Advanced treatment for arthritic diseases based on the capture and inactivation of interleukin-6 by biofunctionalized polymeric nanoparticles

Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, are associated with synovium inflammation (synovitis). Several pro-inflammatory cytokines, especially tumor necrosis factor-α (TNFα) and interleukins (IL), are important mediators of inflammation and articular cartilage destruction, supporting a potential possibility of anticytokine therapy in these diseases. IL-6 is one of the key regulators of the inflammatory response. Thus, human monoclonal antibodies against IL-6 may prevent its action, and consequently reduce inflammation after intra-articular (IA) injection. Indeed, several clinical trials have already demonstrated positive outcomes over disease progression. Although these treatments are very attractive, they are associated with limited efficacy because of the rapid clearance of antibodies by the synovium. A solution to overcome this problem is using nanoparticles (NPs) as a substrate to protect and extend the action of the antibodies. Natural-derived polymers, like chitosan (Ch) and hyaluronic acid (HA), are biocompatible and biodegradable polysaccharides, being HA a natural component of the extracellular matrix of articular cartilage. Therefore, biodegradable polymeric NPs represent a good candidate for IA administration. In the present work we propose natural biodegradable polymeric NPs biofunctionalized with immobilized antibodies that selectively capture and inactivate the pro-inflammatory cytokine IL-6, reducing synovium inflammation. Ch-HA NPs were successfully prepared by polyelectrolyte complexation and further stabilized through carbodiimide chemistry (ethyl(dimethylaminopropyl) carbodiimide (EDC)/Nhydroxysuccinimide (NHS)). The particle size and zeta potential of the NPs were optimized. Stable NPs with 121.8 ± 2.4 of particle diameter, 0.11 ± 0.01 of polydispersity index and +25.12 ± 1.86 mV of zeta potential were produced with 0.25 mg/mL of initial polymers concentrations, at pH 5 and with 50/200 mM of EDC/NHS concentration. The anti-IL-6 antibody was immobilized at the surface of Ch-HA NPs. After determining the maximum antibody immobilization ability (7 µg/mL), the capacity to capture the recombinant IL-6 was evaluated. The efficacy was around 94-97%. Biological assays demonstrated not only the cytocompatibility of the produced NPs with human articular chondrocytes (hACs) (Fig 1) and human macrophages, but also the benefits of the capture and inactivation of IL-6 after stimulation with monocyte-derived macrophage conditioned medium. In conclusion, it is foreseeable that these NPs will overcome the limitations of the abovementioned treatments, since such NPs will increase the therapeutic efficacy due to their subcellular size, non-toxicity and high stability, being a promising approach for the local and sustained treatment of arthritic diseases.info:eu-repo/semantics/publishedVersio

A Comparative Study

This research was funded by EU funds through the FEDER European Regional Development Fund (project LISBOA-02-0145-FEDER-031311) project LA/P/0056/2020 of Institute of Molecular Sciences, and LA/P/0140/2020 of i4HB, project UID/EEA/00066/2020 from the Center of Technology and Systems, and from the Instituto Politécnico de Lisboa with IPL/2018/STREAM_ISEL and IPL/2020/AGE-SPReS_ISEL projects. APCR and AMF thank the Instituto Superior Técnico for the scientific employment contracts IST-ID/119/2018 and IST-ID/131/2018, respectively. Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Aiming to develop a nanoparticle-based optical biosensor using gold nanoparticles (AuNPs) synthesized using green methods and supported by carbon-based nanomaterials, we studied the role of carbon derivatives in promoting AuNPs localized surface plasmon resonance (LSPR), as well as their morphology, dispersion, and stability. Carbon derivatives are expected to work as immobi-lization platforms for AuNPs, improving their analytical performance. Gold nanoparticles (AuNPs) were prepared using an eco-friendly approach in a single step by reduction of HAuCl4·3H2O using phytochemicals (from tea) which act as both reducing and capping agents. UV–Vis spectroscopy, transmission electron microscopy (TEM), zeta potential (ζ-potential), and X-ray photoelectron spectroscopy (XPS) were used to characterize the AuNPs and nanocomposites. The addition of reduced graphene oxide (rGO) resulted in greater dispersion of AuNPs on the rGO surface compared with carbon-based nanomaterials used as a support. Differences in morphology due to the nature of the carbon support were observed and are discussed here. AuNPs/rGO seem to be the most promising candidates for the development of LSPR biosensors among the three composites we studied (AuNPs/G, AuNPs/GO, and AuNPs/rGO). Simulations based on the Mie scattering theory have been used to outline the effect of the phytochemicals on LSPR, showing that when the presence of the residuals is limited to the formation of a thin capping layer, the quality of the plasmonic resonance is not affected. A further discussion of the application framework is presented.publishersversionpublishe

Isolation and selection of campylobacter bacteriophages from poultry carcasses

Fundação para a Ciência e a Tecnologia (FCT

Escherichia coli expressing a fluorescence protein as a model system for the optimization of environmental and processing conditions

The ability to cultivate microbial strains, expressing heterologous recombinant proteins, is an increasingly important technique in biotechnology. Among the many systems available Escherichia coli remains one of the most attractive due to its ability to grow rapidly and at high densities on inexpensive substrates, its well-characterized genetics and the availability of an increasingly large number of cloning vectors and mutant host strains. However, the recombinant proteins produced with E. coli are usually very difficult to measure and so the sampling points along a fermentation run are not sufficient to elucidate some bottlenecks that could exist and are not suitable for process control, modeling and optimization purposes. In this work, a factorial experimental design was used to optimise three variable culture conditions of a model system based on the expression of EYFP protein (Enhanced Yellow Fluorescent Protein): operational temperature, dissolved oxygen and of induction time. During the E. coli fermentation, samples were taken every hour for evaluation of standard state variables. Furthermore, the protein concentration was analysed by spectroflorimetry with no sample processing. A western blotting technique was used to validate the fluorimetric measurements and to determine the subcellular localization of EYFP protein. Other tests were also performed for the validation of this methodology, as linearity analysis and effect of the presence of biomass

2,3-Diphosphoglycerate and the Protective Effect of Pyruvate Kinase Deficiency against Malaria Infection—Exploring the Role of the Red Blood Cell Membrane

Malaria remains a major world public health problem, contributing to poverty and inequality. It is urgent to find new efficacious tools with few adverse effects. Malaria has selected red blood cell (RBC) alterations linked to resistance against infection, and understanding the protective mechanisms involved may be useful for developing host-directed tools to control Plasmodium infection. Pyruvate kinase deficiency has been associated with resistance to malaria. Pyruvate kinase-deficient RBCs display an increased concentration of 2,3-diphosphoglycerate (2,3-DPG).We recently showed that 2,3-DPG impacts in vitro intraerythrocytic parasite growth, induces a shift of the metabolic profile of infected cells (iRBCs), making it closer to that of noninfected ones (niRBCs), and decreases the number of parasite progenies that invade new RBCs. As an increase of 2,3-DPG content may also have an adverse effect on RBC membrane and, consequently, on the parasite invasion, in this study, we explored modifications of the RBC morphology, biomechanical properties, and RBC membrane on Plasmodium falciparum in vitro cultures treated with 2,3-DPG, using atomic force microscopy (AFM)-based force spectroscopy and other experimental approaches. The presence of infection by P. falciparum significantly increased the rigidity of parasitized cells and influenced the morphology of RBCs, as parasitized cells showed a decrease of the area-to-volume ratio. The extracellular addition of 2,3-DPG also slightly affected the stiffness of niRBCs, making it more similar to that of infected cells. It also changed the niRBC height, making the cells appear more elongated. Moreover, 2,3-DPG treatment influenced the cell surface charge, becoming more negative in treated RBCs than in untreated ones. The results indicate that treatment with 2,3-DPG has only a mild effect on RBCs in comparison with the effect of the presence of the parasite on the host cell. 2,3-DPG is an endogenous host metabolite, which may, in the future, originate a new antimalarial tool with few adverse effects on noninfected cells.publishersversionpublishe

Sweet whey cheese matrices inoculated with the probiotic strain Lactobacillus paracasei LAFTI® L26

Consumption of dairy products containing viable probiotic strains has increased dramatically in recent years, owing to general health claims associated therewith. This trend has boosted diversification of the portfolio of said products, including whey cheese matrices. However, taking into account the relatively poor organoleptic and textural features of these matrices, improvement is in order via incorporation of selected additives, provided that viability of the strains is duly assayed. Lactobacillus paracasei LAFTI® L26 was accordingly incorporated into whey protein solid matrices, in the presence of several additives aimed at enhancing their organoleptic appeal and textural performance. These matrices were produced from a combination of either ovine or bovine whey (or a mixture thereof) with ovine milk, and were inoculated at 10% (v/v) with the probiotic strain. Sugar, sugar and aloe vera, sugar and chocolate, and sugar and jam were further added, and the resulting products were then stored at 7 ◦C for 21 d. In general, viable cell numbers remained high in all experimental matrices throughout storage. Despite the observed low extents of breakdown, proteolytic activities by the end of storage were higher in matrices containing jam. Furthermore, L. paracasei partially converted lactose into lactic acid in these matrices. Additives enhanced the organoleptic features of whey cheeses, and produced different textural patterns. The higher sensory scores were attained by matrices containing sugar: sugar and aloe vera received the best scores by 3 d of storage, but these scores decreased as storage time elapsed

Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNF and IFN

In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr−/−, tlr2−/−, trif−/−, and myd88−/− mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon β (IFNβ) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor–knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.—Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ

Actividades experimentais nas escolas através de uma interacção com a universidade

A forma como a ciência é ensinada nas escolas afecta profundamente a percepção que os estudantes têm do mundo que os rodeia e consequentemente a escolha de uma futura carreira nas ciências. Por outro lado, a abertura da escola à comunidade envolvente, proporciona as condições para a sua participação activa na vida escolar, assim como a promoção do sucesso escolar, através da formação integral dos alunos, dotando-os das competências científicas, tecnológicas e sócio-culturais necessárias a sólidas opções futuras. A química como uma ciência de cariz experimental oferece aos estudantes uma melhor compreensão dos conceitos teóricos leccionados. Este trabalho descreve actividades experimentais realizadas no âmbito de um intercâmbio entre a Escola Secundária/3 de Barcelinhos e o Departamento de Química. Os projectos desenvolvidos foram “Museu das Ciências” na disciplina de Área Projecto do 12º ano de escolaridade. A actividade A Ciência na investigação criminal foi baseada nas oficinas apresentadas no projecto Sentidos da Ciência [1]. Estas actividades experimentais executadas em colaboração entre as escolas e a universidade contribuem para uma maior motivação dos alunos, uma formação científica mais sólida e um conhecimento mais integrado

Combining high pressure and electric fields towards nannochloropsis oculata eicosapentaenoic acid-rich extracts

Nannochloropsis oculata is naturally rich in eicosapentaenoic acid (EPA). To turn this microalga into an economically viable source for commercial applications, extraction efficiency must be achieved. Pursuing this goal, emerging technologies such as high hydrostatic pressure (HHP) and moderate electric fields (MEF) were tested, aiming to increase EPA accessibility and subsequent extraction yields. The innovative approach used in this study combined these technologies and associated tailored, less hazardous different solvent mixtures (SM) with distinct polarity indexes. Although the classical Folch SM with chloroform: methanol (PI 4.4) provided the highest yield concerning total lipids (166.4 mglipid/gbiomass), diethyl ether: ethanol (PI 3.6) presented statistically higher values in terms of EPA per biomass, corresponding to 1.3-fold increase. When SM were used in HHP and MEF, neither technology independently improved EPA extraction yields, although the sequential combination of technologies did result in 62% increment in EPA extraction. Overall, the SM and extraction methodologies tested (HHP—200 MPa, 21 °C, 15 min, followed by MEF processing at 40 °C, 15 min) enabled increased EPA extraction yields from wet N. oculata biomass. These findings are of high relevance for the food and pharmaceutical industries, providing viable alternatives to the “classical” extraction methodologies and solvents, with increased yields and lower environmental impact.info:eu-repo/semantics/publishedVersio