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- Exploring bioactivities and peptide content of body mucus from the lusitanian toadfish Halobatrachus didactylusPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel; Emilia Brassesco, M.; Marques, Rita; Neto, José; Almada, Frederico José Oliveira de; Goncalves, David; Pintado, Maria 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 . Fernandez Cunha, Marta; Coscueta, Ezequiel; Emilia Brassesco, M.; Almada, Frederico José Oliveira de; Goncalves, David; Pintado, Maria 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.
- Solution-phase ITC validation of literature-reported Glyphosate DNA Aptamers: Affinity ranking and an operational selectivity boundaryPublication . Sun, Jingchun; Zhang, Linbing; Goncalves, David; Kuang, Shaoping; Yang, HongshengGlyphosate is a highly polar herbicide, the reliable molecular recognition of which is complicated by co-occurring structural analogues, metabolites, and derivatives in real-world samples. Rather than reporting new aptamer discovery, this study establishes a standardized, solution-phase isothermal titration calorimetry (ITC) workflow to thermodynamically reassess two literature-reported glyphosate DNA aptamers, Seq03 and Seq05, under matched buffer composition and instrument settings. After verification of baseline stability and evaluation of heat-of-dilution contributions, ligand-to-aptamer titrations yielded apparent dissociation constants of approximately 8.14 μM for Seq03 and 40.2 μM for Seq05, enabling affinity-based prioritization of these reported candidates within the tested concentration window. To define an application-relevant selectivity boundary, we further constructed a counter-screen panel restricted to glyphosate-related chemicals, including structural analogues, metabolites, and derivatives, and evaluated all candidates using an identical ITC protocol with explicit background handling. None of the counter-screen compounds produced binding-consistent, saturable isotherms after integration and control-based interpretation; instead, their responses remained close to background heat and were therefore operationally classified as having no detectable binding under the tested conditions, including a reverse-titration format check with Glufosinate-N-acetyl. Collectively, these results position ITC as a label-free, platform-independent validation step for small-molecule aptamer benchmarking prior to analytical translation, while also highlighting that the present conclusions are bounded by the tested PBS-based conditions and the sensitivity window of the current ITC configuration.