A New Strategy To Identify Rare Blood Donors: Single Polymerase Chain Reaction Multiplex Snapshot Reaction For Detection Of 16 Blood Group Alleles

Background. As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology. Materials and methods. The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism. Results. We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA. Discussion. This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood. © SIMTI Servizi Srl.12SUPPL.1s256s263Jungbauer, C., Routine use of DNA testing for red cell antigens in blood centres (2011) Transfus Apher Sci, 45, pp. 61-68Nance, S.T., How to find, recruit and maintain rare blood donors (2009) Curr Opin Hematol, 16, pp. 503-508Veldhuisen, B., Van Der Schoot, C.E., De Haas, M., Blood group genotyping: From patient to high-throughput donor screening (2009) Vox Sang, 97, pp. 198-206Moulds, J.M., Future of molecular testing for red blood cell antigens (2010) Clin Lab Med, 30, pp. 419-429Patnaik, S.K., Helmberg, W., Blumenfeld, O.O., BGMUT: NCBI dbRBC database of allelic variations of genes encoding antigens of blood group systems (2012) Nucleic Acids Res, 40, pp. D1023-D1029Vallone, P.M., Butler, J.M., AutoDimer: A screening tool for primer-dimer and hairpin structures (2004) Biotechniques, 37, pp. 226-231Baleotti Jr., W., Rios, M., Reid, M.E., Dombrock gene analysis in Brazilian people reveals novel alleles (2006) Vox Sang, 91, pp. 81-87Rios, M., Hue-Roye, K., Oyen, R., Insights into the Holleyand Joseph- phenotypes (2002) Transfusion, 42, pp. 52-58Baleotti Jr., W., Rios, M., Reid, M.E., A novel DI*A allele without the Band 3-Memphis mutation in Amazonian Indians (2003) Vox Sang, 84, pp. 326-330Arnoni, C., Latini, F.R.M., Person, R.M., Padronização das técnicas de PCR-RFLP para genotipagem dos alelos KEL*3/ KEL*4 e KEL*5/KEL*6 (2011) Rev Bras Hematol Hemoter, 33 (SUPPL.2), pp. 332-488Baleotti Jr., W., Suzuki, R.B., Ruiz, M., A PCR-RFLP strategy for Wright typing (2011) Rev Bras Hematol Hemoter, 33 (SUPPL. 2), pp. 332-488Brazilian Real - United States Dollar Exchange Rate from Central Bank of Brazil, , http://www4.bcb.gov.br/pec/taxas, April 1st to April 30th, 27/03/2013Daniels, G., The molecular genetics of blood group polymorphism (2009) Hum Genet, 126, pp. 729-742Logdberg, L., Reid, M.E., Zelinski, T., Human blood group genes 2010: Chromosomal locations and cloning strategies revisited (2011) Transfus Med Rev, 25, pp. 36-46Di Cristofaro, J., Silvy, M., Chiaroni, J., Bailly, P., Single PCR multiplex SNaPshot reaction for detection of eleven blood group nucleotide polymorphisms: Optimization, validation, and one year of routine clinical use (2010) J Mol Diagn, 12, pp. 453-460Ferri, G., Pelotti, S., Multiplex ABO genotyping by minisequencing (2009) Methods Mol Biol, 496, pp. 51-58Palacajornsuk, P., Halter, C., Isakova, V., Detection of blood group genes using multiplex SNaPshot method (2009) Transfusion, 49, pp. 740-749Silvy, M., Simon, S., Gouvitsos, J., Weak D and DEL alleles detected by routine SNaPshot genotyping: Identification of four novel RHD alleles (2011) Transfusion, 51, pp. 401-411Silvy, M., Di Cristofaro, J., Beley, S., Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by multiplex SNaPshot and fragment assays: A transfusion support for sickle cell disease patients (2011) Br J Haematol, 154, pp. 260-270Pastinen, T., Kurg, A., Metspalu, A., Minisequencing: A specific tool for DNA analysis and diagnostics on oligonucleotide arrays (1997) Genome Res, 7, pp. 606-614Syvanen, A.C., From gels to chips: "Minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms (1999) Hum Mutat, 13, pp. 1-10Information notebook (2011) Blood and Hemoderivates Brasília, , Ministério da Saúde. Secretaria de Atenção à Saúde. Coordenação-Geral de Sangue e Hemoderivados. Hemotherapy production. Unified Health System - SUS Brazil - (Public and private contractors). Private non-contracted services by Unified Health System (SUS Brazil). 4th edSantos, N.P., Ribeiro-Rodrigues, E.M., Ribeiro-Dos-Santos, A.K., Assessing individual interethnic admixture and population substructure using a 48-insertion-deletion (INSEL) ancestry-informative marker (AIM) panel (2010) Hum Mutat, 31, pp. 184-190Storry, J.R., Human blood groups: Inheritance and importance in transfusion medicine (2003) J Infus Nurs, 26, pp. 367-37

Self-similar expansion of the density profile in a turbulent Bose-Einstein condensate

In a recent study we demonstrated the emergence of turbulence in a trapped Bose-Einstein condensate of Rb-87 atoms. An intriguing observation in such a system is the behavior of the turbulent cloud during free expansion.The aspect ratio of the cloud size does not change in the way one would expect for an ordinary non-rotating (vortex-free) condensate. Here we show that the anomalous expansion can be understood, at least qualitatively, in terms of the presence of vorticity distributed throughout the cloud, effectively counteracting the usual reversal of the aspect ratio seen in free time-of-flight expansion of non-rotating condensates.Comment: 8 pages, 4 figure

Arylesterase activity is associated with antioxidant intake and paraoxonase-1 (PON1) gene methylation in metabolic syndrome patients following an energy restricted diet

The arylesterase (ARE) activity linked to the paraoxonase-1 (PON1) gene is known to protect lipoproteins from oxidation and provide defense against metabolic syndrome (MetS) and cardiovascular diseases. The epigenetic regulation of enzymatic activities is gaining importance nowadays. This research aimed to assess the potential relationships between the ARE activity with the methylation levels of the PON1 gene transcriptional regulatory region, anthropometrics, biochemical markers and antioxidant dietary components. Forty-seven subjects (47 ± 10 y.o; BMI 36.2 ± 3.8 kg/m2; 46.8 % female) with MetS features, who followed a sixmonth energy-restricted dietary weight-loss intervention, were included in this study (www.clinicaltrials.gov; NCT01087086). Anthropometric, biochemical, enzymatic and dietary data were assessed using validated procedures. PON1 transcriptional regulatory region methylation was analyzed by a microarray technical approach. Volunteers reduced ARE activity in parallel with body weight (p = 0.005), BMI (p = 0.006), total fat mass (p = 0.020), diastolic blood pressure (p = 0.018), mean blood pressure (p = 0.022) and triglycerides (p = 0.014). Methylation levels of some CpG sites of the PON1 gene correlated negatively with ARE activity (p < 0.05). Interestingly, dietary vitamin C (p = 0.001), tocopherols (p = 0.009) and lycopene (p = 0.038) were positively associated with ARE activity and showed an inverse correlation (p = 0.004, p = 0.029 and p = 0.021, respectively) with the methylation of some selected CpG sites of the PON1 gene. In conclusion, ARE activity decreased in parallel with MetS-related markers associated to the energy restriction, while dietary antioxidants might enhance the ARE activity by lowering the PON1 gene methylation in patients with MetS features

Black Holes from Cosmic Rays: Probes of Extra Dimensions and New Limits on TeV-Scale Gravity

If extra spacetime dimensions and low-scale gravity exist, black holes will be produced in observable collisions of elementary particles. For the next several years, ultra-high energy cosmic rays provide the most promising window on this phenomenon. In particular, cosmic neutrinos can produce black holes deep in the Earth's atmosphere, leading to quasi-horizontal giant air showers. We determine the sensitivity of cosmic ray detectors to black hole production and compare the results to other probes of extra dimensions. With n \ge 4 extra dimensions, current bounds on deeply penetrating showers from AGASA already provide the most stringent bound on low-scale gravity, requiring a fundamental Planck scale M_D > 1.3 - 1.8 TeV. The Auger Observatory will probe M_D as large as 4 TeV and may observe on the order of a hundred black holes in 5 years. We also consider the implications of angular momentum and possible exponentially suppressed parton cross sections; including these effects, large black hole rates are still possible. Finally, we demonstrate that even if only a few black hole events are observed, a standard model interpretation may be excluded by comparison with Earth-skimming neutrino rates.Comment: 30 pages, 18 figures; v2: discussion of gravitational infall, AGASA and Fly's Eye comparison added; v3: Earth-skimming results modified and strengthened, published versio

Sensitivity of the IceCube Detector to Astrophysical Sources of High Energy Muon Neutrinos

We present the results of a Monte-Carlo study of the sensitivity of the planned IceCube detector to predicted fluxes of muon neutrinos at TeV to PeV energies. A complete simulation of the detector and data analysis is used to study the detector's capability to search for muon neutrinos from sources such as active galaxies and gamma-ray bursts. We study the effective area and the angular resolution of the detector as a function of muon energy and angle of incidence. We present detailed calculations of the sensitivity of the detector to both diffuse and pointlike neutrino emissions, including an assessment of the sensitivity to neutrinos detected in coincidence with gamma-ray burst observations. After three years of datataking, IceCube will have been able to detect a point source flux of E^2*dN/dE = 7*10^-9 cm^-2s^-1GeV at a 5-sigma significance, or, in the absence of a signal, place a 90% c.l. limit at a level E^2*dN/dE = 2*10^-9 cm^-2s^-1GeV. A diffuse E-2 flux would be detectable at a minimum strength of E^2*dN/dE = 1*10^-8 cm^-2s^-1sr^-1GeV. A gamma-ray burst model following the formulation of Waxman and Bahcall would result in a 5-sigma effect after the observation of 200 bursts in coincidence with satellite observations of the gamma-rays.Comment: 33 pages, 13 figures, 6 table

Update on the correlation of the highest energy cosmic rays with nearby extragalactic matter

Data collected by the Pierre Auger Observatory through 31 August 2007 showed evidence for anisotropy in the arrival directions of cosmic rays above the Greisen-Zatsepin-Kuz'min energy threshold, \nobreak{$6\times 10^{19}$eV}. The anisotropy was measured by the fraction of arrival directions that are less than $3.1^\circ$ from the position of an active galactic nucleus within 75 Mpc (using the V\'eron-Cetty and V\'eron $12^{\rm th}$ catalog). An updated measurement of this fraction is reported here using the arrival directions of cosmic rays recorded above the same energy threshold through 31 December 2009. The number of arrival directions has increased from 27 to 69, allowing a more precise measurement. The correlating fraction is $(38^{+7}_{-6})$, compared with $21$ expected for isotropic cosmic rays. This is down from the early estimate of $(69^{+11}_{-13})$. The enlarged set of arrival directions is examined also in relation to other populations of nearby extragalactic objects: galaxies in the 2 Microns All Sky Survey and active galactic nuclei detected in hard X-rays by the Swift Burst Alert Telescope. A celestial region around the position of the radiogalaxy Cen A has the largest excess of arrival directions relative to isotropic expectations. The 2-point autocorrelation function is shown for the enlarged set of arrival directions and compared to the isotropic expectation.Comment: Accepted for publication in Astroparticle Physics on 31 August 201

Advanced functionality for radio analysis in the Offline software framework of the Pierre Auger Observatory

The advent of the Auger Engineering Radio Array (AERA) necessitates the development of a powerful framework for the analysis of radio measurements of cosmic ray air showers. As AERA performs "radio-hybrid" measurements of air shower radio emission in coincidence with the surface particle detectors and fluorescence telescopes of the Pierre Auger Observatory, the radio analysis functionality had to be incorporated in the existing hybrid analysis solutions for fluoresence and surface detector data. This goal has been achieved in a natural way by extending the existing Auger Offline software framework with radio functionality. In this article, we lay out the design, highlights and features of the radio extension implemented in the Auger Offline framework. Its functionality has achieved a high degree of sophistication and offers advanced features such as vectorial reconstruction of the electric field, advanced signal processing algorithms, a transparent and efficient handling of FFTs, a very detailed simulation of detector effects, and the read-in of multiple data formats including data from various radio simulation codes. The source code of this radio functionality can be made available to interested parties on request.Comment: accepted for publication in NIM A, 13 pages, minor corrections to author list and references in v

Search for First Harmonic Modulation in the Right Ascension Distribution of Cosmic Rays Detected at the Pierre Auger Observatory

We present the results of searches for dipolar-type anisotropies in different energy ranges above $2.5\times 10^{17}$ eV with the surface detector array of the Pierre Auger Observatory, reporting on both the phase and the amplitude measurements of the first harmonic modulation in the right-ascension distribution. Upper limits on the amplitudes are obtained, which provide the most stringent bounds at present, being below 2% at 99% $C.L.$ for EeV energies. We also compare our results to those of previous experiments as well as with some theoretical expectations.Comment: 28 pages, 11 figure