS13, among which 1G11 and 2F11 showed stronger reaction with ADAMTS13. In addition, under the denatured conditions, 1G11 and 2F11 could inhibit hydrolysis of vWF by ADAMTS13, and that was stronger with the increasing of McAbs concentration., Conclusion: McAbs against ADAMTS13 have been gained, two of which are inhibitory antibodies.

For deep analysis on the regional environmental economic system, the paper analyzes the mutual relation of regional economy development, environmental quality, environmental pollution, and builds the theoretical basis. Then, the economy-pollution-environment quality three-dimensional coupling evaluation model for district is constructed. It includes economic development level index, environmental pollution index, and environmental quality index. The model is a cube, which has spatialization and visualization characteristics. The model includes 8 sub cubes, which expresses 8 types of state, e. g. low pollution-inferior quality-low level of economic development etc. The model can be used to evaluate the status of region, divide development phase, analyze evolution trend etc. It has two ways including relative meaning evaluation (RME) and absolute meaning evaluation (AME). Based on the model, Yinchuan City in the Ningxia Hui Autonomous Region is used as an example for the empirical study. Using RME, compared with Guangzhou city, The result shows that the Yinchuan City has been a high pollution-low quality-low level of economic development state for a long period during 1996-2010. After 2007, the state changed to a high pollution-high quality-low level of economic development. Now, the environmental quality of Yinchuan city gets better, but pollutant discharge pressure is high, and tends to be the break point of high environment quality and low environment. With AME, using national standard, the Yinchuan City remains a high pollution-low quality-low level of economic development state during 1996-2010. Empirical research verifies that different target reference areas and relevant national standards have different main parameters, the evaluating result has an flexible range. The dimensionless data enhances the coupling of index. The data position in model increases the visibility to the environmental management decisions. The model improves mismatches of calculated data size, time asymmetry of spatial data, verification of the former multi-target coupling model.

This study was purposed to investigate the changes of von Willebrand factor cleaving protease (ADAMTS13) activity and vWF antigen level in patients with acute myelogenous leukemia (AML) before and after treatment and evaluate their clinical significance. Seventy-three AML patients were enrolled in this study, the sodium citrate anticoagulated plasma was collected before and after their induction chemotherapy. Fluorescence resonance energy transfer substrate vWF73 (FRETS-vWF73) assay was established to detect the plasma ADAMTS13 activity while vWF antigen level was measured by ELISA. The results showed that the ADAMTS13 activity in newly diagnosed patients with AML before induction therapy was obviously lower than that in normal controls (63.3 ± 25.5)% vs (105.1 ± 37.7)(P < 0.01), while the vWF antigen level was higher than that in normal controls (226.6 ± 127.0)% vs (111.4 ± 39.7)% (P < 0.01). After standard induction chemotherapy, the ADAMTS13 activity of AML patients in complete remission period was higher than that in AML patients before therapy (P < 0.01), and was not significant difference with that in normal controls; the vWF antigen was significantly lower than that in AML patients before therapy (P < 0.01), but it still was higher than that in controls (P < 0.05). The ADAMTS13 activity in newly diagnosed AML patients complicated with infection before therapy was obviously lower than that in AML patients without infection (52.2 ± 20.6)% vs (73.9 ± 24.7)% (P < 0.01), while the vWF antigen level was significantly higher than that in AML patients without infection (262.2 ± 135.7)% vs (193.8 ± 110.2)% (P < 0.05). The ADAMTS13 activity in AML patients with disseminated intravascular coagulation (DIC) was significantly lower than that in AML patients without DIC (42.0 ± 14.5)% vs (73.4 ± 22.7)% (P < 0.01), while the vWF antigen level was obviously higher that in AML patients without DIC (274.2 ± 140.0)% vs (204.7 ± 115.5)% (P < 0.01). It is concluded that the ADAMTS13 activity in newly diagnosed AML patients befor induction therapy has been confiremed to be lower and the vWF antigen level to be higher, especially in AML patients with infection or DIC. The ADAMTS13 and vWF antigen may play a role in the pathogenesis of AML and the formation of infection and DIC.

Compared with endothelial progenitor cells, outgrowth endothelial cells (BOECs) from peripheral blood are rich in protein for blood angiogenesis and cell adhesion, similar to mature endothelial cells in biological characteristics. Moreover, they are now replacing human umbilical vein endothelial cells for the latter's limited life span and drift of phenotype, and might become a new tool for exploring the vascular abnormalities. This study was aimed to establish the protocol of producing BOECs, and then analyze the cell phenotype and function of BOECs. Mononuclear cells were collected from peripheral blood by gradient centrifugation and then seeded on plates and cultivated in EGM-2 medium for 4 weeks. The morphological changes of cells were observed and cell phenotype was examined by flow cytometry. VWF multimers were used to analyse the distribution of vWF multimers in superment of BOECs and the storage of vWF in BOECs, and the secretion of vWF in BOECs under stimulation was detected by confocal fluorescence microscopy. The results showed that after 4-week-culture in vitro, the cell colonies and characteristic cobblestone-like morphology of BOECs were found in plates. For another three weeks of expansion, BOECs expressed CD31, CD34, and EPCR, without the expression of CD14, CD45 and CD133. The vWF from BOECs cell supernatant shared the same multimer pattern as that in normal plasma. By confocal fluorescence microscopy, vWFs were observed in BOECs. The amount of vWF increased in cells, and vWF strings were formed on cell surface by the stimulation of phorbol-12-myristate-13-acetate(PMA). It is concluded that the BOECs are first successfully established, and the phenotype and function of BOECs are analyzed. They are the native cell models for the pathogenesis of von Willebrand diseases (vWD), and may be used as new gene therapy tools for vWD.

Objective: This study aims to retrospectively investigate the contraceptive methods of different reproductive-aged women in two different regions of Yangzhou, China, to understand the changes of the contraceptive methods, and to analyze the targeted improvements of future informed choices of contraception and birth control, and reduce the occurrence of the unintended pregnancies., Materials and Methods: This study included 13,407 and 20,876 married reproductive-aged women (MCW) that were chosen from a city downtown and a county-level city, respectively, for the group-organized random sampling questionnaire survey., Results: There was a significant urban-rural difference in the choice of contraceptive methods in different MCW. The female choices of contraception were single; the accessibility of contraceptive supplies, and personalized services could improve the implementation rate of long-term contraceptive measures. The cooperation of male contraceptive responsibility was related to the education levels and enhanced male-female communication., Conclusions: It was very important for urban and rural women with different ages to choose different contraceptive measures, suggesting that these choices require the development of different educational models towards women of different ages and regions. The accessibility to various aspects of counseling and contraceptive measures should be strengthened, meanwhile male participation should also be promoted to prevent unwanted pregnancies and to reduce the health hazards related to sexual activities.

Objective: To investigate the genetic defect and its mechanism in a patient with congenital afibrinogenemia., Methods: The plasma fibrinogen activity and antigen of the patient was determined using the Clauss method and immuno-nephelometric assay, respectively. Genomic DNA was isolated from peripheral blood of the proband and his related family members. All exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCR followed by direct sequencing. Thrombin fibrin aggregation curve were detected in the plasma of the patient. Wild-type and mutation type fibrinogen vectors were constructed, and then transfected into COS-7 cells. The wild-type and mutant proteins from the culture media and cell lysates were tested by Western blot and ELISA., Results: APTT, PT, TT were significantly longer in the proband. Plasma fibrinogen activity and antigen of the patient could not be detected using the Clauss method and immuno-nephelometry, respectively. Gene analysis revealed that a novel homozygous GTTT insertion between nucleotides 2833 and 2834 in FGB exon 2 in the proband. The proband's father, mother, brother and son were heterozygous. The polymerization curves of the patient did not show a lag phase or final turbidity, compared with the normal controls. Western blot analysis showed the lack of complete half-molecules of the fibrinogen molecule and fibrinogen in patient's plasma under non-reducing conditions. It also could not detect the truncated Bβ chain under reducing conditions. Abnormal fibrinogen molecule (molecule weight>340 000) were found in transfected COS-7 cells by Western blot, which indicated that the mutation caused the abnormal intracellular fibrinogen molecule assembly. The fibrinogen band was absent in culture media transfected by the mutation. Fibrinogen levels of mutant fibrinogen were no significant different from those of wild-type fibrinogen in cell lysates by ELISA analysis [(2.47 ± 0.30) μg/ml vs (2.65±0.60) μg/ml, P=0.0889]; However, the levels of the mutant fibrinogen were statistically significant lower than those of wild type fibrinogen in culture media [(0.01 ± 0.01) μg/ml vs (3.80±0.80) μg/ml, P=0.0001]., Conclusion: Congenital afibrinogenemia was caused by this frameshift mutation in exon 2 of FGB. This novel mutation impaired fibrinogen assembly and secretion.

Unlabelled: Concentrations of Zn, Cu, Cr, Pb and Cd in 96 topsoil samples collected from the northern Hebin Industrial Park were measured by the atomic absorption detector (AAD). Spatial variability was identified by geostatistical analysis with the aid of GS + 3.0 and ArcGIS 9.0. Temporal variability of five heavy metals was contrasted to the concentration in 1985 of the Ningxia second soil census. Google earth maps in years of 2003, 2004, 2009 and 2011 have been analyzed to find the variation of the geographical distribution of factories in study Area. Result showed that mean concentrations of Zn, Cu, Cr, Pb and Cd were 100.29, 352.86, 77.56, 50.52, 4.06 mg x kg(-1), respectively, and were all higher than the background values. Using the second level of environmental quality standard for soils of China as the criterion, Zn, Cr, Pb were not risk, Cu, Cd were mild contamination and serious contamination. Spatial distribution of the concentration of the five heavy metals varies with prevailing wind direction, from the northwest to southeast, increasing gradually. Meanwhile, there were several hotspots. Google earth map analysis showed that in the northeast and southeast part appeared a large number of factories after 2009, which should be the main sources of the five heavy metals in soils. In the lowlying southwest part, although there was no factory, soils still have been polluted heavily because of another adjacent industrial area in Inner Mongolia Autonomous Region and the smoke dust was blocked by the Helan Mountain along its west., Conclusions: (1) The number of the enterprise increased and trans-boundary pollution led to the high heavy metal concentration in surface soil of study area; (2) In northwest China, because of the strong wind, smoke dusts spread very long and distribution range was extensive, and low terrain area and enterprise distribution pattern lead to the island distribution.

This study was aimed to construct a pEGFP-N1 vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studying its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion(®) High-Fidelity (NEB), then the PCR product was double digested with EcoRI and XhoI. After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-N1 vector. The DNA sequence analysis showed that ADAMTS13 was ligated to the pEGFP-N1 vector correctly. After transient expression in HeLa cells, the expression of EGFP could be detected by fluorescent microscopy, and the expression of ADAMTS13 protein could be detected by SDS-PAGE and Western blot. It is concluded that the ADAMTS13-pEGFP-N1 vector is successfully constructed, and it can be widely used in further research on the mechanism of the synthesis and secretion of ADAMTS13.

Objective: To investigate the susceptibility of von Willebrand factor (VWF) type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for von Willebrand disease (VWD) type 2A., Methods: Recombinant wild-type VWF (WT-VWF) and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant ADAMTS13 (rADAMTS13). Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis., Results: In vitro the expression of VWF:Ag in the supernatants of WT-VWF and A1500E mutant VWF were 1.10 U/ml and 0.78 U/ml, respectively, while VWF:Ag in cells lysates of A1500E mutant VWF was 90.6% of that of WT-VWF. The SDS-agarose gel VWF multimers analysis showed that there were no differences between WT-VWF and A1500E mutant VWF. The A1500E mutant VWF could be efficiently cleaved by ADAMTS13 under static condition without denaturants such as urea and guanidine HCl. VWF multimeric analysis showed that high and intermediate molecular weight multimers dramatically decreased while low molecular weight multimers obviously increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under the same condition., Conclusion: The A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.

Aim: To prepare and characterize the mouse monoclonal antibodies against Vibrio parahaemolyticus OmpW., Methods: The OmpW amino acid sequence from three diseased Vibrio was analyzed by Bioinformatics. Mice were immunized with r-OmpW which was highly expressed and purified in E.coli. Five Vibrio(Va, Vp, Vh, Vv, Van) were chosen as antigen for mAb selection.The characters of the anti-OmpW monoclonal antibodies were studied by Western blot, Flow Cytometry, indirect immunofluorescence., Results: OmpW was testified a highly conservative membrane protein.Three clones of anti-OmpW mAb was obtained. The Ig subclass of the mAb secreted from fused cell S5C10 was IgG3, which of the titer was 4.6×10(4);. The mAb could specifically recognize Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, which could not react with Pseudomonas flurosecens, Aeromonas hydrophila, Aeromonas sobria, Aeromonas hydrophila, Escherichia coli., Conclusion: The mAb could specially recognize five diseased Vibrio, which is a useful tool for the further study of the diagnosis of Vibrio.

Objective: To construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates., Methods: The DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 (rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay (ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GST-vWF114-H were used to measure plasma ADAMTS13 activity as substrates., Results: Two small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified, which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GST-vWF73-H and GST-vWF114-H as substrates., Conclusions: Two GST fusion proteins in vWF A2 domain, vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.

Aim: To Prepare recombinant human IL-17F/His protein and investigate its biological activity in vitro., Methods: The gene region of human IL-17F was cloned by RT-PCR. After identification by sequencing, the hIL-17F gene encoding function domain was cloned into expression plasmid PQE3.0 and transfected into E.coli M15. By the induction of Isopropyl-β-D-Thiogalacto-Pyranoside(IPTG), recombinant IL-17F/His protein was effectively expressed in E.coli M15. The recombinant protein was identified by Western blot., Results: After renaturation and purification by HiTrap(TM); affinity column, the recombinant protein can up-regulate macrophages to secret TNF-α, IL-6 and other relative cytokines. It also promoted proliferation of HeLa cells in vitro., Conclusion: hIL-17F/His recombinant protein is of high biological activity, which can be used to make further study of its special characteristic.

Objective: To study the influence of C-terminal domain of ADAMTS13 on its cleaving activity., Methods: The full-length wild-type (WT) and C-terminal domain truncated type (TT, TSP8 + CUB domains were deleted) of human ADAMTS13 recombinant protein were transfected into and permanent expressed on Hela cells. Western blot and R-CBA were used to directly detect the activities of the two recombinant proteins under the static and stressed condition respectively. ELISA was used to compare the binding abilities of the two proteins by coating with vWF., Results: The recombinant proteins were identified by Western blot with anti-his-tag or anti-ADAMTS13 antibodies. With pretreatment of 1.5 M urea, the enzyme activity of TT was significantly higher than that of WT, and so did in binding ability with vWF While, only WT could cleave vWF under high stress., Conclusion: The distal carboxyl-terminal TSP8 together with CUB domains of ADAMTS13 may affect the enzyme activity by regulating the binding of ADAMTS13 to vWF in different conditions, and they are very important for the enzyme activity under high stress force condition.

Background: A severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, so not easily adapted to routine laboratories. ADAMTS13 cleaves von Willebrand factor (VWF) within the domain A2, located between domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structures of VWF before and after the cleavage. Using this hypothesis we try to establish a new and simple method to determine ADAMTS13 activity., Methods: First, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies, SZ-129 and SZ-125, which specifically recognize the VWF A1 and A3 domains by using a two-site sandwich ELISA. Compared with a residual-collagen binding assay (R-CBA), plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibody in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated., Results: Plasma ADAMTS13 activities in normal people and TTP, acute myocardial infarction (AMI), and idiopathic thrombocytopenic purpura (ITP) patients determined by the new assay were (89.75 +/- 7.93)%, (17.63 +/- 18.71)%, (68.55 +/- 18.08)%, (85.83 +/- 9.84)%, respectively. Results were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays. The new assay can easily discriminate a TTP plasma sample from a non-TTP plasma sample (P < 0.01), and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activity of ADAMTS13 autoantibody ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient., Conclusion: This new and simple assay for ADAMTS13 activity could be used routinely in the clinic to determine the activity of ADAMTS13.

Objective: To analyze the phenotype and genotype of a family with inherited dysfibrinogenemia., Methods: Assays of coagulation, including activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT), were carried out with Stago Compact in the proband and his family members. The activity and antigen of fibrinogen in plasma were determined by Clauss and immunoturbidimetry, respectively. Fibrinogen and its constituent were analyzed by Western blot with nonreducing 4%-20% SDS-polyacrylamide gel electrophoresis (PAGE). All exons and exon-intron boundaries of fibringen genes FGA, FGB and FGG were analyzed by PCR and then direct sequencing., Results: The proband had normal APTT and PT, but prolonged TT. The activity of fibrinogen in plasma was decreased while its antigen level was normal. These abnormalities were also found in his mother and a sister. Genetic analysis revealed heterozygous G1233A in the exon 2 of FGA originating from his mother, which resulted in Arg16His missense mutation., Conclusion: Inherited dysfibrinogenemia was caused by Arg16His mutation in exon 2 of FGA, and this is the first case reported in a Chinese family.

Objective: To investigate the effects of Annexin II (AnxA2) gene silencing by siRNA on proliferation and invasive potential of lymphoma cell line Jurkat cells., Methods: A synthesized siRNA duplex targeting to AnxA2 was transfected into Jurkat cells. Transfection efficiency was analyzed by real-time PCR and flow cytometry. MTT assay for cell proliferation and transwell plates for invasive potential were performed., Results: Compared with the negative controls, the cell proliferation inhibitory rate of the AnxA2 siRNA transfected Jurkat cells was significantly increased at 24 h, 48 h and 72 h [(17.4 +/- 2.3)%, (22.4 +/- 3.8)%, (37.6 +/- 1.5)% vs (-1.3 +/- 5.1)%, (-5.5 +/- 4.4)%, (-10.8 +/- 5.5)%, respectively, P<0.05]. The cell invasive potential of the transfected Jurkat cells was inhibited remarkably at 48 h (11.3 +/- 4.2 vs 54.3 +/- 8.7, P<0.01)., Conclusion: AnxA2 gene silenced by siRNA can inhibit the proliferation and the invasive potential of Jurkat cells remarkably.

Aim: To investigate the biological activity of recombinant human IL-17 protein in vitro., Methods: The gene region of human IL-17 was cloned by RT-PCR. After identification by sequencing, the hIL-17 gene encoding function domain was cloned into expression plasmid PQE3.0 and transfect into E.coli M15. By the induction of Isopropyl-beta-D-Thiogalacto-Pyranoside (IPTG), recombinant IL-17/His protein was effectively expressed in E.coli M15. The recombinant protein was identified by Western blot., Results: After denaturation, renaturation and purification by HiTrap affinity column, the recombinant protein stimulated HeLa, a human uterine cervix cancer cell line, to excrete IL-6 and GM-CSF in vitro., Conclusion: IL-17/His recombinant protein is of high biological activity, which can be used to make further study of auto-immune diseases.

Aim: For further investigating of heat shock transcription factor 1 (HSF1) action in thermoregulation and its physiological mechanism., Methods: The relationship among the expression of HSF1 and interleukin 1beta (IL-1beta) mRNA and tumor necrosis factor alpha (TNF-alpha) mRNA in monocytes was studied during the different fever stages in rabbit fever model induced by LPS. The expressions of IL-1beta and TNF-alpha mRNA were measured by RT-PCR assay; HSF1 expression was measured by Western blot., Results: (1) Intravenous injection of LPS produced a double-peak temperature arisen at 60 min and 180 min. (2) The expression of IL-1beta mRNA in monocytes had a peak at 160 min, while the peak of TNF-alpha mRNA expression occurred at 80 min after intravenous injection of LPS. (3) The content of the HSF1 trimer increased gradually after 160 min intravenous injection of LPS. The results indicated that the content of the HSF1 trimer was negative correlation with the expressions of IL-1beta mRNA and TNF-alpha mRNA, and the change in body temperature was a positive correlation with IL-1beta mRNA., Conclusion: It is possible that HSF1 limits the rise in body temperature by repressing the gene expressions of the endogenous pyrogen, IL-1beta and TNF-alpha.