LSRE-LCM - Artigos em Revistas Nacionais e de Circulação Internacional
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- Influence of commensal bacteria on the proteolytic and antigenic profiles of INFOGEST-like digested wheat gliadinPublication . Pereira-Costa, Flávio; Domingues, Vanessa S.; Roque, Ana; Almeida, Zaida L.; Cruz, Pedro F.; Cordeiro, Rachel; Trindade, Daniela; Moura, Carla; Melo, Joana B.; Pereira, Sónia G.; Vaz, Daniela C.Introduction: Celiac disease (CeD) is a chronic autoimmune enteropathy developed by genetically predisposed individuals when exposed to gluten. Gluten gliadins, along with gut microbiota, may influence CeD onset and progression through mechanisms that remain unclear. Methods: Gliadin-degrading bacterial isolates obtained from CeD patients, and their 1st-degree relatives’ stool and blood were identified (Bacillus tropicus, Enterococcus faecalis, Micrococcus sp., Cronobacter sakazakii, Pseudomonas aeruginosa, and Serratia marcescens) and used in an INFOGEST-like protocol to simulate gliadin digestion after 4 h (digested gliadin, d-gliadin). The d-gliadin digesta were analyzed by fast protein liquid chromatography (FPLC), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), fluorescence spectroscopy, and polyclonal and monoclonal (R5 and G12) enzyme-linked immunosorbent assays (ELISA). Results and discussion: In the absence of the bacterial isolates, gliadin is poorly digested and self-assembles within 1 day into intermediate and large protein oligomers/aggregates, enriched in β-sheet structure (FTIR amide I band between 1,600 and 1,700 cm−1) and able to bind thioflavin T and Congo red. Conversely, in the presence of the bacterial isolates, gliadin is further digested, leading to an increase in protein fragments. After 4 h, the P. aeruginosa, C. sakazakii, and B. tropicus d-gliadin digesta presented a mixture of d-gliadin peptides and aggregates that showed higher antigenicity (associated with the exposure of the 5-amino acid QQPFP and 6-amino acid QPQLPY epitopes, present in the 25-mer and 33-mer, respectively) than control digestions (without bacteria), while E. faecalis led to lower antigenicity. In turn, within 24 h of incubation, all bacterial isolates led to the formation of undigested material with lower antigenicity, either due to fewer 33-mers and 25-mers in solution, or to fragment aggregation into amorphous material, not exposing antigenic sequences. Hence, intestinal flora may enhance or diminish the antigenicity of gliadin, thereby modulating the immunogenic response to gliadin/gluten.
- Serum-PEG and BSA-PEG hydrogels as advanced platforms for evaluating plasma protein bindingPublication . Coelho, Carlos D.F.; Paiva, Victor S.; Almeida, Zaida L.; Jesus, João A.; Marteleira, Madalena; Ramos, Cristiana V.; Cruz, Pedro F.; Costa, Telma; Moura, Carla S.; Trindade, Daniela; Brito, Rui M.M.; Lagoa, Ricardo; Vaz, Daniela C.; Moreno, Maria JoãoThe binding of bioactive compounds to proteins is critical for their availability and ADME/Tox profile. Specifically, binding to serum proteins affects both the distribution and elimination of drugs, while permeation through protein-enriched matrices, such as skin, is also influenced by protein interactions. Although several methods exist to evaluate ligand-protein binding, they often fail to replicate the high protein concentrations and molecular crowding conditions found in vivo. In this study, we investigate the use of protein-PEG hydrogels with low crosslinking density as 3D matrices to quantify ligand-protein affinity. Two types of hydrogels were developed: one using bovine serum albumin (BSA) and a more physiologically relevant one using serum. BSA was chosen as a model protein due to its similarity to human serum albumin. The hydrogels were characterized for swelling, stability, mechanical properties, and porosity, and the structural integrity of BSA within the hydrogel was confirmed using circular dichroism, 1H NMR and fluorescence spectroscopy. To assess protein functionality, we evaluated the binding affinity of various ligands, including a homologous series of fluorescent amphiphiles with different hydrophobicity (NBD-Cn, where n = 4, 6, and 8), two pesticides (malathion and chlorpyrifos), and six pharmaceutical drugs (acetaminophen, chlorpromazine, diclofenac, labetalol, salicylic acid, and verapamil). Our results demonstrated that the structural and functional properties of BSA remained intact within the hydrogel, and the large mesh size allowed for rapid and selective ligand binding. A comparison of BSA and serum hydrogels confirmed the major role of serum albumin in ligand binding, while highlighting some differences between cationic and anionic ligands. Altogether, these hydrogels offer an effective and reliable 3D platform for the fast and accurate evaluation of plasma protein binding of drugs and other bioactive compounds.