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  • Open access
  • 191 Reads
First Exploration of the Mesobuthus Cyprius Venom
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

The evolutionary history of the scorpions begun around 425 – 450 million years ago, in the middle Silurian. More than 1500 species have been recognized and classified in different families. Mesobuthus cyprius, one of the two endemic scorpions in Cyprus, belongs in the family of Buthidae which is geographically distributed worldwide and is the largest of the scorpion families. Moreover, from a clinical perspective, Buthidae is the most important scorpion family as several members of this family are toxic to mammals and can be dangerous to humans. Even though Mesobuthus cyprius was discovered in 2000 using molecular phylogenetics there are no other published data regarding the peptide and protein composition, the toxicity, or any other activity of the venom.

For this research work, several specimens were collected, and their venom composition was studied using Liquid Chromatography tandem Mass Spectrometry techniques (LC-PDA-MS and UPLC-TOF-MS). Furthermore, the comparison of the venom of Mesobuthus Cyprius with the venom of Mesobuthus gibbosus was performed, the closest member of the family, common in Greece and Turkey. The same venoms were studied with solution state Nuclear Magnetic Resonance Spectroscopy (NMR). Finally, we tested the venom for its ability to cause cell death in a number of cancer cell lines.

  • Open access
  • 231 Reads
Smartphone-based electrochemical immunosensor for ciguatoxins detection
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Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Ciguatera fish poisoning (CFP) is the most common and one of the most relevant seafood-borne diseases worldwide. CFP is caused by the ingestion of fish contaminated with ciguatoxins (CTX), potent lipophilic marine toxins with complex chemical structures produced by microalgae of the genus Gambierdiscus and Fukuyoa, which are transferred and metabolised through the food webs. The importance of CTXs in seafood safety and their emerging occurrence in locations far away from the tropical areas were they have been historically found, highlight the need for alternative analytical methods for their rapid, simple and cost-effective detection. In this sense, a portable electrochemical biosensor for the detection of CTXs is presented. Two different capture antibodies able to recognise the left wing of CTX1B and 54-deoxyCTX1B and the left wing of CTX3C and 51-hydroxyCTX3C are immobilised on multi-walled carbon nanotube-modified electrodes. A sandwich configuration is adopted by the use of a biotinylated antibody that binds to the right wing of these four CTX congeners. PolyHRP-streptavidin is used as enzymatic label for signal amplification and detection of the biotinylated antibody. Amperometric currents are recorded with a small and ready-to-go potentiostat inserted in a smartphone, providing in situ measurements. A CTX1B calibration curve is obtained, achieving a limit of detection at the pg/mL level. After the evaluation of the matrix effects, the ability of the immunosensor to detect CTX1B contents at the 0.01 µg/kg guidance level proposed by the United States Food and Drug Administration (US FDA) is demonstrated. The biosensor is being applied to the analysis of naturally contaminated fish samples and results will be compared with those obtained by cell-based assay (CBA) and liquid chromatography coupled to mass spectrometry (LC/MS). This portable, easy-to-handle, rapid and low-cost analytical tool will facilitate the monitoring of CTX contents to guarantee seafood safety.

  • Open access
  • 39 Reads
Impaired broiler performance and intestinal integrity as a carry-over effect in broilers fed diets naturally contaminated with moderate levels of deoxynivalenol
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Natural exposure to mycotoxins is a common event in the poultry industry. Deoxynivalenol (DON) is usually detected at levels lower than the maximum recommended ones (5,000 ppb). However, depending on the diet and bird age, such low levels might be sufficient to induce intestinal damage and impair broiler performance. We evaluated the effect of 900 and 2,300 ppb DON with or without activated charcoal as a binding agent on performance and intestinal morphometry and lesions in broilers. 736 day-old male Ross 308 broilers were divided into four treatments with eight replicates. The broilers were fed diets naturally contaminated with low DON (LD; 900 ppb) or moderate DON (MD; 2,300 ppb) with or without activated charcoal, for 28 days. Afterward, all birds were fed a diet without DON or activated charcoal for 7 days. During the first 28 days of the trial, MD without activated charcoal significantly reduced body weight gain and FCR. Even after the 7-d wash-out period, MD resulted in an overall significantly reduced body weight gain and FCR, regardless of the presence of activated charcoal. At 28 d of age, MD diet without activated charcoal caused a decrease in jejunum villus height and an increase in ileum crypt depth, thereby reducing villus:crypt ratio in both intestinal segments. Based on these results, it can be concluded that broiler production and intestinal morphology are negatively affected when feed is contaminated with DON even at moderate levels (2,300 ppb), and performance losses are not recovered even if the broilers are fed a non-contaminated diet afterward.

  • Open access
  • 105 Reads
Expression and purification of rTs7, a recombinant toxin from Tityus serrulatus scorpion venom
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Tityus serrulatus venom is composed of several substances, including the neurotoxins that interact with voltage-gated ion channels. These channels are involved in many diseases, such as arrhythmia, asthma, autoimmune diseases, hypertension and immune response to infection and inflammation, making the T. serrulatus venom an important tool to study them. The Ts7, also called TsTx-K-alpha, acts selectively on potassium channels, and can contribute to the treatment of Kv1.3 channel-related diseases, being this channel a potential therapeutic target in the treatment of autoimmune diseases. In this work, we present the heterologous expression of Ts7 in Pichia pastoris yeast and its purification. The toxin gene was synthesized with TEV (tobacco etch virus) protease cleavage site before N-terminal sequence and cloned into pPICZαA vector. The P. pastoris cells (KM71H strain) were transformed with the linearized plasmid rTs7_pPICZαA, by electroporation. Transformation was confirmed by PCR of selected colonies and 1% agarose gel electrophoresis. Positively transformed colonies were submitted to a screening in a 24-wells plate, under standard conditions (pH6, for 144h), in order to determine the maximum expression rate. The colony showing the highest expression level of the recombinant protein was selected for laboratorial-scale expression, and the progress of expression was monitored by SDS-PAGE. The expressed protein was purified through immobilized metal affinity chromatography (IMAC) followed by reversed-phase chromatography on a C-18 column. In the reversed-phase chromatography, three fractions were observed and, after mass spectrometry analysis, the rTs7 was identified in fraction 3, and fractions 1 and 2 were possibly the cleaved toxin. The rTs7 was successfully expressed and purified, with a satisfactory yield of the recombinant toxin, which showed high similarity with the native toxin. The rTs7 immunosuppressive activity in multiple sclerosis model will be further investigated.

Financial support: CAPES, CNPq, FAPESP.

  • Open access
  • 86 Reads
Heterologous expression of a neurotoxin from Tityus serrulatus scorpion venom in Pichia pastoris yeast and the evaluation of its glycosylation patterns
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Tityus serrulatus is the most dangerous species of scorpion in Brazil. Its venom (TsV) has mainly neurotoxins, which can on sodium or potassium channels and are responsible for most envenoming symptoms. The evaluation of these toxins can elucidate their mechanisms as well as contribute to a more specific therapy. The aim of this study was the expression of Ts15, an α-KTx from TsV, in Pichia pastoris and its characterization. rTs15 gene was synthetized by GenScript® with TEV (tobacco etch virus) protease cleavage site before N-terminal sequence and cloned into pPICZαA vector. The recombinant plasmid was transformed in KM71H Pichia strain and the screening of positive colonies was performed in deep well plate. The laboratorial scale expression was first performed with glycerol medium and methanol medium for the induction. The peptide expression was analysed by SDS-PAGE (16%) with silver stained and Schiff reagent that stain specifically carbohydrates. We also did a spectrometry analysis of toxins in MALDI-TOF equipment, a N-glycosylation reaction with PNGase enzyme and electrophysiological analysis in Kv 1.1, 1.2, 1.3 and 2.1 using the two-microelectrode voltage clamp technique. The SDS-PAGE revealed three bands and their molecular masses by spectrometry analysis were 7.76, 7.5 and 5.5 kDa. The Schiff stain revealed that the toxins with 7.76 and 7.5 kDa are glycosylated and the reaction with PNGase was able to remove part of this glycosylation, indicating that P. pastoris is making N-glycosylation. A preliminary electrophysiological screening with non-glycosylated toxin showed a small inhibition in Kv 1.3. In conclusion, the rTs15 was successfully expressed in P. pastoris yeast, as well as two glycosylated forms of toxin, and the small inhibition in Kv 1.3 is probably due to the recombinant N-terminal. As next steps, the same tests will be performed with glycosylated and after cleaved toxins.

  • Open access
  • 119 Reads
Cloning and expression of a Hemocyanin isolated from the centipede Cryptops iheringi
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Among the centipede of the chilopode Myriapoda class, the Cryptops genus is one of the most associated with accidents in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about Cryptops iheringi toxins. Thus, in this work a transcriptomic analysis of the C. iheringi venom gland was performed to obtain a profile of the toxins of this species. In addition, the crude venom was subjected to mass spectrometry analysis to establish an association between unknown sequences. These approaches for the construction of a general profile of the venom gland expression of this species led to the he identification of a Hemocyanin (Hc) subunit.

Hemocyanins are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hc in the chilopode Myriapoda C. iheringi. Such respiratory proteins have long been considered unnecessary in Myriapoda, due to its tracheal systems.

These respiratory proteins are potent immunogens, which induce the synthesis of large amounts of specific antibodies. Studies pointed out its interaction with polymorphonuclear monocytes and lymphocytes and in vitro tests have shown a potential anticancer activit, with in vitro significant inhibition of the growth of cancerous strains of the breast, pancreas and prostate. Currently scientific data is mostly limited to the study of native Hc of M. crenulata molluscs, therefore, the biotechnological potential of Hcs isolated from centipedes is still unexplored.

Herein, the Hc sequence that was present in both proteome and transcriptome analysis have signal peptide and 76 kDa range. The Hc subunit sequence was synthesized with codon optimization for bacteria expression and the protein expressed as inclusion bodies. Refolding attempts provided soluble forms of Hc. At the moment efforts to access your biological activities are being carried out.

  • Open access
  • 80 Reads
Flavonoids play a key role in resistance to accumulation of aflatoxin in corn.
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Aspergillus flavus is a facultative pathogen capable of producing aflatoxins (AF), potent carcinogens that accumulate in corn kernels, peanuts, cottonseed and tree nuts. To understand resistance mechanisms in corn to AF accumulation we performed a high-throughput genomics study using an in vitro kernel screening assay with A. flavus 3357, resistant corn hybrid TZAR102 and susceptible corn hybrid Va35. Furthermore, we incorporated gene expression data with genomic data to perform redundancy analysis (RDA). We determined that corn genotype, fungal treatment and duration of infection significantly co-vary to influence the overall gene expression patterns. We performed gene ontology enrichment analysis on highly significant genes and found enrichment of pathways linked to fungal and microbial responses such as Pathogenesis-related (PR) proteins. To determine additional genes of interest using field and gene expression data, we linked genome-wide association analysis results with gene expression data, allowing us to detect significant expression quantitative trait loci (eQTL). Our results showed that resistance to aflatoxin contamination is a polygenic trait and found significant association between specific flavonoid biosynthetic pathway genes and infection by A. flavus. Additional experiments including functional genomics analyses and fungal bioassays to identify the role of flavonoids and their contribution to corn resistance to A. flavus growth and AF production will also be presented.

  • Open access
  • 110 Reads
Shedding new lights on the recombinant β-KTx neurotoxin from Tityus serrulatus: heterologous expression, structural and functional characterization.
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Neurotoxins are the major responsible for the symptoms caused by Tityus serrulatus envenoming due to their actions on ion-channels of excitable cells. However, the structural and functional analyses of these toxins is difficult due to the low amount of purified toxin obtained from the crude venom. The combination of “-omics” techniques allows the precise identification of novel components with biotechnological applications enabling its heterologous expression. We reported the heterologous expression of the recombinant Ts19 (rTs19), a β-KTx neurotoxin, and their structural and functional characterizations. The cDNA encoding rTs19 was obtained from Tityus serrulatus venom gland transcriptome, cloned into pPICZαA plasmid and transformed into cells of KM71H Pichia pastoris strain. rTs19 was purified by immobilized metal affinity and C18 chromatography procedures and showed a higher expression after 96h of induction in buffered methanol-complex medium at 30°C. The expression of the toxin was confirmed by western blot using anti-His-tag antibody. In addition, rTs19 showed a molar mass of 6555.05 Da confirmed by FT-ICR high-resolution mass spectrometry (Solarix, Bruker). After reduction and alkylation, MALDI-TOF analyses (Ultraflex II, Bruker) confirmed the three disulfide bridges of the toxin. rTs19 was sequenced by enzymatic digestion using trypsin and MS/MS fragmentation in a Q-TOF mass spectrometer (SynaptG2, Waters). Electrophysiological experiments and voltage-clamp with two microelectrodes on Xenopus laevis oocytes were performed to screen the action of rTs19 over 16 different subtypes of Kv channels. The rTs19 interacts with potassium channels, blocking Kv1.4 and hERG channels with a high potency. These results demonstrated the first recombinant expression of a β-KTx neurotoxin from Tityus serrulatus. P. pastoris expression system seems to be an efficient, rapid and cheap method for obtaining such toxins in a recombinant methodology. Furthermore, these results may open new perspectives of bioprospection of the biological actions of rTs19.

  • Open access
  • 211 Reads
Bothrops moojeni venom: a new tool to investigate osteoclasts differentiation

Bothrops moojeni, a Brazilian lanced-head viper, presents a rich, but not well explored, venom composition. This venom is a powerful tool for the discovery of new molecular targets in many different biological processes. Osteoclasts (OC) are extremely important for bone maintenance, calcium physiology, and balance of tissue regeneration being involved in such diseases as osteoporosis and rheumatoid arthritis. The goal of our study was to evaluate the effect of Bothrops moojeni's venom and its fractions of human peripheral blood mononuclear cells derived OCs in vitro differentiation.

After the induction of OCs differentiation, on day 4 the venom was added at different concentrations (5, 0.5, and 0.05 µg/mL), and the reduction of tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts, which was more prominent at the concentration of 5 µg / mL were observed. In order to evaluate the effect of major venom’s components (metalloproteases and serinoproteases) on TRAP+ OCs, the EDTA, and aprotinin were used to inhibit the catalytic activity of both proteases. The results suggest that proteases were not crucial for the reduction of TRAP+ OCs. Phalloidin staining was used for morphological analyzes of F-actin rings integrity. Venom provoked F-actin ring disruption in treated versus control OCs. We obtain high molecular weight (HW) and low molecular weight (LW) venom fractions. Both fractions induced the reduction of TRAP+ OCs (HW fraction at a concentration of 5 µg / mL and LW fraction at -1 µg / mL, respectively).

We performed a secretome analysis of OCs treated with venom and its fractions using mass spectrometry (LC-MS/IT-Tof). The data obtained demonstrate possible pathways and mechanisms involved in OCs reduction after the treatment. Example giving is catabolic mechanisms for HW and proteins correlated with genetic modifications for LW. New experiments are in progress, aiming to discover the molecules that possibly interfering in the osteoclasts differentiation.

  • Open access
  • 121 Reads
In silico analysis of short linear motifs present in snake venom phospholipases A2

Phospholipases A2 (PLA2s) are important constituents of snake venom that, depending on their amino acid composition, possess several toxic properties whose main ones are neurotoxicity, myotoxicity, cardiotoxicity and impairing of haemostasis. They are proteins of about 120 amino acids, having a structure conserved since basal metazoa, and very similar to that of mammalian secretory PLA2s. Some snake venom PLA2s are heteromultimers, others are monomers or homodimers.

In this work we analyse the sequence alignment of monomeric or homodimeric snake venom PLA2s, grouped according to their myotoxic and neurotoxic properties, and we compare this alignment with that of the most similar mammalian secretory PLA2s. We found short linear motifs, present in three regions of secretory PLA2s that can have a role in their toxic and physiological functions. This work suggests important molecular interactions of secretory PLA2s that can focus and shorten the experimental work of characterization of the mechanism of action of these proteins.