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Background In vitro cell-free systems for protein expression with extracts from prokaryotic ( Escherichia coli ) or eukaryotic (wheat germ) cells coupled to solid matrices have offered a valid approach for antigen discovery in malaria research. However, no comparative analysis of both systems is presently available nor the usage of suspension array technologies, which offer nearly solution phase kinetics. Methods Five Plasmodium vivax antigens representing leading vaccine candidates were expressed in the E. coli and wheat germ cell-free systems at a 50 μl scale. Products were affinity purified in a single-step and coupled to luminex beads to measure antibody reactivity of human immune sera. Results Both systems readily produced detectable proteins; proteins produced in wheat germ, however, were mostly soluble and intact as opposed to proteins produced in E. coli , which remained mostly insoluble and highly degraded. Noticeably, wheat germ proteins were recognized in significantly higher numbers by sera of P. vivax patients than identical proteins produced in E. coli . Conclusions The wheat germ cell-free system offers the possibility of expressing soluble P. vivax proteins in a small-scale for antigen discovery and immuno-epidemiological studies using suspension array technology.

P. vivax infection during pregnancy has been associated with poor outcomes such as anemia, low birth weight and congenital malaria, thus representing an important global health problem. However, no vaccine is currently available for its prevention. Vir genes were the first putative virulent factors associated with P. vivax infections, yet very few studies have examined their potential role as targets of immunity. We investigated the immunogenic properties of five VIR proteins and two long synthetic peptides containing conserved VIR sequences (PvLP1 and PvLP2) in the context of the PregVax cohort study including women from five malaria endemic countries: Brazil, Colombia, Guatemala, India and Papua New Guinea (PNG) at different timepoints during and after pregnancy. Antibody responses against all antigens were detected in all populations, with PNG women presenting the highest levels overall. P. vivax infection at sample collection time was positively associated with antibody levels against PvLP1 (fold-increase: 1.60 at recruitment -first antenatal visit-) and PvLP2 (fold-increase: 1.63 at delivery), and P. falciparum co-infection was found to increase those responses (for PvLP1 at recruitment, fold-increase: 2.25). Levels of IgG against two VIR proteins at delivery were associated with higher birth weight (27 g increase per duplicating antibody levels, p<0.05). Peripheral blood mononuclear cells from PNG uninfected pregnant women had significantly higher antigen-specific IFN-γ TH1 responses (p=0.006) and secreted less pro-inflammatory cytokines TNF and IL-6 after PvLP2 stimulation than P. vivax-infected women (p<0.05). These data demonstrate that VIR antigens induce the natural acquisition of antibody and T cell memory responses that might be important in immunity to P. vivax during pregnancy in very diverse geographical settings.

Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.

O nitrogênio é o nutriente extraído em maior quantidade pelo milho, o de manejo mais complexo e o que mais onera o custo de produção da cultura. O objetivo deste trabalho foi avaliar a viabilidade econômica de diferentes doses e parcelamentos da adubação nitrogenada, na forma de uréia, na cultura do milho em LATOSSOLO VERMELHO Eutrófico. O estudo foi conduzido na Estação Experimental do Pólo Regional do Desenvolvimento dos Agronegócios do Noroeste Paulista (APTA), em Votuporanga, Estado de São Paulo. O delineamento experimental foi o de blocos casualizados com nove tratamentos e quatro repetições, constituído de cinco doses de N: 0, 55, 95, 135 e 175 kg ha¹, aplicandose 15 kg ha¹na semeadura e o restante em cobertura, combinadas a diferentes fracionamentos em cobertura: 40 e 80 kg aos 40 dias após a semeadura (DAS) ou ½ + ½ aos 20 e 40 DAS; 120 kg ha¹ de N fracionados em ½ + ½ ou ⅓ + ⅓ + ⅓ aos 20, 40 ou 60 DAS; 160 kg ha¹ de N parcelado sem ¼ + ⅜ + ⅜ ou ¼ + ¼ + ¼ + ¼ aos 20, 40, 60 ou 80 DAS. A aplicação de 135 kg ha¹ de N parcelada em três vezes proporcionou maior relação benefício/custo. A não aplicação de N proporcionou menor retorno econômico, demonstrando ser inviável.

Nitrogen is the nutrient that is most absorbed by the corn crop, with the most complex management, and has the highest share on the cost of corn production. The objective of this work was to evaluate the economic viability of different rates and split-applications of nitrogen fertilization, as such as urea, in the corn crop in a eutrophic Red Latosol (Oxisol). The study was carried out in the Experimental Station of the Regional Pole of the Sao Paulo Northwest Agribusiness Development (APTA), in Votuporanga, State of Sao Paulo, Brazil. The experimental design was randomized complete blocks with nine treatments and four replications, consisting of five N rates: 0, 55, 95, 135 and 175 kg [ha.sup.-1], 15 kg [ha.sup.-1] applied in the seeding and the remainder in top dressing: 40 and 80 kg [ha.sup.-1] N at forty days after seeding (DAS), or A + A at 20 and 40 DAS; 120 kg [ha.sup.-1] N split in 1/2 + 1/2 or 1/3 + 1/3 + 1/3 at 20, 40 or 60 DAS; 160 kg [ha.sup.-1] N split in 1/4 + 3/8 + 3/8 or 1/4 + 1/4 + 1/4 + 1/4 at 20, 40, 60 and 80 DAS. The application of 135 kg [ha.sup.-1] of N split in three times provided the best benefit/cost ratio. The non-application of N provided the lowest economic return, proving to be unviable. Key words: Zea mays, production cost, gross revenue, beneficial/cost, urea. O nitrogenio e o nutriente extraido em maior quantidade pelo milho, o de manejo mais complexo e o que mais onera o custo de producao da cultura. O objetivo deste trabalho foi avaliar a viabilidade economica de diferentes doses e parcelamentos da adubacao nitrogenada, na forma de ureia, na cultura do milho em LATOSSOLO VERMELHO Eutrofico. O estudo foi conduzido na Estacao Experimental do Polo Regional do Desenvolvimento dos Agronegocios do Noroeste Paulista (APTA), em Votuporanga, Estado de Sao Paulo. O delineamento experimental foi o de blocos casualizados com nove tratamentos e quatro repeticoes, constituido de cinco doses de N: 0, 55, 95, 135 e 175 kg [ha.sup.-1], aplicando-se 15 kg [ha.sup.-1] na semeadura e o restante em cobertura, combinadas a diferentes fracionamentos em cobertura: 40 e 80 kg aos 40 dias apos a semeadura (DAS) ou 1/2 + 1/2 aos 20 e 40 DAS; 120 kg [ha.sup.-1] de N fracionados em 1/2 + 1/2 ou 1/3 + 1/3 + 1/3 aos 20, 40 ou 60 DAS; 160 kg [ha.sup.-1] de N parcelados em 1/4 + 3/8 + 3/8 ou 1/4 + 1/4 + 1/4 + 1/4 aos 20, 40, 60 ou 80 DAS. A aplicacao de 135 kg [ha.sup.-1] de N parcelada em tres vezes proporcionou maior relacao beneficio/custo. A nao aplicacao de N proporcionou menor retorno economico, demonstrando ser inviavel. Palavras-chave: Zea mays, custo de producao, receita bruta, beneficio/custo, ureia.

O nitrogênio é o nutriente extraído em maior quantidade pelo milho, o de manejo mais complexo e o que mais onera o custo de produção da cultura. O objetivo deste trabalho foi avaliar a viabilidade econômica de diferentes doses e parcelamentos da adubação nitrogenada, na forma de uréia, na cultura do milho em LATOSSOLO VERMELHO Eutrófico. O estudo foi conduzido na Estação Experimental do Pólo Regional do Desenvolvimento dos Agronegócios do Noroeste Paulista (APTA), em Votuporanga, Estado de São Paulo. O delineamento experimental foi o de blocos casualizados com nove tratamentos e quatro repetições, constituído de cinco doses de N: 0, 55, 95, 135 e 175 kg ha-1, aplicando-se 15 kg ha-1 na semeadura e o restante em cobertura, combinadas a diferentes fracionamentos em cobertura: 40 e 80 kg aos 40 dias após a semeadura (DAS) ou ½ + ½ aos 20 e 40 DAS; 120 kg ha1 de N fracionados em ½ + ½ ou 1/3 + 1/3 + 1/3 aos 20, 40 ou 60 DAS; 160 kg ha-1 de N parcelados em ¼ + 3/8 + 3/8 ou ¼ + ¼ + ¼ + ¼ aos 20, 40, 60 ou 80 DAS. A aplicação de 135 kg ha-1 de N parcelada em três vezes proporcionou maior relação benefício/custo. A não aplicação de N proporcionou menor retorno econômico, demonstrando ser inviável.

Nitrogen is the nutrient that is most absorbed by the corn crop, with the most complex management, and has the highest share on the cost of corn production. The objective of this work was to evaluate the economic viability of different rates and split-applications of nitrogen fertilization, as such as urea, in the corn crop in a eutrophic Red Latosol (Oxisol). The study was carried out in the Experimental Station of the Regional Pole of the Sao Paulo Northwest Agribusiness Development (APTA), in Votuporanga, State of Sao Paulo, Brazil. The experimental design was randomized complete blocks with nine treatments and four replications, consisting of five N rates: 0, 55, 95, 135 and 175 kg [ha.sup.-1], 15 kg [ha.sup.-1] applied in the seeding and the remainder in top dressing: 40 and 80 kg [ha.sup.-1] N at forty days after seeding (DAS), or A + A at 20 and 40 DAS; 120 kg [ha.sup.-1] N split in 1/2 + 1/2 or 1/3 + 1/3 + 1/3 at 20, 40 or 60 DAS; 160 kg [ha.sup.-1] N split in 1/4 + 3/8 + 3/8 or 1/4 + 1/4 + 1/4 + 1/4 at 20, 40, 60 and 80 DAS. The application of 135 kg [ha.sup.-1] of N split in three times provided the best benefit/cost ratio. The non-application of N provided the lowest economic return, proving to be unviable.