3D hydrogel mimics of the tumor microenvironment: the interplay among hyaluronic acid, stem cells and cancer cells

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)

FROM BIOLOGICAL COLONIZATION TO THE SOIL AND CRUST FORMATION: THE ROLE OF CYANOBACTERIA AND SMALL ANIMALS IN A KARSTIC LANDSCAPE, NORTH OF MINAS GERAIS STATE, BRAZIL

The susceptibility of limestones to biological colonization can be a significant role in the development of karst relief, since the microbial biofilms formation and exopolymeric compounds produced by different metabolic activities, and can influence the dissolution processes and precipitation of minerals, boosting specific transformations at karst. Study about biological performance in limestone outcrops in Brazil are still scarce, so it was aimed to analyze the role of the cyanobacteria and small animals (rodents and marsupials) in the superficial transformation of limestones and soil formation in the North of Minas Gerais state. Thus, the methods used were the total organic carbon content with fractionation of humic substances, potential acidity, and micromorphological and microchemistry characterization. The results showed a low recovery of total organic carbon, with a predominance of humin, followed by humic and fulvic acids. The potential acidity presented values classified as very low to very high, and the micromorphological and microchemistry images showed some specific features of the limestone. The conclusion is that the cyanobacteria, rodents and marsupials are part of the karst relief evolution, and possibly the effects of their colonization were able to promote the formation of biofilms, which through complex metabolic interactions, stimulated the processes of biokarstification, favoring the surface transformations at limestone and the soil and crust formation, that contribute to the karst relie

Bioorthogonal labeling reveals different expression of glycans in mouse hippocampal neuron cultures during their development

The following are available online. Scheme S1: Synthesis of peracetylated azidomannose (Ac4ManNAz); Scheme S2: Alternative synthesis of GlcNAz using chloroacetic anhydride and NaOH as a base; Scheme S3: Synthesis of peracetylated azidofucose (Ac4FucAz); Figure S1: 1H-NMR spectra of ManNAz (D2O, 300 MHz); Figure S2: 1H-NMR spectra of Ac4ManNAz (CDCl3 , 300 MHz), mixture of anomers; Figure S3: HPLC chromatogram of purified Ac4ManNAz showing the two anomers; Figure S4: 1H-NMR spectra of GlcNAz (D2O, 300MHz); Figure S5: 1H-NMR spectra of Ac4GlcNAz (CDCl3 , 300 MHz), mixture of anomers; Figure S6: HPLC chromatogram of purified Ac4GlcNAz showing the two anomers; Figure S7: 1H-NMR spectra of 6-azido-1,2,3,4-tetra-O-acetyl-6-deoxy-α,β-L-galactopyranose Ac4FucAz (CDCl3 , 300 MHz): mixture of anomers; Table S1: Primer sequences used in qRT-PCR.The expression of different glycans at the cell surface dictates cell interactions with their environment and other cells, being crucial for the cell fate. The development of the central nervous system is associated with tremendous changes in the cell glycome that is tightly regulated. Herein, we have employed biorthogonal Cu-free click chemistry to image temporal distribution of different glycans in live mouse hippocampal neurons during their maturation in vitro. We show development-dependent glycan patterns with increased fucose and decreased mannose expression at the end of the maturation process. We also demonstrate that this approach is biocompatible and does not affect glycan transport although it relies on an administration of modified glycans. The applicability of this strategy to tissue sections unlocks new opportunities to study the glycan dynamics under more complex physiological conditions.This research was funded by the European Union Framework Programme for Research and Innovation Horizon 2020 under grant agreement n.º 668983—FoReCaST, by European Union’s Horizon 2020 Research and Innovation programme, under the Grant Agreement number 739572–The Discoveries CTR, and 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)

Evaluation of the potential of fucoidan-based microparticles for diabetes treatment

Abstract INTRODUCTION: Marine organisms have in their constitution materials with a wide range of properties and characteristics inspiring their application within the biomedical field. One important example is fucoidan (Fu), an underexploited sulfated polysaccharide extracted from the cell wall of the brown seaweeds, with high solubility in water1. Fucoidan is composed of L- fucose and glucuronic acid including sulfate groups and has important bioactive properties such as antioxidative, anticoagulant, anticancer and in the reduction of blood glucose1,2. In this work, the biomedical potential of fucoidan was assessed by processing modified fucoidan (MFu) into microparticles by photocrosslinking using superhydrophobic surfaces and visible light3,4. Biological performance on the developed constructs using human pancreatic beta cells is currently under investigation. METHODS: To design the materials structures, fucoidan was modified by methacrylation reaction3. Briefly, Fu aqueous solution 4% w/v was mixed with methacrylated anhydride (MA) in volume of 12% v/v at 50oC to react for 6h. Further, MFu particles with and without insulin (0.5% w/v) were produced by pipetting a solution of 5% MFu v/v with triethanolamine and eosin-y (photoinitiators) onto superhydrophobic surfaces4 (Fig. 1A) and then photocrosslinking using visible light4. MFu and developed particles were characterized using 1HNMR, turbidimetry and SEM to assess their chemistry and morphology, respectively. Moreover, the insulin release was evaluated in phosphate buffered saline (PBS) solution at pH 7and simulated intestinal fluid (SIF) at pH 5. The ability of the developed materials to support adhesion and proliferation of cells was assessed by suspension culture of human pancreatic cells 1.1B4 (3.5x105 cells/ml) in contact with MFu microparticles during up to 7 days. RESULTS: The chemical modification performed on Fu was confirmed by the presence of vinyl and additional methyl peaks in the 1HNMR of modified fucoidan, not present in Fu spectrum. Methacrylated fucoidan was obtained with a methacrylation degree of 17%. The produced fucoidan particles have round shape and average diameter of 1.53 mm (Fig. 1B). The insulin release in PBS and SIF demonstrate that the particles can release insulin in a sustained manner under the studied period. It seems that the insulin release is slower for SIF (pH5, Fig. 1C), than for PBS. The biological tests regarding the culture of pancreatic beta cells demonstrate that cells show a round-like shape and tend to form pseudo-islets during the culture period studied (Fig. 1D). DISCUSSION & CONCLUSIONS: This work demonstrates the successful production of fucoidan- based-microparticles through the methacrylation of fucoidan, using visible light and superhydrophobic surfaces. The covalent crosslinking methacrylated fucoidan through visible light represents a promising method to obtain biocompatible fucoidan particles with a uniform round shape. The obtained insulin release profiles are sensitive to different pH (pH7 and pH5), mimicking the normal physiological pathway for insulin release. Furthermore, the results suggest these systems could be used for treatment of type I diabetes mellitus as they sustain beta cells viability and proliferation. The response also suggested, that the MFu particles could be a good candidate as drug delivery vehicles for the diabetes mellitus treatment. REFERENCES: 1 Silva TH et al (2012), Biomatter 2(4): 278:289. 2Sezer Alidemir et al (2011), Fucoidan: A versatile biopolymer for biomedical applicatons (Springer Ber.Heid).pp377-406. 3Mihaila S.et al (2013), Adv. Health. Mat. 2(6): 895-907. 4Rial Hermida et al, Acta Biomater.(2014) 10(10) 4314-4322. ACKNOWLEDGEMENTS: This work was partially funded by projects 0687_NOVOMAR_1_P (POCTEP), CarbPol_u_Algae (EXPL/MAR- BIO/0165/2013), ComplexiTE (ERC-2012-ADG 20120216-321266). Portuguese Foundation for Science and Technology is also gratefully acknowledged for doctoral grants of L. Reys and N. Oliveira and post- doctoral grants of S.S. Silva and D. Soares da Costafunded by projects 0687_NOVOMAR_1_P (POCTEP), CarbPol_u_Algae (EXPL/MARBIO/0165/2013) , ComplexiTE(ERC-2012-ADG 20120216-321266). Portuguese Foundation for Science and Technologyinfo:eu-repo/semantics/publishedVersio

Toxicological effects of the rare earth element neodymium in Mytilus galloprovincialis

The wide range of applications of rare earth elements (REE) is leading to their occurrence in worldwide aquatic environments. Among the most popular REE is Neodymium (Nd), being widely used in permanent magnets, lasers, and glass additives. Neodymium–iron–boron (NdFeB) magnets is the main application of Nd since they are used in electric motors, hard disk drives, speakers and generators for wind turbines. Recent studies have already evaluated the toxic potential of different REE, but no information is available on the effects of Nd towards marine bivalves. Thus, the present study evaluated the biochemical alterations caused by Nd in the mussel Mytilus galloprovincialis exposed to this element for 28 days. The results obtained clearly demonstrated that Nd was accumulated by mussels, leading to mussel’s metabolic capacity increase and GLY expenditure, in an attempt to fuel up defense mechanisms. Antioxidant and biotransformation defenses were insufficient in the elimination of ROS excess, resulting from the presence of Nd and increased electron transport system activity, which caused cellular damages (measured by lipid peroxidation) and loss of redox balance (assessed by the ratio between reduced and oxidized glutathione). The results obtained clearly highlight the potential toxicity of REEs and, in particular of Nd, with impacts at cellular level, which may have consequences in mussel’s survival, growth and reproduction, affecting mussel’s population.publishe

Impact of adding milk whey, probiotic and prebiotic in passion fruit drinks / Impacto da adição de soro, probiótico e prebiótico em bebidas de maracujá

The objective of this study was to evaluate the impact of adding milk whey, probiotic and prebiotic to passion fruit drinks in order to develop potentially symbiotic foods. Four formulations containing Lacticaseibacillus rhamnosus GG (LGG) and fructooligosaccharide (FOS) were prepared and evaluated. An increase in acidity and reduction in pH were found during storage at 6.5 ºC. The soluble solids, lactose and protein contents of the drinks increased in proportion with the whey concentration used in the formulations. All formulations presented LGG viability above 107 CFU/mL throughout storage (28 days) and can therefore be classified as potentially probiotic products. The addition of LGG and FOS to the drinks did not compromise acceptance of the products, and informing the consumers of the presence of probiotic, prebiotic and milk whey had a positive impact on the evaluation when compared with the tropical passion fruit juice without such additions. These results highlight the potential use of milk whey as an ingredient in the production of a new product, meeting the consumer demand for functional products, as well as minimizing the problem of whey disposal, principally by small scale dairy plants

Biomedical potential of fucoidan, a seaweed sulfated polysaccharide: from a anticancer agent to a building block of cell encapsulating systems for regenerative medicine

Marine macroalgae or seaweeds synthesize a wide variety of polymers and smaller compounds with several bioactivities, among which the sulfated polysaccharides acquire greater relevance not only due to the reported antioxidant, antiviral and anticancer[1] activities, but also to the resemblance of extracellular matrix glycosaminoglycans found in the human body[2]. In this study, the potential of fucoidan (Fu) isolated from brown seaweed Fucus vesiculosus for therapeutical use has been evaluated, focusing in its performance as antitumoral agent (bioactive role) or as building block of cell encapsulating systems (structural role). Materials and Methods: The anticancer activity of Fu extracts was assessed by evaluating the cytotoxic behavior over two human breast cancer cell lines (MCF-7 and MDA-MB-231) in in-vitro culture, using human fibroblasts and endothelial cells (HPMEC-ST1 and MRC-5, respectively) as reference. Regarding the structural role, Fu was modified by methacrylation reaction (MFu) using methacrylic acid and further crosslinked using visible radiation and triethanolamine and eosin-y as photoinitiators. The photocrosslinking was performed on MFu solution droplets placed in a silica-based superhydrophobic surface[3], allowing the formation of particles[4] (since natural Fu is highly soluble in water and ion gelation is not effective). Biological performance of the developed particles was assessed by in vitro culture of fibroblasts and pancreatic cells (L929 and 1.1B4, respectively) in contact with MFu particles, up to 7 days. The ability of the developed materials to support adhesion and proliferation of cells was evaluated for both types of cells. Results and Discussion: The tested anticancer activity is not ubiquitous on Fu extracts, being dependent on its chemical features, with molecular weight (Mw) representing a particular role. Specifically, Mw values around 60 kDa exhibited cytotoxic effects to human breast cancer cell lines, while not affecting normal fibroblasts or endothelial cells (which represent the cells of the healthy tissue that would be closer to the tumor in a real situation). A concentration range of 0.2 to 0.3 mg mL-1 from the selected Fu extract could be considered as the therapeutic window for further studies. Regarding fucoidanâ s role on innovative biomaterials, the developed MFu particles could support the proliferation of fibroblasts (L929), but also of human pancreatic beta cells (1.1B4), which tend to form pseudo-islets after 7 days in culture (Fig. 1). This pancreatic cells could be also successfully encapsulated, opening a new route for a diabetes mellitus type 1 therapeutic approach. Fig. 1: Confocal microscopy images of 1.1B4 cells cultured in the presence of fucoidan-based particles and organized in pseudo-islets (red â actin; blue â nuclei). Conclusion: The present work establishes fucoidan as a high performance building block for the development of advanced therapies for cancer (targeted therapy) or tissue and organ regeneration. It shed light on the relation between chemical structure and biological activity towards anti-cancer effect and proposes novel beta cell laden particles as injectable insulin producing systems to tackle diabetes.Funding from projects 0687_NOVOMAR_1_P (co-funded by INTERREG 2007-2013 / POCTEP), CarbPol_u_Algae (EXPL/MAR-BIO/0165/2013, funded by the Portuguese Foundation for Science and Technology, FCT), POLARIS (FP7-REGPOT-CT2012-316331) and ComplexiTE (ERC-2012-ADG 20120216-321266), funded by the European Union’s Seventh Framework Programme for Research and Development is acknowledged. ASF, SSS, NMO and DSC are also thankful to FCT for their individual fellowships

Alfabetização baseada na ciência: manual do curso ABC

O presente manual faz parte do projeto ABC – Alfabetização Baseada na Ciência, fruto de um Acordo de Cooperação Internacional celebrado entre a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), a Faculdade de Psicologia e de Ciências da Educação da Universidade do Porto (FPCEUP), o Instituto Politécnico do Porto (IPP) e a Universidade Aberta de Portugal (UAb). Essa importante parceria tem o objetivo de contribuir para a formação continuada dos profissionais da educação brasileiros que atuam na área de alfabetização, somando-se aos vários esforços que têm sido envidados pelo Ministério da Educação (MEC) para elevar a qualidade dos processos de alfabetização no Brasil e, consequentemente, os seus resultados. A formação de professores tem sido um dos pilares da Política Nacional de Alfabetização (PNA), instituída pelo MEC por meio do Decreto 9.765/19, a qual destaca entre seus princípios a fundamentação de programas e ações em evidências provenientes das ciências cognitivas, bem como a adoção de referenciais de políticas públicas exitosas, nacionais e estrangeiras, baseadas em evidências científicas.info:eu-repo/semantics/publishedVersio

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio