O papel das ureases de soja (Glycine max (L.)Merr.) no desenvolvimento da planta e na proteção contra nematoide causador de galha

Ureases são tradicionalmente conhecidas por catalisar a hidrólise da ureia em amônia e dióxido de carbono. Em soja, três isoformas de urease foram descritas: 1) urease ubíqua, codificada pelo gene Eu4; 2) urease embrião específica, codificada pelo gene Eu1; 3) urease SBU-III, codificada por Eu5. O nitrogênio (N) é o nutriente mais limitante para o crescimento e desenvolvimento da planta. Portanto, mecanismos eficientes para capturar o N nas suas diversas formas, e realocá-lo, são necessários para otimizar o uso do nutriente pela planta. O produto N da atividade da urease - a amônia é incorporada em compostos orgânicos, principalmente, pela atividade da glutamina sintetase. Assim, a urease está envolvida na remobilização do N, bem como na assimilação do N primário. Um estudo anterior foi realizado por nossa equipe com o objetivo de superexpressar o gene Eu4 em plantas de soja. Inesperadamente, as plantas transgênicas exibiram cosupressão do transgene Eu4 e de todos os genes endógenos que codificam as isoformas de urease. Foi verificada também, uma diminuição da atividade ureolítica. Visando determinar o papel das ureases no desenvolvimento da soja, foram comparadas plantas transgênicas co-suprimidas, plantas mutantes e seus respectivos controles. O desenvolvimento das plantas foi avaliado 7, 14, 21 e 30 dias após a semeadura. As plantas co-suprimidas apresentaram um atraso no desenvolvimento durante o primeiro mês após a germinação. Um desenvolvimento mais lento foi observado para o duplo mutante eu1- a/eu4- e o mutante simples eu3-a (este gene codifica uma proteína acessória inativa). A absorção de N nas plantas transgênicas foi significativamente menor do que a das plantas não transgênicas. Entre os mutantes, eu3-a apresentou o menor e eu1-a o maior conteúdo de N. Um número significativamente menor de sementes foi obtido para as plantas transgênicas. Em conjunto estes resultados indicam que o aconteúdo da urease ou da atividade ureolítica desempenham um papel importante no desenvolvimento da planta. A soja (Glycine max) é afetada por vários estresses bióticos e abióticos, que limitam a distribuição geográfica das culturas e levam a reduções significativas de crescimento e produtividade. No Brasil, as doenças causadas por nematoides são um dos estresses bióticos mais prejudiciais para a soja. As principais espécies encontradas no Brasil são Meloidogyne spp. (formadores de galhas), Heterodera glycines (cisto), Pratylenchus brachyurus (lesões radiculares) e Rotylenchulus reniformis (reniforme). Nematoides formadores de galhas e de cisto (nematóides sedentários), os patógenos mais prejudiciais à soja, são muito difíceis de controlar. Neste estudo, foi identificado um peptídeo derivado da urease de soja (nomeado Soyuretox), que exerce efeito tóxico contra fitonematoides formadores de galhas (M. javanica). Soyuretox foi expresso em raízes de plantas compostas plantas transgênicas estáveis de soja. Raízes de plantas compostas e plantas transgênicas estáveis superexpressando Soyuretox exibiram uma redução significativa (50% e 37.5%, respectivamente) no número médio de nematoides e ovos, quando comparado com raízes não transformadas, 45 dias após a inoculação. Este é o primeiro relato de resistência a nematóides causada por um peptideo derivado de uma urease. Soyuretox pode representar uma ferramenta útil bem como uma nova e eficiente alternativa para o controle de pragas e doenças em culturas economicamente importantes.Ureases are traditionally known for catalyzing the hydrolysis of urea to ammonia and carbon dioxide. In soybean, three urease isoforms have been described: 1) ubiquitous urease, encoded by the Eu4 gene; 2) embryo-specific urease, encoded by Eu1gene; 3) SBU-III urease, encoded by Eu5. Nitrogen (N) is the most limiting nutrient for plant growth and development. Therefore efficient mechanisms both to take up N in its various forms, and to reallocate it, are necessary for optimal N use efficiency. The N product of urease activity- ammonia- is incorporated into organic compounds mainly by glutamine synthase activity. Thus, urease is involved in N remobilization, as well as in primary N assimilation. A previous study was performed by our team aiming to overexpress Eu4 gene in soybean plants. Unexpectedly, the transgenic plants exhibited endogenous (for all three genes) and introduced Eu4 transgene co-suppression and decreased ureolytic activity. Here, we sought to determine urease roles in soybean development by silencing all urease isoforms. Analyses were performed using transgenic co-suppressed and mutant plants. Plant development was evaluated 7, 14, 21 and 30 days after sowing. The cosuppressed plants presented a developmental delay during the first month after germination when compared with control. A slower development was observed for the double eu1-a/eu4-a mutant and the eu3-a (this gene codify an inactive accessory protein) single mutant. The N uptake in transgenic plants was significantly lower than that captured by non-transgenic plants. Among mutants, eu3-a presented the lowest and eu1- a the highest N content. A significantly lower number of seeds was obtained for transgenic plants. Altogether, these results indicate that the urease content and/or ureolytic activity play an important role in plant development. Soybeans (Glycine max) are affected by several abiotic and biotic stresses that limit the geographical distribution of cultures and lead to significant reductions in growth and productivity. In Brazil, the diseases caused by nematodes are one of the most damaging biotic stresses for soybeans.. The main species found in Brazil are Meloidogyne spp. (root-knot), Heterodera glycines (cyst), Pratylenchus brachyurus (root lesion) and Rotylenchulus reniformis (reniform). Root-knot and cyst nematodes (sedentary nematodes), the most damaging soybean pathogens, are very difficult to control. In this study, we identified a soybean urease-derived peptide (named Soyuretox) that exerts toxic effects against the root-knot phytonematode (M. javanica). Soyuretox was expressed in soybean roots of composite plants and complete stable transgenic plants. Roots of composite plants and stable transgenic plants overexpressing Soyuretox exhibited a significant reduction (50% and 37.5%, respectively) in the average number of nematodes and eggs when compared with non-transformed roots, 45 days after inoculation. This is the first report of nematode resistance caused by a urease-derived peptide. Soyuretox may represent a useful tool as a new and efficient alternative to control pests and diseases in economically important crops

Efeito das ureases de soja (Glycine max (L.) Merr.) na germinação de sementes e desenvolvimento das plantas

As ureases produzidas por plantas, fungos e bactérias, são metaloenzimas níquel- dependentes que catalisam a hidrólise da uréia em amônia e dióxido de carbono. Existem três proteínas de urease em soja. A urease ubíqua codificada pelo gene Eu4 é expressa em baixos níveis em todos os tecidos da planta e tem sido considerada responsável pela reciclagem de toda a uréia derivada metabolicamente. A urease embrião-específica codificada pelo gene Eu1 é expressa em níveis elevados em embriões em desenvolvimento e sementes maduras. Um terceiro gene codificando urease foi recentemente identificado. Este gene foi nomeado Eu5, e seu produto SBU-III. Eu5 é expresso nos primeiros estágios de desenvolvimento das raízes, mas o seu nível de expressão é menor se comparado ao de outras isoformas de urease. Plantas transgênicas com a supressão da expressão de Eu4 foram previamente obtidas por nossa equipe. Estas plantas transgênicas representam uma ferramenta poderosa para a genomica funcional, porque mutantes nulos nunca tinham sido obtidos para a urease ubíqua. Os objetivos do presente estudo foram caracterizar em nível molecular as progênies de plantas transgênicas com Eu4 co-suprimido e investigar o papel das ureases na capacidade de germinação de sementes e desenvolvimento das plantas. Sementes T1 e T2 obtidas de plantas transgênicas T0 e T1, respectivamente, foram selecionadas por expressão do gene repórter gfp. A presença de transgenes também foi confirmada por PCR. Níveis de expressão das três isoformas da urease (Eu4, Eu1 e Eu5) foram avaliados por RT-qPCR. Os resultados mostraram que co-supressão de Eu4 foi mantida na progênie transgênica. O silenciamento dos outros dois genes codificando ureases foi também demonstrado nessas plantas. A fim de investigar o papel individual dos genes que codificam urease nos processos de germinação e de desenvolvimento das plantas, mutantes simples eu4, eu1 e duplo eu4/eu1 foram também avaliados. Os resultados sugerem que a ausência de uma ou mais isoformas de urease não afetou a viabilidade das sementes, mesmo depois de armazenadas. Por outro lado, uma grande contribuição de Eu1 e Eu5 no padrão temporal de desenvolvimento das plantas foi demonstrada. A ausência de Eu1 determinou um atraso nos primeiros estágios de desenvolvimento, enquanto que nas plantas com silenciamento de todos os três genes que codificam ureases este fenótipo foi estendido para estágios de desenvolvimento subsequentes. Há sugestão na literatura de que Eu5 não seja funcional uma vez que elevado número de mutações têm sido detectados neste gene. No entanto, de acordo com nossos resultados, o produto deste gene pode estar envolvido no desenvolvimento da planta.Urease, produced by plants, fungi and bacteria, is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide. There are three urease structural proteins in soybean. The ubiquitous urease encoded by Eu4 gene is expressed at low levels in all plant tissues and has been considered to be responsible for recycling all metabolically-derived urea. The embryo-specific urease encoded by Eu1 gene is expressed in high levels in developing embryos and ripe seeds. A third urease encoding gene was recently identified. This gene was named Eu5, and its product SBU-III. Eu5 is expressed in the first stages of root development, but its expression level is lower if compared to that of other urease isoforms. Transgenic plants with suppression of Eu4 expression were previously obtained by our team. These transgenic plants represent a powerful tool for functional genomics, because null mutants have never been obtained for the ubiquitous urease. The goals of the present study were to characterize at molecular level progenies of transgenic Eu4 co-suppressed plants and investigate the role of ureases on seed germination capacity and plant development. T1 and T2 seeds obtained from T0 and T1 transgenic plants, respectively, were screened for gfp-reporter gene expression. Presence of transgenes was also confirmed by PCR. Expression levels of the three urease isoforms (Eu4, Eu1 and Eu5) were evaluated by RT-qPCR. Results showed that Eu4 co-suppression was maintained in the transgenic progeny. Silencing of the other two ureases encoding genes was also demonstrated in those plants. In order to investigate the role of individual urease encoding genes on germination and plant developmental processes, eu4, eu1 single and eu4/eu1 double mutants were also evaluated. Results suggest that lack of one or more ureases isoforms did not affect seed capacity even after stored. On the other hand, a great contribution of Eu1 and Eu5 on temporal plant developmental pattern was demonstrated. The lack of Eu1 determined a delay in the first developmental stages, whereas silencing of all three soybean ureases encoding genes distended this phenotype for further growth stages. Eu5 has been suggested not to be functional as it presents high number of mutations. However, according to our results the product of this gene might be involved on plant development

Are there regional variations in the presentation of childhood leukemia?

Introduction: Treatment of childhood acute lymphoblastic leukemia (ALL) is basedon risk stratification. This study aimed to assess the agreement between risk groupclassifications in the different childhood ALL treatment protocols used in a referralhospital in southern Brazil.Methods: We retrospectively reviewed the medical records of patients aged 1 to18 years with B-cell ALL treated at a hospital from January 2013 to April 2017. Agreementbetween risk classifications was assessed by the kappa coefficient.Results: Seventy-five patients were analyzed. There was poor agreement betweenrisk stratification by GBTLI 2009 and BFM 95 protocols (kappa=0.22; p = 0.003) andby GBTLI 2009 and IC-BFM 2002 protocols (kappa=0.24; p = 0.002). Risk groupdistribution was 13.3% for low risk, 32.0% for intermediate risk, and 54.7% for highrisk based on stratification by the GBTLI 2009 protocol, and 28.0% for low risk, 42.7%for intermediate risk, and 29.3% for high risk based on stratification by the IC-BFM2002 protocol. Overall survival was 68.6%.Conclusion: This study provides numerous points to ponder about the treatmentof leukemia in Brazil. The percentage of patients classified as high risk in oursample was higher than that reported in the international literature. This difference,however, had no impact on overall survival, which was shorter than that reportedin the international literature

From H1N1 to COVID-19 : what we have seen in children with hemoglobinopathies

This work aimed to better understand the impact of pandemics of respiratory viruses on children with hemoglobinopathies through a comprehensive review of the literature. MEDLINE, SCIELO, LILACS, and PUBMED were used as data sources to find articles without time period restrictions. Previous observations suggest that patients with hemoglobinopathies are a group especially susceptible to the complications of viral respiratory infections, with greater morbidity and mortality related to them. Within this context, this review found that, during the 2009 H1N1 pandemic, the risk of hospitalization in children and adults increased, especially in patients with a history of complications such as acute chest syndrome. In addition, the Coronavirus Disease 2019 (COVID-19) pandemic appears to have less repercussion among children with hemoglobinopathies compared to adults, similar to what is seen in the general population. In the H1N1 pandemic, patients with hemoglobinopathies behaved as a group more susceptible to complications, with increased morbidity and mortality. However, for COVID-19, the existing data to date on these patients do not show the same clinical impact. Thus, although these children deserve attention in case of infection due to their potential risks, they seem to have a favorable evolution

Are there regional variations in the presentation of childhood leukemia?

Introduction: Treatment of childhood acute lymphoblastic leukemia (ALL) is based on risk stratification. This study aimed to assess the agreement between risk group classifications in the different childhood ALL treatment protocols used in a referral hospital in southern Brazil. Methods: We retrospectively reviewed the medical records of patients aged 1 to 18 years with B-cell ALL treated at a hospital from January 2013 to April 2017. Agreement between risk classifications was assessed by the kappa coefficient. Results: Seventy-five patients were analyzed. There was poor agreement between risk stratification by GBTLI 2009 and BFM 95 protocols (kappa=0.22; p = 0.003) and by GBTLI 2009 and IC-BFM 2002 protocols (kappa=0.24; p = 0.002). Risk group distribution was 13.3% for low risk, 32.0% for intermediate risk, and 54.7% for high risk based on stratification by the GBTLI 2009 protocol, and 28.0% for low risk, 42.7% for intermediate risk, and 29.3% for high risk based on stratification by the IC-BFM 2002 protocol. Overall survival was 68.6%. Conclusion: This study provides numerous points to ponder about the treatment of leukemia in Brazil. The percentage of patients classified as high risk in our sample was higher than that reported in the international literature. This difference, however, had no impact on overall survival, which was shorter than that reported in the international literature

Breast milk leptin concentrations and infant anthropometric indicators in SGA versus non-SGA breastfed infants born at term

Leptin concentrations in breast milk can influence metabolic programming during the first months of life. Small for gestational age (SGA) newborns show a peculiar growth pattern after birth, which can lead to adulthood diseases. This study aims to assess an association between leptin concentration in mature breast milk and the infant anthropometric indicators of the SGA and the non-SGA groups, in addition, to comparing the hormone level between these groups. A longitudinal study was performed with mother-infant pairs. The maternal sociodemographic information was collected in the first 48 h postpartum. Breast milk was collected at one month postpartum and leptin concentrations were obtained by immunoassays. The infant anthropometric measurements were collected at three and six months postpartum and included weight, height (to body mass index-BMI calculated), triceps skinfold (TSF), and subscapular skinfold (SSF). The BMI for age (BMI/A), TSF, and SSF were calculated by Z-score indicators. Data from 67 mother-infant pairs (n = 16 SGA and n = 51 non-SGA) were analyzed. In univariate analyses, the breast milk of the SGA group had lower leptin concentrations than the non-SGA group (p = 0.006), however, after adjustment, there was no difference between groups (p = 0.181). In the SGA group, there was a significant association between leptin concentrations and lower SSF at six months in infants, after adjustment (p = 0.003). In the non-SGA group, the breast milk leptin was associated with lower BMI/A at three and six months in infants, after adjustment (p = 0.002 and p = 0.010, respectively). The association between breast milk leptin concentrations with SSF in the SGA group and BMI/A in the non-SGA group suggests that leptin may be a modulating factor in infant growth in the first months of life