Exosome Liberation by Human Neutrophils under L-Amino Acid Oxidase of Calloselasma rhodostoma Venom Action.

L-Amino acid oxidase (LAAO) is an enzyme found in snake venom that has multifaceted effects, including the generation of hydrogen peroxide (H2O2) during oxidative reactions, leading to various biological and pharmacological outcomes such as apoptosis, cytotoxicity, modulation of platelet aggregation, hemorrhage, and neutrophil activation. Human neutrophils respond to LAAO by enhancing chemotaxis, and phagocytosis, and releasing reactive oxygen species (ROS) and pro-inflammatory mediators. Exosomes cellular nanovesicles play vital roles in intercellular communication, including immune responses. This study investigates the impact of Calloselasma rhodostoma snake venom-derived LAAO (Cr-LAAO) on human neutrophil exosome release, including activation patterns, exosome formation, and content. Neutrophils isolated from healthy donors were stimulated with Cr-LAAO (100 µg/mL) for 3 h, followed by exosome isolation and analysis. Results show that Cr-LAAO induces the release of exosomes with distinct protein content compared to the negative control. Proteomic analysis reveals proteins related to the regulation of immune responses and blood coagulation. This study uncovers Cr-LAAO's ability to activate human neutrophils, leading to exosome release and facilitating intercellular communication, offering insights into potential therapeutic approaches for inflammatory and immunological disorders.

Preliminary Insights of Brazilian Snake Venom Metalloproteomics.

Snakebite envenoming is one of the most significantly neglected tropical diseases in the world. The lack of diagnosis/prognosis methods for snakebite is one of our motivations to develop innovative technological solutions for Brazilian health. The objective of this work was to evaluate the protein and metallic ion composition of Crotalus durissus terrificus, Bothrops jararaca, B. alternatus, B. jararacussu, B. moojeni, B. pauloensis, and Lachesis muta muta snake venoms. Brazilian snake venoms were subjected to the shotgun proteomic approach using mass spectrometry, and metal ion analysis was performed by atomic spectrometry. Shotgun proteomics has shown three abundant toxin classes (PLA2, serine proteases, and metalloproteinases) in all snake venoms, and metallic ions analysis has evidenced that the Cu2+ ion is present exclusively in the L. m. muta venom; Ca2+ and Mg2+ ions have shown a statistical difference between the species of Bothrops and Crotalus genus, whereas the Zn2+ ion presented a statistical difference among all species studied in this work. In addition, Mg2+ ions have shown 42 times more in the C. d. terrificus venom when compared to the average concentration in the other genera. Though metal ions are a minor fraction of snake venoms, several venom toxins depend on them. We believe that these non-protein fractions are capable of assisting in the development of unprecedented diagnostic devices for Brazilian snakebites.

The effects of mercury exposure on Amazonian fishes: An investigation of potential biomarkers.

Exposure to mercury can interfere with the expression of proteins and enzymes, compromise important pathways, such as apoptosis and glucose metabolism, and even induce the expression of metallothioneins. In this study, analytical techniques were used to determine the concentration of total mercury (THg) in muscle and liver tissue, protein pellets, and spots [using graphite furnace atomic absorption spectrometry (GFAAS)], and molecular techniques were used to identify metalloproteins present in mercury-associated protein spots. Thirty individuals from three different fish species, Cichla sp. (n = 10), Brachyplatystoma filamentosum (n = 10), and Semaprochilodus sp. (n = 10) from the Brazilian Amazon were used. Oxidative stress indicators [such as glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), a marker of lipid peroxidation (LPO)] and the possible expression of metallothioneins in muscle and liver tissues were investigated. The two piscivorous species, Cichla sp. and B. filamentosum, presented the highest concentrations of mercury in their hepatic tissue, 1219 ± 15.00 and 1044 ± 13.6 µg kg-1, respectively, and in their muscle tissue, 101 ± 1.30 µg kg-1 and 87.4 ± 0.900 µg kg-1, respectively. The non-carnivorous species Semaprochilodus sp. had comparatively low concentrations of mercury in both its hepatic (852 ± 11.1 µg kg-1) and muscle (71.4 ± 0.930 µg kg-1) tissues. The presence of mercury was identified in 24 protein spots using GFAAS; concentrations ranged from 11.5 to 787 µg kg-1, and mass spectrometry identified 21 metal-binding proteins. The activities of GSH-Px, CAT, and SOD, related to oxidative stress, decreased proportionally as tissue Hg concentrations increased, while the levels of LPO markers increased, indicating the presence of stress. Our study results demonstrate possible mercury interference in oxidative stress markers (GSH-Px, CAT, SOD, and LPO), in addition to the identification of 21 metal-binding proteins as possible biomarkers of mercury exposure in fish.

Metalloproteomic Strategies for Identifying Proteins as Biomarkers of Mercury Exposure in Serrasalmus rhombeus from the Amazon Region.

This manuscript describes the results of a metalloproteomic study of mercury in samples of muscle and liver tissue of the species Serrasalmus rhombeus, popularly known as black piranha and characterised as the most voracious and aggressive predator in the Brazilian Amazon. The metalloproteomic study involved using two-dimensional electrophoresis (2D PAGE) to fractionate the proteome of the muscle and liver tissue samples, along with atomic absorption spectrometry in a graphite furnace (GFAAS) to identify mercury associated with protein SPOTs and mass spectrometry with electrospray ionisation (ESI-MS/MS) to characterise the mercury-binding proteins. The protein SPOTs characterised showed concentrations in the order of 156 mg kg-1, which ranks as the highest concentrations of mercury determined so far in metalloproteomic studies involving fish species in the Amazon region. Based on FASTA sequences of proteins characterised by ESI-MS/MS, bioinformatics studies were performed that allowed identifying nine proteins with characteristics of biomarkers of mercury exposure. Of those proteins, glutathione peroxidase stands out as an enzyme of great importance in the antioxidant defence of organisms subjected to oxidative stress caused by xenobiotics.

Identification of Zinc Absorption Biomarkers in Muscle Tissue of Nile Tilapia Fed with Organic and Inorganic Sources of Zinc Using Metallomics Analysis.

The development of metallomics techniques has allowed for metallomics analysis of biological systems, enabling a better understanding of the response mechanisms for different stimuli, their relationship to metallic species, and the characterization of biomarkers. In this study, a metallomics analysis of the muscle tissue of Nile tilapia was used to aid the understanding of the molecular mechanisms involved in zinc absorption in this fish species when fed organic and/or inorganic sources of zinc and to identify possible biomarkers for the absorption of this micromineral. To accomplish this, the fish were separated into three groups of 24 g, 74 g, and 85 g initial weights, and each group, respectively, was fed a zinc-free diet (control group, G1), a diet containing zinc found in organic sources (treatment 1, G2), and a diet containing zinc from an inorganic source (treatment 2, G3). Two-dimensional polyacrylamide (2D PAGE) gel electrophoresis was used to separate the proteins of the muscle tissue. Subsequently, the expression profiles of protein spots in the samples where zinc was applied in different concentrations were compared, using the software ImageMaster 2D Platinum version 7.0, to identify proteins that were differentially expressed. The identified proteins were then exposed to atomic absorption spectrometry in a graphite furnace to determine zinc mapping and were subsequently characterized via electrospray ionization tandem mass spectrometry (ESI-MS/MS). The metallomic analysis identified 15 proteins differentially expressed and associated with zinc, leading to the conclusion that three metal-binding proteins presented as possible biomarkers of zinc absorption in fish.

Study of proteins with mercury in fish from the Amazon region.

The high concentrations of mercury found in Amazon have been intensively studied by the scientific community in the last decades. These mercurial species bind preferentially to proteins. Therefore, this work proposal sought to obtain the fractionation, identification and study of mercury - bound proteins present in samples of muscular and hepatic tissue from fish collected in the reservoir of the Jirau Hydroelectric Power Plant - on the Madeira River. Two-dimensional electrophoresis (2D-PAGE) for protein fractionation, graphite furnace atomic absorption spectrometry (GFAAS) for the quantification of mercury and Mass Spectrometry (ESI-MS/MS) were used for the identification of proteins. Concluding the work with analysis of graphics from the Blast2go program. Two mercury - bound proteins were identified as triosephosphate isomerase A and Protein FAM45A. The data generated by the bioinformatics programs confirm the tendency of these proteins to be linked to mercury and elucity the possibles existing physiological and cellular interactions.