Glutamine ameliorates Bungarus multicinctus venom-induced lung and heart injury through HSP70: NF-κB p65 and P53/PUMA signaling pathways involved

Background: Bungarus multicinctus is one of the most dangerous venomous snakes prone to cardiopulmonary damage with extremely high mortality. In our previous work, we found that glutamine (Gln) and glutamine synthetase (GS) in pig serum were significantly reduced after Bungarus multicinctus bite. In the present study, to explore whether there is a link between the pathogenesis of cardiopulmonary injury and Gln metabolic changes induced by Bungarus multicinctus venom. We investigated the effect of Gln supplementation on the lung and heart function after snakebite. Methods: We supplemented different concentrations of Gln to mice that were envenomated by Bungarus multicinctus to observe the biological behavior, survival rate, hematological and pathological changes. Gln was supplemented immediately or one hour after the venom injection, and then changes in Gln metabolism were analyzed. Subsequently, to further explore the protective mechanism of glutamine on tissue damage, we measured the expression of heat-shock protein70 (HSP70), NF-κB P65, P53/PUMA by western blotting and real-time polymerase in the lung and heart. Results: Gln supplementation delayed the envenoming symptoms, reduced mortality, and alleviated the histopathological changes in the heart and lung of mice bitten by Bungarus multicinctus. Additionally, Gln increased the activity of glutamine synthetase (GS), glutamate dehydrogenase (GDH) and glutaminase (GLS) in serum. It also balanced the transporter SLC7A11 expression in heart and lung tissues. Bungarus multicinctus venom induced the NF-κB nuclear translocation in the lung, while the HO-1 expression was suppressed. At the same time, venom activated the P53/PUMA signaling pathway and the BAX expression in the heart. Gln treatment reversed the above phenomenon and increased HSP70 expression. Conclusion: Gln alleviated the glutamine metabolism disorder and cardiopulmonary damage caused by Bungarus multicinctus venom. It may protect lungs and heart against venom by promoting the expression of HSP70, inhibiting the activation of NF-κB and P53/PUMA, thereby delaying the process of snake venom and reducing mortality. The present results indicate that Gln could be a potential treatment for Bungarus multicinctus bite.

Taiwan cobra envenoming: serum venom concentration before and after specific treatment and relationship with debridement of necrotic wound tissue

Background: Bivalent freeze-dried neurotoxic (FN) antivenom has been the primary treatment since the 1980s for Taiwan cobra (Naja atra) envenomation in Taiwan. However, envenomation-related wound necrosis is a significant problem after cobra snakebites. In the present study, we analyzed the changes in serum venom concentration before and after antivenom administration to discover their clinical implications and the surgical treatment options for wound necrosis. Methods: The patients were divided into limb swelling and wound necrosis groups. The clinical outcome was that swelling started to subside 12 hours after antivenom treatment in the first group. Serum venom concentrations before and after using antivenoms were measured to assess the antivenom's ability to neutralize the circulating cobra venom. The venom levels in wound wet dressing gauzes, blister fluids, and debrided tissues were also investigated to determine their clinical significance. We also observed the evolutional changes of wound necrosis and chose a better wound debridement timing. Results: We prospectively enrolled 15 Taiwan cobra snakebite patients. Males accounted for most of this study population (n = 11, 73%). The wound necrosis group received more antivenom doses than the limb swelling group (4; IQR:2-6 vs 1; IQR:1-2, p = 0.05), and less records of serum venom concentrations changed before/after antivenom use (p = 0.0079). The necrotic wound site may release venom into circulation and cause more severe envenomation symptoms. Antivenom can efficiently diminish limb swelling in cobra bite patients. However, antivenom cannot reduce wound necrosis. Patients with early debridement of wound necrosis had a better limb outcome, while late or without debridement may have long-term hospital stay and distal limb morbidity. Conclusions: Antivenom can efficiently eliminate the circulating cobra venom in limb swelling patients without wound necrosis. Early debridement of the bite site wound and wet dressing management are suggestions for preventing extended tissue necrosis and hospital stay.(AU)

Comparing modified Lee and White method against 20-minute whole blood clotting test as bedside coagulation screening test in snake envenomation victims

Abstract Background: Twenty-minute whole blood clotting test (20WBCT) and Modified Lee and White (MLW) method are the most routinely employed bedside tests for detecting coagulopathic snake envenomation. Our study compared the diagnostic utility of MLW and 20WBCT for snakebite victims at a tertiary care hospital in Central Kerala, South India. Methods: This single-center study recruited 267 patients admitted with snake bites. 20WBCT and MLW were performed simultaneously at admission along with the measurement of Prothrombin Time (PT). The diagnostic utility of 20WBCT and MLW was determined by comparing the sensitivity (Sn), specificity (Sp), positive and negative predictive values, likelihood ratios, and accuracy at admission with an INR value > 1.4. Results: Out of 267 patients, 20 (7.5%) patients had VICC. Amongst those who had venom-induced consumption coagulopathy (VICC), MLW was prolonged for 17 patients, (Sn 85% 95% confidence interval [CI]: 61.1-96.0) whereas 20WBCT was abnormal for 11 patients (Sn 55%, 95% CI: 32.04-76.17). MLW and 20WBCT were falsely positive for the same patient (Sp 99.6%, 95% CI: 97.4-99.9%). Conclusion: MLW is more sensitive than 20WBCT to detect coagulopathy at the bedside amongst snakebite victims. However, further studies are necessary for standardizing bedside coagulation tests in snakebite cases.

Transcriptome analysis of the spider Phonotimpus pennimani reveals novel toxin transcripts

Background: Phonotimpus pennimani (Araneae, Phrurolithidae) is a small-sized (3-5 mm) spider endemic to the Tacaná volcano in Chiapas, Mexico, where it is found in soil litter of cloud forests and coffee plantations. Its venom composition has so far not been investigated, partly because it is not a species of medical significance. However, it does have an important impact on the arthropod populations of its natural habitat. Methods: Specimens were collected in Southeastern Mexico (Chiapas) and identified taxonomically by morphological characteristics. A partial sequence from the mitochondrial gene coxI was amplified. Sequencing on the Illumina platform of a transcriptome library constructed from 12 adult specimens revealed 25 toxin or toxinlike genes. Transcripts were validated (RT-qPCR) by assessing the differential expression of the toxin-like PpenTox1 transcript and normalising with housekeeping genes. Results: Analysis of the coxI-gene revealed a similarity to other species of the family Phrurolithidae. Transcriptome analysis also revealed similarity with venom components of species from the families Ctenidae, Lycosidae, and Sicariidae. Expression of the toxinlike PpenTox1 gene was different for each developmental stage (juvenile or adult) and also for both sexes (female or male). Additionally, a partial sequence was obtained for the toxin-like PpenTox1 from DNA. Conclusion: Data from the amplification of the mitochondrial coxI gene confirmed that P. pennimani belongs to the family Phrurolithidae. New genes and transcripts coding for venom components were identified.(AU)

O1-conotoxin Tx6. 7 cloned from the genomic DNA of Conus textile that inhibits calcium currents

Background: Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. Methods: In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDW CPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Results: Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 µM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Conclusion: Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.(AU)

Cone snail species off the Brazilian coast and their venoms: a review and update

The genus Conus includes over 900 species of marine invertebrates known as cone snails, whose venoms are among the most powerful described so far. This potency is mainly due to the concerted action of hundreds of small bioactive peptides named conopeptides, which target different ion channels and membrane receptors and thus interfere with crucial physiological processes. By swiftly harpooning and injecting their prey and predators with such deadly cocktails, the slow-moving cone snails guarantee their survival in the harsh, competitive marine environment. Each cone snail species produces a unique venom, as the mature sequences of conopeptides from the venoms of different species share very little identity. This biochemical diversity, added to the numerous species and conopeptides contained in their venoms, results in an immense biotechnological and therapeutic potential, still largely unexplored. That is especially true regarding the bioprospection of the venoms of cone snail species found off the Brazilian coast - a region widely known for its biodiversity. Of the 31 species described in this region so far, only four - Conus cancellatus, Conus regius, Conus villepinii, and Conus ermineus - have had their venoms partially characterized, and, although many bioactive molecules have been identified, only a few have been actually isolated and studied. In addition to providing an overview on all the cone snail species found off the Brazilian coast to date, this review compiles the information on the structural and pharmacological features of conopeptides and other molecules identified in the venoms of the four aforementioned species, paving the way for future studies.(AU)

Scorpions trapped in amber: a remarkable window on their evolution over time from the Mesozoic period to present days

This synoptic review aims to bring some general information on fossil scorpions, namely those trapped in amber - fossilized resin - ranging from Lower Cretaceous through the Palaeocene and up to the Miocene. The question to be addressed is how the study of these fossils can be connected with possible present scorpionism problems. A precise knowledge of these ancient lineages provides information about the evolution of extant lineages, including the buthoids, which contain most known noxious species. Among the Arthropods found trapped in amber, scorpions are considered rare. A limited number of elements have been described from the Late Tertiary Dominican and Mexican amber, while the most ancient Tertiary amber from the Baltic region produced more consistent results in the last 30 years, primarily focusing on a single limited lineage. Contrarily, the Cretaceous amber from Myanmar, also called Burmite, has yielded and continues to yield a significant number of results represented by several distinct lineages, which attest to the considerable degree of diversity that existed in the Burmese amber-producing forests. As in my previous similar contributions to this journal, the content of this note is primarily addressed to non-specialists whose research embraces scorpions in various fields such as venom toxins and public health. An overview knowledge of at least some fossil lineages can eventually help to clarify why some extant elements associated with the buthoids represent dangerous species while others are not noxious.(AU)

Scorpions trapped in amber: a remarkable window on their evolution over time from the Mesozoic period to present days

This synoptic review aims to bring some general information on fossil scorpions, namely those trapped in amber - fossilized resin - ranging from Lower Cretaceous through the Palaeocene and up to the Miocene. The question to be addressed is how the study of these fossils can be connected with possible present scorpionism problems. A precise knowledge of these ancient lineages provides information about the evolution of extant lineages, including the buthoids, which contain most known noxious species. Among the Arthropods found trapped in amber, scorpions are considered rare. A limited number of elements have been described from the Late Tertiary Dominican and Mexican amber, while the most ancient Tertiary amber from the Baltic region produced more consistent results in the last 30 years, primarily focusing on a single limited lineage. Contrarily, the Cretaceous amber from Myanmar, also called Burmite, has yielded and continues to yield a significant number of results represented by several distinct lineages, which attest to the considerable degree of diversity that existed in the Burmese amber-producing forests. As in my previous similar contributions to this journal, the content of this note is primarily addressed to non-specialists whose research embraces scorpions in various fields such as venom toxins and public health. An overview knowledge of at least some fossil lineages can eventually help to clarify why some extant elements associated with the buthoids represent dangerous species while others are not noxious.(AU)

Cardiorespiratory/sedative effects of a peptide identified in crotalic venom compared to acepromazine and xylazine in horses / Efeitos cardiorrespiratórios/sedativos de um peptídeo identificado no veneno crotálico comparado à acepromazina e à xilazina em equinos

The purpose of this study was to investigate whether intravenous crotalphine produces significant sedation, as well as physiological changes, in healthy standing horses. Six mares, aged 8 years and weighing 415kg underwent three different treatments in a crossover design: TA (acepromazine: 50µg.kg-1), TC (crotalphine: 0.01µg.kg-1) and TX (xylazine: 1000µg.kg-1), intravenously. At various time points over 60 minutes, physiologic variables were recorded: heart rate, respiratory rate, and rectal temperature. The head height from the ground (HHG) was evaluated in centimeters. Data were analyzed using ANOVA followed by Dunnett's test or Friedman followed by Dunn's test, under 5% significance. Heart rate decreased significantly at M5 and M10 compared with Mb in TX (28±7, 26±6 and 40±8 beats/minute-1, respectively; p=0.0004). Respiratory rate and rectal temperature did not differ among groups or time points. The HHG significantly decreased in all groups compared with Mb at various time points (p<0.0001). In conclusion, crotalphine did not produce reliable and durable sedation in healthy standing mares and did not influence cardiorespiratory variables in a clinically relevant manner.

O objetivo deste estudo foi investigar se a administração de crotalfina intravenosa produz sedação significativa e alterações fisiológicas em equinos saudáveis. Seis éguas, idade média de oito anos e peso médio de 415kg, foram submetidas a três tratamentos distintos: TA (acepromazina: 50µg/kg), TC (crotalfina: 0,01µg/kg) e TX xilazina: 1000µg/kg), por via intravenosa. Em vários momentos, ao de longo de 60 minutos, as variáveis fisiológicas registradas foram frequência cardíaca, frequência respiratória e temperatura retal. A altura de cabeça ao solo (ACS) foi avaliada em centímetros. Os dados foram analisados pela ANOVA, seguida pelo teste de Dunnett ou de Friedman e, depois, pelo teste de Dunn, sob 5% de significância. A frequência cardíaca diminuiu significativamente em M5 e M10 em comparação com Mb em TX (28±7, 26±6 e 40±8 bpm, respectivamente; P=0,0004). A frequência respiratória e a temperatura retal não diferiram entre os grupos ou os pontos de tempo. O HHG diminuiu significativamente em todos os grupos em comparação com Mb em vários momentos (P <0,0001). Em conclusão, a crotalfina não produziu sedação confiável e durável em éguas saudáveis e não influenciou as variáveis cardiorrespiratórias de maneira clinicamente relevante.

Compositional and toxicological investigation of pooled venom from farm-raised Naja atra

Abstract Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.

Venom composition of Trimeresurus albolabris, T. insularis, T. puniceus and T. purpureomaculatus from Indonesia

Background: Several studies have been published on the characterization of Trimeresurus venoms. However, there is still limited information concerning the venom composition of Trimeresurus species distributed throughout Indonesia, which contributes to significant snakebite envenomation cases. The present study describes a comparative on the composition of T. albolabris, T. insularis, T. puniceus, and T. purpureomaculatus venoms originated from Indonesia. Methods: Protein content in the venom of four Trimeresurus species was determined using Bradford assay, and the venom proteome was elucidated using one-dimension SDS PAGE nano-ESI- LCMS/MS shotgun proteomics. Results: The venom of T. albolabris contained the highest protein content of 11.1 mg/mL, followed by T. puniceus, T. insularis and T. purpureomaculatus venom with 10.7 mg/mL, 8.9 mg/mL and 5.54 mg/mL protein, respectively. In total, our venomic analysis identified 65 proteins belonging to 16 protein families in T. purpureomaculatus; 64 proteins belonging to 18 protein families in T. albolabris; 58 different proteins belonging to 14 protein families in T. puniceus; and 48 different proteins belonging to 14 protein familiesin T. insularis. Four major proteins identified in all venoms belonged to snake venom metalloproteinase, C-type lectin, snake venom serine protease, and phospholipase A2. There were 11 common proteins in all venoms, and T. puniceus venom has the highest number of unique proteins compared to the other three venoms. Cluster analysis of the proteins and venoms showed that T. puniceus venom has the most distinct venom composition. Conclusions: Overall, the results highlighted venom compositional variation of four Trimeresurus spp. from Indonesia. The venoms appear to be highly similar, comprising at least four protein families that correlate with venom's toxin properties and function. This study adds more information on venom variability among Trimeresurus species within the close geographic origin and may contribute to the development of optimum heterologous antivenom.(AU)

Toxinology in the proteomics era: a review on arachnid venom proteomics

The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000's. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms "(scorpion venom) AND (proteome)" for scorpion venomics, and "(spider venom) AND (proteome)" for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.(AU)

S- and P-type cobra venom cardiotoxins differ in their action on isolated rat heart

Background: The cardiovascular system is one of the first systems to be affected by snake toxins; but not many toxins exert a direct effect on the heart. Cobra venom cardiotoxins are among those few toxins that attack the heart. Although the two cardiotoxin types (S and P) differ in their central-loop structure, it is not known whether they differ in their effect on the mammalian heart. We compared the effects of S- and P-type cardiotoxins, CTÐ¥-1 and CTÐ¥-2, respectively, from the cobra Naja oxiana, on the isolated rat heart. Methods: An isolated rat heart perfused according to the Langendorff technique was used in this study to investigate the activity of cardiotoxins CTX-1 and CTX-2. The following parameters were registered: the left ventricular developed pressure, calculated as the difference between systolic and diastolic pressure in the left ventricle, the end-diastolic pressure, the heart rate, time to maximal end-diastolic pressure (heart contracture), and time to depression of the heart contraction. Results: Both cardiotoxins at the concentration of 5 µg/mL initially produce a slight increase in systolic intraventricular pressure, followed by its rapid decrease with a simultaneous increase in diastolic intraventricular pressure until reaching contracture. CTX-2 blocks cardiac contractions faster than CTX-1; in its presence the maximum diastolic pressure is reached faster and the magnitude of the developed contracture is higher. Conclusion: The P-type cardiotoxin CTX-2 more strongly impairs rat heart functional activity than the S-type cardiotoxin CTX-1, as expressed in its faster blockage of cardiac contractions as well as in more rapid development and greater magnitude of contracture in its presence.(AU)

Comparative compositional and functional venomic profiles among venom specimens from juvenile, subadult and adult Russell's viper ( Daboia siamensis ): correlation with renal pathophysiology in experimental rabbits

Abstract Background: Eastern Russell's viper (Daboia siamensis) is one of the most medically significant snakes responsible for the development of acute renal failure. However, variation of the clinical picture and renal pathophysiology following bites by young and adult D. siamensis have not been elucidated. Methods: In this study, we analyzed the venomic profiles of D. siamensis at different maturation stages of juvenile, subadult and adult groups. The same pooled venom from each group was subjected to enzymatic, electrophoretic and proteomic analysis, including sublethal toxicity (0.1 mg/kg iv.) examined on bodily functions by comparing the venom compositional and functional profiles among venom specimens from juvenile, subadult and adult D. siamensis by correlating them with the renal pathophysiology in experimental rabbits. Results: The comparative studies revealed that juvenile venom possessed higher phospholipase A2 , metalloproteinase and serine proteinase levels, while subadult and adult venoms contained more L-amino acid oxidase, phosphodiesterase, the Kunitz-type serine protease inhibitor, disintegrin families and endothelial growth factor. An in vivo study revealed that the adult and subadult venoms caused persistent hypotension and bradycardia, while thrombocytopenia was a more characteristic effect of juvenile venom. All venom age groups showed significant reductions in renal hemodynamics and electrolyte excretions. The juvenile venom caused a higher tubulonephrosis lesion score than adult and subadult venoms. Conclusions: The D. siamensis venom shows an ontogenetic shift in its compositions and activities. Renal function alterations after envenomation depend on either the synergistic actions of different venom components or the disproportionate expression between the concentrations of enzymatic and non-enzymatic proteins in each age venom group. The high proportion of enzymatic toxin proteins in the juvenile venom results in greater nephrotoxicity.(AU)

New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2

Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

Isolation and characterization of five novel disulfide-poor conopeptides from Conus marmoreus venom

Background: Conopeptides from cone snail venom have aroused great interest related to the discovery of novel bioactive candidates, due to their excellent prospects for the treatment of various health problems such as pain, addiction, psychosis and epilepsy. In order to explore novel biopeptides, we investigated the structure and function of five novel conopeptides isolated from the venom of Conus marmoreus from South China Sea. Methods: C. marmoreus crude venom was prepared, fractionated and purified by HPLC system. The primary sequences of the five novel disulfide-poor conopeptides Mr-1 to Mr-5 were identified by comprehensive analysis of de novo MALDI-TOF tandem mass spectrometry and Edman degradation data. In order to investigate their function, these five conopeptides were synthesized by Fmoc-SPPS chemistry, and their biological effects at several heterologous rat nicotinic acetylcholine receptor (nAChR) subtypes (α1β1δε, α3β2, α3β4, α4β2) were determined by electrophysiological technique. Results: Five novel disulfide-poor conopeptides were identified and named as follows: Mr-1 (DWEYHAHPKPNSFWT), Mr-2 (YPTRAYPSNKFG), Mr-3 (NVIQAPAQSVAPP NTST), Mr-4 [KENVLNKLKSK(L/I)] and Mr-5 [NAVAAAN(L/I)PG(L/I)V]. None of them contains a disulfide bond. The sequences of conopeptides Mr-2 to Mr-5 do not belong to any category of the known disulfide-poor conopeptides. No significant activity against the above nAChR subtypes were observed for the five conopeptides at 100 µM. Conclusion: We purified and structurally characterized five novel disulfide-poor conopeptides from C. marmoreus crude venom and first investigated their nAChR inhibitory effects. This work expanded our knowledge on the structure and function of disulfide-poor conopeptides from this cone snail venom.(AU)

Biochemical and proteomic analyses of venom from a new pit viper, Protobothrops kelomohy

Background: A new pit viper, Protobothrops kelomohy, has been recently discovered in northern and northwestern Thailand. Envenoming by the other Protobothrops species across several Asian countries has been a serious health problem since their venom is highly hematotoxic. However, the management of P. kelomohy bites is required as no specific antivenom is available. This study aimed to investigate the biochemical properties and proteomes of P. kelomohy venom (PKV), including the cross-neutralization to its lethality with antivenoms available in Thailand. Methods: PKV was evaluated for its neutralizing capacity (ER50), lethality (LD50), procoagulant and hemorrhagic effects with three monovalent antivenoms (TAAV, DSAV, and CRAV) and one polyvalent (HPAV) hematotoxic antivenom. The enzymatic activities were examined in comparison with venoms of Trimeresurus albolabris (TAV), Daboia siamensis (DSV), Calloselasma rhodostoma (CRV). Molecular mass was separated on SDS-PAGE, then the specific proteins were determined by western blotting. The venom protein classification was analyzed using mass spectrometry-based proteomics. Results: Intravenous LD50 of PKV was 0.67 µg/g. ER50 of HPAV, DSAV and TAAV neutralize PKV at 1.02, 0.36 and 0.12 mg/mL, respectively. PKV exhibited procoagulant effect with a minimal coagulation dose of 12.5 ± 0.016 µg/mL and hemorrhagic effect with a minimal hemorrhagic dose of 1.20 ± 0.71 µg/mouse. HPAV was significantly effective in neutralizing procoagulant and hemorrhagic effects of PKV than those of TAAV, DSAV and CRAV. All enzymatic activities among four venoms exhibited significant differences. PKV proteome revealed eleven classes of putative snake venom proteins, predominantly metalloproteinase (40.85%), serine protease (29.93%), and phospholipase A2 (15.49%). Conclusions: Enzymatic activities of PKV are similarly related to other viperid venoms in this study by quantitatively hematotoxic properties. Three major venom toxins were responsible for coagulopathy in PKV envenomation. The antivenom HPAV was considered effective in neutralizing the lethality, procoagulant and hemorrhagic effects of PKV.(AU)

Toxinology in the proteomics era: a review on arachnid venom proteomics

Abstract The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000s. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms (scorpion venom) AND (proteome) for scorpion venomics, and (spider venom) AND (proteome) for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Abstract Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.

Proteinograma de ovelhas imunizadas com veneno detoxificado do escorpião Tityus serrulatus / Proteinogram of immunized sheep with detoxified Tityus serrulatus scorpion venom

Background: Scorpionism is a worldwide medical issue, especially relevant in the tropical and subtropical countries. Tityusserrulatus is the species responsible for most cases in Brazil. Antivenom administration to victims is the sole specific therapyobtained from donor animals. Most of these donors suffer with symptoms of the poisoning, debilitating their health andreducing their life expectancy. The aim of the present research was to evaluate whether the immunogens prepared fromthe crude and detoxified venom of T. serrulatus promoted different changes in fractionated sheep plasma proteins, duringa scorpion antivenom serum production.Materials, Methods & Results: Twelve sheep, healthy, mean weight of 30 kg, were distributed into 3 groups (n = 4): G1(control), G2 (crude venom) and G3 (detoxified venom). The adopted immunization protocol (first cycle) had 6 doses, 3using Freund’s adjuvant, with a 21-day interval between each one (day 0, 22 and 43), and 3 doses with no adjuvant (booster)and 0.2 mg of antigen (reinforcement), spaced 3 days between each other (day 50, 53 and 56). Group control (G1) received6 immunizations with phosphate buffered saline (PBS) associated with Freund’s adjuvant (1:1), while the other 2 groupsreceived 0.5 mg of venom (G2) and detoxified venom (G3), respectively, diluted in PBS, associated with the Freund adjuvant. The boosters were 1/3 of the initial dose, diluted only PBS. At baseline (T0) and at 24 and 48 h after immunization,all animals underwent clinical examinations. Blood samples were collected at day 0, 22, 43, 53 and 56 for proteinogramanalysis. Total protein, albumin and globulins fractions were measured. Plasma albumin concentration at T0 ranged from3.41-4.86 g/dL, with a mean value of 4.12 g/dL. There was no statistical difference between...