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  • Open access
  • 10 Reads

Fumonisins are known as secondary metabolites of the mold Fusarium verticillioides that most often contaminate cereals and animal feed. According to recent research, these mycotoxins have also been detected in the aquatic environment, which may be the result of washing off the crops during rainy periods, or because of their possible production in the water. Since fumonisin B1 (FB1) is highly toxic and has dangerous effects on the health of living organisms, in this research the influence of different water matrix on the effectiveness of UV and UV/H2O2 treatments was investigated. Namely, different water types (water from Danube River, tap, and underground) were simulated with the addition of humic acid and major ions for each type of water into ultrapure water (UPW). Results showed a lower removal efficiency of FB1 by direct UV photolysis in the simulated water samples in comparison to the UPW. In the case of UV/H2O2 treatment, the efficiency of FB1 removal in simulated waters was also lower compared to UPW. However, this treatment was more effective than direct photolysis. Namely, in UPW 100% of FB1 removal was achieved after 90 min of irradiation, while among the investigated simulated water samples the highest removal efficiency was reached in tap water, when 50% of FB1 was degraded after the same time of irradiation. These results provide insight into the influence of the matrix of different water types on the efficiency of FB1 removal and contribute to the development of adequate water purification methods for potentially carcinogenic fumonisins.

  • Open access
  • 16 Reads
Inflammatory effect of a PLA2 isolated from Bothrops diporus venom

In northeastern Argentina the vast majority of snakebite envenomings are caused by Bothrops diporus. Proteomic studies have shown that about 24.1% of its venom consists of phospholipases A2 (PLA2s) that induce inflammatory events. However, there are no previous reports on the specific inflammatory mediators released by immune and endothelial cells in response to these toxins. Thus, in this work we quantified a panel of cytokines on peripheral blood mononuclear cells (PBMC) previously incubated with a PLA2 isoform from B. diporus venom. Briefly, PLA2 was isolated by reverse phase chromatography (RP-HPLC) on a C18 column. B. diporus venom (2 mg) was dissolved in 200 μL of 0.1% trifluoroacetic acid (TFA) and elution was performed at 1 mL/min in acetonitrile gradient with 0.1% TFA. Specific phospholipase activity, concentration at 280nm and molecular mass by MALDI-TOF MS (Shimadzu MALDI-8030) were determined. In order to analyze the inflammatory response induced by this toxin on human PBMC, Luminex multiplex technology was used. After 10h incubation of 1x106 PBMCs, from three different donors in technical duplicate, with PLA2 (25 μg/mL) or positive control (PMA/Ionomycin) the Human Panel Th17 for GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, IL-23, IL-27, IL-28A, IL-31, IL-33, MIP-3α, TNFα and TNFβ was used. The statistical analysis was performed using the Two-Way ANOVA and FDR Benjamini-Hochberg method. Results clearly showed that the PLA2 (14,048Da) isoform isolated from B. diporus venom, induced a significant increase in the release of the pro-inflammatory cytokines Il6 and TNFa and the macrophage inflammatory chemokine MIP3a, after 10h incubation. Even the IL-10, an anti-inflammatory cytokine, was also over expressed; the predominantly inflammatory effect induced by PLA2 was confirmed. Further studies, eg. on oxidative stress, will complete this information that could be useful to develop new strategies in anti-venom therapy.

  • Open access
  • 11 Reads
Nigella Sativa: A Potential Natural Antidote for Poisoning Cases
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In several cultures, black cumin, also known as Nigella sativa, has long been used medicinally. Recent research has revealed that this plant has potent anti-inflammatory and antioxidant qualities, making it a possible treatment for several medical conditions. Additionally, because of its capacity to detoxify the liver and protect it from harm, Nigella sativa has demonstrated positive results as an antidote for poisoning. Consumption of hazardous substances by accident or planned poisoning are two prevalent causes of poisoning. The liver is the primary organ in detoxification. According to studies, nigella sativa can help the liver operate better and defend it from toxins' harmful effects. Additionally, it has been demonstrated that Nigella sativa protects against heavy metals toxicity. Thymoquinone and thymohydroquinone, the plant's active components, have been demonstrated to bond with heavy metals and stop the body from absorbing them. Further studies are needed to evaluate the efficacy and safety of Nigella sativa as an antidote for poisoning cases. Nigella sativa presents an interesting natural alternative for treating poisoning cases, potentially complementing traditional medical approaches. The main goal of this review is to explore the potential application of Nigella sativa as an antidote for poisoning cases. The article discusses the plant's strong ability to detoxify and protect the liver. The review highlights preclinical studies that have shown promising results but also emphasizes the need for further clinical trials to determine the efficacy and safety of Nigella sativa as a natural alternative for treating poisoning cases.

  • Open access
  • 3 Reads
The rapid detection of botanical natural toxins using probe ESI unit combined with quadrupole time-of-flight mass spectrometer

Many animals and plants are known to have poisonous components (natural toxins) in their bodies. Food poisoning caused by animals and plants containing these natural toxins occurs every year, although the number of cases and patients are not so high compared to bacterial food poisoning. It is extremely important from the viewpoint of food sanitation because some poisons have a high fatality rate. Therefore, rapid and effective screening methods are required. In this study, we report a method for rapid detection of natural plant toxins in Narcissus tazetta and Colchicum autumnale by utilizing the quadrupole time-of-flight (Q-TOF) mass spectrometer with probe electrospray ionization (PESI) unit , one of ultra-high speed analysis screening. The leaves and bulbs of Narcissus tazetta and the bulbs of Colchicum autumnale were cut and analyzed by using the PESI technique. These resemble vegetables such as chinese chives and onions, and it has been reported that accidental ingestion can cause food poisoning. From sample processing to completion of MS analysis, it took about 5 minutes or less. In the positive mode mass spectrum of the Narcissus tazetta bulb, characteristic natural toxins tazettine, lycorine, and galanthamine were detected with high accuracy within 1 mDa compared to theoretical mass values. In addition to these toxins, other components such as choline, arginine and saccharide were also detected. Similarly, these toxins were detected in the positive mode mass spectrum from leaves. In the case of the Colchicum autumnale bulb, representative natural toxins colchicine and demecolcine were detected in the positive mode mass spectrum. Some toxins could also be confirmed at the MS/MS level using standards. LC and LC/MS are commonly used to measure several toxins simultaneously, but they require a lot of separation time using a column to remove contaminants. This problem can be solved by PESI technique.

  • Open access
  • 8 Reads
Exploring the Cross-Reactivity of a Monoclonal Antibody against Tetrodotoxin Analogues

The implementation of immunoassays in the monitoring of toxins is essential for food safety management and public health protection, being an outstanding bioanalytical tool for its specific, sensitive, rapid and user-friendly intrinsic characteristics. However, their applicability could be questioned considering that the detection principle is based on structural recognition and no toxicological information can be obtained from the analysis. The toxic potential of a sample is product of all the different analogues present, which sometimes have notorious structural differences. For that reason, is important to know which analogues the immunosystem can detect, being the most useful the one able to detect the most toxic ones. With this aim, we evaluated the cross-reactivity of an anti-tetrodotoxin (TTX) monoclonal antibody against different TTX analogues using a competitive magnetic bead-based immunoassay. All TTX analogues have been detected with the immunoassay, but different affinities were observed. 5-deoxyTTX and 11-norTTX-6(S)-ol showed high cross-reactivity while 5,11-dideoxyTTX, 6,11-dideoxyTTX and 5,6,11-trideoxyTTX showed much lower cross-reactivity with the antibody. Considering the toxicity of each TTX analogue, we conclude that the anti-TTX antibody is able to detect the most toxic TTX analogues with high affinity. Therefore, the proposed magnetic bead-based immunoassay is very promising as a bioanalytical tool for TTX risk assessment.

  • Open access
  • 7 Reads
Metabolism and mitochondria alterations in melanoma cells induced by Octpep-1.

Octpep-1, a venom-derived tachykinin-peptide from the Octopus kaurna, impairs melanoma cellular viability in human melanoma BRAF(V600E)-mutated cells, while is innocuous in healthy fibroblasts. Due to the selectivity of this peptide towards melanoma of BRAF mutations, we explored its mechanism of action in tumor cells. We performed a metabolic characterization of cells treated with Octpep-1 using Seahorse Flux technology and microscopy. Specifically, we investigated its effect in glycolysis and OXPHOS as well as changes in mitochondria and mitochondrial dynamics. In addition, we studied the possible effects that these changes may induce in the behavior of melanoma cells and the possible involvement of senescence in the antiproliferative capacity of Octpep-1 by MitoTracker™ Red staining and senescence markers, respectively. Our results showed that Octpep-1 increases oxygen consumption rates, and overall, mitochondrial respiration, without changes in glycolysis in melanoma cells. Hyperactivity of mitochondrial metabolism relates to higher levels of reactive oxygen species without leading to cell apoptosis. Remarkably, dynamic analysis of mitochondria indicated that Octpep-1 does not change the number of mitochondria in cells, but promotes their aggregation in clusters, altering mitochondrial dynamics. These changes could be related to the migratory and proliferative capacities of melanoma cells. Altogether, our results suggest that the antiproliferative profile of Octpep-1 in melanoma BRAF(V600E)-mutated cells may be due to alterations in cellular metabolism. These changes are accompanied by alterations in mitochondrial dynamics in melanoma cells.

  • Open access
  • 2 Reads
Distribution analysis of galanthamine, a plant alkaloid, by MS imaging
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Plant alkaloids are used in various pharmaceuticals, such as anticancer drugs and analgesics. Among these plant alkaloids, galanthamine an Amaryllidaceae-type alkaloid with acetylcholinesterase inhibitor for the treatment of neurological diseases such as Alzheimer's disease. Although the chemical synthesis of galanthamine has been successfully achieved, Narcissus is the main source of its production. Research indicates that galanthamine content varies not only with the type of Narcissus, but also with the developmental stage and the part of the plant. Pharmaceutical companies are pursuing plant species with higher galanthamine content to increase pharmaceutical productivity. Therefore, we quickly confirmed the Narcissus in our study contained galanthamine using DPiMS QT system featuring a high-resolution quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an ion source utilizing probe electrospray ionization (PESI) method. Subsequently, we analyzed the distribution of galanthamine using MS imaging (MSI) system consisting of an Q-TOF mass spectrometer connected to an iMScope QT atmospheric MALDI unit with built-in microscope.

Galanthamine was rapidly detected in the leaves extract solution using the DPiMS QT in only 30 seconds. Other compounds were also able to be detected and identified: lycorine and tazettine, bothalkaloids found in Narcissus.

MS Imaging was performed on sections of leaves (in two locations) and bulbs with a spatial resolution of 25 um for the entire section and 10 um for the area observed under a microscope with a 5x objective lens. As a result, good MS images of galantamine, lycorine, and tazettine were obtained from all samples. The MSI of the bulbs showed that galantamine was distributed more in the leaves, but MSI of lycorine and tazettine did not confirm such a distribution, indicating that the regions of distribution differ depending on the type of plant alkaloid. In addition, this analysis of galanthamine distribution by MSI can identify regions with higher galantamine content and may contribute to an efficient determination of extraction regions for pharmaceutical manufacturing processes.

  • Open access
  • 9 Reads
Comparative analysis and in-silico studies of Oecophylla smaragdina collected from the states of Chhattisgarh and Maharashtra, India
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Background: The consumption of red weaver ant and their eggs have claimed a wide medicinal properties to treat a variety of ailments like cold, whooping cough, high fever, malarial fever, ear pain, etc by the tribal groups across the country. However, it still lacks a scientific evidence over the use.

Objective: The goal of the study was to perform and analyze the biochemical, antimicrobial, anti-inflammatory and cytotoxicity effects of the consumption of the red weaver ant and their eggs (O. smaragdina) and whether they are fit to consume by the humans imparting the potential to the treatment of claimed diseases.

Methodology: In the present study, two samples of the red weaver ants, namely from Bastar (Chhattisgarh) and Gadchirolli (Maharashtra) are compared. Both the sample were dried, grinded and soxhlet based extraction was carried out in the methanol solvent respectively. Crude proteins and elements (Zinc and Calcium) were determined at Bombay Test House, Vashi. Total phenolic content and DPPH radical scavenging was carried out. The antimicrobial potential of the extract was tested against E.coli, S. aureus, K. pneumoniae and S. pyogenes. Nitric oxide assay and RBC membrane stabilization test was performed to observe the inhibition of the inflammatory actions. MTT assay was performed for the analysis of cytotoxicity in the sample using PBMNCs cell culture. To deeply understand the pharmacological process, in-silico based modelling and analysis was performed. The genes and proteins were retrieved from the NCBI database, protein 3D structure were modelled using SWISS-model server, and the modelled 3D structures were assessed for their ProSA and Ramchandran Plot analysis. The 3D structure were optimized using GROMACS package and protein-protein docking was carried out with the drug target of the gastrointestinal (E.coli), respiratory (S. aureus, S. pyogenes and K. pneumoniae), Malarial (Plasmodium), Dengue, Jaundice and COVID-19 (SARS CoV-2) infectious pathogens using the computational tool, Hex 8.0.0.


Biochemical: The proximate analysis revealed a significant presence of Zinc in both the samples. The DPPH assay revealed former to have higher % inhibition and lower antioxidant activity i.e., 54.35 mg/AAE ml whereas later sample showed lower % inhibition and higher antioxidant activity i.e., 86.9 mg/AAE ml. The total phenolic contents (Catechol equivalents, mg/g) in the samples were calculated to be 3.9853 and 5.228 mg Catechol eq/ml, respectively.

Anti-microbial: The ant sample from Gadchirolli demonstrated significant results and clearer zone of inhibition against all the four bacteria, while the former sample gave clear zone of inhibition only for E.coli and bit for K. pneumoniae.

In-vitro: The cytotoxicity of the samples was evaluated in-vitro on PBMNCs culture. The percentage survival of the cell was recorded as 76.76% (Bastar) and 78.26% (Gadchirolli), with IC50 values of 23.5111± 0.4474 & 23.0719 ± 0.4425 respectively. No other hyperactivation of other cells in blood culture were recorded, indicating that these samples do not alleviate any hyper cell activity and are safe for utilization on human blood cells. Furthermore, analysis of the RBC membrane stabilization demonstrated the 46.36%) & 63.12% stabilization potentials of both the samples, indicating the anti-inflammatory potential of these samples.

In-silico: In all the modelled protein 3D structures a maximum number of residues were identified within the allowed regions of Ramachandran Plot, followed by energy minimization in Gromacs. The structures are reported with a good degree of stability according to the potential energy of the protein. The outcome from the protein-protein docking studies reported a strong affinity between both the proteins, supported by numerous hydrogen bonds formation between them. From the in-silico analysis, here we predict the plausible interaction between the proteins from the red weaver ants that interacts to the proteins known as drug target from the selected pathogen and hence may be able to inhibit their pathogenicity too in future.

  • Open access
  • 8 Reads
Study of nematocyst stimulation and venom toxicity of Physalia physalis (Cnidaria, Hydrozoa)

Cnidaria include the most venomous animals of the world, among them Physalia physalis that is high abundant in the North Portugal. The venom of P. physalis is a complex mixture of bioactive substances that are only partially characterized. The study of the jellyfish venom requires the release of venom from the interior of the nematocyst or from the tissue samples. However, the methods for stimulation or inactivation nematocytes lack consensus with a gap in successful methods employed the discharge of the nematocysts, mostly due to the differences observed among species. We tested a range of physical and chemical approaches regarding nematocyte stimulation, including different chemical solvents, ionic solutions and electrical stimulation and we analyzed nematocyst discharge under light microscopy. Venoms released in each situation were analyzed for protein profile and enzymatic activities. Artemia was used as a tool for the preliminary tests of toxicity, paralyzes and modification on the nociception.

According to our observations, nematocyst discharge is not stimulated by seawater; 5% acetic acid solution induces 15 to 30% nematocyst discharge after 30 min exposure; 100% ethanol induces 40 to 60%; 0.9% NaCl for 30 min results in 90% discharge; 0.3M KCl solution induces 80% discharge in less than 15 minutes. Remarkably, it was observed, for the first time, that electrical stimulation with 8 V pulse for 30 seconds of a tentacle immersed in 0.9% NaCl resulted in 80% discharge.

The SDS-page profile of venom proteins released after stimulation with ethanol and ionic solutions showed a similar pattern with a broad MW distribution range and thicker bands at 55 kDa, 40 kDa and 20 kDa. These mixtures had reveals high proteolytic activities that are inhibited differently by PMSF, phenanthroline and EDTA, thus indicating the presence of serine- and metallo- proteases.

Toxicity tests using hatched nauplii revealed a dose-dependent effect on mortality with an LD90 of 17 mg/ml and an LD50 of 7.2 mg/ml. The paralysis was observed 2 hours after exposition whereas, the loose of light nociception was registered after 1 hour of exposition. To be noticed, the venom obtained by stimulation with ethanol loses enzymatic activity, but still can kill and paralyze hatched nauplii.

The present study provides methodologies for venom extraction prior to proteomic analysis and toxicity assays. They also provide useful to understanding on nematocysts stimulation, which is essential for the first rescue into accidental contact, preventing the discharge of nematocysts that may be adhering to the skin.

  • Open access
  • 27 Reads
Gene expression profile of Neutrophil Extracellular Traps (NETs) stimulated by L-amino acid oxidase from Calloselasma rhodostoma venom
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The focus of this study is to investigate the gene expression related to the formation of neutrophil extracellular traps (NETs) stimulated by Calloselasma rhodostoma L-amino acid oxidase (Cr-LAAO). LAAOs found in snake venom have been shown to activate human neutrophils, leading to the production of ROS, chemotaxis, phagocytosis, and the release of pro-inflammatory cytokines and lipid mediators. Additionally, it has been found that Cr-LAAO activates NADPH oxidase, which is responsible for the release of ROS. Neutrophils are known to release NETs to combat pathogens, and this process involves the migration of DNA from the nucleus to the cytoplasm, where it merges with the contents of the granules to produce NETs. Initially, the formation of NETs was associated with cell death, and this process was known as NETosis. However, two forms of NETosis have now been identified: classical or suicidal NETosis, which results in cell death, and vital NETosis, in which the cell retains its viability and many of its effector functions. To evaluate the gene expression related to the formation of NETs, a microarray assay was performed on human neutrophils stimulated with Cr-LAAO. The results show that Cr-LAAO stimulates the expression of important genes for the formation of NETs, such as TXNIP, FOXO3, PPARA, ELANE, CXCL8, and PADI4. This is the first report that shows the transcriptome of neutrophils related to Cr-LAAO-stimulated NETosis, which may lead to the development of local inflammatory effects observed in snakebite victims.