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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. ...

Many questions regarding proteins involved in microbial sulfur metabolism remain unsolved. For sulfur respiration at low pH, the terminal electron acceptor is still unclear. Desulfurella amilsii is a sulfur-reducing bacterium that respires elemental sulfur (S0) or thiosulfate, and grows by S0 disproportionation. Due to its versatility, comparative studies on D. amilsii may shed light on microbial sulfur metabolism. Requirement of physical contact between cells and S0 was analyzed. Sulfide production decreased by around 50\% when S0 was trapped in dialysis membranes, suggesting that contact between cells and S0 is beneficial, but not strictly needed. Proteome analysis was performed under the aforementioned conditions. A Mo-oxidoreductase suggested from genome analysis to act as sulfur reductase was not detected in any growth condition. Thiosulfate and sulfite reductases showed increased abundance in thiosulfate-reducing cultures, while rhodanese-like sulfurtransferases were highly abundant in all conditions. DsrE and DsrL were abundantly detected during thiosulfate reduction, suggesting a modified mechanism of sulfite reduction. Proteogenomics suggest a different disproportionation pathway from what has been reported. This work points to an important role of rhodaneses in sulfur processes and these proteins should be considered in searches for sulfur metabolism in broader fields like meta-omics. ; This research was supported by the organization of the Brazilian Government for the development of Science and Technology CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), by ERC project 323009 and a Gravitation project from the Netherlands Ministry of Education, Culture and Science (024.002.002). The authors thank Monika Jarzembowska (WUR, Wageningen, The Netherlands) for the support with the scanning electron microscopy analysis ...

Fat, oils, and grease present in complex wastewater can be readily converted to methane, but the energy potential of these compounds is not always recyclable, due to incomplete degradation of long chain fatty acids (LCFA) released during lipids hydrolysis. Oleate (C18:1) is generally the dominant LCFA in lipid-containing wastewater, and its conversion in anaerobic bioreactors results in palmitate (C16:0) accumulation. The reason why oleate is continuously converted to palmitate without further degradation via β-oxidation is still unknown. In this work, the influence of methanogenic activity in the initial conversion steps of unsaturated LCFA was studied in 10 bioreactors continuously operated with saturated or unsaturated C16- and C18-LCFA, in the presence or absence of the methanogenic inhibitor bromoethanesulfonate (BrES). Saturated Cn-2-LCFA accumulated both in the presence and absence of BrES during the degradation of unsaturated Cn-LCFA, and represented more than 50\% of total LCFA. In the presence of BrES further conversion of saturated intermediates did not proceed, not even when prolonged batch incubation was applied. As the initial steps of unsaturated LCFA degradation proceed uncoupled from methanogenesis, accumulation of saturated LCFA can be expected. Analysis of the active microbial communities suggests a role for facultative anaerobic bacteria in the initial steps of unsaturated LCFA biodegradation. Understanding this role is now imperative to optimize methane production from LCFA. ; European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement No 323009, and 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), and Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). We also thank the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science ...

Capsicum products, such as chilli powder, paprika, sauces, and spice mix, have highly been consumed worldwide.In Chile, berry fruits of Capsicum annuum L. cv. "Cacho de Cabra" are used for the manufacture of a traditional pepper powder known as Merkén. Two alert notifications regarding the presence of Ochratoxin A in Merkén have been reported by the Chilean Ministry of Health. These alerts have triggered concern mainly due to the food safety of Capsicum derivative products and the health risks to which the Chilean population consumers of Merkén may be subjected. In this context, the main aim of this work was to review the critical points of mycotoxin contamination in the Capsicum production chain used for Merkén manufacture in Chile; outlining the possible causal agents of mycotoxin contamination in Merkén production chain. Current national and international legislation on mycotoxin contamination in pepper and its derivative are here discussed. Moreover, preventive and corrective measures adopted by the Chilean regulators' agencies to control mycotoxin in this spice are also discussed. ; Chilean National Commission for Scientific and Technological Research (CONICYT) which supports the J.C. PhD grant no 21181445; Universidad de La Frontera (Temuco, Chile) with partial funding from the Project DIUFRO PIA 19-0001 lead by C.S.; Portuguese Foundation for Science and Technology (FCT) (UID/BIO/04469/2013 unit), COMPETE 2020 (POCI-01- 0145-FEDER-006684) and the BioTecNorte operation ...

Determination of material properties and functions is a crucial step towards optimization of fabrication methods as well as the development of electrospun nanofibers for use in e.g. food engineering applications. This work focused in evaluating physical and mass transfer properties of simple poly -caprolactone nanofibers (PCL membrane), and poly ε-caprolactone nanofibers with encapsulated trypsin (E-PCL membrane), in view of their future use in a catalytic filter reactor. PCL membranes registered high hydrophobicity values, while E-PCL membranes revealed stronger mechanical properties and an increase of mass due to water incorporation. A decrease of average pore size in the range of 30% to 40% was observed for E-PCL membranes and an average pore diameter of 1/3 of the size was registered when compared to the PCL membrane; this difference was shown to be significant enough to influence the transport of larger molecules (e.g. bovine serum albumin). Release experiments of active compounds (lysozyme, bovine serum albumin and lactoferrin) were successfully described by a model which accounts for both Fick and Case II transport the linear superimposition model. Results show that the transport mechanism is influenced by the type of active compound and by membranes physical properties. ; The present study was developed under the scope of the NanoBioCats project (PTDC/CTM-POL/112289/2009), funded by the Portuguese Science and Technology Foundation (Fundação para a Ciência e a Tecnologia – FCT). This work was also supported by the COMPETE program (funded by the European Union fund, FEDER under the framework of QREN (Programa Operacional Regional do Norte (ON.2 – O Novo Norte; FEDER))) through projects NORTE-07-0124-FEDER-000028 and NORTE-07-0124-FEDER-000069. Also, the authors thank Strategic Projects PEst-OE/EQB/LA0023/2013, PEst-C/EQB/LA0006/2013 – FCOMP-01-0124-FEDER-37285 and PEst-C/QUI/UI0062/2013 – FCOMP-01-0124-FEDER-037296. The Fundação para a Ciência e Tecnologia (FCT, Portugal) through grant ...

Coloured and conductive fabrics were obtained through in situ laccase polymerization of catechol and p-phenylenediamine under high-pressure homogenization. Both monomers, catechol and p-phenylenediamine, were polymerized by different laccase forms, namely native, PEGylated and Epoxy-PEGylated. All the catalysts were placed inside a textile fabric bag which served simultaneously as enzyme support and as substrate for coating with the newly produced polymers. The PEGylated laccase forms gave rise to higher amount of oligomers/polymers and higher colouration level of polyethylene terephthalate (PET), cotton and wool fabrics compared to native laccase. Both functional polymers were able to confer conductivity to the substrates however in a different extent. Fabrics coated with poly(p-phenylenediamine) present higher conductivity, rather due to its polymerized structure than to the amount of polymer produced by enzyme catalysis. Herein a green approach was presented to produce polyphenols with increased fixation onto different textile substrates. These substrates reach high levels of colouration and good fastness behaviour after washing. ; Chinese Government Scholarship under China Scholarship Council [grant No. 201606790036]; and by the Chinese Foundation Key projects of governmental cooperation in international scientific and technological innovation [grant No. 2016 YFE0115700]; 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) 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 work was also supported by the European Union through the European Regional Development Fund (ERDF) under the Competitiveness Operational Program [grant COP-A1.1.4-E-2015nr.30/01.09.2016]; and by the Project "Search-ON2: Revitalization of HPC infrastructure of UMinho" [grant ...

Tyrosine was explored as a cross-linking agent to form cross-linked bovine serum albumin (BSA) using laccase as a catalyst. Liquid chromatography-mass spectrometry (LC-MS) and fluorescence spectra indicated that tyrosine can be mainly oxidized to be dityrosine. Spectra analysis and molecular weight were used to characterize the BSA treated with tyrosine and laccase. Both SDS-PAGE and size exclusion chromatography confirmed the formation of cross-linked BSA, while most of the protein products existed as BSA–tyrosine conjugates. The MALDI-TOF analysis revealed that five tyrosine units were grafted on one BSA monomer, however one cross-linked BSA consists of two BSA monomers and 18 tyrosine. Furthermore, the content of the amino acid of BSA was identified using amino acid analysis, among those the percentage of lysine presented a visible decline from 12.36% to 11.43%, corresponding to 4-5 lysine residues. The pure and modified BSA were hydrolyzed by trypsin and the corresponding peptides were obtained. Different mass of five peptides from LC-MS spectra after hydrolysis indicated that tyrosine could react with Lys-136, Lys-204, Lys-224, Lys-322 and Lys-537 in BSA, promoting the formation of BSA–tyrosine conjugates and cross-linked BSA. ; This study was supported by Chinese Government Scholarship under China Scholar Council (NO. 201906790043) and "the Fundamental Research Funds for the Central Universities (NO. JUSRP52007A). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 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 ...

Conductive cotton fabrics were obtained via in situ aniline polymerization by laccase from Myceliophthora thermophila under mild reaction conditions without the addition of strong proton acids. The reactions were conducted using two types of reactors, namely a water bath (WB) and an ultrasonic bath (US), and the role of a mediator, 1-hydroxybenzotriazol (HBT), on the laccase-assisted polymerization of aniline was investigated. A similar polymerization degree was obtained when using both reactors—however, the ultrasonic bath allowed the experiments to be conducted in shorter periods of time (24 h for WB vs. 2 h for US). The data obtained also revealed that the mediator (1-hydroxybenzotriazol-HBT) played a crucial role in aniline oxidation. A higher conversion yield and polymerization degree were obtained when the reaction was conducted in the presence of this compound, as confirmed by MALDI-TOF analysis. The cotton fabrics coated with polyaniline presented deep coloration and conductivity, especially when the mediator was included on the reactional system. The results obtained are a step forward in the enzymatic polymerization of aniline with the purpose of obtaining coloured conductive textile surfaces, with potential applications in wearable electronics. ; Chinese Government Scholarship under China Scholarship Council [grant No. 201606790036]; and by the Chinese Foundation Key projects of governmental cooperation in international scientific and technological innovation [grant No. 2016 YFE0115700]; 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) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte. The workwas also supported by the FCT- Fundação para a Ciência e a Tecnologia [grant SFRH/BD/121673/2016 and IF/00186/2018]. ...

A new simple and easy-to-prepare imidazole-based probe 1 was synthesized and used to detect Cu(II) and biothiols (Cys, Hcy and GSH) in aqueous environments. Addition of increasing amounts of Cu(II) to water (pH 7.4)-acetonitrile 90:10 v/v solutions of probe 1 induced the apperance of a red-shifted absorption together with a marked colour change from colorless to deep blue. In addition, probe 1 was fluorescent and a marked emission quenching in the presence of Cu(II) was observed. The optical response is selective and other cations tested do not induce significant chromo-fluorogenic modulations. Limits of detection for Cu(II) of 0.7 and 3.2 μM using UV–visible and fluorescence data were determined. On the other hand, addition of Cys, Hcy and GSH to the deep-blue water (pH 7.4)-acetonitrile 90:10 v/v solutions of the 1-Cu(II) complex reulted in a marked bleaching together with the appearance of a highly emissive band centred at 475 nm. Other amino acids tested induced negligible response. The limits of detection for Cys, Hcy and GSH using 1-Cu(II) and emission data are 6.5, 5.0 and 10.2 μM, respectively. These optical changes were ascribed to a biothiol-induced demetallation process of the 1-Cu(II) complex that released the free probe. Besides, probe 1 is non-toxic and can be used for Cu(II) detection in HeLa cells. ; FCT - Generalitat Valenciana(PROMETEOII/2014/047); Spanish Government (projects MAT2015-64139-C4-1-R and AGL2015-70235-C2-2-R) and the Generalitat Valenciana (project PROMETEOII/2014/047). Thanks are also due to Fundação para a Ciência e a Tecnologia for financial support to the Portuguese NMR network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER (European Fund for Regional Development)-COMPETEQREN-EU for financial support to the research centre CQ/UM (Ref. UID/QUI/00686/2013 and UID/QUI/0686/2016), and a PhD grant to R. C. M. Ferreira (SFRH/BD/86408/2012) ...

O texto mostra a evolução recente da biotecnologia agrícola, sua situação no Brasil atual, e faz um balanço de seus impactos econômicos e sociais. Chama atenção para o fato de esta tecnologia ter passado rapidamente do estágio de ciência para o de inovação, com impactos significativos na economia nacional.

Haloalkaliphilic microorganisms that grow optimally at high-pH and high-salinity conditions can be found in natural environments such as soda lakes. These globally spread lakes harbour interesting anaerobic microorganisms that have the potential of being applied in existing technologies or create new opportunities. In this review, we discuss the potential application of haloalkaliphilic anaerobic microbial communities in the fermentation of lignocellulosic feedstocks material subjected to an alkaline pre-treatment, methane production and sulfur removal technology. Also, the general advantages of operation at haloalkaline conditions, such as low volatile fatty acid and sulfide toxicity, are addressed. Finally, an outlook into the main challenges like ammonia toxicity and lack of aggregation is provided. ; This work was performed in the TTIW- cooperation framework of Wetsus, European Centre of Excel- lence for Sustainable Water Technology (www.wetsus.nl). Wetsus is funded by the Dutch Ministry of Economic Affairs, the European Union Regional Development Fund, the Province of Fryslân, the City of Leeuwarden and the EZ/Kompas program of the" Samenwerkingsverband Noord-Nederland". The authors would like to thank the participants of the research theme "Sulfur", namely Paqell, for fruitful discussions and financial ...

Background One of the European Union directives indicates that 10% of all fuels must be bio-synthesized by 2020. In this regard, biobutanolnatively produced by clostridial strainsposes as a promising alternative biofuel. One possible approach to overcome the difficulties of the industrial exploration of the native producers is the expression of more suitable pathways in robust microorganisms such as Escherichia coli. The enumeration of novel pathways is a powerful tool, allowing to identify non-obvious combinations of enzymes to produce a target compound. Results This work describes the in silico driven design of E. coli strains able to produce butanol via 2-oxoglutarate by a novel pathway. This butanol pathway was generated by a hypergraph algorithm and selected from an initial set of 105,954 different routes by successively applying different filters, such as stoichiometric feasibility, size and novelty. The implementation of this pathway involved seven catalytic steps and required the insertion of nine heterologous genes from various sources in E. coli distributed in three plasmids. Expressing butanol genes in E. coli K12 and cultivation in High-Density Medium formulation seem to favor butanol accumulation via the 2-oxoglutarate pathway. The maximum butanol titer obtained was 85±1 mg L1 by cultivating the cells in bioreactors. Conclusions In this work, we were able to successfully translate the computational analysis into in vivo applications, designing novel strains of E. coli able to produce n-butanol via an innovative pathway. Our results demonstrate that enumeration algorithms can broad the spectrum of butanol producing pathways. This validation encourages further research to other target compounds. ; This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of a Ph.D. Grant (PD/BD/52366/2013) from MIT Portugal Program and the strategic funding of UID/BIO/04469 unit. Additional support was received by COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte ...

Tau is a microtubule-associated protein that promotes microtubule assembly and stability. This protein is implicated in several neurodegenerative diseases, including Alzheimer's. To date, the three-dimensional (3D) structure of tau has not been fully solved, experimentally. Even the most recent information is sometimes controversial in regard to how this protein folds, interacts, and behaves. Predicting the tau structure and its profile sheds light on the knowledge about its properties and biological function, such as the binding to microtubules (MT) and, for instance, the effect on ionic conductivity. Our findings on the tau structure suggest a disordered protein, with discrete portions of well-defined secondary structure, mostly at the microtubule binding region. In addition, the first molecular dynamics simulation of full-length tau along with an MT section was performed, unveiling tau structure when associated with MT and interaction sites. Electrostatics and conductivity were also examined to understand how tau affects the ions in the intracellular fluid environment. Our results bring a new insight into tau and tubulin MT proteins, their characteristics, and the structure–function relationship. ; European Union through the European Regional Development Fund (ERDF) under the Competitiveness Operational Program (BioCell-NanoART = Novel Bio-inspired Cellular Nano-architectures, POC-A1.1.4-E-2015 nr. 30/01.09.2016). Artur Cavaco-Paulo thanks the support received from the Portuguese Foundation for Science and Technology (FCT) through 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; Project "Search-ON2: Revitalization of HPC infrastructure of UMinho" (NORTE-07-0162-FEDER-000086), co-founded by the North Portugal Regional Operational Programme (ON.2 –O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European ...

Atlantic cod is processed industrially for food purposes, with several by-products being directed to animal feed and other ends. Looking particularly into swim bladders, the extraction of collagen can be a valuable strategy for by-product valorization, explored in the present work for the first time. Collagen was extracted using acetic acid (ASCsb) and pepsin (PSCsb) with yields of 5.72% (w/w) and 11.14% (w/w), respectively. SDS-PAGE profile showed that the extracts were compatible with type I collagen. FTIR, CD and XRD results suggest that the PSCsb structure underwent partial denaturation, with microDSC showing a band at 54 ºC probably corresponding to a melting process, while ASCsb structure remained intact, with preserved triple helix and a denaturation temperature of 29.6 ºC. Amino acid composition indicates that the total content of proline-like amino acids was 148/1000 residues for ASCsb and 141/1000 residues for PSCsb, with a hydroxylation degree of about 37%. The extracts exhibited a typical shear thinning behavior, interesting property regarding their further processing toward the development of biomaterials. In this regard, assessment of metabolic activity of human fibroblast cells cultured in the presence of collagen extracts with concentrations up to 3mg/mL revealed the absence of cytotoxic behavior. Collagen extracts obtained from Atlantic cod swim bladders shown attractive properties regarding their use in cosmetic or biomedical applications. ; The authors would like to acknowledge to European Union for the financial support under the scope of European Regional Development Fund (ERDF) through the Structured Project NORTE-01-0145-FEDER-000021 (Norte2020) and under the scope of the European Union Seventh Framework Programme (FP7/2007-2013) through grant agreement ERC-2012-ADG 20120216-321266 (ERC Advanced Grant ComplexiTE). The Portuguese Foundation for Science and Technology is also acknowledged for the PhD grant of A. L. A under Doctoral Programme Do ~ Mar (PD/BD/127995/2016), as well as ...

Liposomes are one of the most important and extensively studied drug delivery system due to their ability to encapsulate different kinds of drugs. Exploiting the advantages of 1H Nuclear Magnetic Resonance (NMR) spectrometry, we established a rapid and easy method for quantification of drugs encapsulated in liposomes. An internal standard, pyridine, was used for quantitative determination of drug concentration. Two different drugs were involved in this work, one hydrophilic, methotrexate disodium salt, and another hydrophobic, tamoxifen. The specificity and selectivity of the suggested method were evaluated by the absence of overlapping of at least one signal of each drug with pyridine in the NMR spectrum. The accuracy and precision of the method were assessed by adding a known amount of each drug to unloaded liposomes. Results obtained by quantitative NMR (qNMR) were validated and confirmed by comparing with two other traditional techniques, Ultraviolet - Visible (UV-Vis) spectrophotometry and High-Performance Liquid Chromatography (HPLC). It was found that the results were consistent with the ones obtained from our proposed qNMR method. Considering all the experiments conducted in this study, we deliberate that qNMR can be a suitable tool for the determination of drugs encapsulated in liposomes. ; This work has received funding from the European Union Horizon 2020 research andinnovation programme undergrantagreement NMP06-2015-683356 FOLSMART. This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 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. Diana Guimarães (SFRH/BD/140321/ 2018) and Jennifer Noro (SFRH/BD/121673/2016) holds a scholarship from FCT. SFRH/BD/121673/2016 ...