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

Horizontal Branch Stars: The Interplay between Observations and Theory, and Insights into the Formation of the Galaxy

We review HB stars in a broad astrophysical context, including both variable and non-variable stars. A reassessment of the Oosterhoff dichotomy is presented, which provides unprecedented detail regarding its origin and systematics. We show that the Oosterhoff dichotomy and the distribution of globular clusters (GCs) in the HB morphology-metallicity plane both exclude, with high statistical significance, the possibility that the Galactic halo may have formed from the accretion of dwarf galaxies resembling present-day Milky Way satellites such as Fornax, Sagittarius, and the LMC. A rediscussion of the second-parameter problem is presented. A technique is proposed to estimate the HB types of extragalactic GCs on the basis of integrated far-UV photometry. The relationship between the absolute V magnitude of the HB at the RR Lyrae level and metallicity, as obtained on the basis of trigonometric parallax measurements for the star RR Lyrae, is also revisited, giving a distance modulus to the LMC of (m-M)_0 = 18.44+/-0.11. RR Lyrae period change rates are studied. Finally, the conductive opacities used in evolutionary calculations of low-mass stars are investigated. [ABRIDGED]Comment: 56 pages, 22 figures. Invited review, to appear in Astrophysics and Space Scienc

Efeitos da nutrição mineral sobre o crescimento, aspecto, composição elementar e fixação de nitrogênio em Azolla

The mineral nutrition of Azolla feliculoides Lam was studied in solutions deficient in phosphorus, potassium, calcium, magnesium, sulfur, iron and molybdenum, and in excess of Mn and Al. Dry weight, N2 fixation and mineral composition of Azolla were determined after 3 weeks. Phosphorus, potassium, calcium and magnesium deficiencies and excess of manganese and aluminium decressed growth severely and also depressed the activity of nitrogenase. Phosphorus deficiency improved the uptake of iron and zinc. Potassium deficiency increased the levels of phosphorus in dry matter. Magnesium deficiency caused lower uptake of K and better uptake of Ca, Fe and Mn. Sulfur deficiency reduced aluminium uptake and promoted the best growth. Positive correlations were found between: N content and dry matter, nitrogenase activity and N content.Azolla filiculoides Lam foi cultivado em solução nutritiva arejada, sempre desprovida de N combinado, sendo submetida aos seguintes tratamentos: omissão de P, K, Ca, Mg, S, Fe e Mo, excesso de Mn e Al. As plantas foram colhidas depois de 3 semanas da inoculação. Verificou-se que as deficiências de P, K, Ca e Mg provocaram diminuição na produção de matéria seca e na atividade de nitrogenase. A análise mineral mostrou que: a falta de um elemento provoca redução no seu teor; grande acumulo de Mo; diminuição no teor de Al (do inóculo ou contaminação) no tratamento menos S que garantiu o maior crescimento; efeitos inibitórios ou sinergísticos semelhantes aos descritos no caso de plantas superiores. A toxidez de Al e Mn causou, principalmente a primeira, redução no crescimento e na atividade da nitrogenase. Houve correlações positivas entre: N total e crescimento, atividade de nitrogenase e N total

Macrófitas aquáticas do sistema lacustre do Vale do Rio Doce, Minas Gerais, Brasil

Resumo Esta pesquisa trata da composição e da ocorrência de espécies de macrófitas aquáticas em área de proteção ambiental e áreas não protegidas, que compõem o conjunto de lagos do Vale do Rio Doce em Minas Gerais, terceiro maior sistema lacustre brasileiro. As informações foram levantadas a partir de publicações, material depositado em herbários e coletas botânicas entre os anos de 2007 e 2010, em ambientes aquáticos localizados no Parque Estadual do Rio Doce (PERD) e zona de amortecimento. Foram registradas 184 espécies pertencentes a distintos grupos taxonômicos, hábitos e formas biológicas, sendo aqui proposta a criação de uma nova categoria destas, designada embalsada, para contemplar plantas que se estabelecem em ilhas flutuantes. A pesquisa contribuiu com 152 novas citações para o Vale do Rio Doce em Minas Gerais, com dois primeiros registros nesse estado e com a descrição de uma espécie inédita para a ciência. A similaridade florística entre áreas protegidas e não protegidas indicou que o PERD guarda 74% das espécies de macrófitas aquáticas encontradas. Entretanto, 26% do total de espécies estão desprotegidas, pois não ocorrem nessa unidade de conservação