Zhang Y.,Shenzhen Blood Center Institute of Transfusion Medicine |
Xu H.,Shaanxi Blood Center |
Zhou H.,Guangzhou University of Chinese Medicine |
Wu F.,Shenzhen Blood Center Institute of Transfusion Medicine |
And 3 more authors.
Analytical Biochemistry | Year: 2015
Abstract The quality and yield of single-stranded DNA (ssDNA) play key roles in ssDNA aptamer selection. However, current methods for generating and purifying ssDNA provides either low yield due to ssDNA loss during the gel purification process or low specificity due to tertiary structural damage of ssDNA by alkaline or exonuclease treatment in removing dsDNA and by-products. This study developed an indirect purification method that provides a high yield and quality ssDNA sublibrary. Symmetric PCR was applied to generate a sufficient template, while asymmetric PCR using an excessive nonbiotinylated forward primer and an insufficient biotinylated reverse primer combined with a biotin-strepavidin system was applied to eliminate dsDNA, hence, leading to ssDNA purification. However, no alkaline or exonuclease were involved in treating dsDNA, so as to warrant the tertiary structure of ssDNA for potential aptamer SELEX selection. Agarose gel imaging indicated that no dsDNA or by-product contamination was detected in the ssDNA sublibrary generated by the indirect purification method. Purified ssDNA concentration reached 1020 ± 210 nM, which was much greater than previous methods. In conclusion, this novel method provided a simple and fast tool for generating and purifying a high yield and quality ssDNA sublibrary. © 2015 Elsevier Inc. All rights reserved.
PubMed | Red Cross, Affiliated Hospital of Academy of Military Medical science, Beijing Institute of Transfusion Medicine and Shaanxi Blood Center
Type: Journal Article | Journal: Blood transfusion = Trasfusione del sangue | Year: 2016
Enzymatic conversion of blood group A1B red blood cells (RBC) to group O RBC (ECO) was achieved by combined treatment with -galactosidase and -N-acetylgalactosaminidase. The aim of this study was to evaluate the function and safety of these A1B-ECO RBC in vitro.A 20% packed volume of A1B RBC was treated with enzymes in 250 mM glycine buffer, pH 6.8. The efficiency of the conversion of A and B antigen was evaluated by traditional typing in test tubes, gel column agglutination technology and fluorescence-activated cell sorting (FACS) analysis. The physiological and metabolic parameters of native and ECO RBC were compared, including osmotic fragility, erythrocyte deformation index, levels of 2,3-diphosphoglycerate, ATP, methaemoglobin, free Na(+), and free K(+). The morphology of native and ECO RBC was observed by scanning electron microscopy. Residual -galactosidase or -N-acetylgalactosaminidase in A1B-ECO RBC was detected by double-antibody sandwich ELISA method. Manual cross-matching was applied to ensure blood compatibility.The RBC agglutination tests and FACS results showed that A1B RBC were efficiently converted to O RBC. Functional analysis suggested that the conversion process had little impact on the physiological and metabolic parameters of the RBC. The residual amounts of either -galactosidase or -N-acetylgalactosaminidase in the A1B-ECO RBC were less than 10 ng/mL of packed RBC. About 18% of group B and 55% of group O sera reacted with the A1B-ECO RBC in a sensitive gel column cross-matching test.The conversion process does not appear to affect the morphological, physiological or metabolic parameters of A1B-ECO RBC. However, the A1B-ECO RBC still reacted with some antigens. More research on group O and B sera, which may partly reflect the complexity of group A1 the safety of A1B-ECO RBC is necessary before the application of these RBC in clinical transfusion.
PubMed | Guangzhou University of Chinese Medicine, Shaanxi Blood Center and Shenzhen Blood Center Institute of Transfusion Medicine
Type: | Journal: Analytical biochemistry | Year: 2015
The quality and yield of single-stranded DNA (ssDNA) play key roles in ssDNA aptamer selection. However, current methods for generating and purifying ssDNA provides either low yield due to ssDNA loss during the gel purification process or low specificity due to tertiary structural damage of ssDNA by alkaline or exonuclease treatment in removing dsDNA and by-products. This study developed an indirect purification method that provides a high yield and quality ssDNA sublibrary. Symmetric PCR was applied to generate a sufficient template, while asymmetric PCR using an excessive nonbiotinylated forward primer and an insufficient biotinylated reverse primer combined with a biotin-strepavidin system was applied to eliminate dsDNA, hence, leading to ssDNA purification. However, no alkaline or exonuclease were involved in treating dsDNA, so as to warrant the tertiary structure of ssDNA for potential aptamer SELEX selection. Agarose gel imaging indicated that no dsDNA or by-product contamination was detected in the ssDNA sublibrary generated by the indirect purification method. Purified ssDNA concentration reached 1020210nM, which was much greater than previous methods. In conclusion, this novel method provided a simple and fast tool for generating and purifying a high yield and quality ssDNA sublibrary.
PubMed | Red Cross, Xiamen Blood Center, Shaanxi Blood Center, CapitalBio Corporation and 22 more.
Type: Journal Article | Journal: PloS one | Year: 2015
Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.
Zheng L.-R.,Xi'an Jiaotong University |
Zheng L.-R.,Key Laboratory of Environment and Genes Related to Diseases |
Wang X.-F.,ShaanXi Blood Center |
Zhou D.-X.,Xi'an Jiaotong University |
And 4 more authors.
Reproductive BioMedicine Online | Year: 2012
X-ray repair cross-complementing group 1 (XRCC1) is a scaffold protein that plays a critical role in DNA base excision repair. To explore the association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in a northern Chinese Han population, PCR restriction fragment length polymorphism was used to genotype a SNP locus (rs25487) of XRCC1 in 112 patients with idiopathic azoospermia and 156 healthy controls. Furthermore, nucleotide sequences were sequenced. The results showed that, compared with GG genotype, the GA and GA + AA genotypes showed a significant association with an increased risk of idiopathic azoospermia (OR 2.119, 95% CI 1.245-3.606, P = 0.005), (OR 2.052, 95% CI 1.227-3.431, P = 0.006) respectively. Meanwhile, the A allele frequency was significantly higher in azoospermic patients than that in controls (OR 1.472, 95% CI 1.029-2.105, P = 0.034). The substitutions bring about an amino acid alteration: G → A changes the arginine residue into glutamine. In conclusion, the SNP locus rs25487 of XRCC1 could be a marker for genetic susceptibility to idiopathic azoospermia and the A allele might be a risk gene of idiopathic azoospermia in the northern Chinese Han population. © 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Zhou D.-X.,Xi'an Jiaotong University |
Zhou D.-X.,Key Laboratory of Environment and Genes Related to Diseases |
Huang X.-C.,The Fourth Hospital of Xian City |
Wang X.-F.,Shaanxi Blood Center |
And 3 more authors.
Andrologia | Year: 2012
Human leucocyte antigen (HLA) is a complex gene family that contains several highly polymorphic genes. Some studies have reported HLA-A gene to have a strong role in idiopathic male infertility in Japanese and Yugoslavia populations. Prompted by these findings, we investigated the distributions of HLA-A gene to ascertain their associations with idiopathic male infertility in Chinese population. Polymerase chain reaction-sequence-based typing (PCR-SBT) method was used for DNA typing at HLA-A locus in 109 patients with idiopathic male infertility and 152 healthy controls in Han male population of Shaanxi Province, situated in north-western China. In total, we detected 23 HLA-A alleles in all infertile patients, 22 HLA-A alleles in control subjects. However, no significant differences of these allelic frequencies were found between the patients and the control subjects, suggesting that the HLA-A gene was unlikely a major risk factor of idiopathic male infertility in this sample population. As different populations have different HLA polymorphisms, investigation into the relationship of other HLA genes and idiopathic male infertility in our population is needed in the future. © 2011 Blackwell Verlag GmbH.
PubMed | Xi'an Jiaotong University and Shaanxi Blood Center
Type: Journal Article | Journal: International journal of experimental pathology | Year: 2015
Cortactin, the cytoplasmic substrate of HDAC6, is known to play an actin cytoskeletal regulatory role which is implicated in the motility of cancer cells, and thus in cancer progression. Its activity is found to be regulated by HDAC6. However, the significance of cortactin and HDAC6 remains unclear in uncommon histologic variant human prostatic foamy gland carcinoma (PfCa). In this study, we aimed to identify the expression and potential role of cortactin and HDAC6 in PfCa. Therefore, 16 PfCa specimens containing 48 foci with distinctive lesions were collected to identify the status of cortactin and HDAC6 by immunohistochemistry. Their correlation between clinicopathological characteristics and prognostic values were further analysed. The effect of cortactin and HDAC6 on prostate cancer cell migration and invasion was then evaluated in IA8 cells. The results showed that expression of cortactin and HDAC6 was significantly higher in PfCa foci, compared to that of high-grade prostatic intraepithelial neoplasia (HGPIN) foci and benign foci (P < 0.05). Cortactin and HDAC6 were associated with poor prognosis of patients with PfCa (P < 0.05). Multivariable Cox regression analysis showed HDAC6 level was a significant prognostic factor for survival of patients with PfCa ( = 1.200, Wald value = 7.282, P = 0.007, 95% CI = 1.389-7.941, P < 0.01, > 0). Both knocking down cortactin and inhibition of HDAC6 activity with tubacin reduced in vitro migration and invasion ability of IA8 cells substantially. Furthermore, HDAC6 has prognostic value for patients with PfCa. Dysregulation of cortactin and HDAC6 is implicated in the invasiveness and migration of prostate cancer cells.
PubMed | Xian Medical College, Xi'an Jiaotong University and Shaanxi Blood Center
Type: Journal Article | Journal: Zhongguo shi yan xue ye xue za zhi | Year: 2016
To study the antiapoptotic effect of leukemia-associated gene MLAA-34 in HeLa cells.The MLAA-34 recombinant lentiviral expression vector was constructed, and the stably transfected HeLa cell line with high expression of MLAA-34 was set up; As(2)O(3) was used to induce apoptosis; the MTT assay, colony formation test and flow cytometry were used to detect the ability of cell proliferation, colong formation, apoptosis and cell cycle changes respectively.After treatment with As(2)O(3), the survival rate of HeLa cells with MLAA-34 overexpression was significantly higher than that of the control cells, and the colony formation ability of MLAA-34 significantly increased, and the high expression of MLAA-34 gene significantly decreased the apoptosis rate of HeLa cells, and decreased the proportion of G(2)/M phase cells.The leukemia-associated gene MLAA-34 has been comfirmed to show antiapoptotic effect in HeLa cells which are induced by As(2)O(3).
Ye S.-H.,Shaanxi Blood Center |
Wu D.-Z.,Shaanxi Blood Center |
Wang M.-N.,Shaanxi Blood Center |
Wu X.-Y.,Shenzhen Blood Center |
And 3 more authors.
Blood Transfusion | Year: 2014
Background. This study is a comprehensive analysis of RHD in D-negative phenotypes in saline, in Xi'an, Shanxi province, central China. Material and methods. DCcEe in saline was measured for each blood sample from every donor between January 2008 and June 2012 in the Xi'an Blood Centre, China. D-negative results were confirmed by an indirect antiglobulin test and further investigated by adsorption-elution as required. The initial step of molecular analysis was RHD zygosity testing. Then RHD was detected by a sequence-specific polymerase chain reaction system for RHD(1227G>A), weak D type 15, and RHD(711delC) alleles for the samples carrying at least one RHD. For the remaining non-identified samples, ten RHD exons were amplified using a previously widely used RHD coding region sequencing method. Some RHD/ RHCE conversion alleles were identified while those remaining were submitted to direct sequencing. Results. Overall, 2,493 D-negative samples in saline were detected in a total of 890,403 donors (D-negative rate, 0.28%). Among the D-negative individuals, RHD deletion (d/d) was assessed in 1685 donors (67.59%). Non-functional RHD alleles were detected in 184 donors (7.38%), the most common being the RHD-CE(2-9)-RHD and RHD(711delC) alleles. Two new alleles were observed and family investigations were performed; RHD(1227G>A) DEL was detected in 516 individuals (20.70%), and weak D or partial D variants were identified in 108 donors (4.33%). The most common alleles were weak D type 15, DVI type 3 and DV type 2. Four new weak D alleles were noted, and two cases of RHD(1227G>A)/weak D type 15 heterozygosity were confirmed. Conclusions. Currently, it seems to be difficult to observe any new RHD alleles in the Han Chinese population. D prediction in this population is easier because popular alleles are dominant, accounting for about 99.80% of alleles in D-negative people. Weak D types and partial D variants are rare and occur in approximately 0.01% of the population. © SIMTI Servizi Srl.
PubMed | Institute of Transfusion Medicine, Southern Medical University and Shaanxi Blood Center
Type: | Journal: Blood transfusion = Trasfusione del sangue | Year: 2016
Rhesus (Rh) D antigen is the most important antigen in the Rh blood group system because of its strong immunogenicity. When RhD-negative individuals are exposed to RhD-positive blood, they may produce anti-D alloantibody, potentially resulting in delayed haemolytic transfusion reactions and Rh haemolytic disease of the foetus and newborn, which are difficult to treat. Inhibition of the binding of anti-D antibody with RhD antigens on the surface of red blood cells may effectively prevent immune haemolytic diseases.In this study, single-stranded (ss) DNA aptamers, specifically binding to anti-D antibodies, were selected via systematic evolution of ligands by exponential enrichment (SELEX) technology. After 14 rounds of selection, the purified ssDNA was sequenced using a Personal Genome Machine system. Haemagglutination inhibition assays were performed to screen aptamers for biological activity in terms of blocking antigen-antibody reactions: the affinity and specificity of the aptamers were also determined.In addition to high specificity, the aptamers which were selected showed high affinity for anti-D antibodies with dissociation constant (KOur results demonstrate that ssDNA aptamers may be a novel, promising strategy for the treatment of delayed haemolytic transfusion reactions and Rh haemolytic disease of the foetus and newborn.