Venom diversity in Naja mossambica: Insights from proteomic and immunochemical analyses reveal intraspecific differences.

BACKGROUND: Intraspecific variations in snake venom composition have been extensively documented, contributing to the diverse clinical effects observed in envenomed patients. Understanding these variations is essential for developing effective snakebite management strategies and targeted antivenom therapies. We aimed to comprehensively investigate venoms from three distinct populations of N. mossambica from Eswatini, Limpopo, and KwaZulu-Natal regions in Africa in terms of their protein composition and reactivity with three commercial antivenoms (SAIMR polyvalent, EchiTAb+ICP, and Antivipmyn Africa). METHODOLOGY/PRINCIPAL FINDINGS: Naja mossambica venoms from Eswatini region exhibited the highest content of neurotoxic proteins, constituting 20.70% of all venom proteins, compared to Limpopo (13.91%) and KwaZulu-Natal (12.80%), and was characterized by the highest diversity of neurotoxic proteins, including neurotoxic 3FTxs, Kunitz-type inhibitors, vespryns, and mamba intestinal toxin 1. KwaZulu-Natal population exhibited considerably lower cytotoxic 3FTx, higher PLA2 content, and significant diversity in low-abundant proteins. Conversely, Limpopo venoms demonstrated the least diversity as demonstrated by electrophoretic and mass spectrometry analyses. Immunochemical assessments unveiled differences in venom-antivenom reactivity, particularly concerning low-abundance proteins. EchiTAb+ICP antivenom demonstrated superior reactivity in serial dilution ELISA assays compared to SAIMR polyvalent. CONCLUSIONS/SIGNIFICANCE: Our findings reveal a substantial presence of neurotoxic proteins in N. mossambica venoms, challenging previous understandings of their composition. Additionally, the detection of numerous peptides aligning to uncharacterized proteins or proteins with unknown functions underscores a critical issue with existing venom protein databases, emphasizing the substantial gaps in our knowledge of snake venom protein components. This underscores the need for enhanced research in this domain. Moreover, our in vitro immunological assays suggest EchiTAb+ICP's potential as an alternative to SAIMR antivenom, requiring confirmation through prospective in vivo neutralization studies.

The Application of MALDI-TOF MS for a Variability Study of Paenibacillus larvae.

In recent decades, the significant deterioration of the health status of honey bees has been observed throughout the world. One of the most severe factors affecting the health of bee colonies worldwide is American foulbrood disease. This devastating disease, with no known cure, is caused by the Gram-positive spore-forming bacteria of Paenibacillus larvae species. At present, DNA-based methods are being used for P. larvae identification and typing. In our study, we compare two of the most advanced DNA-based technologies (rep-PCR and 16S rRNA analyses) with MALDI-TOF MS fingerprinting to evaluate P. larvae variability in Central Europe. While 16S rRNA analysis presents a very limited variation among the strains, MALDI-TOF MS is observed to be more efficient at differentiating P. larvae. Remarkably, no clear correlation is observed between whole-genome rep-PCR fingerprinting and MALDI-TOF MS-based typing. Our data indicate that MALDI-TOF protein profiling provides accurate and cost-effective methods for the rapid identification of P. larvae strains and provides novel perspectives on strain diversity compared to conventional DNA-based genotyping approaches. The current study provides a good foundation for future studies.

Development and Characterization of Anti-Naja ashei Three-Finger Toxins (3FTxs)-Specific Monoclonal Antibodies and Evaluation of Their In Vitro Inhibition Activity.

Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.

Development of an Inhibition Enzyme-Linked Immunosorbent Assay (ELISA) Prototype for Detecting Cytotoxic Three-Finger Toxins (3FTxs) in African Spitting Cobra Venoms.

The administration of toxin-specific therapy in snake envenoming is predicated on improved diagnostic techniques capable of detecting specific venom toxins. Various serological tests have been used in detecting snakebite envenoming. Comparatively, enzyme-linked immunosorbent assay (ELISA) has been shown to offer a wider practical application. We report an inhibition ELISA for detecting three-finger toxin (3FTx) proteins in venoms of African spitting cobras. The optimized assay detected 3FTxs in N. ashei (including other Naja sp.) venoms, spiked samples, and venom-challenged mice samples. In venoms of Naja sp., the assay showed inhibition, implying the detection of 3FTxs, but showed little or no inhibition in non-Naja sp. In mice-spiked samples, one-way ANOVA results showed that the observed inhibition was not statistically significant between spiked samples and negative control (p-value = 0.164). Similarly, the observed differences in inhibition between venom-challenged and negative control samples were not statistically significant (p-value = 0.9109). At an LOD of 0.01 µg/mL, the assay was able to confirm the presence of 3FTxs in the samples. Our results show a proof of concept for the use of an inhibition ELISA model as a tool for detecting 3FTxs in the venoms of African spitting cobra snakes.

A Comprehensive Study Monitoring the Venom Composition and the Effects of the Venom of the Rare Ethiopian Endemic Snake Species Bitis parviocula.

The Ethiopian endemic snake of the species Bitis parviocula, recognized for its colorful patterns, might be more interesting as we look deeper into the venom activity. We assayed the effects of venoms from the most widespread venomous African Bitis arietens and closely related species Bitis parviocula using The Hen's Egg Test-Chorioallantoic membrane test (HET-CAM) and Chicken embryotoxicity screening test (CHEST), acetylcholinesterase (AChE) analysis, cytotoxicity assay performed on cell lines and protein analysis of selected venoms. Our results indicated that B. parviocula venom contains vasoactive compounds that have a direct effect on blood vessels. The AChE analysis showed significant ability inhibiting AChE activity in embryonic tissue. Cytotoxicity observed on A549 ATCC® CCL-185™ cells indicates the possible presence of cytotoxic agents in B. parviocula venom. We proved previously described differences in the composition of venom obtained from B. arietans and B. parviocula by using electrophoresis and total protein concentration. Based on similarities in vasoactive effects observed after administration of venoms onto a chicken chorioallantoic membrane, we suggest that venom from B. arietans and B. parviocula might share certain venom proteins responsible for haemotoxicity. The main active components of B. parviocula venom are unknown. Our results suggest that it might be worth performing proteomic analysis of B. parviocula venom as it might contain medically valuable compounds.

Effects of 3FTx Protein Fraction from Naja ashei Venom on the Model and Native Membranes: Recognition and Implications for the Mechanisms of Toxicity.

Three-finger toxins are naturally occurring proteins in Elapidae snake venoms. Nowadays, they are gaining popularity because of their therapeutic potential. On the other hand, these proteins may cause undesirable reactions inside the body's cells. A full assessment of the safety of Naja ashei venom components for human cell application is still unknown. The aim of the study was to determine the effect of the exogenous application of three-finger toxins on the cells of monocytes (U-937) and promyelocytes (HL-60), with particular emphasis on the modification of their membranes under the influence of various doses of 3FTx protein fraction (0-120 ng/mL). The fraction exhibiting the highest proportion of 3FTx proteins after size exclusion chromatography (SEC) separation was used in the experiments. The structural response of cell membranes was described on the basis of single-component and multi-component Langmuir monolayers that mimicked the native membranes. The results show that the mechanism of protein-lipid interactions depends on both the presence of lipid polar parts (especially zwitterionic type of lipids) and the degree of membrane saturation (the greatest-for unsaturated lipids). The biochemical indicators reflecting the tested cells (MDA, LDH, cell survival, induction of inflammation, LD50) proved the results that were obtained for the model.

Analysis of Cytotoxicity of Selected Asteraceae Plant Extracts in Real Time, Their Antioxidant Properties and Polyphenolic Profile.

Plants from Asteraceae family are widely used for their therapeutic effects in the treatment of gastrointestinal diseases, but the consequences of excessive intake still need to be studied. The aims of this study were the evaluation of cytotoxicity, measurement of antioxidant properties and determination of polyphenolic profile of Tanacetum vulgare L. (tansy), Achillea millefolium L. (yarrow) and Solidago gigantea Ait. (goldenrod) ethanolic extracts. The cytotoxicity of extracts was monitored by xCELLigence system in real time by using porcine intestinal epithelial cell line (IPEC-1) and by measurement of changes in metabolic activity ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay). The antioxidant properties were measured by spectrophotometric methods and polyphenolic profiles were determined by HPLC-DAD for 50% ethanol extracts (10% w/v). Strong cytotoxic effect was recorded for tansy and yarrow extracts (125-1000 µg/mL) by xCELLigence system and MTS assay. Conversely, a supportive effect on cell proliferation was recorded for goldenrod extracts (125 µg/mL) by the same methods (p < 0.001). The antioxidant activity was in good correlation with total polyphenolic content, and the highest value was recorded for goldenrod leaves, followed by tansy leaves, goldenrod flowers and yarrow leaf extracts. The goldenrod extracts were abundant with flavonoids, whereas phenolic acid derivatives predominated in the polyphenolic profile of tansy and yarrow.

Different Research Approaches in Unraveling the Venom Proteome of Naja ashei.

The dynamic development of venomics in recent years has resulted in a significant increase in publicly available proteomic data. The information contained therein is often used for comparisons between different datasets and to draw biological conclusions therefrom. In this article, we aimed to show the possible differences that can arise, in the final results of the proteomic experiment, while using different research workflows. We applied two software solutions (PeptideShaker and MaxQuant) to process data from shotgun LC-MS/MS analysis of Naja ashei venom and collate it with the previous report concerning this species. We were able to provide new information regarding the protein composition of this venom but also present the qualitative and quantitative limitations of currently used proteomic methods. Moreover, we reported a rapid and straightforward technique for the separation of the fraction of proteins from the three-finger toxin family. Our results underline the necessary caution in the interpretation of data based on a comparative analysis of data derived from different studies.

Evaluation of Antifungal Activity of Naja pallida and Naja mossambica Venoms against Three Candida Species.

In contrast to comprehensively investigated antibacterial activity of snake venoms, namely crude venoms and their selected components, little is known about antifungal properties of elapid snake venoms. In the present study, the proteome of two venoms of red spitting cobra Naja pallida (NPV) and Mozambique spitting cobra Naja mossambica (NMV) was characterized using LC-MS/MS approach, and the antifungal activity of crude venoms against three Candida species was established. A complex response to venom treatment was revealed. NPV and NMV, when used at relatively high concentrations, decreased cell viability of C. albicans and C. tropicalis, affected cell cycle of C. albicans, inhibited C. tropicalis-based biofilm formation and promoted oxidative stress in C. albicans, C. glabrata and C. tropicalis cells. NPV and NMV also modulated ammonia pulses during colony development and aging in three Candida species. All these observations provide evidence that NPV and NMV may diminish selected pathogenic features of Candida species. However, NPV and NMV also promoted the secretion of extracellular phospholipases that may facilitate Candida pathogenicity and limit their usefulness as anti-candidal agents. In conclusion, antifungal activity of snake venoms should be studied with great caution and a plethora of pathogenic biomarkers should be considered in the future experiments.

Comparison of Methods for Measuring Protein Concentration in Venom Samples.

Snake venom is an extremely interesting natural mixture of proteins and peptides, characterized by both high diversity and high pharmacological potential. Much attention has been paid to the study of venom composition of different species and also detailed analysis of the properties of individual components. Since proteins and peptides are the active ingredients in venom, rapidly developing proteomic techniques are used to analyze them. During such analyses, one of the routine operations is to measure the protein concentration in the sample. The aim of this study was to compare five methods used to measure protein content in venoms of two snake species: the Viperids representative, Agkistrodon contortrix, and the Elapids representative, Naja ashei. The study showed that for A. contortrix venom, the concentration of venom protein measured by four methods is very similar and only the NanoDrop method clearly stands out from the rest. However, in the case of N. ashei venom, each technique yields significantly different results. We hope that this report will help to draw attention to the problem of measuring protein concentration, especially in such a complex mixture as animal venoms.

Antimicrobial Activity of Protein Fraction from Naja ashei Venom Against Staphylococcus epidermidis.

One of the key problems of modern infectious disease medicine is the growing number of drug-resistant and multi-drug-resistant bacterial strains. For this reason, many studies are devoted to the search for highly active antimicrobial substances that could be used in therapy against bacterial infections. As it turns out, snake venoms are a rich source of proteins that exert a strong antibacterial effect, and therefore they have become an interesting research material. We analyzed Naja ashei venom for such antibacterial properties, and we found that a specific composition of proteins can act to eliminate individual bacterial cells, as well as the entire biofilm of Staphylococcus epidermidis. In general, we used ion exchange chromatography (IEX) to obtain 10 protein fractions with different levels of complexity, which were then tested against certified and clinical strains of S. epidermidis. One of the fractions (F2) showed exceptional antimicrobial effects both alone and in combination with antibiotics. The protein composition of the obtained fractions was determined using mass spectrometry techniques, indicating a high proportion of phospholipases A2, three-finger toxins, and L-amino acids oxidases in F2 fraction, which are most likely responsible for the unique properties of this fraction. Moreover, we were able to identify a new group of low abundant proteins containing the Ig-like domain that have not been previously described in snake venoms.

An Effective Method of Isolating Honey Proteins.

Honey is a natural sweetener composed mostly of sugars, but it contains also pollen grains, proteins, free amino acids, and minerals. The amounts and proportions of these components depend on the honey type and bee species. Despite the low content of honey protein, they are becoming a popular study object, and have recently been used as markers of the authenticity and quality of honey. Currently, the most popular methods of protein isolation from honey are dialysis against distilled water, lyophilization of dialysate, or various precipitation protocols. In this work, we propose a new method based on saturated phenol. We tested it on three popular polish honey types and we proved its compatibility with both 1D and 2D polyacrylamide gel electrophoresis (PAGE) and MS (mass spectrometry) techniques. The elaborated technique is also potentially less expensive and less time-consuming than other previously described methods, while being equally effective.

Toxicity of oxalic acid and impact on some antioxidant enzymes on in vitro-reared honeybee larvae.

Nowadays, Varroa destructor is considered as a serious pest of honeybees (Apis mellifera) and its resistance to acaricides has been reported in Europe since the early 1990s. That is why new methods of treatment for Varroa mites are still in focus of many scientists. In our study, we determined the lethal concentration LC50 (72 h) of 2.425% oxalic acid solution following single spray exposure of honeybee larvae under laboratory conditions (Guideline OECD 237 2013). Potential sublethal effects of oxalic acid were monitored through the determination of the activity of antioxidant enzymes. Activation of primary antioxidant enzymes was observed at 1.75% of oxalic acid; 3.5% of oxalic acid brought on a statistically significant increase of glutathione S-transferase activity. This change was accompanied by an increase in thiobarbituric acid reactive substances, products of lipid peroxidation. Our results indicate that oxalic acid may be harmful to bee brood when present during application.

Polyphenols Content, Antioxidant Activity, and Cytotoxicity Assessment of Taraxacum officinale Extracts Prepared through the Micelle-Mediated Extraction Method.

This experiment was conducted with extracts prepared from dandelion (Taraxacum officinale F. H. Wigg) leaves and flowers, using the micelle-mediated extraction method, with the surface active compound Triton X-100 and water⁻acetone as the extraction solvents. Extracts were, first, examined for the content of total phenols and the antioxidant capacity. All extracts showed good anti-radical properties, especially for leaves, in comparison to the flower samples. Flavonoids (mainly luteolin derivatives) and phenolic acids, predominated among the determined polyphenols. Quantitative analyses indicated acetone extract to be the richest in phenols (up to 0.535 mg/mL), in the case of dandelion leaves, and Triton X-100 extract in the case of flowers (0.385 mg/mL). Extracts were also evaluated for cytotoxicity to the model cell line (epithelial rabbit kidney cells RK13), using the colorimetric 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and the real-time cell analysis method ((RTCA); xCELLigence system). The obtained results indicated that surfactants, especially non-ionic ones, can be effectively used as modifiers in the aqueous extraction of phenolic compounds from plant materials. An advantage over the traditional organic solvents is their non-flammability. Furthermore, surfactants might also be used at low concentrations. Studies on cell lines, however, indicated the cytotoxic effect of this type of compound, even in the trace amounts present in the extracts.

First Look at the Venom of Naja ashei.

Naja ashei is an African spitting cobra species closely related to N. mossambica and N. nigricollis. It is known that the venom of N. ashei, like that of other African spitting cobras, mainly has cytotoxic effects, however data about its specific protein composition are not yet available. Thus, an attempt was made to determine the venom proteome of N. ashei with the use of 2-D electrophoresis and MALDI ToF/ToF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry techniques. Our investigation revealed that the main components of analysed venom are 3FTxs (Three-Finger Toxins) and PLA2s (Phospholipases A2). Additionally the presence of cysteine-rich venom proteins, 5'-nucleotidase and metalloproteinases has also been confirmed. The most interesting fact derived from this study is that the venom of N. ashei includes proteins not described previously in other African spitting cobras-cobra venom factor and venom nerve growth factor. To our knowledge, there are currently no other reports concerning this venom composition and we believe that our results will significantly increase interest in research of this species.

Effect of pyrethroids on female genital system. Review.

Pyrethroids have been associated with a range of toxicological effects on various organs in animals.Recent animal studies suggest that neurodevelopmental, reproductive, and immunological effects may result following exposure to some pyrethroids at levels below those that induce overt signs of neurotoxicity. A variety of pyrethroids and their metabolites have the potential to affect the reproductive system. Dose-dependent effects on reproduction are associated with exposure across pyrethroid types. In mammals, permethrin and tetramethrin and cypermethrin have been found to be associated with adverse effects at high doses. Fenvalerate, deltamethrin, cypermethrin, caused morphometric and structural changes in the female genital organs. These pyrethroids affect ovulation, cause atresia of follicles, decrease the number of follicular cells, oocytes and corpora lutea and induce vesicular atrophy of the endometrial glands. The potential hormonal activity of pyrethroids showed that certain pyrethroids and their metabolites have multiple effects on the endocrine system. The level of steroid hormones, such as progesterone and estradiol, was inhibited. The pyrethorids may have the potential to mimic estrogens or to inhibit estrogen action. Some metabolites of pyrethroids, in particular permethrin and cypermethrin, are more likely to interact with the cellular estrogen receptors than the parent pyrethroids. Though several pyrethroids posses low toxicity, some pyrethroids, such as deltamethrin, cypermethrin, fenvalerate and bifenthrin have showed considerable toxicity.

Formetanate toxicity and changes in antioxidant enzyme system of Apis mellifera larvae.

Substantial percentage of world food production depends on pollinating service of honeybees that directly depends on their health status. Among other factors, the success of bee colonies depends on health of developed larvae. The crucial phase of larval development is the first 6 days after hatching when a worker larva grows exponentially and larvae are potentially exposed to xenobiotics via diet. In the present study, we determined the lethal concentration LC50 (72 h) following single dietary exposure of honeybee larvae to formetanate under laboratory conditions, being also the first report available in scientific literature. Activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) were also measured in the homogenates of in vitro reared honeybee larvae after single formetanate exposure. Decreased specific activity of SOD and increased activities of CAT and GST suggest the induction of oxidative stress. Higher levels of thiobarbituric reactive species in all samples supported this fact. Comparing determined larval toxicity (LC50 of 206.01 mg a.i./kg diet) with adult toxicity data, we can suppose that the larvae may be less sensitive to formetanate than the adult bees.

Tauopathies - Focus on Changes at the Neurovascular Unit.

In the past, the blood-brain barrier (BBB) has been characterized mainly as a layer of endothelial cells forming the vessel/capillary wall of the brain. More recently, the BBB is considered to be a part of a highly dynamic and interactive system called the neurovascular unit (NVU), consisting of vascular cells, glial cells, and neurons. The list of central nervous system (CNS) pathologies involving BBB dysfunction is rapidly growing. The opening of the BBB and subsequent infiltration of serum components to the brain can lead to a host of processes resulting in progressive synaptic and neuronal dysfunction and loss. Such processes have been implicated in different diseases, including vascular dementias, stroke, Alzheimer´s disease (AD), Parkinson´s disease, multiple sclerosis, amyotrophic lateral sclerosis, hypoxia, ischemia, and diabetes mellitus. Tauopathies represent a heterogeneous group of around 20 different neurodegenerative diseases characterized by abnormal deposition of microtubule-associated protein tau in cells of the nervous system. Increased microvascular permeability has been more typically related to cerebrovascular deposition of amyloid-ß (Aß), but in contrast very little is known about the connection between functional impairment of the BBB and the misfolded tau proteins. Here, we review what is known about tauopathies, the BBB, and the NVU.

Proteomic Analyses of Agkistrodon contortrix contortrix Venom Using 2D Electrophoresis and MS Techniques.

Snake venom is a complex mixture of proteins and peptides which in the Viperidae is mainly hemotoxic. The diversity of these components causes the venom to be an extremely interesting object of study. Discovered components can be used in search for new pharmaceuticals used primarily in the treatment of diseases of the cardiovascular system. In order to determine the protein composition of the southern copperhead venom, we have used high resolution two dimensional electrophoresis and MALDI ToF/ToF MS-based identification. We have identified 10 groups of proteins present in the venom, of which phospholipase A2 and metalloprotease and serine proteases constitute the largest groups. For the first time presence of 5'-nucleotidase in venom was found in this group of snakes. Three peptides present in the venom were also identified. Two of them as bradykinin-potentiating agents and one as an inhibitor.

Proteome and Peptidome of Vipera berus berus Venom.

Snake venom is a rich source of peptides and proteins with a wide range of actions. Many of the venom components are currently being tested for their usefulness in the treatment of many diseases ranging from neurological and cardiovascular to cancer. It is also important to constantly search for new proteins and peptides with properties not yet described. The venom of Vipera berus berus has hemolytic, proteolytic and cytotoxic properties, but its exact composition and the factors responsible for these properties are not known. Therefore, an attempt was made to identify proteins and peptides derived from this species venom by using high resolution two-dimensional electrophoresis and MALDI ToF/ToF mass spectrometry. A total of 11 protein classes have been identified mainly proteases but also l-amino acid oxidases, C-type lectin like proteins, cysteine-rich venom proteins and phospholipases A2 and 4 peptides of molecular weight less than 1500 Da. Most of the identified proteins are responsible for the highly hemotoxic properties of the venom. Presence of venom phospholipases A2 and l-amino acid oxidases cause moderate neuro-, myo- and cytotoxicity. All successfully identified peptides belong to the bradykinin-potentiating peptides family. The mass spectrometry data are available via ProteomeXchange with identifier PXD004958.