EFEITO DA FERTILIZAÇÃO AZOTADA NA DINÂMICA DE

RESUMO Este trabalho teve como objectivo estudar a influência da aplicação de diferentes quantidades de azoto, repartidas por quatro aplicações, na disponibilidade de azoto nítrico no solo, no comprimento radical, na concentração de azoto nas folhas e na produção comercial de cebola de dias médios (cv. Gilmar) no Alentejo. O ensaio decorreu na Centro de estudos e experimentação da Mitra da Universidade de Évora e foi delineado em blocos casualizados com quatro repetições. Os tratamentos consistiram em 4 níveis de adubação azotada (0, 37, 74 e 111 kg N ha-1), repartidos por quatro aplicações. A disponibilidade de azoto nítrico no solo, o comprimento radical e a concentração de azoto nas folhas foram avaliados aos 33, 57, 96 e 127 dias após a plantação. A densidade radical (cm cm-3) sob o bolbo e a 4 cm da linha de cultura, nas diferentes datas e profundidades de amostragem, não foi afectada pelos níveis de azoto. Ao longo ciclo, 65 a 100 % das raízes, em termos de comprimento radical, concentraram-se sob o bolbo e a densidade radical máxima alcançada foi de 1,88 cm cm-3. A profundidade máxima de enraizamento situou-se entre os 20 e 30 cm, não ultrapassando os 10 cm de profundidade até aos 32 dias após a plantação. Nas condições do ensaio, os resultados indicam como recomendável uma aplicação de 30 kg ha– 1 de azoto à plantação e um aumento da quantidade de azoto aplicado (16,2% do total de N aplicado), no início da formação do bolbo. A produção comercial aumentou com o nível de azoto, mas as produções obtidas com a aplicação de 74 kg ha-1 (5,12 kg m-2) e de 111 Kg N ha-1 (6,59 kg m-2) não diferiram significativamente. Palavras-chave: Allium cepa L, azoto nítr

Methionine and Tryptophan Play Different Modulatory Roles in the European Seabass (Dicentrarchus labrax) Innate Immune Response and Apoptosis Signaling—An In Vitro Study

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes’ immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1ß and nf-¿b expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-¿b and p65. This study showed the ability of methionine surplus to improve leucocytes’ response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.This work was partially supported by UIDB/04423/2020, UIDP/ 04423/2020 and INFLAMMAA (reference PTDC/CVT-CVT/ 32349/2017), financed by Portugal and the European Union through FEDER and COMPETE 2020, and national funds through Fundação para a Ciência e a Tecnologia (FCT, Portugal). MM and BC were supported by FCT, Portugal (SFRH/BD/108243/2015 and IF/00197/2015, respectively)

Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi

The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1-/- (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H2O2). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H2O2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER. © 2012 Furtado et al

Diagnostic value of CSF protein profile in a Portuguese population of sCJD patients

The clinical diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) is difficult, and reliable markers are highly desired. In this work we assess the value of several cerebrospinal fluid (CSF) markers for sCJD diagnosis. Within the framework of the Portuguese Epidemiological Surveillance Program for Human Prion Diseases, CSF samples from 71 patients with clinically suspected sCJD, 30 definite sCJD and 41 non-CJD patients, were analysed for the presence of 14-3-3 protein. CSF levels of tau (t-tau), and phosphorylated tau (p-tau181), S-100b and beta amyloid (Abeta42) proteins were determined. The influence of clinical and genetic characteristics on CSF markers sensitivity was also evaluated. Protein 14-3-3 was detected in 29/30 sCJD patients and 9/41 non-CJD patients. Extremely elevated t-tau and S-100b protein levels were found in sCJD patients, while p-tau181 levels were only slightly elevated and Abeta42 showed no differences compared to controls. 14-3-3 was the most sensitive parameter (97%), but its specificity was low (78%); sensitivity/specificity for other proteins were: S-100b-93/93%, t-tau-93/95%, with maximum accuracy being obtained by a combination of tests (14-3-3 combined with either t-tau or S-100b, or combining S-100b with t-tau/Abeta42 or p-tau/t-tau ratios). The sensitivity of 14-3-3, as well as of p-tau181/t-tau ratio, was decreased in younger patients with long disease duration, with the PrP-2 isotype and MV genotype. Both 14-3-3, t-tau and S-100b are sensitive markers for sCJD, but 14-3-3 specificity seems to be lower in this special clinical setting of rapidly progressing dementias. We propose that in cases with a 14-3-3 weak positive result, or in young patients with long disease duration, a second CSF marker would be valuable for the diagnosis of sCJD

Performance of IFAT, ELISA, direct parasitological examination and PCR on lymph node aspirates for canine visceral leishmaniasis diagnosis

Canine visceral leishmaniasis (CVL) is endemic in numerous Brazilian regions. The greatest difficulty in controlling the disease is the diagnostic limitation. In the present study, the most common tests employed for visceral leishmaniasis diagnosis were compared: immunofluorescence antibody test (IFAT), immunoenzymatic assay (ELISA), direct parasitological examination and polymerase chain reaction (PCR). Samples of lymph node aspirates and blood were collected from 100 dogs that lived in an endemic area (Bauru city, São Paulo state) and from 100 negative controls from a non-endemic area (Botucatu city, São Paulo state). Specificity of both IFAT and PCR was 100% whereas ELISA was 99%. Sensitivities were 97.77, 93.33 and 91.11% respectively for IFAT, ELISA and PCR

Genetic screening of Alzheimer's disease genes in Iberian and African samples yields novel mutations in presenilins and APP

Mutations in three genes (PSEN1, PSEN2, and APP) have been identified in patients with early-onset (<65 years) Alzheimer's disease (AD). We performed a screening for mutations in the coding regions of presenilins, as well as exons 16 and 17 of the APP gene in a total of 231 patients from the Iberian peninsular with a clinical diagnosis of early-onset AD (mean age at onset of 52.9 years; range 31-64). We found three novel mutations in PSEN1, one novel mutation in PSEN2, and a novel mutation in the APP gene. Four previously described mutations in PSEN1 were also found. The same analysis was carried in 121 elderly healthy controls from the Iberian peninsular, and a set of 130 individuals from seven African populations belonging to the Centre d'Etude du Polymorphisme Humain-Human Genome Diversity Panel (CEPH-HGDP), in order to determine the extent of normal variability in these genes. Interestingly, in the latter series, we found five new non-synonymous changes in all three genes and a presenilin 2 variant (R62H) that has been previously related to AD. In some of these mutations, the pathologic consequence is uncertain and needs further investigation. To address this question we propose and use a systematic algorithm to classify the putative pathology of AD mutations

Neural field model for measuring and reproducing time intervals

The continuous real-time motor interaction with our environment requires the capacity to measure and produce time intervals in a highly flexible manner. Recent neurophysiological evidence suggests that the neural computational principles supporting this capacity may be understood from a dynamical systems perspective: Inputs and initial conditions determine how a recurrent neural network evolves from a “resting state” to a state triggering the action. Here we test this hypothesis in a time measurement and time reproduction experiment using a model of a robust neural integrator based on the theoretical framework of dynamic neural fields. During measurement, the temporal accumulation of input leads to the evolution of a self-stabilized bump whose amplitude reflects elapsed time. During production, the stored information is used to reproduce on a trial-by-trial basis the time interval either by adjusting input strength or initial condition of the integrator. We discuss the impact of the results on our goal to endow autonomous robots with a human-like temporal cognition capacity for natural human-robot interactions.The work received financial support from FCT through the PhD fellowship PD/BD/128183/2016, the project ”Neurofield” (POCI-01-0145-FEDER-031393) and the research centre CMAT within the project UID/MAT/00013/2013

Immunomodulatory Protective Effects of Rb9 Cyclic-Peptide in a Metastatic Melanoma Setting and the Involvement of Dendritic Cells

The cyclic VHCDR3-derived peptide (Rb9) from RebMab200 antibody, directed to a NaPi2B phosphate-transport protein, displayed anti-metastatic melanoma activity at 50-300 mu g intraperitoneally injected in syngeneic mice. Immune deficient mice failed to respond to the peptide protective effect. Rb9 induced increased CD8+ T and low Foxp3+ T cell infiltration in lung metastases and high IFN-gamma and low TGF-beta in lymphoid organs. The peptide co-localized with F-actin and a nuclear site in dendritic cells and specifically bound to MIF and CD74 in a dot-blot setting. Murine bone-marrow dendritic cells preincubated with Rb9 for 6 h were treated with MIF for short time periods. The modulated responses showed stimulation of CD74 and inhibition of pPI3K, pERK, and pNF-kappa B as compared to MIF alone. Rb9 in a melanoma-conditioned medium, stimulated the M1 type conversion in bone marrow-macrophages. Functional aspects of Rb9 in vivo were studied in therapeutic and prophylactic protocols using a melanoma metastatic model. In both protocols Rb9 exhibited a marked anti-melanoma protection. Human dendritic cells were also investigated showing increased expression of surface markers in response to Rb9 incubation. Rb9 either stimulated or slightly inhibited moDCs submitted to inhibitory (TGF-beta and IL-10) or activating (LPS) conditions, respectively. Lymphocyte proliferation was obtained with moDCs stimulated by Rb9 and tumor cell lysate. In moDCs from cancer patients Rb9 exerted immunomodulatory activities depending on their functional status. The peptide may inhibit over-stimulated cells, stimulate poorly activated and suppressed cells, or cause instead, little phenotypic and functional alterations. Recently, the interaction MIF-CD74 has been associated to PD-L1 expression and IFN-gamma, suggesting a target for melanoma treatment. The effects described for Rb9 and the protection against metastatic melanoma may suggest the possibility of a peptide reagent that could be relevant when associated to modern immunotherapeutic procedures

BiofOmics: A Web Platform for the Systematic and Standardized Collection of High-Throughput Biofilm Data

Background: Consortia of microorganisms, commonly known as biofilms, are attracting much attention from the scientific community due to their impact in human activity. As biofilm research grows to be a data-intensive discipline, the need for suitable bioinformatics approaches becomes compelling to manage and validate individual experiments, and also execute inter-laboratory large-scale comparisons. However, biofilm data is widespread across ad hoc, non-standardized individual files and, thus, data interchange among researchers, or any attempt of cross-laboratory experimentation or analysis, is hardly possible or even attempted. Methodology/Principal findings This paper presents BiofOmics, the first publicly accessible Web platform specialized in the management and analysis of data derived from biofilm high-throughput studies. The aim is to promote data interchange across laboratories, implementing collaborative experiments, and enable the development of bioinformatics tools in support of the processing and analysis of the increasing volumes of experimental biofilm data that are being generated. BiofOmics data deposition facility enforces data structuring and standardization, supported by controlled vocabulary. Researchers are responsible for the description of the experiments, their results and conclusions. BiofOmics curators interact with submitters only to enforce data structuring and the use of controlled vocabulary. Then, BiofOmics search facility makes publicly available the profile and data associated with a submitted study so that any researcher can profit from these standardization efforts to compare similar studies, generate new hypotheses to be tested or even extend the conditions experimented in the study. Significance BiofOmics novelty lays on its support to standardized data deposition, the availability of computerizable data files and the free-of-charge dissemination of biofilm studies across the community. Hopefully, this will open promising research possibilities, namely: the comparison of results between different laboratories, the reproducibility of methods within and between laboratories, and the development of guidelines and standardized protocols for biofilm formation devices and analytical methods.The financial support from the Institute of Biotechnology and Bioengineering - Center of Biological Engineering (IBB-CEB), Fundacao para a Ciencia e Tecnologia (FCT) and European Community fund FEDER (Program COMPETE), project PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480 and PhD grant of Idalina Machado (SFRH/BD/31065/2006) are gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript