Browsing by Author "Cunha, Marta Fernandez"
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- Bioprospecting bioactive peptides in halobatrachus didactylus body mucus: from in silico insights to essential in vitro validationPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, Maria ManuelaFish body mucus plays a protective role, especially in Halobatrachus didactylus, which inhabits intertidal zones vulnerable to anthropogenic contaminants. In silico predicted bioactive peptides were identified in its body mucus, namely, EDNSELGQETPTLR (HdKTLR), DPPNPKNL (HdKNL), PAPPPPPP (HdPPP), VYPFPGPLPN (HdVLPN), and PFPGPLPN (HdLPN). These peptides were studied in vitro for bioactivities and aggregation behavior under different ionic strengths and pH values. Size exclusion chromatography revealed significant peptide aggregation at 344 mM and 700 mM ionic strengths at pH 7.0, decreasing at pH 3.0 and pH 5.0. Although none exhibited antimicrobial properties, they inhibited Pseudomonas aeruginosa biofilm formation. Notably, HdVLPN demonstrated potential antioxidant activity (ORAC: 1.560 μmol TE/μmol of peptide; ABTS: 1.755 μmol TE/μmol of peptide) as well as HdLPN (ORAC: 0.195 μmol TE/μmol of peptide; ABTS: 0.128 μmol TE/μmol of peptide). Antioxidant activity decreased at pH 5.0 and pH 3.0. Interactions between the peptides and mucus synergistically enhanced antioxidant effects. HdVLPN and HdLPN were non-toxic to Caco-2 and HaCaT cells at 100 μg of peptide/mL. HdPPP showed potential antihypertensive and antidiabetic effects, with IC50 values of 557 μg of peptide/mL for ACE inhibition and 1700 μg of peptide/mL for α-glucosidase inhibition. This study highlights the importance of validating peptide bioactivities in vitro, considering their native environment (mucus), and bioprospecting novel bioactive molecules while promoting species conservation.
- Exploring bioactivities and peptide content of body mucus from the lusitanian toadfish Halobatrachus didactylusPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Marques, Rita; Neto, José; Almada, Frederico; Gonçalves, David; Pintado, ManuelaIdentifying bioactive molecules from marine organisms is still vastly understudied. Fish remain an untapped source of bioactive molecules, even when considering species whose toxicity to other fish species has been noticed before. We assessed potential applications of crude body mucus of the Lusitanian toadfish (Halobratachus didactylus) and characterized its peptide fraction composition. Mucus samples from three individuals (two wild and one captive) revealed potential antioxidant, antihypertensive, and antimicrobial activities. For antioxidant activity, the best results of 2371 ± 97 µmol Trolox Equivalent/g protein for ORAC and 154 ± 6 µmol Trolox Equivalent/g protein for ABTS were obtained. For antihypertensive activity, the relevant inhibitory activity of ACE resulted in IC50 of 60 ± 7 µg protein/mL. Antimicrobial activity was also identified against the pathogenic bacteria Escherichia coli and Listeria monocytogenes. The peptide profile of the crude body mucus was obtained through size exclusion chromatography, with a conspicuous peak at ca. 800 Da. LC-MS/MS allowed the detection of the most probable peptide sequences of this dominant peptide. This is the first study where the bioactive potential of mucus from the Lusitanian toadfish is demonstrated. Peptides with such properties can be applied in the food and pharmaceutical industries.
- Methods for the collection of fish mucus: a systematic reviewPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe aquatic environment holds a vast source of organisms that provide numerous opportunities to bioprospect new molecules. Notably, fish are producers of an epidermal mucus that offers protection against pathogens, making it a promising source of bioactive molecules. This source of molecules, however, has yet to be thoroughly explored, and particularly, optimization of methods for collection and study is needed. This review concentrates on the methods of mucus collection employed to secure high-quality samples, enabling the extraction and characterization of molecules with bioactive potential. A comprehensive search was conducted, and publications were selected based on the following criteria: (i) the mucus has been collected from the external body of the fish, not involving dissection or damage; (ii) mucus crude extracts have undergone a chemical or genetic characterization; (iii) mucus was used in bioactivity assays (e.g., antimicrobial or immune-related). Scraping, bagging, and absorption are the primary methods for collecting fish mucus. They were assessed based on fish handling, sample volume, and processing, including anesthesia and starvation. Scraping with a soft tool, such as cotton balls or sponges, proved most effective and minimized contamination, dilution, and injury risk. This review aids future studies of mucus composition and properties.
- NanoLC-MS/MS sequencing and in silico analysis of bioactive peptides from Lusitanian toadfish halobatrachus didactylus body mucusPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe mucus covering a fish's body works as a protective barrier and contains molecules, such as antimicrobial peptides, that defend against pathogens. In this study, two adult Halobatrachus didactylus were captured from the wild in Sesimbra, and mucus was collected by scraping the dorsal-lateral body with a sponge. Our objective was to identify new bioactive peptides in mucus samples via chromatography. SE-HPLC revealed a prominent peak at 775 Dalton. The mucus samples were pooled, fractionated, and analyzed by nanoLC-MS/MS. Database identification was unreliable, so de novo sequencing with PEAKS Studio was used, resulting in five peptides selected for their predicted bioactivities.
- NanoLC-MS/MS sequencing and in silico analysis of bioactive peptides from Lusitanian Toadfish Halobatrachus didactylus body mucusPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe mucus covers the fish's body, working as a protective barrier. Besides physical protection, mucus provides molecules that protect the fish from damaging pathogens [1,2]. Has been reported that antimicrobial peptides are secreted in the mucus, which play an essential role in defense against microbial pathogens since these belong to the innate immune system [2,3].
- Sustainable bioprospecting and circular economy approach: fractionation and identification of bioactive peptides from lusitanian toadfish (halobatrachus didactylus) mucusPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaBioactive molecules from marine organisms, especially the Halobatrachus didactylus, also known as the Lusitanian toadfish, native to the Portuguese coast, remains largely unexplored. It is important to use sustainable bioprospecting practices to protect both the species and its ecosystem (1). Therefore, by adopting a circular economy approach, we can maximize the sustainable use of marine resources by sampling fish in a non-harmful way (e.g. mucus collection) and returning them to their natural habitat, thus allowing these organisms to thrive in their ecosystems. Fish mucus acts as a natural barrier from the surrounding environment, acting as a first line of defense, for instance, against pathogens (2,3). In this study, we carefully collected H. didactylus mucus using a synthetic sponge to ensure no physical harm to the fish and to minimize stress by reducing handling time. We evaluated the potential bioactivities of the body mucus and characterized its peptide fraction composition. The body mucus of H. didactylus exhibited promising antioxidant, antihypertensive, and antimicrobial activities (4). The body mucus peptide profile was obtained using size exclusion chromatography, revealing a prominent peak ca. 800 Da. Using LC-MS/MS, we identified several peptide sequences from the mucus peptide fraction and selected five of them based on their predicted bioactivities in silico. We then validated the bioactivities through in vitro assays. Peptides with the amino acid sequences EDNSELGQETPTLR (HdKTLR), DPPNPKNL (HdKNL), PAPPPPPP (HdPPP) and VYPFPGPLPN (HdVLPN) showed antibiofilm potential against P. aeruginosa. HdVLPN and PFPGPLPN (HdLPN) exhibit significant antioxidant activity, while HdPPP demonstrates antihypertensive and antidiabetic activities. In conclusion, this study not only underscores the therapeutic potential of H. didactylus peptides but also serves as a model for sustainable bioprospecting practices that align with circular economy principles.