Distinct regulatory networks control toxin gene expression in elapid and viperid snakes.

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

An agonist of CXCR4 induces a rapid recovery from the neurotoxic effects of Vipera ammodytes and Vipera aspis venoms.

People bitten by Alpine vipers are usually treated with antivenom antisera to prevent the noxious consequences caused by the injected venom. However, this treatment suffers from a number of drawbacks and additional therapies are necessary. The venoms of Vipera ammodytes and of Vipera aspis are neurotoxic and cause muscle paralysis by inducing neurodegeneration of motor axon terminals because they contain a presynaptic acting sPLA2 neurotoxin. We have recently found that any type of damage to motor axons is followed by the expression and activation of the intercellular signaling axis consisting of the CXCR4 receptor present on the membrane of the axon stump and of its ligand, the chemokine CXCL12 released by activated terminal Schwann cells. We show here that also V. ammodytes and V. aspis venoms cause the expression of the CXCL12-CXCR4 axis. We also show that a small molecule agonist of CXCR4, dubbed NUCC-390, induces a rapid regeneration of the motor axon terminal with functional recovery of the neuromuscular junction. These findings qualify NUCC-390 as a promising novel therapeutics capable of improving the recovery from the paralysis caused by the snakebite of the two neurotoxic Alpine vipers.

Paranannizziopsis spp. Infection in Wild Vipers, Europe.

We describe the detection of Paranannizziopsis sp. fungus in a wild population of vipers in Europe. Fungal infections were severe, and 1 animal likely died from infection. Surveillance efforts are needed to better understand the threat of this pathogen to snake conservation.

Phylogenomic insights into the diversity and evolution of Palearctic vipers.

Despite decades of molecular research, phylogenetic relationships in Palearctic vipers (genus Vipera) still essentially rely on a few loci, such as mitochondrial barcoding genes. Here we examined the diversity and evolution of Vipera with ddRAD-seq data from 33 representative species and subspecies. Phylogenomic analyses of âˆ¼ 1.1 Mb recovered nine major clades corresponding to known species/species complexes which are generally consistent with the mitochondrial phylogeny, albeit with a few deep discrepancies that highlight past hybridization events. The most spectacular case is the Italian-endemic V. walser, which is grouped with the alpine genetic diversity of V. berus in the nuclear tree despite carrying a divergent mitogenome related to the Caucasian V. kaznakovi complex. Clustering analyses of SNPs suggest potential admixture between diverged Iberian taxa (V. aspis zinnikeri and V. seoanei), and confirm that the Anatolian V. pontica corresponds to occasional hybrids between V. (ammodytes) meridionalis and V. kaznakovi. Finally, all analyzed lineages of the V. berus complex (including V. walser and V. barani) form vast areas of admixture and may be delimited as subspecies. Our study sets grounds for future taxonomic and phylogeographic surveys on Palearctic vipers, a group of prime interest for toxinological, ecological, biogeographic and conservation research.

Hidden in the sand: Phylogenomics unravel an unexpected evolutionary history for the desert-adapted vipers of the genus Cerastes.

The desert vipers of the genus Cerastes are a small clade of medically important venomous snakes within the family Viperidae. According to published morphological and molecular studies, the group is comprised by four species: two morphologically similar and phylogenetically sister taxa, the African horned viper (Cerastes cerastes) and the Arabian horned viper (Cerastes gasperettii); a more distantly related species, the Saharan sand viper (Cerastes vipera), and the enigmatic Böhme's sand viper (Cerastes boehmei), only known from a single specimen in captivity allegedly captured in Central Tunisia. In this study, we sequenced one mitochondrial marker (COI) as well as genome-wide data (ddRAD sequencing) from 28 and 41 samples, respectively, covering the entire distribution range of the genus to explore the population genomics, phylogenomic relationships and introgression patterns within the genus Cerastes. Additionally, and to provide insights into the mode of diversification of the group, we carried out niche overlap analyses considering climatic and habitat variables. Both nuclear phylogenomic reconstructions and population structure analyses have unveiled an unexpected evolutionary history for the genus Cerastes, which sharply contradicts the morphological similarities and previously published mitochondrial approaches. Cerastes cerastes and C. vipera are recovered as sister taxa whilst C. gasperettii is a sister taxon to the clade formed by these two species. We found a relatively high niche overlap (OI > 0.7) in both climatic and habitat variables between C. cerastes and C. vipera, contradicting a potential scenario of sympatric speciation. These results are in line with the introgression found between the northwestern African populations of C. cerastes and C. vipera. Finally, our genomic data confirms the existence of a lineage of C. cerastes in Arabia. All these results highlight the importance of genome-wide data over few genetic markers to study the evolutionary history of species.

Isolation and characterization of polymorphic microsatellite loci for the three Iberian vipers, Vipera aspis, V. Latastei and V. seoanei by Illumina MiSeq sequencing.

BACKGROUND: European vipers (genus Vipera) are a well-studied taxonomic group, but the low resolution of nuclear sanger-sequenced regions has precluded thorough studies at systematic, ecological, evolutionary and conservation levels. In this study, we developed novel microsatellite markers for the three Iberian vipers, Vipera aspis, V. latastei and V. seoanei, and assessed their polymorphism in north-central Iberian populations. METHODS AND RESULTS: Genomic libraries were developed for each species using an Illumina Miseq sequencing approach. From the 70 primer pairs initially tested, 48 amplified reliably and were polymorphic within species. Cross-species transferability was achieved for 31 microsatellites loci in the three target species and four additional loci that were transferable to one species only. The 48 loci amplified in average seven alleles, and detected average expected and observed heterozygosities of 0.7 and 0.55, in the three genotyped populations/species (26 V. aspis, 20 V. latastei and 10 V. seoanei). CONCLUSIONS: Our study provides a selection of 48 polymorphic microsatellite markers that will contribute significantly to current knowledge on genetic diversity, gene flow, population structure, demographic dynamics, systematics, reproduction and heritability in these species, and potentially in other congeneric taxa.

Pro-inflammatory cytokines gene expression in liver and kidneys of rats exposed to a sub-lethal dose of Bitis arietans snake venom.

Bitis arietans (Puff adder) is a poisonous snake and its bite causes pain, edema, blistering, tissue damage and neutrophilia. There are limited studies on inflammatory process involved in Bitis arietans envenomation. We therefore investigated the role of proinflammatory cytokines in Bitis arietans venom (BAV)-induced liver and kidney toxicities in rats. Adult male Sprague Dawley rats were treated with BAV (0.5 mg/kg) and were sacrificed after specific time intervals (2 h, 24 h, 1 week). Blood samples were collected for liver and renal function tests and tissues were collected for histopathology and gene expression analysis of IL-1ß, IL-6, and TNF-α in liver and kidneys. There was no significant difference in serum ALT activities among different treatment groups. Serum AST was significantly increased at 24 h following BAV injection. In both organs, injection of BAV resulted in mild inflammatory cell infiltration at 2 h post-dosing which normalized after 1 week. In liver, there was a significant increase in IL-1ß expression in BAV-treated rats at 2 and 24 h post-dosing that reduced after one week. Significant increases in IL-6 and TNF-α were observed at 24 h and 1 week after BAV exposure. In kidneys, there were significant increases in IL-1ß and TNF-α expression at 24 h that subsided after 1 week. In conclusion, a single sub-lethal dose of BAV caused an acute phase inflammation in liver and kidneys. It is most probable that a higher dose of BAV may result in greater and irreversible damage to these organs.

Pit Viper Envenomation in Two Pregnant Bitches.

Snake envenomation is relatively common in small animals, particularly in endemic areas. Effects and outcomes of envenomation during pregnancy are poorly described in humans and more so in veterinary patients. Two young pregnant female dogs presented to a university teaching hospital with a history of acute soft tissue swelling and bleeding. History, physical examination findings, and diagnostics were consistent with envenomation by crotalid snakes. Medical management of one of the dogs included administration of antivenin. Both dogs survived envenomation with minimal complications and went on to whelp without complications, and all fetuses survived. This is the first description of the management of pit viper envenomation in pregnant dogs.

Serpentirhabdias mexicanus n. sp. (Nematoda: Rhabdiasidae), a parasitic lungworm of the nauyaca viper Bothrops asper (Serpentes: Viperidae) in the Mexican Neotropics.

Serpentirhabdias mexicanus n. sp. (Nematoda: Rhabdiasidae) is described from the lung of the nauyaca viper Bothrops asper in Puebla State, central Mexico. This new species is the fifth of the genus described having onchia. Among the species included in this group, the new species is morphologically closest to S. viperidicus and S. atroxi. However, it differs from both species mainly by having only one excretory gland (compared to two present in S. viperidicus and S. atroxi). In addition, S. mexicanus n. sp. can be separated of S. viperidicus by tail length, shape of vulval lips, geographic distribution and host species and from S. atroxi by body length, number of papillae in the cephalic region, as well as the host species and geographic distribution. In the present study, we propose the new species based on morphological, host spectrum and genetic evidence. Phylogenetic analysis indicated Serpentirhabdias as a monophyletic group, with two subgroups that are congruent with the presence/absence of onchia in the esophagostome, host association and other relevant morphological characters.

High-Throughput Venomics.

In this study, we present high-throughput (HT) venomics, a novel analytical strategy capable of performing a full proteomic analysis of a snake venom within 3 days. This methodology comprises a combination of RP-HPLC-nanofractionation analytics, mass spectrometry analysis, automated in-solution tryptic digestion, and high-throughput proteomics. In-house written scripts were developed to process all the obtained proteomics data by first compiling all Mascot search results for a single venom into a single Excel sheet. Then, a second script plots each of the identified toxins in so-called Protein Score Chromatograms (PSCs). For this, for each toxin, identified protein scores are plotted on the y-axis versus retention times of adjacent series of wells in which a toxin was fractionated on the x-axis. These PSCs allow correlation with parallel acquired intact toxin MS data. This same script integrates the PSC peaks from these chromatograms for semiquantitation purposes. This new HT venomics strategy was performed on venoms from diverse medically important biting species; Calloselasma rhodostoma, Echis ocellatus, Naja pallida, Bothrops asper, Bungarus multicinctus, Crotalus atrox, Daboia russelii, Naja naja, Naja nigricollis, Naja mossambica, and Ophiophagus hannah. Our data suggest that high-throughput venomics represents a valuable new analytical tool for increasing the throughput by which we can define venom variation and should greatly aid in the future development of new snakebite treatments by defining toxin composition.

Sequence Divergence in Venom Genes Within and Between Montane Pitviper (Viperidae: Crotalinae: Cerrophidion) Species is Driven by Mutation-Drift Equilibrium.

Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis.

RESUMEN: El veneno de las serpientes puede variar entre y dentro de las especies. Mientras algunos grupos de viperidos del Nuevo Mundo­como las cascabeles­han sido bien estudiadas, muy poco se sabe acerca del veneno de las nauyacas de frío (Cerrophidion) que se encuentran en las zonas altas de Mesoamérica. Comparadas con las extensamente estudiadas cascabeles, que estan ampliamente distribuidas, las poblaciones de Cerrophidion, aisladas en montañas, pueden poseer trayectorias evolutivas y diferenciación en su veneno unicos. En el presente trabajo, describimos el transcriptoma de las glándulas de veneno de poblaciones de C. petlalcalensis, C. tzotzilorum, y C. godmani de México, y un individuo de C. sasai de Costa Rica. Exploramos la variación en la expresión de toxinas en Cerrophidion y la evolución en las secuencias geneticas en C. godmani específicamente. El transcriptoma de la glándula de veneno de Cerrophidion esta compuesto principalmente de Metaloproteinasas de Veneno de Serpiente, Fosfolipasas A[Formula: see text] (PLA[Formula: see text]s), y Serin Proteasas de Veneno de Serpiente. Cerrophidion petlalcalensis presenta poca variación intraespecífica; sin embargo, los transcriptomas de la glandula de veneno de C. godmani y C. tzotzilorum difieren significativamente entre poblaciones geográficamente aisladas. Curiosamente, la variación intraespecífica estuvo atribuida principalmente a la expresión de las toxinas ya que no encontramos señales de selección en las toxinas de C. godmani. Adicionalmente, encontramos miotoxinas similares a PLA[Formula: see text] en todas las especies excepto C. petlalcalensis, y PLA[Formula: see text]s similares a crotoxina en la población sureña de C. godmani. Nuestros resultados demuestran la presencia de variacion intraespecífica presente en el veneno de C. godmani y C. tzotzilorum. Las toxinas de Cerrophidion godmani muestran poca evidencia de selección direccional, y la variación en la secuencias de las toxinas es consistente con evolucion bajo un modelo de equilibrio de mutación-deriva. Algunos individuos de C. godmani de la población del sur potencialmente tienen un veneno neurotóxico dada la presencia de PLA[Formula: see text]s similares a la crotoxina, sin embargo, se necesita más evidencia para corroborar esta hipótesis.

Polyandry and non-random fertilisation maintain long-term genetic diversity in an isolated island population of adders (Vipera berus).

Conservation genetic theory suggests that small and isolated populations should be subject to reduced genetic diversity i.e., heterozygosity and allelic diversity. Our 34 years study of an isolated island population of adders (Vipera berus) in southern Sweden challenges this notion. Despite a lack of gene flow and a yearly mean estimated reproductive adult population size of only 65 adult adders (range 12-171), the population maintains high levels of heterozygosity and allelic diversity similar to that observed in two mainland populations. Even a 14-year major "bottleneck" i.e., a reduction in adult adder numbers, encompassing at least four adder generations, did not result in any reduction in the island adders' heterozygosity and allelic diversity. Female adders are polyandrous, and fertilisation is non-random, which our empirical data and modelling suggest are underpinning the maintenance of the population's high level of heterozygosity. Our empirical results and subsequent modelling suggest that the positive genetic effects of polyandry in combination with non-random fertilisation, often overlooked in conservation genetic analyses, deserve greater consideration when predicting long-term survival of small and isolated populations.

Total-evidence phylogeny and evolutionary morphology of New World pitvipers (Serpentes: Viperidae: Crotalinae).

Crotalines (pitvipers) in the Americas are distributed from southern Canada to southern Argentina, and are represented by 13 genera and 163 species that constitute a monophyletic group. Their phylogenetic relationships have been assessed mostly based on DNA sequences, while morphological data have scarcely been used for phylogenetic inquiry. We present a total-evidence phylogeny of New World pitvipers, the most taxon/character comprehensive phylogeny to date. Our analysis includes all genera, morphological data from external morphology, cranial osteology and hemipenial morphology, and DNA sequences from mitochondrial and nuclear genes. We performed analyses with parsimony as an optimality criterion, using different schemes for character weighting. We evaluated the contribution of the different sources of characters to the phylogeny through analyses of reduced datasets and calculation of weighted homoplasy and retention indexes. We performed a morphological character analysis to identify synapomorphies for the main clades. In terms of biogeography, our results support a single colonization event of the Americas by pitvipers, and a cladogenetic event into a Neotropical clade and a North American/Neotropical clade. The results also shed light on the previously unstable position of some taxa, although they could not sufficiently resolve the position of Bothrops lojanus, which may lead to the paraphyly of either Bothrops or Bothrocophias. The morphological character analyses demonstrated that an important phylogenetic signal is contained in characters related to head scalation, the jaws and the dorsum of the skull, and allowed us to detect morphological convergences in external morphology associated with arboreality.

First helminthological data on the Iberian adder, Vipera seoanei.

The helminth fauna present in the gut contents of Iberian adders, Vipera seoanei (Squamata: Viperidae), were characterised and analysed in respect to biological and eco-geographic factors that may affect the occurrence and diversity of helminths in this species. A total of 317 samples of preserved stomachs and intestines, covering the distributional range of V. seoanei, were examined. Similar to other Vipera species from the Iberian Peninsula, the helminth fauna was also impoverished in V. seoanei, but unlike other Vipera species from Central and East Europe, helminths were mostly found in adult vipers, and occurred in vipers located at the periphery of the species range, characterised by low elevation, high temperature and precipitation levels, and abundant pastures.

The Brazilian Atlantic Bushmaster Lachesis (Linnaeus, 1766) Mitogenome With Insights On Snake Evolution And Divergence (Serpentes: Viperidae: Crotalinae).

This study presents the first complete mitogenome of the Brazilian Atlantic bushmaster Lachesis with insights into snake evolution. The total length was 17,177 bp, consisting of 13 PCGs, 22 tRNAs, two rRNAs and a duplicate control region (CRs). Almost all genes were encoded by the heavy-strand, except for the ND6 gene and eight tRNAs (tRNA-Gln, Ala, Asn, Cys, Tyr, Ser[TGA anticodon], Glu, Pro). Only ATG, ATA, and ATC were starting codons for protein-coding sequences. Stop codons mainly were TAA, AGA, AGG, and TAG; whereas ND1, ND3, and CYTB terminated with incomplete stop codons. Phylogeny retrieved Lachesis within the Crotalinae as the sister group of Agkistrodon; and the Lachesis+Agkistrodon clade as the sister group of (Sistrurus+Crotalus)+Bothrops. The tree supports Crotalinae, Viperinae, and Azemiopinae in the Viperidae family, being sister taxa of Colubridae+(Elapidae+Psammophiidae). The mean genetic distance across 15 snake families and 57 nucleotide sequences was 0.37. The overall mean value of genetic distance across the Crotalinae was 0.23, with Lachesis muta exhibiting the shortest distance of 0.2 with Agkistrodon piscivorus, Protobothrops dabieshanensis and P. flavoviridis and the greatest 0.25 with Gloydius blomhoffii, Trimeresurus albolabris, S. miliarius, and Deinagkistrodon acutus. The complete Atlantic L. muta mitogenome presented herein is only the third annotated mitogenome from more than 430 described Brazilian snake species.

Fangs in the Ghats: Preclinical Insights into the Medical Importance of Pit Vipers from the Western Ghats.

The socioeconomic impact of snakebites in India is largely attributed to a subset of snake species commonly known as the 'big four'. However, envenoming by a range of other clinically important yet neglected snakes, a.k.a. the 'neglected many', also adds to this burden. The current approach of treating bites from these snakes with the 'big four' polyvalent antivenom is ineffective. While the medical significance of various species of cobras, saw-scaled vipers, and kraits is well-established, the clinical impact of pit vipers from regions such as the Western Ghats, northeastern India, and the Andaman and Nicobar Islands remains poorly understood. Amongst the many species of snakes found in the Western Ghats, the hump-nosed (Hypnale hypnale), Malabar (Craspedocephalus malabaricus), and bamboo (Craspedocephalus gramineus) pit vipers can potentially inflict severe envenoming. To evaluate the severity of toxicity inflicted by these snakes, we characterised their venom composition, biochemical and pharmacological activities, and toxicity- and morbidity-inducing potentials, including their ability to damage kidneys. Our findings highlight the therapeutic inadequacies of the Indian and Sri Lankan polyvalent antivenoms in neutralising the local and systemic toxicity resulting from pit viper envenomings.

CC5 and CC8, Two Disintegrin Isoforms from Cerastes cerastes Snake Venom Decreased Inflammation Response In Vitro and In Vivo.

Inflammation is associated with many pathology disorders and the malignant progression of most cancers. Therefore, targeting inflammatory pathways could provide a promising strategy for disease prevention and treatment. In this study, we experimentally investigated the anti-inflammatory effect of CC5 and CC8, two disintegrin isoforms isolated from Cerastes cerastes snake venom, on LPS-stimulated macrophages, both on human THP-1 and mouse RAW264.7 cell adherence and their underlying mechanisms by measuring cytokine release levels and Western blot assay. Equally, both molecules were evaluated on a carrageenan-induced edema rat model. Our findings suggest that CC5 and CC8 were able to reduce adhesion of LPS-stimulated macrophages both on human THP-1 and mouse RAW264.7 cells to fibrinogen and vitronectin through the interaction with the αvß3 integrin receptor. Moreover, CC5 and CC8 reduced the levels of reactive oxygen species (ROS) mediated by the NF-κB, MAPK and AKT signaling pathways that lead to decreased production of the pro-inflammatory cytokines TNF-α, IL-6 and IL-8 and increased secretion of IL-10 in LPS-stimulated THP-1 and RAW264.7 cells. Interestingly, both molecules potently exhibited an anti-inflammatory effect in vivo by reducing paw swelling in rats. In light of these results, we can propose the CC5 and CC8 disintegrins as interesting tools to design potential candidates against inflammatory-related diseases.

Functional Characterization and Anti-Tumor Effect of a Novel Group II Secreted Phospholipase A2 from Snake Venom of Saudi Cerastes cerates gasperetti.

Secreted phospholipases A2 are snake-venom proteins with many biological activities, notably anti-tumor activity. Phospholipases from the same snake type but different geographical locations have shown similar biochemical and biological activities with minor differences in protein sequences. Thus, the discovery of a new phospholipase A2 with unique characteristics identified in a previously studied venom could suggest the origins of these differences. Here, a new Group II secreted phospholipase A2 (Cc-PLA2-II) from the snake venom of Saudi Cerastes cerastes gasperetti was isolated and characterized. The purified enzyme had a molecular weight of 13.945 kDa and showed high specific activity on emulsified phosphatidylcholine of 1560 U/mg at pH 9.5 and 50 °C with strict calcium dependence. Interestingly, stability in extreme pH and high temperatures was observed after enzyme incubation at several pH levels and temperatures. Moreover, a significant dose-dependent cytotoxic anti-tumor effect against six human cancer cell lines was observed with concentrations of Cc-PLA2 ranging from 2.5 to 8 µM. No cytotoxic effect on normal human umbilical-vein endothelial cells was noted. These results suggest that Cc-PLA2-II potentially has angiogenic activity of besides cytotoxicity as part of its anti-tumor mechanism. This study justifies the inclusion of this enzyme in many applications for anticancer drug development.

Delayed Compression Paralysis Following an Iliopsoas Hematoma 30 Days After Saw-Scaled Viper (Echis carinatus sochureki) Envenoming: A Case Report.

Snakebite envenoming is a neglected tropical disease disproportionately affecting the rural and marginalized population in low-middle-income countries. The saw-scaled viper (Echis carinatus) is a clinically important snake that causes serious morbidity and mortality in the Indian subcontinent. Even though it is within the so-called big-four snakes against which polyvalent antivenom is available throughout India, reports of antivenom ineffectiveness are emerging in saw-scaled viper envenoming, especially around Jodhpur, Rajasthan, India. This case report highlights a patient with saw-scaled viper envenoming with an ineffective antivenom response complicated by acute kidney injury as well as local and systemic bleeding complications, which subsequently resulted in a pelvic hematoma that compressed the lumbosacral nerves, causing lower-limb weakness and sensory deficits. He was successfully managed with hematoma aspiration and supportive care. This case brings into focus the challenges of managing saw-scaled viper envenoming in this region with antivenom ineffectiveness, resulting in delayed and significant coagulopathy and its complications leading to prolonged hospital stay and morbidity. Our report spotlights less emphasized aspects of long-term morbidity in snakebite survivors, such as loss of working days and productivity. We also highlight the need for an organized system of long-term follow-up of snakebite survivors to screen for possible complications and manage them early.

Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain.

Ecarin is one of the most widely used drug compounds in blood clotting experiments and is used to monitor and treat many diseases such as cancer, liver, lupus, and cardiovascular disease. The metalloproteinase domain is known as the active site of ecarin. In this study, an ecarin metalloproteinase cassette was designed and synthesized in the pUC57 vector. The gene fragment was released and cloned into the pET-28a vector and expressed in Escherichia coli. The recombinant protein was confirmed by western blotting. Enzyme activity was estimated by a laboratory coagulation test, and prothrombin time and tertiary structure were determined by using the Iterative Threading ASSEmbly Refinement (I-TASSER) server. Data from blood clotting tests for the produced ecarin activity were analyzed using an independent t test. As per I-TASSER server prediction, model 1 with the highest confidence score 0.95, template modeling score (0.84 ± 0.08), and root mean square deviation (3.5 ± 2.4 Å) was considered as the best model, and the 2e3xA enzyme was more similar to the target protein. The predictive results helped to better understand the relationship between the structure and function of the ecarin metalloproteinase domain. Also, the production of this active site in the prokaryotic expression system, which is simpler and more cost-effective than the production of the eukaryotic system, showed that this recombinant ecarin could be used as a substitute for the raw snake venom of Echis carinatus because it converts prothrombin into thrombin, and its activity, as estimated using the prothrombin time test, was found to be faster than normal ecarin.