Diabetes mellitus is a major public health concern on a global scale and warrants medical attention due to rapidly soaring figures of diabetics. It is a group of multifactorial disorders characterized by chronic elevated blood glucose levels (hyperglycemia), and impaired metabolism of key biomolecules such as carbohydrates, proteins and lipids . According to both molecular and pathological research, some environmental toxins have impact on insulin production by interfering with the activity of cells belonging to the pancreatic islets of Langerhans. Environmental factors like chronic exposure to arsenic, persistent organic pollutants (POPs), soil, unhealthy food, psychological stress, obesity, vitamin D deficiency, and immune system impairment etc have a major role in the etiology of diabetes mellitus as well as in multiple health conditions. Plant based diet, lowering stress, changing lifestyle habits along with focus on proper physical activity improves the body's glucose response, insulin signalling and insulin sensitivity. It is, therefore, necessary to conduct more sustained, long-term research to assess the significance of such environmental risk factors with reference to their implications in the prognosis of Diabetes mellitus.

Bacillus thuringiensis (Bt) is a gram-positive bacterium that produces parasporal crystal proteins showing biocidal activity against different invertebrate species. In addition, other gram-positive bacteria such as Lysinibacillus sphaericus, Paenibacillus spp. and Brevibacillus laterosporus have been described for showing insecticidal activity. In 2022, a Bacillus toyonensis strain has been described for the first time as producing parasporal crystals with insecticidal activity and designated Bacillus toyonensis biovar Thuringiensis. In this study we report the isolation and characterization of another Bacillus toyonensis strain showing typical Bt colony morphology on nutrient agar plates and bipyramidal parasporal crystals under phase contrast microscopy. A major ca. 130 kDa protein band was revealed when the parasporal crystals were analyzed using SDS-PAGE gel electrophoresis. The strain was isolated from a soil sample obtained from Río Tercero city at the province of Córdoba, Argentina. The genome sequence was obtained and assembled into 93 contigs with a total genomic size of 6,072,356 bp and 34.9 % G+C. The genome analysis performed with the Type (Strain) Genome Server showed that this strain belongs to B. toyonensis species, and therefore was designated as Bacillus toyonensis biovar Thuringiensis strain Bto_100. Its genome sequence harbors five coding sequences showing homology with Cry7Bb1, Cry7Ga2, Cry73Aa1, Mpp2Aa4 and Xpp22Aa3 proteins. In order to determine the biocidal activity of this novel strain, bioassays were conducted with Cydia pomonella (Lepidoptera: Tortricidae), Anthonomus grandis (Coleoptera: Curculionidae), Spodoptera frugiperda (Lepidoptera: Noctuidae), Panagrellus redivivus (Nematoda: Panagrolaimidae) and Caenorhabditis elegans (Nematoda: Rhabditidae). Bioassays showed that this strain was very toxic for P. redivivus and C. elegans (100% mortality) but low toxic for C. pomonella (10% mortality) and A. grandis (14% mortality) showing no toxicity for S. frugiperda. Further studies need to be developed to determine the real biocidal potential of this novel strain.

Snake venoms are a complex mixture of toxins, mostly of enzymatic nature, such as Phospholipases A₂ (PLA₂), Metalloproteinases (SVMP), Serine proteases (SVSP), others. At present, there is a growing interest in the search for natural or synthetic inhibitors acting on these components and thus allow mitigating their local and systemic effects. The main damage induced by the venom of B. alternatus is caused by SVMP, for this reason in the present work we proposed the use of 6-o-palmitoyl-l-ascorbic acid (ASC16) as an inhibitor of the toxic effects induced by the SVMP of B. alternatus venom. For this purpose, ASC16 (0.24 mM) was incubated with crude venom (1µg/µL) for 30 min at 37 °C. Proteolytic activity was measured using azocasein as substrate, and tissue injury (edema, hemorrhage and necrosis) was evaluated in a murine model (CF01). ASC16 inhibited 49.6% proteolytic activity, 78.06 % hemorrhagic activity, also the edema produced by the venom in the presence of ASC16 showed significant difference (p<0.05) compared to the whole venom control without inhibitor. Histological results showed that, as it known, crude venom provoked severe tissue injury, with extensive hemorrhagic areas, abundant inflammatory infiltrate and necrosis. In contrast, the B. alternatus venom treated with ASC16 did not cause hemorrhages and it caused mild lesions with little necrosis of muscle fibers and moderate inflammatory infiltrate. The results obtained indicate that compound ASC16 has potential as an inhibitor of SVMP enzymatic activity, suggesting its possible application as a local therapeutic agent. However, to understand in depth the mechanisms of ASC16 inhibition, further studies are required.

The venom proteomics of Indian Naja oxiana was characterized by SDS-PAGE, reverse-phase HPLC and mass spectrometry-based proteomics. The venom proteome of N. oxiana was found to be unique among other Naja species (N. naja and N. kaouthia). Three-finger toxins (85.3%) and snake venom metalloproteinase (9.1%) were the most dominant toxin families. Less abundant proteins included cardiotoxins, 5’-nucleotidases, cysteine rich secretory proteins, cobra venom factor, l-amino acid oxidase, phosphodiesterases. The immunoreactivity of two Indian polyvalent antivenoms (VINS and Premium Serums) was investigated by immunoblotting and immunoaffinity chromatography profiling. Immunoblotting analysis revealed that both antivenoms more effectively recognize higher molecular weight venom proteins than the lower mass. Further, immunological profiling of antivenoms revealed quantitative differences in immunorecognition capacity between the two antivenoms. The unbound proteins eluted from immunochromatography columns were characterized as non-immunodepleted toxins, and identified as three-finger toxins. These neglected toxins could limit antivenom efficacy in treating N. oxiana envenomings.

In South America, most snakebites are caused by genus Bothrops. The B. diporus venom is composed mainly of metalloproteinases (SVMPs) responsible for local effects such as hemorrhage, edema, myotoxicity and systemic bleeding. The only treatment for snakebite is antivenom, produced by immunizing animals with snake venom using Freund's adjuvant, which causes local damage at the injection site and affects the welfare of producer animals. To address this issue, B. diporus venom was treated with Na₂EDTA and used as inmmunogen in combination with CpG-ODN/Coa-ASC16 adjuvant. Previous works demonstrated that CpG-ODN/Coa-ASC16 adjuvant causes very few local reactions when used with other antigens. For this proposal, B. diporus venom (1.8 mg) was incubated with Na₂EDTA (0.2 M, BdV/Na₂EDTA) for 1 h at 37°C and then it was subjected to Sephadex G-25 chromatography to remove the excess of chelator. Likewise, B. diporus venom without chelating agent (BdV) underwent the same process. In both samples, the effective inhibition of SVMPs using azocasein as substrate was determined. CF-1 mice (5/group) were immunized subcutaneously on days 0, 15 and 30 with BdV or BdV/Na₂EDTA (7-30 μg) formulated with CpG-ODN/Coa-ASC16. On day 45, the final bleeding was performed and sera were obtained for antibody detection. The enzyme-linked immunosorbent assay results indicated that anti-BdV and anti-BdV/EDTA exhibited similar antibody titers (~2.56 x 10⁴) against Bothropic venom. Western Blot analysis revealed that the anti-BdV/EDTA serum recognized the main venom proteins (15-150 kDa) similarly to the anti-BdV serum. Finally, both experimental sera displayed neutralizing abilities against the proteolytic, indirect hemolytic, and coagulant activity tested in vitro. These findings suggest that Na₂EDTA does not impair protein immunogenicity, and BdV/Na₂EDTA together with CpG-ODN/Coa-ASC16 adjuvant was an appropriate immunogen since the animals immunized with it showed an adequate immune response to B.diporus venom similar to that of animals immunized with venom without an inhibitor.

1. Snake bite is a neglected public health issue in many tropical and subtropical countries. Each year, about 5.4 million snake bites occur, resulting in 138,000 deaths, and over 400,000 amputations and other permanent disabilities. The bite causes severe neurotoxic, hemorrhagic and myotoxic damage, the extent of which depends on the toxicity and venom composition of the snake species. Therefore, predicting the toxicity from snake venom composition would vastly improve diagnosis, antivenom treatment, saving lives and limbs. Herein, we investigate the potential of Machine Learning (ML) in venomics, by training several models to predict Lethal Dose (LD50) from venom composition. The analysis was conducted on 130 snake species (15% of all venomous species), using five ML models: Support Vector Machine (SVM), Multilayer Perceptron (MLP), Linear Regression, Decision Tree, Random Forest, and four ensemble learning methods: Stacking, Voting, Bagging, AdaBoost, trained to predict LD50 from relative protein abundance. Although, data from 14 proteins and enzymes were combined, results showed an overall weak correlation between model prediction and LD50 (correlations ranging from 0.48 to 0.55, R2 ranging from 0.16 to 0.23), even when considering only the highly significant proteins and enzymes: SVMP, SVSP, 3FTx, and PLA2. These results, challenge the assumption that relative protein abundance is the main driver of toxicity. They suggest that toxicity is a multi-factor phenomenon influenced by different biological aspects, such as protein 3D structure and potential binding sites. This in turn highlights the need for high quality multi-modal venomics databases, combining toxicity with several biological factors such as protein structure and metabolic data to better understand the nature of snake venom toxicity.

Pakistan is third in worldwide dairy production ranking, behind India and USA. The climatic conditions of South Asia favour the contamination of crops with mycotoxins, such as aflatoxins, which can impair human/animal health. This study targeted the analysis of a broad range of mycotoxins and fungal secondary metabolites (>500) in total mixed rations (TMR) (n=30) using a validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI–MS/MS). The samples were collected in big commercial dairy farms (> 200 lactating cows) in Punjab, Pakistan, detecting 96 mycotoxins/fungal metabolites. Contamination with multiple mycotoxins was ubiquitous. On average, the samples contained 14 mycotoxins/sample (range: 11 - 20). Metabolites derived from “other fungi” and Fusarium spp. showed the highest occurrences, concentration, and diversity among the detected fungal compounds. Aflatoxin B1 (AFB1) occurred in 40% of the samples, and 7% exceeded the EU maximum limit for feeding dairy cattle (5 μg/kg). The maximum concentration of this potent carcinogenic was 33.8 μg/kg (more than 7 times over the EU maximum limit). No other mycotoxin than AFB1 exceeded the EU guidance values. Excepting for ergot alkaloids (73%), all the groups of metabolites (i.e., derived from Alternaria spp., Aspergillus spp., Fusarium spp., Penicillium spp., and other fungi) occurred in 100% of the TMR samples. Although the contamination levels of single mycotoxins are relatively low, the effects on animal health, reproduction, and productivity under the detected realistic scenario (“cocktails effect”) are still unpredictable. Thus, future toxicological studies should address interactions (additivity, potentiation, synergism, and antagonism) as well as the long-term exposure effects of “mycotoxin mixtures”.

Paralytic shellfish poisoning (PSP) is the human illness associated with the consumption of contaminated seafood products with the toxins knows as saxitoxins. Saxitoxin (STX) are produced by dinoflagellates, generally by the genus Gymnodinium, Alexandrium and Pyrodinium. But between 2007 and 2008 episodes of PSPs contaminations in bivalves has occurred in Angola where there is not monitoring program for shellfish contamination with marine biotoxins. Ten samples extracted from the Semele proficua from Luanda Bay and Senilia senilis from Mussulo Bay, were analyzed by HPLC and STX, dcSTX and other four compounds that have an unusual profile different to the known hydrophilic PSP toxins were found in different amounts and combinations. These unknown compounds were not autofluorescent, and they presented much stronger response after peroxide oxidation than after periodate oxidation. Electrophysiological studies developed to elucidate their effects at cellular level lead to conclude that they have different activities, some of the isolated compounds bind to voltage-gated sodium channels and block the passage of sodium ions across the membrane, similarly to STX, thus, contributing to the STX activity while others did not show activity on sodium channels

Voltage-gated potassium Kv1.3 channel play a role in many physiological processes and is a prospective target for the treatment of neurological and autoimmune diseases. Many peptides from scorpion venoms are highly specific and potent blockers of Kv1.3 and other channels from Kv1 family, that makes them important pharmacological compounds. Agitoxin 2 (AgTx2) is one of these blockers with a subnanomolar affinity to eukaryotic channels Kv1.1, Kv1.3 and Kv1.6. Fluorescently labeled peptide blockers are useful tools for studying channels by the means of fluorescence microscopy techniques. These tagged blockers can be obtained either by chemical synthesis or by genetically encoding a peptide fused to a fluorescent protein. Previously studied conjugates of AgTx2 with tetramethylrhodamine, as well as bioengineered fusions with GFP and RFP showed high affinity to Kv1 channels [1].

The aim of the present study was to characterize properties of A-AgTx2, a new fluorescently labeled ligand of Kv1 channels, and its possible applications. A-AgTx2 (AgTx2 N-terminally labeled with the fluorophore Atto488) was produced by Smartox (France) and studied by us using laser scanning confocal microscopy and E.coli spheroplasts that express hybrid channels KcsA-Kv1.x (x=1, 3, 6) in the plasma membrane [2]. As shown earlier, hybrid channels KcsA-Kv1.x (x=1, 3, 6) mimic corresponding eukaryotic Kv1.x channels in the ability to bind peptide blockers[3].

In contrast to AgTx2, the A-AgTx2 ligand was determined to bind to KcsA-Kv1.3, but not to KcsA-Kv1.1 and KcsA-Kv1.6 channels. Our studies have shown that N-terminal labeling AgTx2 modifies its pharmacological profile, as it was showed previously for GFP-AgTx2 ligand [1]. Thus, a new selective and high-affinity ligand of the Kv1.3 channel was obtained with the dissociation constant value 4.3±0.2 nM. A-AgTx2 can be used in a combination with KcsA-Kv1.3 hybrid channel for the recognition of unlabeled Kv1.3 blockers and for the quantitative analysis of their affinity to Kv1.3 channel.

The studies were supported by the Russian Science Foundation (grant no. 22-14-00406).

References

1. Nekrasova, O.V.; Primak, A.L.; Ignatova, A.A.; Novoseletsky, V.N.; Geras’kina, O.V.; Kudryashova, K.S.; Yakimov, S.A.; Kirpichnikov, M.P.; Arseniev, A.S.; Feofanov, A.V. N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site. Toxins 2020, 12, doi:10.3390/toxins12120802.
2. Denisova, K.R.; Orlov, N.A.; Yakimov, S.A.; Kirpichnikov, M.P.; Feofanov, A.V.; Nekrasova, O.V. Atto488-Agitoxin 2—A Fluorescent Ligand with Increased Selectivity for Kv1.3 Channel Binding Site. Bioengineering 2022, 9, 295, doi:10.3390/bioengineering9070295.
3. Kudryashova, K.S.; Nekrasova, O.V.; Kirpichnikov, M.P.; Feofanov, A.V. Chimeras of KcsA and Kv1 as a Bioengineering Tool to Study Voltage-Gated Potassium Channels and Their Ligands. Biochem. Pharmacol. 2021, 190, doi:10.1016/j.bcp.2021.114646.

In the last decade, Common Gulls from the Iberian Peninsula have been observed to suffer from a paralyzing syndrome of unknown origin, affecting mostly adults in the west and south regions. This disease has been attributed to different causes, such as nutritional deficiencies, avian botulism, and exposure to algal toxins. In the quest for the causative agent, we selected twelve individuals of Larus michahellis, L. fuscus, L. ridibundus and Ichthyaetus melanocephalus that succumbed to this illness concomitantly with marine algal blooms in the nearby coasts of Málaga (Spain). Amnesic and paralytic shellfish toxins were analyzed in the gull´s liver, kidney and feces. Furthermore, the neurotoxin β-Methylamino-L-alanine, and its analogues were measured in the liver, brain and blood serum of the same individuals by UPLC-MS/MS. Liver and brain were also used to determine stable isotope (SI) signature as a trophic marker. None of the analyzed neurotoxins were detected in any of the samples, indicating that poisoning due to blooms caused by the algae producers of these neurotoxins was unlikely cause of the bird death. Moreover, despite similar symptoms, the SI signatures revealed significant differences in the feeding grounds between the species. These results point to the causative agent not being related to the food sources of the birds.