Exploring venom diversity in Mixcoatlus browni and Mixcoatlus barbouri: A comparative analysis of two rare Mexican snake species with crotoxin-like presence.

The genus Mixcoatlus is composed of three species: Mixcoatlus barbouri, M. browni, and M. melanurus, of which the venom composition of M. melanurus, the most common species of the three, has only recently been described. However, very little is known about the natural history of M. barbouri and M. browni, and the venom composition of these two species has remained thus far unexplored. In this study we characterize the proteomic profiles and the main biochemical and toxic activities of these two venoms. Proteomic data obtained by shotgun analysis of whole venom identified 12 protein families for M. barbouri, and 13 for M. browni. The latter venom was further characterized by using a quantitative 'venomics' protocol, which revealed that it is mainly composed of 51.1 % phospholipases A2 (PLA2), 25.5 % snake venom serine proteases (SVSP), 4.6 % l-amino oxidases (LAO), and 3.6 % snake venom metalloproteases (SVMP), with lower percentages other six protein families. Both venoms contained homologs of the basic and acidic subunits of crotoxin. However, due to limitations in M. barbouri venom availability, we could only characterize the crotoxin-like protein of M. browni venom, which we have named Mixcoatlutoxin. It exhibited a lethal potency in mice like that described for classical rattlesnake crotoxins. These findings expand knowledge on the distribution of crotoxin-like heterodimeric proteins in viper snake species. Further investigation of the bioactivities of the venom of M. barbouri, on the other hand, remains necessary.

Quantifying venom production: A study on Micrurus snakes in Mexico.

Our study quantifies venom production in nine Mexican coral snake species (Micrurus), encompassing 76 specimens and 253 extractions. Noteworthy variations were observed, with M. diastema and M. laticollaris displaying diverse yields, ranging from 0.3 mg to 59 mg. For animals for which we have length data, there is a relationship between size and venom quantity. Twenty-eight percent of the observed variability in venom production can be explained by snake size, suggesting that other factors influence the amount of obtained venom. These findings are pivotal for predicting venom effects and guiding antivenom interventions. Our data offer insights into Micrurus venom yields, laying the groundwork for future research and aiding in medical response strategies. This study advances understanding coral snake venom production, facilitating informed medical responses to coral snake bites.

Antimicrobial, toxicological, and antigenic characteristics of three scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus and Tityus n. sp. aff. metuendus.

The venom fractions of three buthid scorpion species from Colombia, C. margaritatus, T. pachyurus and T. n. sp. aff. metuendus, were examined for antimicrobial and toxicity toward mice and insects. The three venoms were separated into individual fractions using RP-HPLC, resulting in 85 fractions from C. margaritatus, 106 from T. pachyurus, and 70 from T. n. sp. aff. metuendus. The major fractions from the three scorpion venoms, which were eluted between 35 and 50 min, were tested for antimicrobial activity and toxicity. It was confirmed that the venom of the three species contains fractions with antimicrobial peptides that were evaluated against two bacterial strains of public health importance, Pseudomonas aeruginosa and Staphylococcus aureus. The venom of C. margaritatus had two antimicrobial fractions that showed activity against the named tested strains. The venom of T. pachyurus had three fractions that showed activity against S. aureus and two against both bacterial strains. Finally, the venom of T. n. sp. aff. metuendus had one fraction that showed activity against S. aureus, and five fractions showed activity against both bacterial strains. Also, some peptide fractions from the three venoms were toxic to mice. Last, the venoms of C. margaritatus and T. pachyurus were used as immunogens to obtain neutralizing antibodies against its respective venoms and to observe antibody recognition to related and unrelated scorpion venoms. A total of 15 mg of lyophilized antibodies were able to neutralize 1.5⋅LD50 of the venoms from T. n. sp. aff. metuendus, T. pachyurus and C. margaritatus, respectively. This information provides valuable insights into the diversity of each species' venom and their potential role in antimicrobial and venom toxicity.

Quantitative proteomic analysis reveals high interference on protein expression of H9c2 cells activated with glucose and cardiotonic steroids.

In recent decades, the incidence of death and morbidity due to diabetes has increased worldwide, causing a high social and economic impact. Diabetes is a major cause of blindness, kidney failure, heart attack, stroke and lower limb amputation. However, the molecular mechanisms that make the heart and kidneys the main targets of diabetes are not completely understood. To better understand the complex biochemical mechanism of diabetic cardiomyopathy, we investigated the effects of hyperglycemia with concomitant digoxin and ouabain stimulation in H9c2 cells. Total extracted proteins were analyzed by label-free LC-MS/MS, quantified by Scaffold software and validated by parallel reaction monitoring (PRM) methodology. Here, we show that the eukaryotic initiation factors (Eifs) and elongation factors (Eefs) Eif3f, Eef2 and Eif4a1 are overexpressed following cardiotonic steroid (CTS) stimulation. Similarly, the expression of four 14-3-3 proteins that play a key role in cardiac ventricular compaction was altered after CTS stimulation. In total, the expression of nine protein groups was altered in response to the stimulation of H9c2 cells. Here, the biological consequences of these changes are discussed in depth. SIGNIFICANCE: Hyperglycemia is the main physiological condition that provokes tissue and vascular injuries in heart of diabetic patients. However, the changings at large scale in the expression of proteins of cardiomyocytes generated by this condition was not yet studied. Here we report for the first time the altered biosynthesis of nine groups of proteins of H9c2 cells activated by high glucose concentrations and by cardiotonic steroids (CTS). Furthermore, the increased biosynthesis of Eifs, Eefs and 14-3-3 protein groups by CTS, which play a crucial role in cardiomyopathies are original data reported in this work. These findings not only enhance our knowledge concerning to the effects of hyperglycemia and CTS on H9c2 cells but also indicate potential molecular targets to interfere in diabetes cardiomyopathy progression.

A Comprehensive Proteomic Study of the Skin Secretions of the Frog Lithobates spectabilis.

Disulfide C-terminal loop fragments derived from AMPs and the presence of peptidases have been previously reported in the skin secretions of different amphibians. However, there are only a few studies on the identification of enzymes in frog skin secretion based on the primary structure of these proteins. Similarly, little data exist regarding the identification of disulfide C-terminal loops at large scale. Therefore, a comprehensive study on this issue certainly could bring in much more information for understanding this molecular process and its biochemical consequences. Thus, the aim of this work was to characterize the presence of disulfide C-terminal loop fragments of AMPs and identify the proteins and probable enzymes present in the completely unknown secretion contents of the frog Lithobates spectabilis. For this purpose, high-resolution mass spectrometry was applied to analyze the skin secretions processed by two different protocols: (1) using a cocktail of enzymatic inhibitors and 2) without any protease inhibitors, maintaining the solution for 2 hours at 10°C. Results from procedure-1, revealed 122 molecular masses, whereas procedure-2 permitted 253 different molecular masses to be identified. Fifty-nine peptides including 22 disulfide C-terminal loop-containing peptides were obtained following procedure-2. Polyacrylamide gel electrophoresis separation, tryptic digestion and LCMS/ MS were used for "de novo" sequencing of 111 different peptides and the unequivocal identification of fifteen proteins including at least three different peptidases. Additionally, it was possible to fully sequence eight peptides, including a ranatuerin-related peptide identified here as Spectabilin, that was subsequently chemically synthesized and showed high antibacterial, antiparasitic and cytotoxic activities.

Comparative proteomic analysis of male and female venoms from the Cuban scorpion Rhopalurus junceus.

A complete mass spectrometry analysis of venom components from male and female scorpions of the species Rhophalurus junceus of Cuba is reported. In the order of 200 individual molecular masses were identified in both venoms, from which 63 are identical in male and females genders. It means that a significant difference of venom components exists between individuals of different sexes, but the most abundant components are present in both sexes. The relative abundance of identical components is different among the genders. Three well defined groups of different peptides were separated and identified. The first group corresponds to peptides with molecular masses of 1000-2000 Da; the second to peptides with 3500-4500 Da molecular weight, and the third with 6500-8000 Da molecular weights. A total of 86 peptides rich in disulfide bridges were found in the venoms, 27 with three disulfide bridges and 59 with four disulfide bridges. LC-MS/MS analysis allowed the identification and amino acid sequence determination of 31 novel peptides in male venom. Two new putative K(+)-channel peptides were sequences by Edman degradation. They contain 37 amino acid residues, packed by three disulfide bridges and were assigned the systematic numbers: α-KTx 1.18 and α-KTx 2.15.

Analysis of sulfated peptides from the skin secretion of the Pachymedusa dacnicolor frog using IMAC-Ga enrichment and high-resolution mass spectrometry.

Immobilized metal ion affinity chromatography (IMAC) has been widely used for the enrichment of phosphopeptides, whereas no report exists describing the use of IMAC columns for the enrichment of sulfopeptides. In this study, we used IMAC-Ga microcolumns for the enrichment of sulfopeptides from a complex mixture of peptides, extracted from skin secretions of the Pachymedusa dacnicolor frog. The enriched fraction obtained by IMAC-Ga was analyzed by liquid chromatograpy/electrospray ionization mass spectrometry (LC/ESI-MS) in an Orbitrap XL and by matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF/TOF) in an ABI 4800 instrument. From this fraction, different sulfated and non-sulfated peptides belonging to the caerulin and bradykinin families were structurally characterized. Other interesting negatively charged groups, such as phosphate adducts of dermaseptins and pyridoxal phosphate attached to a protease inhibitor, were also characterized. Unexpectedly, some dermaseptin antimicrobial peptides were also enriched by IMAC-Ga and a Sauvatine-like peptide was also fully sequenced. Furthermore, neutral loss of sulfated peptides and their fragmentation patterns in the gas phase were also compared using collision-induced dissociation (CID) and high-energy collision dissociation (HCD). Our present study provides evidence that IMAC-Ga enrichment is a fast, useful and promising method for high-throughput analysis of sulfated-peptides, since high-resolution mass spectrometers can be used for this purpose.

Peptidomic analysis of the skin secretions of the frog Pachymedusa dacnicolor.

High-resolution mass spectrometry-based peptidomics has been used to characterize several components in electro-stimulated skin secretions of the endemic Mexican frog Pachymedusa dacnicolor. Peptide mass screening performed in an Orbitrap-XL mass spectrometer showed that P. dacnicolor skin secretions possess 194 different components with molecular masses ranging mainly from 500 to 6,000 Da. Dozens of molecules were partially sequenced including two novel protease inhibitors. Additionally, one posttranslationally modified bradykinin and two novel dermaseptin-like antimicrobial peptides were fully sequenced. The novel peptide named here DMS-DA5 was fully characterized and showed potent antibacterial activity against various bacteria such as Escherichia coli, Bacillus subtilis, Salmonella enterica serovar typhimurium, and Pseudomonas aeruginosa with minimal inhibitory concentrations from 3.10 to 25.0 microM.

Novel antimicrobial peptides isolated from skin secretions of the Mexican frog Hyla eximia.

In this work, we describe the original characterization of peptides and proteins present in the skin secretions of the Mexican amphibian Hyla eximia. To this purpose, a novel water/dark extraction method, as well as the classic electrical stimulation procedure, was applied in order to extract the skin secretion. Two novel antimicrobial peptides He-1 and He-2 were sequenced. In addition, a molecular mass fingerprint revealed more than one hundred different molecules. Eight peptides in homogeneous form were assayed against five species of bacteria. Thereafter, the peptide He-2 demonstrated high antiparasitic activity against ookinete forms of malaria parasites at low concentration.