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EFG1 is a central transcriptional regulator of filamentation that is an important virulence factor of Candida albicans. This study serves to assess in vivo the applicability of the anti-EFG1 2-OMethylRNA oligomer for inhibiting Candida albicans filamentation and to attenuate candidiasis, using the Galleria mellonella model. For that, larvae infected with a lethal concentration of C. albicans cells were treated with a single-dose and with a double-dose of the anti-EFG1 2OMe oligomer (at 40 and 100 nM). The anti-EFG1 2OMe oligomer toxicity and effect on larvae survival was evaluated. No evidence of anti-EFG1 2OMe oligomer toxicity was observed and the treatment with double-dose of 2OMe oligomer empowered the larvae survival over 24 h (by 90-100%) and prolonged its efficacy until 72 h of infection (by 30%). Undoubtedly, this work validates the in vivo therapeutic potential of anti-EFG1 2OMe oligomer for controlling C. albicans infections., This study was 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 Norte2–20 – Programa Operacional Regional do Norte and Daniela Eira Araújo [ SFRH/BD/121417/2016 ] PhD grant. The authors also acknowledge the project funding by the “ 02/SAICT/2017 – Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT) – POCI-01-0145-FEDER-028893.” Funding received by IBB – Institute for Bioengineering and Biosciences from FCT ( UID/BIO/04565/2020 ) and Programa Operacional Regional de Lisboa 2020 (project no. 007317 ) is also acknowledged. We acknowledge Dr Lucília Goreti Pinto, Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho , for processing and sectioning tissue samples of G. mellonella., info:eu-repo/semantics/publishedVersion

Nanocomplexes systems made up natural poylymers have pharmacotechnical advantages such as increase of water solubility and a decrease of drugs toxicity. Amphotericin B (AmB) is a drug apply as anti-leishmanial and anti-fungal, however it has low water solubility and high toxicity, limiting its therapeutic application. With this in mind, the present study aimed to produce nanocomplexes composed by alginate (Alg), a natural polymer, with AmB covered by nanocrystals from bacterial cellulose (CNC). For this reason, the nanocomplexes were produced utilizing sodium alginate, amphotericin B in a borate buffer (pH 11.0). The CNC was obtained by enzymatic hydrolysis of the bacterial cellulose. To CNC cover the nanocomplexes 1 ml of the nanocomplexes was added into 1 ml of 0.01% CNC suspension. The results showed an ionic adsorption of the CNC into the Alg-AmB nanocomplexes surface. This phenomena was confirmed by an increase in the particle size and PDI decrease. Besides, nanocomplexes samples covered by CNC showed uniformity. The amorphous inclusion of AmB complex into the polysaccharide chain network in both formulations. AmB in the nanocomplexes was in supper-aggregated form and showed good biocompatibility, being significantly less cytotoxic in vitro against kidney cells and significantly less hemolytic compared to the free-drug. The in vitro toxicity results indicated the Alg-AmB nanocomplexes can be considered a non-toxic alternative to improve the AmB therapeutic effect. All process to obtain nanocomplexes and it coat was conduce without organic solvents, can be considered a green process, and allowed to obtain water soluble particles. Furthermore, CNC covering the nanocomplexes brought additional protection to the system can contribut advancement in the pharmaceutical., The authors acknowledge financial support from Coordination for Higher Level Graduate Improvements (CAPES/Brazil, finance code 001), National Council for Scientific and Technological Development (CNPq/Brazil Process #428751/2016-4), and the State of São Paulo Research Foundation (FAPESP/Brazil, processes #2019/22626-5 and #2018/10508-5)., info:eu-repo/semantics/publishedVersion

Magnetoliposomes have gained increasing attention as delivery systems, as they surpass many limitations associated with liposomes. The combination with magnetic nanoparticles provides a means for development of multimodal and multifunctional theranostic agents that enable on-demand drug release and real-time monitoring of therapy., This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CF-UM-UP (UIDB/04650/2020). S. R. S. Veloso acknowledges FCT for a PhD grant (SFRH/BD/144017/2019)

We generated stable amphiphilic copolymer-based polymeric micelles (PMs) with temperature-responsive properties utilizing Pluronic® L35 and a variety of ionic liquids (ILs) to generate different aqueous two-phase micellar systems (ATPMSs). The partitioning of the hydrophobic model compound curcumin (CCM) into the PM-rich phase and the drug delivery capabilities of the PMs were investigated. ATPMSs formed using more hydrophobic ILs (i.e., [Ch][Hex] [Ch][But] > [Ch][Pro] > [Ch][Ac] [Ch]Cl) were the most effective in partitioning (KCCM) and recovering (RECRich) CCM into the PM-rich phase (15.2 < KCCM < 22.0 and 90% < RECRich < 95%, respectively). Moreover, using 1.2 M [Ch][But] and 0.2 M [Ch][Hex] ILs yielded higher encapsulation efficiency (EE) (94.1 and 96.0%, respectively) and drug loading (DL) capacity (14.8 and 16.2%, respectively), together with an increase in the average hydrodynamic diameter of the PMs (DH) (42.5 and 45.6 nm, respectively). The CCMPM formulations were stable at 4.0, 25.0, and 37.0 °C and the release of CCM was faster with the less hydrophobic ILs (i.e., [Ch]Cl and [Ch][Ac]). Furthermore, due to the lower critical solution temperature properties of Pluronic® L35, the PMs exhibit temperature responsiveness at 37.0 °C. In vitro cytotoxicity assays were also performed to determine the potency of CCMPM formulations, and a 1.8-fold decrease in IC50 values was observed between the CCMPMs/[Ch][Hex] and CCMPMs/[Ch]Cl formulations for PC3 cells. The lower IC50 value for the [Ch][Hex] version corresponded to a greater potency compared to the [Ch]Cl version, since a lower concentration of CCM was required to achieve the same therapeutic effect. The ATPMSs investigated in this study serve as a novel platform for Pluronic® L35/PBS buffer (pH 7.4) + IL-based ATPMS development. The unique properties reported here may be useful in applications such as controlled-release drug delivery systems (DDS), encapsulation, and bioseparations., This study was funded by the Coordination for Higher Level Graduate Improvements (CAPES/Brazil, finance code 001), the National Council for Scientific and Technological Development (CNPq/Brazil) and the State of São Paulo Research Foundation (FAPESP/Brazil, processes #2014/16424-7, #2017/10789-1, #2018/10799-0, #2018/05111-9, #2019/05624-9, #2019/08549-8, and #2020/03727-2)., info:eu-repo/semantics/publishedVersion

The increasing consumption of plantain fruits with specific quality standards generates high agricultural waste. This work aimed at valorising the rejected unripe pulp of Dominico-Hartón plantain fruits (Musa AAB Simmonds). The pulp was characterised physico-chemically, thermally and functionally. The data gathered experimentally and collected from different databases was used to design a production process of isomalto-oligosaccharides (IMO) syrup. The plantain flour contains high levels of starch (87 ± 2 %) and amylose (31.2 ± 0.8 %). The flour showed stability at high temperatures (pasting temperature of 79.26 ± 0.02 ºC), allowing its use in high temperature processes. In vitro gastrointestinal digestion of the plantain flour showed that when cooked, the glycemic index of the flour increases from 47.7 ± 2.2 to 84.2 ± 1.8, while its resistant starch content only slightly decreases from 71.7 ± 1 % to 52.6 ± 2 %, suggesting that this type of flour preserves high content of dietary fibre after digestion. The conceptual process design showed that 18.79 g of IMO are theoretically obtained from 53.24 g of plantain flour maltose. Results suggest that the rejected plantain pulp holds high potential as an ingredient for the production of prebiotic compounds such as IMO., James A. Gómez S. acknowledges the Ministry of Science Technology and Innovation of Colombia (MinCiencias) for the financial support to carry out this work and the Engineering Faculty of the Universidad de Caldas for its administrative and logistic assistance. This work was supported by the MinCiencias through the research project “Design of a Biorefinery for the Use of the Lignocellulosic and Starchy Waste of the Plantain Agribusiness” (Call 757 for Funding of National Doctorates) [grant 1640318]; and the Universidad de Caldas through the research project “Basic Process Development for the Use of Agro-industrial and Urban Waste Under the Concept of Biorefineries” [grant 0240518]. Enrique Pino-Hernández acknowledges the European Social Fund (call NORTE-69-2015-15) for the financial support provided through the fellowship NORTE-08-5369-FSE-000036. The 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, Project ColOsH (POCI-01-0145-FEDER-030071) and Project AgriFood XXI (NORTE-01-0145-FEDER-000041) funded by the European Regional Development Fund under the scope of Norte 2020-Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

The understanding of microbial susceptibility to disinfectants is an important step to provide drinking water (DW) of adequate microbiological quality. In drinking water distribution systems (DWDS) the application of disinfectants is the main approach to control microorganisms. Although chlorine has been commonly used for DW treatment, the increase of microbial resistance and the production of harmful disinfection by-products promote the necessity to seek new alternatives. This study evaluated the antimicrobial activity of sodium dichloroisocyanurate (NaDCC), trichloroisocyanuric acid (TCCA), and pentapotassium bis(peroxymonosulphate) bis(sulphate) (OXONE) against two emerging pathogens isolated from DW, Acinetobacter calcoaceticus and Stenotrophomonas maltophilia. Free chlorine from calcium hypochlorite was used for comparison. The dose and time-responses against planktonic bacteria were performed as well as the assessment of the effects on membrane integrity. Moreover, the effects against 48 h-old biofilms formed on polyvinyl chloride and stainless steel were evaluated in terms of biofilm culturability and removal. Minimum bactericidal concentrations of 2.1 and 3.1 mg/L for NaDCC, 2.5 and 3.8 mg/L for TCCA, 340 and 690 mg/L for OXONE, and 0.80 and 1.0 mg/L for free chlorine alone were obtained against S. maltophilia and A. calcoaceticus, respectively. The kinetic modeling revealed that NaDCC and TCCA caused similar inactivation rates and the time for first log reduction by OXONE was less than 10 min, for both bacteria. All the disinfectants triggered significant bacterial cytoplasmic membrane destabilization, even at sub-lethal concentrations. A 30 min treatment with the disinfectants allowed a reduction in the biofilm culturability up to 5 log. OXONE was the disinfectant with the best efficiency against both bacterial biofilms. However, none of the disinfectants caused significant biofilm removal (reduction < 1 log cells/cm2). This study highlights NaDCC, TCCA, and OXONE as promising alternatives to free chlorine for DW disinfection, particularly for planktonic growth control and biofilm culturability reduction., This work was financially supported by: Base Funding - UIDB/00511/2020 of LEPABE funded by national funds through the FCT/MCTES (PIDDAC); Project Biocide_for_Biofilm - PTDC/BII-BTI/30219/2017 - POCI-01-0145-FEDER-030219; Germirrad - POCI-01-0247-FEDER 072237, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; Project PRESAGE (Aquatic/0007/2020) Joint Programming Initiative Water challenges for a changing world; Project “HealthyWaters – Identification, Elimination, Social Awareness and Education of Water Chemical and Biological Micropollutants with Health and Environmental Implications”, with reference NORTE-01-0145-FEDER-000069, supported by Norte Portugal Regional Operational Pro gramme (NORTE 2020), under the PORTUGAL 2020 Partnership Agree ment, through the European Regional Development Fund (ERDF). This study was further supported by the 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 Norte; and FCT PhD grant awarded to Isabel Maria Oliveira (SFRH/BD/138117/2018), info:eu-repo/semantics/publishedVersion

High-pressure processing (HPP) is an emergent technology for nonthermal pasteurization of foods, which here demonstrates the potential to be used by the chestnut industry. This study evaluated HPP as a post-harvest processing technology to control molds and insect larvae proliferation in chestnuts (Castanea sativa Mill.) and improve the fruit's shelf-life. Chestnuts were processed by HPP (400, 500, and 600 MPa for 5.0 min at 20 °C) and the conventional hydrothermal technology (HT, 50 °C for 45 min). Untreated chestnuts were also used as a control group. Shelf-life studies were conducted for 60 d at i) 25 °C and 40 % relative humidity (RH); and ii) 5 °C and 70 % RH. After the treatment and over the storage period, parameters such as color, texture, weight loss, nutritional composition, ascorbic acid, and fungal load were evaluated. The results showed that immediately after the HPP treatments, the chestnuts became slightly softer than HT samples. Fruit treated by HPP maintained their nutritional composition and ascorbic acid content. During the first 30 d of storage, no changes of color were observed in treated fruit. Also, the shelf-life of chestnuts treated at 600 MPa was extended by 40 d, compared to HT, where no molds at a detectable level and insect larvae were observed when stored at 5 °C. This storage condition also reduced the fruit weight losses. Compared with HT, HPP has short processing times, which is an important advantage. In addition, the observed changes of texture as softening and easy peeling can be useful for obtaining ready-to-eat products since chestnuts looked partially cooked. However, its possible application at the industrial level should be adequately investigated based on the promising results obtained., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and by the University of Aveiro and FCT/MCT for the financial support for LAQV-REQUIMTE research Unit (FCT UIDB/50006/2020) through national funds and, where applicable, co financed by the FEDER, within the PT2020 Partnership Agreement. Enrique Pino-Hernandez ´ is a recipient of a fellowship supported by an advanced doctoral training program (call NORTE-69-2015-15), funded by the European Social Fund under the scope of Norte2020 – Programa Operacional Regional do Norte (NORTE-08-5369-FSE-000036). Finan cial support for author Carlos A. Pinto was provided by FCT through fellowship SFRH/BD/137036/2018. Luís Abrunhosa acknowledges FCT for the assistant research contract CEECIND/00728/2017., info:eu-repo/semantics/publishedVersion

Cisplatin is a widely used antineoplastic agent that has DNA as the main target, though cellular resistance hampers its therapeutic efficacy. An emerging hallmark of cancer cells is their altered metabolism, characterized by increased glycolysis even under aerobic conditions, with increased lactate production (known as the Warburg effect). Although this altered metabolism often results in increased resistance to chemotherapy, it also provides an opportunity for targeted therapeutic intervention. It has been suggested that cisplatin cytotoxicity can be affected by tumor metabolism, though with varying effects. We therefore sought to better characterize how lactate affects cisplatin sensitivity in the simplified Saccharomyces cerevisiae model. We show that lactate renders yeast cells resistant to cisplatin, independently of growth rate or respiration ability. We further show that histone acetylation is not affected, but histone phosphorylation is decreased in lactate-containing media. Finally, we show that Rad4p, essential for nucleotide excision repair, is required for the observed phenotype and thus likely underlies the mechanism responsible for lactate-mediated resistance to cisplatin. Overall, understanding how lactate modulates cisplatin sensitivity will aid in the development of new strategies to overcome drug resistance., This work was supported by the "Contrato-Programa" (UID/BIA/04050/2020) funded by national funds through FCT I.P., as well as a Post-doc fellowship to S. Chaves (SFRH/BPD/89980/2012).

Microplastics are one of the main environmental pollutants in marine ecosystems, and their presence in seawater is a consequence of the widespread use of plastic materials in modern commodities. This wide usage of plastics includes the employment of microspheres in common personal care products, which end up being ultimately released into the aquatic compartment. Known ecotoxicological effects of microplastics favoured the search for technologically viable and environmentally safer alternatives, such as paraffin wax microparticles, whose ecotoxicological risks have not been entirely characterized. To address this gap, the present study exposed mussels (Mytilus sp.) for 96 h to three densities (5 mg/L, 20 mg/L and 80 mg/L) of four size ranges (100–300 μm, 300–500 μm, 400–850 μm, and 800–1200 μm) of paraffin wax particles. Toxicological endpoints were the activities of four enzymes involved in key cellular processes, including antioxidant defence (catalase (CAT), glutathione reductase (GRed) and glutathione peroxidase (GPx)) and phase II metabolism (glutathione S-transferases (GSTs)), as well as lipid content and fatty acid profiles of the digestive gland. Significant interactions between the presence/absence of food and paraffin particle density were found, as food sometimes worked as a confounding factor in the analysed biomarkers. Despite this uncertainty, some overall patterns emerged. In general, smaller paraffin wax particles (100–300 μm) caused little effects on the activity of the four enzymes tested, whereas larger particles (800–1200 μm) caused significant effects on almost all biomarkers. CAT activity was enhanced in animals exposed to larger paraffin particles, whilst GPx activity was depressed; GRed activity was not affected by the exposure to paraffin particles. The activity of GSTs was enhanced, but only in one tested condition. No effects were observed in terms of the total lipid content and fatty acids of exposed animals. Overall, data obtained in this work suggest that, at densities of paraffin wax particles comparable to the levels found in the environment for microplastic beads, no toxicity is expected to occur in the tested mussel species, by measuring the here-assessed toxicological endpoints., This study received financial support from CESAM (UID/AMB/50017/2019) and from FCT/MEC through national funds, and the co-funding by the ERDF, within the PT2020 Partnership Agreement and Compete 2020.

Edible and medicinal fungi are one of the major sources for extraction and identification of polysaccharides, which are important biological response modifiers with notable antitumor, hepatoprotective effect and other pharmacological activities. This study aimed to evaluate the hepatoprotective effect of isolated Phellinus linteus polysaccharide (PL-N1) against acetaminophen (APAP) induced liver injury in mice. Mice were treated intragastrical with PL-N1 (10, 50 and 100 mg/kg) and APAP (300 mg/kg) injection. APAP alone caused increased serum aminotransferase levels and changes in hepatic histopathology, promoted oxidative stress by increasing lipid peroxidation and decreasing anti-oxidant enzyme activities, leading to hepatocellular necrosis and reduced liver function. PL-N1 decreased cytochrome P450 2E1 (CYP2E1) expression and hepatic release of cytokines to enhance the level of phase II enzymes. Also, PL-N1 obviously accelerates the metabolism of APAP in the rat model. Molecular docking analysis revealed the α-d-glucopyranosyl exhibit maximum interaction (-8.099) against CYP2E1 as comparably less than standard drug silibinin (-13.767). PL-N1 could be a promising natural substance for ameliorating acute APAP-induced oxidative stress and hepatic injury., This work was supported by the national Key Research and Development Program of China [grant number 2017YFD0501400], Innovation Capability Support Program of Shaanxi [grant number 2019XY-04], as well as the strategic programme UID/BIA/04050/2013 [grant number POCI-01-0145-FEDER-007569] funded by national funds through the FCT, Ministerio da Ciencia, Tecnologia e Ensino Superior (MCTES) and by the ERDF through the COMPETE 2020- Programa Operacional Competitividade e Internacionalizacao (POCI), Portugal. We are grateful to Mr. A. Aierken for his technical work.

1. Coextinction is the simplest form of secondary extinction and freshwater mussels (Bivalvia, Unionida) may be particularly prone to this phenomenon as their life cycle includes an obligatory parasitic larval stage on fish hosts. 2. The main aims of this study were to determine the possible ecological fish hosts of Anodonta anatina (Linnaeus, 1758) in several rivers of the Douro basin in north ern Portugal and to assess possible spatial and temporal differences in glochidial (larval) loads. In order to achieve this, electrofishing was conducted from December to April, the fish fauna was characterized, and levels of infestation with A. anatina glochidia were determined. 3. Native cyprinid species, mainly Luciobarbus bocagei (Iberian barbel) and Squalius carolitertii (northern Iberian chub), together with the non-native Lepomis gibbosus (pumpkinseed sunfish) and Alburnus alburnus (common bleak), were found to have the highest glochidial loads. Clear differences in infestation between rivers and throughout time were detected, with an infestation period from January to March, and with the Tâmega River having the highest prevalence. 4. Anodonta anatina is able to infest a variety of fish species, and this together with earlier studies showed that the metamorphosis into juveniles occurs mainly in native cyprinid species, although non-native species like common bleak can also be considered suitable hosts. However, the larvae infesting other non-native spe cies, such as the pumpkinseed sunfish, do not metamorphose and can be consid ered ‘dead ends’. 5. Overall, the results reported here are important for the conservation of A. anatina (and other unionoid species) because several Iberian rivers (and worldwide) have been subjected to the extirpation of native fish species and the introduction of non-native fish species. Therefore, careful assessments of fish communities should be conducted to assess possible negative interactions with freshwater mussels, Fundação para a Ciência e a Tecnologia, Grant/Award Number: PTDC/AGRFOR/1627/2014

The exponential growth and ubiquitous use of mobile electronics supported by the Internet of Things (IoT) is leading to an increasing need of self-powered wearable electronic devices. In particular, converting electrical energy from magnetic fields is very appealing for the development of a sustainable power sources/sensing tools. Magneto-mechano-electric energy conversion is an effective way to convert magnetic fields into electricity for low power applications. The successful printing of such materials allows the design of customized structures, improved integration into devices and reduced materials waste. Here, a self-powered printed sensing device is presented, by exploiting piezoelectric/magnetic composites in response to magnetic stimuli via magneto-mechano-electric energy conversion. The printed magnetoactive device has the capabilities of harvesting magnetic energy with maximum output power density of 9.7 mW cm3 as well as detecting magnetic fields with a r2 of 0.9991 and a sensitivity of 30 V T1. Such sensing/harvesting performance allied to the Bluetooth connectivity offer great potential for applications in wearable electronics devices and IoT-related applications., The authors thank the FCT- Fundação para a Ciência e Tecnologia- for financial support in the framework of the Strategic Funding UID/FIS/04650/2019 and under projects PTDC/EEI-SII/5582/2014, PTDC/BTM-MAT/28237/2017 and PTDC/EMD-EMD/28159/2017. P. M. and C. R. thank the FCT for the contract under the Stimulus of Scientific Employment (Individual Support – 2017 Call (CEECIND/03975/2017) and Individual Support – 2020 Call (2020.04163.CEECIND), respectively). R. B-P. and N. P. acknowledge also support from FCT (SFRH/BD/140698/2018 and SFRH/BD/131729/2017 grants, respectively). Finally, the authors acknowledge funding by the Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2016-76039-C4-3-R (AEI/FEDER, UE) and from the Basque Government Industry and Education Department under the ELKARTEK, HAZITEK and PIBA (PIBA-2018-06) programs, respectively. Funding from European Union’s Horizon 2020 Program for Research, ICT-02-2018 - Flexible and Wearable Electronics. Grant agreement no. 824339 – WEARPLEX is acknowledged., info:eu-repo/semantics/publishedVersion

Whereas locked nucleic acid (LNA) has been extensively used to control gene expression, it has never been exploited to control Candida virulence genes. Thus, the main goal of this work was to compare the efficacy of five different LNA-based antisense oligonucleotides (ASO) with respect to the ability to control EFG1 gene expression, to modulate filamentation and to reduce C. albicans virulence. In vitro, all LNA-ASOs were able to significantly reduce C. albicans filamentation and to control EFG1 gene expression. Using the in vivo Galleria mellonella model, important differences among the five LNA-ASOs were revealed in terms of C. albicans virulence reduction. The inclusion of PS-linkage and palmitoyl-2-amino-LNA chemical modification in these five LNA gapmers proved to be the most promising combination, increasing the survival of G. mellonella by 40%. Our work confirms that LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field., This study was supported by the Portuguese Foundation forScience and Technology (FCT) under the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European RegionalDevelopment Fund under the scope of Norte2020-ProgramaOperacional Regional do Norte and Daniela Eira Araújo [SFRH/BD/121417/2016] PhD grant. The authors also acknowledge theproject funding by the“02/SAICT/2017–Projetos de Investiga-ção Científica e Desenvolvimento Tecnológico (IC&DT)–POCI-01-0145-FEDER-028893”. VILLUM Fonden is acknowledgedfor funding the Biomolecular Nano-scale Engineering Center(BioNEC), a Villum center of excellence, grant numberVKR18333. Funding received by iBB-Institute for Bioengineer-ing and Biosciences from FCT (UID/BIO/04565/2020) andPrograma Operacional Regional de Lisboa 2020 (Project No.007317) is also acknowledged. We acknowledge Dr. LucíliaGoreti Pinto, Life and Health Sciences Research Institute(ICVS), School of Medicine, University of Minho, forprocessing and sectioningG. mellonellatissue samples.The authors declare no conflict of interest., info:eu-repo/semantics/publishedVersion

Zymomonas mobilis is a well-recognized ethanologenic bacterium with outstanding characteristics which make it a promising platform for the biotechnological production of relevant building blocks and fine chemicals compounds. In the last years, research has been focused on the physiological, genetic, and metabolic engineering strategies aiming at expanding Z. mobilis ability to metabolize lignocellulosic substrates toward biofuel production. With the expansion of the Z. mobilis molecular and computational modeling toolbox, the potential of this bacterium as a cell factory has been thoroughly explored. The number of genomic, transcriptomic, proteomic, and fluxomic data that is becoming available for this bacterium has increased. For this reason, in the forthcoming years, systems biology is expected to continue driving the improvement of Z. mobilis for current and emergent biotechnological applications. While the existing molecular toolbox allowed the creation of stable Z. mobilis strains with improved traits for pinpointed biotechnological applications, the development of new and more flexible tools is crucial to boost the engineering capabilities of this bacterium. Novel genetic toolkits based on the CRISPR-Cas9 system and recombineering have been recently used for the metabolic engineering of Z. mobilis. However, they are mostly at the proof-of-concept stage and need to be further improved., This review is supported by Portuguese Foundation for Science and Technol ogy (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the project FoSynBio (POCI-01-0145-FEDER-029549). SFRH/BD/138325/2018, SFRH/BD/132324/2017, SFRH/BD/04433/2020, Fundação para a Ciência e a Tecnologia, UMINHO/BPD/4/2019., info:eu-repo/semantics/publishedVersion

The climate changes expected for the next decades will expose plants to increasing occurrences of combined abiotic stresses, including drought, higher temperatures, and elevated CO2 atmospheric concentrations. These abiotic stresses have significant consequences on photosynthesis and other plants’ physiological processes and can lead to tolerance mechanisms that impact metabolism dynamics and limit plant productivity. Furthermore, due to the high carbohydrate content on the cell wall, plants represent a an essential source of lignocellulosic biomass for biofuels production. Thus, it is necessary to estimate their potential as feedstock for renewable energy production in future climate conditions since the synthesis of cell wall components seems to be affected by abiotic stresses. This review provides a brief overview of plant responses and the tolerance mechanisms applied in climate change scenarios that could impact its use as lignocellulosic biomass for bioenergy purposes. Important steps of biofuel production, which might influence the effects of climate change, besides biomass pretreatments and enzymatic biochemical conversions, are also discussed. We believe that this study may improve our understanding of the plant biological adaptations to combined abiotic stress and assist in the decision-making for selecting key agronomic crops that can be efficiently adapted to climate changes and applied in bioenergy production., info:eu-repo/semantics/publishedVersion

Spinal Cord Injury (SCI) promotes a cascade of inflammatory events that are responsible for neuronal death and glial scar formation at the site of the injury, hindering tissue neuroregeneration. Among the main approaches for the treatment of SCI, the use of biomaterials, especially gelatin methacryloyl (GelMA), has been proposed because it is biocompatible, has excellent mechanical properties, favoring cell adhesion and proliferation. In addition, it can act as a carrier of anti-inflammatory drugs, preventing the formation of glial scars. The present work presents the development and in situ application of a light-curing formulation based on GelMA containing a natural extract rich in anti-inflammatory, antioxidant and neuroprotective substances (hydroalcoholic extract of red propolisHERP) in an experimental model of SCI in rats. The formulations were prepared and characterized by time of UV exposition, FTIR, swelling and degradation. The hydrogels containing 1 mg/mL of HERP were obtained by the exposure to UV radiation of 2 L of the formulation for 60 s. The locomotor evaluation of the animals was performed by the scale (BBB) and demonstrated that after 3 and 7 days of the injury, the GelMA-HERP group (BBB = 5 and 7) presented greater recovery compared to the GelMA group (BBB = 4 and 5). Regarding the inflammatory process, using histomorphological techniques, there was an inflammation reduction in the groups treated with GelMA and GelMA-HERP, with decreases of cavitation in the injury site. Therefore, it is possible to conclude that the use of GelMA and GelMA-HERP hydrogel formulations is a promising strategy for the treatment of SCI when applied in situ, as soon as possible after the injury, improving the clinical and inflammatory conditions of the treated animals. View Full-Text, This work was supported by the Banco do Nordeste (grant FUNDECI/2016.0015), National Council for Scientific and Technological (CNPq), Research and Technological Innovation Support Foundation of the State of Sergipe (Fapitec) and Coordination for the Improvement of Higher Education Personnel (CAPES). RLCAJ (309458/2019-6) and PS (301964/2019-0) thank CNPq for the financial support of the PQ scholarship. JCC thanks CNPq for the financial support of the DT scholarship (312451/2021/2). EBS acknowledges the Portuguese Science and Technology Foundation (FCT) for the project UIDB/04469/2020 (strategic fund) funded by national funds, and co-financed Education (FCT/MEC) from national funds and FEDER, under the Partnership Agreement PT2020., info:eu-repo/semantics/publishedVersion

Tomato bacterial spot (Bs), caused by Xanthomonas spp., including X. euvesicatoria (Xeu) remains a major threat for tomato production. The emergence of copper resistance strains of Xeu calls urgently for eco-friendly phytosanitary treatments as sustainable green alternatives for disease control. Satureja spp. essential oil (EO) has antimicrobial activity against xanthomonads and combined with zein nanoparticles (ZNPs), might offer a viable option for field applications. This study aims to evaluate the effects of S. montana EO, of ZNPs, and their combination in a nanoformulation, on Xeu quantity, and how these compounds modulate molecular and physiological changes in the pathosystem. Uninfected and infected tomato plants (var. Oxheart) were treated with EO; ZNPs and nanoformulation (EO + ZNPs). Treatments reduced Xeu amount by a minimum of 1.6-fold (EO) and a maximum of 202-fold (ZNPs) and improved plants health. Nanoformulation and ZNPs increased plants phenolic content. ZNPs significantly increased GPX activity and reduced CAT activity. Overall treatments upregulated transcripts of the phenylpropanoid pathway in infected plants, while ZNPs and nanoformulation upregulated those transcripts in uninfected plants. Both sod and aao transcripts were downregulated by treatments in infected plants. These findings demonstrate that S. montana EO, ZNPs and their nanoformulation are suitable to integrate tomato bacterial spot management strategies, mainly due to their antimicrobial activity on Xeu, however further field studies clarifying the long-term action of these products are required. These results also support the prophylactic potential of ZNPs on tomato bacterial spot., The authors would like to thank other funding sources that supported the researchers involved in this work, such as, the European Investment Funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI) and National Funds by the Portuguese Foundation for Science and Technology (FCT) (02/SAICT/2017, Ref. PTDC/BAAAGR/31131/2017, Acronym EOIS-CropProt). National Funds through FCT (Portugal) also funded this work within the scope of GreenUPorto, FCUP research unit: UIDB/05748/2020 and UIDP/05748/2020 projects; CITAB research unit, UTAD: project UIDB/04033/2020). Furthermore, N.M-P. was funded by a PhD grant SFRH/BD/138187/2018. This study was also supported by the European Union’s Horizon 2020 Research and Innovation Program (Grant Agreement Num ber 857251)., info:eu-repo/semantics/publishedVersion

The observation that exogenous Ca shifted the polyphenolic profile of grape berries and conferred a glossy appearance to mature fruits led us to hypothesize that the composition of grape berry waxes and thus surface microbiota are modified. In two cultivars sharing the same microclimate, the triterpenoid and steroid profile of berry cuticle was characterized by a targeted metabolomic approach, and surface microbial communities were surveyed by ITS and 16S metabarcoding. Results showed that Ca strongly decreased the levels of oleanolic acid, while steroids and neutral triterpenoids were affected in a cultivar-dependent manner. A total of 174 fungi and 192 bacteria OTUs were identified, with Dothideomycetes, Leotiomycetes, Alphaproteobacteria and Gammaproteobacteria comprising the most abundant classes. Ca decreased fungi biodiversity, favoring the growth of Basidiomycetes, and shifting fungi-bacteria relationships. Metabolite-microbiota networks revealed a tight relationship between microbial communities and triterpenoid components of fruit waxes, mainly stigmasterol, tremulone and oleanolic acid., The authors thank Dr Ana Garcia (CIIMAR, Portugal) for providing access to the experimental fields used in this study. This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by portuguese national funds through the FCT I.P. The work was also supported by FCT, CCDR-N (Norte Portugal Regional Coordination and Development Commission) and European Funds (FEDER/POCI/COMPETE2020) through the project AgriFood XXI (NORTE-01-0145-FEDER-000041), EcoAgriFood (NORTE-01-0145-FEDER-000009), and the research projects GrapeMicrobiota (PTDC/BAA-AGR/2691/2020), BerryPlastid (PTDC/BIA-FBT/28165/2017 and POCI-01-0145-FEDER-028165) and MitiVineDrought (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341). A.T. was supported by a postdoctoral researcher contract/position within the project BerryPlastid. GC-MS analyses were carried out with the use of CePT infrastructure financed by the European Union-the European Regional Development Fund (Agreement POIG.02.02.00-14-024/08-00)., info:eu-repo/semantics/publishedVersion

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.708911/full#supplementary-material, We established a syntrophic coculture of Syntrophobacter fumaroxidans MPOBT (SF) and Geobacter sulfurreducens PCAT (GS) growing on propionate and Fe(III). Neither of the bacteria was capable of growth on propionate and Fe(III) in pure culture. Propionate degradation by SF provides acetate, hydrogen, and/or formate that can be used as electron donors by GS with Fe(III) citrate as electron acceptor. Proteomic analyses of the SF-GS coculture revealed propionate conversion via the methylmalonyl-CoA (MMC) pathway by SF. The possibility of interspecies electron transfer (IET) via direct (DIET) and/or hydrogen/formate transfer (HFIT) was investigated by comparing the differential abundance of associated proteins in SF-GS coculture against (i) SF coculture with Methanospirillum hungatei (SF-MH), which relies on HFIT, (ii) GS pure culture growing on acetate, formate, hydrogen as propionate products, and Fe(III). We noted some evidence for DIET in the SF-GS coculture, i.e., GS in the coculture showed significantly lower abundance of uptake hydrogenase (43-fold) and formate dehydrogenase (45-fold) and significantly higher abundance of proteins related to acetate metabolism (i.e., GltA; 62-fold) compared to GS pure culture. Moreover, SF in the SF-GS coculture showed significantly lower abundance of IET-related formate dehydrogenases, Fdh3 (51-fold) and Fdh5 (29-fold), and the rate of propionate conversion in SF-GS was 8-fold lower than in the SF-MH coculture. In contrast, compared to GS pure culture, we found lower abundance of pilus-associated cytochrome OmcS (2-fold) and piliA (5-fold) in the SF-GS coculture that is suggested to be necessary for DIET. Furthermore, neither visible aggregates formed in the SF-GS coculture, nor the pili-E of SF (suggested as e-pili) were detected. These findings suggest that the IET mechanism is complex in the SF-GS coculture and can be mediated by several mechanisms rather than one discrete pathway. Our study can be further useful in understanding syntrophic propionate degradation in bioelectrochemical and anaerobic digestion systems., This work was performed in the TTIW-cooperation framework of Wetsus, European Centre of Excellence for Sustainable Water Technology (www.wetsus.nl). Wetsus was 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”. Research of AJMS was financed by an advanced grant of the European Research Council under the European Union’s Seventh Framework Programme (FP/2007e2013)/ERC Grant Agreement (project 323009) and a Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science., info:eu-repo/semantics/publishedVersion

Staphylococcus epidermidis is one of the most important commensal microorganisms of human skin and mucosae. However, this bacterial species is also the cause of severe infections in immunocompromised patients, specially associated with the utilization of indwelling medical devices, that often serve as a scaffold for biofilm formation. S. epidermidis strains are often multidrug resistant and its association with biofilm formation makes these infections hard to treat. Their remarkable ability to form biofilms is widely regarded as its major pathogenic determinant. Although a significant amount of knowledge on its biofilm formation mechanisms has been achieved, we still do not understand how the species survives when exposed to the host harsh environment during invasion. A previous RNA-seq study highlighted that iron-metabolism associated genes were the most up-regulated bacterial genes upon contact with human blood, which suggested that iron acquisition plays an important role in S. epidermidis biofilm development and escape from the host innate immune system. In this perspective article, we review the available literature on the role of iron metabolism on S. epidermidis pathogenesis and propose that exploiting its dependence on iron could be pursued as a viable therapeutic alternative., FO is supported by the Fundação para a Ciência e a Tecnologia research project with reference PTDC/BIA-MOL/29553/2017, under the scope of COMPETE2020 (POCI-01-0145-FEDER-029553)., info:eu-repo/semantics/publishedVersion

The growth location and plant variety may influence the active components and biological activities of plants used in phytomedicine. In this study, nine sets of different Epimedii Folium, from different representative cultivation locations and Epimedium species, were collected for comparison, using HPLC-DAD combined with multivariate analysis. The objective was to investigate the influence of geographical origin and Epimedium species on the quality of Epimedii Folium, and provide applicable guidance for cultivation and quality control of Epimedii Folium. Several Epimedium spp. sets were used to establish the HPLC-DAD fingerprints and 91 peaks (compounds) were selected for the multivariate analysis. Major compounds were analyzed by HPLC-DAD combined with principal component analysis (PCA). HPLC quantitative analysis of known bioactive compounds was performed. Application of PCA to HPLC data showed that Epimedium samples sharing the same geographical origin or species clustered together, indicating that both species and geographical origin have impacts on the quality of Epimedii Folium. The major bioactive flavonoid compounds, epimedin C, icariin and baohuoside I, were identified and quantified. The concentration of bioactive compounds was significantly influenced both by species and geographical origin. E. sagittatum from Sichuan showed the highest content of bioactive compounds. The results showed that both Epimedium species and geographical origin have strong impact into quality of Epimedii Folium. HPLC data combined with multivariate analysis is a suitable approach to inform the selection of cultivation areas and choose Epimedium spp. most suitable for different geographical areas, resulting in improved quality of Epimedii Folium., This work was supported by Incubation Project on State Key Laboratory of Biological Resources and Ecological Environment of Qinba Areas (SLGPT2019KF04-04), China, and the ERDF through the COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI), Portugal., info:eu-repo/semantics/publishedVersion

Brewers spent grain (BSG) is the largest by-product originated from the brewery industry with a high potential for producing carbohydrases by solid-state fermentation. This work aimed to test the efficacy of a carbohydrases-rich extract produced from solid-state fermentation of BSG, to enhance the digestibility of a plant-based diet for European seabass (Dicentrarchus labrax). First, BSG was fermented with A. ibericus to obtain an aqueous lyophilized extract (SSF-BSG extract) and incorporated in a plant-based diet at increasing levels (0control; 0.1%, 0.2%, and 0.4%). Another diet incorporating a commercial carbohydrases-complex (0.04%; Natugrain; BASF) was formulated. Then, all diets were tested in in vitro and in vivo digestibility assays. In vitro assays, simulating stomach and intestine digestion in European seabass, assessed dietary phosphorus, phytate phosphorus, carbohydrates, and protein hydrolysis, as well as interactive effects between fish enzymes and dietary SSF-BSG extract. After, an in vivo assay was carried out with European seabass juveniles fed selected diets (0control; 0.1%, and 0.4%). In vitro digestibility assays showed that pentoses release increased 45% with 0.4% SSF-BSG extract and 25% with Natugrain supplemented diets, while amino acids release was not affected. A negative interaction between endogenous fish enzymes and SSF-BSG extract was observed in both diets. The in vivo digestibility assay corroborated in vitro data. Accordingly, the dietary supplementation with 0.4% SSF-BSG increased the digestibility of dry matter, starch, cellulose, glucans, and energy and did not affect protein digestibility. The present work showed the high potential of BSG to produce an added-value functional supplement with high carbohydrases activity and its potential contribution to the circular economy by improving the nutritional value of low-cost and sustainable ingredients that can be included in aquafeeds. © 2021, The Author(s)., Te authors thank UNICER, a sof-drink company (Porto, Portugal), for providing the brewer’s spent grain used in this study. Tis study was supported by the project “SPO3-Development of innovative sustainable protein and omega-3 rich feedstufs for aquafeeds, from local agro-industrial by-products”, reference POCI-01- 0145-FEDER-030377, funded by European Regional Development Fund (ERDF) and Portuguese Foundation for Science and Technology (FCT) and by strategic funding of UIDB/04423/2020 by UIDB/04469/2020 unit, the BioTecNorte operation (NORTE-01-0145-FEDER-000004) through national funds provided by FCT, and under the scope of the strategic funding of UIDB/04469/2020 unit. Te H.F. and C.C were supported by grants SFRH/BD/131219/2017 and SFRH/BPD/114942/2016, respectively, from FCT, MCTES, FSE, and UE under the North Portugal Regional Operational Programme (NORTE2020). José Manuel Salgado was supported by Beatriz Galindo contract of Ministry of Education (Spain)., info:eu-repo/semantics/publishedVersion

The interpretation of salt´s effects on protein stability discriminates between low concentration regimes, dominated by ion specific-binding or Debye-Hückel screening, and high concentration regimes, generally described by Hofmeister effects. However, predicting a critical concentration at which one effect overcomes the other is still challenging. Reasonable quantitative estimates can be obtained resorting to surface/bulk solvent partition models developed for non- Coulombic effects, though these are limited by the availability of reliable structures representative of the unfolded state. Here, we use myoglobin as a model to explore how ion-dependency on the nature of the unfolded state affects protein stability, combining spectroscopic techniques with molecular dynamic simulations. To this end, the thermal and chemical stability of myoglobin was assessed, in the presence of three different salts (NaCl, (NH4)2SO4 and Na2SO4), at physiologically relevant concentrations (0-0.3 M). We observed a mild destabilization of the native state induced by each ion, attributed to unfavorable screening and hydrogen-bonds with the protein side-chains. Both effects, combined with binding of Na+, Cl- and SO42- to the thermal-unfolded state, resulted in an overall destabilization of the protein. Contrastingly, ion binding was hindered in the chemically-unfolded conformation, due to occupation of the binding sites by urea molecules. Such mechanistic action led to a lower degree of destabilization, promoting surface tension effects that overall stabilized myoglobin following the Hofmeister series. Therefore, we demonstrate that the transition concentration from Coulombic to Hofmeister effects is modulated by the nature of the unfolded state. Altogether, our findings evidence the need to characterize the structure of the unfolded state when trying to dissect the molecular mechanisms underlying effects of salts on protein stability., The authors thank to Nirnay Samanta for the useful discussions during the preparation of the manuscript. The Portuguese Foundation for Science and Technology (FCT) is gratefully acknowledged for financial support through Centre of Chemistry of University of Minho (CQ-UM) (project UID/QUI/00686/2020). Access to computing resources funded by the Project “Search-ON2: Revitalization of HPC infrastructure of UMinho” (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), is also acknowledged. TGC thanks FCT under the scope of the strategic funding of UIDB/04469/2020 unit. Grant UIDP/Multi/04046/2020 (to BioISI), is also acknowledged., info:eu-repo/semantics/publishedVersion

Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2021.132773., Quantitative image analysis (QIA) is a simple and automated method for process monitoring, complementary to chemical analysis, that when coupled to mathematical modelling allows associating changes in the biomass to several operational parameters. The majority of the research regarding the use of QIA has been carried out using synthetic wastewater and applied to activated sludge systems, while there is still a lack of knowledge regarding the application of QIA in the monitoring of aerobic granular sludge (AGS) systems. In this work, chemical oxygen demand (COD), ammonium (NNH4+), nitrite (NNO2-), nitrate (NNO3-), salinity (Cl), and total suspended solids (TSS) levels present in the effluent of an AGS system treating fish canning wastewater were successfully associated to QIA data, from both suspended and granular biomass fractions by partial least squares models. The correlation between physical-chemical parameters and QIA data allowed obtaining good assessment results for COD (R2 of 0.94), NNH4+ (R2 of 0.98), NNO2- (R2 of 0.96), NNO3- (R2 of 0.95), Cl (R2 of 0.98), and TSS (R2 of 0.94). While the COD and NNO2- assessment models were mostly correlated to the granular fraction QIA data, the suspended fraction was highly relevant for NNH4+ assessment. The NNO3-, Cl and TSS assessment benefited from the use of both biomass fractions (suspended and granular) QIA data, indicating the importance of the balance between the suspended and granular fractions in AGS systems and its analysis. This study provides a complementary approach to assess effluent quality parameters which can improve wastewater treatment plants monitoring and control, with a more cost-effective and environmentally friendly procedure, while avoiding daily physical-chemical analysis., The authors thank the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding to the research units CEB (UIDB/04469/2020) and CBQF (UIDB/50016/2020) and the project AGeNT - PTDC/BTA-BTA/31264/2017 (POCI-01-0145-FEDER-031264). The authors wish to thank the company Aguas do Tejo Atlantico, S.A. for supplying the granules. Daniela P. Mesquita and Catarina L. Amorim thank FCT for funding through program DL 57/2016 – Norma transitoria., info:eu-repo/semantics/publishedVersion

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2021.779376/full#supplementary-material, Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and has been associated with serious health complications, mainly in pregnant women. It is characterized by a decrease in the number of Lactobacillus species in the healthy vaginal microbiota and an overgrowth of strict and facultative anaerobic bacteria that develop a polymicrobial biofilm. Despite over 60 years of research investigating BV, its etiology is not fully understood. Gardnerella spp. is a crucial microorganism that contributes to the formation of the biofilm and the development of BV, but the role of other BV-associated bacteria is not clear. Nevertheless, Fannyhessea vaginae (previously known as Atopobium vaginae) is a highly specific species for BV, and co-colonization with Gardnerella is thought to be a very specific diagnostic marker. The diagnosis of BV still presents some limitations, since currently used methods often fail to accurately detect BV. This work aims to develop a novel peptide nucleic acid (PNA) probe targeting F. vaginae. This probe was further validated in a multiplex assay, which included a Gardnerella-specific PNA probe, as a possible method for diagnosis of BV, and was compared with quantification by qPCR. The new PNA probe showed excellent sensitivity and specificity and could discriminate F. vaginae-Gardnerella biofilms, confirming the potential to be used for the detection of BV-associated pathogens., This work was funded by the National Institute of Allergy and Infectious Diseases (R01AI146065-01A1). It was also partially funded by the Portuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding of unit (UIDB/04469/2020)., info:eu-repo/semantics/publishedVersion

Glycoside hydrolases are responsible for the enzymatic deconstruction of complex carbohydrates. Most of the families are known to conserve the catalytic machinery and molecular mechanisms. This work introduces a new method to predict glycolytic abilities in sequenced genomes and thus, gain a better understanding of how to target specific carbohydrates and identify potentially interesting sources of specialised enzymes. Genome sequences are aligned to those of organisms with expertly curated glycolytic abilities. Clustering of homology scores helps identify organisms that share common abilities and the most promising organisms regarding specific glycolytic abilities. The method has been applied to members of the bacterial families Ruminococcaceae (39 genera), Eubacteriaceae (11 genera) and Lachnospiraceae (59 genera), which hold major representatives of the human gut microbiota. The method predicted the potential presence of glycoside hydrolases in 1701 species of these genera. Here, the validity and practical usefulness of the method is discussed based on the predictions obtained for members of the genus Ruminococcus. Results were consistent with existing literature and offer useful, complementary insights to comparative genomics and physiological testing. The implementation of the Gleukos web portal (http://sing-group.org/gleukos) offers a public service to those interested in targeting microbial carbohydrate metabolism for biotechnological and health applications., SING group thanks CITI (Centro de Investigacion, Transferencia e Innovacion) from the University of Vigo for hosting its IT infrastructure. Funding: This work was funded by the Spanish “Ministerio de Economıa y Competitividad—Agencia Estatal de Investigacion” (grant AGL2016-78311-R; AEI/ FEDER, UE) and by the grant IDI/2018/000236 from the Government of the Principality of Asturias. 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 the Consellerıa de Educacion, Universidades e Formacion Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group. Conflict of interest: Abelardo Margolles and Borja Sanchez are scientific founders of Microviable Therapeutics SL. The rest of the authors have no conflicts of interest to declare, info:eu-repo/semantics/publishedVersion

Electrohydrodynamic processing (EHDP) allows the use of a wide range of biopolymers and solvents, including food-grade biopolymers and green solvents, for the development of micro- and nanostructures. These structures present a high surface-area-to-volume ratio and different shapes and morphologies. The aim of this work was to design and produce hydroxypropyl methylcellulose (HPMC)-based micro- and nanostructures through EHD processing using green solvents, while exploring the influence of process and solution parameters, and incorporating a bioactive extracted from a food by-product. Low (LMW) and high (HMW) molecular weight HPMC have been used as polymers. The design-of-experiments methodology was used to determine the effects of process parameters (polymer concentration, flow rate, tip-to-collector distance, and voltage) of EHDP on the particle and fibre diameter, aspect ratio, diameter distribution, aspect ratio distribution, and percentage of fibre breakage. Additionally, melanoidins extracted from spent coffee grounds were encapsulated into the HPCM-based structures at a concentration of 2.5mg melanoidins/mL of the polymer solution. Polymer solutions were characterised regarding their viscosity, surface tension and conductivity, and showed that the incorporation of melanoidins increased the viscosity and conductivity values of the polymer solutions. The developed structures were characterised regarding their thermal, crystallinity and morphological properties before and after melanoidin incorporation and it was observed that melanoidin incorporation did not significantly influence the characteristics of the produced micro- and nanostructures. Based on the results, it is possible to envision the use of the produced micro- and nanostructures in a wide range of applications, both in food and biomedical fields., We would like to acknowledge the European Commission through the H2020-MSCA-RISE project FODIAC—Food for Diabetes and Cognition (reference number 778388), Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and by the I&D&I AgriFood XXI project, operation number NORTE-01-0145-FEDER-000041, co-financed by the European Regional Development Fund (FEDER) under the scope of NORTE 2020 (Programa Operacional Regional do Norte 2014/2020). The author Pedro Silva is the recipient of a fellowship (SFRD/BD/130247/2017) supported by Fundação para a Ciência e a Tecnologia, (FCT, Portugal)., info:eu-repo/semantics/publishedVersion

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fenvs.2021.735607/ full#supplementary-material, Aerobic granular sludge (AGS) processes are among the most robust wastewater treatments. One of their greatest advantages is related to the granules multi-layered structure, which creates a protective barrier against organic shock loads and variable wastewater composition, particularly attractive for the treatment of industrial wastewater. However, when treating a wastewater with variable and complex composition, the difficulty in identifying factors that most affect a specific biological process increases. In this study, the effect of organic loading rate (OLR), namely carbon content, on nitrification in an AGS process treating fish canning wastewater was investigated. Besides process performance, also biomass structural changes, and microbial community composition were analysed. Reactor operation lasted for 107 days and was divided in three phases during which different OLR and C/N ratios were applied. A higher OLR was applied during the first two phases (ca. 1.1 and 1.5 kg COD m3 day1, respectively) compared to the third phase (between 0.12 and 0.78 kg COD m3 day1) and the C/N ratios also varied (ca. 4.4, 7.8, and 2.9, respectively). Throughout the operation, COD concentration in the outlet was lower than 100 mg O2 L1. Nitrification was inhibited during the second phase and recovered afterwards. Principal component analysis (PCA) of quantitative image analysis (QIA) and performance data allowed to distinguish process changes over the three operational phases. During the first two phases, the decrease in the biomass robustness occurred, but recovered during the last phase, indicating that the high content of organic matter had possibly an effect on the aerobic granules structural characteristics. The composition of the AGS microbiome did not change substantially after the end of the higher OLR periods. The main microbial diversity shifts were mostly associated to adaptation to higher or lower carbon availability. Bacteria and inferred enzymes associated to nitrogen and phosphorous removal were identified. Chryseobacterium, a bacterium with high metabolic versatility, was able to adapt to the organic shock load, becoming dominant over operation. Despite the variable composition of the fish canning wastewater, carbon was identified as the main driver for nitrification inhibition, while promoting changes in the physical characteristics and on the microbial community of granules., This work was supported by National Funds from FCT—Fundação para a Ciência e a Tecnologia, through the project GReAT-PTDC/BTA-BTA/29970/2017 (POCI-01-0145-FEDER-029970)., We would like to thank the scientific collaboration of CBQF under the FCT project UIDB/50016/2020 and of CEB under the FCT project UIDB/04469/2020. We would like also to thank “A Poveira S.A.” (Póvoa de Varzim, Portugal) for providing the fish canning wastewater., info:eu-repo/semantics/publishedVersion

Ionic liquids (ILs) have been extensively explored and implemented in different areas, ranging from sensors and actuators to the biomedical field. The increasing attention devoted to ILs centers on their unique properties and possible combination of different cations and anions, allowing the development of materials with specific functionalities and requirements for applications. Particularly for biomedical applications, ILs have been used for biomaterials preparation, improving dissolution and processability, and have been combined with natural and synthetic polymer matrixes to develop IL-polymer hybrid materials to be employed in different fields of the biomedical area. This review focus on recent advances concerning the role of ILs in the development of biomaterials and their combination with natural and synthetic polymers for different biomedical areas, including drug delivery, cancer therapy, tissue engineering, antimicrobial and antifungal agents, and biosensing., The authors thank the FCT (Fundacao para a Ciencia e Tecnologia) for financial support under the framework of the Strategic Funding UID/FIS/04650/2019 and pro-jects PTDC/BTM-MAT/28237/2017, PTDC/EMD-EMD/28159/2017, and PTDC/FIS-MAC/28157/2017. Funds provided by FCT in the framework of EuroNano-Med 2016 call, Project LungChek ENMed/0049/2016 are also gratefully acknowledged. D.M.C., L.C.F., R.M.M., and M.M.F. also thank the FCT for the grants SFRH/BPD/121526/2016, SFRH/BD/145345/2019, and FRH/BD/148655/2019, SFRH/BPD/121464/2016, respectively and CR thanks the FCT for the contract under the Stimulus of Scientific Employment (2020.04163.CEECIND). S.L.M. and J.R. acknowledge the funding support from the Spanish State Research Agency (AEI) through the project PID2019-106099RB-C43/AEI/10.13039/501100011033 and from the Basque Government Industry Department under the ELKARTEK program., info:eu-repo/semantics/publishedVersion

A novel electrochemical biosensor based on a molecularly imprinted polymer (MIP) was developed for the impedimetric determination of Tau protein, a biomarker of Alzheimers disease (AD). Indeed, a recent correlation between AD symptoms and the presence of Tau proteins in their aggregated form made hyperphosphorylated Tau protein (Tangles) a promising biomarker for Alzheimers diagnosis. The MIP was directly assembled on a screen-printed carbon electrode (C-SPE) and prepared by electropolymerization of 3-aminophenol (AMP) in the presence of the protein template (p-Tau-441) using cyclic voltammetry. The p-Tau-441 protein bound to the polymeric backbone was digested by the action of the proteolytic activity of proteinase K in urea and then washed away to create vacant sites. The performances of the corresponding imprinted and non-imprinted electrodes were evaluated by electrochemical impedance spectroscopy. The detection limit of the MIP-based sensors was 0.02 pM in PBS buffer pH 5.6. Good selectivity and good results in serum samples were obtained with the developed platform. The biosensor described in this work is a potential tool for screening Tau protein on-site and an attractive complement to clinically established methodologies methods as it is easy to fabricate, has a short response time and is inexpensive., 0624_2IQBIONEURO_6_E, 2QBioneuro, Impulso de una red de I + i en química biológica para diagnóstico y tratamiento de enfermedades neurológicas EP-INTERREG V A España Portugal (POCTEP)., info:eu-repo/semantics/publishedVersion

Metal nanoparticles have been extensively investigated for different types of pharmaceutical applications. However, their use has raised some concerns about their toxicity involving the increase of reactive oxygen species causing cellular apoptosis. Therefore, in this review we summarize the most relevant toxicity mechanisms of gold, silver, copper and copper oxide nanoparticles as well as production methods of metal nanoparticles. Parameters involved in their toxicity such as size, surface charge and concentration are also highlighted. Moreover, a critical revision of the literature about the strategies used to reduce the toxicity of this type of nanoparticles is carried out throughout the review. Additionally, surface modifications using different coating strategies, nanoparticles targeting and morphology modifications are deeply explained., This work was funded by the Portuguese Science and Technology Foundation (FCT) from the Ministry of Science and Technology (MCTES), European Social Fund (FSE) of EU, through the project UIDB/04469/2020 (CEB strategic fund), co-funded by European Funds (PRODER/COMPETE) and FEDER, under the Partnership Agreement PT2020., info:eu-repo/semantics/publishedVersion

Background: Candida albicans is the most prevalent fungi isolated in endodontic infections. In this study, the ability of C. albicans biofilm to tolerate the novel solvent mixtures methyl ethyl ketone (MEK)/tetrachloroethylene (TCE) and MEK/orange oil (OOil) sequentially to the standard irrigation of sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic (EDTA) was evaluated. Methods: Biofilm cell cultures of C. albicans SC 5314 were treated sequentially with NaOCl and EDTA and exposed to MEK/TCE or MEK/OOil. The effect of the antimicrobial treatment was evaluated using the disk diffusion method for planktonic culture, and the enumeration of colony-forming units (CFUs) and scanning electron microscope (SEM) for biofilm cell culture. Results: C. albicans mature biofilm (24 h) was significantly reduced in biomass and cell viability after solvent mixtures exposure, compared with the previous NaOCl and EDTA treatments. MEK/OOil combination caused a total reduction of biofilm, while with MEK/TCE, there was a 3-log (CFU/cm2) reduction compared with the sequence NaOCl and EDTA, and a 4-log (CFU/cm2) reduction compared with the control. Conclusions: The additional exposure of a preformed 24 h C. albicans biofilm to novel solvent mixtures MEK/TCE and MEK/OOil caused a positive antibiofilm impact, overcoming the performance of the conventional endodontic irrigating protocol., This article was supported by National Funds through FCT-Fundação para a Ciência e a Tecnologia, I.P., within CINTESIS, R&D Unit (reference UIDB/4255/2020). This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and grant ref 2020.05720.BD for Liliana Fernandes; 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

Micropatterned pores in scaffolds have been extensively used to promote mass transport, tissue integration, and vascularization. However, the combined effects of microchannels and biomimetic structure on cell functions have not been investigated. Herein, microchannels with varying diameters of about 100, 200, and 400 $\mu$m were fabricated in the scaffolds composed of nanofibrous (diameter\thinspace, This work was supported by the National Natural Science Foundation of China (Grant Nos. 31870963 and 51973058), the Key Research and Development Program of Jiangxi Province (No. 20192ACB80008), and the Key Project of Natural Science Foundation of Jiangxi Province (20202ACBL204013), info:eu-repo/semantics/publishedVersion

The main goal of this study was to chemically characterize an aqueous S. nigra flower extract and validate it as a bioactive agent. The elderflower aqueous extraction was performed at different temperatures (50, 70 and 90 °C). The extract obtained at 90 °C exhibited the highest phenolic content and antiradical activity. Therefore, this extract was analyzed by GC-MS and HPLC-MS, which allowed the identification of 46 compounds, being quercetin and chlorogenic acid derivatives representative of 86% of the total of phenolic compounds identified in hydrophilic fraction of the aqueous extract. Naringenin (27.2%) was the major compound present in the lipophilic fraction. The antiproliferative effects of the S. nigra extract were evaluated using the colon cancer cell lines RKO, HCT-116, Caco-2 and the extracts antigenotoxic potential was evaluated by the Comet assay in RKO cells. The RKO cells were the most susceptible to S. nigra flower extract (IC50=1250 µg mL1). Moreover, the extract showed antimicrobial activity against Gram-positive bacteria, particularly Staphylococcus aureus and S. epidermidis. These results show that S. nigra-based extracts can be an important dietary source of bioactive phenolic compounds that contribute to health-span improving life quality, demonstrating their potential as nutraceutical, functional foods and/or cosmetic components for therapeutic purposes., This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and for Scientific Employment Stimulus 2017 (CEECIND/01507/2017) provided to Ana M. Sousa., info:eu-repo/semantics/publishedVersion

Surfactants are amphiphilic compounds having hydrophilic and hydrophobic moieties in their structure. They can be of synthetic or of microbial origin, obtained respectively from chemical synthesis or from microorganisms activity. A new generation of ecofriendly surfactant molecules or biobased surfactants is increasingly growing, attributed to their versatility of applications. Surfactants can be used as drug delivery systems for a range of molecules given their capacity to create micelles which can promote the encapsulation of bioactives of pharmaceutical interest; besides, these assemblies can also show antimicrobial properties. The advantages of biosurfactants include their high biodegradability profile, low risk of toxicity, production from renewable sources, functionality under extreme pH and temperature conditions, and long-term physicochemical stability. The application potential of these types of polymers is related to their properties enabling them to be processed by emulsification, separation, solubilization, surface (interfacial) tension, and adsorption for the production of a range of drug delivery systems. Biosurfactants have been employed as a drug delivery system to improve the bioavailability of a good number of drugs that exhibit low aqueous solubility. The great potential of these molecules is related to their auto assembly and emulsification capacity. Biosurfactants produced from bacteria are of particular interest due to their antibacterial, antifungal, and antiviral properties with therapeutic and biomedical potential. In this review, we discuss recent advances and perspectives of biosurfactants with antimicrobial properties and how they can be used as structures to develop semisolid hydrogels for drug delivery, in environmental bioremediation, in biotechnology for the reduction of production costs and also their ecotoxicological impact as pesticide alternative., This work was supported by the Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/COMPETE) through the grants M-ERA-NET/0004/2015 (PAIRED), UIDB/04469/2020 (strategic fund), and UIDB/04033/2020 (CITAB), cofinanced by FEDER, under the Partnership Agreement PT2020. T. Pashirova acknowledges the financial support received from the government assignment for FRC Kazan Scientific Center of Russian Academy of Sciences., info:eu-repo/semantics/publishedVersion

Viticulture practices that change the light distribution in the grapevine canopy can interfere with several physiological mechanisms, such as grape berry photosynthesis and other metabolic pathways, and consequently impact the berry biochemical composition, which is key to the final wine quality. We previously showed that the photosynthetic activity of exocarp and seed tissues from a white cultivar (Alvarinho) was in fact responsive to the light microclimate in the canopy (low and high light, LL and HL, respectively), and that these different light microclimates also led to distinct metabolite profiles, suggesting a berry tissue-specific interlink between photosynthesis and metabolism. In the present work, we analyzed the transcript levels of key genes in exocarps and seed integuments of berries from the same cultivar collected from HL and LL microclimates at three developmental stages, using real-time qPCR. In exocarp, the expression levels of genes involved in carbohydrate metabolism (VvSuSy1), phenylpropanoid (VvPAL1), stilbenoid (VvSTS1), and flavan-3-ol synthesis (VvDFR, VvLAR2, and VvANR) were highest at the green stage. In seeds, the expression of several genes associated with both phenylpropanoid (VvCHS1 and VvCHS3) and flavan-3-ol synthesis (VvDFR and VvLAR2) showed a peak at the véraison stage, whereas that of RuBisCO was maintained up to the mature stage. Overall, the HL microclimate, compared to that of LL, resulted in a higher expression of genes encoding elements associated with both photosynthesis (VvChlSyn and VvRuBisCO), carbohydrate metabolism (VvSPS1), and photoprotection (carotenoid pathways genes) in both tissues. HL also induced the expression of the VvFLS1 gene, which was translated into a higher activity of the FLS enzyme producing flavonol-type flavonoids, whereas the expression of several other flavonoid pathway genes (e.g., VvCHS3, VvSTS1, VvDFR, and VvLDOX) was reduced, suggesting a specific role of flavonols in photoprotection of berries growing in the HL microclimate. This work suggests a possible link at the transcriptional level between berry photosynthesis and pathways of primary and secondary metabolism, and provides relevant information for improving the management of the light microenvironment at canopy level of the grapes., This research and the APC was funded by FCT—Portuguese Foundation for Science and Technology, grant provided to Andreia Garrido (PD/BD/128275/2017), under the Doctoral Programme “Agricultural Production Chains—from fork to farm” (PD/00122/2012), and by the European Social Funds and the Regional Operational Programme Norte 2020., info:eu-repo/semantics/publishedVersion

Resveratrol is a phenolic compound with strong antioxidant activity, being promising for several applications in health, food, and cosmetics. It is generally extracted from plants or chemically synthesized, in both complex and not sustainable processes, but microbial biosynthesis of resveratrol can counter these drawbacks. In this work, resveratrol production by microbial biosynthesis from lignocellulosic materials was assessed. Three robust industrial Saccharomyces cerevisiae strains known for their thermotolerance and/or resistance to inhibitory compounds were identified as suitable hosts for de novo resveratrol production from glucose and ethanol. Through the CRISPR/Cas9 system, all industrial strains, and a laboratory one, were successfully engineered with the resveratrol biosynthetic pathway via the phenylalanine intermediate. All strains were further screened at 30 °C and 39 °C to evaluate thermotolerance, which is a key feature for Simultaneous Saccharification and Fermentation processes. Ethanol Red RBP showed the best performance at 39 °C, with more than 2.6-fold of resveratrol production in comparison with the other strains. This strain was then used to assess resveratrol production from glucose and ethanol. A maximum resveratrol titer of 187.07 ± 19.88 mg/L was attained from a medium with 2\% glucose and 5\% ethanol (w/v). Lastly, Ethanol Red RBP produced 151.65 ± 3.84 mg/L resveratrol from 2.95\% of cellulose from hydrothermally pretreated Eucalyptus globulus wood, at 39 °C, in a Simultaneous Saccharification and Fermentation process. To the best of our knowledge, this is the first report of lignocellulosic resveratrol production, establishing grounds for the implementation of an integrated lignocellulose-to-resveratrol process in an industrial context., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UIDB/04469/2020 unit and a Ph.D. grant SFRH/BD/130739/2017 to C.E.C. I.B. and I.M.-H. acknowledge financial support from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (YEAST-TRANS project, Grant Agreement No. 757384), the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 814408 (SHIKIFACTORY100 project), and The Novo Nordisk Foundation (Grant agreement No. NNF20CC0035580 and No. NNF20OC0060809)., info:eu-repo/semantics/publishedVersion

Tese de doutoramento em Biologia (especialização em Biotecnologia Molecular), Organic acids are recognized as versatile chemical compounds with a vast variety of applications in sectors ranging from food and beverages, pharmaceutical, personal care, cosmetic products, consumer goods to the chemical industry. Due to the strong demand for these compounds, alternative approaches to non-sustainable processes, e.g. chemical synthesis from petroleum derivatives, are being developed. Sustainable strategies rely on the utilization of microbial cell factories, where transporter proteins play a crucial role through the control of substrate influx and product efflux. In particular, the expression of suitable carboxylic acid exporters avoids the internal cell toxicity due to the accumulation of these compounds, while facilitating its purification from the culture broth. The biodiversity of the microbial world is an excellent pool to uncover new organic acid transporters. In this study, wild yeast species isolated from acidic environments were explored regarding their ability to utilize organic acids. The yeast Cyberlindnera jadinii was selected for further studies due to its ability to utilize a vast range of organic acids as sole carbon and energy source, to synthesize a variety of valuable compounds for the food and pharmaceutical sectors, its intrinsic robust characteristics, and its capacity to use inexpensive media with high productivity levels. Using an in silico approach, we have explored the predicted transportome of C. jadinii and selected putative carboxylate transporters for functional characterization by heterologous expression in Saccharomyces cerevisiae. A total of 16 plasma membrane carboxylate transporters, members of the AceTr, SHS, SSS, TDT, DASS, and MCT families, were characterized in terms of transport activity and specificity, structural features, and evolutionary relationships. S. cerevisiae was also used as expression host to deepen the knowledge of the citrate exporter CexAp from Aspergillus niger, a member of the DHA1 transporter family. The structural characterization of AceTr family members Ato1 and SatP led to the identification of a conserved motif essential for protein function. In addition, the plasma membrane proteins Gpr1 from Yarrowia lipolytica and AceP from the archaea Methanosarcina acetivorans were functionally characterized as acetate transporters. The role of AceTr members from S. cerevisiae, Ato1, Ato2, and Ato3, in the transport of monocarboxylic acids was evaluated via a combination of directed evolution, whole-genome resequencing, and reverse engineering, leading to the discovery of Ato2 and Ato3 mutants as two novel lactic acid transport proteins. Structural insights were also provided using 3D structure predictions combined with molecular docking., Os ácidos orgânicos são reconhecidos como compostos versáteis com uma vasta aplicabilidade em sectores desde a área alimentar à indústria química. A forte procura por estes compostos exige abordagens alternativas à sua produção convencional, como a síntese química a partir de derivados de petróleo. As estratégias sustentáveis incluem a utilização de fábricas celulares microbianas nas quais as proteínas transportadoras desempenham um papel crucial no controlo do importe de substratos e o exporte de produtos. O exporte de ácidos carboxílicos evita a sua toxicidade intracelular, facilitando a sua purificação do meio de cultura. A biodiversidade microbiana é um excelente reservatório para a descoberta de novos transportadores de ácidos orgânicos. Neste estudo, foram estudadas espécies de leveduras provenientes de ambientes ácidos e avaliada a sua capacidade de utilização de ácidos orgânicos. A levedura Cyberlindnera jadinii foi selecionada para estudos posteriores dada a sua capacidade de utilização de uma gama alargada de ácidos orgânicos como fonte única de carbono e energia, para além de sintetizar vários compostos relevantes para os setores industriais. Esta levedura apresenta ainda uma grande robustez e tem capacidade para crescer em meios de cultura baratos apresentando elevados níveis de produtividade. Numa abordagem in silico, explorámos o transportoma inferido de C. jadinii, selecionando vários potenciais transportadores de carboxilatos para uma caracterização funcional por expressão heteróloga em Saccharomyces cerevisiae. Um total de 16 proteínas-alvo, membros das famílias AceTr, SHS, SSS, TDT, DASS e MCT, foram caracterizadas quanto à sua capacidade transporte de carboxilatos, especificidade, características estruturais e relações evolutivas. O exportador de citrato CexA de Aspergillus niger, membro da família DHA1, foi também caracterizado por expressão heteróloga na levedura S. cerevisiae. A caracterização estrutural dos membros da família AceTr, Ato1 e SatP, levou à identificação de um motivo conservado nestas proteínas transportadoras, essencial para a sua função. As proteínas Gpr1 da levedura Yarrowia lipolytica e AceP da archaea Methanosarcina acetivorans foram ainda caracterizadas como transportadores de acetato. O papel no transporte de ácidos carboxílicos de outros membros desta família, Ato1, Ato2 e Ato3 de S. cerevisiae, foi avaliado por uma abordagem combinada de evolução dirigida, ressequenciação do genoma e engenharia reversa, levando à descoberta dos mutantes Ato2 e Ato3 como dois novos transportadores de lactato. As características estruturais destas proteínas foram avaliadas por modelação de estruturas 3D e estudos de docking molecular.

Hypericum perforatum L. (St. John's wort) is a well-known medicinal plant that possesses secondary metabolites with beneficial pharmacological properties. However, improvement in the production of secondary metabolites via genetic manipulation is a challenging task as H. perforatum remains recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, the transcripts of key genes involved in several plant defence responses (secondary metabolites, RNA silencing, reactive oxygen species (ROS) and specific defence genes) were investigated in H. perforatum suspension cells inoculated with A. tumefaciens by quantitative real-time PCR. Results indicated that key genes from the xanthone, hypericin and melatonin biosynthesis pathways, the ROS-detoxification enzyme HpAOX, as well as the defence genes Hyp-1 and HpPGIP, were all upregulated to rapidly respond to A. tumefaciens elicitation in H. perforatum. By contrast, expression levels of genes involved in hyperforin and flavonoid biosynthesis pathways were markedly downregulated upon A. tumefaciens elicitation. In addition, we compared the expression patterns of key genes in H. perforatum leaf tissues with and without dark glands, a major site of secondary metabolite production. Overall, we provide evidence for the upregulation of several phenylpropanoid pathway genes in response to elicitation by Agrobacterium, suggesting that production of secondary metabolites could modulate H. perforatum recalcitrance to A. tumefaciens-mediated transformation., The author acknowledges the financial support provided by the Fundação para a Ciência e a Tecnologia (FCT) project (PTDC/AGR-GPL/119211/2010), to G.F., grant UID/BIA/04050/2013 (POCI-01-0145FEDER-007569), PhD grant (SFRH/BD/52561/2014), under the Doctoral Programme “Agricultural Production Chains – from fork to farm” (PD/00122/2012) to A.C.P., and by Ministry of Economy and Competitiveness of Spain (grant BIO2016-75619-R [AEI/FEDER, UE]) to F.T., info:eu-repo/semantics/publishedVersion

Riparian areas in the Cantabrian Atlantic ecoregion (northwest Portugal) play a key role in soil formation and conservation, regulation of nutrient and water cycle, creation of landscape aesthetic value and the preservation of biodiversity. The maintenance of their ecological integrity is crucial given the ever increase in multiple anthropogenic (water demand and agriculture) and climatic pressures (droughts and extreme events). We developed a transferable remote sensing approach, taking advantage of the latest freely available technologies (Sentinel-2 and Copernicus Land products), to detect intra-annual and inter-annual changes in riparian vegetation productivity at the river basin scale related to water stress. This study has used the normalized difference vegetation index (NDVI) to investigate riparian vegetation productivity dynamics on three different vegetation types (coniferous, broadleaved and grassland) over the past 5 years (2015-2019). Our results indicated that inter-annual seasonality differed between drier (2017) and wetter (2016) years. We found that intra-annual dynamics of NDVI were influenced by the longitudinal river zonation. Our model ranked first (r2m = 0.73) showed that the productivity of riparian vegetation during the dry season was positively influenced by annual rainfall and by the type of riparian vegetation. The emergent long lags between climatic variation and riparian plant productivity provides opportunities to forecast early warnings of climatically-driven impacts. In addition, the different average productivity levels among vegetation types should be considered when assessing climatic impacts on riparian vegetation. Future applications of Sentinel 2 products could seek to distinguish riparian areas that are likely to be more vulnerable to changes in the annual water balance from those that are more resistant under longer-term changes in climate., Contrato-Programa UIDB/04050/2020. ERA4CS/0004/2016. CLIMALERT: Climate Alert Smart System for Sustainable Water and Agriculture, an ERA-NET initiated by JPI Climate (ERA4CS programme) co-funded by the EU commission (Grant Agreement 690462) and FCT (ERA4CS/0004/2016). This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by national funds through the FCT I.P. (GP)

The life cycle of Unio foucauldianus Pallary, 1936, a critically endangered freshwater mussel species (Bivalvia: Unionida), includes a parasitic phase using fish as hosts. Therefore, to develop more efficient conservation strategies it is essential to know which are the suitable fish hosts of U. foucauldianus. In this study, two approaches were used to assess the fish hosts of U. foucauldianus: the determination of infestation rates of fishes under natural conditions through monthly sampling (from January to June) in the Laabid River (Oum Rbia basin) and the N'Fis River (Tensift basin), and artificial infestation in laboratory trials using fish species from both rivers. The natural infestation of fish was detected from February to June, with a peak in May. Fully metamorphosed juveniles were only detected in native fish species, i.e. Luciobarbus ksibi (Boulenger, 1905), Carasobarbus fritschii (Gunther, 1874), Luciobarbus zayanensis Doadrio, Casal-lopez & Yahyaoui, 2016, Labeobarbus maroccanus (Gunther, 1874), and Luciobarbus magniatlantis (Pellegrin, 1919). The two non-native fish species used do not function as effective hosts. Given the increasing human pressure on native fish species in the Mediterranean biodiversity hotspot, including the increased number of non-native fish introductions, urgent conservation measures are discussed for this and other freshwater mussel species., The authors would like to express their thanks to the High Commission for Water and Forests (HCEFLCD) for granting permission to use electrofishing in the Moroccan basins. This study was conducted within the scope of the project ‘Biodiversity and conservation of the critically endangered freshwater mussels in Morocco: ecogeographic, genetic and physiological information’, funded by the Mohamed Bin Zayed Species Conservation Fund (ref. 15256799), and the project ‘Breeding the most endangered bivalve on Earth: argaritifera marocana’, funded by the IUCN SOS (Save our Species) fund (ref. 2015B‐015).

Mitigating climate change effects involves strategic decisions by individuals that may choose to limit their emissions at a cost. Everyone shares the ensuing benefits and thereby individuals can free ride on the effort of others, which may lead to the tragedy of the commons. For this reason, climate action can be conveniently formulated in terms of Public Goods Dilemmas often assuming that a minimum collective effort is required to ensure any benefit, and that decision-making may be contingent on the risk associated with future losses. Here we investigate the impact of reward and punishment in this type of collective endeavors — coined as collective-risk dilemmas — by means of a dynamic, evolutionary approach. We show that rewards (positive incentives) are essential to initiate cooperation, mostly when the perception of risk is low. On the other hand, we find that sanctions (negative incentives) are instrumental to maintain cooperation. Altogether, our results are gratifying, given the a-priori limitations of effectively implementing sanctions in international agreements. Finally, we show that whenever collective action is most challenging to succeed, the best results are obtained when both rewards and sanctions are synergistically combined into a single policy., SCOPUS: ar.j, info:eu-repo/semantics/published

Bivalve shells can persist over a geological time, acting as important physical resources to the associated fauna. However, few studies have investigated their relevance as persistent long-term ecological attributes to the ecosystem. As such, it is relevant to investigate the shell decays in riverine systems subjected to different environmental conditions. Towards this end, shells of four bivalve species (Anodonta anatina, Corbicula fluminea, Potomida littoralis and Unio delphinus) were made available individually and in clusters of different sizes. The effects of river flow and seasonality were assessed by recording the decay rates of shells in lentic and lotic habitats throughout the year. Our results evidenced that the decays varied among species and depend on shell size, water flow and season. Thin shelled species (A. anatina and U. delphinus) showed the highest mean percentage of decay per month, 3.17% (lotic) and 2.77% (lotic), respectively, and thick shelled species (C. fluminea and P. littoralis) the lowest, 2.02% (lotic) and 1.83% (lotic), respectively. Size was a relevant variable explaining decays, with the smallest shells presenting the highest values, 1.2–2.0 times higher compared to the other size classes. Also, robustness showed to be the most relevant feature explaining the decays in thick shelled species. River flow was also a relevant descriptor of the decays, with higher decays observed in the lotic compared to the lentic habitats. Furthermore, lower decays were observed mainly during summer (lentic site), and autumn (lotic site) associated to the burial effect of leaves. In summary, shells of the native species A. anatina and U. delphinus are expected to persist and contribute less as habitat engineering species, than shells of the native P. littoralis and invasive C. fluminea species. This is especially valid to lotic habitats where the decays were up to 2.13 times higher than in lentic habitats., This study was supported by the FCT (Foundation for Science and Technology, Portugal) strategic fund UID/Multi/04423/2019.

Wickerhamomyces anomalus strain LBCM1105 was originally isolated from the wort of cachaça (the Brazilian fermented sugarcane juice-derived Brazilian spirit) and has been shown to grow exceptionally well at high amounts of glycerol. This paramount residue from the biodiesel industry is a promising cheap carbon source for yeast biotechnology. The assessment of the physiological traits underlying the W. anomalus glycerol consumption ability in opposition to Saccharomyces cerevisiae is presented. A new WaStl1 concentrative glycerol-H+ symporter with twice the affinity of S. cerevisiae was identified. As in this yeast, WaSTL1 is repressed by glucose and derepressed/induced by glycerol but much more highly expressed. Moreover, LBCM1105 aerobically growing on glycerol was found to produce ethanol, providing a redox escape to compensate the redox imbalance at the level of cyanide-resistant respiration (CRR) and glycerol 3P shuttle. This work is critical for understanding the utilization of glycerol by non-Saccharomyces yeasts being indispensable to consider their industrial application feeding on biodiesel residue., This work was supported by grants from Fundacao de Capacitacao de Pessoal de Nivel Superior from the Ministry of Education-CAPES/Brazil (PNPD 2755/2011; PCF-PVE 021/2012), from FEDER through POFC-COMPETE and by FCT through strategic funding (UID/BIA/04050/2013), from Universidade Federal de Ouro Preto, and a research fellowship from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq (Brazil) Process 304815/2012 (research grant) and Process 305135/2015-5 (research fellowship to R.L.B.). C.L. is supported by the strategic program UID/BIA/04050/2013 [POCI-01-0145-FEDER-007569] funded by national funds through the FCT I. P. and by the ERDF through the COMPETE2020-Programa Operacional Competitividade e (POCI). The AUXPE-PVES 1801/2012 (Process 23038.015294/2016-18) from Brazilian Government supported a grant of Visiting Professor to C.L. and a research fellowships to A.C.C. and to F.F.O.

In this article, a set of Prussian Blue (PB) thin films with different electrodeposition times (25 s, 50 s, 75 s, 100 s and 150 s) in air at ambient temperature was prepared. The layers were characterized by a variety of techniques which include, field effect scanning electron microscopy, energy-dispersive X-ray spectrometer, X-ray diffraction, Fourier transform infared spectroscopy, UV-Vis spectrophotometry, and electrochemical analysis. A simple and exact electrochemical method was used to estimate the optimal voltages for coloring and bleaching of different PB layers. Controlling electrodeposition time along with applying suitable voltage enabled us to investigate and improve electrochromic properties of PB layer. The sample prepared under 75 s deposition time showed probably a composition intermediate between the insoluble and soluble form. Furthermore, this sample (S75) shows a better electrochromic properties. High value of electrochromic contrast 55.36% at 555 nm and well stability of ion exchange by cycling are the characteristics of this layer. The control of the deposition time resulted in an increase of 9.38 times of the contrast ratio and corresponding values for optical density (Delta OD) of the PB layers., The authors also acknowledgement FEDER, through COMPETE and Fundacao para a Ciencia e a Tecnologia (FCT) Pest-OE/QUI/UI0616/2014 and the project LUMECD (POCI-01-0145-FEDER-016884).

The increased use of silver nanoparticles (AgNPs) raises concerns about their impacts on aquatic ecosystems. The impacts of Ag+ and AgNPs were assessed on proteomic and antioxidant enzymatic responses of Pseudomonas sp. M1. The effects of Ag+ on bacterial growth were stronger than those of AgNPs (EC20 = 107.1 μg L−1 for Ag+; EC20 = 307.2 μg L−1 for AgNPs), indicating the lower toxicity of the latter. At EC20, the activities of antioxidant enzymes increased more under exposure to Ag+ than to AgNPs, particularly for superoxide dismutase and glutathione peroxidase (stimulation of 667% and 433%, respectively). A total of 166 proteins were identified by SWATH-MS; among these, only 59 had their content significantly altered by one or both forms of silver. Exposure to AgNPs resulted in an increase of about 54% of these proteins, whereas 54% decreased under exposure to Ag+. Gene Ontology enrichment analysis revealed that protein folding and transmembrane transport were the most relevant processes affected by Ag+ exposure, whereas AgNPs mostly affected translation. Also, results suggest that each form of silver induced different adaptive responses. Furthermore, the low levels of Ag+ released from AgNPs (, ERDF through the COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by the Portuguese Foundation for Science and Technology I.P. (FCT) through the strategic funding UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569), PTDC/AAC-AMB/121650/2010, POCI-01-0145-FEDER-007440 (ref. UID/NEU/04539/2013), the National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER402-022125 (ref. ROTEIRO/0028/2013), and PTDC/BIA-BMA/ 30922/2017

Endoplasmic reticulum-mitochondria contact sites have been a subject of increasing scientific interest since the discovery that these structures are disrupted in several pathologies. Due to the emerging data that correlate endoplasmic reticulum-mitochondria contact sites function with known events of the apoptotic program, we aimed to dissect this interplay using our well-established model of acetic acid-induced apoptosis in Saccharomyces cerevisiae. Until recently, the only known tethering complex between ER and mitochondria in this organism was the ER-mitochondria encounter structure (ERMES). Following our results from a screening designed to identify genes whose deletion rendered cells with an altered sensitivity to acetic acid, we hypothesized that the ERMES complex could be involved in cell death mediated by this stressor. Herein we demonstrate that single ablation of the ERMES components Mdm10p, Mdm12p and Mdm34p increases the resistance of S. cerevisiae to acetic acid-induced apoptosis, which is associated with a prominent delay in the appearance of several apoptotic markers. Moreover, abrogation of Mdm10p or Mdm34p abolished cytochrome c release from mitochondria. Since these two proteins are embedded in the mitochondrial outer membrane, we propose that the ERMES complex plays a part in cytochrome c release, a key event of the apoptotic cascade. In all, these findings will aid in targeted therapies for diseases where apoptosis is disrupted, as well as assist in the development of acetic acid-resistant strains for industrial processes., This work was funded by the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) financed by national funds through Fundação para a Ciência e a Tecnologia (FCT) and by Fundo Europeu de Desenvolvimento Regional (FEDER) through COMPETE 2020 – Programa Operacional Competitividade e Internacionalização (POCI).

The synthesis of silver nanoparticles using plant extracts is known as a green approach, as it does not require the use of high pressure, energy, high temperature, or toxic chemicals. The approach makes use of plant extracts in a process called bioreduction, which is mediated by enzymes, proteins, amino acids, and metabolites found in bark, seed, and leaf extracts, transforming silver ions into metallic silver. This work aimed at developing silver nanoparticles (AgNPs) from Brazilian pepper, applying this green methodology. Hydroalcoholic extract of leaves of Schinus terebinthifolius Raddi was prepared and its concentration of polyphenols, tannins, and saponins quantified. The produced nanoparticles were characterized by UV-Vis spectroscopy, thermogravimetric analysis (TG), dynamic light scattering (DLS), and zeta potential (ZP). AgNPs were formulated in sodium alginate hydrogels to obtain a nano-based semi-solid formulation for skin application. The obtained silver nanoparticles of mean size between 350 and 450?nm showed no cytotoxicity against L929 mouse fibroblasts within the concentration range of 0.025?mg/mL and 10?mg/mL. Schinus terebinthifolius Raddi was found to enhance microbial inhibition against the tested strains, especially against gram-negative bacteria. Its potential use as an alternative to overcome bacterial resistance can be expected., This work was supported by the Banco do Nordeste do Brazil [FUNDECI/2016.0015], Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq), [443238/2014-6 and 470388/ 2014-5], Fundação de Apoio a Pesquisa e a Inovação Tecnológica do Estado de Sergipe (Fapitec), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Fundação para a Ciencia e a Tecnologia (FCT/MCT) and from European Funds (PRODER/COMPETE) for the projects M-ERA-NET/0004/2015-PAIRED and UIDB/04469/2020 (strategic fund), co-financed by FEDER, under the Partnership Agreement PT2020., info:eu-repo/semantics/publishedVersion

In grape cell cultures cv. Gamay Fréaux var. Teinturier, Ca was shown to decrease cell pigmentation through the inhibition of anthocyanin biosynthesis, while stimulating stilbenoids accumulation. Because methyl jasmonate (MeJA) is a well-known inducer of secondary metabolism in grape cells, and Ca antagonizes its stimulatory effect over several enzymes of core metabolic branches, in the present study we hypothesized that Ca and MeJA signaling pathways interact to regulate specific secondary metabolism routes. Grape cultured cells were elicited with MeJA or with MeJA + Ca and an UPLC-MS-based targeted metabolomic method was implemented to characterize their polyphenolic profiles. Results were compared with the profile of cells elicited with Ca only, previously reported. Data was complemented with gene expression analysis, allowing the assembly of a metabolic map that unraveled routes specifically regulated by both elicitors. MeJA + Ca specifically boosted E-resveratrol and E--viniferin levels by 180% and 140%, respectively, in comparison to cells treated with MeJA only, while the stimulatory effect of MeJA over flavonoid synthesis was inhibited by Ca. In parallel, Ca downregulated most flavonoid pathway genes, including LAR1, ANS, BAN and ANR. Ca was able to mimic or potentiate the effect of MeJA on the expression of JA signaling genes, including JAR1, PIN and PR10. Transcript/metabolite correlation networks exposed the central influence of FLS1, STS, CDPK17 and COI1 in polyphenolic biosynthetic routes. This study highlights the potential of the MeJA-Ca combination for diverting polyphenolic metabolism towards the production of specific metabolites of interest, highly relevant in a biotechnological perspective., This work was supported by the “Contrato-Programa” UIDB/04050/2020 funded by portuguese national funds through the FCT I.P. The work was also supported by FCT, CCDR-N (Norte Portugal Regional Coordination and Development Commission) and European Funds (FEDER/POCI/COMPETE2020) through the project AgriFood XXI (NORTE-01-0145-FEDER-000041) and the research projects BerryPlas tid (PTDC/BIA-FBT/28165/2017 and POCI-01-0145-FEDER-028165) and MitiVineDrought (PTDC/BIA-FBT/30341/2017 and POCI-01-0145- FEDER-030341). A.T. was supported by a postdoctoral researcher con tract/position within the project “BerryPlastid”. This article/publication is based upon work from COST Action CA 17111 INTEGRAPE, supported by COST (European Cooperation in Science and Technology) – in the context of a STSM to V.M. The R´egion-Centre Val de Loire (France) supported this work under the grant agreement to Projects CEPATLAS and VINODRONE to A.L., info:eu-repo/semantics/publishedVersion

Saccharomyces cerevisiae is the most commonly used yeast in wine, beer, and bread fermentations. However, Torulaspora delbrueckii has attracted interest in recent years due to its properties, ranging from its ability to produce flavor- and aroma-enhanced wine to its ability to survive longer in frozen dough. In this work, publicly available genomes of T. delbrueckii were explored and their annotation was improved. A total of 32 proteins were additionally annotated for the first time in the type strain CBS1146, in comparison with the previous annotation available. In addition, the annotation of the remaining three T. delbrueckii strains was performed for the first time. eggNOG-mapper was used to perform the functional annotation of the deduced T. delbrueckii coding genes, offering insights into its biological significance, and revealing 24 clusters of orthologous groups (COGs), which were gathered in three main functional categories: information storage and processing (28% of the proteins), cellular processing and signaling (27%), and metabolism (23%). Small intraspecies variability was found when considering the functional annotation of the four available T. delbrueckii genomes. A comparative study was also conducted between the T. delbrueckii genome and those from 386 fungal species, revealing a high number of homologous genes with species from the Zygotorulaspora and Zygosaccharomyces genera, but also with Lachancea and S. cerevisiae. Lastly, the phylogenetic placement of T. delbrueckii was clarified using the core homologs that were found across 204 common protein sequences of 386 fungal species and strains., This work was supported by the UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and UID/BIA/04050/2019 strategic programs and by the project PTDC/BIA-MIC/32059/2017 (TODOMICS: Omics approaches towards the industrial exploitation of Torulaspora delbrueckii: Elucidation of the molecular basis underlying complex cellular traits), which is funded by national funds through the FCT-Fundação para a Ciência e Tecnologia and by the ERDF- European Regional Development Fund through the COMPETE2020–Programa Operacional Competitividade e Internacionalização (POCI) and Sistema de Apoio à Investigação Científica e Tecnológica (SAICT).