The efficacy of ethnic specific blood groups genotyping for routine donor investigation and rare donor identification in Taiwan.

BACKGROUND: Large-scale single nucleotide variation (SNV)-based blood group genotyping assays have been made available for over a decade. Due to differences in ethnic groups, there is much diversity in clinically important blood group antigens and genetic variants. Here, we developed a robust matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based blood group genotyping method on MassARRAY system. STUDY DESIGN AND METHODS: A total of 1428 donors were enrolled into three groups: (a) reagent red cell donors; (b) rare donor or common antigen-negative donors; and (c) group O, R1 R1 /R2 R2 donors. Forty-two SNVs were designed for determining nine blood groups, with X/Y chromosome in two multiplex reactions, on MassARRAY 96-well format system. Further targeted sequence analyses were performed by Sanger sequencing. RESULTS: WHO reference reagent (NIBSC code: 11/214) was tested for concordance with the provided genotype results. Among the donors, concordance rate was over 99%. Alleles of important phenotypes such as Mi(a+), Di(a+), and Asian-type DEL and alleles of rare blood groups such as Fy(a-), Jk(a-b-) and s- were screened. Three types of discrepancies were found. Serologically, the 'N' antigen was expressed on genetically MM with GYP*Mur red blood cells and caused genuine discrepancies (9.5%). Genetically, allele dropout (ADO) was caused by rare SNV in the primer for Ss genotype (2.1%) and partial insertion of RHD genes (0.9%) led to difficulties in predicting phenotypes. CONCLUSION: Hemo panel module and MassARRAY System in 96-well format showed good performance in terms of large-scale blood group genotyping and phenotype predictions. Implementation of this method is effective for routine blood group genotype screening of donors.

The Influences of Sports Psychological Capital to University Baseball Athletes' Life Stress and Athlete Burnout.

Previous studies suggested that athletes' psychological capital level is related to life stress and burnout. Therefore, the purpose of this study was to explore the influences of university baseball athletes' psychological capital on their life stress and burnout and provide practical suggestions for athletes and coaches to reduce their life stress and burnout. In this study, we used athletes' control variables (grade, year of training experience, and training days per week) and psychological capital (self-efficacy, hope, optimism, and resilience) to predict their life stress and burnout. A total of 428 division I baseball athletes from 16 teams of the national college baseball sports league in Taiwan participated in this survey, with a return rate of 89.2%. Partial least squares structural equation modeling was used to test the relationships among the above-mentioned variables. The results showed that the athletes demographics such as grade (ß = 0.03, p > 0.05) and years of baseball training experience (ß = 0.00, p > 0.05) had no significant influences on athlete burnout, while the days of baseball training per week (ß = 0.32, p < 0.05) had a positive influence on athlete burnout. As for psychological capital, self-efficacy (ß = -0.09, p < 0.05), hope (ß = -0.27, p < 0.05), and optimism (ß = -0.20, p < 0.05) had negative influences on life stress, while resilience (ß = -0.07, p > 0.05) had no significant influences on life stress. Hope (ß = -0.20, p < 0.05) had negative influences on athlete burnout, while self-efficacy (ß = -0.00, p > 0.05), optimism (ß = -0.06, p > 0.05), and resilience (ß = -0.01, p > 0.05) had no significant influences on athlete burnout. Life stress (ß = 0.52, p < 0.05) had significant influences on the burnout. Based on our research findings, suggestions were made to reduce the athletes' life stress and athlete burnout.

Sinorhizobium meliloti ExoR is the target of periplasmic proteolysis.

Sinorhizobium meliloti ExoR regulates the production of succinoglycan and flagella through the ExoS/ChvI two-component regulatory system. ExoR has been proposed to inhibit the ExoS sensor through direct interaction in the periplasm. To understand how ExoR suppression of ExoS is relieved, which is required for the expression of ExoS/ChvI-regulated symbiosis genes, we characterized wild-type ExoR and ExoR95 mutant proteins. In addition to the previously identified precursor and mature forms of ExoR (designated ExoR(p) and ExoR(m), respectively), we detected a 20-kDa form of ExoR (designated ExoR(c20)) derived from the wild-type ExoR protein, but not from the ExoR95 mutant protein. ExoR(c20) was isolated directly from S. meliloti periplasm to identify its N-terminal amino acids and the site of the proteolysis, which is highly conserved among ExoR homologs. ExoR(c20) retains the C terminus of the wild-type ExoR. When expressed directly, ExoR(c20) did not complement the exoR95 mutation, suggesting that ExoR(c20) does not function directly in the ExoR-ExoS/ChvI regulatory pathway and that ExoR(m) is the functional form of ExoR. A single-amino-acid change (ExoRL81A) at the site of ExoR periplasmic proteolysis resulted in the reduction of the amount of ExoR(m) and the loss of the regulatory function of the ExoR protein. These findings suggest that ExoR(m) is a target of periplasmic proteolysis and that the amount of ExoR(m) could be reduced through effective proteolysis to relieve its suppression of ExoS.

The Relationships among Sport Participation Level, Flow Experience, Perceived Health Status and Depression Level of College Students.

The purpose of this study was to examine the relationships between sport participation level, flow, perceived health status and depression using gender and grades as control variables of college students in Taiwan. Based on previous research, the study established the proposed model: using sport participation level and flow experience as predicting variables, perceived health status and depression as dependent variables, and gender and grades as control variables. A total of 700 structured questionnaires were distributed to college students using convenience sampling among seven universities in Taiwan with a valid return rate of 86.5%. Structural equation modeling was used to test the relationships among the above-mentioned variables. The study found: 1. Male students had higher self-rated health perception than female students. 2. Students with higher grades perceived higher levels of depression than those with lower grades. 3. Among all variables, the level of sport participation had a positive predicting power of perceived health status and a negative predicting power of depression level; perceived health status had a negative predicting power of depression; while flow had no moderating effect among sport participation level, perceived health status and depression. In the model, the predicting variables had a predicting power of 0.58 (R2) for depression, indicating a good model. Conclusions and implications were made according to the findings of the study.

Universal Detection of Mia Antigen and Frequencies of Glycophorin Hybrids among Blood Donors in Taiwan by Human Monoclonal Antibodies against Mia (MNS7), Mur (MNS10), and MUT (MNS35) Antigens.

Glycophorin hybrids such as GP.Mur are common in Southeast Asians. In Taiwan, clinically significant alloantibodies to the GP.Mur phenotype are the most important issue in blood banks. A large-scale screening of glycophorin hybrids in the Taiwanese population is urgently needed to ensure transfusion safety. Four clones of human hybridomas that secrete anti-Mia, anti-MUT, and anti-Mur were established by fusing human B-lymphocytes and myeloma cells (JMS-3). The specificity of each monoclonal antibody (MoAb) was characterized. Three MoAbs were applied on an Automated Pretransfusion Blood Testing Analyzer (PK7300/PK7400) for donor screening. Genotyping was performed to determine the detailed subgrouping of glycophorin hybrids. Four MoAbs are IgM antibodies. Anti-Mia (377T) binds to 46DXHKRDTYA54, 48HKRDTYAAHT57 peptides, and anti-Mia (367T) binds to 43QTNDXHKRD51 peptides (X indicates T, M, or K). Anti-Mur is reactive with 49KRDTYPAHTA58 peptides. Anti-MUT is reactive with 47KHKRDTYA54. A total of 78,327 donors were screened using three MoAbs, and 3690 (4.71%) were GP.Mur, 20 (0.025%) were GP.Hut, and 18 (0.022%) were GP.Vw. When the Mia antigen was introduced as routine screening, the frequency of Mi(a+) among blood donors in Taiwan was 4.66% (67,348/1,444,541). Mia antigen was implemented as a routine blood testing, and the results were labeled on all red blood cell (RBC) units.

The efficacy of individual-donation and minipool testing to detect low-level hepatitis B virus DNA in Taiwan.

BACKGROUND: Financial constraints are the main concern in implementing nucleic acid testing (NAT) as routine blood screening in Taiwan. The PROCLEIX ULTRIO assay (Ultrio) on the TIGRIS System (Novartis Diagnostics) was evaluated for its operational performance both for individual-donation testing (IDT) and in minipools of 4 (MP4) to develop a feasible solution. STUDY DESIGN AND METHODS: Analytical sensitivity was determined by testing WHO international standards. We tested 10,290 blood donors, 4210 in IDT and 6080 in MP4. Potential hepatitis B virus (HBV) yield donors (hepatitis B surface antigen [HBsAg] negative/NAT reactive) were evaluated for up to 9 months' follow-up. Discordant results between the Ultrio assay and the HBsAg tests were further analyzed by HBV antibody serology, alternative NATs, HBV DNA quantification, and sequencing. RESULTS: The 95% limits of detection in IU/mL (95% confidence interval) were as follows: human immunodeficiency virus Type 1 (HIV-1), 18 (12-34); hepatitis C virus (HCV), 4.4 (2.8-8.9); and HBV, 6.3 (4.4-11). The retest rates were 0.55% for IDT and 0.33% for MP4. No HIV or HCV yield cases were found, while there were 12 potential HBV yield cases, nine from IDT and three from MP4 testing. Eleven of them were successfully genotyped as B2. Ten of them returned for follow-up and mostly were determined as occult HBV infection (OBI). The IDT yield rate of 9 in 4210 (0.21%) was fourfold greater than the MP4 yield rate of 3 in 6080 (0.05%; p < 0.05). CONCLUSION: The higher yield rate for IDT versus MP4 demonstrates the benefit to implement a more sensitive NAT strategy in regions having significant OBI carriers such as Taiwan.

[Screening for autoinducer synthase gene in Sinorhizobium meliloti and analysis of the autoinducer produced by recombinant expression in Escherichia coli].

The plasmid pJZ290 containing mariner transposon was used to mutagenize Sinorhizobium meliloti and 3000 transposon insertion mutants were subsequently screened for autoinducer-deficient (AI-deficient) mutants. One AI-deficient mutant YW1 was obtained by quantitative activity assay and qualitative TLC detection. In an effort to characterize the transposon insertions of autoinducer-deficient mutant YW1, we performed an arbitrary PCR and sequencing techniques to identify insertion sites. The sequence analysis result showed that the transposon inserted between the 277th bp and the 278th bp of one 621bp ORF in autoinducer-deficient mutant YW1. The 612-bp ORF encodes a putative protein of 206 amino acids that is highly homologous (99% identity) to AHL-synthase traI of Sinorhizobium medicae WSM419. Cloned into the broad host range expression vectors pYC12 and transformed into Escherichia coli DH5alpha, the putative AI synthase gene was overexpressed in E. coli, and four different autoinducers could be detected in the supernatant of the positive recombinant strain by TLC, among which the two AHL molecules that were deficient in AI-deficient mutant YW1 could be found. All of these showed that the 621bp ORF was an AI synthase gene. This study paved the way of further studying quorum sensing systems in S. meliloti.

[Recombinant expression of an autoinducer synthase of Mesorhizobium loti in Escherichia coli and analysis of the autoinducer produced in recombinant strain].

Although the autoinducers of Mesorhizobium loti could not be detected under the conditions of experimentally pure culture, and even adding some root exact of Lotus japonicus into the medium in which M. loti grew to detect the acyl-homoserine lactone (AHL) production, no positive result has been available. However, analysis of homologous comparison showed that there were at least four annotated acylated homoserine lactone (AHL) synthase genes from M. loti genome sequences. The amino acids of all these four genes showed different homologous to those of lux I , tra I and lax I all of which were AHL synthase genes and have been studied most before. By cloned into the expression vector of pET30a and transformed into Escherichia coli BL21, one of these synthase genes, m14543 was overexpressed in E. coli, and a strong 20kDa protein band could be got by SDS-PAGE of the recombinant cells. It has been detected that at least three different AHLs were produced in the recombinant strain with IPTG induced. These results indicated that M. loti has the quorum sensing system and it may work under some specific growth conditions, most likely involved in the microbe-plant interaction. This study means to give some clues to any further study of the quorum sensing system of M. loti .

[High-level expression of an extreme-thermostable xylanase B from Thermotoga maritima MSB8 in Escherichia coli and Pichia pastoris].

The family 10 xylanase (XynB) from Thermotoga maritima MSB8 is extremely thermostable and great potential in the applications of various fields of industry. The gene mxynB(64) was amplified by the method of PCR with the template of the genomic DNA of Thermotoga maritima MSB8, and cloned into the expression vectors of Escherichia coli and Pichia pastoris respectively. Xylanase B(40kD) was successfully expressed by the two heterologous protein expression systems with high-level production. The recombinant protein of XynB expressed in Pichia pastoris showed extreme thermostability and pH stability, which was optimally active at 90 degrees C and quite stable over the pH range of pH 5.0-10.8 at 70 degrees C. After incubation of the enzyme at 100 degrees C for 30 min, XynB retained 70% higher residual activity. The recombinant XynB expressed in Pichia pastoris is of great use in a variety of industrial and agricultural applications.

[Increased expression of heparanase mRNA is correlated with metastasis of gastric carcinoma and is probably correlated with c-met protein].

OBJECTIVE: To explore the expression of human heparanase mRNA and its relationship with the clinicopathological characteristics and angiogenesis and its possible mechanism in gastric carcinomas. METHODS: Expression of heparanase mRNA in gastric cancerous tissues and its corresponding adjacent tissues were detected by RT-PCR in forty-seven patients. Expression of c-met protein and microvessel density (MVD) were examined by immunohistochemcal staining. RESULTS: Expression of heparanse mRNA was significantly higher in gastric cancerous tissues than in its adjacent tissues (74.5% vs. 19.1%, P < 0.01). MVD was significantly higher in heparanase positive group than its counterpart (51 +/- 15 vs. 31 +/- 10, P < 0.01). Expression of heparanase mRNA was correlated with expression of c-met (r = 0.714, P < 0.01). The positive rate of heparanse expression in gastric carcinomas were correlated with tumor diameter, invasive depth, regional lymph nodes metastasis and TNM stage, whereas there were no relation with gender, age, tumor differentiation and remote metastasis of the patient. CONCLUSION: Increased heparanase expression contributes to metastasis and angiogenesis in gastric cancer. Heparanase mRNA expression correlates with poor clinicopathologic characteristics in its prognosis. C-met is correlated with heparanase expression.

Inhibition of tankyrase 1 in human gastric cancer cells enhances telomere shortening by telomerase inhibitors.

Telomere stability is believed to be related to aging and tumorigenesis. Besides telomerase, telomere length is also regulated by several telomere-specific binding proteins. Tankyrase 1, a telomeric poly(ADP-ribose) polymerase (PARP), elongates telomere length by inhibiting TRF1 binding to telomeres. In order to study the synergistic action of tankyrase 1 and telomerase in the maintenance of telomere length in mammalian cells, we constructed anti-sense tankyrase 1 (aTNKS) eukaryotic expression vectors and then transfected them into the SGC-7901 human gastric cancer cell line, as well as SGC-7901 cells that had been transfected with antisense hTR (7901-ahTR) and antisense hTERT (7901-ahTERT) with DOTAP liposomes. The activity of telomerase, telomere length and telomerase-associated protein activities were measure by TRAP-ELISA, Southern blot and western blot analysis, respectively, in aTNKS transfected and untransfected cells. The results demonstrated that telomere length was significantly shorter in cells with concomitant tankyrase 1 and telomerase inhibition than by either tankyrase 1 or telomerase inhibition alone, in SGC-7901 cells. We also found that aTNKS had no effect on telomerase activity. These results reveal that inhibition of tankyrase 1 could shorten telomere length and play a synergistic role with telomerase inhibitors in telomere length shortening in the SGC-7901 gastric cancer cell line. Co-inhibition of tankyrase 1 and telomerase activity may be a rational strategy for telomere-directed gastric cancer therapeutics.

Three missense mutations, including a novel 860C>T transition, and allelic enhancement phenomenon associated with ABO blood subgroups A in Taiwan.

BACKGROUND: It was estimated that approximately 25 percent of Taiwanese residents were ABO blood group A. Many subgroups of A, however, revealed ambiguous serologic typing results. This study aimed to delineate the molecular basis of the A3, Ax, and weak A phenotypes. STUDY DESIGN AND METHODS: Serologic analyses including adsorption and elution assay, serum transferases activity assay, and saliva test were performed to determine the unique phenotypes of these samples. DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism were performed to further investigate the relationships between the genetic characteristics and phenotypic features of these samples. RESULTS: Three single-nucleotide transitions (745C>T, 820G>A, and a novel 860C>T) were found in nine A3/A3B cases. In addition, the Ax and A3B subjects shared the same 860C>T mutation. This A(x) allele with 860C>T transition expressed A3B phenotype in A(x)/B101 heterozygote but Ax phenotype in A(x)/O01 heterozygote. This allelic enhancement was also observed in the weak A family with Aw05 allele, which was previously not found in Taiwan. CONCLUSION: This allelic enhancement phenomenon was prone to cause serologic discrepancy between parents and children. Genotyping could help us to resolve this problem. Thus, a novel mutation is reported among Taiwanese blood donors.