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 Comparison of Honey Enriched with Laboratory Fermented Pollen vs. Natural Bee Bread in Terms of Nutritional and Antioxidant Properties, Protein In Vitro Bioaccessibility, and Its Genoprotective Effect in Yeast Cells.

The aim of the study was to compare the nutritional value and bioactivity of honey enriched with a 10% addition of natural bee bread and its substitutes obtained as a result of laboratory fermentation of bee pollen. Physicochemical parameters, antioxidant properties, as well as the bioaccessibility of proteins using an in vitro static digestion model were analyzed. The bioactivity of the obtained enriched honeys was tested using the yeast model. The research indicates the similarity of honeys with the addition of "artificial bee bread" to honey with natural ones. During in vitro digestion, good bioaccessibility of the protein from the tested products was demonstrated. The ability of the products to protect yeast cells against hydrogen superoxide-induced oxidative stress was demonstrated using a qualitative spot test, which was stronger in the case of enriched honey than in pure rapeseed control honey. Significant inhibition of the growth of both strains of yeast exposed to bee pollen-enriched honeys was also demonstrated. Furthermore, all tested samples showed significant genoprotective activity against the genotoxic effect of zeocin and the reduction of the number of DNA double-strand breaks by a minimum of 70% was observed.

SDS-PAGE Protein and HPTLC Polyphenols Profiling as a Promising Tool for Authentication of Goldenrod Honey.

The aim of the study was to use protein and polyphenolic profiles as fingerprints of goldenrod honey and to apply them for verification of the labeled variety. The markers for 10 honey samples were correlated with the standard physicochemical parameters and biological activity measured in vitro as antioxidant, antifungal and antibacterial activities. Honey proteins were examined regarding soluble protein, diastase and SDS-PAGE protein profile. The polyphenolic profile was obtained with the use of the HPTLC and the antioxidant activity was detected with standard colorimetric methods. The antimicrobial effect of representative honey samples of different chemical profiles was verified against E. coli and budding yeast. It was found that the SDS-PAGE technique allows for creating the protein fingerprint of the goldenrod honey variety which was consistent for 70% of tested samples. At the same time, the similarity of their polyphenolic profile was observed. Moreover, specific chemical composition resulted in higher bioactivity of honey against tested bacteria and yeast. The study confirmed the usefulness of both SDS-PAGE and HPTLC techniques in honey authentication, as an initial step for selection of samples which required pollen analysis.

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.

The Use of HPTLC and SDS-PAGE Methods for Coniferous Honeydew Honey Fingerprinting Compiled with Mineral Content and Antioxidant Activity.

Fir honeydew honey is a uniquely beneficial product which is often subjected to adulteration; however, pollen analysis is not useful to verify this honey type. Fourteen samples of EU protected designation of origin fir honeydew honey gathered directly from apiaries were studied. Standards of legal requirements and additional parameters, i.e., specific optical rotation, mineral content, and antioxidant activity, were tested. Five nectar honeys of different varieties were used as a comparative material. HPTLC and SDS-PAGE methods were used to fingerprint the honey types. All honeys tested fulfilled the quality requirements in terms of water content, pH, total acidity, conductivity, HMF, and diastase number. They were defined as dark amber on the Pfund scale and exhibited positive specific rotation (+2.5 to 25). Honeydew honey surpassed the tested nectar honeys in terms of mineral content and antioxidant activity as well as total polyphenolic content, except for buckwheat honey. The sugar and polyphenolic profile obtained by HPTLC allowed to distinguish honeydew from nectar honeys. The same was achieved by SDS-PAGE protein profiling. Both techniques seem to be cheap and quick tools for precisely distinguishing honeydew honey.

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.

Searching for Differences in Chemical Composition and Biological Activity of Crude Drone Brood and Royal Jelly Useful for Their Authentication.

Drone brood is a little-known bee product which is frequently considered as a male equivalent of royal jelly and is sometimes used as its adulterant. The aim of the study was to compare the chemical composition and biological activity of both bee products originated from the same apiaries (n = 3) limiting the influence of genetic and environmental factors. Moreover, for drone brood study covered testing three stages of larval development (days 7, 11, and 14). The comparison included mineral composition (ICP-OES method), protein content and protein profile (SDS-PAGE), testosterone and estradiol content (ELISA tests). HPTLC method was used to analyze of sugar, amino acids, and polyphenolic profile of drone brood and royal jelly. Moreover, their antioxidant and enzymatic properties were compared. A lot of similarities between drone brood and royal jelly were found in terms of chemical components. However, drone brood was more abundant in iron and manganese, reducing sugars and some amino acids, especially proline, tyrosine, and leucine. It contained more testosterone (especially on the 14th day) and estradiol (on the 7th day). The greatest differences in the enzymatic activities and polyphenolic profile were found. Diastase and α-glucosidase activity were found as specific enzymes of the drone brood. Similarly, ferulic and ellagic acids were characteristic for brood and were not present in royal jelly. The study showed a lot of similar features for both tested bee products, however, some specific markers which can serve to differentiate drone brood and royal jelly were found.

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.

The Comparison of Physicochemical Parameters, Antioxidant Activity and Proteins for the Raw Local Polish Honeys and Imported Honey Blends.

Many imported honeys distributed on the Polish market compete with local products mainly by lower price, which can correspond to lower quality and widespread adulteration. The aim of the study was to compare honey samples (11 imported honey blends and 5 local honeys) based on their antioxidant activity (measured by DPPH, FRAP, and total phenolic content), protein profile obtained by native PAGE, soluble protein content, diastase, and acid phosphatase activities identified by zymography. These indicators were correlated with standard quality parameters (water, HMF, pH, free acidity, and electrical conductivity). It was found that raw local Polish honeys show higher antioxidant and enzymatic activity, as well as being more abundant in soluble protein. With the use of principal component analysis (PCA) and stepwise linear discriminant analysis (LDA) protein content and diastase number were found to be significant (p < 0.05) among all tested parameters to differentiate imported honey from raw local honeys.

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.

Bases, solvates and salts: new benzimidazole- and pyridine-scaffolded ligands.

The intermolecular interactions in the structures of a series of Schiff base ligands have been thoroughly studied. These ligands can be obtained in different forms, namely, as the free base 2-[(2E)-2-(1H-imidazol-4-ylmethylidene)-1-methylhydrazinyl]pyridine, C10H11N5, 1, the hydrates 2-[(2E)-2-(1H-imidazol-2-ylmethylidene)-1-methylhydrazinyl]-1H-benzimidazole monohydrate, C12H12N6·H2O, 2, and 2-{(2E)-1-methyl-2-[(1-methyl-1H-imidazol-2-yl)methylidene]hydrazinyl}-1H-benzimidazole 1.25-hydrate, C13H14N6·1.25H2O, 3, the monocationic hydrate 5-{(1E)-[2-(1H-1,3-benzodiazol-2-yl)-2-methylhydrazinylidene]methyl}-1H-imidazol-3-ium trifluoromethanesulfonate monohydrate, C12H13N6+·CF3O3S-·H2O, 5, and the dicationic 2-{(2E)-1-methyl-2-[(1H-imidazol-3-ium-2-yl)methylidene]hydrazinyl}pyridinium bis(trifluoromethanesulfonate), C10H13N52+·2CF3O3S-, 6. The connection between the forms and the preferred intermolecular interactions is described and further studied by means of the calculation of the interaction energies between the neutral and charged components of the crystal structures. These studies show that, in general, the most important contribution to the stabilization energy of the crystal is provided by π-π interactions, especially between charged ligands, while the details of the crystal architecture are influenced by directional interactions, especially relatively strong hydrogen bonds. In one of the structures, a very interesting example of the nontypical F...O interaction was found and its length, 2.859 (2) Å, is one of the shortest ever reported.

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.

Complex-decorated surfactant-encapsulated clusters (CD-SECs) as novel multidynamic hybrid materials.

Generation of well-defined organic-inorganic hybrid materials with controllable size and morphology is challenging and an active area of modern research in view of their unique properties and thus multifunctional applications. Specifically polyoxometalates (POMs) were recognized as a very promising group for the construction of those systems, nonetheless there are domains where the profound understanding of hierarchical mutual interactions and assembly are lacking. We present an efficient approach towards the synthesis of a novel group of POM-based nanocomposites that we name Complex-Decorated Surfactant Encapsulated-Clusters (CD-SECs). In the investigated system the organic surfactant may behave as a metal-coordinating agent, thus allowing for derivatization of the synthesized SECs via utilization of the non-covalent interactions. We demonstrate possibilities and limitations of three types of hybrid systems (H1-H3) generated via seven distinct constructing approaches (routes A-G). These systems have the potential to exhibit multiresponsive functions depending on the nature of their building blocks and could find many potential applications in biology or materials science.

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.

New Artificial Biomimetic Enzyme Analogues based on Iron(II/III) Schiff Base Complexes: An Effect of (Benz)imidazole Organic Moieties on Phenoxazinone Synthase and DNA Recognition.

Elucidation of the structure and function of biomolecules provides us knowledge that can be transferred into the generation of new materials and eventually applications in e.g., catalysis or bioassays. The main problems, however, concern the complexity of the natural systems and their limited availability, which necessitates utilization of simple biomimetic analogues that are, to a certain degree, similar in terms of structure and thus behaviour. We have, therefore, devised a small library of six tridentate N-heterocyclic coordinating agents (L1-L6), which, upon complexation, form two groups of artificial, monometallic non-heme iron species. Utilization of iron(III) chloride leads to the formation of the 1:1 (Fe:Ln) 'open' complexes, whereas iron(II) trifluoromethanosulfonate allows for the synthesis of 1:2 (M:Ln) 'closed' systems. The structural differences between the individual complexes are a result of the information encoded within the metallic centre and the chosen counterion, whereas the organic scaffold influences the observed properties. Indeed, the number and nature of the external hydrogen bond donors coming from the presence of (benz)imidazole moieties in the ligand framework are responsible for the observed biological behaviour in terms of mimicking phenoxazinone synthase activity and interaction with DNA.

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.

Snake venoms promote stress-induced senescence in human fibroblasts.

Snake venoms are widely studied in terms of their systemic toxicity and proteolytic, hemotoxic, neurotoxic, and cytotoxic activities. However, little is known about snake-venom-mediated effects when used at low, noncytotoxic concentrations. In the current study, two human fibroblast cell lines of different origin, namely WI-38 fetal lung fibroblasts and BJ foreskin fibroblasts were used to investigate snake-venom-induced adaptive response at a relatively noncytotoxic concentration (0.01 µg/ml). The venoms of Indochinese spitting cobra ( Naja siamensis), western green mamba ( Dendroaspis viridis), forest cobra ( Naja melanoleuca), and southern copperhead ( Agkistrodon contortrix) were considered. Snake venoms promoted FOXO3a-mediated oxidative stress response and to a lesser extent DNA damage response, which lead to changes in cell cycle regulators both at messenger RNA and protein levels, limited cell proliferation and migration, and induced cellular senescence. Taken together, we have shown for the first time that selected snake venoms may also exert adverse effects when used at relatively noncytotoxic concentrations.

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.

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.