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The Black mamba, D. polylepis, is one of the many venomous snakes found in Kenya, and known to account for some snakebite incidents. The Kenyan Ministry of Health data reveals annual 15,000 snakebites occurrences. Also, 1 in 15 people in Kenya gets bitten by a snake, and tragically, 1 in 147 of these individuals die of snakebite yearly. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using IgY antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In this current study, D. polylepis specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with D. polylepis venom. These antibodies were subsequently assessed for their in-vivo neutralizing capacity vis-à-vis commercial antivenoms, PANAF-Premium and VINS. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by SDS-PAGE and ELISA. The LD₅₀ of D. polylepis was found to be 0.54 mg/kg in chicks, and 0.34 mg/kg in mice, respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 μg. Anti-D. polylepis IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of D. polylepis venom. This study confirms a successful IgY generation against Black mamba venom for the first time, and observed toxic effects of the venom as well as neutralizing capacity of antivenoms.

Biocrusts dominate the soil surface in deserts and are composed of diverse microbial communities that provide important ecosystem services. Cyanobacteria in biocrusts produce many secondary metabolites, including the neurotoxins BMAA, AEG, DAB, anatoxin-a(S) (guanitoxin), and the microcystin hepatotoxins, all known or suspected to cause disease or illness in humans and other animals. We examined cyanobacterial growth and prevalence of these toxins in biocrusts at millimeter-scales, under a desert-relevant illumination gradient. In contrast to previous work, we showed that hydration had an overall positive effect on growth and toxin accumulation, that nitrogen was not correlated with growth or toxin production, and that phosphorus enrichment negatively affected AEG and BMAA concentrations. Excess illumination positively correlated with AEG, and negatively correlated with all other toxins and growth. Basic pH negatively affected only the accumulation of BMAA. Anatoxin-a(S) (guanitoxin) was not correlated with any tested variables, while microcystins were not detected in any of the samples. Concerning toxin pools, AEG and BMAA were good predictors of the presence of one another. In a newly conceptualized scheme, we integrate aspects of biocrust growth and toxin pool accumulations with arid-relevant desertification drivers.

Nowadays, more than two billion inhabitants of underdeveloped tropical and subtropical countries are at risk of being stung by scorpions. Scorpion stings annually cause 2000–3000 deaths as they can lead to the respiratory and/or cardiovascular complications. Pathogenesis of lung damage under scorpion envenomation is often comprehensive. Respiratory failure can have a cardiogenic origin, associated with venom neurotoxin action. However, some venom components can stimulate pro-inflammatory signaling cascades followed by cytokines synthesis, recruit and activate immune cells, participating in the inflammatory response in lung injury. Scorpions of the Leiurus genus ("deathstalker") are one of the most dangerous Arthropoda. To date, 22 species of this genus have been described, but the venom composition and the mechanisms of tissues damage under envenomation have been studied to some extent only for L. quinquestriatus, L. hebraeus, and L. abdullahbayrami. Scorpions of L. macroctenus species are expected to be very hazardous, but the possibility of their venom cause inflammation in the lung tissue has not been investigated to date. Therefore, in this study, we focused on evaluating the levels of cytokines and their regulators – transcription factors (HIF-1α and NF-κB) and growth factors (FGF-2, VEGF, and EGF) – in rat lung homogenates after L. macroctenus envenomation. The results revealed a decrease in the levels of most pro-inflammatory cytokines (IL-6, IL-8, IL-1β and TNF-α) with simultaneous rise in the content of both anti-inflammatory cytokines (IL-4 and IL-10) and interferon-γ. Furthermore, the levels of all researched transcription factors and growth factors were shown to be increased too. The detected changes peak occurred at 24 h, whereas a tendency towards all indicators values normalization was observed in 72 h after venom injection. Thus, our results did not reveal signs of a classic inflammatory process in the lungs of rats injected with L. macroctenus venom. However, the obtained data indicate venom influence both on cytokine profile and on their regulators content in the rat lungs, which is a feature of certain alterations in the innate immune response, caused by studied venom components. But, the mechanisms of the changes we found require additional researches.

Snakebite envenoming is a global health issue that affects millions of people worldwide, and that causes morbidity rates surpassing 450,000 individuals annually. Patients suffering from snakebite morbidities may experience permanent disabilities such as pain, blindness and amputations. The (local) tissue damage that causes these life-long morbidities is the result of cell- and tissue-damaging toxins present in the venoms. These compounds belong to a variety of toxin classes and may affect cells in various ways, for example, by affecting the cell membrane. In this study, we have developed a high-throughput in vitro assay that can be used to study membrane disruption caused by snake venoms using phospholipid vesicles from egg yolk as a substrate. Resuspended chicken egg yolk was used to form these vesicles, which were fluorescently stained to allow monitoring of the degradation of egg yolk vesicles on a plate reader. The assay proved to be suitable for studying phospholipid vesicle degradation of crude venoms and was also tested for its applicability for neutralisation studies of varespladib, which is a PLA₂ inhibitor. We additionally made an effort to identify the responsible toxins using liquid chromatography, followed by post-column bioassaying and protein identification using high-throughput venomics. We successfully identified various toxins in the venoms of C. rhodostoma and N. mossambica, which are likely to be involved in the observed vesicle-degrading effect. This indicates that the assay can be used for screening the membrane degrading activity of both crude and fractionated venoms as well as for neutralisation studies.

Mice are routinely used in snake venom research but are costly and subject to pain and suffering. The crustacean Artemia salina could be an alternative to mice, but data to support its adoption in snake venom research is limited. The aim of the present study was to evaluate the suitability of A. salina as a surrogate of mice in assessing the toxicity of venoms and the preclinical efficacy of antivenoms. The toxicity of venoms from 22 snakes of medical importance in sub–Saharan Africa was evaluated in mice (intraperitoneally; i.p. and intravenously; i.v.) and in A. salina. Subsequently, the capacity of a commercial antivenom to neutralize the toxicity of these venoms in mice and A. salina was investigated. There was a positive correlation between the i.v. median lethal doses (LD_50s) and the i.p. LD_50s in mice (r = 0.804; p < 0.0001), a moderate correlation between the i.v. LD_50s in mice and the median lethal concentrations (LC_50s) in A. salina (r = 0.606; p = 0.003), and a moderate correlation between the i.p. LD_50s in mice and the LC_50s in A. salina (r = 0.426; p = 0.048). Moreover, there was a strong correlation between the i.p. median effective doses (ED_50s) and the i.v. ED_50s in mice (r = 0.941, p < 0.0001), between the i.p. ED_50s in mice and the ED_50s in A. salina (r = 0.818, p < 0.0001), and between the i.v. ED_50s in mice and the ED_50s in A. salina (r = 0.972, p < 0.0001). These findings present A. salina as a promising candidate for reducing reliance on mice in snake venom research. Future investigations should build upon these findings, addressing potential limitations and expanding the scope of A. salina in venom research and antivenom development.

Snakebite is a neglected public health issue, with many scientific and medical issues to be solved. Cobras are among the most common venomous snakes in Myanmar and are responsible for a considerable number of severe snakebite envenoming. There are three species of cobra (Naja kaouthia, Naja mandalayensis and Ophiophagus hannah) in Myanmar. The study aims to characterize the N. kaouthia and N. mandalayensis venoms and to investigate the efficacy of anti-cobra antivenom (BPI) against the two venoms. Protein components and fibrinogenolytic activity were determined by SDS-PAGE. Enzymatic activities for PLA₂, protease and acetylcholinesterase were determined by spectrophotometric method. Anticoagulant activity was determined by recalcification time of citrated human plasma. Myotoxicity, necrotizing activity, median lethal dose (LD₅₀) and median effective dose (ED₅₀) were determined by WHO recommended methods. The SDS-PAGE displayed the proteins and enzymes containing in two venoms were different. N. kaouthia venom exhibited more in PLA₂, acetylcholinesterase, anticoagulant, fibrinogenolytic and necrotizing activities than N. mandalayensis venom. N. mandalayensis venom had more protease activity and myotoxicity than N. kaouthia venom. The median lethal dose (LD₅₀) of N. kaouthia and N. mandalayensis venom was 4.33 μg/mouse and 5.04 μg/mouse respectively. Both venoms induced fibrinogen Aα chain degradation in 30 min (N. kaouthia) and in 6 h (N. mandalayensis). The same median effective dose (ED₅₀) (19.56 μg/mouse) showed that anti-NK antivenom can neutralize against lethal effect of N. mandalayensis venom. It can also neutralize the protease activity, anticoagulant activity and fibrinogenolytic activity of both venoms. Immunodiffusion and immunoblotting studies showed that the antivenom recognized its homologous venom (N. kaouthia) and cross-reacted against the heterologous venom (N. mandalayensis). The anti-NK antivenom is suitable to use for N. mandalayensis bite if monospecific antivenom is not available.

Snakebite envenoming is a priority Neglected Tropical Disease that causes an estimated 81,000–135,000 fatalities each year. The development of a new generation of safer, affordable, and accessible antivenom therapies is urgently needed. With this goal in mind, rigorous characterisation of the specific toxins in snake venom is key to generating novel therapies for snakebite. Monoclonal antibodies directed against venom toxins are emerging as potentially strong candidates in the development of new snakebite diagnostics and treatment. Venoms comprise many different toxins of which several are responsible for their pathological effects. Due to the large variability of venoms within and between species, formulations of combinations of human antibodies are proposed as the next generation antivenoms. Here a high-throughput screening method employing antibody-based ligand fishing of venom toxins in 384 filter-well plate format has been developed to determine the antibody target/s The approach uses Protein G beads for antibody capture followed by exposure to a full venom or purified toxins to bind their respective ligand toxin(s). This is followed by a washing/centrifugation step to remove non-binding toxins and an in-well tryptic digest. Finally, peptides from each well are analysed by nanoLC-MS/MS and subsequent Mascot database searching to identify the bound toxin/s for each antibody under investigation. The approach was successfully validated to rapidly screen antibodies sourced from hybridomas, derived from venom-immunised mice expressing either regular human antibodies or heavy-chain-only human antibodies (HCAbs).

Alternaria species produce several mycotoxins, such as alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin (ATX), tentoxin (TTX) and tenuazonic acid (TeA). This research aimed to isolate and identify mycotoxins from highly toxic Alternaria alternata (w19) and A. tennuisima isolates and their phytotoxicity effects. Fungal metabolites were extracted from 21-day cultures of Alternaria in a Czapek broth medium with the organic solvent chloroform/acetone and identified using the HPLC method. Alternaria metabolites were infiltrated in vivo into several plant leaves for phytotoxicity detection. The study investigated the impact of temperature, time, and metabolite concentration on phytotoxicity using the detached leaf infiltration technique. Five mycotoxins (TTX, TeA, ALT, AOH, and AME) were detected in A. alternata W19 isolate with 959.24, 102.03, 24.01, 9.04, and 2.44 ppm, respectively. A. tennuisima produce these toxins in a lower concentration. Infiltration of fungal metabolites induced leaf chlorosis and necrosis, which differs based on temperature, concentration and plant species. Based on our knowledge, this is the first report of Alternaria mycotoxins in Iran and a highly toxic isolate of A. alternata with rapid phytotoxicity on a wide range of susceptible hosts.

As injectable therapeutics, snake antivenoms must meet specifications for endotoxin content. The Limulus amebocyte lysate (LAL) test was used to evaluate the endotoxin content in several commercially available antivenoms released for clinical use. It was found that some products have endotoxin concentrations higher than the accepted limit for these contaminants. These results emphasize the need to include endotoxin determination as part of the routine evaluation of antivenoms by manufacturers and regulatory agencies.

Venoms comprise highly sophisticated bioactive molecules modulating ion channels, receptors, coagulation factors, and the cellular membranes. This array of targets and bioactivities requires advanced high-content bioassays to facilitate the development of novel envenomation treatments and biotechnological and pharmacological agents. In response to the existing gap in venom research, we developed a cutting-edge fluorescence-based high-throughput and high-content cellular assay. This assay enables the simultaneous identification of prevalent cellular activities induced by venoms such as membrane lysis, pore formation, and ion channel modulation. By integrating intracellular calcium with extracellular nucleic acid measurements, we have successfully distinguished these venom mechanisms within a single cellular assay. Our high-content bioassay was applied across three cell types exposed to venom components representing lytic, ion pore-forming or ion channel modulator toxins. Beyond unveiling distinct profiles for these action mechanisms, we found that the pore-forming latrotoxin α-Lt1a prefers human neuroblastoma to kidney cells and cardiomyocytes, while the lytic bee peptide melittin is not selective. Furthermore, evaluation of snake venoms showed that Elapid species induced rapid membrane lysis, while Viper species showed variable to no activity on neuroblastoma cells. These findings underscore the ability of our high-content bioassay to discriminate between clades and interspecific traits, aligning with clinical observations at venom level, beyond discriminating among ion pore-forming, membrane lysis and ion channel modulation. We hope our research will expedite the comprehension of venom biology and the diversity of toxins that elicit cytotoxic, cardiotoxic and neurotoxic effects, and assist in identifying venom components that hold the potential to benefit humankind.