Application of genomics tools for cacao disease resistance : S04T07

Biotic plant problems are caused by living organisms, such as fungi, bacteria, viruses, nematodes, insects, mites, and animals. Plant responses to different stresses are highly complex and involve changes at the transcriptome, cellular, and physiological levels. In Theobroma cacao the main biotic stresses are cause by fungi, causing the witches' broom (Moniliophthora perniciosa) disease of cacao, Black pod (Phytophthora spp.), frost pod (Moniliophthora roreri and, recently, Ceratocysts wilt (Ceratocystis cacaofunesta). Durable resistance is the key to hamper the advance of these diseases. The OMICS with the classical phytopatological and breeding approaches have allowed: to identify putative resistance genes; to deciphering the genomics of Thebroma cacao, to discover new microsatellite and SNP markers, and to find new QTLs linked to disease resistance. These informations are being integrating in the CEPLAC' breeding program to accelerate the search for new resistance material that carries different resistant genes. In parallel, we study these diseases at the histopatological level trying to characterizing the mechanisms of resistance underneath the hosts as well gene expression in situ. The adaptability of these plant pathogens has also been considered. Advances in the understanding of the breakdown of witches´ broom resistance have been achieved. It was shown that the fungus has a high ability to evolve towards some genotypes. Partial results of these projects and the overall strategy will be presented. Work supported by CNPq, FINEP, FAPESB,RENORBIO (Texte intégral

Identification, characterization and mapping of EST-derived SSRs from cacao-Ceratocystis cacaofunesta interaction

Ceratocystis cacaofunesta is an ascomycete responsible for the lethal wilt disease of cacao (Theobroma cacao L.). Marker-assisted selection combined with conventional breeding is one way in which the cacao resistance to Ceratocystis wilt can be improved. In this study, we screened a set of ESTs obtained from cacao elicited with C. cacaofunesta to identify ESTSSRs and test their efficacy for mapping. Among the 3,432 ESTs analyzed, 384 contained SSRs and 428 EST-SSRs were identified, mainly dinucleotides (78.5%), com 4 numbers of repeats (75.23%), and preferentially AG/CT motif (25.47%). GO function was assigned to the ESTs containing SSRs: 4.04% belonged to "defense response" category, with 20.69% of them to the sub-category "defense response to fungus". In relation to the ORF, the same amount of EST-SSRs was observed in 5'UTR as well as in the 3'UTR (about 30%). From the 428 EST-SSRs identified, 12 were polymorphic, revealing a total of 41 alleles. The number of alleles per locus ranged from 2 to 6, with an average of 3.41. Four EST-SSRs were mapped on the F2 Sca 6 x ICS 1 population segregating for Ceratocystis wilt, which were distributed on the linkage groups 2, 3, 4 and 8. These markers contributed to saturate the genetic map of the cacao mapping population from CEPEC/ CEPLAC and will be valuable for the research community to improve the cacao breeding program. (Texte intégral

Perspectives et caractérisation des SNP dans les étiquettes de séquences exprimées (EST) à partir de l'interaction Theobroma Cacao-Moniliophthora Perniciosa

Moniliophthora (ex Crinipellis) perruciosa (Stahel) Singer (Aime e1 Phillips-Mora, 2005) est un basidiomycète hémibiotrophe (Theobroma cacao L.). responsable de la maladie des balais de sorcières (WBD) sur les cacaoyers (Theobroma cacao L). Étant donné l'importance des impact socio-économiques et environnementaux de la WBD sur les plantations cacaoyères dans la région de Bahia au Brésil, plusieurs études de génomique fonctionnelle sur l'interaction cacao-Moniliophthora pemiciosa ont été réalisées (Gesteira et al 2007; Argout et al. 2008, projets CEPLAC subventionnés par le FAPESB et CNPq). Ces programmes ont permis l'identification d'EST participant à la résistance du cacaoyer à la WBD, en fournissant un contexte pour détecter les marqueurs de polymorphisme (par ex. SNP) nécessaires pour des études supplémentaires comme des stratégies de sélection génétique, un pyramidage des gènes et une sélection assistée par marqueurs (MAS). Nous rendons compte de la recherche, de la validation et de la caractérisation de polymorphismes de nucléotides simples (SNP) dans les EST de l'interaction cacao - Moniliophthora perniciosa en utilisant le reséquençage de 73 gènes candidats dans des individus Sca 6, JCS 1, TSH 516 et 68 de la F2. Cette analyse a permis l'identification de 185 SNP, dont 57 % correspondent à des cas de transversion, 29% de transition et 14% à des " indels ". Les EST contenant les SNP ont été classés dans 14 catégories fonctionnelles principales. Au travers de la validation, 91 SNP ont été confirmés ; leur fréquence dans les régions codantes et non codant.es a été évaluée, et leurs positions dans l'ORF ainsi que la fréquence de SNP synonymes et non synonymes a été déterminée. Les paramètres de diversité des nucléotides et des haplotypes pour les SNP validés ont été calculés. La diversité génétique basée sur les haplotypes et le contenu d'information polymorphique (PIC) allaient de 0,559 à 0,56 et de 0,115 à 0,12 respectivement De plus, nous avons démontré l'avantage de la prise en compte de la structure des haplotypes pour chaque locus au lieu de S:N""P uniques. Le nombre de haplotypes variait de quatre à seize selon les fragments de gènes, avec une moyenne de huit La plupart des fragments de gènes avaient un haplotype majeur accompagné d'une série de haplotypes de faible fréquence. Ainsi, l'approche de reséquençage s'est avérée déterminante pour identifier des SNP utiles pour de vastes applications génétiques. En outre, la disponibilité de la séquence du génome du cacao devrait permettre de reséquencer une sélection positionnelle de fragments d'ADN, permettant ainsi de renforcer l'utilité des SN""P découverts. En conclusion, nous recommandons ce système de marqueur codominant fonctionnel pour une identification à grande échelle du statut allélique des gènes de résistance du cacao, par une sélection assistée par marqueurs, pour permettre le développement de génotypes prometteurs avec une durabilité de résistance élevée à la maladie des balais de sorcières. (Texte intégral

Obtenção de produtos de PCR dos principais fungos causadores de doenças no cacaueiro visando estudos filogeneticos e taxonômicos : [Resumo]

Onde é cultivado, o cacaueiro (#Theobroma cacao# L.) é alvo de várias doenças de grande impacto econômico. Entre estas estão a vassoura-de-bruxa (#Moniliophthora perniciosa#), podridão-parda (#Phytophthora# spp.), monilíase (#Moniliophthora roreri#) e a murcha de Ceratocystis (#Ceratocystis cacafunesta#). O conhecimento da biologia destes fitopatogenos é essencial para estabelecer um manejo eficiente das doenças. As limitações metodológicas para seqüenciamento de genes de interesse têm sido a extração do DNA e a amplificação de regiões especificas com qualidade satisfatória, ou seja, que permitam o uso com sucesso dos produtos da amplificação em reações de seqüenciamento e em métodos moleculares comparativos. Nesse sentido, nosso objetivo foi determinar as condições ótimas das reações de PCR para amplificação de regiões ribossomais, dentre outras, as quais vêm sendo usadas rotineiramente no FITOMOL no auxilio da taxonomia molecular e filogenia destes fitopatogenos. Isolados de quatro espécies de fungos em estudo (#M. perniciosa#, #Phytophthora# spp., #M. roreri# e #Ceratocystis cacaofunesta#) tiveram seu DNA extraído segundo protocolos específicos para cada espécie. O DNA foi quantificado e determinado sua qualidade utilizando gel de agarose a 1%. A otimização das condições da PCR foi avaliada segundo dois aspectos: concentração final do DNA template na reação (10, 25 e 50 ng/ul) e concentração dos primers (4, 10 e 20 pmol/ul) em reações com volumes finais de 20 ul e 50 ul. Os resultados demonstram que as condições ótimas para amplificação das regiões em estudo é uma reação com volume final de 20 ul, 10 ng/ul de DNA template e concentração do primer a 4 pmol/ul, Os resultados mostram que uma menor concentração de DNA deve ser utilizada, o que é desejável levando em consideração as dificuldades de obtenção de micélio e extração do DNA em fungos, bem como uma economia em relação aos reagentes utilizados na PCR. Esta abordagem provou ser de sucesso para amplificação de regiões ribossomais utilizados na rotina de identificação e genotipagem de fungos no nosso laboratório. Apoio financeiro: Fapesb, CNPq e CEPLAC. (Texte intégral

Graded Poisson-Sigma Models and Dilaton-Deformed 2D Supergravity Algebra

Fermionic extensions of generic 2d gravity theories obtained from the graded Poisson-Sigma model (gPSM) approach show a large degree of ambiguity. In addition, obstructions may reduce the allowed range of fields as given by the bosonic theory, or even prohibit any extension in certain cases. In our present work we relate the finite W-algebras inherent in the gPSM algebra of constraints to algebras which can be interpreted as supergravities in the usual sense (Neuveu-Schwarz or Ramond algebras resp.), deformed by the presence of the dilaton field. With very straightforward and natural assumptions on them --like demanding rigid supersymmetry in a certain flat limit, or linking the anti-commutator of certain fermionic charges to the Hamiltonian constraint-- in the ``genuine'' supergravity obtained in this way the ambiguities disappear, as well as the obstructions referred to above. Thus all especially interesting bosonic models (spherically reduced gravity, the Jackiw-Teitelboim model etc.)\ under these conditions possess a unique fermionic extension and are free from new singularities. The superspace supergravity model of Howe is found as a special case of this supergravity action. For this class of models the relation between bosonic potential and prepotential does not introduce obstructions as well.Comment: 22 pages, LaTeX, JHEP class. v3: Final version, to appear in JHE

Essential oil of Seseli tortuosum L. from Portugal: safety and anti-inflammatory potential evaluation

Several Seseli L. (Apiaceae) species are used in folk medicine for several healing effects, namely herbal remedy for human inflammation, swelling, rheumatism, pain and common cold. In Portugal, there are two taxa usually used in traditional medicine: Seseli tortuosum L. and Seseli montanum subsp. peixotoanum (Samp.). The aim of the present research was to evaluate the anti-inflammatory activity of S. tortuosum and to assess their safety profile in several mammalian cell types at concentrations presenting strong bioactivity. This oil is characterized by high percentage of α-pinene (21.1%), β-pinene (22.6%) and cis-β-ocimene (11.8). The anti-inflammatory potential was investigated in lipopolysaccharide (LPS)-triggered nitric oxide (NO) production by macrophages and microglia concomitantly treated with S. tortuosum essential oil. Our results demonstrated a significant decrease of LPS-induced NO production at concentrations up to 0.16 μL/mL, without affecting cell viability. Our findings confirm the safety of S. tortuosum oil in doses with anti-inflammatory activity. These results support further studies envisaging the use of S. tortuosum oil in pharmaceutical formulations for inhalation, topical application or oral administration

Novel scFv against Notch Ligand JAG1 Suitable for Development of Cell Therapies toward JAG1-Positive Tumors

Funding Information: This research was funded by the Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) grant PTDC/BBB-BMD/4497/2014 (to A.B.), through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). Publisher Copyright: © 2023 by the authors.The Notch signaling ligand JAG1 is overexpressed in various aggressive tumors and is associated with poor clinical prognosis. Hence, therapies targeting oncogenic JAG1 hold great potential for the treatment of certain tumors. Here, we report the identification of specific anti-JAG1 single-chain variable fragments (scFvs), one of them endowing chimeric antigen receptor (CAR) T cells with cytotoxicity against JAG1-positive cells. Anti-JAG1 scFvs were identified from human phage display libraries, reformatted into full-length monoclonal antibodies (Abs), and produced in mammalian cells. The characterization of these Abs identified two specific anti-JAG1 Abs (J1.B5 and J1.F1) with nanomolar affinities. Cloning the respective scFv sequences in our second- and third-generation CAR backbones resulted in six anti-JAG1 CAR constructs, which were screened for JAG1-mediated T-cell activation in Jurkat T cells in coculture assays with JAG1-positive cell lines. Studies in primary T cells demonstrated that one CAR harboring the J1.B5 scFv significantly induced effective T-cell activation in the presence of JAG1-positive, but not in JAG1-knockout, cancer cells, and enabled specific killing of JAG1-positive cells. Thus, this new anti-JAG1 scFv represents a promising candidate for the development of cell therapies against JAG1-positive tumors.publishersversionpublishe

Extract2Chip—Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance

Funding Information: This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). Publisher Copyright: © 2023 by the authors.Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein–protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein–protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.publishersversionpublishe

Identificação de SNPs na população F2 SCA6 x ICS1para obtençao de marcas associadas à resistência do cacaueiro a vassoura-de-bruxa : [Resumo]

A vassoura-de-bruxa causada pelo fungo #Moniliophthora perniciosa# é a doença de maior impacto na cacauicultura no Brasil. Sua principal forma de controle é a utilização de clones resistentes, porém é preciso aumentar a base genética nos plantios comerciais para que se tenha uma resistência mais duradoura. O principal objetivo do Programa de Melhoramento do Cacaueiro do CEPEC/CEPLAC é a piramidação de diferentes genes de resistência, visando aumentar a eficiência e a durabilidade da resistência (PIRES et al., 1996). O objetivo deste estudo foi a busca de polimorfismos de nucleotídeos únicos (SNPs) em genes de resistência a vassoura-de-bruxa, com o intuito de aplicar o mais diretamente possível o resultado dos estudos moleculares à seleção de cacaueiros duravelmente resistentes a M. perniciosa. Os SNPs são resultantes de mutações pontuais e se definem como um loco para o qual encontramos dois alelos em uma freqüência mínima de 1%. A partir de 153 genes de resistência identificados na biblioteca de TSH1188, foram desenhados 73 primers para identificação e validação de SNPs na população F2 SCA6 x ICS1 por seqüenciamento do produto de PCR amplificado. Inicialmente foi feita à otimização da temperatura de 38 primers. Esses primers foram amplificados e seqüenciados nos progenitores SCA6 e ICS1, na F1 TSH516 e no TSH1188. Dos 38 primers,16 foram seqüenciados, sendo 9 monomórficos e 7 polimórficos. O resultado do seqüenciamento foi analisado através do programa CLUSTAL W, onde foi possível a obtenção de 286 SNPs; uma proporção de aproximadamente 51 SNPs para cada gene de resistência onde foi detectado SNP. Foram encontrados SNPs em importantes genes de defesa, como o Cf9_Rapidly Elicited protein, Disease resistance protein, Beta 1,3 - glucanase, e o maior numero de SNPs foi encontrado no gene Pathogenesis-related protein 4B, com 112 SNPs. Este resultado é bastante promissor considerando que SNPs identificados por sequenciamento, normalmente já se encontram validados. Conclui-se que a identificação de SNPs em #Theobroma cacao# mostra-se uma ferramenta promissora para a geração de marcas relacionadas à resistência a vassoura-de-bruxa. (Texte intégral

Software engineering for self-adaptive systems:research challenges in the provision of assurances

The important concern for modern software systems is to become more cost-effective, while being versatile, flexible, resilient, dependable, energy-efficient, customisable, configurable and self-optimising when reacting to run-time changes that may occur within the system itself, its environment or requirements. One of the most promising approaches to achieving such properties is to equip software systems with self-managing capabilities using self-adaptation mechanisms. Despite recent advances in this area, one key aspect of self-adaptive systems that remains to be tackled in depth is the provision of assurances, i.e., the collection, analysis and synthesis of evidence that the system satisfies its stated functional and non-functional requirements during its operation in the presence of self-adaptation. The provision of assurances for self-adaptive systems is challenging since run-time changes introduce a high degree of uncertainty. This paper on research challenges complements previous roadmap papers on software engineering for self-adaptive systems covering a different set of topics, which are related to assurances, namely, perpetual assurances, composition and decomposition of assurances, and assurances obtained from control theory. This research challenges paper is one of the many results of the Dagstuhl Seminar 13511 on Software Engineering for Self-Adaptive Systems: Assurances which took place in December 2013