Towards the metabolic engineering of myrcene pathway of pseudomonas sp. M1 using an integrated omic approach

Pseudomonas sp. M1, isolated from the Rhine River, is able to utilize a large variety of toxic and/or recalcitrant compounds as sole carbon and energy sources, including phenols, benzene and monoterpenes like myrcene [1-3]. Therefore, M1 strain holds great potential as a source of novel biomolecules and cell factories for various biotechnological applications namely in biocatalysis, biosensors, bioremediation and biomedicine. However, the full exploitation of its enzymatic repertoire requires detailed and integrated information about the biomolecular catalog of M1 strain, including genes, proteins and metabolites. In this context, the genome of Pseudomonas sp. M1 was sequenced by NGS technologies, using Illumina Genome Analyser IIx and Roche 454 FLX. The resulting raw data was assembled into 41 contigs and annotated using different pipelines. The current genome draft of Pseudomonas sp. M1 has an estimated GC content of 67%, a size of about 6.9 Mbps and includes 6214 CDS. Importantly, in silico genome analysis predicted a number of metabolic pathways involved in utilization/biotransformation of several unusual carbons sources (e.g. biphenyls, halophenols and different monoterpenes). Proteomic and transcriptomic approaches have been setup envisaging the elucidation of the myrcene stimulon. In 2009, a set of myrcene-dependent proteins has been described using subproteome analysis of the cytoplasmic fraction [3]. More recently, a RNA-seq transcriptome analysis led to the identification of a 28kb genomic island of key importance in the catabolism of myrcene. This island includes genes involved in: i) myrcene oxidation and bioconversion of myrcene derivatives via a beta-oxidation like pathway; ii) regulation of myrcene pathway; iii) myrcene sensing. In addition several other gene clusters spread in the genome of Pseudomonas sp. M1 have been found to be myrcene-dependently expressed and are currently being characterized. Integration of genomic, transcriptomic, proteomic and metabolic data (which is currently being setup) will deliver a very solid and detailed description of the myrcene catabolism (and other monoterpenes), and on the associated molecular mechanisms of adaptation, providing the adequate support for the application of M1 as a biocatalyst in whole-cell biotransformations of plant-derived volatiles.Fundação para a Ciência e a Tecnologia (FCT

An integrated omic approach towards the metabolic engineering of myrcene pathway of pseudomonas sp. M1

Best Poster AwardPseudomonas sp. M1 is able to utilize a large variety of toxic and/or recalcitrant compounds as sole carbon and energy sources, including phenols, benzene and monoterpenes like myrcene [1-3]. Therefore, M1 strain holds great potential as a source of novel biomolecules and cell factories for various biotechnological applications namely in biocatalysis, biosensors, bioremediation and biomedicine. However, the full exploitation of its enzymatic repertoire requires detailed and integrated information about the biomolecular catalog of M1 strain, including genes, proteins and metabolites. In this context, the genome of Pseudomonas sp. M1 was sequenced by NGS technologies, using Illumina GA IIx and Roche 454 FLX. The resulting raw data was assembled and annotated using different pipelines. The current genome draft of Pseudomonas sp. M1 has an estimated GC content of 67%, a size of about 7.1 Mbps and includes 6276 CDS. Importantly, in silico genome analysis predicted a number of metabolic pathways involved in utilization/biotransformation of several unusual carbons sources (e.g. biphenyls, halophenols and different monoterpenes). Proteomic and transcriptomic approaches have been setup envisaging the elucidation of the myrcene stimulon. In 2009, a set of myrcene-dependent proteins has been described using subproteome analysis of the cytoplasmic fraction [3]. In this work, a RNA-seq transcriptome analysis led to the identification of a 28kb genomic island of key importance in the catabolism of myrcene. This island includes genes involved in: i) myrcene oxidation and bioconversion of myrcene derivatives via a beta-oxidation like pathway; ii) regulation of myrcene pathway; iii) myrcene sensing. In addition several other gene clusters spread in the genome of Pseudomonas sp. M1 have been found to be myrcene-dependently expressed and are under investigation. Integration of genomic, transcriptomic, proteomic and metabolic data will deliver a very solid and detailed description of the myrcene catabolism (and other monoterpenes), and on the associated molecular mechanisms of adaptation, providing the adequate support for the application of M1 as a biocatalyst in whole-cell biotransformations of plantderived volatiles.Fundação para a Ciência e a Tecnologia (FCT

Current advances in the bacterial toolbox for the biotechnological production of monoterpene-based aroma compounds

Monoterpenes are plant secondary metabolites, widely used in industrial processes as precursors of important aroma compounds, such as vanillin and (−)-menthol. However, the physicochemical properties of monoterpenes make difficult their conventional conversion into value-added aromas. Biocatalysis, either by using whole cells or enzymes, may overcome such drawbacks in terms of purity of the final product, ecological and economic constraints of the current catalysis processes or extraction from plant material. In particular, the ability of oxidative enzymes (e.g., oxygenases) to modify the monoterpene backbone, with high regio- and stereo-selectivity, is attractive for the production of “natural” aromas for the flavor and fragrances industries. We review the research efforts carried out in the molecular analysis of bacterial monoterpene catabolic pathways and biochemical characterization of the respective key oxidative enzymes, with particular focus on the most relevant precursors, β-pinene, limonene and β-myrcene. The presented overview of the current state of art demonstrates that the specialized enzymatic repertoires of monoterpene-catabolizing bacteria are expanding the toolbox towards the tailored and sustainable biotechnological production of values-added aroma compounds (e.g., isonovalal, α-terpineol, and carvone isomers) whose implementation must be supported by the current advances in systems biology and metabolic engineering approaches.This work was supported by the project VALEU (PTDC/EAM-AMB/30488/2017); by the strategic program UID/BIA/04050/2019 through the Fundação para a Ciência e a Tecnologia (FCT) I.P.; and by the European Regional Development Fund (ERDF) through the COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI). The work was also supported by a Ph.D grant (grant number PD/BD/146184/2019) to F.S

Bioprospection of the bacterial β-myrcene-biotransforming trait in the rhizosphere

The biocatalysis of β-myrcene into value-added compounds, with enhanced organoleptic/therapeutic properties, may be performed by resorting to specialized enzymatic machinery of β-myrcene-biotransforming bacteria. Few β-myrcene-biotransforming bacteria have been studied, limiting the diversity of genetic modules/catabolic pathways available for biotechnological research. In our model Pseudomonas sp. strain M1, the β-myrcene catabolic core-code was identified in a 28-kb genomic island (GI). The lack of close homologs of this β-myrcene-associated genetic code prompted a bioprospection of cork oak and eucalyptus rhizospheres, from 4 geographic locations in Portugal, to evaluate the environmental diversity and dissemination of the β-myrcene-biotransforming genetic trait (Myr+). Soil microbiomes were enriched in β-myrcene-supplemented cultures, from which β-myrcene-biotransforming bacteria were isolated, belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Sphingobacteriia classes. From a panel of representative Myr+ isolates that included 7 bacterial genera, the production of β-myrcene derivatives previously reported in strain M1 was detected in Pseudomonas spp., Cupriavidus sp., Sphingobacterium sp., and Variovorax sp. A comparative genomics analysis against the genome of strain M1 found the M1-GI code in 11 new Pseudomonas genomes. Full nucleotide conservation of the β-myrcene core-code was observed throughout a 76-kb locus in strain M1 and all 11 Pseudomonas spp., resembling the structure of an integrative and conjugative element (ICE), despite being isolated from different niches. Furthermore, the characterization of isolates not harboring the Myr+-related 76-kb locus suggested that they may biotransform β-myrcene via alternative catabolic loci, being thereby a novel source of enzymes and biomolecule catalogue for biotechnological exploitation. KEY POINTS: • The isolation of 150 Myr+ bacteria hints the ubiquity of such trait in the rhizosphere. • The Myr+ trait is spread across different bacterial taxonomic classes. • The core-code for the Myr+ trait was detected in a novel ICE, only found in Pseudomonas spp.Open access funding provided by FCT|FCCN (b-on). This work was supported by the project VALEU (PTDC/EAM-AMB/30488/2017), by the strategic program UID/BIA/04050/2013 (POCI-01–0145-FEDER-007569), by the GenomePT project (POCI-01–0145-FEDER-022184), supported by COMPETE 2020—Opera tional Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the Portugal 2020 Partnership Agreement, funded by national funds through the Fundação para a Ciência e a Tecnologia (FCT) I.P. and the European Regional Development Fund (ERDF), through the COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI). The work was supported as well through the Ph.D. grants to P.S-C (grant number SFRH/BD/76894/2011) and to F.S. (grant number PD/BD/146184/2019)

Outcome of combined peribulbar ropivacaine 0.75% block and general anesthesia for retinal detachment surgery: A randomized controlled study

AbstractBackgroundRetinal detachment surgery (RDS) is frequently associated with a high incidence of significant perioperative pain and oculocardiac reflex (OCR) intra-operatively. The peribulbar block has gained wide acceptance in ophthalmic anesthetic practice in the recent times. However, there is little current knowledge regarding its efficacy in RDS.This prospective randomized clinical study evaluated the effect and feasibility of peribulbar block when used in conjunction with general anesthesia on perioperative outcome.Methods98 patients, ASA II-III, were randomly allocated to one of two groups to receive either peribulbar block in conjunction with general anesthesia (n=49) or general anesthesia alone (n=49).Parameters compared were incidence of OCR, surgical bleeding, duration of surgery, postoperative pain and patient‘s satisfaction.Results and discussionPatients with PB block had a significantly lower incidence of intraoperative OCR (n=4 vs. n=13, p<0.05). It also provided more effective post-operative analgesia with fewer patients requiring rescue analgesia medication (n=19 vs. n=27; p=0.105). Surgical bleeding was more profuse in the general anesthesia group (n=5 vs. n=27, p<0.001), with no cases of bleeding interfering with surgery in the peribulbar group.ConclusionsPB block combined with GA improved significantly operating conditions and lower incidence of OCR. Patients in the block group also had better postoperative analgesia

Políticas públicas de educação: o papel dos diretores na redução da retenção precoce

Portugal regista o segundo pior registo dos países da OCDE relativamente à retenção precoce, apenas ultrapassado pela Bélgica. Este problema dificulta o desenvolvimento social das crianças, uma vez que a retenção precoce trás consigo efeitos que não podem nem devem ser negligenciados. Muitas vezes, a retenção precoce acontece por falta de diagnóstico das dificuldades de aprendizagem, ou por este diagnóstico ser já tardio. No estudo das políticas públicas, ao longo das últimas décadas, tem sido frequente a publicação de artigos que atribuem importância aos atores locais, e que confrontam a tipologia de implementação top-down com a bottom-up. Em Portugal, a área da educação tem sido pioneira no processo de descentralização, estando hoje muitas competências de política educativa confiadas aos organismos de intervenção local. Ao longo deste trabalho, foi analisado, através do estudo Aprender a Ler e Escrever em Portugal, o papel do diretor escolar, figura criada pelo decreto-lei nº 75/2008, de 22 de abril, na redução da retenção precoce. Quando confrontadas 5 escolas que continuaram a ser escolas do insucesso durante os anos em análise e 5 escolas que o deixaram de ser, vemos que a direção escolar desempenha um papel determinante no percurso académico dos alunos, podendo tomar medidas e adotar programas para que a melhoria dos resultados e uma convergência com a média da OCDE seja uma realidade.Portugal registers the second worst OECD registration in relation to early retention, only surpassed by Belgium. This problem hinders the children social development because the retention brings up on the society effects that cannot and should not be neglected. Very often school retention occurs due to lack of diagnosis of learning difficulties, or because this diagnosis is already late. In the public policies study, over the last decades, articles have been written very often showing the importance to the local actors, those who go against the typology of top-down implementation with the bottom-up. In Portugal, the area of education has been a pioneer in the decentralization process, and many educational policy competences are now given to local intervention agencies. Throughout this work, the role of the school director, figure created by decree law number 75/2008, of April 22, was analysed through the study Learning to Read and Write in Portugal, in the cutback of school retention. When we compare 5 schools that carried on to be failure schools during the years under review and 5 schools that no longer have this record, we see that school management plays an important key role in the students’ academic career taking actions and adopting programs to improve the results and a convergence with the OECD average becomes a reality

Sintomatologia do desalinhamento e desajustamento de sistemas de informação

Dissertação de mestrado em Engenharia e Gestão de Sistemas de InformaçãoNo panorama atual das organizações que compõem a nossa sociedade, as Tecnologias e Sistemas de Informação (TSI) desempenham um papel relevante na manipulação da informação e no suporte às variadíssimas atividades organizacionais. O significado atribuído a essa informação constitui a principal base das ações organizacionais, e sendo um ato tipicamente humano, emergem, assim, as pessoas como parte integrante dessas mesmas ações. A forma de planear, decidir, administrar e de atribuir significado não pode ser somente entendida como um exercício formal dado que se poderá inculcar a visão de que tudo é objetivo e apolítico e que as relações e decisões tomadas nas organizações são baseadas somente na racionalidade. Assim, ajustar os Sistemas de Informação (SI) à mudança requer que sejam tidas em conta essas preocupações organizacionais. Considerando as circunstâncias internas e externas a uma organização que influenciam o processo de ajuste à mudança de SI, é coerente diagnosticar problemas na relação entre os vários eixos que desempenham um papel importante na problemática do alinhamento de SI. Assumindo que o alinhamento de SI é uma condição política que conjuga vários interesses constituintes dos vários atores dos SI, emerge a perspetiva do desalinhamento e consequentemente do desajustamento, cujos focos se associam a uma miríade de "maleitas" organizacionais que tornam as organizações menos preparadas para procurar novas formas de acrescentar valor ao negócio. É necessário ter em conta, portanto, atividades como a correção e verificação de discrepâncias de significação e ação organizacionais entre os vários atores, de forma a melhor compreender o fenómeno do desalinhamento e desajustamento de SI. Com efeito, este trabalho de investigação visa auxiliar as organizações a reduzir o seu grau de desalinhamento e desajustamento de SI. Para tal concebeu-se uma sintomatologia, onde se identificaram, caracterizaram e estruturaram sintomas, se associaram causas e se enunciaram ações terapêuticas, adotando-se para esse efeito a Design Science Research como estratégia de investigação. Como resultado principal foi especificado um modelo de desalinhamento e desajustamento de SI, fundado naquela sintomatologia, de modo a auxiliar as organizações a reduzir o seu grau de desalinhamento e desajustamento de SI.In the current scenario of organizations that compounds our society, Information Systems and Technologies (IST) play an important role in information handling and supporting an extensive range of organizational activities. The meaning attributed to such information is the main base of organization actions, and people emerge as an integral part of these same actions. The way of thinking and assigning meaning cannot be understood only as a formal exercise since it can inculcate the view that everything is objective e apolitical, and that organizational relationships and decisions are base solely on rationality. Thus, adjusting Information Systems (IS) to change requires to be taken into account these organizational concerns. Considering the internal and external circumstances to an organization that influence the process of adjusting IS, it is consistent to diagnose relationships problems between the various axis that play an important role in the problem of Business/IS Alignment. Assuming that Business/IS alignment is a political condition needed to combine several constituent interests of various IS actors, emerges the prospect of Business/IS misalignment and hence the prospect of Business/IS maladjustment whose focuses are associated with a myriad of organizational "ailments" that make organizations less prepared to seek new ways of adding value to business. It is necessary to take into account, therefore, activities such as verification and correction of discrepancies of meaning and action among the various organizational actors in order to better understand Business/IS misalignment and maladjustment phenomenon. Indeed, this research work aims to help organizations to reduce their degree of Business/IS misalignment and maladjustment. For such, was conceived a symptomology, where identified, characterized and structured symptoms that were associated causes and enunciated therapeutic actions, adopting for this purpose, the Design Science Research as a research strategy. As primary outcome was specified a Business/IS misalignment and maladjustment model, founded on that simptomology, in order to help organizations reducing their degree of Business/IS misalignment and maladjustment

Insights into the accessory genome of two pseudomonas aeruginosa clinical isolates using comparative genomics

Recently, we have set a collaboration with Hospital de Braga, located in the North of Portugal, that handles over 600 P. aeruginosa isolates per year, aiming to rouse a holistic research approach to provide relevant information and tools to the clinicians to circumvent the multi-resistance phenomena in P. aeruginosa. Since then, we have set procedures aiming a systematic phenotypic characterization of the clinical isolates and developed strategies for the identification of pathogenicity islands and SNPs among the clinical isolates via comparative genomics. In this context, we have determined the full genome sequence of two clinical isolates using the high-throughput system Illumina Genome Analyzer IIx. These two clinical isolates, named 138244 and 152504, are representatives of allelic sequence types ST175 (widely disseminated and associated with multidrug-resistance) and ST560 (rare allele), respectively. Importantly, under standardized experimental procedures, isolate 138244 did not produce pigments and evidenced an antibiotic pan-resistant phenotype whereas 152504 produced a high amount of pyocyanin pigment and was susceptible to all antibiotics tested. A comparative genomic analysis using the genome sequences of both isolates and of all P. aeruginosa strains deposited in Genbank so far, allowed the identification of the accessory genome content of both isolates. Apparently, isolate 152504 harbors in its genome 254 unique genes, often clustered together in the same locus. Based on the genome annotation information, the pool of unique genes mainly encode several virulence factors, chemical stress resistance systems as well as 183 hypothetical proteins, 45 of which predicted members of the secretome of P. aeruginosa 152504. The accessory genome of 138244 mainly includes genes associated with mobile elements (phages, transposases, integrons) and genes encoding for 160 hypothetical proteins. Currently, research approaches are focused on the functional elucidation of sets of genes encoding hypothetical proteins of both isolates and in the description and characterization of their secretomes.Fundação para a Ciência e a Tecnologia (FCT

Comparative genomics of two pseudomonas aeruginosa clinical isolates to elucidate the composition of their mobilomes

Recently, we have set a collaboration with Hospital de Braga, located in the North of Portugal, that handles over 600 P. aeruginosa isolates per year, aiming to rouse a holistic research approach to provide relevant information and tools to the clinicians to circumvent the multi-resistance phenomena in P. aeruginosa. Since then, we have set procedures aiming a systematic phenotypic characterization of the clinical isolates and developed strategies for the identification of pathogenicity islands and SNPs among the clinical isolates via comparative genomics. In this context, we have determined the full genome sequence of two clinical isolates using the high-throughput system Illumina Genome Analyzer IIx. These two clinical isolates, named 138244 and 152504, are representatives of allelic sequence types ST175 (widely disseminated and associated with multidrug-resistance) and ST560 (rare allele), respectively. Importantly, under standardized experimental procedures, isolate 138244 did not produce pigments and evidenced an antibiotic pan-resistant phenotype whereas 152504 produced a high amount of pyocyanin pigment and was susceptible to all antibiotics tested. A comparative genomic analysis using the genome sequences of both isolates and of all P. aeruginosa strains deposited in Genbank so far, allowed the identification of the accessory genome content of both isolates. Apparently, isolate 152504 harbors in its genome 243 unique genes, often clustered together in the same locus. Based on the genome annotation information, the pool of unique genes mainly encode several virulence factors, chemical stress resistance systems as well as 106 hypothetical proteins, some of which predicted members of the secretome of P. aeruginosa 152504. The accessory genome of 138244 mainly includes genes associated with mobile elements (phages, transposases, integrons) and genes encoding for 190 hypothetical proteins. Currently, research approaches are focused on the functional elucidation of sets of genes encoding hypothetical proteins of both isolates and in the description and characterization of their secretomes.Fundação para a Ciência e a Tecnologia (FCT