Key parameters to enhance the antibacterial effect of graphene oxide in solution.

Graphene oxide (GO) has lately become an interesting biomaterial due to its stunning properties and versatility, its claimed antimicrobial activity holds promise for potential health applications. Nonetheless, multiple reports investigating GO antibacterial activity lack rigor and uniformity on several aspects which are crucial when evaluating this effect. In this work, we highlight and address these parameters: morphology of the materials, exposure time, exposure methodology and concentration. We investigate the effect of GO and GO-based metallic composites observing these parameters on two pathogenic bacteria. Our nanomaterials have been characterized by means of SEM, EDX, DLS, FTIR and Raman spectroscopies. Escherichia coli and Salmonella Typhimurium suspended in saline solutions (no growth medium) have been exposed to GO (lateral size = 100 nm), silver nanoparticles, ceria nanoparticles, GO/silver and GO/ceria aqueous solutions for 0, 5, 15, 30, 60 and 90 minutes, before plating. Our experiments indicate that no prior exposure of the materials to bacteria (0 min) results in poor inactivation rates independently of concentration, while increasing times of interaction enhance inactivation. Moreover, our experiments show concentration-dependent results showing higher activity for concentrations of 100 µg mL-1; and prove that 30 minutes of exposure are sufficient to deploy the antimicrobial effects of these materials. GO possesses the lowest inactivation rate, and the presence of silver and ceria nanoparticles in the GO surface boosts its antimicrobial effect. Thus, the enhancement of the antibacterial activity of graphene oxide relies on 30 minutes of interaction in water, concentration of 100 µg mL-1, and its decoration by silver/ceria nanoparticles.

Identification of protective B-cell epitopes of Atroxlysin-I: A metalloproteinase from Bothrops atrox snake venom.

Atroxlysin-I (Atr-I) is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops atrox venom, the snake responsible for the majority of bites in the north region of South America. SVMPs like Atr-I produce toxic effects in victims including hemorrhage, inflammation, necrosis and blood coagulation deficiency. Mapping of B-cell epitopes in SVMPs might result in the identification of non-toxic molecules capable of inducing neutralizing antibodies and improving the anti-venom therapy. Here, using the SPOT-synthesis technique we identified two epitopes located in the N-ter region of Atr-I (AtrEp1-(22)YNGNSDKIRRRIHQM(36); and AtrEp2-(55)GVEIWSNKDLINVQ(68)). Based on the sequence of AtrEp1 and AtrEp2 a third peptide named Atr-I biepitope (AtrBiEp) was designed and synthesized ((23)NGNSDKIRRRIH(34)GG(55)GVEIWSNKDLINVQ(68)). AtrBiEp was used to immunize BALB/c mice. Anti-AtrBiEp serum cross-reacted against Atr-I in western blot and was able to fully neutralize the hemorrhagic activity of Atr-I. Our results provide a rational basis for the identification of neutralizing epitopes on Atr-I snake venom toxin and show that the use of synthetic peptides could improve the generation of immuno-therapeutics.

Serological, biochemical and enzymatic alterations in rodents after experimental envenomation with Hadruroides lunatus scorpion venom.

Toxic effects of Peruvian Hadruroides lunatus scorpion venom on different biochemical and enzymatic parameters in blood serum of Wistar rats and Swiss mice were determined after experimental envenomation. An increase in enzymatic activities of Aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and levels of serum protein and albumin were observed while a decrease in creatinine level in serum was perceived after 30 min of envenomation. No alterations in urea levels and in kidney histology were detected in the envenomed rats. The global leukocytes count was diminished, with decrease in lymphocytes, eosinophils and neutrophils levels in the bloodstream, while no alterations were found in hematological parameters of red series in rats injected with H. lunatus venom. IL-2, IL-4, IL-6, INF-γ, TNF, IL-17A and IL-10 levels were evaluated 0.5, 3 and 6 h after experimental envenomation of mice with H. lunatus venom. From all the analyzed cytokines, only IL-6 showed an increase in serum levels. Taken together, these results point out that envenomation by H. lunatus can impair hematological and immunological parameters and therefore might be monitored in accidents involving this species.

Identification and characterization of B-cell epitopes of 3FTx and PLA(2) toxins from Micrurus corallinus snake venom.

The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.