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The respiratory protection equipment (RPE) used by health professionals consists of an essential device to prevent infectious diseases, especially those caused by biological agents such as the coronavirus (SARS-CoV-2). The current epidemiological panorama is worrying, and the context of creation and production of the mask has emerged as an alternative to RPE to face the public health crisis worldwide. The aim of this work is to present a low-cost alternative as an FFP2-like filter for a reusable respirator face mask. This study presents the comparison of different cellulose-based filtering materials performed by retention testing, time saturation testing, aerosol penetration testing, nanoparticle (~140 nm) filtration testing, bacterial filtration efficiency (BFE), analysis of material morphology and usability. The reusable respirator face mask used in this study is an open-source innovation, using 3D printing. Cotton disc proved to be the best filter material for the reusable mask, with satisfactory results and a performance similar to that shown by the N95-type mask. The cotton disc ensured effectiveness over 6 h of use, and after that, the reusable respirator face mask (here, Delfi-TRON®) needed to be sanitized and replenished with a new cotton disc. Upon preliminary analyses of filtration efficiency, the selected filter was shown to be a low-cost biodegradable and biocompatible alternative., info:eu-repo/semantics/publishedVersion

Troponins are recognized as one of the most specific and sensitive biomarkers for the diagnosis and prognosis of acute coronary syndromes. The use of troponins as acute cardiac isquemic biomarkers impacts positively on the treatment costs and on the mortality rate due to cardiovascular diseases. Nowadays, efforts have been made to develop new methods to detect and quantify troponins in a non-invasive way, reducing the need for blood samples and venipuncture. Saliva started to be increasingly recognized as an attractive diagnostic fluid. However, the challenges in achieving statistically significant correlations between saliva and serum concentrations, the limitations dealing with this high viscosity fluid and the background interferences, have limited the application of biosensors for troponin quantification in saliva. This review briefly approaches the mechanisms of troponin release and reports the methods found in literature for detection of troponin in saliva. Finally, the authors present a discussion of the challenges and perspectives on this topic., This work was supported by Portuguese FCT, under the reference project UIDB/04436/2020, and by Project NORTE-01-0145-FEDER-028178 funded by NORTE 2020 Portugal Regional Operational Program under PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund and the Fundação para a Ciência e Tecnologia

Healthcare-associated infections (HAIs) have been increasing during recent decades, leading to long hospital stays and high morbidity and mortality rates. The usage of antibiotics therapy against these infections is enhancing the emergence of more multiple-drug resistant strains, in particular in Staphylococcus epidermidis. Hence, this study focused on the resistance pattern of S. epidermidis isolates from clinical settings and its association with phenotypic and molecular traits. Our results showed that HAIs were more prevalent among infants and older adults, and the most frequent type of HAI was central line-associated bloodstream infection. Half of the patients received antibiotic therapy before laboratory diagnosis. Preceding microbiological diagnosis, the number of patients receiving antibiotic therapy increased by 29.1%. Eighty-six per cent of the clinical isolates presented a multidrug resistance (MDR) profile, and a quarter were strong biofilm producers. Furthermore, polysaccharide intercellular adhesin (PIA)-dependent biofilms presented higher biomass production (p=0.0041) and a higher rate of antibiotic non-susceptibility than PIA-independent biofilms, emphasizing the role of icaABDC operon in infection severity. Therefore, this study suggests that a thorough understanding of the phenotypic and molecular traits of the bacterial cause of the HAIs may lead to a more suitable selection of antibiotic therapy, improving guidance and outcome assessment., Fundação para a Ciência e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 (FCOMP-01-0124-FEDER-014309). The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), 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)

Platelet-derived products (PDPs) have gained popularity, mainly due to their high concentrations of bioactive molecules such as growth factors and cytokines, which play important roles in tissue healing and regeneration. PDPs are obtained through minimally invasive procedures and their therapeutic effect has been widely recognized. In veterinary medicine, however, the lack of standard protocols to generate PDPs is a major hurdle for assessing the clinical relevance of PDP-based therapies and for their widespread usage. The aim of this review is to analyze the technical and scientific specificities of PDPs in terms of preparation methodologies, classification categorization, nomenclature, and biological proprieties to advance their future biotechnological potential in veterinary contexts., This work was supported by Fundação para a Ciência e Tecnologia, from Portugal SFRH/BD/118871/2016 PhD grant. The authors acknowledge the financial support from the European Union Framework Program for Research and Innovation HORIZON 2020, under the FOOD4CELLS project (PTDC/CTM-BIO/4706/2014 - POCI-01-0145-FEDER 016716)

The present work reports on a 3D model of the tumor microenvironment that contains hyaluronic acid (HA) and alginate, and demonstrates the utility of this model to study the effect of HA size on the crosstalk between cancer cells and mesenchymal stem cells (MSCs). The system incorporates a core that contains HA of specific size (i.e. 6.4, 741 or 1500 kDa) with encapsulated epithelial MKN45 cancer cells and a shell with MSCs that mimic the presence of stem cells next to the tumor site. It was found that short HA (i.e. 6.4 kDa) promotes the invasion of cancer cells from the core to the shell, whereas longer HA (i.e. 741 and 1500 kDa) recruits the MSCs into the core, i.e. the tumor site, where a reduction of the formation of cancer cell aggregates was observed. In summary, the developed 3D model recapitulates some key tumor features related to the effect of HA size on both cancer cell invasiveness and MSC behavior at the tumor site., We acknowledge the financial support from the European Commission and the Horizon 2020 - WIDESPREAD programme, under the grant agreement number #668983-FORECAST. SA acknowledges the Portuguese Foundation for Science and Technology for her PhD grant (SFRH/BD/112075/2015).

Decellularized matrices are attractive substrates, being able to retain growth factors and proteins present in the native tissue. Several biomaterials can be produced by processing these matrices. However, new substrates capable of being injected that reverse local kidney injuries are currently scarce. Herein, we hypothesized that the decellularized particulate kidney porcine ECM (pKECM) could support renal progenitor cell cultures for posterior implantation. Briefly, kidneys are cut into pieces, decellularized by immersion on detergent solutions, lyophilized and reduced into particles. Then, ECM particles are analyzed for nuclear material remaining by DNA quantification and histological examination, molecular conformation by FITR and structural morphology by SEM. Protein extraction is also optimized for posterior identification and quantification by mass spectrometry. The results obtained confirm the collagenous structure and composition of the ECM, the effective removal of nucleic material and the preservation of ECM proteins with great similarity to human kidneys. Human renal progenitor cells (hRPCs) are seeded in different ratios with pKECM, on 3D suspensions. The conducted assays for cell viability, proliferation and distribution over 7 days of culture suggest that these matrices as biocompatible and bioactive substrates for hRPCs. Also, by analyzing CD133 expression, an optimal ratio for specific phenotypic expression is revealed, demonstrating the potential of these substrates to modulate cellular behavior. The initial hypothesis of developing and characterizing a particulate ECM biomaterial as a consistent substrate for 3D cultures is successfully validated. The findings in this manuscript suggest these particles as valuable tools for regenerative nephrology by minimizing surgeries and locally reversing small injuries which can lead to chronic renal disfunction., This work was supported by the European Regional Development Fund (ERDF) on the project FROnTHERA (NORTE-01-0145-FEDER-000023) and the FCT PhD Grant on the Doctoral Program on Advanced Therapies for Health (PATH) (PD/BD/128102/2016). Mass spectrometry and proteomics were performed at the Proteomics i3S Scientific Platform with the assistance of Hugo Osório. This work was supported by the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145- FEDER-022125).

In recent years, the inputs from magnetically assisted strategies have been contributing to the development of more sensitive screening methods and precise means of diagnosis to overcome existing and emerging treatment challenges. The features of magnetic materials enabling in vivo traceability, specific targeting and space- and time-controlled delivery of nanomedicines have highlighted the resourcefulness of the magnetic toolbox for biomedical applications and theranostic strategies. The breakthroughs in magnetically assisted technologies for contact-free control of cell and tissue fate opens new perspectives to improve healing and instruct regeneration reaching a wide range of diseases and disorders. In this review, the contribution of magnetic nanoparticles (MNPs) will be explored as sophisticated and versatile nanotriggers, evidencing their unique cues to probe and control cell function. As cells detect and engage external magnetic features, these approaches will be overviewed considering molecular engineering and cell programming perspectives as well as cell and tissue targeting modalities. The therapeutic relevance of MNPs will be also emphasized as key components of nanostructured systems to control the release of nanomedicines and in the context of new therapy technologies., ERC CoG grant MagTendon (No. 772817), H2020 Twinning project Achilles (No. 810850), FCT Project MagTT PTDC/CTM-CTM/29930/2017 (POCI-01-0145-FEDER29930), and project Norte-01-0145-FEDER-02219015 supported by Norte Portugal Regional Operational Programme (NORTE 2020). Ana F. Almeida is funded by Fundação para a Ciência e Tecnologia (FCT) – Doctoral Grant SFRD/BD/144816/2019

Musculoskeletal interfaces are naturally hypoxic. An understanding of key interactions occurring between different cell populations and their environment is critical for native tissue recapitulation. Here, an enthesis coculture model (preosteoblasts and tendon cells) was used to understand the influence of hypoxia (5% O2) and osteogenic medium (OM) supplementation in cellsâ phenotype modulation. In single cultures, preosteoblasts were found to undergo osteogenic impairment, while tendon cells underwent a maturation process through extracellular matrix (ECM) rescue. When in coâ culture, hypoxia and osteoinduction promoted a temporal chondro/osteogenic pathway activation, as observed by an early deposition of cartilaginous ECM associated with HIF1A stabilization and RUNX2 activation, and later hypertrophic differentiation resulting from HIF2A translocation and SOX9 activation. Moreover, the presence of OM under hypoxia was shown to influence the extracellular ROS/HIF1A interplay. Overall, this study revealed a link between biochemical factors and cellâ cell crosstalk, providing a molecular framework for hypoxic control and modulation of cellsâ fate toward enthesisâ like phenotypes., The authors thank Hospital da Prelada (Porto, Portugal) for providing lipoaspirate tissue (Plastic Surgery Department) and tendon tissue (Orthopedic Department) samples. The authors acknowledge the financial support from the European Union Framework Programme for Research and Innovation HORIZON2020, under the TEAMING Grant agreement No. 739572-The Discoveries CTR, the ERC Grant CoG MagTendon nr 772817, FCT-Fundacao para a Ciencia e a Tecnologia for the PhD grant of IC (PD/BD/128088/2016).

Two novel semicarbazide [HL1, 1, 1-((4-nitrophenyl)(phenyl)methylene)semicarbazide] and thiosemicarbazide [HL2, 2, 1-((4-nitrophenyl)(phenyl)methylene)thiosemicarbazide] ligands were generated from 4-nitrobenzophenone and the corresponding (thio)semicarbazide precursors. Compounds 1 and 2 were fully characterized and their X-ray crystal structures were established. These organic derivatives were further applied as bioactive building blocks for the self-assembly synthesis of silver(i) coordination compounds. The reactions of silver(i) nitrate with HL1/HL2 in the presence of aqueous NH4OH resulted in new silver(i) coordination compounds formulated as [Ag(HL1)(NO3)]·H2O (3) and [Ag6(?3-L2)6] (4). These complexes were characterized by standard methods (FT-IR, NMR, ESI-MS, and elemental analysis) as well as single-crystal X-ray diffraction. The structure of 3 features a two-coordinate silver(i) center with a distorted linear AgNO geometry, wherein an unusual coordination of HL1 through the nitrogen atom of the amine group is realized. The structure of 4 reveals a discrete Ag6 cluster composed of two joined trisilver(i) rings, wherein the three-coordinate Ag(i) centers adopt an AgNS2 environment. The antimicrobial activity of 14 was screened against Gram-positive (S. epidermidis and S. aureus) and Gram-negative (P. aeruginosa and E. coli) bacteria. Compound 4 presented antimicrobial activity against Gram-positive bacteria, including the ability to impair biofilm formation. This study thus contributes to a still limited research area on the development of new silver coordination compounds with potential antibiofilm activity., Fundação para a Ciência e Tecnologia (FCT) and Portugal 2020 (projects PTDC/QUI-QIN/29697/2017, LISBOA-01-0145-FEDER-029697, UIDB/00100/2020, UIDP/00100/2020, SFRH/BSAB/150368/2019 and REM2013). The publication was also prepared with the support of the RUDN University Program 5-100. TAF acknowledges the FCT for BPD grant SFRH/BPD/119980/2016, info:eu-repo/semantics/publishedVersion

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and secA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and cell properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. Significance L. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology., INRAE (“Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement”, previously called INRA, “Institut National de la Recherche Agronomique”), NCN (“Narodowe Centrum Nauki”) National Science Centre Poland (n°2013/09/B/NZ6/00710), ANR (“Agence National de la Recherche”) PathoFood project (n°ANR-17-CE21-0002), COST (European Cooperation in Science and Technology) Action FA1202 BacFoodNet, RMT (“Réseau Mixte Technologique”) CHLEAN (“Conception Hygiénique des Lignes et Equipements et Amélioration de la Nettoyabilité pour une alimentation saine et sure”) and France-Poland CampusFrance EGIDE PHC (“Programme Hubert Curien”) POLONIUM 2013 (n°28298ZE), info:eu-repo/semantics/publishedVersion

Peripheral nerve injury still remains a major clinical challenge, since the available solutions lead to dysfunctional nerve regeneration. Even though autologous nerve grafts are the gold standard, tissue engineered nerve guidance grafts are valid alternatives. Nerve growth factor (NGF) is the most potent neurotrophic factor. The development of a nerve guidance graft able to locally potentiate the interaction between injured neurons and autologous NGF would be a safer and more effective alternative to grafts that just release NGF. Herein, a biofunctional electrospun fibrous mesh (eFM) was developed through the selective retrieval of NGF from rat blood plasma. The neurite outgrowth induced by the eFM-NGF systems was assessed by culturing rat pheochromocytoma (PC12) cells for 7 days, without medium supplementation. The biological results showed that this NGF delivery system stimulates neuronal differentiation, enhancing the neurite growth more than the control condition., This research was funded by Portuguese Foundation for Science and Technology (FCT), grant numbers PTDC/BTM-SAL/28882/2017 (Cells4_Ids) and PTDC/CTM-BIO/4388/2014 (SPARTAN). The PhD grant of M.R.C. (PD/BD/113797/2015) was funded by the FCT Doctoral Program on Advanced Therapies for Health (PATH) (FSE/POCH/PD/169/2013) and the A.M. was financed by the IF grant IF/00376/2014.

Quaternary derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO) and of quinuclidine surfactants were used to develop oil-in-water nanoemulsions with the purpose of selecting the best long-term stable nanoemulsion for the ocular administration of triamcinolone acetonide (TA). The combination of the best physicochemical properties (i.e., mean droplet size, polydispersity index, zeta potential, osmolality, viscoelastic properties, surface tension) was considered, together with the cell viability assays in ARPE-19 and HMC3 cell lines. Surfactants with cationic properties have been used to tailor the nanoemulsions surface for site-specific delivery of drugs to the ocular structure for the delivery of TA. They are tailored for the eye because they have cationic properties that interact with the anionic surface of the eye., Portuguese Science and Technology Foundation (FCT) from the Ministry of Science and Technology (MCTES), European Social Fund (FSE) of the EU, for the scholarship SFRH/BD/130555/2017 granted to A. R. Fernandes, and for the projects UIDB/04469/2020 (CEB strategic fund) and UIDB/04033/2020 (CITAB), co-funded by European Funds (PRODER/COMPETE) and FEDER, under the Partnership Agreement PT2020, info:eu-repo/semantics/publishedVersion

Human mesenchymal stem cells (hMSCs) secretome has been have been at the forefront of a new wave of possible therapeutic strategies for central nervous system neurodegenerative disorders, as Parkinson's disease (PD). While within its protein fraction, several promising proteins were already identified with therapeutic properties on PD, the potential of hMSCs-secretome vesicular fraction remains to be elucidated. Such highlighting is important, since hMSCs secretome-derived vesicles can act as biological nanoparticles with beneficial effects in different pathological contexts. Therefore, in this work, we have isolated hMSCs secretome vesicular fraction, and assessed their impact on neuronal survival, and differentiation on human neural progenitors' cells (hNPCs), and in a 6-hydroxydopamine (6-OHDA) rat model of PD when compared to hMSCs secretome (as a whole) and its protein derived fraction. From the results, we have found hMSCs vesicular fraction as polydispersity source of vesicles, which when applied in vitro was able to induce hNPCs differentiation at the same levels as the whole secretome, while the protein separated fraction was not able to induce such effect. In the context of PD, although distinct effects were observed, hMSCs secretome and its derived fractions displayed a positive impact on animals' motor and histological performance, thereby indicating that hMSCs secretome and its different fractions may impact different mechanisms and pathways. Overall, we concluded that the use of the secretome collected from hMSCs and its different fractions might be active modulators of different neuroregeneration mechanisms, which could open new therapeutical opportunities for their future use as a treatment for PD., Prémios Santa Casa Neurociências Prize Mantero Belard for Neurodegenerative Diseases Research (MB-28-2019). This work was supported by the European Regional Development Fund (FEDER), through the Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of projects UIDB/50026/2020; UIDP/50026/2020 and POCI-01-0145-FEDER-029751. This article has also been developed under the scope of the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI–POCI-01-0145-FEDER-022122), info:eu-repo/semantics/publishedVersion

Leishmaniasis is a neglected parasitic disease for which the conventional treatment can be considered inefficient and extremely aggressive, generating several and severe side effects. Therefore, the discovery of new drug candidates is important for the improvement in the quality of life of patients. Previously, we reported the promising results of isopentyl caffeate (ICaf) against Leishmania chagasi (agent of visceral leishmaniasis) and Leishmania amazonensis (agent of cutaneous leishmaniasis) promastigotes, displaying IC50 of 1.56 and 1.71 M, respectively. Herein, we aimed to decipher the mechanisms of anti-Leishmania action of ICaf. Light and scanning electron microscopy assays showed relevant morphological changes in promastigotes when treated with ICaf, including rounding of the parasite body, shortening of the flagellum, blebs on the plasma membrane and cellular aggregation. The parasite mitochondrion was targeted by ICaf, resulting in a significant reduction in its metabolic activity and electric membrane potential followed by an increase in the production of reactive oxygen species, which culminated in the loss of plasma membrane integrity and parasite death. Relevantly, ICaf also had a potent anti-amastigote action. The IC50 values calculated for intracellular amastigotes of L. amazonensis were 3.27, 1.60 and 1.52 M, while for L. chagasi the values were 2.48, 1.84 and 1.60 M, respectively, after treating the infected macrophages with ICaf for 24, 48 and 72 h. ICaf was well tolerated by THP-1 macrophages, which gave rise to excellent selectivity indexes considering both Leishmania species. The current results suggest that ICaf may emerge as a chemotherapeutic alternative for the treatment of leishmaniasis., Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES – Financial code 001), Fundação de Apoio à Pesquisa e Inovação (FAPITEC/SE), (PROCESSO: 88887.159533/2017-00 extração, encapsulação e caracterização de bioativos para o interesse biotecnologico), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 301964/2019-0 Chamada 06/2019, and Chamada CNPq nº 01/2019), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for financial support. The authors thank Denise da Rocha de Souza, who is supported by a FAPERJ scholarship, for technical assistance. EBS wishes to acknowledge the Portuguese Science and Technology Foundation (FCT/MCT) and European Funds (PRODER/COMPETE) for the project UIDB/04469/2020 (strategic fund), co-financed by FEDER, under the Partnership Agreement PT2020, info:eu-repo/semantics/publishedVersion

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais, Decellularized inner body membranes have been studied for several different tissue engineering applications. However, its availability is limited, except in the case of placental membranes. Placental membranes are composed by the amnion membrane and the chorion membrane. While the human amnion membrane has been widely studied for tissue engineering and regenerative medicine applications, the human chorion membrane was poorly studied, only being subject of research interest in recent years. Envisioning the potential of decellularized human chorion membrane (dHCM) to be used in tissue engineering applications, a decellularization protocol was developed for the human chorion membrane. The dHCM was characterized regarding its composition and mechanical properties. Additionally, the dHCM was used as a substrate to support cell cultures for the creation of a blood brain barrier (BBB) in vitro model, as a surgical patch for the creation of a vascular graft and also for the treatment of congenital diaphragmatic hernias. dHCM showed cytocompatibility with human and mice endothelial cells (EA.hy296, HUVECs, and b.End3), human fibroblasts (MRC5), and mice astrocytes (C8-D1A). The angiogenic properties of the dHCM were also investigated. Moreover, dHCM demonstrated hemocompatibility and antibacterial activity against S. aureus. After in vivo implantation, dHCM integrated well in the host tissue after 28 days when implanted subcutaneously in mice. Nevertheless, when sutured into a pig diaphragm, dHCM was fully integrated by the host tissue after 3 weeks. Altogether, the works developed in this thesis, allowed for a detailed characterization of dHCM for the first time. It was demonstrated that dHCM has unique characteristics that make it a valuable new biomaterial to be use as substrate to support cell cultures and as a surgical patch., As membranas de órgãos internos descelularizadas têm sido estudadas para diferentes aplicações em engenharia de tecidos. No entanto, a disponibilidade destas membranas é limitada, com exceção das membranas da placenta. A placenta é composta por duas membranas, a membrana amniótica e a membrana do córion. Enquanto a membrana amniótica humana descelularizada foi extensivamente estudada para aplicações em engenharia de tecidos e medicina regenerativa, a membrana do córion só recentemente despertou interesse na comunidade científica para estas aplicações. Para estudar o potencial da membrana do córion humano descelularizada (dHCM) em engenharia de tecidos, foi optimizado um protocolo de descelularização eficiente para esta membrana. A dHCM foi caracterizada em relação à sua composição e propriedades mecânicas. Posteriormente, a dHCM foi utilizada como substrato de suporte ao crescimento de células para a criação de um modelo in vitro da barreira hemato-encefálica, como um enxerto cirúrgico para a criação de enxertos vasculares e também para o tratamento de hérnias diafragmáticas. A dHCM mostrou ser citocompatível com células endoteliais de humano e ratinho (EA.hy296; HUVECs, e b.End3), fibroblastos humanos (MRC5) e astrócitos de ratinho (C8-D1A). As propriedades angiogénicas da dHCM foram investigadas e foi demonstrado que a dHCM é compatível com o sangue humano e tem atividade antimicrobiana contra S. aureus. Após implantação in vivo, a dHCM começou a ser integrada pelo tecido hospedeiro após 28 dias de implantação subcutânea em ratinhos. No entanto, quando suturada ao diafragma de porcos, a dHCM mostrou capacidade de integração no tecido hospedeiro após 3 semanas. Os trabalhos desenvolvidos nesta tese permitiram obter uma caracterização detalhada da dHCM, previamente inexistente na literatura. Adicionalmente, foi demonstrado que a dHCM tem características únicas, tornando-a um novo biomaterial promissor como substrato de suporte ao desenvolvimento de culturas de células e como enxerto cirúrgico., This work was supported by the Portuguese Foundation for Science and Technology (FCT) (grant PD/BD/128103/2016 and FSE/POCH/PD/169/2013), by the European Regional Development Fund (ERDF) (grant NORTE-01-0145-FEDER-0000232) and by the project Cells4_IDs (PTDC/BTMSAL/28882/2017)

Tese de doutoramento em Chemical and Biological Engineering, Candida albicans is the main cause of candidiasis and its pathogenicity is supported by a series of virulence factors, including the switch from yeast to filamentous forms. The transcription factor EFG1 plays a central role in C. albicans regulation morphology. In addition, there is rise of C. albicans multidrug resistance accompanied by the scarcity of new classes of fungal drugs. Antisense oligonucleotides (ASOs) can hold great promise as a therapeutic agent, however yet they are poorly explored for controlling Candida infections. Therefore, the main goal of this work was to develop a therapeutic approach based on antisense therapy (AST) to track C. albicans filamentation. To this end, two major objectives were addressed: I) Exploitation and application of ASOs to control C. albicans filamentation; II) Creation of strategies for ASOs cargo and delivery. The first goal of this research was the exploitation of the second and third generations of ASOs to control EFG1 gene and C. albicans filamentation. To this end, the anti-EFG1 2’OMethylRNA was firstly projected based on second generation of ASOs and was proved to have an impact on the reduction of EFG1 gene expression (by 60 %) and on Efg1p protein translation (by 60 %). Interestingly, the ASO was able to inhibit filamentation and to attenuate the virulence of C. albicans on in vivo Galleria mellonella (increasing its survival on 30 % at 72h with a double-dose administration). Based on the third generation of ASOs, a set of anti-EFG1 LNA (Locked nucleic acids) ASOs were developed, and their performance was tested in vitro and in vivo. All LNA-ASOs were able to reduce EFG1 gene expression (by 40-80 %) and consequently filamentation (by 45-55 %) in vitro. The in vivo results revealed that the LNA-ASO modified with PS-linkages and palmitoyl-2’-amino-LNA as the most favorable for an in vivo application (increasing G. mellonella survival by 40 % only with a single-dose administration). The second goal of this research was the development of strategies for anti-EFG1 ASO cargo and delivery. Two distinct approaches were tested: (i) development of polyplexes based on polyamide 4 and 6, that revealed to be feasible carriers, once the ASO maintains its activity against C. albicans cells; (ii) application of lipid-based formulations, where the DOTAP/DOPC 80/20 ρ=3 revealed to be the most promising when tested in a G. mellonella model (25 % of increase on larvae survival in comparison to ASO-free at 72 h). In summary, this research underlines the therapeutic potential of ASOs for controlling C. albicans virulence determinants, as is the case of EFG1 gene, as well as the importance of developing carriers’ systems for ASOs cargo. Considering the future research into the management of C. albicans infections, this research provides valuable information to the development of a credible and alternative method to control C. albicans infections, based on AST., Candida albicans é uma das principais causas de candidíase e a sua patogenicidade é suportada por um conjunto de fatores de virulência, incluindo a capacidade de filamentar. EFG1 é um fator de transcrição com papel fundamental na filamentação. O aumento da multirresistência de C. albicans é acompanhado pela escassez de novos antifúngicos. Os oligonucleotídeos de antisense (OAS) têm revelado potencial como agentes terapêuticos, contudo ainda pouco explorados no controlo da candidíase. Assim, o principal objetivo deste trabalho foi desenvolver uma abordagem terapêutica baseada na Terapia Antisense (TA) para controlar a filamentação de C. albicans. Para tal, dois grandes objectivos complementares foram definidos: I) Exploração e aplicação de OAS para controlar a filamentação e II) Criação de estratégias para o transporte e entrega dos OASs. Como primeiro objetivo explorou-se a segunda e terceira gerações de OASs para controlar o gene EFG1. O anti-EFG1 2’OMethylRNA foi projetado com base na segunda geração, e provou ter um impacto na redução da expressão do gene EFG1 (em 60 %) e na tradução da proteína Efg1p (em 60 %). O OAS foi capaz de inibir a filamentação e atenuar in vivo a virulência de C. albicans em Galleria mellonella (30 % de aumento de sobrevivência às 72 h após dupla administração). Por outro lado, um conjunto de OAS modificados com LNA (terceira geração) foram desenvolvidos e testados in vitro e in vivo. In vitro, os OAS-LNA inibiram a expressão do gene de EFG1 (em 40-80 %) e a filamentação (em 45-55 %). In vivo, o OAS-LNA com as modificações de PS e palmitoyl-2’amino-LNA revelou ser o mais favorável (40 % de aumento de sobrevivência da G. mellonella com uma administração). Como segundo objetivo exploraram-se poliplexos e lipoplexos como estratégias para o transporte e entrega do anti-EFG1 2’OMe. Como primeira abordagem, desenvolveram-se poliplexos à base de poliamida 4 e 6, que revelaram ser veículos viáveis, uma vez que o OAS manteve a sua atividade contra as células de C. albicans. Em seguida, foram exploradas formulações lipídicas, sendo que o DOTAP/DOPC 80/20 ρ=3 revelou ser a mais promissora quando testada in vivo (25 % de aumento de sobrevivência da G. mellonella em comparação com o OAS livre às 72 h). Em suma, esta investigação destaca o potencial terapêutico de OAS para o controlo de determinantes de virulência de C. albicans, como o caso do gene EFG1, bem como a importância do desenvolvimento de sistemas de veículos para o seu transporte. Considerando futuros trabalhos sobre o controlo de infeções por C. albicans, esta investigação fornece informação valiosa para o desenvolvimento de um método credível e alternativo para o controlo destas infeções, com base na TA., This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the PhD grant SFRH/BD/121417/2016 and 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. This study was also supported by the project funding by the “02/SAICT/2017 – Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT) – POCI-01-0145-FEDER-028893”, designated as Antisen4CandiB - Application of antisense oligomers for controlling Candida species biofilm formation on medical surfaces”.

Lactoferrin (Lf), a bioactive milk protein, exhibits strong anticancer and antifungal activities. The search for Lf targets and mechanisms of action is of utmost importance to enhance its effective applications. A common feature among Lf-treated cancer and fungal cells is the inhibition of a proton pump called V-ATPase. Lf-driven V-ATPase inhibition leads to cytosolic acidification, ultimately causing cell death of cancer and fungal cells. Given that a detailed elucidation of how Lf and V-ATPase interact is still missing, herein we aimed to fill this gap by employing a five-stage computational approach. Molecular dynamics simulations of both proteins were performed to obtain a robust sampling of their conformational landscape, followed by clustering, which allowed retrieving representative structures, to then perform protein-protein docking. Subsequently, molecular dynamics simulations of the docked complexes and free binding energy calculations were carried out to evaluate the dynamic binding process and build a final ranking based on the binding affinities. Detailed atomist analysis of the top ranked complexes clearly indicates that Lf binds to the V1 cytosolic domain of V-ATPase. Particularly, our data suggest that Lf binds to the interfaces between A/B subunits, where the ATP hydrolysis occurs, thus inhibiting this process. The free energy decomposition analysis further identified key binding residues that will certainly aid in the rational design of follow-up experimental studies, hence bridging computational and experimental biochemistry., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding UIDP/04378/2020, UIDB/04378/2020, UIDB/04469/2020 and UIDB/04050/2020; and by the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral program in Applied and Environmental Microbiology (DP_AEM). Juliana F. Rocha is the recipient of the SFRH/BD/136746/2018 fellowship supported by FCT. Henrique S. Fernandes acknowledges FCT for his Ph.D. grant SFRH/BD/115396/2016. Some of the calculations were produced with the support of INCD funded by FCT and FEDER under the project 01/SAICT/2016 number 022153., info:eu-repo/semantics/publishedVersion

Background The global population is ageing. Elderly population has surpassed the number of children under 5 years of age, making older adults the fastest-growing age group. The life expectancy increase is the result of scientific, technological and social advances made during recent decades. However, the increase in life expectancy is not accompanied by a healthy ageing, since older adults undergo several physiological changes that may result in the development of several diseases or chronic conditions. Scope and approach Malnutrition of elderly population is one of the most urgent topics in the food industry, costing millions of euros each year to health and social care systems. The development of functional foods with tailored properties for the elderly population, that aim at improving the bioaccessibility and bioavailability of important nutrients in their diets, have been gaining a lot of interest in recent years. Knowledge on which properties are more relevant and how those products behave in older adults gastrointestinal tract is yet to be thoroughly described. Key findings and conclusions Nutrition is the support of human life. Nowadays, through application of novel food processing technologies, older adults health and wellbeing can be greatly improved. These advances, along with the increasing knowledge regarding ageing physiological changes, should be employed for the development of nutritional approaches that aim at preventing diseases and ensure a healthier ageing of the global population., Foundation for Science and Technology (FCT) for their fellowship (SFRH/BD/147286/2019 and SFRH/BD/09849/2020, respectively). 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 I&D&I AgriFood XXI project (NORTE-01-0145-FEDER-000041) co-funded by the European Regional Development Fund under the scope of Norte 2020 (Programa Operacional Regional do Norte 2014/2020), info:eu-repo/semantics/publishedVersion

Exosomes are cell-derived vesicles that act as carriers for proteins and nucleic acids, with therapeutic potential and high biocompatibility. We propose a new concept of exosome-like liposomes for controlled delivery. The goal of this work was to develop a new type of liposomes with a unique mixture of phospholipids, similar to naturally occurring exosomes but overcoming their limitations of heterogeneity and low productivity, for therapeutic delivery of bioactive compounds. Curcumin was chosen as model compound, as it is a phytochemical molecule known to have antioxidant and anti-inflammatory properties, which can protect the brain against oxidative stress and reduce β-amyloid accumulation, major hallmarks of Alzheimer's disease (AD). These new liposomes can efficiently encapsulate hydrophobic curcumin, yielding particles with a size smaller than 200 nm, and a polydispersity index lower than 0.20, which make them ideal for crossing the blood-brain barrier. These particles have a long shelf life, being stable up to 6 months. The curcumin encapsulation efficiency was higher than 85% (up to approximately 94%). Curcumin-loaded liposomes were not cytotoxic (up to 20 μM curcumin, and 200 μM of exo-liposomes), and significantly reduced oxidative stress induced in SH-SY5Y neuronal cells, indicating their potential for neuroprotection. They also do not show any toxicity and are internalized in zebrafish embryos, concentrating in lipid enriched areas, as the brain and the yolk sac. Such innovative carriers are a new effective approach to deliver drugs into the brain, as these are stable, protect the cargo and are uptaken by neuronal cells. Upon internalization, liposomes release the therapeutic biomolecules, resulting in successful neuroprotection, being a positive alternative strategy for AD therapy., Strategic Programme UID/BIA/04050/2020 funded by National funds through the Fundação para a Ciência e Tecnologia I.P., and the project “FUN2CYT: Harnessing the Potential for Biomedical Applications of Pleiotropic Cytokines LIF and Oncostatin M” (POCI-01-0145-FEDER-030568), supported by Programa Operacional Competitividade e Internacionalização (FEDER) and FCT, IP. Mário Fernandes (SFRH/BD/147819/2019) and Cláudia Botelho (SFRH/BPD/111291/2015) hold scholarships from Fundação para a Ciência e Tecnologia, and Ivo Lopes is a recipient of a scholarship from the Direção Regional da Ciência e Tecnologia, Governo Regional dos Açores (M3.1.a/F/128/2015)., info:eu-repo/semantics/publishedVersion

Accepted Manuscript, Marine polymers such as collagen, chitosan, and fucoidan can be combined to form ionic-linked hydrogel networks towards applications in tissue engineering (TE). The use of greener approaches (as determined by green metrics â E-factor), including the absence of external chemical cross-linking agents, has advantages regarding the potential cytotoxicity. By tailoring the formulation of such an ionic-linked hydrogel, it is possible to fine-tune scaffold biofunctionality. In this study, a comparative study of composite hydrogels was accomplished, seeking to understand the correlation between polymer characteristics and physical behaviour to develop the applicability of this technology in soft-to-hard TE. Parameters such as polymer concentration, molecular weight, polymer-biomaterials bonds, biomaterial structural architecture, pore size, and mechanical rheological properties were directly correlated to the hydrogelâ s formulation. The results highlight that the formulation with greatest potential was the 3-component hydrogel (H12, followed by H10, H11), due to its superior mechanical properties, making it suitable for cartilage TE. This research offers a valuable perspective on hydrogel formulation and a new processing methodology, as well as how tailoring the hydrogel composition influences mechanical behaviour to support selecting the best composition for tissue engineering applications., The authors would like to acknowledge to Portuguese Foundation of Science and Technology (FCT) for a Ph.D. fellowship (D. N. Carvalho) under the scope of the doctoral program Tissue Engineering, Regenerative Medicine and Stem Cells, ref. PD/BD/143044/2018, and for Postdoctoral fellowship (C. Goncalves), ref. SFRH/BPD/94277/2013. This work has been partially funded by ERDF under the scope of the Atlantic Area Program through project EAPA_151/2016 (BLUEHUMAN). The authors also thank Jellagen Ltd (UK) for the provision of purified jellyfish collagen. The authors would also like to acknowledge Dr Julio Maroto from the Fundacion CETMAR and Roi Vilela from PESCANOVA S.A, Spain, for the kind offer of squid pens, and to Lara L. Reys for the initial chitosan extraction process.

Background The present study describes the design and production of an oilinwater (o/w) nanoemulsion composed of oil neem as the inner phase to be used as antifungal and antiparasitic control in aquaculture. A 23 Central Composite Rotatable Design (CCRD) adopting surface response was used as the experimental design, setting the neem oil concentration (4%15%), surfactant concentration (0.5%10%) and sonication power (2090%) as the independent variables, while the droplet size, polydispersity index (PDI) and zeta potential were set as the response (dependent) variables. Results Droplet size and PDI showed the best results when using 5 minutes of process using the sonication method. The coefficients of determination were greater than 0.900 for all response. The best formulation was obtained with 9% of neem oil and 5.25 % of polysorbate 20. Conclusion Antifungal activity of the optimized nanoemulsion against the Saprolegnia parasitica has been demonstrated, also attributed to the presence of the surfactant. The concentrations of 200 mg/L of nanoemulsion resulted in 100% parasitic mortality., Banco do Nordeste (grant FUNDECI/2016.0015), Coordenação Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) and Fundação de Ámparo à Pesquisa do Estado de Sergipe (FAPITEC) (PROCESSO: 88887.159533/2017-2100 extração, encapsulação e caracterização de bioativos para o interesse biotecnologico), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 301964/2019-0 Chamada 06/2019 and Chamada CNPq No. 01/2019) and from the Portuguese Science and Technology Foundation (FCT) project UIDB/04469/2020 (strategic fund), info:eu-repo/semantics/publishedVersion

Journal pre-proofs, Curcumin (CUR) is a phenolic compound present in some herbs, including Curcuma longa Linn. (turmeric rhizome), with a high bioactive capacity and characteristic yellow color. It is mainly used as a spice, although it has been found that CUR has interesting pharmaceutical properties, acting as a natural antioxidant, anti-inflammatory, antimicrobial, and antitumoral agent. Nonetheless, CUR is a hydrophobic compound with low water solubility, poor chemical stability, and fast metabolism, limiting its use as a pharmacological compound. Smart drug delivery systems (DDS) have been used to overcome its low bioavailability and improve its stability. The current work overviews the literature from the past 10 years on the encapsulation of CUR in nanostructured systems, such as micelles, liposomes, niosomes, nanoemulsions, hydrogels, and nanocomplexes, emphasizing its use and ability in cancer therapy. The studies highlighted in this review have shown that these nanoformulations achieved higher solubility, improved tumor cytotoxicity, prolonged CUR release, and reduced side effects, among other interesting advantages., This study was funded by the Coordination for Higher Level Graduate Improvements (CAPES/Brazil, finance code 001), National Council for Scientific and Technological Development (CNPq/Brazil, PIBIC process #123483/2020-4), State of São Paulo Research Foundation (FAPESP/Brazil, processes #2017/10789-1, #2018/10799-0, #2018/06475-4, #2018/07707-6, #2019/08549-8, and #2020/03727-2). This work was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the project AgriFood XXI (NORTE-01-0145-FEDER-000041) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Our Figures were created with BioRender, info:eu-repo/semantics/publishedVersion

Several studies report the potential of bacterial cellulose (BC) in the fashion and leather industries. This work aimed at the development of BC-based composites containing emulsified acrylated epoxidized soybean oil (AESO) that are polymerized with the redox initiator system hydrogen peroxide (H2O2) and L-ascorbic acid and ferrous sulfate as a catalyst. BC was fermented under static culture. The polymerization of the emulsified organic droplets was tested before and after their incorporation into BC by exhaustion. The composites were then finished with an antimicrobial agent (benzalkonium chloride) and dyed. The obtained composites were characterized in terms of wettability, water vapor permeability (WVP), mechanical, thermal and antimicrobial properties. When AESO emulsion was polymerized prior to the exhaustion process, the obtained composites showed higher WVP, tensile strength and thermal stability. Meanwhile, post-exhaustion polymerized AESO conferred the composite higher hydrophobicity and elongation. The composites finished with the antimicrobial agent showed activity against S. aureus. Finally, intense colors were obtained more uniformly when they were incorporated simultaneously with the emulsified AESO with all the dyes tested., FEDER funds through the Operational Competitiveness Program—COMPETE and by National Funds through Fundação para a Ciência e Tecnologia (FCT) under the project POCI-01-0145-FEDER-007136 and UID/CTM/00264/2019 and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit., info:eu-repo/semantics/publishedVersion

Tissue engineered (TE) substitutes of clinically relevant sizes need an adequate vascular system to ensure function and proper tissue integration after implantation. However, the predictable vascularization of TE substitutes is yet to be achieved. Molecular weight variations in hyaluronic acid (HA) have been pointed to trigger angiogenesis. Thus, this study investigates HA oligomer immobilization as a promoter for TE construct vascularization. As a proof-of-concept, the surface of methacrylated gelatin (GelMA) hydrogels were functionalized with high molecular weight (HMW; 1.5 to 1.8 MDa) and low molecular weight (LMW; < 10 kDa) HA, previously modified with aldehyde groups to enable the immobilization through Schiffâ s base formation. The ability of A-HA to bind amine-presenting surfaces was confirmed by Surface Plasmon Resonance (SPR). Human Umbilical Vein Endothelial Cells (HUVECs) seeded over hydrogels functionalized with LMW HA showed higher proliferation and expression of angiogenic markers (KDR and CD31), than those grown in HMW HA conjugated- or plain surfaces, in line with the activation of HA ERK1/2 mediated downstream signaling. Moreover, when cocultured with human dental pulp cells (hDPCs) encapsulated into the GelMA, an increase in endothelial cell migration was observed for the LMW HA functionalized formulations. Overall LMW HA functionalization enhanced endothelial cell response showing potential as an angiogenesis inducer for TE applications., Norte Portugal Regional Operational Program (NORTE-01−0145-FEDER-000021) and InjecTE - Injectable biomaterials for dental tissue engineering - Project ID: 287953, financed by Norwegian Research Council (NFR) under program NANO2021

uncorrected proof version, The generation of invasive fluctuating protrusions is a distinctive feature of tumor dissemination. During the invasion, individual cancer cells modulate the morphodynamics of protrusions to optimize their migration efficiency. However, it remains unclear how protrusion fluctuations govern the invasion of more complex multi-cellular structures, such as tumors, and their correlation with the tumor metastatic potential. Herein, a reductionist approach based on 3D tumor cell micro-spheroids with different invasion capabilities is used as a model to decipher the role of tumor-associated fluctuating protrusions in cancer progression. To quantify fluctuations, a set of key biophysical parameters that precisely correlate with the invasive potential of tumors is defined. It is shown that different pharmacological drugs and cytokines are capable of modulating protrusion activity, significantly altering protrusion fluctuations, and tumor invasiveness. This correlation is used to define a novel quantitative invasion index encoding the key biophysical parameters of fluctuations and the relative levels of cell-cell/matrix interactions, which is capable of assessing the tumor's metastatic capability solely based on its magnitude. Overall, this study provides new insights into how protrusion fluctuations regulate tumor cell invasion, suggesting that they may be employed as a novel early indicator, or biophysical signature, of the metastatic potential of tumors., Portuguese Foundation for Science and Technology (FCT) under the program CEEC Individual 2017 (CEECIND/00352/2017). D.C., V.B., A.C.L., C.M.A., and S.C.K. also thank the support from the FCT under the scope of the projects 2MATCH (PTDC/BTM-ORG/28070/2017) and BREAST-IT (PTDC/ BTM-ORG/28168/2017) funded by the Programa Operacional Regional do Norte supported by European Regional Development Funds (ERDF). The authors also thank the financial support from the European Union Framework Program for Research and Innovation Horizon 2020 on FoReCaST project under (Grant Number: 668983), and the Institution of Engineering and Technology (IET) for the funding provided under the ENG ThE CANCER project (Grant Number: IET Harvey Engineering Research Award 2018). J.M.O. is grateful for the FCT distinctions (IF/00423/2012 and IF/01285/2015)

Implantation of biomaterials and hybrid constructs in tissue engineering approaches presents major limitations such as inflammatory reaction and the lack of vasculature integration. Therefore, new strategies are needed to enhance implant function, immune protection, and revascularization. In this work, we developed fibrous meshes composed of fucoidan (Fu), a sulfated polysaccharide extracted from brown algae, and polycaprolactone (PCL), a synthetic biodegradable polymer, using the airbrush technique. The chemical characterization by FTIR, EDS, and XPS confirmed the presence of the two polymers in the structure of airbrushed nanofibrous meshes (ANFM). Moreover, these nanofibrous exhibited good wettability and mechanical properties envisaging their application as templates for biomaterials and cell culture. The developed ANFM were directly cultured with human pulmonary microvascular endothelial (HPMEC-ST1.6R) cells for up to 7 days. Biological results demonstrated that ANFM comprising Fu promoted cellular attachment, spreading, and proliferation of human endothelial cells. The angiogenic potential of ANFM was further evaluated by onplantation of PCL and PCL/Fu ANFM in chick chorioallantoic membrane (CAM). In ovo and ex ovo results showed that the incorporation of Fu increased the proangiogenic potential of ANFM. Altogether, the results suggest that airbrush biocomposite meshes could be used as a biomaterial substrate to promote vascularization., Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through Structured projects NORTE-01-0145-FEDER-000021 and NORTE-01-0145-FEDER-000023. Financial support from the Portuguese Foundation for Science and Technology (FCT) is also acknowledged through PhD fellowship SFRH/BD/112139/2015 and R&D&I projects PTDC/CTM-CTM/29813/2017 and PTDC/BII-BIO/31570/2017.

The bone is a complex and dynamic structure subjected to constant stress and remodeling. Due to the worldwide incidence of bone disorders, engineered bone tissues have emerged as solution for bone grafting, which require sophisticated scaffolding architectures while keeping high mechanical performance. However, the conjugation of bone-like scaffold architecture with efficient mechanical properties is still a critical challenge for biomedical applications.  In this sense, the present study is focused on the development of silk fibroin (SF) scaffolds crosslinked with horseradish peroxidase and mixed with zinc (Zn) and strontium (Sr)-doped β-tricalcium phosphate (ZnSr.TCP) to mimic bone structures. The ZnSr.TCP-SF hydrogels were tuned in programmable ice-templating parameters, and further freeze-dried, to obtain 3D scaffolds with controlled pore orientation. The results showed interconnected channels in the ZnSr.TCP-SF scaffolds that mimic the porous network of the native subchondral bone. The architecture of the scaffolds was characterized by microCT and showing tunable pore size according to freezing temperatures (-196 ºC: ~80.2 ± 20.5 µm; -80 ºC: ~73.1 ± 20.5 µm; -20 ºC: ~104.7 ± 33.7 µm). The swelling ratio, weight loss, and rheological properties were also assessed, revealing that the scaffolds were able to keep their integrity and morphology after aqueous immersion. Thus, the ZnSr.TCP-SF scaffolds made of aligned porous structure were developed as affordable candidates for future applications in clinical osteoregeneration and in vitro bone tissue modelling., FCT for the financial support of the Hierarchitech project (M435 ERA.NET/0001/2014) and for the distinctions attributed to S.P. (CEECIND/03673/2017) and C. 436 G. (SFRH/BPD/94277/2013). R. F. Canadas is also thankful to FCT for the doctoral scholarship 437 (SFRH/BD/92565/2013), Fundo Europeu de Desenvolvimento Regional (FEDER), and Programa 438 Operacional Competitividade e Internacionalização (POCI) for funding the Young Researcher 439 Contract (B-Liver Project, PTDC/EMD-EMD/29139/2017)

PubMed: 31209238, The effectiveness of most cancer targeted therapies is short-lived. Tumors often develop resistance that might be overcome with drug combinations. However, the number of possible combinations is vast, necessitating data-driven approaches to find optimal patient-specific treatments. Here we report AstraZeneca’s large drug combination dataset, consisting of 11,576 experiments from 910 combinations across 85 molecularly characterized cancer cell lines, and results of a DREAM Challenge to evaluate computational strategies for predicting synergistic drug pairs and biomarkers. 160 teams participated to provide a comprehensive methodological development and benchmarking. Winning methods incorporate prior knowledge of drug-target interactions. Synergy is predicted with an accuracy matching biological replicates for >60% of combinations. However, 20% of drug combinations are poorly predicted by all methods. Genomic rationale for synergy predictions are identified, including ADAM17 inhibitor antagonism when combined with PIK3CB/D inhibition contrasting to synergy when combined with other PI3K-pathway inhibitors in PIK3CA mutant cells. © 2019, The Author(s)., National Institute for Health Research, NIHR Wellcome Trust, WT: 102696, 206194 Magyar Tudományos Akadémia, MTA Bayer 668858 PrECISE AstraZeneca, We thank the Genomics of Drug Sensitivity in Cancer and COSMIC teams at the Wellcome Trust Sanger Institute for help with the preparation of the molecular data, Denes Turei for help with Omnipath, and Katjusa Koler for help with matching drug names across combination screens. We thank AstraZeneca for funding and provision of data to the DREAM Consortium to run the challenge, and funding from the European Union Horizon 2020 research (under grant agreement No 668858 PrECISE to J.S.R.), the Joint Research Center for Computational Biomedicine (which is partially funded by Bayer AG) to J.S.R., National Institute for Health Research (NIHR) Sheffield Biomedical Research Center, Premium Postdoctoral Fellowship Program of the Hungarian Academy of Sciences. M.G lab is supported by Wellcome Trust (102696 and 206194)., Competing interests: K.C.B., Z.G., G.Y.D., E.K.Y.T., S.F., and J.R.D. are AstraZeneca employees. K.C.B., Z.G., E.K.Y.T., S.F., and J.R.D. are AstraZeneca shareholders. Y.G. receives personal compensation from Eli Lilly and Company, is a shareholder of Cleerly, Inc., and Ann Arbor Algorithms, Inc. M.G. receives research funding from AstraZeneca and has performed consultancy for Sanofi. The remaining authors declare no competing interests.

The excellent electronic properties of single-layer graphene provide the fundamental basis for its use in advanced electronics applications. Graphene-based biosensors, however, are one of the exceptions among the applications that take advantage of the unique properties of this material, because high hydrophobicity of graphene hinders the controlled immobilization of biomolecular probes, particularly of antibodies for immunoassays. In contrast to methods that rely on aggressive chemical oxidation of graphene, we overcome this challenge by implementing an effective strategy that modifies single-layer graphene to render it hydrophilic and biocompatible without compromising the electronic structure. Graphene produced in-house by chemical vapor deposition (CVD) was modified with a heterobifunctional linker that attached to the surface via a pyrene group and presented a standard N-hydroxysuccinimide (NHS) ester ligand for covalent immobilization of biomolecules. The suitability of thus modified surface for biofunctionalization was then confirmed via real-time monitoring of the immobilization of an antibody by quartz crystal microbalance (QCM) measurements followed by the chemical characterization by x-ray photoelectron spectroscopy (XPS). Finally, as a proof-of-concept for biofunctionalization of graphene without compromising its electronic structure, a graphene immuno-field-effect transistor (immuno-FET) was fabricated, biofunctionalized, and tested for detection of Matrix Metalloproteinase 9 (MMP-9), a biomarker related to clinical diagnostics of ischemic stroke., CNPq 237630/2012-5, SFRH/BD/128579/2017, NORTE-01-0145-FEDER-000023 (FROnTHERA), POCI-01-0145- FEDER-006941. CNPq, Brazil. P.D.C. acknowledges a PhD grant (SFRH/BD/128579/2017) from the Portuguese Foundation for Science and Technology (FCT). J.B. and D.Y.P. acknowledge European funding from FROnTHERA project under contract NORTE-01-0145-FEDER-000023. This work was supported by the FCT in the framework of the Strategic Funding UID/FIS/04650/2013 and UID/EEA/04436/2013, by FEDER funds through the COMPETE 2020 – Programa Operacional Competitividade e Internacionalização (POCI) with the reference projects POCI-01-0145- FEDER-006941 and POCI-01-0145- FEDER-028114 (GRAPHSENS)

Tendon tissue engineering strategies that recreate the biophysical and biochemical native microenvironment have a greater potential to achieve regeneration. Here, we developed tendon biomimetic scaffolds using mechanically competent yarns of poly-ε-caprolactone, chitosan and cellulose nanocrystals to recreate the inherent tendon hierarchy from the nano to macro scale. These were then coated with tropoelastin (TROPO) through polydopamine linking (PDA), to mimic the native extracellular matrix (ECM) composition and elasticity. Both PDA and TROPO coatings decreased surface stiffness without masking the underlying substrate. We found that human adipose-derived stem cells (hASCs) seeded onto these TROPO biomimetic scaffolds more rapidly acquired their spindle-shape morphology and high aspect ratio characteristic of tenocytes. Immunocytochemistry shows that the PDA and TROPO-coated surfaces boosted differentiation of hASCs towards the tenogenic lineage, with sustained expression of the tendon-related markers scleraxis and tenomodulin up to 21 days of culture. Furthermore, these surfaces enabled the deposition of a tendon-like ECM, supported by the expression of collagens type I and III, tenascin and decorin. Gene expression analysis revealed a downregulation of osteogenic and fibrosis markers in the presence of TROPO when compared with the control groups, suggesting proper ECM deposition. Remarkably, differentiated cells exposed to TROPO acquired an elastogenic profile due to the evident elastin synthesis and deposition, contributing to the formation of a more mimetic matrix in comparison with the PDA-coated and uncoated conditions. In summary, our biomimetic substrates combining biophysical and biological cues modulate stem cell behavior potentiating their long-term tenogenic commitment and the production of an elastin-rich ECM., European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 706996, Teaming grant agreement No 739572 – The Discoveries CTR, European Research Council grant agreement No 726178, and ERA Chair grant agreement No 668983 - FORECAST; Biomedical Engineering Australian Mobility (BEAM) Program – Master Joint Mobility Project between EU Commission Australian Government; Fundação para a Ciência e a Tecnologia (FCT) for Post-Doc grant SFRH/BPD/112459/2015 and project SmarTendon (PTDC/NAN-MAT/30595/2017); Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund for NORTE-01-0145-FEDER-000021

Load-bearing implants success is strongly dependent on several physical and chemical properties that are known to drive cellular response. In this work, multi-material β-TCP-Ti6Al4V cellular structures were designed to combine Ti6Al4V mechanical properties and β-Tricalcium Phosphate bioactivity, in order to promote bone ingrowth as the bioactive material is being absorbed and replaced by newly formed bone. In this sense, the produced structures were characterized regarding roughness, wettability, β-TCP quantity and quality inside the structures after fabrication and the pH measured during cell culture (as consequence of β-TCP dissolution) and those aspects were correlated with cellular viability, distribution, morphology and proliferation. These structures displayed a hydrophilic behavior and results showed that the addition of β-TCP to these cellular structures led to an alkalization of the medium, aspect that significantly influences the cellular response. Higher impregnation ratios were found more adequate for lowering the media pH and toxicity, and thus enhance cell adhesion and proliferation., FCT (Fundação para a Ciência e Tecnologia) through the grants SFRH/BD/140191/2018; SFRH/BPD/112111/2015, SFRH/BD/128657/2017, SFRH/BD/141056/2018; SFRH/BPD/97701/2013, PD/BDE/127836/2016, and the projects PTDC/EMS-TEC/5422/2014_ADAPTPROSTHESIS and NORTE-01-0145-FEDER-000018-HAMaBICo. Additionally, this work was supported by FCT with the reference project UID/EEA/04436/2013, by FEDER funds through the COMPETE 2020 – Programa Operacional Competitividade e Internacionalização (POCI) with the reference project POCI-01-0145-FEDER-006941

Medicinal plants always are part of folk medicine and are nowadays receiving worldwide attention for prophylaxis, management, and treatment of several diseases, as an alternative to chemical drugs. The current work provided a comprehensive overview and analysis of the Astragalus and health relationship in literature. The analysis of their therapeutic potential is thus instrumental to understand their bioactivity. Among these, the flowering medicinal plant Astragalus membranaceus has raised interest due to several beneficial health effects. This perspective review discussed the botanical, geographical, historical, and the therapeutic properties of A. membranaceus, with a special focus on its health improving effects and medicinal applications both in vitro and in vivo., Funding provided by Università degli Studi di Napoli Federico II within the CRUI-CARE Agreement, info:eu-repo/semantics/publishedVersion

Plant extracts rich in phenolic compounds have been demonstrated to accelerate wound healing, but their use by oral route has been poorly studied. The leaves of Vitis labrusca are rich in phenolic acids and flavonoids. The goal of this study was to assess the healing properties of the oral administration of hydroalcoholic extract of V. labrusca leaves (HEVL) in a murine model. HEVL was obtained by Soxhlet and dynamic maceration, and their yield and phenolic acids and flavonoid contents were determined. For the wound healing assay, 8 mm wounds were performed on the back of 48 Wistar rats, assigned into four groups (n = 12): CTR (distilled water), HEVL100, HEVL200, and HEVL300 (HEVL at 100, 200, and 300 mg/kg, respectively). On days 7 and 14, wound closure rates were assessed, and the healing wounds were subjected to histological analysis. Soxhlet-obtained extract was selected for the wound healing assay because it provided a higher yield and phenolic acid and flavonoid contents. HEVL significantly reduced leukocytosis in the peripheral blood (p &lt, 0.05), accelerated wound closure (p &lt, 0.05), and improved collagenization (p &lt, 0.05) on day 7, as well as enhanced the epidermal tissue thickness (p &lt, 0.001) and elastic fiber deposition on day 14 (p &lt, 0.01). Furthermore, HEVL promoted an increase in the histological grading of wound healing on both days 7 and 14 (p &lt, 0.01). The doses of 200 and 300 mg/kg provided better results than 100 mg/Kg. Our data provide histological evidence that the oral administration of HEVL improves wound healing in rodents. Therefore, the extract can be a potential oral medicine for healing purposes.

Tendon and ligament (T/L) engineering strategies towards clinical practice have been challenged by a paucity of understanding in the identification and still poorly described characterization of cellular niches. Prospecting how resident cell populations behave in vitro, and how cryopreservation may influence T/ L-promoting factors, can provide insights into T/ L-cellular profiles for novel regenerative solutions. Therefore, we studied human T/ L-derived cells isolated from patellar tendons and cruciate ligaments as suitable cellular models to anticipate tendon and ligament niches responses for advanced strategies with predictive tenogenic and ligamentogenic value. Our results show that the crude populations isolated from tendon and ligament tissues hold a stem cell subset and share a similar behavior in terms of tenogenic/ligamentogenic commitment. Both T/ L-derived cells successfully undergo cryopreservation/thawing maintaining the tenogenic/ligamentogenic profiles. The major differences between cryopreserved and fresh populations were observed at the gene expression of MKX, SCX, and TNMD as well as at the protein levels of collagen type I and III, in which cells from tendon origin (hTDCs) evidence increased values in comparison to the ones from ligament (hLDCs, pâ, Funded by the ERC CoG MagTendon (No. 772817), Fundação para a Ciência e Tecnologia (FCT) Project MagTT PTDC/CTM-CTM/29930/2017 (POCI-01-0145-FEDER-29930), project Norte-01-0145-FEDER-02219015 supported by Norte Portugal Regional Operational Programme (NORTE 2020) and H2020 Twinning project Achilles No. 810850. Doctoral grant PD/BD/128089/2016 of A. Vinhas was provided by FCT. The authors thank Hospital da Prelada (Porto, Portugal) for providing ligament and tendon samples.

Polyelectrolytic complexation has stood out due to its application in the development of drug delivery systems using biopolymers as raw materials. The formation of complexes between cashew gum and chitosan can be intermediated by cross-links, mediated by the action of the sodium tripolyphosphate crosslinking agent. These polymers have been used in the nanotechnological development of formulations to protect peptide drugs, such as insulin, allowing their oral administration. In this work, we describe the development of polyelectrolytic complexes from cashew gum and chitosan as biopolymers for oral administration of insulin. The obtained complexes showed a mean particle size of 234 nm and polydispersity index of 0.2. The complexes were 234 nm in size, PDI 0.2, zeta potential 4.5 mV and 22% trapping. The obtained complexes demonstrated considerable and promising characteristics for use as oral insulin delivery systems., e Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE), Banco do Nordeste (grant FUNDECI/2016.0015), Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/COMPETE) through the project UIDB/04469/2020 (strategic fund), co-financed by FEDER, under the Partnership Agreement PT2020, info:eu-repo/semantics/publishedVersion

Emerging and recurrent outbreaks caused by zoonotic agents pose a public health risk. They result in morbidity and mortality in humans and significant losses in the livestock and food industries. This highlights the need for rapid surveillance methods. Despite the high reliability of conventional pathogen detection methods, they have high detection limits and are time-consuming and not suitable for on-site analysis. Furthermore, the unpredictable spread of zoonotic infections due to a complex combination of risk factors urges the development of innovative technologies to overcome current limitations in early warning and detection. Biosensing, in particular, is highlighted here, as it offers rapid and cost-effective devices for use at the site of infection while increasing the sensitivity of detection. Portuguese research in biosensors for zoonotic pathogens is the focus of this review. This branch of research produces exciting and innovative devices for the study of the most widespread pathogenic bacteria. The studies presented here relate to the different classes of pathogens whose characteristics and routes of infection are also described. Many advances have been made in recent years, and Portuguese research teams have increased publications in this field. However, biosensing still needs to be extended to other pathogens, including potentially pandemic viruses. In addition, the use of biosensors as part of routine diagnostics in hospitals for humans, in animal infections for veterinary medicine, and food control has not yet been achieved. Therefore, a convergence of Portuguese efforts with global studies on biosensors to control emerging zoonotic diseases is foreseen for the future., Centro de Investigação Desenvolvimento e Inovação da Academia Militar (CINAMIL) from Academia Militar/Instituto Universitário Militar by project SIPA (Sistema Integrado de Proteção Alimentar), info:eu-repo/semantics/publishedVersion

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disorder that mostly affects the synovial joints and can promote both cartilage and bone tissue destruction. Several conservative treatments are available to relieve pain and control the inflammation; however, traditional drugs administration are not fully effective and present severe undesired side effects. Hydrogels are a very attractive platform as a drug delivery system to guarantee these handicaps are reduced, and the therapeutic effect from the drugs is maximized. Furthermore, hydrogels can mimic the physiological microenvironment and have the mechanical behavior needed for use as cartilage in vitro model. The testing of these advanced delivery systems is still bound to animal disease models that have shown low predictability. Alternatively, hydrogel-based human dynamic in vitro systems can be used to model diseases, bypassing some of the animal testing problems. RA dynamic disease models are still in an embryonary stage since advances regarding healthy and inflamed cartilage models are currently giving the first steps regarding complexity increase. Herein, recent studies using hydrogels in the treatment of RA, featuring different hydrogel formulations are discussed. Besides, their use as artificial extracellular matrices in dynamic in vitro articular cartilage is also reviewed., Norte2020 project (NORTE-08-5369-FSE000044) and the Portuguese Foundation for Science and Technology (FCT) program (PD/BD/143081/2018). IFC thanks the TERM RES-Hub, Tissue Engineering and Regenerative Medicine Infrastructure project, funded by FCT. The FCT distinction attributed to JMO under the Investigator FCT program (number IF/01285/2015) is also greatly acknowledged

Background Several diseases associated to colon microbial imbalance (dysbiosis), such as obesity, diabetes, inflammatory bowel disease, cardiovascular disease and cancer, are being reverted by modulation of gut microbiota composition through treatment with prebiotics and probiotics. Multiple in vitro models have been developed over the past three decades, with several experimental configurations, as they provide a quick, easy, and cost-effective approach to study the gut microbiome, as compared to troublesome and time-consuming in vivo studies. Scope and approach This review aims to provide an overview of the most relevant available in vitro models used to mimic the human colon microbiome dynamics, including macro-scale and microfluidic-based models. Main characteristics, functionalities, current applications and advantages or disadvantages of the models are discussed in order to provide useful information for end users (namely food and pharmaceutical researchers), when selecting the most appropriated model for assessing health claims and safety of novel functional food and drugs. Finally, the use of these colon models as a tool to study prebiotic and probiotic response in host-microbiota interaction is reviewed. Key findings and conclusions A wide range of in vitro models representing specific colon parts have been developed. However, none of these models can simultaneously cover all the key conditions found in the human colon (namely anatomical, physical, biochemical, and biological characteristics), as well as the complex microbiome-host interaction. Thus, there is a significant opportunity for further improvement of the models experimental setups towards more realistic operating systems, including mucosal surfaces, intestinal cells and tissues allowing microbiomehost crosstalk representation., SFRH/BD/139884/2018 from the Portuguese Foundation for Science and Technology (FCT). This study was supported by the FCT under the scope of the strategic funding of UIDB/04469/2020 unit and the Project ColOsH PTDC/BTM–SAL/30071/2017 (POCI–01–0145–FEDER–030071), info:eu-repo/semantics/publishedVersion

This work reports the design of a novel plastic antibody for cystatin C (Cys-C), an acute kidney injury biomarker, and its application in point-of-care (PoC) testing. The synthetic antibody was obtained by tailoring a molecularly imprinted polymer (MIP) on a carbon screen-printed electrode (SPE). The MIP was obtained by electropolymerizing pyrrole (Py) with carboxylated Py (Py-COOH) in the presence of Cys-C and multiwall carbon nanotubes (MWCNTs). Cys-C was removed from the molecularly imprinted poly(Py) matrix (MPPy) by urea treatment. As a control, a non-imprinted poly(Py) matrix (NPPy) was obtained by the same procedure, but without Cys-C. The assembly of the MIP material was evaluated in situ by Raman spectroscopy and the binding ability of Cys-C was evaluated by the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) electrochemical techniques. The MIP sensor responses were measured by the DPV anodic peaks obtained in the presence of ferro/ferricyanide. The peak currents decreased linearly from 0.5 to 20.0 ng/mL of Cys-C at each 20 min successive incubation and a limit of detection below 0.5 ng/mL was obtained at pH 6.0. The MPPy/SPE was used to analyze Cys-C in spiked serum samples, showing recoveries, POCTEP/FEDER, grant number 0624_2IQBIONEURO_6_E”, by CNPQ (National Council for Scientific and Technological Development, Brazil), grant number 440605/2016-4 and by FCT (Fundação para a Ciência e Tecnologia, Portugal) with the Grant number SAICT-POL/24325/2016, info:eu-repo/semantics/publishedVersion

Accepted Article, Summary Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in cystic fibrosis (CF) patients. Both organisms often cause chronic and recalcitrant infections, in large part due to their ability to form biofilms, being these mixed-species infections correlated with poor clinical outcomes. In this study, the hypothesis that S. aureus adopts phenotypes allowing its coexistence with P. aeruginosa during biofilm growth was put forward. We noticed that S. aureus undergoes a viable but non-cultivable (VBNC) state in the dominated P. aeruginosa dual-species consortia, whatsoever the strains used to form the biofilms. Moreover, an increased expression of genes associated with S. aureus virulence was detected suggesting that the phenotypic switching to VBNC state might account for S. aureus pathogenicity and, in turn, influence the clinical outcome of the mixed-species infection. Thus, P. aeruginosa seems to induce both phenotypic and transcriptomic changes in S. aureus, helping its survival and co-existence in the dual-species biofilms. Overall, our findings illustrate how interspecies interactions can modulate bacterial virulence in vitro, contributing to a better understanding of the behaviour of P. aeruginosa-S. aureus dual-species biofilms. This article is protected by copyright. All rights reserved., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/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. The authors also acknowledge the support, through the Programa Operacional Competitividade e Internacionalização (COMPETE2020) and by national funds, through the Portuguese Foundation for Science and Technology (FCT), of the POLY-PrevEnTT project (PTDC/BTMSAL/29841/2017-POCI-01-0145-FEDER-029841), and for the Scientific Employment Stimulus 2017 (CEECIND/01507/2017), info:eu-repo/semantics/publishedVersion

Atmospheric air is a microbial habitat of pathogenic bioaerosols that may pose serious risks to humans. A commonly laboratory-based approach for the diagnosis of such infections in the bloodstream is the blood culture analysis. Its clinical relevance is attributed to the fact that these infections are characterized by high rates of morbidity and mortality, requiring the need for efficient methods for rapid diagnosis. For this reason, our study aimed to develop a method of manometric monitoring for the rapid detection of viable microorganisms in blood culture vials. A methodology was developed to detect pressure variation in intra-vials through a manometric instrument that was coupled to vials of blood culture containing culture broth that allowed microbial growth. This device allowed the early detection of microbial activity based on the production or use of intra-flask gases as a result of microbial metabolic activity. The analyzed variables were the pressure as a function of time, microbial species, and culture medium. The highest pressure found in the flasks without microorganisms was 40 mmHg between 2 and 6 h, and the lowest pressure was 42 mmHg between 21 and 24 h. The variation of the internal pressure in blood culture flasks according to different groups of microorganisms as a function of time demonstrated that the fermentative gram-negative bacilli and gram-positive cocci exhibited a significant increase in relation to their respective control groups (p < 0.001). The non-fermenting gram-negative bacilli showed expected results in relation to the pressure variation in which the production of negative pressures was noticed during the period of analysis, with a significant difference with respect to their control groups (p < 0.001). The developed methodology for the early detection of microorganisms responsible for bloodstream infection was demonstrated to be effective., e Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe-Program Centelha (FAPITEC/SE) and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Portuguese Science and Technology Foundation (FCT) through 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

The scientific community has been doing significant efforts towards engineering new 3D bone models in recent years. Osteocytes are mechanosensitive cells that play significant roles in the maintenance of bone homeostasis. Currently, as far as we know, there are no 3D models that faithfully recapitulate a bone microenvironment capable of promoting the differentiation of osteoblasts towards osteocytes. Besides, in the existing models, the use of human cells does not prevail over the animal cell lines. For so, we propose a 3D model that may have important implications for ongoing efforts towards a better understanding of bone physiology and disease. The main aim of the current work was the promotion of an effective differentiation of osteoblasts into osteocytes by mean of using a 3D model composed of primary human osteoblasts (hOBs) cultured on Gellan Gum-Hydroxyapatite (GG-HAp) matrix under a long-term osteogenic culture. The results revealed that GG-HAp matrix stimulated a fast cell migration/entrapment, attachment, spreading, and mineralization. Moreover, the transition process from osteoblasts to osteocytes was confirmed by the expression of the osteogenic-related (ALP, Runx2, COL I, OC, OPN and OSX) and osteocyte-related (hPDPN) marker throughout the culture time. Overall, the developed 3D model holds a great promise for the treatment of various bone diseases, namely on diagnostic applications and for bone regeneration purposes., FROnTHERA (NORTE-01-0145-FEDER-0000232). A.R.B. thanks the funds provided by FCT under the doctoral program in Tissue Engineering, Regenerative Medicine and Stem Cells (PD/BD/143043/2018). F.R.M. acknowledges FCT for her contract under the Transitional Rule DL 57/2016 (CTTI-57/18-I3BS(5))

Nosocomial or hospital-acquired infections (HAIs) have a major impact on mortality worldwide. Enterococcus and Staphylococcus are among the leading causes of HAIs and thus are important pathogens to control mainly due to their increased antibiotic resistance. The gold-standard diagnostic methods for HAIs are time-consuming, which hinders timely and adequate treatment. Therefore, the development of fast and accurate diagnostic tools is an urgent demand. In this study, we combined the sensitivity of magnetoresistive (MR) sensors, the portability of a lab-on-chip platform, and the specificity of phage receptor binding proteins (RBPs) as probes for the rapid and multiplex detection of Enterococcus and Staphylococcus. For this, bacterial cells were firstly labelled with magnetic nanoparticles (MNPs) functionalized with RBPs and then measured on the MR sensors. The results indicate that the RBP-MNPS provided a specific individual and simultaneous capture of more than 70\% of Enterococcus and Staphylococcus cells. Moreover, high signals from the MR sensors were obtained for these samples, providing the detection of both pathogens at low concentrations (10 CFU/ml) in less than 2h. Overall, the lab-on-chip MR platform herein presented holds great potential to be used as a point-of-care for the rapid, sensitive and specific multiplex diagnosis of bacterial infections., Fundação para a Ciência e a Tecnologia, Grant/Award Numbers: PTDC/BTM‐SAL/ 32442/2017 (POCI‐01‐0145‐FEDER‐03442), UID /05367/2020, UIDB/04469/2020; European Regional Development Fund, Grant/Award Number: NORTE‐01‐0145‐ FEDER‐000004, info:eu-repo/semantics/publishedVersion

As the major nonpsychotropic constituent of Cannabis sativa, cannabidiol (CBD) is regarded as one of the most promising therapeutic agents due to its proven effectiveness in clinical trials for many human diseases. Due to the urgent need for more efficient pharmacological treatments for several chronic diseases, in this review, we discuss the potential beneficial effects of CBD for Alzheimers disease, epilepsy, multiple sclerosis, and neurological cancers. Due to its wide range of pharmacological activities (e.g., antioxidant, anti-inflammatory, and neuroprotective properties), CBD is considered a multimodal drug for the treatment of a range of neurodegenerative disorders, and various cancer types, including neoplasms of the neural system. The different mechanisms of action of CBD are here disclosed, together with recent progress in the use of this cannabis-derived constituent as a new therapeutic approach. © 2021 by the authors. Licensee MDPI, Basel, Switzerland., Institute of Human Genetics, Polish Academy of Sciences by the internal grant for the implementation of a single scientific activity and by the National Centre for Research and Development by the grant ONKOKAN, INNOMED/I/11/NCBR/2014, info:eu-repo/semantics/publishedVersion

Pathogenic microbes are a major source of health and environmental problems, mostly due to their easy proliferation on most surfaces. Currently, new classes of antimicrobial agents are under development to prevent microbial adhesion and biofilm formation. However, they are mostly from synthetic origin and present several disadvantages. The use of natural biopolymers such as cellulose, hemicellulose, and lignin, derived from lignocellulosic materials as antimicrobial agents has a promising potential. Lignocellulosic materials are one of the most abundant natural materials from renewable sources, and they present attractive characteristics, such as low density and biodegradability, are low-cost, high availability, and environmentally friendly. This review aims to provide new insights into the current usage and potential of lignocellulosic materials (biopolymer and fibers) as antimicrobial materials, highlighting their future application as a novel drug-free antimicrobial polymer., This research was partially funded through the project TERM RES Hub—Infraestrutura Científica para a Engenharia de Tecidos e Medicina Regenerativa, Refª Norte-01-0145-FEDER02219015, co-financed by the European Regional Development Fund (FEDER) through the North Regional Operational Programme (NORTE2020) and national funds, by the Portuguese Foundation for Science and Technology (FCT).

Published online: 14 November 2019, Engineering tissue-like scaffolds that can mimic the microstructure, architecture, topology, and mechanical properties of native tissues while offering an excellent environment for cellular growth has remained an unmet need. To address these challenges, multicompartment composite fibers are fabricated. These fibers can be assembled through textile processes to tailor tissue-level mechanical and electrical properties independent of cellular level components. Textile technologies also allow control of the distribution of different cell types and the microstructure of fabricated constructs and the direction of cellular growth within the 3D microenvironment. Here, we engineered composite fibers from biocompatible cores and biologically relevant hydrogel sheaths. The fibers are mechanically robust to being assembled using textile processes and could support adhesion, proliferation, and maturation of cell populations important for the engineering of skeletal muscles. We also demonstrated that the changes in the coating of the multicompartment fibers could potentially enhance myogenesis in vitro., S.R.S. was partially supported by the National Institutes of Health (R21EB026824) and the Brigham Research Institute Stepping Strong Innovator Award. C.R. would like to thank the funding from the National Centre for Research and Develop ment (STRATEGMED1/233224/10/NCBR/2014, project START). This work was partially supported by the National Institutes of Health (AR066193, AR066193, EB022403, AR057837, HL137193, GM126831, AR073822). M.A. would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC). H.A. thanks the Scientific and Technological Research Council of Turkey (TUBITAK). Additionally, I.K.Y. acknowledges financial support from the NIH through the Organ Design and Engineering Training program (T32 EB16652); R.C.-A. thanks the Portuguese funds through FCT−Fundação para a Ciência e a Tecnologia in the framework of FCT-POPH-FSE, the Ph.D. grant SFRH/BD/96593/2013.

Self-standing nanocomposite films based on biopolymers and functional nanostructures have been widely used due to their potential applications as active elements in biomedical devices. The coupling between chitosan (CHI) and alginate (ALG) multilayered films and magnetic nanoparticles (MNPs) allowed to fabricate magnetic responsive freestanding membranes with a high structural control along the thickness, using the layer-by-layer (LbL) methodology. The mechanical characterization evidenced a trend for an increase of both Young modulus, and ultimate tensile strength with the inclusion of MNPs, or by cross-linking with genipin. Additionally, the multilayered membranes exhibited shape memory properties triggered by hydration. The in vitro biological performance studies showed that cells were more viable and adherent with higher proliferation rates when MNPs were included in the membranes. Our results suggested the potential of the developed magneto-active freestanding membranes for biomedical applications, such as in tissue engineering and biomedical applications., Portuguese Foundation for Science and Technology (FCT) through the Doctoral grants with the reference numbers SFRH/BD/81372/2011 (JMS).This work was financially supported by the FCT, by the project PTDC/FIS/115048/ 2009.

Dissertação de mestrado em Biotechnology, Antibiotic resistance is a big threat to global health, as the treatment of a growing number of bacterial infections is becoming a serious problem. In addition, cancer is responsible for the death of millions of people every year and resistance to available therapies is of big concern. It is well established that Actinobacteria are prolific producers of bioactive compounds with pharmaceutical applications. Bioprospecting unexplored or underexplored environments, like the deepsea, may be a key for the discovery of new bioactive molecules. This work aimed to study the biodiversity of the cultivable Actinobacteria associated with deep-sea samples from Portugal, and investigate the antimicrobial and cytotoxic potential of the isolated strains. Nine deep-sea samples, that included sponges, corals and sediments, were collected at the Madeira archipelago at depths between 463 m and 865 m, using the submersible Lula1000. A heat pre-treatment and three selective culture media supplemented with different antibiotics were used to promote the selection of Actinobacteria. Sixty-eight actinobacterial strains were isolated from the analyzed samples, being affiliated with the genera Brevibacterium, Tsukamurella, Microbacterium, Micrococcus, Leucobacter, Rhodococcus, Brachybacterium and Streptomyces. Two of these actinobacterial isolates may represent new Microbacterium species as their 16S rRNA gene similarity was below the cut-off value of 98.7%, used to discriminate between species. The crude extracts of the isolated actinobacterial strains were screened for antimicrobial and cytotoxic activities, using the disk diffusion method and MTT assay, respectively. Two actinobacterial strains associated with the genera Brevibacterium and Brachybacterium were active against one or more of the reference strains tested, namely Candida albicans, Bacillus subtilis and Staphylococcus aureus, exhibiting MIC values in the range of 1000 μg mL-1. Cytotoxic assays revealed 23 strains capable of reducing the cellular viability of at least one of the cell lines tested (T47-D, HepG2 and hCMEC/D3). This work contributed to increase the knowledge about the diversity of Actinobacteria associated with deep-sea samples of Portuguese environments and of their bioactive potential. In the future, dereplication of the bioactive extracts will be performed to look for the presence of new bioactive compounds., A resistência aos antibióticos é uma ameaça grave à saúde global, uma vez que o tratamento de um número crescente de infeções bacterianas se tem tornado um problema sério. Adicionalmente, o cancro é responsável pela morte de milhões de pessoas todos os anos e a resistência às terapias existentes é preocupante. As Actinobactérias são produtoras prolíficas de compostos bioativos com aplicações farmacêuticas. A bioprospecção de ambientes não explorados ou sub-explorados, como é o caso do mar profundo, poderá ser a chave para a descoberta de novas moléculas bioativas. Este trabalho teve como foco o estudo da biodiversidade de Actinobactérias cultiváveis associadas a amostras de mar profundo de Portugal, e a investigação do potencial antimicrobiano e citotóxico das estirpes isoladas. Nove amostras de mar profundo, incluindo esponjas, corais e sedimentos, foram recolhidas no Arquipélago da Madeira a profundidades entre os 463 m e 865 m, utilizando o submersível Lula1000. De modo a promover a seleção de Actinobactérias, foi utilizado um pré-tratamento com calor e usados três meios de cultura seletivos suplementados com antibióticos. Foram isoladas 68 estirpes de Actinobactérias a partir das amostras analisadas, afiliadas com os géneros Brevibacterium, Tsukamurella, Microbacterium, Micrococcus, Leucobacter, Rhodococcus, Brachybacterium e Streptomyces. Dois destes isolados actinobacterianos poderão representar novas espécies de Microbacterium, uma vez que a similaridade dos respetivos genes 16S rRNA se encontra abaixo do valor de referência de 98.7%, utilizado para distinguir entre espécies. Os extratos brutos das estirpes de Actinobactérias isoladas foram avaliados quanto às suas atividades antimicrobiana e anticancerígena, utilizando o método de difusão em disco e o ensaio MTT, respetivamente. Duas estirpes de Actinobactérias, associadas aos géneros Brevibacterium e Brachybacterium, tiveram atividade contra uma ou mais estirpes de referência testadas, nomeadamente Candida albicans, Bacillus subtilis e Staphylococcus aureus, exibindo valores de MIC de 1000 μg mL-1. Os ensaios de citotoxicidade revelaram 23 estirpes capazes de reduzir a viabilidade celular de pelo menos uma das linhas celulares testadas (T47-D, HepG2 e hCMEC/D3). Este trabalho contribuiu para aumentar o conhecimento sobre a diversidade de Actinobactérias associadas a amostras de mar profundo em Portugal e o seu potencial bioativo. No futuro, será realizada a desreplicação dos extratos bioativos para procurar novos compostos bioativos.

Here is presented a highly sensitive biomimetic sensor for the detection of an Alzheimers Disease (AD) biomarker, interleukin-6 (IL6), for point-of-care (PoC) analysis. The imprinted polymeric film was prepared by the co-electropolymerization of pyrrole (Py) and carboxylated pyrrole in the presence of IL6, on a carbon-screen printed electrode . The biomarker molecule was then removed by oxalic acid , creating the recognition vacant sites. Similarly, a control was also prepared in absense of the IL6 biomarker . The different steps of the sensor fabrication were characterized by Raman Spectroscopy, Electrochemical Impedance Spectroscopy (EIS), and Cyclic Voltammetry. Biomarker recognition and capture capacity of the sensing material was measured by EIS. The biomimetic sensor showed a limit of detection for IL6 of 0.02?pg/mL in spiked serum samples. Overall, the biosensing device showed good sensitivity, reproducibility, accuracy, and rapid response time, which contibute for the development of early diagnostics Poc tools for neurological diseases., POCTEP/INTERREG is acknowledged through the project 2QBioneuro, Impulso de una red de I + i en química biológica para diagnóstico y tratamiento de enfermedades neurológicas. ERA-Net, FCT/JPND cofunded Oligomer-Focused Screening and Individualized Therapeutics to target Neurodegenerative Disorders, info:eu-repo/semantics/publishedVersion