Snakebite and its socio-economic impact on the rural population of Tamil Nadu, India

BACKGROUND: Snakebite represents a significant health issue worldwide, affecting several million people each year with as many as 95,000 deaths. India is considered to be the country most affected, but much remains unknown about snakebite incidence in this country, its socio-economic impact and how snakebite management could be improved. METHODS/PRINCIPAL FINDINGS: We conducted a study within rural villages in Tamil Nadu, India, which combines a household survey (28,494 people) of snakebite incidence with a more detailed survey of victims in order to understand the health and socio-economic effects of the bite, the treatments obtained and their views about future improvements. Our survey suggests that snakebite incidence is higher than previously reported. 3.9% of those surveyed had suffered from snakebite and the number of deaths corresponds to 0.45% of the population. The socio-economic impact of this is very considerable in terms of the treatment costs and the long-term effects on the health and ability of survivors to work. To reduce this, the victims recommended improvements to the accessibility and affordability of antivenom treatment. CONCLUSIONS: Snakebite has a considerable and disproportionate impact on rural populations, particularly in South Asia. This study provides an incentive for researchers and the public to work together to reduce the incidence and improve the outcomes for snake bite victims and their families.

Style and substance: some metal figurines from south-west Britain

A hoard found in Southbroom, Devizes in 1714 contained a group of copper-alloy figurines executed in both classical and local styles and depicting deities belonging to the Graeco-Roman and Gallo-Roman pantheons. The deities in a local style appear to form part of a larger tradition of figurines, predominantly found in the South-West, which are characterised both by a similar artistic style and by the use of Gallo-Roman symbolism and deities, such as the torc, ram-horned snake, carnivorous dog and Sucellus. The unique composition of the hoard in comparison with other hoards of similar date provides insights into the beliefs of Roman Britain.

CLEC-2 is not required for platelet aggregation at arteriolar shear

The C-type lectin receptor CLEC-2 is expressed primarily on the surface of platelets, where it is present as a dimer, and is found at low level on a subpopulation of other hematopoietic cells, including mouse neutrophils [1–4] Clustering of CLEC-2 by the snake venom toxin rhodocytin, specific antibodies or its endogenous ligand, podoplanin, elicits powerful activation of platelets through a pathway that is similar to that used by the collagen receptor glycoprotein VI (GPVI) [4–6]. The cytosolic tail of CLEC-2 contains a conserved YxxL sequence preceded by three upstream acidic amino acid residues, which together form a novel motif known as a hemITAM. Ligand engagement induces tyrosine phosphorylation of the hemITAM sequence providing docking sites for the tandem-SH2 domains of the tyrosine kinase Syk across a CLEC-2 receptor dimer [3]. Tyrosine phosphorylation of Syk by Src family kinases and through autophosphorylation leads to stimulation of a downstream signaling cascade that culminates in activation of phospholipase C γ2 (PLCγ2) [4,6]. Recently, CLEC-2 has been proposed to play a major role in supporting activation of platelets at arteriolar rates of flow [1]. Injection of a CLEC-2 antibody into mice causes a sustained depletion of the C-type lectin receptor from the platelet surface [1]. The CLEC-2-depleted platelets were unresponsive to rhodocytin but underwent normal aggregation and secretion responses after stimulation of other platelet receptors, including GPVI [1]. In contrast, there was a marked decrease in aggregate formation relative to controls when CLEC-2-depleted blood was flowed at arteriolar rates of shear over collagen (1000 s−1 and 1700 s−1) [1]. Furthermore, antibody treatment significantly increased tail bleeding times and mice were unable to occlude their vessels after ferric chloride injury [1]. These data provide evidence for a critical role for CLEC-2 in supporting platelet aggregation at arteriolar rates of flow. The underlying mechanism is unclear as platelets do not express podoplanin, the only known endogenous ligand of CLEC-2. In the present study, we have investigated the role of CLEC-2 in platelet aggregation and thrombus formation using platelets from a novel mutant mouse model that lacks functional CLEC-2.

Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signalling

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

Renal cells activate the platelet receptor CLEC-2 through podoplanin.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

Reproductive ecology of dipsadine snakes, with emphasis on South American species

A relatively large amount of variation occurs in the reproductive ecology of tropical snakes, and this variation is generally regarded as being a consequence of seasonality in climate and prey availability. In some groups, even closely related species may differ in their reproductive ecology; however, in others it seems to be very conservative. Here we explore whether characters related to reproduction are phylogenetically constrained in a monophyletic group of snakes, the subfamily Dipsadinae, which ranges from Mexico to southern South America. We provide original data on reproduction for Leptodeira annulata, Imantodes cenchoa, and three species of Sibynomorphus from southern, southeastern and central Brazil, and data from literature for other species and populations of dipsadines. Follicular cycles were seasonal in Atractus reticulatus, Dipsa, albifrons, Hypsiglena torquata, Leptodeira maculata, L. punctata, Sibynomorphus spp. and Sibon sanniola from areas where climate is seasonal. In contrast, extended or continuous follicular cycles were recorded in Dipsas catesbyi, D. neivai, Imantodes cenchoa, Leptodeira annulata, and Ninia maculata from areas with seasonal and aseasonal climates. Testicular cycles also varied from seasonal (in H. torquiata) to continuous (in Dipsa,5 spp., Leptodeira annulata, L. maculata, N. maculata, and Sibynomorphus spp.). Most dipsadines are small (less than 500 rum SVL), and females attain sexual maturity with similar relative body size than males. Sexual dimorphism occurred in terms of SVL and tail length in most species, and clutch size tended to be small (less than five eggs). Combat behavior occurs in Imantodes cenchoa, which did not show sexual size dimorphism. Reproductive timing, for both females and males, varied among species but in general there were no differences between the tribes of Dipsadinae in most of the reproductive characteristics, such as mean body size, relative size at sexual maturity, sexual size and tail dimorphism, duration of vitellogenesis or egg-carrying in oviducts.

Proteomics of the neurotoxic fraction from the sea anemone Bunodosoma cangicum venom: Novel peptides belonging to new classes of toxins

In contrast to the many studies on the venoms of scorpions, spiders, snakes and cone snails, tip to now there has been no report of the proteomic analysis of sea anemones venoms. In this work we report for the first time the peptide mass fingerprint and some novel peptides in the neurotoxic fraction (Fr III) of the sea anemone Bunodosoma cangicum venom. Fr III is neurotoxic to crabs and was purified by rp-HPLC in a C-18 column, yielding 41 fractions. By checking their molecular masses by ESI-Q-Tof and MALDI-Tof MS we found 81 components ranging from near 250 amu to approximately 6000 amu. Some of the peptidic molecules were partially sequenced through the automated Edman technique. Three of them are peptides with near 4500 amu belonging to the class of the BcIV, BDS-I, BDS-II, APETx1, APETx2 and Am-II toxins. Another three peptides represent a novel group of toxins (similar to 3200 amu). A further three molecules (similar to similar to 4900 amu) belong to the group of type 1 sodium channel neurotoxins. When assayed over the crab leg nerve compound action potentials, one of the BcIV- and APETx-like peptides exhibits an action similar to the type 1 sodium channel toxins in this preparation, suggesting the same target in this assay. On the other hand one of the novel peptides, with 3176 amu, displayed an action similar to potassium channel blockage in this experiment. In summary, the proteomic analysis and mass fingerprint of fractions from sea anemone venoms through MS are valuable tools, allowing us to rapidly predict the occurrence of different groups of toxins and facilitating the search and characterization of novel molecules without the need of full characterization of individual components by broader assays and bioassay-guided purifications. It also shows that sea anemones employ dozens of components for prey capture and defense. (C) 2008 Elsevier Inc. All rights reserved.

Bunodosine 391: An Analgesic Acylamino Acid from the Venom of the Sea Anemone Bunodosoma cangicum

A new acylamino acid, bunodosine 391 (BDS 391), was isolated from the venom of the sea anemone Bunodosoma cangicum. The structure was elucidated by spectroscopic analyses (2D NMR, ESIMS/MS) and verified by its synthesis. Intraplantar injection of BDS 391 into the hind paw of a rat induced a potent analgesic effect. This effect was not altered by naloxone (an opioid receptor antagonist), but was completely reversed by methysergide (a serotonin receptor antagonist), indicating that the effect is mediated by activation of serotonin receptors:

Notes on distribution, variation and characterization of Erythrolamprus pseudocorallus Roze, 1959 (Serpentes: Colubridae) with the first records from Colombia

The Maracaibo false coral snake Erythrolamprus pseudocorallus, previously known only from Venezuela, is recorded from five departments in Colombia. These new data include the westernmost and the southernmost records presently known for the species. Two specimens previously identified as E. aesculapii, from the localities of El Valle, Distrito Federal, Venezuela, and Yarumal, Antioquia, Colombia, are now attributed to E. pseudocorallus, the first one representing the northeasternmost record of the species. Morphological characterization of E. pseudocorallus is expanded based on the new specimens.

Bothrops insularis venomics: A proteomic analysis supported by transcriptomic-generated sequence data

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A(2) and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and M(r) of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies. (c) 2009 Elsevier B.V. All rights reserved.

On the Generic Identity of Siagonodon brasiliensis, with the Description of a New Leptotyphlopid from Central Brazil (Serpentes: Leptotyphlopidae)

The geographic variation and hemipenial morphology of Siagonodon brasiliensis are described based on a comprehensive sample, allowing the reappraisal of its generic identity, and the proposal of a new nomenclatural combination. We suggest that the presence of two supralabials, as mentioned in the original description of S. brasiliensis, is not a common feature for this species, occurring at low frequencies throughout its geographic distribution. Based on a diagnosis presented in a recently published paper, as well as on additional external traits and on hemipenial characters, we recognize Siagonodon brasiliensis as a species of the genus Tricheilostoma. In addition, a new species of worm snake of the genus Siagonodon is described from the savannas of the state of Tocantins, Brazil. The new species differs from other congeners by having a slightly acuminate snout in lateral and ventral views, subcircular rostral in dorsal view, and 12 scale rows around middle of tail. The diagnosis of the genus Siagonodon is revised and expanded based on direct observation of morphological characters.

Crotalphine induces potent antinociception in neuropathic pain by acting at peripheral opioid receptors

Neuropathic pain is an important clinical problem and it is usually resistant to the current therapy. We have recently characterized a novel analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in an experimental model of neuropathic pain induced in rats by chronic constriction, of sciatic nerve. The effect of the peptide was compared to that induced by the crude venom, which confirmed that crotalphine is responsible for the antinociceptive effect of the crotalid venom on neuropathic pain. For characterization of neuropathic pain, the presence of hyperalgesia, allodynia and spontaneous pain was assessed at different times after nerve constriction. These phenomena were detected 24 h after surgery and persisted at least for 14 days. The pharmacological treatments were performed on day 14 after surgery. Crotalphine (0.2-5 mu g/kg) and the crude venom (400-1600 mu g/kg) administered p.o. inhibited hyperalgesia, allodynia and spontaneous pain induced by nerve constriction. The antinociceptive effect of the peptide and crude venom was long lasting, since it was detected up to 3 days after treatment. Intraplantar injection of naloxone (1 mu g/paw) blocked the antinociceptive effect, indicating the involvement of opioid receptors in this phenomenon. Gabapentin (200 mg/kg, p.o.), and morphine (5 mg/kg, s.c.), used as positive controls, blocked hyperalgesia and partially inhibited allodynia induced by nerve constriction. These data indicate that crotalphine induces a potent and long lasting opioid antinociceptive effect in neuropathic pain that surpasses that observed with standard analgesic drugs. (C) 2008 Elsevier B.V. All rights reserved.

Crotoxin alters lymphocyte distribution in rats: Involvement of adhesion molecules and lipoxygenase-derived mediators

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom and modulates immune and inflammatory responses, interfering with the activity of leukocytes. In the present work, the effects of crotoxin on the number of blood and lymphatic leukocytes and on lymph nodes and spleen lymphocytes population were investigated. The toxin s.c. administered to male Wistar rats, decreases the number of lymphocytes in blood and lymph circulation and increases the content of B and T-lymphocytes in lymph nodes. These effects were detected 1-2 h after treatment. The crotoxin molecule is composed of two subunits, an acidic non-toxic polypeptide, named crotapotin and a toxic basic phospholipase A(2) (PLA(2)). PLA(2), but not crotapotin, decreased the number of circulating blood and lymph lymphocytes. Crotoxin promotes leukocyte adherence to endothelial cells of blood microcirculation and to lymph node high endothelial venules, which might contribute to the drop in the number of circulating lymphocytes. Crotoxin increases expression of the adhesion molecule LFA-1 in lymphocytes. The changes in the expression of the adhesion molecule might contribute, at least in part, for the increased leukocyte adhesion to endothelium. Zileuton, a 5-lipoxygenase inhibitor, blocked the decrease in the number of circulating leukocytes induced by crotoxin and also abolished the changes observed in leukocyte-endothelial interactions, suggesting the involvement of lipoxygenase-derived mediators in the effects of the toxin. (c) 2008 Elsevier Ltd. All rights reserved.

Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar bristle extract induces direct lysis by cleaving erythrocyte membrane glycoproteins

Lonomia obliqua caterpillar bristle extract induces hemolysis in vitro on washed human and rat erythrocytes, in either the absence or presence of exogenous lecithin. In the former condition, phospholipases A(2) are key enzymes involved in hemolysis. However, the mechanism whereby this extract causes direct hemolysis is not known. Thus, the aim of this study was to investigate the hemolytic mechanism of the crude extract of the caterpillar L obliqua on human erythrocytes in the absence of lecithin. The extract significantly increased the erythrocyte osmotic fragility and promoted the removal of glycophorins A and C, and band 3 from the erythrocyte membrane. The use of Ca(2+) and Mg(2+) ions significantly potentiated glycoprotein removal, remarkably of erythrocyte band 3. The composition of fatty acids was analyzed by HPLC in both L obliqua caterpillar bristle extract and human erythrocyte membranes incubated with the extract. The levels of unsaturated fatty acids were remarkably augmented in erythrocytes incubated with the extract than in control erythrocytes, modifying thereby the saturated/unsaturated fatty acid ratio. Altogether, evidence is provided here that the interplay of at least three mechanisms of action accounts for the direct activity of the bristle extract on erythrocyte membrane, leading to hemolysis: the removal of glycoproteins and band 3; the insertion of fatty acids; and the action of phospholipases. Such mechanisms might affect erythrocyte flexibility and deformability, which may induce hemolysis by increasing erythrocyte fragility. However, whether the direct hemolytic activity of L obliqua caterpillar is the major cause of intravascular hemolysis during envenomation still needs further investigation. (C) 2010 Elsevier Ltd. All rights reserved.

Delayed local inflammatory response induced by Thalassophryne nattereri venom is related to extracellular matrix degradation

Symptoms evoked by Thalassophryne nattereri fish envenomation include local oedema, severe pain and intense necrosis with strikingly inefficient healing, continuing for several weeks or months. Investigations carried out in our laboratory showed that, in the venom-induced acute inflammation, thrombosis in venules and constrictions in arterioles were highly visible, in contrast to a notable lack of inflammatory cell. Nevertheless, the reason that the venom toxins favour delayed local inflammatory response is poorly defined. In this study, we analysed the movement of leucocytes after T. nattereri venom injection in the intraplantar region of Swiss mice, the production of pro-inflammatory mediators and the venom potential to elicit matrix metalloproteinase production and extracellular matrix degradation. Total absence of mononuclear and neutrophil influx was observed until 14 days, but the venom stimulates pro-inflammatory mediator secretion. Matrix metalloproteinases (MMP)-2 and MMP-9 were detected in greater quantities, accompanied by tissue degradation of collagenous fibre. An influx of mononuclear cells was noted very late and at this time the levels of IL-6, IL-1 beta and MMP-2 remained high. Additionally, the action of venom on the cytoskeletal organization was assessed in vitro. Swift F-actin disruption and subsequent loss of focal adhesion was noted. Collectively these findings show that the altered specific interaction cell-matrix during the inflammatory process creates an inadequate environment for infiltration of inflammatory cells.