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- The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficitsPublication . Koppenol, Rebekah; Conceição, André; Afonso, Inês T.; Afonso-Reis, Ricardo; Costa, Rafael G; Tomé, Sandra; Teixeira, Diogo; Pinto-da-Silva, Joana; Codêsso, José Miguel; Brito, David V.C.; Mendonça, Liliana; Marcelo, Adriana; Pereira de Almeida, Luís; Matos, Carlos A; Nóbrega, ClévioKoppenol et al. show that overexpression of G3BP1 in cell models of SCA2 and SCA3 leads to a reduction in ataxin-2 and ataxin-3 aggregation. G3BP1 lentiviral delivery reduces motor deficits and neuropathology in preclinical models, suggesting that G3BP1 may be a potential therapeutic target for polyQ disorders. Polyglutamine diseases are a group of neurodegenerative disorders caused by an abnormal expansion of CAG repeat tracts in the codifying regions of nine, otherwise unrelated, genes. While the protein products of these genes are suggested to play diverse cellular roles, the pathogenic mutant proteins bearing an expanded polyglutamine sequence share a tendency to self-assemble, aggregate and engage in abnormal molecular interactions. Understanding the shared paths that link polyglutamine protein expansion to the nervous system dysfunction and the degeneration that takes place in these disorders is instrumental to the identification of targets for therapeutic intervention. Among polyglutamine diseases, spinocerebellar ataxias (SCAs) share many common aspects, including the fact that they involve dysfunction of the cerebellum, resulting in ataxia. Our work aimed at exploring a putative new therapeutic target for the two forms of SCA with higher worldwide prevalence, SCA type 2 (SCA2) and type 3 (SCA3), which are caused by expanded forms of ataxin-2 (ATXN2) and ataxin-3 (ATXN3), respectively. The pathophysiology of polyglutamine diseases has been described to involve an inability to properly respond to cell stress. We evaluated the ability of GTPase-activating protein-binding protein 1 (G3BP1), an RNA-binding protein involved in RNA metabolism regulation and stress responses, to counteract SCA2 and SCA3 pathology, using both in vitro and in vivo disease models. Our results indicate that G3BP1 overexpression in cell models leads to a reduction of ATXN2 and ATXN3 aggregation, associated with a decrease in protein expression. This protective effect of G3BP1 against polyglutamine protein aggregation was reinforced by the fact that silencing G3bp1 in the mouse brain increases human expanded ATXN2 and ATXN3 aggregation. Moreover, a decrease of G3BP1 levels was detected in cells derived from patients with SCA2 and SCA3, suggesting that G3BP1 function is compromised in the context of these diseases. In lentiviral mouse models of SCA2 and SCA3, G3BP1 overexpression not only decreased protein aggregation but also contributed to the preservation of neuronal cells. Finally, in an SCA3 transgenic mouse model with a severe ataxic phenotype, G3BP1 lentiviral delivery to the cerebellum led to amelioration of several motor behavioural deficits. Overall, our results indicate that a decrease in G3BP1 levels may be a contributing factor to SCA2 and SCA3 pathophysiology, and that administration of this protein through viral vector-mediated delivery may constitute a putative approach to therapy for these diseases, and possibly other polyglutamine disorders.
- Pontastacus leptodactylus (Eschscholtz, 1823) and Faxonius limosus (Rafinesque, 1817) as new, alternative sources of chitin and chitosanPublication . Nuc, Zofia; Brusotti, Gloria; Catenacci, Laura; Grenha, Ana; Pontes, Jorge Filipe; Pinto-da-Silva, Joana; Rosa Da Costa, Ana; Moro, Paola; Milanese, Chiara; Grisoli, Pietro; Sorrenti, Milena; Dobrzycka-Krahel, Aldona; Bonferoni, Maria Cristina; Caramella, Carla MarcellaThe growing demand for chitin and chitosan makes it necessary to look for new sources of these polymers and to develop more environmentally friendly methods for their isolation. The subjects of the current study were chitin and chitosan extracted from shells of two crayfish species: P. leptodactylus and F. limosus. The obtained polymers were characterized by physicochemical properties (molecular weight, thermal stability, and structure). The obtained chitosan was evaluated regarding biocompatibility and antimicrobial activity. The yield of chitin obtained from P. leptodactylus and F. limosus with a standard method was 22 ± 2.7% and 20 ± 3.6% (w/w), respectively (a preliminary extraction with a natural deep eutectic solvent was performed successfully only for P. leptodactylus). The yield of chitosan production was 15 ± 0.3% and 14 ± 4.2%, respectively. Both chitosan samples showed antimicrobial activity against E. coli and S. aureus. Cytotoxicity assays revealed a time- and concentration-dependent effect, with a milder impact at concentrations up to 250 µg/mL. A more favourable profile was observed for chitosan from F. limosus shells.
- Improving benzyl – isothiocyanate bioaccessibility in white mustard (Sinapis alba) sauce through spray – drying microencapsulation and pickering emulsionsPublication . Rincón, E.; Grenha, Ana; da Silva, Joana Pinto; Espinosa, E.; Lafont-Déniz, F.; Almeida, M. P.; Cámara-Martos, F.This study compared the bioaccessibility and behaviour of different formulations of benzyl - isothiocyanate (benzyl - ITC) prepared using different approaches and the INFOGEST in vitro digestion protocol. The aim was to improve the low bioaccessibility of this bioactive compound caused by its lipophilic properties. Spray-dried microparticles were prepared using a matrix of either mannitol or maltodextrin, and compared with Pickering emulsions produced with cellulose nanofibres. The different systems were characterised and their ability to associate benzyl - ITC determined. The in vitro digestion characteristics provided by the different systems was evaluated. Microencapsulation of benzyl-ITC by spray-drying was not successful when mannitol was used as excipient, while maltodextrin resulted in a production yield around 70 % with an ITC association efficiency up to 75 %. Nevertheless, significant improvement of benzyl- ITC bioaccessibility in a mustard sauce was not achieved. In contrast, the formulation of benzyl - ITC in a Pickering emulsion prepared with cellulose nanofibres showed an association efficiency of around 100 % and high bioaccessibility with values up to 77 %. The chemical similarity between the mixed micelles formed for lipid absorption in the small intestine, and the structure of cellulose nanofibre emulsion could justify the observed improvement.
- Locust bean gum (LBG) – a potential excipient for inhalation purposes: excipient characterisation and in vitro and in vivo toxicological evaluationPublication . Pontes, Jorge Filipe; Guerreiro, Filipa; da Silva, Joana Pinto; Almeida, Maria; Rosso, Annalisa; Rosa da Costa, Ana M; Agusti, Géraldine; Lollo, Giovanna; Gaspar, Maria Manuela; Grenha, AnaDry powders proposed for lung drug delivery typically involve excipients not approved for inhalation. The physicochemical characterisation of excipients is informative regarding their interaction with different body structures. Locust Bean Gum (LBG) has been proposed for diverse applications, including inhalation, benefiting from targeting ability towards phagocytic cells owing to the presence of mannose moieties. The aim of the present study was to characterise the physicochemical parameters of LBG and, for the first time, draw a toxicological profile for this excipient. LBG from three different suppliers was evaluated and eventual changes on polymer characteristics induced by purification and microparticle production were assessed, which were observed to not occur. The commercial samples showed identical FTIR spectra, as well as TGA and DSC profiles, Mw around 3.60 × 106 Da, and Tg near − 39 ◦C. All microparticles presented similar morphology and Feret diameters around 4 μm. In vitro assays performed on respiratory (A549) cells evidenced no impact of LBG microparticles on cell viability (> 80 %) when biorelevant concentrations (≈ 0.1 mg/mL) were used. Additionally, inhalation of LBG microparticles by mice provided indications of a safe profile, without induction of allergic reactions. The basis is laid for further exploration of this material in inhalation.