Introduction and Goals: Cancer pain produces severe distress and disrupt the life quality of patients and very often is not effectively treated. Opioids are practically the only analgesics capable of controlling cancer pain but this therapy leads to distinct side effects that limit its use. Methadone is a valuable opioid analgesic, which can be administered in case of cancer pain and can reverse other opioids tolerance like morphine. However, methadone has some side effects as other opioids. Phα1β toxin from the spider Phoneutria nigriventer has an antinociceptive action in several models of pain in rodents and it is known that it induces analgesic effect in a model of cancer pain in mice. This toxin is a dual blocker of TRPA1 channels and voltage-gated calcium channels and exhibits greater selectivity for N-type channels. One strategy to improve the therapeutic utility of opioids is to co-administer with other analgesic agents, such as Phα1β toxin, looking for overall dose reduction and also reducing side effects to improving the quality of analgesia. This work aims to analyze by an Isobolographic analysis whether antinociceptive interaction of Methadone and Phα1β is subadditive, additive, or synergic. Methodology: B16F10 cells inoculation was performed on the right paw on C57BL/6J for tumor induction. The PWT (Von-Frey filaments) was measured before (baseline), at day 7 and at day 14 before and after drug treatment. N= 6-9 per group. Dose-response curves of drugs alone or in combination were performed using a fixed proportion design. Data interpretation was performed using isobolographic analysis to determine the interaction index of the combination. To evaluate the possible side effects from this combination, the Open Field test, Rotarod test and quantification of the gastrointestinal transit were performed. To check whether the combination is capable of reversing morphine tolerance induced by several morphine doses, the protocol with tail-flick apparatus was used. All the procedures were authorized by CEPEEA, the Ethics Committee in animals’ experimentation from Santa Casa of Belo Horizonte Education and Research (Protocol 002/2018). Results: Fourteen days after B16-F10 right hind paw inoculation, marked hyperalgesia was induced measured by Von Frey filaments. This hyperalgesia was reversed by the i.t. treatment with Phα1β at 100 pmol/site and also by the methadone s.c injection at 1mg/kg, other doses were tested and the final dose used to combine these two drugs were made as described by Tallarida in a protocol of fixed proportions by two components. The antinociceptive effect of Phα1β and Methadone was dose-dependent, with ED50 values of 1.076 pmol/site for Phα1β and 86.849 pmol/site for Methadone. The combination of Phα1β + Methadone has an ED50 which is lower than the theoretical additive ED50 (p<0.05), indicating synergism. The required dose to reach synergism is 20 pmol/site for Phα1β (IC95%: 7-58pmol/site) and 15.410 pmol/site for Methadone (IC95%: 5.480-41.680pmol/site). Since we have observed strong potentiation in the analgesic effect, we assess the animal behavior in an open field, as well as motor impairment checking fall latency and intestinal motility after the administration of drugs alone or in combination at ED50 doses. No changes in animal behavior were observed in an open field (traveled distance, movement number and movement time duration) after drug administration, p>0,05; the same occurs in latency to fall, no difference was seen between the groups p>0,05. Combined drugs reduced 27% of gastrointestinal transit compared with the control group (p=0,02), whereas the two drugs alone did not significantly differ with the control group (p<0,05). Methadone is currently used to reverse morphine tolerance, methadone given alone at its ED50 dose was able to reverse morphine-induced tolerance in animals with significant difference (p<0,05) to control (PBS). Phα1β also reversed morphine tolerance as described earlier. At lower doses, Phα1β + Methadone ED50 value reversed morphine tolerance without significant difference (p>0,05) to isolated compounds but statistically different to the control group (p<0,05). Conclusions: Our data show that synergism occurred when s.c methadone was administered simultaneously with i.t Phα1β suggesting potentiation on the analgesic effect of these drugs when both are added together. Even with strong potentiation in the analgesic effect, no relevant side effects associated with this combination were observed. In addition to producing an antinociceptive effect, the combination of these compounds was able to reverse morphine-induced tolerance.

Herbal supplements are natural products, which are traditionally considered "helpful or at least harmless" for health promotion. Consumption of herbal products increases, but their safety, especially mycotoxin contamination, is still poorly controlled. Recent surveys report the occurrence of Aspergillus and Penicillium metabolites (aflatoxins (AFLs), ochratoxin A (OTA), cyclopiazonic and mycophenolic acids (MPA), sterigmatocystin (STE), citrinin), Fusarium (trichothecenes, zearalenone (ZEA), fumonisins (FBs), enniatins (ENNs)) and Alternaria (alternariol (AOH), its methyl ether (AME), tentoxin (TTX) and tenuazonic acid) toxins. A significant part of herbal supplements is consumed in the form of infusion. Thus correct risk assessment needs evaluation of mycotoxins transfer rates from the herbal matrix into the solution. We have studied the transfer of AFLs, OTA, STE, deoxynivalenol (DON), ZEA, FBs, T-2 and HT-2 toxins, AOH, AME, TE, ENNs, beauvericin and MPA from the spiked herbal matrix into infusion at different pH and total dissolved solids (TDS) characteristics of the water used for its preparation. Analytes were detected by HPLC-MS/MS. Transfer rate proved to be dependent on mycotoxins polarity and pH of the resulting infusion. TDS did not affect transfer significantly. ENNs, BEA, STC, ZEA and AOH transfer into infusion was below 25%; AFLs – 25-45%; DON, TTX and T-2 toxins – 60-90%, FB1 – 80-100%. The concentration of OTA, MPA and FB2 in the infusion depended on its pH. At pH about 4, it proved to be about 20%, 40% and 60% correspondingly. The increase of infusion pH led to almost complete transfer of these mycotoxins into the solution. The study of naturally contaminated samples supported the results of the model experiments.

Scorpionic poisoning is a public health problem, due to the high number of cases registered not only in Brazil but in the world, mainly in tropical and subtropical areas. It is known that scorpion poisoning can cause problems ranging from simple local manifestations, such as small edema, to serious problems, such as cardiocirculatory complications, which can lead to death. In the case of poisoning of women during pregnancy, there are risks for both the mother and the fetus, causing the death of both in extreme cases. In previous studies, we observed that when the venom of the scorpion Tityus bahiensis is administered to rats during pregnancy or lactation, changes in the physical, reflexological and behavioral development of the offspring occur, both in the perinatal phase and in adulthood, as well as changes in levels of some cytokines and neurotransmitters. Serotherapy is the most suitable method for treating scorpion poisoning. However, there are very few studies regarding the effects that antivenom can have on the fetus, whether beneficial or not. Therefore, this project aims to study and elucidate the effects of perinatal scorpion serotherapy, checking if there is any physiological change in the fetus, as well as if there is a reversal of the changes caused by the poisoning of their mothers.

Non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is a bioactive molecule synthesized by various phytoplankton species, such as cyanobacteria, diatoms and dinoflagellates, and is known to be a causative agent of human neurodegeneration diseases. The ability of different microalgae to synthesize BMAA may be an indicator of the importance of this molecule in the interaction of phytoplankton organisms in nature. We were interested in the question: what kinds of mechanisms underline BMAA’s action on cyanobacterial cells under different nitrogen supply conditions. To answer this question we have performed molecular studies using a model cyanobacterial strain Nostoc (Anabaena) sp. PCC 7120. We have experimentally shown that the action of BMAA on nitrogen-fixing filamentous cyanobacteria changes nitrogen-carbon balance regulation, and differs under nitrogen starvation and in nitrogen-replete conditions. The primary main targets of BMAA’s action in cyanobacteria cells are, apparently, metabolic processes, such as nitrogen fixation, photosynthesis, carbon fixation and different biosynthetic processes, the regulation of which involves 2-oxyglutarate and glutamate. Our proteomic study has demonstrated that under BMAA-treatment the most significant difference lies in the expression change of a key nitrogen regulatory protein PII. This protein is downregulated in nitrogen-starving conditions and it is upregulated in nitrogen-replete conditions in the presence of BMAA. This could be the main reason behind a specific regulatory effect on heterocyst formation and heterocyst- and nitrogenase-related gene expression that this amino acid causes in Nostoc sp. PCC 7120. Due to the fact that all metabolic processes are interconnected and well balanced in cyanobacteria cells, the disturbance in nitrogen metabolism leads to changes in carbon metabolism and photosynthesis. This explains the severe changes of CO₂ fixation proteins and photosystem reaction centre proteins that were found in our proteomics studies. BMAA addition leads to disorder in both amino acid synthesis and in purine synthesis, as well as disturbs DNA transcription and protein translation. Finally, many enzymes of oxidative stress, chaperones and SOS-response proteins are upregulated under such metabolic stress conditions. Therefore we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as: starvation-inducible DNA-binding protein, stress-response enzymes, proteases and SOS-response and DNA repair enzymes. It can be hypothesized that BMAA could be used by phytoplankton representatives (cyanobacteria, diatom, dinoflagelates) as a possible allelopatic tool to control cyanobacteria cell populations during their competition for nitrogen and other resources.

Ontogenetic changes in venom composition have been described in Bothrops snakes but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in venom modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic PLA2s were the most abundant components in larger snakes while in venoms from smaller snakes, PIII-class SVMPs were the major components. The SVMPs identified corresponded to novel sequences and conferred both pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may be related to the advantage of coagulant and hemorrhagic venoms from small snakes to predatory function.

In the Brazilian Amazon, there is a significant occurrence of snake bites, predominated by Bothrops atrox. Tissue damage is one of the hallmarks in B. atrox envenoming. Interestingly, many snakebites patients have a delayed onset of blistering with a concomitant increase of the risk of infections. We hypothesize that blister fluid may represent a window into the pathophysiology at the injured tissues. In this study, we examined blister fluid by proteomics from 5 patients hospitalized with B. atrox envenomation, who were successfully treated with antivenom with no long-lasting effects nor morbidities. The proteomic data of the blister fluids correlated with previous blister fluid studies showing the presence of DAMPs and immunomodulators. The blister composition was observed to be similar among the patients regardless of the clinical severity of envenomation. An unprecedented additional finding was that we identified venom and antivenom proteins in the bite site by ELISA. Venom was quantified in the fluid a significant time after envenommation (up to135 hours), suggesting a slow clearance of venom at the site of bite, which might have influence on local tissue well after the time of envenomation. Antibodies from the administered antivenom identified in the blister fluids were shown capable of binding venom proteins by Western blotting. Thus, blister fluid antibodies should be capable of neutralizing the any venom components in the fluid. Taken together, these findings suggest that although blistering is a delayed phenomenon of envenomation, its likely pathophysiological origins occur in advance of antivenom administration and venom neutralization at the site of envenomation and continues despite the eventual neutralization of venom. This evidence confirms previous reports that the early events in envenomation pathophysiology give rise to endogenous factors that over time, contribute to the development of blisters which are not attenuated even by prompt antivenom administration.

Hemolysin II of Bacillus cereus sensu lato is synthesized in a bacterial cell in the form of a water-soluble secreted monomer and penetrates into eukaryotic membranes. The HlyII protein has a C-terminal extension (HlyIICTD), includes 94 amino acid residues [1]. Removal of HlyIICTD from HlyII significantly complicates transfer of the deletion variant HlyIIDCTD to E. coli cells, possibly due to the attack of the bacterial membrane. Additional deletion of the signal peptide, which excludes the penetration of the protein into the periplasm, provides E. coli cells survival carrying this gene with two deletions. Using monoclonal antibodies against recombinant HlyIICTD [2], showed a similar the binding effectively to red blood cells of various origins and noticeably differ for cells of the J774 and Jurkat lines. HlyIICTD in water solution is able to form oligomeric structures. In the presence of membrane HlyIICTD exits in oligomeric form while monomeric forms are almost completely absent. HlyIICTD trimerized in the presence of 4M urea, forming a possibly some structure that can be integrated into the artificial bilayer membrane with the formation of pores. The current-voltage characteristic of these channels was determined. Such protein structures are characteristic of trimeric autotransporter proteins [3]. In this case, the secreted full-sized monomeric form of hemolysin II acts as a passenger, and HlyIICTD acts as an element involved in adhesion to membrane and secretion from bacterial cells. The materials presented in this paper demonstrate suggests that hemolysin II may belong to trimeric autotransporter proteins – the first case of the description of this family of molecules among Gram positive microorganisms.

Acknowledgments. The study was supported by a grant from the Russian foundation for basic research (no. 14-04-00592) and by the Ministry of Science and Higher Education of the Russian Federation (unique project number RFMEFI60419X0218).

1. Baida G., Budarina Z. I., Kuzmin N.P., Solonin A.S. Complete nucleotide sequence and molecular characterization of hemolysin II gene from Bacillus cereus, FEMS Microbiol. 1999, V.180. pp. 7–14.
2. Rudenko, N.V., Karatovskaya, A.P., Zamyatina, A.V., Siunov, A.V., Andreeva-Kovalevskaya, Zh.I., Nagel, A.S., Brovko, F.A., and Solonin, A.S., Russ. J. Bioorg. Chem., 2020, V. 46, pp. 321–326. doi.org/ https://doi.org/10.1134/S1068162020030188
3. Kiessling A.R., Malik A., Goldman A. Recent advances in the understanding of trimeric autotransporter adhesins. Medical Microbiology and Immunology 2020, V. 209, pp 233–242 https://doi.org/10.1007/s00430-019-00652-3

Ciguatera fish poisoning (CFP) is one of the most relevant seafood-borne diseases worldwide. It is caused by the ingestion of fish containing ciguatoxins (CTXs), lipophilic marine toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa that accumulate into fish flesh and through the food webs. CFP is characterized by severe neurological, gastrointestinal, and cardiovascular disorders and affects approximately between 50,000 and 500,000 consumers annually worldwide. Real incidence of CFP is difficult to ascertain, due to under-reporting and misdiagnosis.

Here, the first electrochemical immunosensor for the detection of CTXs is presented. Three different monoclonal antibodies (mAbs), two capture (3G8, 10C9) and a detector (8H4), were merged in a sandwich configuration for the combined detection of two main groups of CTX congeners (CTX1B and CTX3C ). Initially, the applicability of the immunosensor has been demonstrated with the analysis of fish samples coming from La Réunion island, providing results that correlate with mouse bioassay and cell-based assay. Then, fish coming from Mediterranean waters were analysed, giving promising results. Finally, extracts from Gambierdiscus and Fukuyoa were screened, allowing the separate detection of the two groups of CTX congeners, and giving new information regarding the toxin production of the genera.

The developed bioanalytical tool is user-friendly, and can help to mitigate ciguatera risk, contributing to the protection of consumers health.

Venomous mammals are rare and their venoms have not been comprehensively investigated. Among shrews, only venoms of the short-tailed shrew Blarina brevicauda and the Eurasian water shrew Neomys fodiens have been characterized thus far. Neomys fodiens employs its venom to hunt on larger prey and store it in a comatose state. Recently, the potent paralytic activity of its venom has been confirmed. Here we assayed the hemolytic effects of crude extracts of salivary glands of N. fodiens and the common shrew Sorex araneus in the red blood cells of frog. Toxins present in saliva were identified by high-performance liquid chromatography coupled to tandem mass spectrometry. For both shrew species we found significant concentration-dependent effects of venom on hemolysis in erythrocytes evaluated as hemoglobin release. Hemolytic effects of N. fodiens saliva were stronger than those produced by S. araneus. We identified four toxins in N. fodiens venom and five in the saliva of S. araneus. Some of them are likely to produce hemolysis in frog’s erythrocytes. Our results show that shrew venoms, in addition to potent paralytic properties, possess also hemolytic activity that may allow them to hunt larger prey as frogs. Additionally, because S. araneus saliva exhibits toxic activity we propose to add the common shrew to the list of venomous mammals.

Between 2010 and 2019, 2.432 gulls (Larus michahellis and Larus fuscus) with paretic syndrome were received at RIAS Wildlife Rehabilitation and Research Centre. The clinical signs included weakness, anorexia, paralysis, diarrhoea (flaccid cloacae), dyspnoea and, in some cases, death. Several biotic contaminants are among the potential cause of this syndrome: marine biotoxins, Clostridium botulinum, cyanotoxins and virus. This presentation compiles the results of the Clostridium botulinum and marine biotoxins analysis conducted at the French National Reference Centre for anaerobic bacteria and botulism, Pasteur Institute (Paris) and the Vigo Centre of the Spanish Oceanographic Institute, respectively.
C. botulinum analyses were conducted in livers and intestines from 5 gulls with paretic syndrome symptoms admitted at RIAS Wildlife Rehabilitation and Research Centre. Samples were pooled in two groups according to the tissue and analysed by targeted Real Time Polymerase Chain Reaction (PCR) on neurotoxin genes after sample enrichment culture under anaerobic conditions. The presence of botulinum toxin was confirmed by a lethality test on mice (mouse bioassay). Mice were intraperitoneally injected with filtered supernatant of the culture.
Paralytic shellfish toxins (PSTs) were analysed by Liquid Chromatography with Fluorescence Detection and Post-column Oxidation in samples from ten gull kidneys and in the cloacae contents from another gull. Domoic acid (DA) analysis was conducted following a procedure that involved a methanolic extraction and analysis by Liquid Chromatography coupled to High Resolution Mass Spectrometry. DA was analysed in twenty three gull samples: ten livers, ten intestines and three cloacae contents. PSTs and DA were not detected in any of the samples tested.
Results obtained so far point to C. botulimum type C/D as the causative agent of the paretic syndrome in gulls.