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Three-finger proteins are the most abundant toxins in the venom of Naja ashei, a snake species from the Elapidae family. This research aimed to describe the effects of varying charges of these proteins, isolated from Naja ashei venom using SEC and IEX chromatography. The study examined how differently charged three-finger toxin fractions interact with and affect neuroblastoma (SK-N-SH) and promyeloblast (HL-60) cells, as well as model Langmuir membranes and liposomes designed to mimic cellular lipid composition. Findings revealed that protein surface charges significantly impact cell survival (MTT assay), membrane damage (lactate dehydrogenase release, malondialdehyde formation), and the structural and electrochemical properties of model membranes (Langmuir membranes and zeta potential for liposomes and cancer cell lines). Results indicated that SK-N-SH cells, characterized by a higher negative charge on their cell membranes, interacted more effectively with positively charged toxins than HL-60 cells. However, the mechanism of these electrostatic interactions is complex. The research demonstrated that electrostatic and mechanical membrane modifications induced by venom proteins can significantly affect cell metabolism. Additionally, the total charge of the membrane, influenced by polar lipid components and phospholipid saturation, plays a decisive role in toxin interaction. [ABSTRACT FROM AUTHOR]

Simple Summary: Crotalus snake envenomation poses a serious challenge due to its diverse toxicological effects, including neurological, myotoxic, and cytotoxic symptoms, often leading to death. The aim of the study was to elucidate the physiological effects of exposure to Crotalus atrox and Crotalus adamanteus venoms and to assess toxicity using chicken embryo models. Currently, there is not a lot of research demonstrating the physiological effects of venom, including its potential impact on embryos, in accordance with the 3R rules. The applied research model consisted of the chick embryotoxicity screening test (CHEST) and the chick chorioallantoic membrane (CAM) test, which allowed for (i) a demonstration of the greater toxicity of C. adamanteus venom and (ii) an observation of the embryotoxic effect and vasoactive nature of the tested venom species. Additionally, (iii) morphological abnormalities (such as Siamese twins) emerged, and (iv) changes in the activity of acetylcholinesterase (AChE) were identified (resulting solely from its presence in the examined tissues due to the lack of this component in exogenously applied venom). These results provide crucial insights into the mechanisms of Crotalus venom toxicity and their potential biomedical applications. Crotalus snakebites induce various toxicological effects, encompassing neurological, myotoxic, and cytotoxic symptoms, with potentially fatal outcomes. Investigating venom toxicity is essential for public health, and developing new tools allows for these effects to be studied more comprehensively. The research goals include the elucidation of the physiological consequences of venom exposure and the assessment of toxicity using animal models. Chicken embryos serve as valuable models for assessing venom toxicity through the chick embryotoxicity screening test (CHEST) and the chick chorioallantoic membrane (CAM) assay, particularly useful for evaluating vascular impacts. C. adamanteus venom application resulted in higher embryotoxicity and morphological abnormalities, such as Siamese twins. The CAM assay demonstrated the hemorrhagic effects of venom, varying with venom type and concentration. The irritant potential of both venom types was classified as slight or moderate depending on their concentration. Additionally, acetylcholinesterase (AChE) activity was performed to receive information about organ toxicity. The results show that both venoms induced changes in the whole embryo, heart, and liver weights, but the C. adamanteus venom was identified as more toxic. Specific venom concentrations affected AChE activity in embryonic tissues. These findings underscore the embryotoxic and vasoactive properties of Crotalus venoms, providing valuable insights into their mechanisms of toxicity and potential applications in biomedicine. [ABSTRACT FROM AUTHOR]

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. Author summary: Snakebite envenoming is a pervasive global health concern, posing substantial risks, particularly in less developed regions. The intricate variations in venom composition within a single species have been well-documented, contributing significantly to the varied clinical effects experienced by envenomed patients. It is imperative to unravel these variations, as they are pivotal in the formulation of effective snakebite management strategies and the development of targeted antivenom therapies. In this study, our focus rested on the venom of the Naja mossambica species, dwelling in diverse African regions. Our objective was to delve into the toxin composition of these venoms and understand how these toxins interact with commercially available antivenoms. This exploration was aimed to uncover which toxins, despite antivenom application, evade neutralization. This information becomes a cornerstone in the design of more potent and efficacious antivenoms, contributing to a nuanced approach in combating the complex landscape of snakebite envenoming. [ABSTRACT FROM AUTHOR]

The effect of 10% dietary flaxseed fed for 3 and 6 weeks on serum hormone levels of fattening gilts, the fatty acid (FA) follicular fluid (FF) composition of small and large antral follicles, and the steroidogenesis and IGF-I secretion by isolated small antral follicles and their response to regulatory hormones (LH, FSH, IGF-I) was studied using immunoassay and gas chromatography analyses. Both supplemental periods increased levels of P4 and IGF-I in blood serum. A shorter period inhibited steroidogenesis (P4, T, E2) and IGF-I secretion by small antral follicles, which was associated with decreased levels of monounsaturated FAs (MUFA) and preferred n-6 polyunsaturated FA (PUFA) metabolism. A longer period stimulated hormone secretion at elevated levels of saturated FAs (SFA) at the expense of MUFAs and PUFAs preferring the n-3 PUFA metabolism. Out of ovarian regulators, only LH and IGF-I were able to alter the secretion of steroids and IGF-I by small follicles of fattening pigs fed a basal diet. The effect of flaxseed on the secretion of follicular hormones after both supplemental periods was altered by all regulatory hormones in a dose-dependent manner. The level of SFAs and PUFAs in FF of large follicles increased with the length of flaxseed feeding, suggesting the suppression of ovulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The effect of combining probiotics (Lactobacillus plantarum and Lactobacillus fermentum) with flaxseed (a source of n-3 PUFAs) on the lipid metabolism and long-chain fatty acid profile of conventional piglets after weaning was studied. The levels of total lipids and high-density lipoproteins cholesterol decreased from Day 7 post-weaning, whereas levels of low-density lipoproteins cholesterol, total cholesterol and triglycerides did not change significantly in piglets with supplemented diet. The levels of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased seven days post-weaning; however, the levels of dihomogamma-linolenic acid and arachidonic acid (AA) were lower and linoleic acid (LA) higher in synbioticsfed piglets compared with controls. This study demonstrates the efficacy of conversion of ALA to EPA and DHA, where delta-6-desaturase was predominantly used for n-3 polyunsaturated fatty acid synthesis from ALA at the expense of n-6 PUFAs from LA, which caused rapid increase in EPA/AA ratio on Day 14 after weaning. Combination of probiotic cheese and flaxseed is a good dietary supplement for piglets before weaning, helping them to adapt to change in diet more easily by regulating changes in lipid metabolism and vitality, and reducing the likelihood of chronic diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The aim of our experiment was to study how synbiotics are able to deal with the problems of post-weaning piglets. Lactobacillus plantarum - BiocenolTM LP96 (CCM 7512), Lactobacillus fermentum - BiocenolTM LF99 (CCM 7514) and flaxseed (rich in n-3 polyunsaturated fatty acids) were administered to 36 conventional piglets from a problematic breed with confirmed presence of enterotoxigenic Escherichia coli and Coronavirus. The experimental piglets were supplied with probiotic cheeses and crushed flax-seed in the period starting 10 days before weaning and lasting up to 14 days post-weaning. Piglets in the control group were supplied only control cheese. The impact of such additives on the release of lactate dehydrogenase (LDH; spectroscopic and electrophoretic assay), alteration of immunity (index of metabolic activity), jejunum histology (light microscopy), and health of conventional piglets from a problematic breed (monitoring of hematology, consistency and moisture of feces and body temperature) were examined. We found significant decrease in LDH leakage in the blood serum and tissue extracts, indicating better cell membrane integrity in the individual organs of animals. Probiotics and flaxseed applied together seem to be a good source of nutrients to improve the immune status and the integrity of jejunum mucosa during infection. © 2015 Japanese Society of Animal Science [ABSTRACT FROM AUTHOR]

A novel approach to the study of venoms characterizes and compares the venom structure of genusDendroaspispresented in this work. The complex molecular structure of individual venoms of genusDendroaspiswas defined graphically as a record of synchronous fluorescence fingerprint and atomic force microscopy. Simple comparison of these methods mentioned above of individual analyzed venom samples will immediately reveal changes in each venom composition. Application of these methods with electrophoresis and total protein concentration are new alternatives that were used for monitoring venom composition in selected snakes. Electrophoretogram of black mamba with very low content of total proteins contained the largest number of separated fractions, whereas synchronous fluorescence analysis showed that the highest endogenous fluorescence was found in the venom of black mamba compared with the venom of green mambas, respectively. Our results confirmed different molecular structure in the venom of the genusDendroaspis. The practical advantages of the selected techniques are high sensitivity and minimal quantity of venom required for the assay. These novel methods show that the least toxicDendroaspis intermediuscontained the greatest amount of proteins contrary toDendroaspis polylepiswith a considerably lower content of proteins but the highest toxic bioactivity. [ABSTRACT FROM AUTHOR]

The aim of this paper was to characterize and compare spitting cobras of Africa. The autofluorescence ofNaja asheiandNaja nigricollisvenoms was investigated by atomic force microscopy and fluorescence analysis. The synchronous fluorescence analysis showed higher endogenous fluorescence ofNaja asheivenom in comparison withNaja nigricollisvenom. The atomic force microscopy revealed different surfaces of compared venoms as a result of different amounts of proteins. Our results confirmed different structures ofNaja nigricollisandNaja asheivenom. The practical advantages of selected techniques are high sensitivity, minimal quantity of venom, and capability to measure the spitting cobra by autofluorescence. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]