Genotyping of the ABCG2 gene using Matrix-Associated Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry.

OBJECTIVES: The aim of this study was to evaluate the applicability of genotyping of the ABCG2 gene using MALDI-TOF MS and to estimate the allele frequency in the Japanese population. BACKGROUND: Jr (a-) phenotype has a prevalence of approximately 0·05% among Japanese blood donors; DNA-based genotyping was conducted to investigate the molecular basis of the Jr (a-) phenotype along with serological typing. To detect all SNPs of the ABCG2 gene, a high-throughput SNP genotyping platform is needed. METHODS: Overall, 1004 Jr (a-) blood samples were collected from blood donors in Japan and pre-genotyped. To detect the SNPs of the ABCG2 gene using MALDI-TOF MS, polymerase chain reaction and unextend primer were designed. In total, 205 Jr (a-) samples were genotyped using MALDI-TOF MS analysis. RESULTS: The SNPs of 1004 Jr (a-) samples were identified using the HRM analysis and DNA sequencing, and 799 of 1004 (80%) Jr (a-) samples had the homozygous for c.376 T. The designed primers for MALDI-TOF MS perfectly detected the SNPs of the ABCG2 gene. A total of 205 Jr (a-) samples were genotyped using MALDI-TOF MS. Calling failures occurred in only two samples with the mutations c.736CT to c.376C and c.421C to c.421CA. The concordance rate between the pre-genotyped and MALDI-TOF MS-based genotyping results was very high (99·02%) for all ABCG2 alleles. CONCLUSIONS: Jr (a-) Japanese donors had almost the homozygous for c.376 T. However, detections of more than 20 SNPs of the ABCG2 gene for the JR blood group genotyping are needed. MALDI-TOF MS-based genotyping was highly concordant with the pre-genotyped results for all ABCG2 alleles.

International society of blood transfusion working party on red cell immunogenetics and terminology: report of the Seoul and London meetings.

The Working Party has met twice since the last report: in Seoul, South Korea 2014, and in London, UK 2015, both in association with the International Society of Blood Transfusion (ISBT) Congress. As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. Eleven new blood group antigens were added to seven blood group systems. This brings the current total of blood group antigens recognized by the ISBT to 346, of which 308 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known blood group system.

Angular Structure of the In-Medium QCD Cascade.

We study the angular broadening of a medium-induced QCD cascade. We derive the equation that governs the evolution of the average transverse momentum squared of the gluons in the cascade as a function of the medium length, and we solve this equation analytically. Two regimes are identified. For a medium of a not too large size, and for not too soft gluons, the transverse momentum grows with the size of the medium according to standard momentum broadening. The other regime, visible for a medium of a sufficiently large size and very soft gluons, is a regime dominated by multiple branchings: there, the average transverse momentum saturates to a value that is independent of the size of the medium. This structure of the in-medium QCD cascade is, at least qualitatively, compatible with the recent LHC data on dijet asymmetry.

Comparison of neutralization profiles for anti-HCV reactive donor samples with or without detectable HCV RNA.

BACKGROUND AND OBJECTIVES: At Japanese Red Cross (JRC) Blood Centers, all donated blood is screened for hepatitis C virus (HCV) by serological and nucleic acid amplification testing. Donor plasma that tested reactive for anti-HCV by serological test is disqualified even if the donor tests negative for HCV RNA. These test results reflect both true-positive results because of past HCV infection and false-positive results because the cross-reactivity of plasma IgG, which current testing methods are unable to distinguish. To characterize these antibody test results, we examined the neutralizing activity of these plasma samples. MATERIAL AND METHODS: Donor plasma samples that tested reactive for anti-HCV by serological test but negative for HCV RNA (n = 43) were analysed for determining their neutralizing activities measured by the inhibition of the cellular entry of pseudoparticles harbouring HCV envelope glycoproteins (HCVpp). RESULTS: Strong and broad neutralizing activities against HCVpp entry similar to the samples that tested reactive for anti-HCV serological test and positive for HCV RNA (considered to be derived from individuals with chronic HCV infection) were observed in three of 43 plasma samples from donors who tested anti-HCV reactive but HCV RNA negative. CONCLUSION: By examining the neutralizing activities of plasma samples, we identified individuals with a past HCV infection from those in whom we were unable to confirm HCV infection according to the current testing algorithms of JRC, which do not perform anti-HCV confirmatory tests.

A hollow-fibre column system to effectively prepare washed platelets.

BACKGROUND AND OBJECTIVES: We developed a hollow-fibre column system specifically adapted to prepare washed platelet concentrates (WPCs). This study was performed to evaluate the efficacy of the hollow-fibre column system for preparing WPCs. MATERIALS AND METHODS: First, the percentages of platelet (PLT) recovery and remaining plasma proteins were calculated by determining the PLT count, volume and plasma protein levels in both the prewash and postwash. Secondly, washed PLTs and unwashed control PLTs were stored for 5 days, and the changes during this 5-day storage of in vitro PLT characteristics were determined. RESULTS: The hollow-fibre column system effectively removed >98% of plasma in platelet concentrates (PCs), and the PLT recovery was 97% on an average. The CD62P-expression level on washed PLTs immediately after washing was approximately twofold higher than that on prewashed PLTs as well as on PLTs washed via manual methods or cell washing devices. Until day 5 during storage, PLT aggregability, hypotonic shock response and swirling scores of washed PLTs were not significantly different from those of the control PCs. CONCLUSION: Our novel hollow-fibre column system proved valuable in preparing washed PLTs with <2% of residual plasma proteins and high recovery of PLTs.

The influence of season and air temperature on water intake by food groups in a sample of free-living Japanese adults.

BACKGROUND/OBJECTIVES: To examine the influence of season and climate (air temperature and humidity) on water intake by the food group in a sample of free-living Japanese adults. SUBJECTS/METHODS: Four-nonconsecutive-day, semi-weighed dietary records were collected from each of the four seasons in a single 12-month period (16 days in total). The influence of season and climate on individual water intake by the food group was analyzed using a mixed linear model. Participants were 242 healthy adults (121 women aged 30-69 years and 121 men aged 30-76 years) from four areas in Japan. RESULTS: For women and men together, the mean total water intake was 2230 g/day (highest in summer: 2331 g/day; lowest in winter: 2134 g/day). Fifty-one percent of water was derived from foods and the rest from beverages. In a mixed linear model adjusted for sex, age and body mass index, intake of water from foods decreased by 3.1 g/day and that from beverages increased by 8.4 g/day, with an increase in the mean outdoor air temperature on the survey day of 1 °C (both P < 0.0001). The influence of humidity was nonsignificant. CONCLUSIONS: In contrast to previous findings in Western countries, half of water intake in Japanese adults was derived from foods. Water intake from beverages was positively associated with air temperature, whereas that from foods was inversely associated with air temperature.

International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Cancun report (2012).

The International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology convened during the International congress in Cancun, July 2012. This report details the newly identified antigens in existing blood group systems and presents three new blood group systems.

Evaluation of a blood group genotyping platform (BLOODchip(®) Reference) in Japanese samples.

BACKGROUND: Blood-group genotyping arrays have been widely used in Caucasian and African American populations, but have not been thoroughly tested in Japanese subjects. AIM: To evaluate, using the BLOODchip(®) Reference genotyping system, the concordance of previously typed samples with expected phenotypes and the coverage of the Japanese variants. METHODS: Blood samples from 100 Japanese donors were obtained. DNA was extracted with QIAsymphony (Qiagen, Hilden, Germany). Samples were typed by serological methods and processed with the BLOODchip(®) . When a non-concordant result was identified, further sequencing by polymerase chain reaction-single specific primer (PCR-SSP) was performed. RESULTS: Concordance between systems was 98% (736/751), and 98.8% (742/751) if only non-software-related non-concordances were considered. In the ABO group, 6 'No Call' (NC, inability of the BLOODchip(®) to assign a result) were ascribed to a variant of blood subtype A1 (A102; 467C>T), a common subtype in Asian populations, whereas three NC presented additional polymorphisms not contained in the BLOODchip(®) (A102/A205, A102/O06 and A204/O02). In the RhD group, one discrepancy was correctly genotyped as RHD*1227A (Del phenotype) by the BLOODchip(®) (phenotyped as partial D, RHD*DIVb). Another was phenotyped as D+ by the BLOODchip(®) (phenotyped weak D by serology) and confirmed as RHD*D-CE(2)-D heterozygous by sequencing. The 3 RhD NC can be solved by further software update. For RhCE, one discrepancy was correctly genotyped for both systems; however, only the BLOODchip(®) was able to detect RHCE*CX allele. CONCLUSIONS: By programming the A102 ABO variant into the system software with the new allele combinations, the BLOODchip(®) Reference is a suitable genotyping tool to be applied to Asian samples.

Medium-induced QCD cascade: democratic branching and wave turbulence.

We study the average properties of the gluon cascade generated by an energetic parton propagating through a quark-gluon plasma. We focus on the soft, medium-induced emissions which control the energy transport at large angles with respect to the leading parton. We show that the effect of multiple branchings is important. In contrast with what happens in a usual QCD cascade in vacuum, medium-induced branchings are quasidemocratic, with offspring gluons carrying sizable fractions of the energy of their parent gluon. This results in an efficient mechanism for the transport of energy toward the medium, which is akin to wave turbulence with a scaling spectrum ~1/sqrt[ω]. We argue that the turbulent flow may be responsible for the excess energy carried by very soft quanta, as revealed by the analysis of the dijet asymmetry observed in Pb-Pb collisions at the LHC.

Skin autofluorescence is associated with severity of vascular complications in Japanese patients with Type 2 diabetes.

AIMS: Skin autofluorescence, a non-invasive measure of the accumulation for advanced glycation end products, has been reported to be a useful marker for diabetic vascular risks in the Caucasian population. The aim of this study was to evaluate associations between skin autofluorescence and vascular complications in non-Caucasian patients with Type 2 diabetes. METHODS: Subjects in this cross-sectional study comprised 130 Japanese patients with Type 2 diabetes. Skin advanced glycation end products were assessed by skin autofluorescence using an autofluorescence reader. Association between skin autofluorescence and severity of vascular complications was evaluated. RESULTS: Of the 130 patients, 60 (46.2%) had microvascular complications such as diabetic retinopathy, neuropathy and nephropathy, 10 (7.7%) had macrovascular complications and 63 (48.5%) had micro- and/or macrovascular complications. Skin autofluorescence increased with severity of vascular complications. Independent determinants of skin autofluorescence were age (ß = 0.24, P < 0.01), mean HbA(1c) in previous year (ß = 0.17, P = 0.03), microvascular complications (ß = 0.44, P < 0.01) and macrovascular complications (ß = 0.27, P < 0.01). Multiple logistic regression analysis revealed that diabetes duration (odds ratio 1.15, P < 0.01), systolic blood pressure (odds ratio 1.04, P = 0.01), skin autofluorescence (odds ratio 3.62, P = 0.01) and serum albumin (odds ratio 0.84, P < 0.01) were independent factors for the presence of vascular complications in these patients. CONCLUSIONS: Skin autofluorescence had independent effects on vascular complications in Japanese patients with Type 2 diabetes. This indicates that skin advanced glycation end products are a surrogate marker for vascular risk and a non-invasive autofluorescence reader may be a useful tool to detect high-risk cases in non-Caucasian patients with diabetes.

Significant background rates of HBV and HCV infections in patients and risks of blood transfusion from donors with low anti-HBc titres or high anti-HBc titres with high anti-HBs titres in Japan: a prospective, individual NAT study of transfusion-transmitted HBV, HCV and HIV infections.

BACKGROUND: The Japanese Red Cross (JRC) conducted a prospective study to evaluate the frequency of transfusion-transmitted HBV, HCV and HIV infections to assess the risk of transfusion of blood components routinely supplied to hospitals. STUDY DESIGN AND METHODS: Post-transfusion specimens from patients at eight medical institutes were examined for evidence of infection with HBV (2139 cases), HCV (2091) and HIV (2040) using individual nucleic acid amplification testing (NAT). If these specimens were reactive, pre-transfusion specimens were also examined for the virus concerned by individual NAT. In the event that the pre-transfusion specimen was non-reactive, then all repository specimens from implicated donors were tested for the viruses by individual donation NAT. In addition, a further study was carried out to evaluate the risk of transfusion of components from donors with low anti-HBc titres or high anti-HBc with high anti-HBs titres. RESULTS: Transfusion-transmitted HCV and HIV infections were not observed. One case of post-transfusion HBV infection was identified (rate, 0·0004675; 95% CI for the risk of transmission, 1 in 451-41,841). The background rates of HBV, HCV and HIV infections in patients prior to transfusion were 3·4% (72/2139), 7·2% (150/2091) and 0% (0/2040), respectively. Sixty-four anti-HBc- and/or anti-HBs-reactive blood components were transfused to 52 patients non-reactive for anti-HBc or anti-HBs before and after transfusion (rate, 0; 95% CI for the risk of transmission, <1 in 22). CONCLUSION: This study demonstrated that the current criteria employed by JRC have a low risk, but the background rates of HBV and HCV infections in Japanese patients are significant.

Processing of high-molecular-weight form adrenocorticotropin in human adrenocorticotropin-secreting tumor cell line (DMS-79) after transfection of prohormone convertase 1/3 gene.

Ectopic ACTH-producing tumors preferentially secrete biologically inactive ACTH precursors and ACTH-related fragments. DMS-79 is known to secrete unprocessed high-molecular-weight (HMW) form ACTH. To determine whether prohormone convertase (PC) 1/3 is involved in the abnormal processing of proopiomelanocortin (POMC), we studied whether PC1/3 and 2 genes are expressed in DMS-79, and whether overexpression of PC1/3 gene affects POMC processing pattern. Steady-state mRNA levels of PC1/3 and 2 were determined by real-time RT-PCR. Molecular weights of ACTH-related peptides were determined by chromatographical analyses coupled with ACTH and beta-endorphin (beta-END) radioimmunoassays. PC1/3 gene was transfected into DMS-79 by retrovirus transduction using pMX-IP vector encoding PC1/3 cDNA. The steady-state mRNA levels of PC1/3 and 2 in DMS-79 were lower than those in ACTH-secreting and nonfunctioning pituitary tumors. DMS-79 predominantly secreted HMW form with both ACTH and beta-END immunoreactivities by size-exclusion chromatography. After purification by immunoaffinity chromatography with anti-ACTH antibody, the apparent molecular weight of HMW form ACTH was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining. After retroviral transfection of PC1/3 cDNA into DMS-79 and puromycin selection, PC1/3 stably-expressing cell line (DMS-79T) secreted two immunoreactive ACTH components, a major one coeluting with ACTH(1-39) and a minor one as a HMW form as well as two beta- END immunoreactive components coeluting with beta-lipotropic hormone and beta-END, respectively. Thus, we have established PC1/3 stably-expressing cell line (DMS-79T) capable of proteolytically processing ACTH precursor molecule(s) into mature ACTH and beta-END.