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The potential of ionic liquids (ILs) to be used as antimicrobial agents for biomedical applications has been hindered by the fact that most of them are cytotoxic toward mammalian cells. Understanding the mechanism of bacterial and mammalian cellular damage of ILs is key to their safety design. In this work, we evaluate the antimicrobial activity and mode of action of several ILs with varying anions and cations toward the clinically relevant Gram-negative Escherichia coli. Langmuir monolayer technique was used to evaluate if the IL’s mode of action was related to the bacterial cell membrane interaction for an effective E. coli killing. 1-Decyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide [DMIM][TFSI] and trihexyltetradecyl phosphonium bis(trifluoromethylsulfonyl) imide [P6,6,6,14][TFSI] were surface-active and induced bacterial cell lysis, through a membrane-disruption phenomenon on bacteria, in a mechanism that was clearly related to the long alkyl chains of the cation. 1-Ethyl-3-methylimidazolium hydrogen sulfate [EMIM][HSO4] was highly antimicrobial toward E. coli and found suitable for biological applications since it was harmless to mammalian cells at most of the tested concentrations. The results suggest that the imidazolium cation of the ILs is mostly responsible not only for their antimicrobial activity but also for their cytotoxicity, and the inclusion of different anions may tailor the ILs’ biocompatibility without losing the capacity to kill bacteria, as is the case of [EMIM][HSO4]. Importantly, this IL was found to be highly antimicrobial even when incorporated in a polymeric matrix., The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for the post-doctoral research grant SFRH/BPD/121464/2016 (MMF), the PhD research grants SFRH/BD/145455/2019 (EOC) and the strategic funding UID/FIS/04650/2021 and UID/BIO/04469/2021 and projects PTDC/BTM-MAT/28237/2017, PTDC/EMDEMD/28159/2017, UIDB/50006/2020 and UIDP/50006/2020. The authors acknowledge funding by Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the project PID2019-106099RBC43/AEI/10.13039/501100011033 and from the Basque Government Industry Departments under the ELKARTEK program.

Wound infection hinders adequate healing, being particularly grievous and prevalent in burn wounds and chronic wounds. Wound infection extends inflammation, preventing epithelialization and angiogenesis. Therefore, infection prolongs healing time, steeply increases treatment costs and degrades patients wellbeing. One successful strategy to control wound infection is to apply an active wound dressing, able to eliminate or significantly reduce the microbial population present at the infection site. Silver nanoparticles (AgNPs) are a multipurpose antimicrobial agent with a wide scope of applications which include wound dressings. Nevertheless, several studies denote AgNPs dose-dependent cytotoxicity, and their capability to bypass the blood-brain barrier and induce a neurotoxic effect. Hence, we propose to adopt two different strategies to attempt the simultaneously immobilize and increase the load of AgNPs within the wound dressing fabric. Thus, the envisaged objective is to prevent potential systemic cytotoxicity /through immobilization and to improve its antimicrobial capability due to the higher concentration of AgNPs. Two different approaches were used: i. AgNPs were suspended in an alginate (ALG) solution, ii. AgNPs were embedded in Mordenite (MOR) zeolite, followed by addition of an ALG solution. Both suspensions were incorporated into polyester fabric assisted by its surface activation by dielectric barrier discharge (DBD) plasma treatment. The bactericidal and virucidal effectiveness of each composite was tested against bacteria species known to induce nosocomial infections and a bacteriophage that is a potential surrogate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two distinct antimicrobial analysis were used to provide insights on the antimicrobial effectiveness of the obtained composites and to indirectly assess the release of AgNPs.

The functionalization of polyester fabric (PES) with antimicrobial agents presents huge number of potential applications in advanced products. However, the lack of functional groups and the high PES hydrophobicity make the functionalization processes costly, prolonged and requires the use of polluting chemical compounds. In this work, dielectric barrier discharge (DBD) plasma treatment, an affordable and environmental-friendly method, was used to introduce new chemical groups, increase the surface energy and roughness of PES in order to improve the adhesion of silver nanoparticles (AgNPs) in its surface. The PES functionalization was evaluated by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and antimicrobial efficacy against Staphylococcus aureus and Escherichia coli. Despite some additional oxidation, the DBD plasma- treated PES showed superior adhesion of AgNPs and excellent antimicrobial efficacy even after 10 washing cycles (WC)., (undefined)

Polyester (PET) fabrics are widely applied in functional textiles due to their outstanding properties such as high strength, dimensional stability, high melting point, low cost, recyclability, and flexibility. Nevertheless, the lack of polar groups in the PET structure makes its coloration and functionalization difficult. The present work reports the one-step in situ synthesis of copper nanoparticles (CuNPs) onto the PET fabric employing sodium hypophosphate and ascorbic acid as reducing and stabilizing agents, at acidic (pH 2) and alkaline pH (pH 11). This synthesis (i) used safer reagents when compared with traditional chemicals for CuNP production, (ii) was performed at a moderate temperature (85 °C), and (iii) used no protective inert gas. The dielectric barrier discharge (DBD) plasma was used as an environmentally friendly method for the surface functionalization of PET to enhance the adhesion of CuNPs. The size of the CuNPs in an alkaline reaction (76–156 nm for not treated and 93.4–123 nm for DBD plasma-treated samples) was found to be smaller than their size in acidic media (118–310 nm for not treated and 249–500 nm for DBD plasma-treated samples), where the DBD plasma treatment promoted some agglomeration. In acidic medium, metallic copper was obtained, and a reddish color became noticeable in the textile. In alkaline medium, copper(I) oxide (Cu2O) was detected, and the PET samples exhibited a yellow color. The PET samples with CuNPs presented improved ultraviolet protection factor values. Finally, a minimal concentration of copper salt was studied to obtain the optimized antibacterial effect against Staphylococcus aureus and Escherichia coli. The functionalized samples showed strong antibacterial efficacy using low-concentration solutions in the in situ synthesis (2.0 mM of copper salt) and even after five washing cycles. The DBD plasma treatment improved the antibacterial action of the samples prepared in the alkaline medium., This research was funded by the project PLASMAMED─PTDC/CTM-TEX/28295/2017 and ARCHKNIT─039733 financed by FCT, FEDER, and POCI of Portugal 2020, the project UID/CTM/00264/2021 of 2C2T under the COMPETE, and FCT/MCTES (PIDDAC) cofinanced by FEDER through the PT2020 program., A.I.R. acknowledges FCT for the Ph.D. scholarship (SFRH/BD/137668/2018)

DEPTAL MCL® is a professional cleaning agent approved by the Portuguese Food Regulatory Authority and is used in agro-food industries, namely in fish canning industries in the north of Portugal. Its extensive use during cleaning procedures results in potential significant negative impacts on the performance of the downstream municipal wastewater treatment plant (WWTP). A lab-scale extended areation activated sludge wastewater treatment system, continuously fed by influent collected at a municipal WWTP, was used to assess the impact of a range of DEPTAL MCL® concentrations during 72h. Despite distinct activated sludge community composition (due to its dynamic nature) and variations in real influent characteristics, a relevant impact was observed. DEPTAL MCL® effect was underscored through the use of a multivariate analysis using seventeen physicochemical operational factors and nineteen quantitative image analysis (QIA) parameters. DEPTAL MCL® exerted a severe negative impact on phosphorous (P-PO4) removal, total nitrogen (TN) removal and sludge volume index (SVI). With increasing DEPTAL MCL® concentrations, both P-PO4 and TN removal were affected and diminished proportionally. Moreover, several QIA parameters indicate defloculation when DEPTAL MCL® was present, in particular for intermediate size aggregates with significant impacts. Optical density of the effluent (Ode), displayed an increase of effluent turbidity. Percentage of area covered by small aggregates (%Areasml) was also significantly higher for the intermediate and higher DEPTAL MCL® concentrations tested. Principal component analysis exhibited 3 distinct ordenations: (i) control without addition of DEPTAL MCL®; (ii) addition of 0.03% and 0.06% and of (iii) 0.13 and 0.26% (v DEPTAL MCL®/v aeration tank). Canonical correspondence analysis (CCA) was used to correlate the physicochemical data, QIA and the filamentous bacteria species prevalence to DEPTAL MCL® concentration and incubation time. A time persistent DEPTAL MCL® effect was observed, underscoring the need of a pretreatment of wastewater containing this cleaning agent., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This work was also supported by the project Innovative strategies to control filamentous overgrowth in activated sludge wastewater treatment (INOVCONTROLFIL) (PTDC/AAG-TEC/3331/2014). In addition, it was also supported Universidad de la Frontera DI21-1004, ANID/REDES 180003 and by FEDER funds through the Operational Competitiveness Program COMPETE and FCT under the projects PTDC/CTM-TEX/28295/2017 and UID/CTM/00264/2019., info:eu-repo/semantics/publishedVersion

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/eca2022-12708/s1, Burn wounds can lead to numerous severe complications including bacterial infections causing patient morbidity and mortality, mostly in low- and middle-income countries. The considerable increase in microbial resistance against traditional antibiotics is leading towards alternative strategies to treat bacterial infections. Nisin Z is an antimicrobial peptide which exhibits a significant antibacterial activity against Gram-positive bacteria. Its efficacy against Gram-negative bacteria is limited, nonetheless it can be improved with the addition of surfactants, such as ethylenediaminetetraacetic acid (EDTA). The incorporation of peptide and other biomolecules within a biopolymer matrix provides protection maintaining their antimicrobial potential. Bacterial nanocellulose (BNC) has been widely used as wound dressings. Its impressive water retention capacity (>99%) and porosity are beneficial to manage wounds due to its potential to absorb exudates, providing a breathable and humid environment. In this work, the functionalization of BNC with Nisin Z (BNC-NZ) via vacuum filtration is reported. The entrapment of the peptide inside the BNC films was confirmed through morphological characterization using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometry. Typical absorbance peaks of Nisin Z are easily identifiable at 1647 cm−1 (amide group) and 1520 cm−1 (bending of primary amines). Thermal gravimetric analysis (TGA) suggested that Nisin Z did not interfere with the BNC matrix. The antimicrobial activity of Nisin Z against five of the most common bacteria found in burn wounds was verified by minimum bactericidal concentration (MBC) ranging 8.0–256.0 µg/mL. Agar diffusion and shake flask methods revealed the potential of BNC-NZ for prospective applications in burn wound dressings., This research was funded by FEDER funds through COMPETE and by national funds through FCT via the projects POCI-01-0145-FEDER-028074 and UID/CTM/00264/2020. L.M. and T.D.T. also acknowledge FCT for their Ph.D. scholarships with references 2020.04919.BD and 2020.06046.BD., info:eu-repo/semantics/publishedVersion

[Excert] The extraction and exploration of cellulose-based polymers is an exciting area of research. For many years, wood (especially from bleached kraft wood pulp) was considered the main source of cellulosic compounds because of its abundance in nature [1,2]. However, in the past decade, researchers have been devoted to finding alternatives to extract cellulose from byproducts of agricultural crops and/or textile wastes, both highly available at a very reduced raw material cost., This research was funded by the Portuguese Foundation for Science and Technology (FCT) grants PTDC/CTMTEX/28074/2017, PTDC/CTM TEX/28295/2017 and UID/CTM/00264/2021.

Almost two years have passed since COVID-19 was officially declared a pandemic by the World Health Organization. However, it still holds a tight grasp on the entire human population. Several variants of concern, one after another, have spread throughout the world. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant may become the fastest spreading virus in history. Therefore, it is more than evident that the use of personal protective equipment (PPE) will continue to play a pivotal role during the current pandemic. This work depicts an integrative approach attesting to the effectiveness of ultra-violet-C (UV-C) energy density for the sterilization of personal protective equipment, in particular FFP2 respirators used by the health care staff in intensive care units. It is increasingly clear that this approach should not be limited to health care units. Due to the record-breaking spreading rates of SARS-CoV-2, it is apparent that the use of PPE, in particular masks and respirators, will remain a critical tool to mitigate future pandemics. Therefore, similar UV-C disinfecting rooms should be considered for use within institutions and companies and even incorporated within household devices to avoid PPE shortages and, most importantly, to reduce environmental burdens., This research was funded by the Portuguese Foundation for Science and Technology (FCT), European Regional Development Fund (FEDER), Operational Program for Competitive ness Factors (COMPETE), and Portuguese Ministry of Science, Technology and Higher Education (MCTS) [UID/CTM/00264/2021]; [PTDC/CTM TEX/28295/2017]; [PTDC/CTM-TEX/1213/2020]; [UIDB/50026/2020]; [NORTE-01-0145-FEDER-072555]; [NORTE-01-0145-FEDER-000039] and con tract funding [2020.03113.CEECIND] to M.I.V. This work is a result of a project with the EXMA company in co-promotion with the University of Minho [NORTE-01-02B7-FEDER-048968] and supported by the Northern Portugal Regional Operational Program (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the FEDER. Finally, this project was funded by the FCT Research4COVID-19 special fund [011_595803006UV-Fast].

The application of light‐emitting diodes (LEDs) has been gaining popularity over the last decades. LEDs have advantages compared to traditional light sources in terms of lifecycle, robustness, compactness, flexibility, and the absence of non‐hazardous material. Combining these advantages with the possibility of emitting Ultraviolet C (UVC) makes LEDs serious candidates for light sources in decontamination systems. Nevertheless, it is unclear if they present better decontamination effectiveness than traditional mercury vapor lamps. Hence, this research uses a systematic literature review (SLR) to enlighten three aspects: (1) UVC LEDs’ application according to the field, (2) UVC LEDs’ application in terms of different biological indicators, and (3) the decontamination effectiveness of UVC LEDs in comparison to conventional lamps. UVC LEDs have spread across multiple areas, ranging from health applications to wastewater or food decontamination. The UVC LEDs’ decontamination effectiveness is as good as mercury vapor lamps. In some cases, LEDs even provide better results than conventional mercury vapor lamps. However, the increase in the targets’ complexity (e.g., multilayers or thicker individual layers) may reduce the UVC decontamination efficacy. Therefore, UVC LEDs still require considerable optimization. These findings are stimulating for developing industrial or final users’ applications., This work was funded by the R&D project of EXMA company in co‐promotion with the University of Minho NORTE‐01‐02B7‐FEDER‐048968 supported by Norte Portugal Regional Oper‐ ational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), by the UV‐Fast project funded by the Portu‐ guese Science and Technology Foundation (FCT—Fundação para a Ciência e Tecnologia) through UID/CTM/00264/2021 and the Research4COVID‐19 special fund (Project 011_595803006), and PLASMAMED project (ref. PTDC/CTM‐TEX/28295/2017) co‐financed by COMPETE2020, under Portugal 2020 through the ERDF and FCT.

Antimicrobial textiles are helpful tools to fight against multidrug-resistant pathogens and nosocomial infections. The deposition of silver nanoparticles (AgNPs) onto textiles has been studied to achieve antimicrobial properties. Yet, due to health and environmental safety concerns associated with such formulations, processing optimizations have been introduced: biocompatible materials, environmentally friendly agents, and delivery platforms that ensure a controlled release. In particular, the functionalization of polyester (PES) fabric with antimicrobial agents is a formulation in high demand in medical textiles. However, the lack of functional groups on PES fabric hinders the development of cost-effective, durable systems that allow a controlled release of antimicrobial agents. In this work, PES fabric was functionalized with AgNPs using one or two biocompatible layers of chitosan or hexamethyldisiloxane (HMDSO). The addition of organo-matrices stabilized the AgNPs onto the fabrics, protected AgNPs from further oxidation, and controlled their release. In addition, the layered samples were efficient against Staphylococcus aureus and Escherichia coli. The sample with two layers of chitosan showed the highest efficacy against S. aureus (log reduction of 2.15 ± 1.08 after 3 h of contact). Against E. coli, the sample with two layers of chitosan showed the best properties. Chitosan allowed to control the antimicrobial activity of AgNPs, avoid the complete loss of AgNPs after washings and act in synergy with AgNPs. After 3 h of incubation, this sample presented a log reduction of 4.81, and 7.27 of log reduction after 5 h of incubation. The antimicrobial results after washing showed a log reduction of 3.47 and 4.88 after 3 h and 5 h of contact, respectively. Furthermore, the sample with a final layer of HMDSO also presented a controlled antimicrobial effect. The antimicrobial effect was slower than the sample with just an initial layer of HMDSO, with a log reduction of 4.40 after 3 h of incubation (instead of 7.22) and 7.27 after 5 h. The biocompatibility of the composites was confirmed through the evaluation of their cytotoxicity towards HaCaT cells (cells viability > 96% in all samples). Therefore, the produced nanocomposites could have interesting applications in medical textiles once they present controlled antimicrobial properties, high biocompatibility and avoid the complete release of AgNPs to the environment., This research was funded by the projects PLASMAMED-PTDC/CTM-TEX/28295/2017 and MEDCOR-PTDC/CTM-TEX/1213/2020, the project UID/CTM/00264/2021 of 2C2T under the COMPETE and FCT/MCTES (PIDDAC) co-financed by FEDER through the PT2020 program.

One of the most important measures implemented to reduce SARS-CoV-2 transmission has been the use of face masks. Yet, most mask options available in the market display a passive action against the virus, not actively compromising its viability. Here, we propose to overcome this limitation by incorporating antiviral essential oils (EOs) within polycaprolactone (PCL) electrospun fibrous mats to be used as intermediate layers in individual protection masks. Twenty EOs selected based on their antimicrobial nature were examined for the first time against the

Cellulose is recurrently defined as the most abundant biopolymer on planet Earth, displaying an overall estimated production rate of more than 0.2 billion tons in a single day. Cellulose prompt availability allied to it its mechanical properties made it virtually indissociable from the majority of anthropogenic commodities. Nevertheless, continuous progress demands superior features form daily common materials, being most of them adequately suited by low-cost petrochemical polymers. Fortunately, the higher Environmental awareness of the global population as well as 1 the remarkable properties of biosynthesized polymers, has driven an extensive research on a plethora of biopolymers, including cellulose. Cellulose most notable features are associated to its crystal domains, which are impressively underscored with the development of cellulose nanotechnology. Moreover, at nanoscale the cellulose surface richness in hydroxyl groups is comprehensively more available, considerably t broadening he effectiveness and potential of their interaction per se, but also by enhancing the efficacy of surface modification and functionalization. Nanocellulose surface modification was implemented almost contemporary to its discovery and characterization, and its objectives ranged between improving yield of nanocellulose production, lower its production costs, and to provide nanocellulose a completely distinct surface properties by changing its polarity, generating different functional groups, decorating it with adsorbed or tightly bound nanoparticles, and to provide additional chemical compatibility with distinct compounds to generate advanced nanocomposites. The plethora of successfully reported modifications and functionalizations underscore notable properties of both modified nanocellulose and its composites. This Chapter intends to highlight these remarkable features, hopefully widening the scope of novel applications of these impressive bio-based polymers.

The COVID-19 pandemic has had a negative impact on education. The restrictions imposed have undoubtedly led to impairment of the psychological well-being of both teachers and students, and of the way they experience interpersonal relationships. As reported previously in the literature, adverse effects such as loneliness, anxiety, and stress have resulted in a decrease in the cognitive performance of school and higher education students. Therefore, the objective of this work is to present a general overview of the reported adverse effects of the COVID-19 pandemic which may potentially influence the learning performance of students. Some neuroscientific findings related to memory and cognition, such as neuroplasticity and long-term potentiation, are also shown. We also discuss the positive effects of the practice of mindfulness, as well as other simple recommendations based on neuroscientific findings such as restful sleep, physical activity, and nutrition, which can act on memory and cognition. Finally, we propose some practical recommendations on how to achieve The COVID-19 pandemic has had a negative impact on education. The restrictions imposed have undoubtedly led to impairment of the psychological well-being of both teachers and students, and of the way they experience interpersonal relationships. As reported previously in the literature, adverse effects such as loneliness, anxiety, and stress have resulted in a decrease in the cognitive performance of school and higher education students. Therefore, the objective of this work is to present a general overview of the reported adverse effects of the COVID-19 pandemic which may potentially influence the learning performance of students. Some neuroscientific findings related to memory and cognition, such as neuroplasticity and long-term potentiation, are also shown. We also discuss the positive effects of the practice of mindfulness, as well as other simple recommendations based on neuroscientific findings such as restful sleep, physical activity, and nutrition, which can act on memory and cognition. Finally, we propose some practical recommendations on how to achieve more effective student learning in the context of the pandemic. The aim of this review is to provide some assistance in this changing and uncertain situation in which we all find ourselves, and we hope that some of the information could serve as a starting point for hypotheses to be tested in educational research and their association with neuroscience., This research was funded by the Dirección de Investigación-Universidad de La Frontera (DI20-1003) and Project Covid (73) Process number: 23006.002357/2020-20—Universidade Federal do ABC. In addition, this work was partially funded by FEDER funds through the Operational Competitiveness Program—COMPETE and by National Funds through Fundação para a Ciência e Tecnologia (FCT)—under the projects PTDC/CTM-TEX/28295/2017 and UID/CTM/00264/2019.

Ectomycorrhizal fungi are crucial for forests sustainability. For Castanea sativa, ectomycorrhizal fungus Pisolithus tinctorius is an important mutualist partner. Saprotrophic fungi Hypholoma fasciculare, although used for biocontrol of Armillaria root disease, it negatively affected the interaction between the P. tinctorius and plant host roots, by compromise the formation of P. tinctorius-C. sativa mycorrhizae. In this work, fungal morphology during inhibition of H. fasciculare against P. tinctorius was elucidated. P. tinctorius growth was strongly affected by H. fasciculare, which was significantly reduced after six days of co-culture and become even more significant through time. During this period, P. tinctorius developed vesicles and calcium oxalate crystals, which were described as mechanisms to stress adaption by fungi. H. fasciculare produced different volatile organic compounds in co-cultures over time and differ between single or in dual-species. H. fasciculare highly produced sesquiterpenes (namely, alpha-muurolene) and nitrogen-containing compounds, which are recognised as having antimicrobial activity., This work was funded by FEDER through the Operational Competitiveness Program (COMPETE) and by Portuguese national funds through the Foundation for Science and Technology (FCT) within the scope of project PTDC/AGR-AAM/099556/2008, UID/MULTI/04046/2013, and the CIMO (UIDB/00690/2020) and the Applied Molecular Biosciences Unit-UCIBIO (UID/Multi/04378/2020). Nathalie Moreira acknowledge FCT for her grant, SFRH/BDP/109668/2015.

Surfactants are multipurpose active compounds and are ubiquitously present in detergents. Detergent demand spiked due to the current COVID-19 pandemic, further alerting to the need to replace petrochemical synthetic surfactants with natural and renewable surfactants to mitigate further environmental damage. The neem tree (Azadirachta indica A. Juss) is a fast-growing tree that provides a multitude of commodities, namely neem oil. Neem oil possesses insecticidal and medicinal activity. This work reports the extraction and characterization of a surfactant from neem oil (SNO), displaying a yield of approximately 100%. SNO exhibited suitable detergent characteristics with a high potential to be used as a cleansing agent for textile applications, such as high pH value (10.1), suitable foaming of 1.5 cm and a critical micelle concentration of nearly 0.12 g mL−1. In addition, SNO showed a moderate bactericidal activity against Escherichia coli and bacteriostatic activity against Staphylococcus aureus, both common nosocomial pathogens. Therefore, SNO has a good potential to be used in medical textile applications due to its detergent and bactericidal properties. Finally, an economical overview of the SNO production process was assessed, underscoring its viability., The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Brazilian Coordenacao de Aperfeioamento de Pessoal de Nivel Superior and by the Portuguese Fundacao para Ciencia e a Tecnologia, Fundo Europeu de Desenvolvimento Regional and Programa Operacional Competitividade e Internacionalizacao in the frame of the Portugal 2020 program for the project PLASMAMED PTDC/CTM-TEX/28295/2017.

The increased resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious health concerns. This has stimulated interest in the development of bio-based therapeutics with limited resistance, namely, essential oils (EOs) or antimicrobial peptides (AMPs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control compounds for comparison purposes. The biomolecules were initially screened for their antibacterial efficacy using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000–1250 µg/mL) to induce the same effects as the AMPs (500–7.8 µg/mL). Pexiganan was the most effective biomolecule, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5-7.8 µg/mL), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were observed to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these new biomolecules as replacements for the conventional antibiotics and allowed us to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.

A previously developed fiber-based polyester (PES) stent, with mechanical properties comparable to commercial nitinol stents, was coated with metallic silver (Ag0) and silver oxides (AgxO) thin films through direct current (DC) magnetron sputtering. Ag0 and AgxO coatings provide antimicrobial properties to the stents to minimize the occurrence of coronary stent infections. Nevertheless, the stent interacts with the atmosphere and then with the biological fluids and may lead to the generation of silver species with diminished antimicrobial efficiency and/or prone to induce cytotoxicity. Therefore, stent coating nanostructures aged 3 months were thoroughly analyzed by X-ray photoelectron spectroscopy (XPS) and their antimicrobial and cytotoxicity properties were assessed. Aging led to the presence of silver carbonate and bicarbonate as well as chemisorbed oxygen species in Ag0 and AgxO coatings. Bactericidal efficacy was tested against an important nosocomial bacterium, particularly associated to indwelling devices: Staphylococcus epidermidis. Aged Ag0 and AgxO coating presented a Log reduction of 1 and 2 at their surface, respectively. However, aged stents were able to induce a Log reduction of 2 (Ag0) and 4 (AgxO) on the surrounding medium. Only aged AgxO stent was able to provide a mild reduction of the bacterium at its surface and a clear antimicrobial effect (Log reduction &gt, 3) within its vicinity. More importantly, both aged Ag0 and AgxO stents were shown to be compatible with fibroblasts cells indicating that they can be safely used as indwelling devices, despite the aging effect.

The increased resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious health concerns. This has stimulated interest in the development of bio-based therapeutics with limited resistance, namely, essential oils (EOs) or antimicrobial peptides (AMPs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control compounds for comparison purposes. The biomolecules were initially screened for their antibacterial efficacy using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000‐1250 &micro, g/mL) to induce the same effects as the AMPs (500‐7.8 &micro, g/mL) or EOs (365.2‐19.7 &micro, g/mL). Pexiganan and CLO were the most effective biomolecules, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5‐7.8 &micro, g/mL and 39.3‐19.7 &micro, g/mL, respectively), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were observed to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these new biomolecules as replacements for the conventional antibiotics and allowed us to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.

Bovine lactoferrin (bLF) has been extensively described as a wide spectrum antimicrobial protein. bLF bactericidal activity has been mainly attributed to two different mechanisms: environmental iron depletion and cell membrane destabilization. Due to its antimicrobial properties, bLF has been included in the formulation nutraceutical food products and edible active packages. This work comprises the experimental evidence of the requirement of bLF unrestricted mobility (free bLF) to effectively perform its bactericidal action. To assess the unrestricted and restricted bLF activity, a nontoxic matrix of bacterial nanocellulose (BNC) was used as carrier, and as an anchoring scaffold, respectively. Therefore, BNC was functionalized with bLF through two different methodologies: (i) bLF was embedded within the three-dimensional structure of BNC and; (ii) bLF was covalently bounded to the nanofibrils of BNC. bLF efficiency was tested against two bacteria isolated from clinical specimens, Escherichia coli and Staphylococcus aureus. bLF concentration after covalent binding to BNC was two-fold higher in comparison to the embedding method. Nevertheless, only the embedded bLF exhibited a significant bactericidal activity, due to bLF ability to permeate the BNC matrix and execute its bactericidal action., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/ 04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER- 000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of UID/FIS/04650/2020 and projects PTDC/BTM-MAT/28237/ 2017 and PTDC/EMDEMD/28159/2017, Portuguese Foundation for Science and Technology (FCT) SFRH/BD/64901/2009 and SFRH/BD/111478/2015, Spanish State Research Agency (AEI) and the European Regional Development Fund through the project PID2019-106099RBC43/ AEI/10.13039/501100011033 and from the Basque Government Industry and Education Department under the ELKARTEK, HAZITEK and PIBA (PIBA-2018-06) programs, respectively, info:eu-repo/semantics/publishedVersion

Bacteria are simple organisms with a remarkable capacity for survival by adapting to different environments, which is a result of their long evolutionary history. Taking into consideration these adapting mechanisms, this work now investigates the effect of electrically active microenvironments on bacteria and on how this stimulation may trigger bacteria growth inhibition or proliferation. Electrical microenvironments are generated via stimulation of a piezoelectric polymer with a mechanical cue, thus developing an electrical response and a variation on the surface charge of the polymeric material. Specifically, Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli were grown overnight under static and dynamic conditions on piezoelectric poly(vinylidene) fluoride (PVDF) films to further study bacteria behavior under: (i) the effect of the material surface charge in static conditions, (ii) the mechanical effect, and (iii) the piezoelectric effect, the last two performed under dynamic conditions. Bacteria viability in planktonic and biofilm forms was measured, and the microorganism morphology was characterized. Whereas E. coli responds little to any of the stimuli application, S. epidermidis growth can be regulated through the material surface charge and by the applied frequency. Positively charged PVDF induces bacterial growth inhibition in planktonic and adhered cells in static conditions, whereas antifouling properties are obtained when a mechanical or piezoelectric effect at 4 Hz stimuli is applied. By increasing the stimuli to 40 Hz, however, the adhesion of bacteria is promoted. In conclusion, the behavior of certain bacteria species is tailored through the application of piezoelectric materials, which provide sufficient mechanoelectrical stimuli for growth or inhibition of bacteria, allowing for the design of suitable anti- and promicrobial strategies. Such strategies are only found in studies related to mammalian cells, whereas in bacterial cells this type of stimuli are still unknown. Thus, this work provides one of the first insights on the effect of piezoelectric stimuli on bacterial cells., FCT(Fundação para a Ciência e Tecnologia) and by ERDF through COMPETE2020 Programa Operacional Competitividade e Internacionalização (POCI) in the framework of the Strategic Programs UID/FIS/04650/2019 as well as under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 Programa Operacional Regional do Norte. The work was also supported by FCT through project LungChek ENMed/0049/2016. M.M.F. and C.R. thank FCT for the grant SFRH/BPD/121464/2016 and SFRH/BPD/ 90870/2012, respectively. Finally, the authors acknowledge funding by the Spanish Ministry of Economy and Com- petitiveness (MINECO) through the project MAT2016- 76039-C4-3-R (AEI/FEDER, UE) and from the Basque Government Industry and Education Departments under the ELKARTEK and PIBA (PIBA-2018-06) programs, respectively, info:eu-repo/semantics/publishedVersion

Nitrosomonas europaea is the most extensively studied ammonia-oxidizing bacterium (AOB), being a ubiquitous player in the conversion of ammonia in the soil environment. The precipitation of constituents of the standard culture medium, American Type Culture Collection (ATCC) \#2265, further hinders both the study of N. europaea vital role in the nitrogen cycle and the development of biotechnological applications, as the presence of inorganic debris severely compromises scale-up procedures and downstream processing. The standard formulation was analyzed; the precipitate was identified as being struvite. Struvite is spontaneously formed when magnesium, phosphate, and ammonium are available in a high pH solution. This combination is common and transversal to a series of AOB media formulations. Therefore, a non-precipitating medium was developed. The modified medium does not require filter sterilization, which reduces 99\\% of its production cost. The kinetic performance of N. europaea was evaluated in the standard and modified medium. A precise and direct quantification of the bacterium cell growth profile was achieved using fluorescent in situ hybridization (FISH) and flow cytometry, which was correlated with other metabolic parameters. The performance of N. europaea was not impaired in the modified medium., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Susana Cortez would like to acknowledge the research grant funded by FCT (SFRH/BPD/73720/2010). Gonzalo Tortella would like to acknowledge the FONDECYT project 1161713., info:eu-repo/semantics/publishedVersion

Bacterial cellulose (BC) films from two distinct sources (obtained by static culture with Gluconacetobacter xylinus ATCC 53582 (BC1) and from a commercial source (BC2)) were modified by bovine lactoferrin (bLF) adsorption. The functionalized films (BC+bLF) were assessed as edible antimicrobial packaging, for use in direct contact with highly perishable foods, specifically fresh sausage as a model of meat products. BC+bLF films and sausage casings were characterized regarding their water vapour permeability (WVP), mechanical properties, and bactericidal efficiency against two food pathogens, Escherichia coli and Staphylococcus aureus. Considering their edibility, an in vitro gastrointestinal tract model was used to study the changes occurring in the BC films during passage through the gastrointestinal tract. Moreover, the cytotoxicity of the BC films against 3T3 mouse embryo fibroblasts was evaluated. BC1 and BC2 showed equivalent density, WVP and maximum tensile strength. The percentage of bactericidal efficiency of BC1 and BC2 with adsorbed bLF (BC1+bLF and BC2+bLF, respectively) in the standalone films and in inoculated fresh sausages, was similar against E. coli (mean reduction 69 % in the films per se versus 94 % in the sausages) and S. aureus (mean reduction 97 % in the films per se versus 36 % in the case sausages). Moreover, the BC1+bLF and BC2+bLF films significantly hindered the specific growth rate of both bacteria. Finally, no relevant cytotoxicity against 3T3 fibroblasts was found for the films before and after the simulated digestion. BC films with adsorbed bLF may constitute an approach in the development of bio-based edible antimicrobial packaging systems., The authors would like to acknowledge Portuguese Foundation for Science and Technology (Fundação para a Ciência e Tecnologia) for the research grants: Jorge Padrão SFRH/BD/64901/2009, Sara Gonçalves SFRH/BD/63578/2009, João Pedro Silva SFRH/BPD/ 64958/2009, Ana Cristina Pinheiro SFRH/BPD/101181/2014. V. Sencadas thanks support from the COST Action MP1206: “Electrospun nano-fibres for bio inspired composite materials and innovative industrial applications” and MP1301: “New Generation Biomimetic and Customized Implants for Bone Engineering”. The authors would also like to thank the co-funded by the Programa Operacional Regional do Norte (ON.2 e O Novo Norte), QREN, FEDER Projects “BioHealth e Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124- FEDER-000027; “BioInd e Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07- 0124-FEDER-000028; Strategic Project PEST-C/FIS/UI607/2014; Matepro eOptimizing Materials and Processes”, ref. NORTE-07- 0124-FEDER-000037; Strategic Project PEst-OE/EQB/LA0023/2013 and project ref. RECI/BBB-EBI/0179/2012 (project number FCOMP- 01-0124-FEDER-027462). Finally, the authors thank the Fundação para a Ciência e Tecnologia for the strategic funding from the UID/ BIO/04469/2013 unit.

The increase of antibiotic resistant microorganisms urged the development and synthesis of novel antimicrobial biomaterials to be employed in a broad range of applications, ranging from food packaging to medical devices. This work describes the production and characterization of a protein-based electrospun fibrous membranes bearing antimicrobial properties. Its composition is exclusively comprised of proteins, with fish gelatine as structural matrix and bovine lactoferrin (bLF) as the active antimicrobial agent. The bLF bactericidal effect was determined against clinical isolates of Escherichia coli and Staphylococcus aureus through microdilution assays. Two distinctive methods were used to incorporate bLF into the fish gelatine nanofibres: (i) as a filler in the electrospinning formulation with concentrations of 2, 5 and 10 (wt%), and cross-linked with glutaraldehyde vapour, in order to achieve stability in aqueous solution; and (ii) through adsorption in a solution with 40 mg mL1 bLF. Fourier transform infrared spectroscopy analysis showed that the structure of both proteins remained intact through the electrospinning blending and cross-linking procedure. Remarkable antibacterial properties were obtained with membranes containing 5% and 10% bLF with a bacterial reduction of approximately 90% and 100%, respectively., Portuguese Foundation for Science and Technology (FCT) Strategic Project PEst-OE/EQB/LA0023/ 2013 UID/BIA/04050/2013, UID/BIO/04469/2013, and for the research grants funding through POPH/FSE. FEDER funds through the “Programa Operacional Fatores de Competitividade – COMPETE” and by national funds arranged by FCT FCT, project ref- erences PEST-C/FIS/UI607/2014, Matepro – Optimizing Materials and Processes”, ref. NORTE-07-0124-FEDER-000037”, RECI/BBB- EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the project “BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07-0124-FEDER-000028, co-funded by the “Programa Operacional Regional do Norte” (ON.2 – O Novo Norte), under the “Quadro de Referência Estratégico Nacional” (QREN), through the “Fundo Europeu de Desenvolvimento Regional” (FEDER). COST Action MP1206 “Electrospun Nano-fibres for bio inspired composite materials and innovative industrial applications”.

The feasibility of bacterial cellulose (BC) as a novel substrate for retinal pigment epithelium (RPE) culture was evaluated. Thin (41.6 ± 2.2 m of average thickness) and heatdried BC substrates were surface modified via acetylation and polysaccharide adsorption, using chitosan and carboxymethyl cellulose. All substrates were characterized according to their surface chemistry, wettability, energy, topography and also regarding their permeability, dimensional stability, mechanical properties and endotoxin content. Then, their ability to promote RPE cell adhesion and proliferation in vitro was assessed. All surface-modified BC substrates presented similar permeation coefficients with solutes of up to 300 kDa. Acetylation of BC decreased its swelling and the amount of endotoxins. Surface modification of BC greatly enhanced the adhesion and proliferation of RPE cells. All samples showed similar stress-strain behavior; BC and acetylated BC showed the highest elastic modulus, but the latter exhibited a slightly smaller tensile strength and elongation at break as compared to pristine BC. Although similar proliferation rates were observed among the modified substrates, the acetylated ones showed higher initial cell adhesion. This difference may be mainly due to the moderately hydrophilic surface obtained after acetylation., The authors acknowledge the Portuguese Foundation for Science and Technology (FCT, Portugal) for the financial support provided by the Research Grants SERH/BD/63578/2009, SFRH/BD/64901/2009, SFRH/BPD/64958/2009, and SFRH/BPD/63148/2009 for S.G., J.P., J.P.S., and V.S., respectively. The authors also acknowledge the Projects PEst-OE/EQB/LA0004/2013, PEst-OE/EQB/LA0023/2013, PTDC/BBB-BQB/2450/2012, and RECI/BBB-EBI/0179/2012 (Number: FCOMP-01-0124-FEDER-027462), cofunded by QREN, FEDER.

Which one of us never noticed the emerging trend of natural products? The presented case study aims to study the increasing demand for natural and organic cosmetics and how can Biolage R.A.W. leverage from it. Biolage R.A.W. is a natural professional haircare brand taking its first steps in the Portuguese market. The brand is already facing a lack of awareness which, alongside with the higher prices of its products can leave the brand more exposed. So, how can Biolage R.A.W. come up with a launch strategy to solve these main problems? To better understand the market and brand, an exhaustive qualitative and quantitative analysis was developed that allowed to draw some primary conclusions: Millennials are the healthy beauty products generation and are willing to pay more for natural products. Regarding the launch communication strategy, Biolage R.A.W should follow an integrated communication strategy, using different communication options, creating synergies, to obtain different outcomes in a more cost-efficient way. Taking into consideration millennials’ characteristics, online platforms and e-influencers should take a big part of the brands’ communication. Although recent in the market, Biolage R.A.W. can easily be aware of the growing demand for natural cosmetics and take it as an opportunity to develop and expand the brand. In this case study is clear that the brand has four large strategic core areas to develop, of which two are remarkable for their potential for expansion: distribution channels and innovation. The brand must in the future explore new distribution channels such as natural supermarkets and prioritize a brand extension at the product and category level by launching a line of natural dyes for the hair. Quem de nós nunca notou a tendência emergente de produtos naturais? O estudo de caso apresentado visa estudar a crescente procura por cosméticos naturais e orgânicos e perceber como pode Biolage R.A.W. beneficiar com isso. Biolage R.A.W. é uma marca natural de cuidados capilares profissionais naturais que está atualmente a dar os primeiros passos no mercado português. Enfrenta já alguma falta de awereness que, juntamente com os preços altos praticados quando comparados com os seus concorrentes, a deixam mais suscetível a eventuais. Então, como pode Biolage R.A.W. encontrar uma estratégia de lançamento para resolver esses principais problemas? Para entender melhor o mercado e a marca, uma análise qualitativa e quantitativa exaustiva foi realizada de forma a retirar algumas conclusões principais: os millennials são uma geração de produtos de beleza saudáveis e estão dispostos a pagar mais por produtos naturais. Em relação à estratégia de comunicação de lançamento, Biolage R.A.W deve seguir uma comunicação integrada usando diferentes opções de comunicação, criando sinergias para obter diferentes resultados e de forma eficiente. Tendo em consideração as características dos millennials, as plataformas on-line e os influenciadores digitais devem ter parte fulcral da comunicação da marca. Embora recente no mercado, Biolage R.A.W. pode facilmente estar ciente da crescente procura por cosméticos naturais e encará-la como uma oportunidade para desenvolver e expandir a marca. Neste estudo de caso fica claro que a marca tem quatro grandes áreas estratégicas principais para desenvolver, das quais duas são notáveis pelo seu potencial de expansão: canais de distribuição e inovação. A marca deve, no futuro, explorar novos canais de distribuição, como os supermercados naturais, e priorizar a extensão da marca ao nível do produto e categoria, lançando uma linha de coloração natural para o cabelo

This work studied the physical immobilization of a commercial laccase on bacterial nanocellulose (BNC) aiming to identify the laccase antibacterial properties suitable for wound dressings. Physico-chemical analysis demonstrates that the BNC structure is manly formed by pure crystalline I cellulose. The pH optimum and activation energy of free laccase depends on the substrate employed corresponding to pH 6, 7, 3 and 57, 22, 48 kJ mol1 for 2,6-dimethylphenol (DMP), catechol and 2,2 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), respectively. The Michaelis-Menten constant (Km) value for the immobilized laccase (0.77 mM) was found to be almost double of that of the free enzyme (0.42 mM). However, the specific activities of immobilized and free laccase are similar suggesting that the cage-like structure of BNC allows entrapped laccase to maintain some flexibility and favour substrate accessibility. The results clearly show the antimicrobial effect of laccase in Gram-positive (92%) and Gram-negative (26%) bacteria and cytotoxicity acceptable for wound dressing applications., Andrea Zille (C2011-UMINHO-2C2T-01) acknowledges FCT funding from Programa Compromisso para a Ciência 2008, Portugal, FEDER funding on the Programa Operacional Factores de Competitividade - COMPETE and by national funds through FCT – Foundation for Science and Technology within the scope of the project POCI-01-0145-FEDER-007136 and UID/CTM/00264. Jorge Padrão and João P. Silva acknowledge Portuguese Foundation for Science and Technology (FCT) grants SFRH/BD/64901/2009 and SFRH/BPD/64958/2009 respectively. The authors Jorge Padrão, JoãoP. Silva and Fernando Dourado would like to thank the FCT StrategicProject PEst-OE/EQB/LA0023/2013, also to the Project “BioHealth −Biotechnology and Bioengineering approaches to improve healthquality”, Ref. NORTE-07-0124-FEDER-000027, co-funded by thePrograma Operacional Regional do Norte (ON.2-O Novo Norte),QREN, FEDER, and finnally to RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462).

This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of Retinal Pigment Epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.52.0 swelling/mm3), high mechanical strength (2048 MPa) and non-pyrogenicity (2.12 EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored., The authors would also like to acknowledge José Ramalho for kindly supplying the RPE65 primary antibody. The authors acknowledge the Portuguese Foundation for Science and Technology (FCT, Portugal) for the financial support provided through the doctoral and posdoctoral grants SFRH/BD/63578/2009, SFRH/BD/64901/2009, SFRH/BPD/64958/2009 and SFRH/BPD/63148/2009 for Sara Gonc¸ alves, Jorge Padrão, João Pedro Silva, and Vitor Sencadas, respectively. The authors also acknowledge the projects PEst-OE/EQB/LA0004/2013, PEst-OE/EQB/LA0023/2013, PEST-C/FIS/UI607/2014, PTDC/BBB-BQB/2450/2012 and PTDC/SAU-ORG/118694/2010, co-funded by QREN, FEDER. Finally, the authors thank the FCT strategic project UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124- FEDER-027462) and the project “BioInd—Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07-0124-FEDER-000028 co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER.

Genetic engineering was used to produce an elastin-like polymer (ELP) with precise amino acid composition, sequence and length, resulting in the absolute control of MW and stereochemistry. A synthetic monomer DNA sequence encoding for (VPAVG)20, was used to build a library of concatemer genes with precise control on sequence and size. The higher molecular weight polymer with 220 repeats of VPAVG was biologically produced in Escherichia coli and purified by hot and cold centrifugation cycles, based on the reversible inverse temperature transition property of ELPs. The use of low cost carbon sources like lactose and glycerol for bacteria cells culture media was explored using Central Composite Design approach allowing optimization of fermentation conditions. Due to its self-assembling behaviour near 33 oC stable spherical microparticles with a size ~ 1μm were obtained, redissolving when a strong undercooling is achieved. The polymer produced showed hysteresis behaviour with thermal absorbing/releasing components depending on the salt concentration of the polymer solution., This work was supported by the ElastM Project, POCI/CTM/57177/2004 from Fundação para a Ciência e Tecnologia (Portugal) and FEDER, “Junta de Castilla y Leon” (VA087A06, VA016B08 and VA030A08), MEC (MAT2007-66275-C02-01 and NAN2004-08538), and by the Marie Curie RTN Biopolysurf (MRTN-CN-2004-005516). Raul Machado received a PhD grant: SFRH/BD/36754/2007 from Fundação para a Ciência e Tecnologia (Portugal). The authors would like to thank Magda Graça (CBMA) for all the technical support in sequencing, Lígia Rodrigues for the CCD enlightenment and Dan W. Urry for the stimulating discussions and encouragement to pursue the studies on protein-based polymers.

"Available online 6 May 2016", An effective tissue engineering approach requires adjustment according to the target tissue to be engineered. The possibility of obtaining a protein-based formulation for the development of multivalent tunable scaffolds that can be adapted for several types of cells and tissues is explored in this work. The incremental substitution of bovine serum albumin (BSA) by human serum albumin (HSA), changing the scaffolds hydrophilic/hydrophobic ratio, on a previously optimized scaffold formulation resulted in a set of uniform porous scaffolds with different physical properties and associated cell proliferation profile along time. There was a general trend towards an increase in hydrophilicity, swelling degree and in vitro degradation of the scaffolds with increasing replacement of BSA by HAS. The set of BSA/HSA scaffolds presented distinct values for the storage (elastic) modulus and loss factor which were similar to those described for different native tissues such as bone, cartilage, muscle, skin and neural tissue. The preferential adhesion and proliferation of skin fibroblasts on the BSA25%HSA75% and HSA100% scaffolds, as predicted by their viscoelastic properties, demonstrate that the BSA/HSA scaffold formulation is promising for the development of scaffolds that can be tuned according to the tissue to be repaired and restored., This work was supported by FCT I.P through the strategic funding UID/BIO/04469/2013 and the strategic funding UID/BIA/04050/2013. Artur Ribeiro thanks FCT for the SFRHBPD983882013 grant.

Silk-elastin-like proteins (SELPs) have enormous potential for use as customizable biomaterials in numerous biomedical and materials applications yet success in harnessing this potential has been limited by the lack of a commercially viable industrially relevant production process. We have developed a scalable fed-batch production approach which enables a SELP volumetric productivity of 4.3 g L-1 with E. coli BL21(DE3). This is the highest SELP productivity reported to date and is 50-fold higher than that reported by other groups. As compared to typical fed-batch processes, high pre-induction growth rates and low inducer and oxygen concentrations are allowed whereas reduced post-induction feeding rates are preferred. Limiting factors were identified and productivity was found to be strongly influenced by a trade-off between the rate of production and plasmid stability. The process developed is robust, reproducible and applicable to scale up to the industrial level and moves these biopolymers a step closer to the marketplace., Fundação para a Ciência e a Tecnologia (FCT), This work was financed by the European Commission, via the 7th Framework Programme Project EcoPlast (FP7-NMP-2009- SME-3, collaborative project number 246176), by FEDER through POFC − COMPETE and by national funds from Fundação para a Ciência e Tecnologia (FCT) through PEst- OE/BIA/U14050/2014. RM acknowledges FCT for SFRHBPD/ 86470/2012 grant. TC is thankful to FCT for its support through Programa Ciência 2008.

Elastin isolated from fresh bovine ligaments was dissolved in a mixture of 1,1,1,3,3,3-Hexafluoro-2-propanol and water and electrospun into fiber membranes under different processing conditions. Fiber mats of randomly and aligned fibers were obtained with fixed and rotating ground collectors and fibrils were composed by thin ribbons whose width depends on electrospinning conditions; fibrils with 721 nm up to 2.12 m width were achieved. After cross-linking with glutaraldehyde, -elastin can uptake as much as 1700 % of PBS solution and a slight increase on fiber thickness was observed. The glass transition temperature of electrospun fiber mats was found to occur at ~ 80 ºC. Moreover, -Elastin showed to be a perfect elastomeric material, and no mechanical hysteresis was found in cycle mechanical measurements. The elastic modulus obtained for oriented and random fibers mats in a PBS solution was 330 ± 10 kPa and 732 ± 165 kPa, respectively. Finally, the electrospinning and cross-linking process does not inhibit MC-3T3-E1 cell adhesion. Cell culture results showed good cell adhesion and proliferation in the cross-linked elastin fiber mats., This work is funded by FEDER funds through the "Programa Operacional Factores de Competitividade – COMPETE" and by national funds arranged by FCT- Fundação para a Ciência e a Tecnologia, project references NANO/NMed-SD/0156/2007, PTDC/CTM-NAN/112574/2009 and PEST-C/FIS/UI607/2011. The authors also thank funding from Matepro –Optimizing Materials and Processes”, ref. NORTE-07-0124-FEDER-000037”, co-funded by the “Programa Operacional Regional do Norte” (ON.2 – O Novo Norte), under the “Quadro de Referência Estratégico Nacional” (QREN), through the “Fundo Europeu de Desenvolvimento Regional” (FEDER). The authors also thank support from the COST Action MP1003, 2010 ‘European Scientific Network for Artificial Muscles’. VS, JP, JS and DMC thank the FCT for the SFRH/BD/48708/2008, SFRH / BD / 64901 / 2009, SFRH / BPD / 64958 / 2009 and SFRH/BPD/63148/2009, and SFRH/BD/ 82411/2011 grants, respectively. JLGR acknowledges the support of the Spanish Ministry of Science and Innovation through project No. MAT2010-21611-C03-01 (including the FEDER financial support). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

Development of suitable membranes is a fundamental requisite for tissue and biomedical engineering applications. This work presents fish gelatin random and aligned electrospun membranes cross-linked with glutaraldehyde (GA). It was observed that the fiber average diameter and the morphology is not influenced by the GA exposure time and presents fibers with an average diameter around 250 nm. Moreover, when the gelatin mats are immersed in a phosphate buffered saline solution (PBS), they can retain as much as 12 times its initial weight of solution almost instantaneously, but the material microstructure of the fiber mats changes from the characteristic fibrous to an almost spherical porous structure. Cross-linked gelatin electrospun fiber mats and films showed a water vapor permeability of 1.37 ± 0.02 and 0.13 ± 0.10 (g.mm)/(m2.h.kPa), respectively. Finally, the processing technique and cross-linking process does not inhibit MC-3T3-E1 cell adhesion. Preliminary cell culture results showed good cell adhesion and proliferation in the cross-linked random and aligned gelatin fiber mats., This work is funded by FEDER funds through the "Programa Operacional Factores de Competitividade - COMPETE" and by the national funds arranged by FCT-Fundacao para a Ciencia e a Tecnologia, project references NANO/NMed-SD/0156/2007, PTDC/CTM-NAN/112574/2009, and PEST-C/FIS/UI607/2011. The authors also thank funding from Matepro - Optimizing Materials and Processes," ref. NORTE-07-0124-FEDER-000037," co-funded by the "Programa Operacional Regional do Norte" (ON.2 - O Novo Norte), under the "Quadro de Referencia Estrategico Nacional" (QREN), through the "Fundo Europeu de Desenvolvimento Regional" (FEDER). The authors also thank support from the COST Action MP1003, 2010 "European Scientific Network for Artificial Muscles." V. Sencadas, J. Padrao, and J. Silva thank the FCT for the SFRH/BPD/64958/2009, SFRH/BD/64901/2009, and SFRH/BPD/63148/2009 grants, respectively.

Fiber meshes of poly(hydroxybutyrate) (PHB) and poly(hydroxybutyrate)/poly(ethylene oxide) (PHB/PEO) with different concentrations of chlorhexidine (CHX) were prepared by electrospinning for assessment as a polymer based drug delivery system. The electrospun fibers were characterized at morphological, molecular and mechanical levels. The bactericidal potential of PHB and PHB/PEO electrospun fibers, with and without CHX, was investigated against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) by disk diffusion susceptibility tests. Electrospun fibers containing CHX exhibited bactericidal activity. PHB/PEO-1%CHX displayed higher CHX release levels and equivalent antibacterial activity when compared to PHB/PEO with 5 and 10 wt% CHX. Bactericidal performance of samples with 1 wt% CHX was assessed by Colony Forming Units (CFU), where reductions of 100% and 99.69% against E. coli and S. aureus were achieved, respectively., This work was supported by FEDER through the COMPETE Program and by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Project PEST-C/FIS/UI607/2011 and PEST-C/QUI/UIO686/2011. The authors also thank funding from Matepro - Optimizing Materials and Processes", ref. NORTE-07-0124-FEDER-000037", co-funded by the "Programa Operacional Regional do Norte" (ON.2 - O Novo Norte), under the "Quadro de Referencia Estrategico Nacional" (QREN), through the "Fundo Europeu de Desenvolvimento Regional" (FEDER). D.M.C, JP and VS thanks the FCT for the, SFRH/BD/82411/2011, SFRH/BD/64901/2009 and SFRH/BPD/63148/2009 grants respectively. The authors also thank support from the COST Action MP1003, 2010 'European Scientific Network for Artificial Muscles' and to the COST Action MP1206 'Electrospun Nano-fibres for Bio inspired Composite Materials and Innovative Industrial Applications'. The authors also thank prof. Jose Luis Gomez Ribelles from the Unversidad Politecnica de Valencia, Spain, for interesting discussions on these issues.

The thermal and hydrolytic degradation of electrospun gelatin membranes cross-linked with glutaraldehyde in vapor phase has been studied. In vitro degradation of gelatin membranes was evaluated in phosphate buffer saline solution at 37 ºC. After 15 days under these conditions, a weight loss of 68 % was observed, attributed to solvation and depolymerization of the main polymeric chains. Thermal degradation kinetics of the gelatin raw material and as-spun electrospun membranes showed that the electrospinning processing conditions do not influence polymer degradation. However, for cross-linked samples a decrease in the activation energy was observed, associated with the effect of glutaraldehyde cross-linking reaction in the inter- and intra-molecular hydrogen bonds of the protein. It is also shown that the electrospinning process does not affect the formation of the helical structure of gelatin chains., This work was supported by FEDER through the COMPETE Program and by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Project PEST-C/FIS/UI607/2011 and by projects project references NANO/NMed-SD/0156/2007 and PTDC/CTM-NAN/112574/2009. The authors also thank support from the COST Action MP1003, 2010 ‘European Scientific Network for Artificial Muscles’. DMC, JP and VS would like to acknowledge the FCT for the SFRH/BD/ 82411/2011, SFRH/BD/64901/2009 and SFRH/BD/64901/2009 grants respectively.

Protein-based polymers are present in a wide variety of organisms fulfilling structural and mechanical roles. Advances in protein engineering and recombinant DNA technology allow the design and production of recombinant protein-based polymers (rPBPs) with an absolute control of its composition. Although the application of recombinant proteins as biomaterials is still an emerging technology, the possibilities are limitless and far superior to natural or synthetic materials, as the complexity of the structural design can be fully customized. In this work, we report the electrospinning of two new genetically engineered silk-elastin-like proteins (SELPs) consisting of alternate silk- and elastin-like blocks. Electrospinning was performed with formic acid and aqueous solutions at different concentrations without addition of further agents. The size and morphology of the electrospun structures was characterized by scanning electron microscopy showing its dependence on the concentration and solvent used. Treatment with methanol-saturated air was employed to stabilize the structure and promote water insolubility through a time-dependent conversion of random coils into β-sheets (FTIR). The resultant methanol-treated electrospun mats were characterized for swelling degree (570–720%), water vapour transmission rate (1083 g/m2/day) and mechanical properties (modulus of elasticity ∼126 MPa). Furthermore, the methanol-treated SELP fibre mats showed no cytotoxicity and were able to support adhesion and proliferation of normal human skin fibroblasts. Adhesion was characterized by a filopodia-mediated mechanism. These results demonstrate that SELP fibre mats can provide promising solutions for the development of novel biomaterials suitable for tissue engineering applications., This work was financially supported by the European Commission via the 7th Framework Programme project EcoPlast (FP7-NMP-2009-SME-3, collaborative project number 246176), by Portuguese funding from FEDER through POFC-COMPETE and PEst project C/BIA/UI4050/2011 (Portugal), PEST-C/FIS/UI607/2011 and PEST-C/QUI/UIO686/2011. By MICINN (MAT 2009-14195-C03-03, IT2009-0089, ACI2009-0890, MAT201015310 and MAT2010-15982), the JCyL (VA034A09, VA030A08 and VA049A11-2) and CIBER-BBN. EC (Spain). The authors also thank funding from Matepro-Optimizing Materials and Processes", ref. NORTE-07-0124-FEDER-000037", co-funded by the 'Programa Operacional Regional do Norte' (ON.2-O Novo Norte), under the 'Quadro de Referencia Estrategico Nacional' (QREN), through the 'Fundo Europeu de Desenvolvimento Regional' (FEDER). RM, AC, CMC and VS acknowledge FCT for SFRH-BPD/86470/2012, SFRH/BD/75882/2011, SFRH/BD/68499/2010 and SFRH/BPD/63148/2009 grants, respectively., info:eu-repo/semantics/publishedVersion

A laccase from Ascomycetemyceliophthora thermophila was used to assist the binding of chitosan and catechin onto a previous enzymatically oxidized linen surface. The process consists of the pre-treatment of the linen with laccase followed by the application of chitosan in a first step and catechin plus laccase in a second step. The results presented here support the conclusion that laccase is able to oxidize phenols naturally existing in flax fibres, and that the o-quinones formed promote the attachment of chitosan or/and catechin. The pre-treatment of linen with laccase is therefore the key factor for the success of catechin and chitosan grafting. A multifunctional linen product with both antioxidant and antibacterial properties was obtained with an acceptable level of durability in terms of end user requirements., Carla Silva would like to acknowledge the Portuguese Fundacao para a Ciencia e a Tecnologia (FCT) for funding under the scholarship SFRH/BPD/46515/2008.