Profiling and Localization of Stilbene Phytoalexins Revealed by MALDI-MSI during the Grapevine-Botrytis cinerea Interaction.

Stilbene phytoalexins are among the most accumulated compounds during grapevine-pathogen interactions. However, their steady-state accumulation level and spatial distribution within the tissues to counteract Botrytis cinerea infection remain to be explored. In this work, matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to determine the spatial distribution of different phytoalexins in grapevine leaves upon infection with B. cinerea. Ultraperformance liquid chromatography-fluorescence (UPLC-FL) was also employed to monitor the accumulation pattern of these phytoalexins. This study showed that stilbene compounds accumulate in areas close to the pathogen infection sites. It was also revealed that the accumulation patterns of the stilbene phytoalexins can vary from one time point postinfection to another with specific accumulation patterns within each time point. To the best of our knowledge, this is the first time that the separate localization of grapevine stilbene phytoalexins has been revealed following B. cinerea infection.

A Metabolome Analysis and the Immunity of Phlomis purpurea against Phytophthora cinnamomi.

Phlomis purpurea grows spontaneously in the southern Iberian Peninsula, namely in cork oak (Quercus suber) forests. In a previous transcriptome analysis, we reported on its immunity against Phytophthora cinnamomi. However, little is known about the involvement of secondary metabolites in the P. purpurea defense response. It is known, though, that root exudates are toxic to this pathogen. To understand the involvement of secondary metabolites in the defense of P. purpurea, a metabolome analysis was performed using the leaves and roots of plants challenged with the pathogen for over 72 h. The putatively identified compounds were constitutively produced. Alkaloids, fatty acids, flavonoids, glucosinolates, polyketides, prenol lipids, phenylpropanoids, sterols, and terpenoids were differentially produced in these leaves and roots along the experiment timescale. It must be emphasized that the constitutive production of taurine in leaves and its increase soon after challenging suggests its role in P. purpurea immunity against the stress imposed by the oomycete. The rapid increase in secondary metabolite production by this plant species accounts for a concerted action of multiple compounds and genes on the innate protection of Phlomis purpurea against Phytophthora cinnamomi. The combination of the metabolome with the transcriptome data previously disclosed confirms the mentioned innate immunity of this plant against a devastating pathogen. It suggests its potential as an antagonist in phytopathogens' biological control. Its application in green forestry/agriculture is therefore possible.

FT-ICR-MS-based metabolomics: A deep dive into plant metabolism.

Metabolomics involves the identification and quantification of metabolites to unravel the chemical footprints behind cellular regulatory processes and to decipher metabolic networks, opening new insights to understand the correlation between genes and metabolites. In plants, it is estimated the existence of hundreds of thousands of metabolites and the majority is still unknown. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a powerful analytical technique to tackle such challenges. The resolving power and sensitivity of this ultrahigh mass accuracy mass analyzer is such that a complex mixture, such as plant extracts, can be analyzed and thousands of metabolite signals can be detected simultaneously and distinguished based on the naturally abundant elemental isotopes. In this review, FT-ICR-MS-based plant metabolomics studies are described, emphasizing FT-ICR-MS increasing applications in plant science through targeted and untargeted approaches, allowing for a better understanding of plant development, responses to biotic and abiotic stresses, and the discovery of new natural nutraceutical compounds. Improved metabolite extraction protocols compatible with FT-ICR-MS, metabolite analysis methods and metabolite identification platforms are also explored as well as new in silico approaches. Most recent advances in MS imaging are also discussed.

Untargeted Metabolomics Reveals Antidepressant Effects in a Marine Photosynthetic Organism: The Diatom Phaeodactylum tricornutum as a Case Study.

The increased use of antidepressants, along with their increased occurrence in aquatic environments, is of concern for marine organisms. Although these pharmaceutical compounds have been shown to negatively affect marine diatoms, their mode of action in these non-target, single-cell phototrophic organisms is yet unknown. Using a Fourier-transform ion cyclotron-resonance mass spectrometer (FT-ICR-MS) we evaluated the effects of fluoxetine in the metabolomics of the model diatom Phaeodactylum tricornutum, as well as the potential use of the identified metabolites as exposure biomarkers. Diatom growth was severely impaired after fluoxetine exposure, particularly in the highest dose tested, along with a down-regulation of photosynthetic and carbohydrate metabolisms. Notably, several mechanisms that are normally down-regulated by fluoxetine in mammal organisms were also down-regulated in diatoms (e.g., glycerolipid metabolism, phosphatidylinositol signalling pathway, vitamin metabolism, terpenoid backbone biosynthesis and serotonin remobilization metabolism). Additionally, the present work also identified a set of potential biomarkers of fluoxetine exposure that were up-regulated with increasing fluoxetine exposure concentration and are of high metabolic significance following the disclosed mode of action, reinforcing the use of metabolomics approaches in ecotoxicology.

Grapevine leaf MALDI-MS imaging reveals the localisation of a putatively identified sucrose metabolite associated to Plasmopara viticola development.

Despite well-established pathways and metabolites involved in grapevine-Plasmopara viticola interaction, information on the molecules involved in the first moments of pathogen contact with the leaf surface and their specific location is still missing. To understand and localise these molecules, we analysed grapevine leaf discs infected with P. viticola with MSI. Plant material preparation was optimised, and different matrices and solvents were tested. Our data shows that trichomes hamper matrix deposition and the ion signal. Results show that putatively identified sucrose presents a higher accumulation and a non-homogeneous distribution in the infected leaf discs in comparison with the controls. This accumulation was mainly on the veins, leading to the hypothesis that sucrose metabolism is being manipulated by the development structures of P. viticola. Up to our knowledge this is the first time that the localisation of a putatively identified sucrose metabolite was shown to be associated to P. viticola infection sites.

More than Just Wine: The Nutritional Benefits of Grapevine Leaves.

The domesticated species Vitis vinifera L. harbours many cultivars throughout the world that present distinctive phenology and grape quality. Not only have the grapes been used for human consumption, but the leaves are also used as a source of bioactive compounds and are present in the diets of several Mediterranean countries. We have selected seven different cultivars and performed elemental, fatty acid (FA) and pigment profiling. Total reflection X-ray fluorescence (TXRF) enabled the identification of 21 elements. Among them, Na, Ca and K were highly represented in all the cultivars and Zn was prevalent in V. vinifera cv. 'Pinot noir' and 'Cabernet sauvignon'. Through gas chromatography, six FAs were identified, including omega-3 and omega-6 FA, the most abundant mainly in V. vinifera cv. 'Tinta barroca', 'Pinot noir' and 'Cabernet sauvignon'. FA composition was used to determine nutritional quality parameters, namely atherogenic, thrombogenic, hypocholesterolemic/hypercholesterolemic and peroxidisability indexes as well as oxidability and oxidative susceptibility. Grapevine leaves were highlighted as a suitable source of health-promoting lipids. Given the popularity of "green" diets, we have also performed a leaf pigment analysis. Seventeen pigments including chlorophylls, trans-lutein, b-carotene and zeaxanthins were detected. 'C19' presented the highest content of most of the detected pigments.

Integrating metabolomics and targeted gene expression to uncover potential biomarkers of fungal/oomycetes-associated disease susceptibility in grapevine.

Vitis vinifera, one of the most cultivated fruit crops, is susceptible to several diseases particularly caused by fungus and oomycete pathogens. In contrast, other Vitis species (American, Asian) display different degrees of tolerance/resistance to these pathogens, being widely used in breeding programs to introgress resistance traits in elite V. vinifera cultivars. Secondary metabolites are important players in plant defence responses. Therefore, the characterization of the metabolic profiles associated with disease resistance and susceptibility traits in grapevine is a promising approach to identify trait-related biomarkers. In this work, the leaf metabolic composition of eleven Vitis genotypes was analysed using an untargeted metabolomics approach. A total of 190 putative metabolites were found to discriminate resistant/partial resistant from susceptible genotypes. The biological relevance of discriminative compounds was assessed by pathway analysis. Several compounds were selected as promising biomarkers and the expression of genes coding for enzymes associated with their metabolic pathways was analysed. Reference genes for these grapevine genotypes were established for normalisation of candidate gene expression. The leucoanthocyanidin reductase 2 gene (LAR2) presented a significant increase of expression in susceptible genotypes, in accordance with catechin accumulation in this analysis group. Up to our knowledge this is the first time that metabolic constitutive biomarkers are proposed, opening new insights into plant selection on breeding programs.

Vitis vinifera 'Pinot noir' leaves as a source of bioactive nutraceutical compounds.

Agricultural by-products are often hidden sources of healthy plant ingredients. The investigation of the nutritional values of these by-products is essential towards sustainable agriculture and improved food systems. In the vine industry, grape leaves are a bulky side product which is strategically removed and treated as waste in the process of wine production. In this work we performed an untargeted metabolomic profiling of the methanol extract of the leaves of Vitis vinifera cultivar 'Pinot noir', analysed their fatty acid content, and estimated their antioxidative capacity, with the purpose of investigating its nutritional and medicinal value. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) analysis identified the presence of numerous compounds which are known to possess diverse nutritional and pharmacological properties, particularly polyphenols and phenolic compounds (e.g. caffeic acid, catechin, kaempferol and quercetin), several phytosterols and fatty acids. Fatty acids were the most represented lipids' secondary class, with the essential alpha-linolenic acid being the most abundant in 'Pinot noir' leaves, with a relative content of 42%. Also, we have found that 'Pinot noir' leaves present a high antioxidant capacity, putting grapevine leaves at the top of the list of foods with the highest antioxidative activity. Our findings scientifically confirmed that 'Pinot noir' leaves have a high content and diversity of biologically active phytochemical compounds which make it of exceptional interest for pharmaceutical and food industries.

Early stage metabolic events associated with the establishment of Vitis vinifera - Plasmopara viticola compatible interaction.

Grapevine (Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world, with 7.5 million of production area in 2017. The domesticated varieties of grapevine are highly susceptible to many fungal infections, of which downy mildew, caused by the biotrophic oomycete Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is one of the most threatening. In V. vinifera, several studies have shown that a weak and transient activation of a defense mechanism occurs, but it is easily overcome by the pathogen leading to the establishment of a compatible interaction. Major transcript, protein and physiologic changes were shown to occur at later infection time-points, but comprehensive data on the first hours of interaction is scarce. In the present work, we investigated the major physiologic and metabolic changes that occur in the first 24 h of interaction between V. vinifera cultivar Trincadeira and P. viticola. Our results show that there was a negative modulation of several metabolic classes associated to pathogen defense such as flavonoids or phenylpropanoids as well as an alteration of carbohydrate content after inoculation with the pathogen. We also found an accumulation of hydrogen peroxide and increase of lipid peroxidation but to a low extent, that seems to be insufficient to restrain pathogen growth during the initial biotrophic phase of the interaction.

Grapevine Subtilase Family: Update on New Sequences and Nomenclature Proposal.

In grapevine, serine peptidases from the subtilase family were recently associated to Plasmopara viticola resistance. This family in grapevine, first characterized in 2014, was re-analyzed last year and 82 subtilase genes were identified. However, in November of 2016, the National Center for Biotechnology Information database (NCBI) made a new public release of the grapevine genome annotation based on new sequencing data and better prediction algorithms. As a consequence, some gene annotations and lengths changed. Here we present an update to the grapevine subtilase gene family sequences (SBT), namely sequence identifiers, bioinformatic predictions and recommend a nomenclature for the grapevine SBT genes. Our results show that grapevine subtilase gene family is now constituted by 87 subtilase genes encoding for 109 subtilase proteins and, despite the reported alterations, expression data on subtilases associated to grapevine resistance to P. viticola pathosystem did not suffer any alteration.

Revisiting Vitis vinifera Subtilase Gene Family: A Possible Role in Grapevine Resistance against Plasmopara viticola.

Subtilisin-like proteases, also known as subtilases, are a very diverse family of serine peptidases present in many organisms. In grapevine, there are hints of the involvement of subtilases in defense mechanisms, but their role is not yet understood. The first characterization of the subtilase gene family was performed in 2014. However, simultaneously, the grapevine genome was re-annotated and several sequences were re-annotated or retrieved. We have performed a re-characterization of this family in grapevine and identified 82 genes coding for 97 putative proteins, as result of alternative splicing. All the subtilases identified present the characteristic S8 peptidase domain and the majority of them also have a pro-domain I9 inhibitor, a protease-associated (PA) domain, and a signal peptide for targeting to the secretory pathway. Phylogenetic studies revealed six subtilase groups denominated VvSBT1 to VvSBT6. As several evidences have highlighted the participation of plant subtilases in response to biotic stimulus, we have investigated subtilase participation in grapevine resistance to Plasmopara viticola, the causative agent of downy mildew. Fourteen grapevine subtilases presenting either high homology to P69C from tomato, SBT3.3 from Arabidopsis thaliana or located near the Resistance to P. viticola (RPV) locus were selected. Expression studies were conducted in the grapevine-P. viticola pathosystem with resistant and susceptible cultivars. Our results may indicate that some of grapevine subtilisins are potentially participating in the defense response against this biotrophic oomycete.

Crianças brincam! Considerações sobre o desenvolvimento emocional infantil e a linguagem lúdica / Children play! Considerations about the child's emotional development and playful language

A partir da teoria de Donald Winnicott, traça-se uma descrição sucinta de aspectos essenciais do desenvolvimento emocional infantil. Confere-se à linguagem lúdica da criança um papel nuclear para o desenvolvimento emocional saudável

From Donald Winnicott's theory, we draw up a brief description of essential aspects of the child's emotional development. It is given to the playful language of the child a central role for the healthy emotional development

Construção de confiança e reconhecimento no processo de formação ministrado pela Estratégia Brasileirinhas e Brasileirinhos Saudáveis aos consultores estaduais da saúde da criança do Ministério da Saúde / Trust building and recognition in the training process lectured by the Estratégia Brasileirinhas e Brasileirinhos Saudáveis to state consultants of child health of the Ministry of Health

Analisamos neste artigo as dimensões éticas e subjetivas da noção de reconhecimento implicadas no processo de formação conferida pela Estratégia Brasileirinhas e Brasileirinhos Saudáveis aos consultores estaduais do Ministério da Saúde. Nosso principal objetivo foi contribuir para a formulação e implantação da Política Nacional de Atenção Integral à Saúde da Criança. Conclui-se que trabalhar a 'escuta implicada' como habilidade específica, a qual estimula o reconhecimento das potencialidades e das fragilidades próprias e do outro durante o processo de formação, foi um agente facilitador para a construção de redes de confiança, desdobrando-se como importante ferramenta para incentivar o processo cooperativo no campo de trabalho

We analyze in this article the ethical and subjective dimensions of the notion of recognition implicated in the training process granted by Estratégia Brasileirinhas e Brasileirinhos Saudáveis to the state consultants of the Ministry of Health. Our main goal wasto contribute to the formulation and implementation of the National Policy for Comprehensive Child Health Care. We conclude that working the 'involved listening' as a specific skill, which stimulates the recognition of one's own potential and weaknesses and of the other during the training process was a facilitator for building trust networks, unfolding as an important tool toencourage the cooperative process at the work field

Cartografia, grupalidade e cuidado: operadores conceituais do processo de formação da Estratégia Brasileirinhas e Brasileirinhos Saudáveis / Cartography, groupality and care: conceptual operators of the process of formation of the Estratégia Brasileirinhas e Brasileirinhos Saudáveis

A partir da experiência da Estratégia Brasileirinhas e Brasileirinhos Saudáveis, pretende-se descrever e sistematizar sua proposta de formação no campo da saúde, encarnada aqui no processo de formação dos consultores estaduais de saúde da criança. Para tal,desenvolveu-se uma metodologia baseada em três operadores conceituais, dinamicamente interligados: a cartografia como processo de entrada, conhecimento e reconhecimento do território, a grupalidade como dispositivo de ação e o cuidado em sua dimensão ética.Sublinha-se o modo de fazer específico que prioriza a inclusão da dimensão sensível no processo de formação...

This article aims at describing and synthesizing, within the experience of the Estratégia Brasileirinhas e Brasileirinhos Saudáveis, the proposal of training in the health field, incarnated here in the process of training state consultants for child health. For this purpose, a methodology was developed based in three conceptual operators dynamically interlinked: cartography as aprocess of entry, knowledge and recognition of the territory; groupality as an action device; and care in its ethical dimension. We highlight the specific way of doing that prioritizes the inclusion of the sensible dimension in the training process...

Metabolite extraction for high-throughput FTICR-MS-based metabolomics of grapevine leaves.

In metabolomics there is an ever-growing need for faster and more comprehensive analysis methods to cope with the increase of biological studies. Direct infusion Fourier-transform ion cyclotron-resonance mass spectrometry (DI-FTICR-MS) is used in non-targeted metabolomics to obtain high-resolution snapshots of the metabolic state of a system. In any metabolic profiling study, the establishment of an effective metabolite extraction protocol is paramount. We developed an improved metabolite extraction method, compatible with DI-FTICR-MS-based metabolomics, using grapevine leaves. This extraction protocol allowed the extraction of polar and non-polar compounds, covering all major classes found in plants and increasing metabolome coverage.

Por uma ética do cuidado

Os estudos reunidos nesta coletânea partem da vulnerabilidade da criança e do adolescente na sua saúde física e mental e recolhem contribuições que ampliam esse escopo para os campos do direito, da sociologia, da filosofia e da segurança pública. Articulando o trabalho dessas áreas do conhecimento com a prática clínica diária da Maternidade Escola da UFRJ, foi implementada uma grande rede interdisciplinar para pensar este cotidiano clínico e social. Apesar de boa parte dos autores serem ligados à área de Saúde, este não é um livro voltado para especialistas. O objetivo dos escritos aqui reunidos é provocar uma iniciativa de cuidado, buscando barrar o sentimento desolador de descuido.

Da dor de existir ao insuportável da dor psíquica / From existencial pain, to the unbearable of physic pain

O objetivo deste artigo é estabelecer um diálodo entre alguns aspectos dos ideais de subjetivação na cultura contemporânea e a experiência da dor segundo um recorte psicanalítico. Para tal, distingue-se três especificidades de dor: dor física, dor de existir e dor psíquica. A partir deste debate, faz-se uma pergunta: o que pode acontecer quando o ideário cultural de um tempo afirma que a dor de existir, afeto fundamental para a experiência subjetiva, não pode existir?(AU)