Myocardial proteome changes in aortic stenosis rats subjected to long-term aerobic exercise.

The effects of exercise training (ET) on the heart of aortic stenosis (AS) rats are controversial and the mechanisms involved in alterations induced by ET have been poorly clarified. In this study, we analyzed the myocardial proteome to identify proteins modulated by moderate-intensity aerobic ET in rats with chronic supravalvular AS. Wistar rats were divided into four groups: sedentary control (C-Sed), exercised control (C-Ex), sedentary aortic stenosis (AS-Sed), and exercised AS (AS-Ex). ET consisted of five treadmill running sessions per week for 16 weeks. Statistical analysis was performed by ANOVA or Kruskal-Wallis and Goodman tests. Results were discussed at a significance level of 5%. At the end of the experiment, AS-Ex rats had higher functional capacity, lower blood lactate concentration, and better cardiac structural and left ventricular (LV) functional parameters than the AS-Sed. Myocardial proteome analysis showed that AS-Sed had higher relative protein abundance related to the glycolytic pathway, oxidative stress, and inflammation, and lower relative protein abundance related to beta-oxidation than C-Sed. AS-Ex had higher abundance of one protein related to mitochondrial biogenesis and lower relative protein abundance associated with oxidative stress and inflammation than AS-Sed. Proteomic data were validated for proteins related to lipid and glycolytic metabolism. Chronic pressure overload changes the abundance of myocardial proteins that are mainly involved in lipid and glycolytic energy metabolism in rats. Moderate-intensity aerobic training attenuates changes in proteins related to oxidative stress and inflammation and increases the COX4I1 protein, related to mitochondrial biogenesis. Protein changes are combined with improved functional capacity, cardiac remodeling, and LV function in AS rats.

SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources.

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

Variant expression signatures of microRNAs and protein related to growth in a crossbreed between two strains of Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is a species of worldwide importance for aquaculture. A crossbred lineage was developed through introgressive backcross breeding techniques and combines the high growth performance of the Chitralada (CHIT) lwith attractive reddish color of the Red Stirling (REDS) strains. Since the crossbreed has an unknown genetically improved background, the objective of this work was to characterize expression signatures that portray the advantageous phenotype of the crossbreeds. We characterized the microRNA transcriptome by high throughput sequencing (RNA-seq) and the proteome through mass spectrometry (ESI-Q-TOF-MS) and applied bioinformatics for the comparative analysis of such molecular data on the three strains. Crossbreed expressed a distinct set of miRNAs and proteins compared to the parents. They comprised several microRNAs regulate traits of economic interest. Proteomic profiles revealed differences between parental and crossbreed in expression of proteins associated with glycolisis. Distinctive miRNA and protein signatures contribute to the phenotype of crossbreed.

Good management practices of venomous snakes in captivity to produce biological venom-based medicines: achieving replicability and contributing to pharmaceutical industry.

One of the factors responsible for lack of reproducible findings may be attributed to the raw material used. To date, there are no apparent studies examining reproducibility using venoms for the development of new toxin-based drugs with respect to regulatory agencies' policies. For this reason, protocols were implemented to produce animal toxins with quality, traceability, and strict compliance with Good Manufacturing Practices. This required validation of the production chain from the arrival of the animal to the vivarium, followed by handling, housing, as well as compliance with respect to extraction, freeze-drying, and, finally, storage protocols, aimed at generating compounds to serve as candidate molecules applicable in clinical trials. Currently, to produce quality snake venoms to support reproductive studies, the Center for the Study of Venoms and Venomous Animals (CEVAP) from São Paulo State University (UNESP), São Paulo, Brazil has 449 microchipped snakes through rigid and standardized operating procedures for safety, health, and welfare of animals. Snakes were frequently subjected to vet clinical examination, anthelmintic, and antiparasitic treatment. Venom milk used to destroy prey was collected from each animal in individual plastic microtubes to avoid contamination and for traceability. In addition, venoms were submitted to microbiological, and biochemical toxicological analyses. It is noteworthy that investigators are responsible for caring, maintaining, and manipulating snakes and ensuring their health in captivity. This review aimed to contribute to the pharmaceutical industry the experimental experience and entire snake venom production chain required to generate quality products for therapeutic human consumption.

Influence of apitoxin and melittin from Apis mellifera bee on Staphylococcus aureus strains.

The antibacterial activities of apitoxin, a venom produced by Apis mellifera bee, and melittin, an antimicrobial peptide from apitoxin, were tested against planktonic and biofilm states of Staphylococcus aureus methicillin-resistant (MRSA), including clinical, and enterotoxin-producing isolates. Also, the synergism of apitoxin and melittin in combination with oxacillin were evaluated as well. The induced morphological changes on S. aureus cells of both products were detected by transmission electronic microscopy (TEM). The minimum inhibitory concentration (MIC) values were 7.2 µg/mL, and 6.7 µg/mL, for apitoxin and melittin, respectively. The minimum bactericidal concentration (MBC) values were 28.7 µg/mL, and 26 µg/mL for apitoxin and melittin, respectively. The time-kill curve assays of apitoxin or melittin with oxacillin exhibited bactericidal synergism against MRSA isolates. TEM images showed cell distortion, cell disintegration with leakage of cytoplasmic content and loss of cytoplasm content. However, apitoxin and melittin did not interfere with staphylococcal enterotoxin production or release. Thus, apitoxin and melittin are potential agents against MRSA that can serve as possible models for new antibacterial drugs.

Proteomic analyses unraveling water stress response in two Eucalyptus species originating from contrasting environments for aridity.

Eucalyptus are widely cultivated in several regions of the world due to their adaptability to different climatic conditions and amenable to tree breeding programs. With changes in environmental conditions pointing to an increase in aridity in many areas of the globe, the demand for genetic materials that adapt to this situation is required. Therefore, the aim of this work was to identify contrasting differences between two Eucalyptus species under water stress through the identification of differentially abundant proteins. For this, total protein extraction was proceeded from leaves of both species maintained at 40 and 80% of field capacity (FC). The 80% FC water regime was considered as the control and the 40% FC, severe water stress. The proteins were separated by 2-DE with subsequent identification of those differentially abundant by liquid nanocromatography coupled to high resolution MS (Q-Exactive). Comparative proteomics allowed to identify four proteins (ATP synthase gamma and alpha, glutamine synthetase and a vacuolar protein) that were more abundant in drought-tolerant species and simultaneously less abundant or unchanged in the drought- sensitive species, an uncharacterized protein found exclusively in plants under drought stress and also 10 proteins (plastid-lipid, ruBisCO activase, ruBisCO, protease ClpA, transketolase, isoflavone reductase, ferredoxin-NADP reductase, malate dehydrogenase, aminobutyrate transaminase and sedoheptulose-1-bisphosphatase) induced exclusively in the drought-tolerant species in response to water stress. These results suggest that such proteins may play a crucial role as potential markers of water stress tolerance through the identification of species-specific proteins, and future targets for genetic engineering.

Development and comparative analysis of yeast protein extraction protocols for mass spectrometry.

Mass spectrometry is the most used method for protein identification and quantification. Here we developed four protein extraction protocols precisely for mass spectrometry, and we compared with other ones already published. The best protocol developed by us consists on a simple extraction solution, a heat-shock step, and does not use protease inhibitor; moreover, it is the most efficient and uniform among replicates, besides to be safe, cheap and fast. That method also provided the highest number of proteins uniquely identified and allows finding a diversity of protein classes, which their absence is a problem to be avoided.

Inferring putative virulence factors for Pythium insidiosum by proteomic approach.

Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening disease that affects human and animals, has difficult diagnosis, and therapy. Studies on protein characterization of P. insidiosum are scarce, so we aimed to determine the protein profile of P. insidiosum by mass spectrometry and bioinformatics strategies targeting in proteins that may act as putative virulence factors. Therefore, an extraction protocol was standardized to obtain the total proteins of P. insidiosum. By the analysis of Image Master 2D Platinum software, it was found that 186 spots ranging between 12 and 89 KDa and isoelectric point from 4 to 7. By the analysis of 2D-SDS-PAGE it was possible to visualize and excise 103 spots, which were hydrolyzed with trypsin and submitted to mass spectrometry, resulting in the identification of 36 different proteins. Three of them were classified as proteins supposedly related to virulence factors due to its functions, such as glucan 1,3-beta glucosidase, Heat shock protein (Hsp) 70 and enolase. These results may contribute to a better understanding of the virulence factors of this medically important oomycete, as well as to subsidize new studies on diagnosis and therapeutic approaches.

Molecular identification and phylogenetic analysis of Bothrops insularis bacterial and fungal microbiota.

Bothrops insularis, known as the golden lancehead snake, has its natural habitat restricted to Queimada Grande Island on the southern coast of Brazil. This culture-dependent study aimed to identify microorganisms obtained from the mouth, eyes, and cloaca of this species. Swabs from 20 snakes were collected for fungal and bacterial isolation. DNA was extracted from all samples, and identification was performed by amplifying the ITS1-5.8S-ITS2 regions and the 16S rDNA gene, respectively. All strains were identified and deposited in the GenBank nucleotide database. MEGA v6.0 software was utilized to construct phylogenetic trees. In total, 100 strains were isolated and characterized, from which 42 fungi were distributed into 23 species and 58 bacteria into 13 species. The genus Fusarium was predominant since 11 strains and probably a new species was isolated from this fungus. Pseudomonas aeruginosa and Enterococcus faecalis were the predominant groups of aerobic bacteria isolated. Phylogenetic analyses between bacterial and fungal sequences suggest a similarity between the microorganisms found on the island and on the continent. These findings may be attributed to anthropic actions resulting from both expeditions to the island and actions of migratory birds, which are the main sources of food for snakes.

Protein Content of Cervicovaginal Fluid Is Altered During Bacterial Vaginosis.

OBJECTIVES: The aim of the study was to compare, using a proteomic approach, cervicovaginal fluid (CVF) proteins of women with bacterial vaginosis (BV) with those presenting normal microbiota. MATERIALS AND METHODS: A total of 309 reproductive-aged women were cross-sectionally enrolled. Participants were tested for vaginal candidosis, Trichomonas vaginalis, Chlamydia trachomatis, and Neisseria gonorrhoeae and excluded if positive. Vaginal microbiota was classified microscopically according to Nugent criteria in normal, intermediate, and BV. Randomly selected CVF samples of 29 women with BV and an equal number with normal microbiota were subjected to proteomic analysis. Thus, a total of 58 CVF samples were evaluated using shotgun liquid chromatography-tandem mass spectrometry in a Q-Tof PREMIER API mass spectrometer (MicroMass/Waters) for peptide detection and relative quantification. RESULTS: Of the 309 women enrolled, 63 (20.4%) were excluded after testing positive for at least one of the tested co-infections or because of low-quality samples. Microscopic classification of vaginal microbiota on the remaining 246 samples revealed that 132 women (53.6%) had normal microbiota, 33 (13.4%) had intermediate microbiota, and 81 (33.0%) had BV. Proteomic analysis of CVF of 58 randomly selected women with normal microbiota (n = 29) or BV (n = 29) successfully identified 74 proteins. In addition, the comparison of abundance of those proteins between the groups showed that the following five (6.7%) were enriched in BV: neutrophil elastase, kaliocin-1, neutrophil defensin-1, Ig lambda-2 chain C regions, and protein S100-A7. All of which have a recognized role in host's immunity. CONCLUSIONS: Exclusive finding of BV affects immunity-related CVF components of reproductive-aged women.

Reassessment of the taxonomic position of Burkholderia andropogonis and description of Robbsia andropogonis gen. nov., comb. nov.

The phylogenetic classification of the species Burkholderia andropogonis within the Burkholderia genus was reassessed using 16S rRNA gene phylogenetic analysis and multilocus sequence analysis (MLSA). Both phylogenetic trees revealed two main groups, named A and B, strongly supported by high bootstrap values (100%). Group A encompassed all of the Burkholderia species complex, whi.le Group B only comprised B. andropogonis species, with low percentage similarities with other species of the genus, from 92 to 95% for 16S rRNA gene sequences and 83% for conserved gene sequences. Average nucleotide identity (ANI), tetranucleotide signature frequency, and percentage of conserved proteins POCP analyses were also carried out, and in the three analyses B. andropogonis showed lower values when compared to the other Burkholderia species complex, near 71% for ANI, from 0.484 to 0.724 for tetranucleotide signature frequency, and around 50% for POCP, reinforcing the distance observed in the phylogenetic analyses. Our findings provide an important insight into the taxonomy of B. andropogonis. It is clear from the results that this bacterial species exhibits genotypic differences and represents a new genus described herein as Robbsia andropogonis gen. nov., comb. nov.

Quantitative Proteomic Profiling Reveals That Diverse Metabolic Pathways Are Influenced by Melatonin in an in Vivo Model of Ovarian Carcinoma.

To obtain more information into the molecular mechanisms underlying ovarian cancer (OC), we proposed a comparative proteomic analysis in animals receiving long-term melatonin as therapy or only vehicle using multidimensional protein identification combined with mass spectrometry. To induce tumor, a single dose of 100 µg 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in 10 µL of sesame oil was injected under the left ovarian bursa of 20 Fischer 344 rats. The right ovaries were injected with sesame oil only. After tumors were developed, half of the animals received intraperitoneal administration of melatonin (200 µg/100g body weight/day) for 60 days. Melatonin therapy promoted down-regulation in numerous proteins involved in OC signaling pathways. The most significant portion of these proteins are involved in several metabolic processes, mainly those associated with mitochondrial systems, generation of metabolites and energy, hypoxia-inducible factor-1 signaling, antigen processing and presentation, endoplasmic reticulum stress-associated pathways, and cancer-related proteoglycans. A small number of proteins that were overexpressed by melatonin therapy included ATP synthase subunit ß, fatty acid-binding protein, and 10-kDa heat shock protein. Taken together, our findings suggest that melatonin therapy efficiently modulated important signaling pathways involved in OC, and these proteins might be further targets that should be explored in new therapeutic opportunities for OC.

Melatonin attenuates the TLR4-mediated inflammatory response through MyD88- and TRIF-dependent signaling pathways in an in vivo model of ovarian cancer.

BACKGROUND: Toll-like receptors (TLRs) are effector molecules expressed on the surface of ovarian cancer (OC) cells, but the functions of the TLR2/TLR4 signaling pathways in these cells remain unclear. Melatonin (mel) acts as an anti-inflammatory factor and has been reported to modulate TLRs in some aggressive tumor cell types. Therefore, we investigated OC and the effect of long-term mel therapy on the signaling pathways mediated by TLR2 and TLR4 via myeloid differentiation factor 88 (MyD88) and toll-like receptor-associated activator of interferon (TRIF) in an ethanol-preferring rat model. METHODS: To induce OC, the left ovary of animals either consuming 10% (v/v) ethanol or not was injected directly under the bursa with a single dose of 100 µg of 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in 10 µL of sesame oil. The right ovaries were used as sham-surgery controls. After developing OC, half of the animals received i.p. injections of mel (200 µg/100 g b.w./day) for 60 days. RESULTS: Although mel therapy was unable to reduce TLR2 levels, it was able to suppress the OC-associated increase in the levels of the following proteins: TLR4, MyD88, nuclear factor kappa B (NFkB p65), inhibitor of NFkB alpha (IkBα), IkB kinase alpha (IKK-α), TNF receptor-associated factor 6 (TRAF6), TRIF, interferon regulatory factor 3 (IRF3), interferon ß (IFN-ß), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-6. In addition, mel significantly attenuated the expression of IkBα, NFkB p65, TRIF and IRF-3, which are involved in TLR4-mediated signaling in OC during ethanol intake. CONCLUSION: Collectively, our results suggest that mel attenuates the TLR4-induced MyD88- and TRIF-dependent signaling pathways in ethanol-preferring rats with OC.

Using proteomic strategies for sequencing and post-translational modifications assignment of antigen-5, a major allergen from the venom of the social wasp Polybia paulista.

Antigen-5 is one of the major allergens identified in wasp venoms, and despite the fact that its biological function is still unknown, many studies have demonstrated its allergenicity. In this study, the biochemical and structural characterization of antigen-5 from the venom of the social wasp Polybia paulista are reported. A gel-based mass spectrometry strategy with CID fragmentation methods and classical protocols of protein chemistry, which included N- and C-terminal sequencing, were used to assign the complete sequence and determine the presence/location of the post-translational modifications (PTMs) of this protein. Six different isoforms of antigen-5 were identified in the crude venom of P. paulista ; the most abundant, which corresponds to the intact form of this protein, was recognized by the pool of human specific-IgE. This protein was extensively sequenced through CID mass spectrometry, and a series of PTMs were observed such as hydroxylation, phosphorylation, and glycosylation. Sequence data revealed that this protein has 59.3-93.7% identity with antigen-5 proteins from other known vespid venoms. The molecular model of P. paulista antigen-5 shows that this protein has three α-helices, one 310 helix, and four ß-sheets covering 28 and 17.9% of the sequence, respectively. The identification and characterization of allergenic compounds is essential for the development of advanced component-resolved allergy diagnostics and treatment.

Epstein-Barr Virus miR-BARTs 7 and 9 modulate viral cycle, cell proliferation, and proteomic profiles in Burkitt lymphoma.

The Epstein-Barr Virus (EBV) encodes viral microRNAs (miRs) that have been implicated in the pathogenesis of nasopharyngeal and gastric carcinomas, yet their potential roles in lymphomas remain to be fully elucidated. This study evaluated the impact of CRISPR/Cas9-mediated knockdown of EBV miRs BART-7 and BART-9 in EBV-positive Burkitt lymphoma cells Akata. As anticipated, the Akata cells subjected to CRISPR/Cas9-mediated knockdown of either EBV BART-7 or BART-9 exhibited a significant reduction in the expression of these viral miRs compared to cells with wild-type (wt) EBV genomes. This outcome effectively validates the experimental model employed in this study. Knocking down either BART-7 or BART-9 resulted in a notable reduction in cell viability and proliferation rates, alongside an elevation in the expression of EBV lytic genes. Global proteomic analysis revealed that the knockdown of EBV BART-7 significantly decreased the expression of ubiquitin/proteasome proteins while concurrently increasing RNA binding proteins (RBPs). Conversely, BART-9 knockdown reduced proteins associated with oxidoreductase activity, particularly those involved in fatty acid metabolism. Our findings unveil previously undiscovered EBV miRs BARTs 7 and 9 roles in cellular pathways relevant to both viral biology and lymphomagenesis.

Digestion of whey peptide induces antioxidant and anti-inflammatory bioactivity on glial cells: Sequences identification and structural activity analysis.

Whey derived peptides have shown potential activity improving brain function in pathological condition. However, there is little information about their mechanism of action on glial cells, which have important immune functions in brain. Astrocytes and microglia are essential in inflammatory and oxidative defense that take place in neurodegenerative disease. In this work we evaluate antioxidant and anti-inflammatory potential bioactivity of whey peptide in glial cells. Peptides were formed during simulated gastrointestinal digestion (Infogest protocol), and low molecular weight (<5kDA) peptides (WPHf) attenuated reactive oxygen species (ROS) production induced by hydrogen peroxide stimulus in both cells in dose-dependent manner. WPHf induced an increase in the antioxidant glutathione (GSH) content and prevented GSH reduction induced by lipopolysaccharides (LPS) stimulus in astrocytes cells in a cell specific form. An increase in cytokine mRNA expression (TNFα and IL6) and nitric oxide secretion induced by LPS was attenuated by WPHf pre-treatment in both cells. The inflammatory pathway was dependent on NFκB activation. Bioactive peptide ranking analysis showed positive correlation with hydrophobicity and negative correlation with high molecular weights. The sequence identification revealed 19 peptides cross-referred with bioactive database. Whey peptides were rich in leucine, valine and tyrosine in the C-terminal region and lysine in the N-terminal region. The anti-inflammatory and antioxidant potential of whey peptides were assessed in glia cells and its mechanisms of action were related, such as modulation of antioxidant enzymes and anti-inflammatory pathways. Features of the peptide structure, such as molecular size, hydrophobicity and types of amino acids present in the terminal region are associated to bioactivity.

The combination of omics strategies to evaluate starter and probiotic strains in the Catharina sour Brazilian-style beer.

Catharina sour, the first internationally recognized Brazilian beer, is characterized by fermentation with lactic acid bacteria (LAB), which may have probiotic potential, and the addition of fruit juice. This study aimed to evaluate the use of the starter Streptococcus thermophilus TH-4 (TH-4) and the probiotics Lacticaseibacillus paracasei F19 and 431, associated with Saccharomyces cerevisiae US-05, in the absence (control)/presence of passion fruit or peach juices. Evaluation proceeded during fermentation and storage by enumeration using pour-plate and qPCR; gene expressions of hop resistance; proteome by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS); and odor, flavor, and metabolome by Headspace Solid-Phase Microextraction (HS-SPME), coupled with the gas chromatography-mass spectrometry (GC-MS) analysis. We concluded that the strains studied are recommended for applications in sour beers, due to the presence of defense mechanisms like membrane adhesion and H + pump. Furthermore, HS-SPME/GC-MS indicated that the strains may contribute to the beer flavor and odor.

Digestion-resistant whey peptides promote antioxidant effect on Caco-2 cells.

Enteric endothelial cells are the first structure to come in contact with digested food and may suffer oxidative damage by innumerous exogenous factors. Although peptides derived from whey digestion have presented antioxidant potential, little is known regarding antioxidant pathways activation in Caco-2 cell line model. Hence, we evaluated the ability to form whey peptides resistant to simulated gastrointestinal digestive processes, with potential antioxidant activity on gastrointestinal cells and associated with sequence structure and activity. Using the INFOGEST method of simulated static digestion, we achieved 35.2% proteolysis, with formation of peptides of low molecular mass (<600 Da) evaluated by FPLC. The digestion-resistant peptides showed a high proportion of hydrophobic and acidic amino acids, but with average surface hydrophobicity. We identified 24 peptide sequences, mainly originated from ß-lactoglobulin, that exhibit various bioactivities. Structurally, the sequenced peptides predominantly contained the amino acids lysine and valine in the N-terminal region, and tyrosine in the C-terminal region, which are known to exhibit antioxidant properties. The antioxidant activity of the peptide digests was on average twice as potent as that of the protein isolates for the same concentration, as evaluated by ABTS, DPPH and ORAC. Evaluation of biological activity in Caco-2 intestinal cells, stimulated with hydrogen peroxide, showed that they attenuated the production of reactive oxygen species and prevented GSH reduction and SOD activity increase. Caco-2 cells were not responsive to nitric oxide secretion. This study suggests that whey peptides formed during gastric digestion exhibit biological antioxidant activity, without the need for previously hydrolysis with exogenous enzymes for supplement application. The study's primary contribution was demonstrating the antioxidant activity of whey peptides in maintaining the gastrointestinal epithelial cells, potentially preventing oxidative stress that affects the digestive system.

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.

Synergistic Antibacterial Efficacy of Melittin in Combination with Oxacillin against Methicillin-Resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) often cause infections with high mortality rates. Antimicrobial peptides are a source of molecules for developing antimicrobials; one such peptide is melittin, a fraction from the venom of the Apis mellifera bee. This study aimed to evaluate the antibacterial and antibiofilm activities of melittin and its association with oxacillin (mel+oxa) against MRSA isolates, and to investigate the mechanisms of action of the treatments on MRSA. Minimum inhibitory concentrations (MICs) were determined, and synergistic effects of melittin with oxacillin and cephalothin were assessed. Antibiofilm and cytotoxic activities, as well as their impact on the cell membrane, were evaluated for melittin, oxacillin, and mel+oxa. Proteomics evaluated the effects of the treatments on MRSA. Melittin mean MICs for MRSA was 4.7 µg/mL and 12 µg/mL for oxacillin. Mel+oxa exhibited synergistic effects, reducing biofilm formation, and causing leakage of proteins, nucleic acids, potassium, and phosphate ions, indicating action on cell membrane. Melittin and mel+oxa, at MIC values, did not induce hemolysis and apoptosis in HaCaT cells. The treatments resulted in differential expression of proteins associated with protein synthesis and energy metabolism. Mel+oxa demonstrated antibacterial activity against MRSA, suggesting a potential as a candidate for the development of new antibacterial agents against MRSA.