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Obstructive Sleep Apnea (OSA) is one of the most common sleep disorders, and invokes numerous negative health-related outcomes and physiopathological processes. Understanding the mechanisms by which OSA potentiates cardiometabolic risk of patients remains a current challenge. Sleep disruption is highly prevalent among shift workers and shift work (SW) is an important modulator of circadian rhythms, with health consequences intimately related to cardiometabolic health. Since the circadian timing system (CTS) interacts with the normal functioning of the ANS, CTS impact on OSA patients should be closely assessed. This review raises the question of whether SW-induced circadian misalignment potentiates the cardiometabolic risk of OSA patients, providing a novel perspective on the role of autonomic modulation as a key downstream mechanism linking cardiometabolic risk with both OSA and CTS misalignment.

Supplementary information is available for this paper at https://doi.org/10.1038/s41587-020-0446-y, Reconstructing metabolic reaction networks enables the development of testable hypotheses of an organisms metabolism under different conditions1. State-of-the-art genome-scale metabolic models (GEMs) can include thousands of metabolites and reactions that are assigned to subcellular locations. Geneproteinreaction (GPR) rules and annotations using database information can add meta-information to GEMs. GEMs with metadata can be built using standard reconstruction protocols2, and guidelines have been put in place for tracking provenance and enabling interoperability, but a standardized means of quality control for GEMs is lacking3. Here we report a community effort to develop a test suite named MEMOTE (for metabolic model tests) to assess GEM quality., We acknowledge D. Dannaher and A. Lopez for their supporting work on the Angular parts of MEMOTE; resources and support from the DTU Computing Center; J. Cardoso, S. Gudmundsson, K. Jensen and D. Lappa for their feedback on conceptual details; and P. D. Karp and I. Thiele for critically reviewing the manuscript. We thank J. Daniel, T. Kristjánsdóttir, J. Saez-Saez, S. Sulheim, and P. Tubergen for being early adopters of MEMOTE and for providing written testimonials. J.O.V. received the Research Council of Norway grants 244164 (GenoSysFat), 248792 (DigiSal) and 248810 (Digital Life Norway); M.Z. received the Research Council of Norway grant 244164 (GenoSysFat); C.L. received funding from the Innovation Fund Denmark (project “Environmentally Friendly Protein Production (EFPro2)”); C.L., A.K., N. S., M.B., M.A., D.M., P.M, B.J.S., P.V., K.R.P. and M.H. received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 686070 (DD-DeCaF); B.G.O., F.T.B. and A.D. acknowledge funding from the US National Institutes of Health (NIH, grant number 2R01GM070923-13); A.D. was supported by infrastructural funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Cluster of Excellence EXC 2124 Controlling Microbes to Fight Infections; N.E.L. received funding from NIGMS R35 GM119850, Novo Nordisk Foundation NNF10CC1016517 and the Keck Foundation; A.R. received a Lilly Innovation Fellowship Award; B.G.-J. and J. Nogales received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no 686585 for the project LIAR, and the Spanish Ministry of Economy and Competitivity through the RobDcode grant (BIO2014-59528-JIN); L.M.B. has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 633962 for project P4SB; R.F. received funding from the US Department of Energy, Offices of Advanced Scientific Computing Research and the Biological and Environmental Research as part of the Scientific Discovery Through Advanced Computing program, grant DE-SC0010429; A.M., C.Z., S.L. and J. Nielsen received funding from The Knut and Alice Wallenberg Foundation, Advanced Computing program, grant #DE-SC0010429; S.K.’s work was in part supported by the German Federal Ministry of Education and Research (de.NBI partner project “ModSim” (FKZ: 031L104B)); E.K. and J.A.H.W. were supported by the German Federal Ministry of Education and Research (project “SysToxChip”, FKZ 031A303A); M.K. is supported by the Federal Ministry of Education and Research (BMBF, Germany) within the research network Systems Medicine of the Liver (LiSyM, grant number 031L0054); J.A.P. and G.L.M. acknowledge funding from US National Institutes of Health (T32-LM012416, R01-AT010253, R01-GM108501) and the Wagner Foundation; G.L.M. acknowledges funding from a Grand Challenges Exploration Phase I grant (OPP1211869) from the Bill & Melinda Gates Foundation; H.H. and R.S.M.S. received funding from the Biotechnology and Biological Sciences Research Council MultiMod (BB/N019482/1); H.U.K. and S.Y.L. received funding from the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (grants NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea; H.U.K. received funding from the Bio & Medical Technology Development Program of the NRF, the Ministry of Science and ICT (NRF-2018M3A9H3020459); P.B., B.J.S., Z.K., B.O.P., C.L., M.B., N.S., M.H. and A.F. received funding through Novo Nordisk Foundation through the Center for Biosustainability at the Technical University of Denmark (NNF10CC1016517); D.-Y.L. received funding from the Next-Generation BioGreen 21 Program (SSAC, PJ01334605), Rural Development Administration, Republic of Korea; G.F. was supported by the RobustYeast within ERA net project via SystemsX.ch; V.H. received funding from the ETH Domain and Swiss National Science Foundation; M.P. acknowledges Oxford Brookes University; J.C.X. received support via European Research Council (666053) to W.F. Martin; B.E.E. acknowledges funding through the CSIRO-UQ Synthetic Biology Alliance; C.D. is supported by a Washington Research Foundation Distinguished Investigator Award. I.N. received funding from National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) (grant P20GM125503)., info:eu-repo/semantics/publishedVersion

First-principle metabolic modelling holds potential for designing microbial chassis that are resilient against phenotype reversal due to adaptive mutations. Yet, the theory of model-based chassis design has rarely been put to rigorous experimental test. Here, we report the development of Saccharomyces cerevisiae chassis strains for dicarboxylic acid production using genome-scale metabolic modelling. The chassis strains, albeit geared for higher flux towards succinate, fumarate and malate, do not appreciably secrete these metabolites. As predicted by the model, introducing product-specific TCA cycle disruptions resulted in the secretion of the corresponding acid. Adaptive laboratory evolution further improved production of succinate and fumarate, demonstrating the evolutionary robustness of the engineered cells. In the case of malate, multi-omics analysis revealed a flux bypass at peroxisomal malate dehydrogenase that was missing in the yeast metabolic model. In all three cases, flux balance analysis integrating transcriptomics, proteomics and metabolomics data confirmed the flux re-routing predicted by the model. Taken together, our modelling and experimental results have implications for the computer-aided design of microbial cell factories., We would like to acknowledge the support of R. Mattel and F. Stein from the Proteomics Core Facility and the Genomics Core Facility at the European Molecular Biology Laboratory (EMBL Heidelberg, Germany). This study was supported by national funds through FCT/MCTES (Portugal, Ref. ERA-IB-2/0003/2013) and BMBF (Germany, Grant number: 031A343A, Ref. ERA-IB-2/0003/2013). The Portuguese Foundation for Science and Technology (FCT) supported HL through grant ref. PD/BD/52336/2013. FCT also supported this study under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and through the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). Open Access funding enabled and organized by Projekt DEAL., info:eu-repo/semantics/publishedVersion

Chinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by parsimonious flux balance analysis (pFBA) using the CHO GSMM and a wide range of 13C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function., COMET center acib: Next Generation Bioproduction, which is funded by BMK, BMDW, SFG, Standortagentur Tirol, Government of Lower Austria and Vienna Business Agency in the framework of COMET - Competence Centers for Excellent Technologies. The COMET-Funding Program is managed by the Austrian Research Promotion Agency FFG; D.S., J.S., M.W., M.H., D. E.R. This work has also been supported by the PhD program BioToP of the Austrian Science Fund (FWF Project W1224), info:eu-repo/semantics/publishedVersion

Background: As genome sequencing projects grow rapidly, the diversity of organisms with recently assembled genome sequences peaks at an unprecedented scale, thereby highlighting the need to make gene functional annotations fast and efficient. However, the (high) quality of such annotations must be guaranteed, as this is the first indicator of the genomic potential of every organism. Automatic procedures help accelerating the annotation process, though decreasing the confidence and reliability of the outcomes. Manually curating a genome-wide annotation of genes, enzymes and transporter proteins function is a highly time-consuming, tedious and impractical task, even for the most proficient curator. Hence, a semi-automated procedure, which balances the two approaches, will increase the reliability of the annotation, while speeding up the process. In fact, a prior analysis of the annotation algorithm may leverage its performance, by manipulating its parameters, hastening the downstream processing and the manual curation of assigning functions to genes encoding proteins. Results: Here SamPler, a novel strategy to select parameters for gene functional annotation routines is presented. This semi-automated method is based on the manual curation of a randomly selected set of genes/proteins. Then, in a multi-dimensional array, this sample is used to assess the automatic annotations for all possible combinations of the algorithm’s parameters. These assessments allow creating an array of confusion matrices, for which several metrics are calculated (accuracy, precision and negative predictive value) and used to reach optimal values for the parameters. Conclusions: The potential of this methodology is demonstrated with four genome functional annotations performed in merlin, an in-house user-friendly computational framework for genome-scale metabolic annotation and model reconstruction. For that, SamPler was implemented as a new plugin for the merlin tool., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of [UID/BIO/ 04469] unit and COMPETE 2020 [POCI-01-0145-FEDER-006684] and BioTecNorte operation [NORTE-01-0145-FEDER-000004] funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors thank the project DDDeCaF - Bioinformatics Services for Data-Driven Design of Cell Factories and Communities, Ref. H2020-LEIT-BIO-2015-1 686070–1, funded by the European Commission., info:eu-repo/semantics/publishedVersion

Supine hypertension commonly occurs in patients with neurogenic orthostatic hypotension due to autonomic failure. Supine hypertension promotes nocturnal sodium excretion and orthostatic hypotension, thus, interfering with quality of life. Perusal of the literature on essential hypertension and smaller scale investigations in autonomic failure patients also suggest that supine hypertension may predispose to cardiovascular and renal disease. These reasons provide a rationale for treating supine hypertension. Yet, treatment of supine hypertension, be it through nonpharmacological or pharmacological approaches, may exacerbate orthostatic hypotension when patients get up during the night. Fall-related complications may occur. More research is needed to define the magnitude of the deleterious effects of supine hypertension on cardiovascular, cerebrovascular, and renal morbidity and mortality. Integration of more precise cardiovascular risk assessment, efficacy, and safety data, and the prognosis of the underlying condition causing autonomic failure is required for individualized management recommendations.

In the last years, Salmonella has been extensively studied not only due to its importance as a pathogen, but also as a host to produce pharmaceutical compounds. However, the full exploitation of Salmonella as a platform for bioproduct delivery has been hampered by the lack of information about its metabolism. Genome-scale metabolic models can be valuable tools to delineate metabolic engineering strategies as long as they closely represent the actual metabolism of the target organism. In the present study, a 13C-MFA approach was applied to map the fluxes at the central carbon pathways of S. typhimurium LT2 growing at glucose-limited chemostat cultures. The experiments were carried out in a 2L bioreactor, using defined medium enriched with 20% 13C-labeled glucose. Metabolic flux distributions in central carbon pathways of S. typhimurium LT2 were estimated using OpenFLUX2 based on the labeling pattern of biomass protein hydrolysates together with biomass composition. The results suggested that pentose phosphate is used to catabolize glucose, with minor fluxes through glycolysis. In silico simulations, using Optflux and pFBA as simulation method, allowed to study the performance of the genome-scale metabolic model. In general, the accuracy of in silico simulations was improved by the superimposition of estimated intracellular fluxes to the existing genome-scale metabolic model, showing a better fitting to the experimental extracellular fluxes, whereas the intracellular fluxes of pentose phosphate and anaplerotic reactions were poorly described.

In the last decade, genome-scale metabolic models have been increasingly used to study plant metabolic behavior at the tissue and multi-tissue level under different environmental conditions. Quercus suber, also known as the cork oak tree, is one of the most important forest communities of the Mediterranean/Iberian region. In this work, we present the genome-scale metabolic model of the Q. suber (iEC7871), the first of a woody plant. The metabolic model comprises 7871 genes, 6231 reactions, and 6481 metabolites across eight compartments. Transcriptomics data was integrated into the model to obtain tissue-specific models for the leaf, inner bark, and phellogen, with specific biomass compositions. The tissue-specific models were merged into a diel multi-tissue metabolic model to predict interactions among the three tissues at the light and dark phases. The metabolic models were also used to analyze the pathways associated with the synthesis of suberin monomers. Nevertheless, the models developed in this work can provide insights into other aspects of the metabolism of Q. suber, such as its secondary metabolism and cork formation.

The current survey aims to describe the main methodologies for extending the reconstruction and analysis of genome-scale metabolic models and phenotype simulation with Flux Balance Analysis mathematical frameworks, via the integration of Transcriptional Regulatory Networks and/or gene expression data. Although the surveyed methods are aimed at improving phenotype simulations obtained from these models, the perspective of reconstructing integrated genome-scale models of metabolism and gene expression for diverse prokaryotes is still an open challenge., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04 469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 -Programa Operacional Regional do Norte. Fernando Cruz holds a doctoral fellowship (SFRH/BD/139198/2018) funded by the FCT. This study was supported by the European Commission through project SHIKIFACTORY100 -Modular cell factories for the production of 100 compounds from the shikimate pathway (Reference 814408). The submitted manuscript has been created by UChicago Argonne, LLC as Operator of Argonne National Laboratory (`Argonne') under Contract No. DE-AC02-06CH11357 with the U.S. Department of Energy. The U.S. Government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan., info:eu-repo/semantics/publishedVersion

Candida albicans is one of the most impactful fungal pathogens and the most common cause of invasive candidiasis, which is associated with very high mortality rates. With the rise in the frequency of multidrug-resistant clinical isolates, the identification of new drug targets and new drugs is crucial in overcoming the increase in therapeutic failure. In this study, the first validated genome-scale metabolic model for Candida albicans, iRV781, is presented. The model consists of 1221 reactions, 926 metabolites, 781 genes, and four compartments. This model was reconstructed using the open-source software tool merlin 4.0.2. It is provided in the well-established systems biology markup language (SBML) format, thus, being usable in most metabolic engineering platforms, such as OptFlux or COBRA. The model was validated, proving accurate when predicting the capability of utilizing different carbon and nitrogen sources when compared to experimental data. Finally, this genome-scale metabolic reconstruction was tested as a platform for the identification of drug targets, through the comparison between known drug targets and the prediction of gene essentiality in conditions mimicking the human host. Altogether, this model provides a promising platform for global elucidation of the metabolic potential of C. albicans, possibly guiding the identification of new drug targets to tackle human candidiasis., “Fundação para a Ciência e a Tecnologia” (FCT) [Contract PTDC /BII-BIO/28216/2017] and by Programa Operacional Regional de Lisboa 2020 [LISBOA-01-0145-FEDER-022231], through the Biodata.pt Research Infrastructure. Funding received by iBB-Institute for Bioengineering and Biosciences from FCT [Contract UIDB/04565/2020], info:eu-repo/semantics/publishedVersion

The global market of butanol is increasing due to its growing applications as solvent, flavoring agent and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through Acetone-Butanol-Ethanol fermentation, at non-competitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates.. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism; elimination of competing pathways; cofactor balancing; development of new pathways; expression of homologous enzymes; consumption of different substrates and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify and exploit the outstanding challenges. This article is protected by copyright. All rights reserved., Ministério da Ciência, Tecnologia e Ensino Superior, Grant/Award Number: ERA‐IB‐2/0002/2014; Fundação para a Ciência e a Tecnologia, Grant/Award Numbers: ERA‐IB‐2/0002/2014, LISBOA¬01¬0145¬FEDER¬007660, PD/BD/52366/2013; European Commission, Grant/Award Number: H2020‐LEIT‐BIO‐2015‐1 686070–1, info:eu-repo/semantics/publishedVersion

The cerebrovascular effects of a failing heart-pump are largely unknown. Chronic heart failure (HF) might cause pre-conditioning effect on cerebral hemodynamics but not study so far in acute stroke. We aimed to investigate if HF induces effects in dynamic cerebral autoregulation (CA), within 6 h of symptom-onset through chronic stage of ischemic stroke. We enrolled 50 patients with acute ischemic stroke. Groups with (N = 8) and without HF and 20 heathy controls were compared. Arterial blood pressure (Finometer) and cerebral blood flow velocity (transcranial Doppler) were monitored within 6 and at 24 h from symptom-onset and at 3 months. We assessed dynamic CA by transfer function analysis and cardiac disease markers. HF associated with higher phase (better dynamic CA) at ischemic hemisphere within 6 (p = 0.042) and at 24 h (p = 0.006) but this effect was not evident at 3 months (p > 0.05). Gain and coherence trends were similar between groups. We found a positive correlation between phase and admission troponin I levels (Spearman’s r = 0.348, p = 0.044). Our findings advances on the knowledge of how brain and heart interact in acute ischemic stroke by showing a sustained dynamic cerebral autoregulation response in HF patients mainly with severe aortic valve disease. Understanding the physiological mechanisms that govern this complex interplay can be useful to find novel therapeutic targets which can improve outcome in ischemic stroke.

© 2020 Springer Nature Switzerland AG. Part of Springer Nature., Long-term lead (Pb) exposure alters the normal development of the nervous system and physiology. It affects multiple organ systems, causing hypertension, cardiorespiratory dysfunction, being a well-known neurotoxin, inducing changes in neurogenesis, neurodegeneration, and glial cells. However, studies of the developmental effects of lead and its outcomes throughout life are lacking. Determine morphofunctional, behavioral, and cognitive developmental effects of long-term lead exposure at three different ages. Wistar rats were exposed to a Pb-acetate solution from fetal period until adulthood and compared to a non-exposed control group. General behavior and cognitive skills were evaluated by behavioral tests and physiological data and cardiorespiratory reflexes measured. Neurodegeneration, neuroinflammation, and synaptic activity were assessed by immunohistochemistry. Lead exposure caused long-lasting anxiety-like behavior and strong long-term memory impairment without changes in locomotor and exploratory activity. Hypertension was observed at all time points, concomitant with baroreflex impairment and increased chemoreflex sensitivity. Persistent neuroinflammation, transient synaptic overexcitation without neurodegeneration was observed. Long-term Pb exposure, since fetal period, causes long-lasting anxiety-like behavior, concomitant with hypertension, without general motor skills impairment. Synaptic overexcitation, reactive astrogliosis, and microgliosis could underlie behavioral and long-term memory changes, which might have been caused during developmental phases and consolidated during adulthood. Also, alterations observed in the cardiorespiratory reflexes can explain persistent hypertension. This longitudinal study identifies and characterizes lead toxicity nature and magnitude, important to devise and test potential interventions to attenuate the long-term harmful effects of lead on the nervous and cardiovascular systems., Vera Geraldes received the post-doctoral fellowship given by the Fundação para a Ciência e Tecnologia (FCT) (Ref: SFRH/BPD/119315/2016).

O processo de fabricacao de alimentos produz grande quantidade de residuos orgânicos. Uma industria de extracao de aromas, por exemplo, pode gerar, anualmente, 104 e 92 toneladas de residuo de erva-mate e guarana, respectivamente. Uma alternativa para o reaproveitamento desse material lignocelulosico seria utilizalos na producao de biocompositos com micelio fungico atuando como ligante das particulas do substrato. Sendo assim, objetivou-se nesse trabalho a producao de biocompositos a partir de Pleurotus sajor-caju utilizando os residuos de erva-mate e guarana na proporcao (1:1) (m/m), verificando a influencia da fracao de inoculo 10, 20 e 30% e da temperatura de secagem de 40 e 60°C. Para cada condicao testada foi avaliado o tempo de crescimento micelial, o teor de umidade inicial, a velocidade inicial de secagem, a resistencia a compressao, absorcao de umidade do ar e absorcao de agua. A condicao determinada foi a de 10% de inoculo e secagem a 60°C, pois apresentou maior resistencia a compressao (0,094 MPa), menor absorcao de agua (91%), maior velocidade de secagem (27,7 g/dia). Os biocompositos utilizam o crescimento biologico e nao processos de fabricacao com intenso desperdicio de energia, ainda exigem apenas residuos orgânicos de baixo custo como materia-prima, podendo preencher geometrias complexas apresentando potencial para a confeccao de embalagens, pois sao materiais seguros que apresentaram boa resistencia a compressao e baixa absorcao de umidade do ar e de agua. Palavras-chave: Pleurotus sajor-caju, residuos industriais, cultivo solido, biocompositos.

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Following the success of and the high demand for recombinant protein-based therapeutics during the last 25 years, the pharmaceutical industry has invested significantly in the development of novel treatments based on biologics. Mammalian cells are the major production systems for these complex biopharmaceuticals, with Chinese hamster ovary (CHO) cell lines as the most important players. Over the years, various engineering strategies and modeling approaches have been used to improve microbial production platforms, such as bacteria and yeasts, as well as to create pre-optimized chassis host strains. However, the complexity of mammalian cells curtailed the optimization of these host cells by metabolic engineering. Most of the improvements of titer and productivity were achieved by media optimization and large-scale screening of producer clones. The advances made in recent years now open the door to again consider the potential application of systems biology approaches and metabolic engineering also to CHO. The availability of a reference genome sequence, genome-scale metabolic models and the growing number of various “omics” datasets can help overcome the complexity of CHO cells and support design strategies to boost their production performance. Modular design approaches applied to engineer industrially relevant cell lines have evolved to reduce the time and effort needed for the generation of new producer cells and to allow the achievement of desired product titers and quality. Nevertheless, important steps to enable the design of a chassis platform similar to those in use in the microbial world are still missing. In this review, we highlight the importance of mammalian cellular platforms for the production of biopharmaceuticals and compare them to microbial platforms, with an emphasis on describing novel approaches and discussing still open questions that need to be resolved to reach the objective of designing enhanced modular chassis CHO cell lines., This work has been supported the Federal Ministry for Digital and Economic Affairs (bmwd), the Federal Ministry for Transport, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, Government of Lower Austria and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. A.H. has been supported by the Portuguese NORTE-08-5369-FSE-000053 operation. Additional funding came from the PhD program BioToP (Biomolecular Technology of Proteins) of the Austrian Science Fund (FWF Project W1224) and MIT-Portugal PhD program (Bioengineering Systems). The funding agencies had no influence on the conduct of this research. Open Access Funding by the University of Vienna., info:eu-repo/semantics/publishedVersion

Background Actinobacillus succinogenes is a promising bacterial catalyst for the bioproduction of succinic acid from low-cost raw materials. In this work, a genome-scale metabolic model was reconstructed and used to assess the metabolic capabilities of this microorganism under producing conditions. Results The model, iBP722, was reconstructed based on the functional reannotation of the complete genome sequence of A. succinogenes 130Z and manual inspection of metabolic pathways, covering 1072 enzymatic reactions associated with 722 metabolic genes that involve 713 metabolites. The highly curated model was effective in capturing the growth of A. succinogenes on various carbon sources, as well as the SA production under various growth conditions with fair agreement between experimental and predicted data. Calculated flux distributions under different conditions show that a number of metabolic pathways are affected by the activity of some metabolic enzymes at key nodes in metabolism, including the transport mechanism of carbon sources and the ability to fix carbon dioxide. Conclusions The established genome-scale metabolic model can be used for model-driven strain design and medium alteration to improve succinic acid yields., Financially supported by BRIGIT (KBBE-2012-6-311935, FP7 project Contract nr 311935) and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), in addition to the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

We would like to thank the European Union Framework Program 7 ‘BacHBerry’ (www.bachberry.eu), Project No. FP7- 613793 for financial support, the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01- 0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.

The lipid nanoparticles, namely Nanostructured Lipid Carriers (NLC), as drug delivery systems have been investigated for several years. One of the delivery routes for which these carriers can be applied is buccal administration. However, the liquid dispersions of lipid nanoparticles can be rapidly removed from oral cavity by saliva. Thus, the development of a system that allows increased retention time on the mucosa is necessary. For this reason, the development of mucoadhesive preparations for buccal administration of lipid nanoparticles becomes important. Hydrogels prepared with mucoadhesive polymers (Carbopol® 980 and polycarbophil) constitute a promising option. The aim of this work was to develop mucoadhesive buccal hydrogels with NLC, using ibuprofen as a model drug. The obtained results showed that the developed NLC dispersions presented particles in the nanometric size range, with low polydispersity index values and efficient ability for the entrapment of the model drug. Moreover, the incorporation of NLC in hydrogels of mucoadhesive polymers resulted in preparations with desirable rheological features as well as texture (firmness and adhesiveness) and mucoadhesive properties, which could benefit the therapeutic efficacy, by increasing the residence time and easiness for topical application in the buccal mucosa. Additionally, the developed preparations exhibited sustained drug release as intended for these systems.

© 2017 Sociedade Portuguesa de Cardiologia. Published by Elsevier España, S.L.U. All rights reserved, Knowledge of the physiology underlying the autonomic nervous system is pivotal for understanding autonomic dysfunction in clinical practice. Autonomic dysfunction may result from primary modifications of the autonomic nervous system or be secondary to a wide range of diseases that cause severe morbidity and mortality. Together with a detailed history and physical examination, laboratory assessment of autonomic function is essential for the analysis of various clinical conditions and the establishment of effective, personalized and precise therapeutic schemes. This review summarizes the main aspects of autonomic medicine that constitute the background of cardiovascular autonomic dysfunction., O conhecimento subjacente à fisiologia do sistema nervoso autónomo é fundamental para se entender a disfunção autonómica na prática clinica. A disfunção autonómica pode resultar primariamente de modificações do sistema ou, secundariamente, a uma série de patologias conducentes a morbilidade ou mortalidade. Juntamente com a colheita detalhada da história clínica e do exame objetivo, a avaliac¸ão autonómica laboratorial torna-se essencial na análise de algumas condições clínicas e no estabelecimento de esquemas terapêuticos mais eficazes, refinados e personalizados. Assim, nesta revisão sumarizam-se os aspetos mais relevantes da fisiologia autonómica subjacente a disfunção autonómica cardiovascular.

Cigarette smoke is associated to pathological weakening of bone tissue, being considered an important playmaker in conditions such as osteoporosis and periodontal bone loss. In addition, it is also associated with an increased risk of failure in bone regeneration strategies. The present work aimed to characterize the effects of nicotine on human osteoclastogenesis over a hydroxyapatite substrate. Osteoclast precursors were maintained in the absence or presence of the osteoclastogenesis enhancers M-CSF and RANKL, and were further treated with nicotine levels representative of the concentrations observed in the plasma and saliva of smokers. It was observed that nicotine at low concentrations elicit an increase in osteoclast differentiation, but only in the presence of M-CSF and RANKL it was also able to significantly increase the resorbing ability of osteoclasts. A slight downregulation of NFkB pathway and an increase in the production of TNF-α and, particularly PGE2, were involved in the observed effects of nicotine. At high concentrations, nicotine revealed cytotoxic effects, causing a decrease in cell density. In conclusion, nicotine at levels found in the plasma of the smokers, has the ability to act directly on osteoclast precursors, inducing its osteoclastogenic differentiation. The stimulatory behavior appears to be dependent on the stage of osteoclastic differentiation of the precursor cells, which means, in the absence of M-CSF and RANKL, it only favors the initial stages of osteoclast differentiation, while in the presence of the growth factors, a significant increase in their resorbing ability is also achieved.

Background Hemorrhagic transformation and cerebral edema are feared complications of acute ischemic stroke but mechanisms are poorly understood and reliable early markers are lacking. Early assessment of cerebrovascular hemodynamics may advance our knowledge in both areas. We examined the relationship between dynamic cerebral autoregulation (CA) in the early hours post ischemia, and the risk of developing hemorrhagic transformation and cerebral edema at 24 h post stroke Methods We prospectively enrolled 46 patients from our center with acute ischemic stroke in the middle cerebral artery territory. Cerebrovascular resistance index was calculated. Dynamic CA was assessed by transfer function analysis (coherence, phase and gain) of the spontaneous blood flow velocity and blood pressure oscillations. Infarct volume, hemorrhagic transformation, cerebral edema, and white matter changes were collected from computed tomography performed at presentation and 24 h. Results At admission, phase was lower (worse CA) in patients with hemorrhagic transformation [6.6 ± 30 versus 45 ± 38°; adjusted odds ratio 0.95 (95% confidence internal 0.94–0.98), p = 0.023] and with cerebral edema [6.6 ± 30 versus 45 ± 38°, adjusted odds ratio 0.96 (0.92–0.999), p = 0.044]. Progression to edema was associated with lower cerebrovascular resistance (1.4 ± 0.2 versus 2.3 ± 1.5 mm Hg/cm/s, p = 0.033) and increased cerebral blood flow velocity (51 ± 25 versus 42 ± 17 cm/s, p = 0.033) at presentation. All hemodynamic differences resolved at 3 months Conclusions Less effective CA in the early hour post ischemic stroke is associated with increased risk of hemorrhagic transformation and cerebral edema, possibly reflecting breakthrough hyperperfusion and microvascular injury. Early assessment of dynamic CA could be useful in identifying individuals at risk for these complications.

The composition of a cell in terms of macromolecular building blocks and other organic molecules underlies the metabolic needs and capabilities of a species. Although some core biomass components such as nucleic acids and proteins are evident for most species, the essentiality of the pool of other organic molecules, especially cofactors and prosthetic groups, is yet unclear. Here we integrate biomass compositions from 71 manually curated genome-scale models, 33 large-scale gene essentiality datasets, enzyme-cofactor association data and a vast array of publications, revealing universally essential cofactors for prokaryotic metabolism and also others that are specific for phylogenetic branches or metabolic modes. Our results revise predictions of essential genes in Klebsiella pneumoniae and identify missing biosynthetic pathways in models of Mycobacterium tuberculosis. This work provides fundamental insights into the essentiality of organic cofactors and has implications for minimal cell studies as well as for modeling genotype-phenotype relations in prokaryotic metabolic networks., J.C.X. was sponsored by Fundação para a Ciência e Tecnologia, Portugal [Grant SFRH/BD/81626/2011]. This study was supported by the European Molecular Biology Laboratory, the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01–0145-FEDER-006684) and BioTecNorte operation (NORTE-01–0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This project has received funding from the European Union's Horizon 2020 research and innovation programme under Grant agreement no 686070.

Genome-scale metabolic models (GEMs) can be used to evaluate genotype-phenotype relationships and their application to microbial strain engineering is increasing in popularity. Some of the algorithms used to simulate the phenotypes of mutant strains require the determination of a wild-type flux distribution. However, the accuracy of this reference, when calculated with flux balance analysis, has not been studied in detail before. Here, the wild-type simulations of selected GEMs for Saccharomyces cerevisiae have been analysed and most of the models tested predicted erroneous fluxes in central pathways, especially in the pentose phosphate pathway. Since the problematic fluxes were mostly related to areas of the metabolism consuming or producing NADPH/NADH, we have manually curated all reactions including these cofactors by forcing the use of NADPH/NADP+ in anabolic reactions and NADH/NAD+ for catabolic reactions. The curated models predicted more accurate flux distributions and performed better in the simulation of mutant phenotypes., FCT -Fuel Cell Technologies Program(SFRH/BD/51111/2010)

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.01283/full#supplementary-material, A high-quality GSM model for Streptococcus pneumoniae R6 model strain (iDS372), comprising 372 genes and 529 reactions, was developed. The construction of this model involved performing a genome-wide reannotation to identify the metabolic capacity of the bacterium. A reaction representing the abstraction of the biomass composition was reconciled from several studies reported in the literature and previous models, and included in the model. The final model comprises two compartments and manifold automatically generated gene rules. The validation was performed with experimental data from recent studies, regarding the usability of carbon sources, the effect of the presence of oxygen and the requirement of amino acids for growth. This model can be used to better understand the metabolism of this major pathogen, provide clues regarding new drug targets and eventually design strategies for fighting infections by these bacteria., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional doNorte. This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreementno.686070., info:eu-repo/semantics/publishedVersion

Computational strain optimisation methods (CSOMs) have been successfully used to exploit genome-scale metabolic models, yielding strategies useful for allowing compound overproduction in metabolic cell factories. Minimal cut sets are particularly interesting since their definition allows searching for intervention strategies that impose strong growth-coupling phenotypes, and are not subject to optimality bias when compared with simulation-based CSOMs. However, since both types of methods have different underlying principles, they also imply different ways to formulate metabolic engineering problems, posing an obstacle when comparing their outputs., “DeYeastLibrary – Designer yeast strain library optimized for metabolic engineering applications”, Ref.ERA-IB-2/0003/2013, funded by national funds through FCT/MCTES, DD-DeCaf and SHIKIFACTORY100, both funded by the European Union through the Horizon 2020 research and innovation programme (grant agreements no. 686070 and 814408). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors acknowledge the use of computing facilities within the scope of the Search-ON2: Revitalization of HPC infrastructure of UMinho” project (NORTE-07-0162-FEDER-000086), co-funded by the North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). VV also thanks funding from FCT/MCTES for the PhD studentship with reference SFRH/BD/118657/2016., info:eu-repo/semantics/publishedVersion

The uncertain relationship between genotype and phenotype can make strain engineering an arduous trial and error process. To identify promising gene targets faster, constraint-based modelling methodologies are often used, although they remain limited in their predictive power. Even though the search for gene knock-outs is fairly established in constraint-based modelling, most strain design methods still model gene up/down-regulations by forcing the corresponding flux values to fixed levels without taking in consideration the availability of resources. Here, we present a constraint-based algorithm, the Turnover Dependent Phenotypic Simulation (TDPS) that quantitatively simulates phenotypes in a resource conscious manner. Unlike other available algorithms, TDPS does not force flux values and considers resource availability, using metabolite production turnovers as an indicator of metabolite abundance. TDPS can simulate up-regulation of metabolic reactions as well as the introduction of heterologous genes, alongside gene deletion and down-regulation scenarios. TDPS simulations were validated using engineered Saccharomyces cerevisiae strains available in the literature by comparing the simulated and experimental production yields of the target metabolite. For many of the strains evaluated, the experimental production yields were within the simulated intervals and the relative strain performance could be predicted with TDPS. However, the algorithm failed to predict some of the production changes observed experimentally, suggesting that further improvements are necessary. The results also showed that TDPS may be helpful in finding metabolic bottlenecks, but further experiments would be required to confirm these findings., ortuguese Foundation forScience and Technology (FCT) under the scope of thestrategic funding of UID/BIO/04469/2019 unit and Bio-TecNorte operation (NORTE-01-0145-FEDER-000004)funded by the European Regional Development Fund under the scope of Norte2020−Programa Operacional Regional doNorte. This project has received funding from the EuropeanUnion’s Horizon 2020 research and innovation programmeunder Grant Agreement No. 686070 and from the NovoNordisk Foundation. The work of Rui Pereira was supported by a PhD grant from FCT (ref SFRH/BD/51111/2010), info:eu-repo/semantics/publishedVersion

Portugal presents the highest incidence of stage 5 chronic kidney disease in Europe. It is speculated that a high consumption of non-steroidal anti-inflammatory drugs (NSAIDS) may contribute to this high incidence. Our aim was to characterize the prescription of non-steroidal anti-inflammatory drugs to patients with diabetes mellitus in Portugal.We analyzed the national prescription database in triennium 2015 - 2017. In patients with diabetes mellitus, we evaluated the prescription of non-steroidal anti-inflammatory drugs according to age, gender and region of the patient and specialty of the prescribing physician. We evaluated the prescription of non-steroidal anti-inflammatory drugs in all patients with diabetes mellitus, in patients with presumed renal impairment, and in those with concomitant prescription of angiotensin converting enzyme inhibitors or angiotensin receptor antagonists.We analyzed 23 320 620 prescriptions, corresponding to 610 157 adults, including 104 306 patients with diabetes mellitus. The most prescribed non-steroidal anti-inflammatory drugs were ibuprofen (20.1%), metamizole (14.7%), and diclofenac (11.4%). The prescription of non-steroidal anti-inflammatory drugs was higher in females, in patients aged 51 - 70 years and in the Alentejo region. Non-steroidal anti-inflammatory drugs were prescribed to 70.6% of patients with diabetes mellitus, from which 10.6% were prescribed ≥ 10 packages during the three years. Among patients with diabetes mellitus on angiotensin converting enzyme inhibitors/angiotensin receptor antagonists and with presumed reduction in kidney function, 69.3% were prescribed non-steroidal anti-inflammatory drugs and 11.5% were prescribed ≥ 10 packages during the three years.The level of prescribing of non-steroidal anti-inflammatory drugs to patients with diabetes mellitus is high. The concern of reducing non-steroidal anti-inflammatory drugs prescription to patients already on angiotensin converting enzyme inhibitors/angiotensin receptor antagonists and/or decreased renal function does not seem to exist.In Portugal, the level of prescribing of non-steroidal anti-inflammatory drugs to patients with diabetes mellitus should be reduced, particularly in the subgroups identified with higher prescription and with higher risk of progression to stage 5 chronic kidney disease.Introdução: Portugal apresenta a incidência mais elevada de doença renal crónica estádio 5 na Europa. Especula-se que o elevado consumo de anti-inflamatórios não esteroides possa contribuir para esta incidência. O objetivo do presente estudo foi caracterizar a prescrição de anti-inflamatórios não esteroides a doentes com diabetes mellitus em Portugal. Material e Métodos: Na Base de Dados Nacional de Prescrições do Ministério da Saúde, triénio 2015 - 2017, analisámos a prescrição de anti-inflamatórios não esteroides em doentes com diabetes mellitus, de acordo com a idade, género e região do doente e a especialidade do médico prescritor. Avaliámos a prescrição de anti-inflamatórios não esteroides no total de doentes com diabetes mellitus, em doentes com diminuição presumida da função renal e naqueles com prescrição concomitante de inibidores da enzima de conversão da angiotensina ou antagonistas dos recetores da angiotensina. Resultados: Analisámos 23 320 620 prescrições, correspondendo a 610 157 adultos, dos quais 104 306 doentes com diabetes mellitus. Os anti-inflamatórios não esteroides mais prescritos foram ibuprofeno (20,1%), metamizol (14,7%) e diclofenac (11,4%). A prescrição foi mais frequente nas mulheres, nos doentes com 51 - 70 anos e no Alentejo. Foram prescritos anti-inflamatórios não esteroides a 70,6% dos doentes com diabetes mellitus, dos quais 10,6% receberam prescrições de ≥ 10 embalagens durante os três anos. Dos doentes com diabetes mellitus medicados com inibidores da enzima de conversão da angiotensina ou antagonistas dos receptores da angiotensina e com diminuição presumida da taxa de filtração glomerular, 69,3% receberam prescrição de anti-inflamatórios não esteroides e 11,5% receberam ≥ 10 embalagens durante os três anos. Discussão: A prescrição de anti-inflamatórios não esteroides na diabetes mellitus é elevada. Não parece existir uma preocupação na menor utilização de anti-inflamatórios não esteroides em doentes simultaneamente medicados com inibidores da enzima de conversão da angiotensina ou antagonistas dos recetores da angiotensina e/ou com diminuição da função renal. Conclusão: A prescrição de anti-inflamatórios não esteroides em Portugal a doentes com diabetes mellitus deverá ser reduzida, particularmente nos subgrupos identificados com prescrição mais elevada e com maior risco de progressão para doença renal crónica estádio 5.

Plants synthesize a variety of different secondary metabolites, such as polyphenols, terpenoids, alkaloids, etc., with pharmaceutical and nutraceutical importance. Polyphenols have shown numerous health benefits with rare side effects. However, the extraction of these compounds from natural sources cannot meet the increasing consumer demand for natural products, and its purification is often difficult, making the overall process too expensive. In contrast, microbial production of polyphenols is a powerful alternative to produce natural products in large amounts, in an environmentally sustainable way. Nevertheless, plant-derived polyphenols are not naturally produced by microorganisms and therefore require the integration of the heterologous pathway from plants through genetic engineering techniques. In the present chapter, the recent advances in microbial production of plant-derived polyphenols, emphasizing on flavonoids, anthocyanins, curcuminoids, and stilbenes, have been summarized. In addition, different strategies used to increase the product yield, and the production processes are also highlighted., We would like to thank the European Union Framework Program 7 “BacHBerry” (www.bachberry.eu), Project No. FP7- 613793 for financial support, the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), and BiotecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

In recent years microorganisms have been engineered towards synthesizing interesting plant polyphenols such as flavonoids and stilbenes from glucose. Currently, low endogenous supply of malonylCoA, indispensable for plant polyphenol synthesis, impedes high product titers. Usually, limited malonylCoA availability during plant polyphenol production is avoided by supplementing fatty acid synthesisinhibiting antibiotics such as cerulenin, which are known to increase the intracellular malonylCoA pool as a side effect. Motivated by the goal of microbial polyphenol synthesis being independent of such expensive additives, we used rational metabolic engineering approaches to modulate regulation of fatty acid synthesis and flux into the citric acid cycle in Corynebacterium glutamicum strains capable of flavonoid and stilbene synthesis. Initial experiments showed that sole overexpression of genes coding for the native malonylCoAforming acetylCoA carboxylase is not sufficient for increasing polyphenol production in C. glutamicum. Hence, the intracellular acetylCoA availability was also increased by reducing the flux into the citric acid cycle through reduction of citrate synthase activity. In defined cultivation medium, the constructed C. glutamicum strains accumulated 24 mg/L (0.088 mM) naringenin or 112 mg/L (0.49 mM) resveratrol from glucose without supplementation of phenylpropanoid precursor molecules or any inhibitors of fatty acid synthesis., We would like to thank the European Union Framework Program 7 ''BacHBerry'' (www.bachberry.eu), Project No. FP7‐ 613793 for financial support and also the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469 unit, COMPETE 2020 (POCI‐01–0145‐FEDER‐006684) and BiotecNorte operation (NORTE‐01–0145‐FEDER‐000004) funded by the European Regional Development Fund under the scope of Norte2020 ‐ Programa Operacional Regional do Norte. Furthermore, we would like to thank Dr. Tino Polen for providing the plasmids to replace the gltA promoter by dapA promoter variants., info:eu-repo/semantics/publishedVersion

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

If we tried to list every known chemical reaction within an organismhuman, plant or even bacteriawe would get quite a long and confusing read. But when this information is represented in so-called genome-scale metabolic networks, we have the means to access computationally each of those reactions and their interconnections. Some parts of the network have alternatives, while others are unique and therefore can be essential for growth. Here, we simulate growth and compare essential reactions and genes for the simplest type of unicellular speciesprokaryotesto understand which parts of their metabolism are universally essential and potentially ancestral. We show that similar patterns of essential reactions echo phylogenetic relationships (this makes sense, as the genome provides the building plan for the enzymes that perform those reactions). Our computational predictions correlate strongly with experimental essentiality data. Finally, we show that a crucial step of protein synthesis (tRNA charging) and the synthesis and transformation of small molecules that enzymes require (cofactors) are the most essential and conserved parts of metabolism in prokaryotes. Our results are a step further in understanding the biology and evolution of prokaryotes but can also be relevant in applied studies including metabolic engineering and antibiotic design., This work was supported by grants from: the Fundac ¸ão para a Ciência e a Tecnologia (http:// www.fct.pt) with award number UID/BIO/04469/2013, the European Regional Development Fund (http://www.norte2020.pt) with award number NORTE-01-0145-FEDER-000004 (https://www. ceb.uminho.pt/Projects/Details/6040), Horizon 2020 (https://ec.europa.eu/programmes/ horizon2020) with award number 686070 (http:// dd-decaf.eu/) and COMPETE2020 with award number POCI-01-0145-FEDER-006684 to JCX and IR and the Fundação para a Ciência e a Tecnologia (http://www.fct.pt) with award number SFRH/BD/81626/2011 to JCX. The funders had no role in study design, data collectionand analysis, decision to publish, or preparation of the manuscript., info:eu-repo/semantics/publishedVersion

Mannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward. However, the current prohibitive production costs impose severe constraints towards large-scale applications. All known microbial producers synthesize MG from GDP-mannose and 3-phosphoglycerate via a two-step pathway in which mannosyl-3-phosphoglycerate is the intermediate metabolite. In an early work, this pathway was expressed in Saccharomyces cerevisiae with the goal to confirm gene function (Empadinhas et al. in J Bacteriol 186:4075--4084, 2004), but the level of MG accumulation was low. Therefore, in view of the potential biotechnological value of this compound, we decided to invest further effort to convert S. cerevisiae into an efficient cell factory for MG production., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and also by project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI). Cristiana Faria was supported by a Ph.D. Grant from FCT (Ref. SFRH/ BD/79552/2011)., info:eu-repo/semantics/publishedVersion

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated., Background: Depressive symptoms are common in individuals suffering from severe somatic conditions. There is a lack of interventions and evidence-based interventions aiming to reduce depressive symptoms in patients with severe somatic conditions. The aim of the NEVERMIND project is to address these issues and provide evidence by testing the NEVERMIND system, designed to reduce and prevent depressive symptoms in comparison to treatment as usual. Methods: The NEVERMIND study is a parallel-groups, pragmatic randomised controlled trial to assess the effectiveness of the NEVERMIND system in reducing depressive symptoms among individuals with severe somatic conditions. The NEVERMIND system comprises a smart shirt and a user interface, in the form of a mobile application. The system is a real-time decision support system, aiming to predict the severity and onset of depressive symptoms by modelling the well-being condition of patients based on physiological data, body movement, and the recurrence of social interactions. The study includes 330 patients who have a diagnosis of myocardial infarction, breast cancer, prostate cancer, kidney failure, or lower limb amputation. Participants are randomised in blocks of ten to either the NEVERMIND intervention or treatment as usual as the control group. Clinical interviews and structured questionnaires are administered at baseline, at 12 weeks, and 24 weeks to assess whether the NEVERMIND system is superior to treatment as usual. The endpoint of primary interest is Beck Depression Inventory II (BDI-II) at 12 weeks defined as (i) the severity of depressive symptoms as measured by the BDI-II. Secondary outcomes include prevention of the onset of depressive symptoms, changes in quality of life, perceived stigma, and self-efficacy. Discussion: There is a lack of evidence-based interventions aiming to reduce and prevent depressive symptoms in patients with severe somatic conditions. If the NEVERMIND system is effective, it will provide healthcare systems with a novel and innovative method to attend to depressive symptoms in patients with severe somatic conditions., The NEVERMIND project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 689691. The funding body had no role in the design of the study and collection as well as in writing the manuscript. Open access funding provided by Karolinska Institute.

In the era of next-generation sequencing and ubiquitous assembly and binning of metagenomes, new putative genome sequences are being produced from isolate and microbiome samples at ever-increasing rates. Genome-scale metabolic models have enormous utility for supporting the analysis and predictive characterization of these genomes based on sequence data. As a result, tools for rapid automated reconstruction of metabolic models are becoming critically important for supporting the analysis of new genome sequences. Many tools and algorithms have now emerged to support rapid model reconstruction and analysis. Here, we are comparing and contrasting the capabilities and output of a variety of these tools, including ModelSEED, Raven Toolbox, PathwayTools, SuBliMinal Toolbox and merlin.FBA, Flux Balance Analysis; GEM, genome-scale metabolic model; GPR, gene\textendashprotein\textendashreaction association; GUI, graphical user interfaces; KBase, DOE Systems Biology Knowledgebase; LP, linear programing; MILP, Mixed-Integer Linear Programing; SBML, Systems Biology Markup Language, This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research; under contract DE-AC02-06CH11357 as a part of the DOE Knowledgebase project., info:eu-repo/semantics/publishedVersion

Introduction Microbial cells secrete many metabolites during growth, including important intermediates of the central carbon metabolism. This has not been taken into account by researchers when modeling microbial metabolism for metabolic engineering and systems biology studies. Materials and Methods The uptake of metabolites by microorganisms is well studied, but our knowledge of how and why they secrete different intracellular compounds is poor. The secretion of metabolites by microbial cells has traditionally been regarded as a consequence of intracellular metabolic overflow. Conclusions Here, we provide evidence based on time-series metabolomics data that microbial cells eliminate some metabolites in response to environmental cues, independent of metabolic overflow. Moreover, we review the different mechanisms of metabolite secretion and explore how this knowledge can benefit metabolic modeling and engineering., The authors are thankful to Mia Jullig for assistance with Fig. 2. Callaghan Innovation and Bioresource Processing Alliance provided PhD stipends for James Daniell and Ninna Granucci respectively., info:eu-repo/semantics/publishedVersion

Background Chronic kidney disease increases stroke incidence and severity but the mechanisms behind this cerebro-renal interaction are mostly unexplored. Since both vascular beds share similar features, microvascular dysfunction could be the possible missing link. Therefore, we examined the relationship between renal function and cerebral autoregulation in the early hours post ischemia and its impact on outcome. Methods We enrolled 46 ischemic strokes (middle cerebral artery). Dynamic cerebral autoregulation was assessed by transfer function (coherence, phase and gain) of spontaneous blood pressure oscillations to blood flow velocity within 6 h from symptom-onset. Estimated glomerular filtration rate (eGFR) was calculated. Hemorrhagic transformation (HT) and white matter lesions (WML) were collected from computed tomography performed at presentation and 24 h. Outcome was evaluated with modified Rankin Scale at 3 months. Results High gain (less effective autoregulation) was correlated with lower eGFR irrespective of infarct side (p

©Springer Science+Business Media, LLC, part of Springer Nature 2018, Hypertension is a prevalent and major health problem, involving a complex integration of different organ systems, including the central nervous system (CNS). The CNS and the hypothalamus in particular are intricately involved in the pathogenesis of hypertension. In fact, evidence supports altered hypothalamic neuronal activity as a major factor contributing to increased sympathetic drive and increased blood pressure. Several mechanisms have been proposed to contribute to hypothalamic-driven sympathetic activity, including altered ion channel function. Ion channels are critical regulators of neuronal excitability and synaptic function in the brain and, thus, important for blood pressure homeostasis regulation. These include sodium channels, voltage-gated calcium channels, and potassium channels being some of them already identified in hypothalamic neurons. This brief review summarizes the hypothalamic ion channels that may be involved in hypertension, highlighting recent findings that suggest that hypothalamic ion channel modulation can affect the central control of blood pressure and, therefore, suggesting future development of interventional strategies designed to treat hypertension., Dr. Rocha reports a grant from Portuguese Foundation for Science and Technology [2010–2013–FCT/PTDC/SAUOSM/109081/2008].

Purpose: Patients suffering from cardiovascular autonomic failure often develop neurogenic supine hypertension (nSH), i.e., high blood pressure (BP) in the supine position, which falls in the upright position owing to impaired autonomic regulation. A committee was formed to reach consensus among experts on the definition and diagnosis of nSH in the context of cardiovascular autonomic failure. Methods: As a first and preparatory step, a systematic search of PubMed-indexed literature on nSH up to January 2017 was performed. Available evidence derived from this search was discussed in a consensus expert round table meeting in Innsbruck on February 16, 2017. Statements originating from this meeting were further discussed by representatives of the American Autonomic Society and the European Federation of Autonomic Societies and are summarized in the document presented here. The final version received the endorsement of the European Academy of Neurology and the European Society of Hypertension. Results: In patients with neurogenic orthostatic hypotension, nSH is defined as systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg, measured after at least 5 min of rest in the supine position. Three severity degrees are recommended: mild, moderate and severe. nSH may also be present during nocturnal sleep, with reduced-dipping, non-dipping or rising nocturnal BP profiles with respect to mean daytime BP values. Home BP monitoring and 24-h-ambulatory BP monitoring provide relevant information for a customized clinical management. Conclusions: The establishment of expert-based criteria to define nSH should standardize diagnosis and allow a better understanding of its epidemiology, prognosis and, ultimately, treatment.

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project., The authors would like to thank the European Union’s Seventh Framework Programme (BacHBerry, Project No. FP7-613793, and FP7-PEOPLE2013-COFUND, Project No. FP7-609405) for the financial support. AD, RF, MHHN, PG, SGS, and JF would also like to acknowledge the Novo Nordisk Foundation. We express our gratitude to Dr. Martha Cyert (Stanford School of Medicine, EUA), Dr. Hitoshi Shimoi (National Research Institute of Brewing, Japan) and Dr. Yoshio Araki (Graduate School of Biosphere Science, Hiroshima University, Japan) for providing the yeast strain YAA5. We also thank Dr. Ian Macraedie, RMIT University, Australia) for providing the plasmid p416_GPDprGFP-A42., info:eu-repo/semantics/publishedVersion

© 2018 Elsevier B.V. All rights reserved., Background: Exposures to lead (Pb) during developmental phases can alter the normal course of development, with lifelong health consequences. Permanent Pb exposure leads to behavioral changes, cognitive impairment, sympathoexcitation, tachycardia, hypertension and autonomic dysfunction. However, the effects of an intermittent lead exposure are not yet studied. This pattern of exposure has been recently increasing due to migrations, implementation of school exchange programs and/or residential changes. Objective: To determine and compare lead effects on mammal’s behavior and physiology, using a rat model of intermittent and permanent Pb exposures. Methods: Fetuses were intermittently (PbI) or permanently (PbP) exposed to water containing lead acetate (0.2% w/v) throughout life until adulthood (28 weeks of age). A control group (CTL) without any exposure to lead was also used. Anxiety was assessed by elevated plus maze (EPM) and locomotor activity and exploration by open field test (OFT). Blood pressure (BP), electrocardiogram (ECG), heart rate (HR), respiratory frequency (RF), sympathetic and parasympathetic activity and baro- and chemoreceptor reflex profiles were evaluated. Immunohistochemistry protocol for the assessment of neuroinflammation, neuronal loss (NeuN), gliosis and synaptic alterations (Iba-1, GFAP, Syn), were performed at the hippocampus. One-way ANOVA with Tukey’s multiple comparison between means were used (significance p < 0.05) for statistical analysis. Results: The intermittent lead exposure produced a significant increase in diastolic and mean BP values, concomitant with a tendency to sympathetic overactivity (estimated by increased low-frequency power) and without significant changes in systolic BP, HR and RF. A chemoreceptor hypersensitivity and a baroreflex impairment were also observed, however, less pronounced when compared to the permanent exposure. Regarding behavioral changes, both lead exposure profiles showed an anxiety-like behavior without changes in locomotor and exploratory activity. Increase in GFAP and Iba-1 positive cells, without changes in NeuN positive cells were found in both exposed groups. Syn staining suffered a significant decrease in PbI group and a significant increase in PbP group. Conclusion: This study is the first to show that developmental Pb exposure since fetal period can cause lasting impairments in physiological parameters. The intermittent lead exposure causes adverse health effects, i.e, hypertension, increased respiratory frequency and chemoreflex sensitivity, baroreflex impairment, anxiety, decreased synaptic activity, neuroinflammation and reactive gliosis, in some ways similar to a permanent exposure, however some are lower-grade, due to the shorter duration of exposure. This study brings new insights on the environmental factors that influence autonomic and cardiovascular systems during development, which can help in creating public policy strategies to prevent and control the adverse effects of Pb toxicity., Vera Geraldes acknowledges the post-doctoral fellow given by the Fundação para a Ciência e Tecnologia (FCT) (Ref: SFRH/BPD/119315/ 2016).

OptFlux was launched in 2010 as the first open-source and user-friendly platform containing all the major methods for performing metabolic engineering tasks in silico. Main features included the possibility of performing microbial strain simulations with widely used methods such as Flux Balance Analysis and strain design using Evolutionary Algorithms. Since then, OptFlux suffered a major re-factoring to improve its efficiency and reliability, while many features were added in the form of novel plug-ins, such as the BioVisualizer and the over/under expression plug-ins. The current chapter described the main mathematical formulations of the major methods implemented within OptFlux, also providing a detailed guide on the usage of those functionalities., info:eu-repo/semantics/publishedVersion

Metabolic Engineering (ME) aims to design microbial cell factories towards the production of valuable compounds. In this endeavor, one important task relates to the search for the most suitable heterologous pathway(s) to add to the selected host. Different algorithms have been developed in the past towards this goal, following distinct approaches spanning constraint-based modelling, graph-based methods and knowledge-based systems based on chemical rules. While some of these methods search for pathways optimizing specific objective functions, here the focus will be on methods that address the enumeration of pathways that are able to convert a set of source compounds into desired targets and their posterior evaluation according to different criteria. Two pathway enumeration algorithms based on (hyper)graph-based representations are selected as the most promising ones and are analyzed in more detail: the Solution Structure Generation and the Find Path algorithms. Their capabilities and limitations are evaluated when designing novel heterologous pathways, by applying these methods on three case studies of synthetic ME related to the production of non-native compounds in E. coli and S. cerevisiae: 1-butanol, curcumin and vanillin. Some targeted improvements are implemented, extending both methods to address limitations identified that impair their scalability, improving their ability to extract potential pathways over large-scale databases. In all case-studies, the algorithms were able to find already described pathways for the production of the target compounds, but also alternative pathways that can represent novel ME solutions after further evaluation., The work is partially funded by ERDF - European Regional Development Fund through the COMPETE Programme (operational programme for competitiveness) and by National Funds through the FCT (Portuguese Foundation for Science and Technology) within projects ref. COMPETE FCOMP-01-0124-FEDER-015079 and Strategic Project PEst-OE/EQB/LA0023/2013, and also by Project 23060, PEM - Technological Support Platform for Metabolic Engineering, co-funded by FEDER through Portuguese QREN under the scope of the Technological Research and Development Incentive system, North Operational.

[EN] Chagas disease (CD), which mostly affects those living in deprived areas, has become one of Latin America's main public health problems. Effective prevention of the disease requires early diagnosis, initiation of therapy, and regular blood monitoring of the infected individual. However, the majority of the Trypanosoma cruzi infections go undiagnosed because of mild symptoms, limited access to medical attention and to a high variability in the sensitivity and specificity of diagnostic tests. Consequently, more affordable and accessible detection technologies capable of providing early diagnosis and T. cruzi load measurements in settings where CD is most prevalent are needed to enable enhanced intervention strategies. This work analyzes the potential contribution of biosensing technologies, reviewing examples that have been tested and contrasted with traditional methods, both serological and parasitological (i.e., molecular detection by PCR), and discusses some emerging biosensing technologies that have been applied for this public health issue. Even if biosensing technologies still require further research efforts to develop portable systems, we arrive at the conclusion that biosensors could improve the accuracy of CD diagnosis and the follow-up of patients' treatments in terms of the rapidity of results, small sample volume, high integration, ease of use, real-time and low cost detection when compared with current conventional technologies., M.I.R.-G., F.S. and D.B. thank the Agence National de la Recherche (ANR), for its financial support by granting the international AWESOM project. The authors of this work are also grateful to Janine W. Ramsey for her useful comments and suggestions about Figure 2

This work reports the nutritional profile and in vitro neuroprotective properties of leaves of Carpobrotus edulis L, a medicinal and edible succulent species native to the coast of South Africa. Biomass was evaluated for proximate composition and for contents in carotenoids, liposoluble pigments and minerals. Hexane, dichloromethane, ethyl acetate and methanol extracts were prepared by Soxhlet extraction from dried biomass and evaluated for in vitro inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), capacity to attenuate hydrogen peroxide (H2O2)-induced injury in the human dopaminergic cell line SH-SY5Y and for anti-neuroinflammatory potential on lipopolysaccharide (LPS)-stimulated microglia cells. Extracts were evaluated for antioxidant activity by four complementary methods, total content of phenolics, tannins and flavonoids. Finally the profile of the main phenolic compounds was determined by high performance liquid chromatography with diode array detection (HPLC-DAD). C edulis has a high moisture content, high levels of crude protein, fibre, ash, carotenoids, calcium and iron and a low fat level. The extracts were able to efficiently scavenge the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), reduce iron and chelate copper and iron ions, and exhibited different levels of phenolic compounds in the order ethyl acetate > methanol > dichloromethane > hexane. The main compounds detected were gallic and salicylic acids and quercetin, all in the ethyl acetate extract. The extracts allowed a dual and potent inhibition of AChE and BuChE. The dichloromethane and methanol extracts had the strongest capacity to prevent cell death induced by H2O2, and the methanol extract had anti-neuronflammatory properties. All together our results suggest that consumption of leaves of C edulis can contribute for a balanced diet, and that they may add to the improvement of cognitive functions. It also suggests possible novel biotechnological applications of C. edulis such as source of molecules and/or products for the food and/or pharmaceutical industries. Studies aiming to the isolation and identification of the bioactive compounds are already in progress. (C) 2017 SAAB. Published by Elsevier B.V. All rights reserved. Portuguese National Budget XtremeGourmet project [ALG-01-0247-FEDER-017676] FCT Investigator Programme [IF/00049/2012] info:eu-repo/semantics/publishedVersion

Over the last 15 years, several genome-scale metabolic models (GSMMs) were developed for different yeast species, aiding both the elucidation of new biological processes and the shift toward a bio-based economy, through the design of in silico inspired cell factories. Here, an historical perspective of the GSMMs built over time for several yeast species is presented and the main inheritance patterns among the metabolic reconstructions are highlighted. We additionally provide a critical perspective on the overall genome-scale modeling procedure, underlining incomplete model validation and evaluation approaches and the quest for the integration of regulatory and kinetic information into yeast GSMMs. A summary of experimentally validated model-based metabolic engineering applications of yeast species is further emphasized, while the main challenges and future perspectives for the field are finally addressed, This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of a Ph.D. grant (PD/BD/52336/2013), of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01–0145FEDER-006684) and also in the context of the EU-funded initiative ERA-NET for Industrial Biotechnology (ERA-IB-2/0003/2013), in addition to the BioTecNorte operation (NORTE-01–0145FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Although little is known about their nutritional composition, Sarcocornia perennis subsp. perennis, S. perennis subsp. alpini and Salicornia ramosissima (Salicorniaceae) as well as Arthrocnemum macro-stachyum (Amaranthaceae) are consumed in gourmet cuisine. In spite of belonging to different families, these halophytes share morphological and organoleptic characteristics. This work explored the nutritional properties and the antioxidant potential of these species using five integrative methods. All species had a nutritional profile suitable for human consumption with high levels of protein (5.20-13.2 g/100 g dw) and n-3 polyunsaturated fatty acids (FA), particularly or alpha-linolenic acid (19.3-25.9% of total FA), and low concentration of toxic metals (below the limits imposed by the European Commission). These halophytes are also a good source of minerals, particularly sodium (64.1-109 mg/gdw), and S. ramosissima is an excellent source of manganese (204 mu g/g dw). However, due care should be taken not to exceed the legal limits for sodium ingestion. These plants showed also significant antioxidant potential, with high radical scavenging activity (RSA), iron reducing power and total phenolics content (20.5-49.2 mg GAE/g). A. macrostachyum had the highest RSA (lC(50-DPPH) = 0.84 mg/mL; IC50-NO = 0.60 mg/mL), and iron reducing potential (IC50= 0.84 mg/mL) along with high levels of alpha- and gamma-tocopherol (8.74 and 4.71 mg/100 g dw, respectively). (C) 2017 Elsevier Inc. All rights reserved. XtremeBio project - Portuguese Foundation for Science and Technology (FCT) [PTDC/MAR-EST/4346/2012] XtremeGourmet project from the Portuguese National Budget [ALG-01-0247-FEDER-017676] FCT Investigator Programme [IF/00049/2012] FCT project [CCMAR/Multi/04326/2013] [FCTFRH/BD/105541/2014] info:eu-repo/semantics/publishedVersion

Genome-scale metabolic models (GSMMs) have become important assets for rational design of compound overproduction using microbial cell factories. Most computational strain optimization methods (CSOM) using GSMMs, while useful in metabolic engineering, rely on the definition of questionable cell objectives, leading to some bias. Metabolic pathway analysis approaches do not require an objective function. Though their use brings immediate advantages, it has mostly been restricted to small scale models due to computational demands. Additionally, their complex parameterization and lack of intuitive tools pose an important challenge towards making these widely available to the community. Recently, MCSEnumerator has extended the scale of these methods, namely regarding enumeration of minimal cut sets, now able to handle GSMMs. This work proposes a tool implementing this method as a Java library and a plugin within the OptFlux metabolic engineering platform providing a friendly user interface. A standard enumeration problem and pipeline applicable to GSMMs is proposed, making use by the community simpler. To highlight the potential of these approaches, we devised a case study for overproduction of succinate, providing a phenotype analysis of a selected strategy and comparing robustness with a selected solution from a bi-level CSOM., The authors thank the project “DeYeastLibrary—Designer yeast strain library optimized for metabolic engineering applications”, Ref. ERA-IB-2/0003/2013, funded by national funds through “Fundação para a Ciência e Tecnologia / Ministério da Ciência, Tecnologia e Ensino Superior”., info:eu-repo/semantics/publishedVersion

Myocardial infarction (MI) is a high prevalence cardiovascular disease with a tendency to increase. Although MI therapy has increased its effectiveness, the loss of myocardial function in consequence of MI is still responsible for major subsequent complications as rhythm changes and heart failure. In human hearts, MI is characterized by cardiomyocyte death and fibrin deposition, which is later replaced by scar tissue. In opposition, zebrafish heart undergoes minimal scaring and cardiac tissue can undergo full functionally regeneration in 60 to 120 days. Therefore, this animal model has been used in cardiovascular research as a model for studying mechanisms of cardiac regeneration. The hearts of zebrafish is anatomically different from the human heart, though the electrocardiogram (ECG) pattern and the critical conduction pathways are comparable to the human's. The objective of our full project is the implementation of an ECG recording system with 3 orthogonal electrodes in 2 planes, in order to create a 4D recording of the zebrafish cardiac electrical activity. Here we present our preliminary work related to a non-invasive ECG monitoring with two electrodes.