Exploring the Potential Biological Activities of Pyrazole-Based Schiff Bases as Anti-Diabetic, Anti-Alzheimer's, Anti-Inflammatory, and Cytotoxic Agents: In Vitro Studies with Computational Predictions.

In this innovative research, we aim to reveal pyrazole-based Schiff bases as new multi-target agents. In this context, we re-synthesized three sets of pyrazole-based Schiff bases, 5a-f, 6a-f, and 7a-f, to evaluate their biological applications. The data from in vitro biological assays (including antioxidant and scavenging activities, anti-diabetes, anti-Alzheimer's, and anti-inflammatory properties) of the pyrazole-based Schiff bases 5a-f, 6a-f, and 7a-f showed that the six pyrazole-based Schiff bases 5a, 5d, 5e, 5f, 7a, and 7f possess the highest biological properties among the compounds evaluated. The cytotoxicity against lung (A549) and colon (Caco-2) human cancer types, as well as normal lung (WI-38) cell lines, was evaluated. The data from the cytotoxicity investigation demonstrated that the three Schiff bases 5d, 5e, and 7a are active against lung (A549) cells, while the two Schiff bases 5e and 7a exhibited the highest cytotoxicity towards colon (Caco-2) cells. Additionally, the enzymatic activities against caspase-3 and Bcl-2 of the six pyrazole-based Schiff bases 5a, 5d, 5e, 5f, 7a, and 7f were evaluated. Furthermore, we assessed the in silico absorption, distribution, metabolism, and toxicity (ADMT) properties of the more potent pyrazole-based Schiff bases. After modifying the structures of the six pyrazole-based Schiff bases, we plan to further extend the studies in the future.

Prolidase activity assays. A survey of the reported literature methodologies.

Prolidase (EC.3.4.13.9) is a dipeptidase known nowadays to play a pivotal role in several physiological and pathological processes. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), thus finely regulating the homeostasis of free proline and hydroxyproline. Abnormally high or low levels of prolidase have been found in numerous acute and chronic syndromes affecting humans (chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection, and others) for which the content of proline is well recognized as a clinical marker. As a consequence, the accurate analytical determination of prolidase activity is of greatly significant importance in clinical diagnosis and therapy. Apart from the Chinard's assay, some other more sensitive and well validated methodologies have been published. These include colorimetric and spectrophotometric determinations of free proline produced by enzymatic reactions, capillary electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, electrochemoluminescence, thin layer chromatography, and HPLC. The aim of this comprehensive review is to make a detailed survey of the in so far reported analytical techniques, highlighting their general features, as well as their advantages and possible drawbacks, providing in the meantime suggestions to stimulate further research in this intriguing field.

Multidimensional analysis of wheat original crucial endogenous enzymes driving microbial communities metabolism during high-temperature Daqu fermentation.

Knowledge of the metabolism of functional enzymes is the key to accelerate the transformation and utilization of raw materials during high temperature Daqu (HTD) manufacturing. However, the metabolic contribution of raw materials-wheat is always neglected. In this research, the relationship between the metabolism of wheat and microorganisms was investigated using physicochemical and sequencing analysis method. Results showed that the process of Daqu generation was divided into three stages based on temperature. In the early stage, a positive correlation was found between Monascus, Rhizopus and glucoamylase metabolism (r > 0.8, p < 0.05). Meanwhile, the glucoamylase metabolism in wheat occupied 63.8 % of the total matrix at the day 4. In the middle to later stages, the wheat metabolism of proteases, α-amylases and lipases in gradually reached their peak. Additionally, Lactobacillus and α-amylases presented a positive correlation (r > 0.7, p < 0.05), and the α-amylases metabolism in wheat occupied 22.18 % of the total matrix during the same time period. More importantly, the changes of enzyme activity metabolic pathway in wheat and microorganism were reflected by respiratory entropy (RQ). Overall, these results guide the choice of substrate during Daqu production.

Total and chemoenzymatic synthesis of the lipodepsipeptide rhizomide A.

Rhizomides are a family of depsipeptide macrolactones synthesized by a non-ribosomal peptide synthetase (NRPS) encoded in the genome of Paraburkholderia rhizoxinica str. HKI 454. In this study, the total and chemoenzymatic synthesis of the depsipeptide rhizomide A is described. Rhizomide A was generated through macrolactamization while thelinear C-terminal N-acetylcysteamine (SNAC) thioester substrate was synthesized through a C-terminal thioesterification strategy. It was shown that the rhizomide A thioesterase (RzmA-TE) is an active macrocyclization catalyst, allowing the chemoenzymatic synthesis of rhizomide A.This work further showcases the biocatalytic power of TEs in accessing complex macrocyclic natural products.

Novel Thiazole-Based SIRT2 Inhibitors Discovered via Molecular Modelling Studies and Enzymatic Assays.

Recently, the development of sirtuin small molecule inhibitors (SIRTIs) has been gaining attention for the treatment of different cancer types, but also to contrast neurodegenerative disease, diabetes, and autoimmune syndromes. In the search for SIRT2 modulators, the availability of several X-crystallographic data regarding SIRT2-ligand complexes has allowed for setting up a structure-based study, which is herein presented. A set of 116 SIRT2 inhibitors featuring different chemical structures has been collected from the literature and used for molecular docking studies involving 4RMG and 5MAT PDB codes. The information found highlights key contacts with the SIRT2 binding pocket such as Van der Waals and π-π stacking with Tyr104, Phe119, Phe234, and Phe235 in order to achieve high inhibitory ability values. Following the preliminary virtual screening studies, a small in-house library of compounds (1a-7a), previously investigated as putative HSP70 inhibitors, was described to guide the search for dual-acting HSP70/SIRT2 inhibitors. Biological and enzymatic assays validated the whole procedure. Compounds 2a and 7a were found to be the most promising derivatives herein proposed.

Design of a structure-activity relationship model of vitamin K epoxide reductase (VKORC1) inhibitors combining chemical synthesis of new compounds, enzymatic assays and molecular modelling.

Vitamin K antagonists (VKAs) anticoagulants have been used since the 1950s as medicines and rodenticides. These molecules are mainly 4-hydroxycoumarin derivatives and act by inhibiting the vitamin K epoxide reductase (VKORC1), an endoplasmic reticulum membrane resident enzyme. However, many VKORC1 mutations have been reported over the last decade, inducing VKAs resistances and thus treatments failures. Although studies have reported experimental and computational investigations of VKAs based on VKORC1 structural homology models, the development of new effective anticoagulants has been quite complex due to the lack of structural data and reliable structure-activity relationships. However, the recent publication of VKORC1 crystal structure provides new information for further studies. Based on these findings, we combined chemical synthesis, enzymatic assays and molecular modelling methods to design a structure-activity relationship (SAR) model. Our results proved that the lipophilicity, the membrane permeability of inhibitors and their affinity towards human VKORC1 enzyme are the main characteristics for potent anticoagulants. Our SAR model managed to rank compounds according to their ability to inhibit the human VKORC1. Such a tool might constitute an alternative to evaluate new molecules potency before their chemical synthesis and biological assessment and might assist the development of new VKAs.

Evaluation and Comparison of Dermo-Cosmetic Activities of Three Oak Species by Targeting Antioxidant Metabolites and Skin Enzyme Inhibitors.

The two main species, sessile oak (Quercus petraea Liebl.) and pedunculate oak (Quercus robur L.), predominant in French forests, are mainly used for aging wines and spirits; however, the potential of oak wood extract as a source of natural antioxidants, due to its high polyphenol content, could be more widely exploited. This study focuses on three oak species, the two that are well-known, namely, sessile and pedunculate oak, and a third that has seldom been described and valorized, namely, pubescent oak (Quercus pubescens). Water extracts of these three species were fractionated by semi-preparative HPLC. The antioxidant activities of crude extracts and fractions were measured by colorimetric and enzymatic tests. The anti-elastase and anti-collagenase activities of the extracts and their fractions were also evaluated. In parallel, samples were analyzed by UHPLC-HRMS to correlate the activity with the molecular composition using molecular networks. The results obtained for the total extract of the three species were compared to determine if the activity depended on the species. The results within the same species were also compared to highlight which fraction and, therefore, which molecular family was involved in the activity of the total extract. The various antioxidant tests showed good activity of the total extract for the three species of oak and a very good anti-collagenase activity. The antioxidant activity of oak extract has already been proven in the literature and this is correlated with its richness in polyphenols. This study shows that each molecular family of the extract contributes to the activities of the total extract. Oak extract can be used to neutralize the ROS produced during oxidative stress and to prevent the degradation of collagen and elastase during skin aging. Its complementary properties make oak extract a valuable ingredient to act against skin aging.

A Prototype Assay Multiplexing SARS-CoV-2 3CL-Protease and Angiotensin-Converting Enzyme 2 for Saliva-Based Diagnostics in COVID-19.

With the current state of COVID-19 changing from a pandemic to being more endemic, the priorities of diagnostics will likely vary from rapid detection to stratification for the treatment of the most vulnerable patients. Such patient stratification can be facilitated using multiple markers, including SARS-CoV-2-specific viral enzymes, like the 3CL protease, and viral-life-cycle-associated host proteins, such as ACE2. To enable future explorations, we have developed a fluorescent and Raman spectroscopic SARS-CoV-2 3CL protease assay that can be run sequentially with a fluorescent ACE2 activity measurement within the same sample. Our prototype assay functions well in saliva, enabling non-invasive sampling. ACE2 and 3CL protease activity can be run with minimal sample volumes in 30 min. To test the prototype, a small initial cohort of eight clinical samples was used to check if the assay could differentiate COVID-19-positive and -negative samples. Though these small clinical cohort samples did not reach statistical significance, results trended as expected. The high sensitivity of the assay also allowed the detection of a low-activity 3CL protease mutant.

The Venom Composition of the Snake Tribe Philodryadini: 'Omic' Techniques Reveal Intergeneric Variability among South American Racers.

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Genotypic and phenotypic insights into virulence factors of nosocomial Stenotrophomonas maltophilia isolates collected in Bulgaria (2011-2022).

The present study aimed to explore the virulence characteristics in 221 Bulgarian nosocomial Stenotrophomonas maltophilia isolates (2011-2022) via screening for the presence of virulence genes, their mutational variability, and the corresponding enzyme activity. PCR amplification, enzymatic assays, whole-genome sequencing (WGS), and biofilm quantification on a polystyrene plate were performed. The incidence of virulence determinants was as follows: stmPr1 (encoding for the major extracellular protease StmPr1) 87.3%, stmPr2 (minor extracellular protease StmPr2) 99.1%, Smlt3773 locus (outer membrane esterase) 98.2%, plcN1 (non-hemolytic phospholipase C) 99.1%, and smf-1 (type-1 fimbriae, biofilm-related gene) 96.4%. The 1621-bp allele of stmPr1 was most frequently found (61.1%), followed by the combined allelic variant (17.6%), stmPr1-negative genotype (12.7%), and 868-bp allele (8.6%). Protease, esterase, and lecithinase activity was observed in 95%, 98.2%, and 17.2% of the isolates, respectively. The WGS-subjected isolates (n = 9) formed two groups. Five isolates possessed only the 1621-bp variant of stmPr1, higher biofilm formation ability (Optical Density at λ = 550 nm (OD550): 1.253-1.789), as well as a low number of mutations in the protease genes and smf-1. Three other isolates had only the 868-bp variant, weaker biofilm production (OD550: 0.788-1.108), and higher number of mutations within these genes. The only weak biofilm producer (OD550 = 0.177) had no stmPr1 alleles. In conclusion, the similar PCR detection rates did not allow differentiation of the isolates. In contrast, WGS permitted stmPr1 alleles-based differentiation. To the best of our knowledge, this is the first Bulgarian study presenting genotypic and phenotypic insights into virulence factors of S. maltophilia isolates.

Isolation and Characterisation of Electrogenic Bacteria from Mud Samples.

To develop efficient microbial fuel cell systems for green energy production using different waste products, establishing characterised bacterial consortia is necessary. In this study, bacteria with electrogenic potentials were isolated from mud samples and examined to determine biofilm-formation capacities and macromolecule degradation. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identifications have revealed that isolates represented 18 known and 4 unknown genuses. They all had the capacities to reduce the Reactive Black 5 stain in the agar medium, and 48 of them were positive in the wolfram nanorod reduction assay. The isolates formed biofilm to different extents on the surfaces of both adhesive and non-adhesive 96-well polystyrene plates and glass. Scanning electron microscopy images revealed the different adhesion potentials of isolates to the surface of carbon tissue fibres. Eight of them (15%) were able to form massive amounts of biofilm in three days at 23 °C. A total of 70% of the isolates produced proteases, while lipase and amylase production was lower, at 38% and 27% respectively. All of the macromolecule-degrading enzymes were produced by 11 isolates, and two isolates of them had the capacity to form a strong biofilm on the carbon tissue one of the most used anodic materials in MFC systems. This study discusses the potential of the isolates for future MFC development applications.

The venom composition of the snake tribe Philodryadini: ‘Omic’ techniques reveal intergeneric variability among South American racers

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Enantiocontrolled Preparation of ϒ-Substituted Cyclohexenones: Synthesis and Kinase Activity Assays of Cyclopropyl-Fused Cyclohexane Nucleosides.

The enantioselective preparation of the two isomers of 4-hydroxy-2-cyclohexanone derivatives 1a,b was achieved, starting from a common cyclohexenone, through asymmetric transfer hydrogenation (ATH) reactions using bifunctional ruthenium catalysts. From these versatile intermediates, a stereoselective route to a cytosine analogue built on a bicyclo [4.1.0]heptane scaffold is described. Nucleoside kinase activity assays with this cyclopropyl-fused cyclohexane nucleoside, together with other related nucleosides (2a-e), were performed, showing that thymine- and guanine- containing compounds have affinity for herpes simplex virus Type 1 (HSV-1) thymidine kinase (TK) but not for human cytosolic TK-1, thus pointing to their selectivity for herpetic TKs but not cellular TKs.

Methods to Analyze the Redox Reactivity of Plant Proteins.

Proteins can be covalently modified by a broad range of highly reactive chemicals and redox mechanisms. Reversible redox-mediated post-translational modifications of sensitive cysteine thiol groups in proteins impact protein characteristics such as interaction behavior and activity state. Evaluating the response of proteins to redox perturbation or reactive chemical species is critical for understanding the underlying mechanisms involved and their contribution to plant stress physiology. Here we provide a detailed workflow that includes procedures for (i) purification, processing, and analysis of protein samples with redox agents, (ii) determining redox-modulated monomer to oligomer transitions using size exclusion chromatography, and (iii) activity assays for monitoring the impact of redox agents on purified enzymes and in crude extracts from plants subjected to oxidative stress. We exemplified how to apply several of the methods discussed for analyzing redox-sensing metallopeptidases, such as thimet oligopeptidases. We anticipate that these protocols should find broad applications in monitoring biochemical properties of other classes of redox-sensitive plant proteins.

Repurposing of FDA Approved Drugs Against SARS-CoV-2 Papain-Like Protease: Computational, Biochemical, and in vitro Studies.

The pandemic caused by SARS-CoV-2 (SCoV-2) has impacted the world in many ways and the virus continues to evolve and produce novel variants with the ability to cause frequent global outbreaks. Although the advent of the vaccines abated the global burden, they were not effective against all the variants of SCoV-2. This trend warrants shifting the focus on the development of small molecules targeting the crucial proteins of the viral replication machinery as effective therapeutic solutions. The PLpro is a crucial enzyme having multiple roles during the viral life cycle and is a well-established drug target. In this study, we identified 12 potential inhibitors of PLpro through virtual screening of the FDA-approved drug library. Docking and molecular dynamics simulation studies suggested that these molecules bind to the PLpro through multiple interactions. Further, IC50 values obtained from enzyme-inhibition assays affirm the stronger affinities of the identified molecules for the PLpro. Also, we demonstrated high structural conservation in the catalytic site of PLpro between SCoV-2 and Human Coronavirus 229E (HCoV-229E) through molecular modelling studies. Based on these similarities in PLpro structures and the resemblance in various signalling pathways for the two viruses, we propose that HCoV-229E is a suitable surrogate for SCoV-2 in drug-discovery studies. Validating our hypothesis, Mefloquine, which was effective against HCoV-229E, was found to be effective against SCoV-2 as well in cell-based assays. Overall, the present study demonstrated Mefloquine as a potential inhibitor of SCoV-2 PLpro and its antiviral activity against SCoV-2. Corroborating our findings, based on the in vitro virus inhibition assays, a recent study reported a prophylactic role for Mefloquine against SCoV-2. Accordingly, Mefloquine may further be investigated for its potential as a drug candidate for the treatment of COVID.

Enhanced perchloroethene dechlorination by humic acids via increasing the dehalogenase activity of Dehalococcoides strains.

Perchloroethene (PCE) is a widely used chlorinated solvent. PCE is toxic to humans and has been identified as an environmental contaminant at thousands of sites worldwide. Several Dehalococcoides mccartyi strains can transform PCE to ethene, and thus contribute to bioremediation of contaminated sites. Humic acids (HA) are ubiquitous redox-active compounds of natural aquatic and soil systems and have been intensively studied because of their effect in electron transfer. In this study, we observed the dechlorination of PCE was accelerated by HA in mixed cultures containing Dehalococcoides strains. Anthraquinone-2,6-disulfonic acid (AQDS), a humic acid analogue, inhibited PCE dechlorination in our cultures and thus induced an opposite effect on PCE dehalogenation than HA. We observed the same effect on PCE dechlorination with the pure culture of Dehalococcoides mccartyi strain CBDB1. Not only in mixed cultures but also in pure cultures, growth of Dehalococcoides was not influenced by HA but inhibited by AQDS. Enzymatic activity tests confirmed the dehalogenating activity of strain CBDB1 was increased by HA, especially when using hydrogen as electron donor. We conclude that HA enhanced PCE dechlorination by increasing the reaction speed between hydrogen and the dehalogenase enzyme rather than acting as electron shuttle through its quinone moieties.

Measurement of Hepatic Lipids.

Liver plays a central role in lipid metabolism, uptake of lipoproteins and lipids from the circulation (e.g., chylomicron remnant), and secretions of very low-density lipoproteins (VLDL). Therefore, measurements of lipid levels in the liver have been broadly used to check hepatic function, especially in subjects who have chronic liver diseases, such as nonalcoholic steatohepatitis (NASH), in which there is accumulation of fat, inflammation, and damage to liver cells. In this chapter, we describe the processes of extracting hepatic lipids by the method of Folch et al., and measuring the levels of cholesterol, triglycerides, phospholipids, and non-esterified fatty acids using enzymatic assays.

Measurement of neutral ceramidase activity in vitro and in vivo.

Neutral ceramidase is a hydrolase of ceramide that has been implicated in multiple biologic processes, including inflammation and oncogenesis. Ceramides and other sphingolipids, belong to a family of N-acyl linked lipids that are biologically active in signaling, despite their limited structural functions. Ceramides are generally pro-apoptotic, while sphingosine and sphingosine-1-phosphate (S1P) exert proliferative and pro-oncogenic effects. Ceramidases are important regulators of ceramide levels that hydrolyze ceramide to sphingosine. Thus, ceramidase inhibition significantly increases the quantities of ceramide and its associated signaling. To better understand the function of ceramide, biochemical and cellular assays for enzymatic activity were developed and validated to identify inhibitors of human neutral ceramidase (nCDase). Here we review the measurement of nCDase activity both in vitro and in vivo.

Shedding Lights on Crude Venom from Solitary Foraging Predatory Ant Ectatomma opaciventre: Initial Toxinological Investigation.

Some species of primitive predatory ants, despite living in a colony, exercise their hunting collection strategy individually; their venom is painful, paralyzing, digestive, and lethal for their prey, yet the toxins responsible for these effects are poorly known. Ectatomma opaciventre is a previously unrecorded solitary hunting ant from the Brazilian Cerrado. To overcome this hindrance, the present study performed the in vitro enzymatic, biochemical, and biological activities of E. opaciventre to better understand the properties of this venom. Its venom showed several proteins with masses ranging from 1-116 kDa, highlighting the complexity of this venom. Compounds with high enzymatic activity were described, elucidating different enzyme classes present in the venom, with the presence of the first L-amino acid oxidase in Hymenoptera venoms being reported. Its crude venom contributes to a state of blood incoagulability, acting on primary hemostasis, inhibiting collagen-induced platelet aggregation, and operating on the fibrinolysis of loose red clots. Furthermore, the E. opaciventre venom preferentially induced cytotoxic effects on lung cancer cell lines and three different species of Leishmania. These data shed a comprehensive portrait of enzymatic components, biochemical and biological effects in vitro, opening perspectives for bio-pharmacological application of E. opaciventre venom molecules.

Shedding lights on crude venom from solitary foraging predatory ant Ectatomma opaciventre: initial toxinological investigation

Some species of primitive predatory ants, despite living in a colony, exercise their hunting collection strategy individually; their venom is painful, paralyzing, digestive, and lethal for their prey, yet the toxins responsible for these effects are poorly known. Ectatomma opaciventre is a previously unrecorded solitary hunting ant from the Brazilian Cerrado. To overcome this hindrance, the present study performed the in vitro enzymatic, biochemical, and biological activities of E. opaciventre to better understand the properties of this venom. Its venom showed several proteins with masses ranging from 1–116 kDa, highlighting the complexity of this venom. Compounds with high enzymatic activity were described, elucidating different enzyme classes present in the venom, with the presence of the first L-amino acid oxidase in Hymenoptera venoms being reported. Its crude venom contributes to a state of blood incoagulability, acting on primary hemostasis, inhibiting collagen-induced platelet aggregation, and operating on the fibrinolysis of loose red clots. Furthermore, the E. opaciventre venom preferentially induced cytotoxic effects on lung cancer cell lines and three different species of Leishmania. These data shed a comprehensive portrait of enzymatic components, biochemical and biological effects in vitro, opening perspectives for bio-pharmacological application of E. opaciventre venom molecules.