Discovery of broadly-neutralizing antibodies against brown recluse spider and Gadim scorpion sphingomyelinases using consensus toxins as antigens.

Broadly-neutralizing monoclonal antibodies are becoming increasingly important tools for treating infectious diseases and animal envenomings. However, designing and developing broadly-neutralizing antibodies can be cumbersome using traditional low-throughput iterative protein engineering methods. Here, we present a new high-throughput approach for the standardized discovery of broadly-neutralizing monoclonal antibodies relying on phage display technology and consensus antigens representing average sequences of related proteins. We showcase the utility of this approach by applying it to toxic sphingomyelinases from the venoms of species from very distant orders of the animal kingdom, the recluse spider and Gadim scorpion. First, we designed a consensus sphingomyelinase and performed three rounds of phage display selection, followed by DELFIA-based screening and ranking, and benchmarked this to a similar campaign involving cross-panning against recombinant versions of the native toxins. Second, we identified two scFvs that not only bind the consensus toxins, but which can also neutralize sphingomyelinase activity of native whole venom in vitro. Finally, we conclude that the phage display campaign involving the use of the consensus toxin was more successful in yielding cross-neutralizing scFvs than the phage display campaign involving cross-panning.

Identifying the geographic distribution pattern of venomous snakes and regions of high snakebite risk in Iran.

We describe species richness patterns of venomous snakes in Iran in order to produce snakebite risk prediction maps and identify gaps in regional health care centers capable of managing snakebites. We digitized distribution maps from the literature, Global Biodiversity Information Facility (GBIF), and the results of our own field studies of 24 terrestrial venomous snake species (including 4 endemic to Iran). Species richness patterns were associated with eight environmental factors. The variables have been extracted from the WorldClim dataset (bio12 = annual precipitation, bio15 = precipitation seasonality, bio17 = precipitation of the driest quarter, bio2 = mean diurnal range, bio3 = isothermality (bio2/bio7), bio4 = temperature seasonality, bio9 = mean temperature of the driest quarter and slope). Based on spatial analyses, species richness in Iran is highly affected by three environmental variables (bio12, 15, and 17) associated with precipitation. The relationship patterns among these predictors and species richness were strong and linear. The hotspot regions for venomous snakes species are concentrated on the western to southwestern and north to northeastern regions of Iran, which is partially consistent with the known Irano-Anatolian biodiversity hotspot. Because of the high number of endemic species and climatic conditions on the Iranian Plateau, the venoms of snakes distributed in those areas may contain novel properties and components.

Local envenoming by the coinsnake or Asian racer, Hemorrhois nummifer and mountain racer or leopard snake, Hemorrhois ravergieri (Serpentes: Colubridae, Colubrinae) in Iran: A reminder of the importance of species identification in the medical management of snakebites.

In Iran, there are approximately 4500-6500 snakebites per year, but fortunately only 3-9 of these are fatal. However, in some population centers such as Kashan city (Isfahan Province, central Iran), approximately 80% of snakebites are attributed to "non-venomous" snakes that are often comprised of several species of non-front-fanged snakes (NFFS). NFFS comprise a diverse group that constitute approximately 2900 species belonging to an estimated 15 families. We report here two cases of local envenoming from H. ravergieri, and one from H. nummifer that occurred in Iran. The clinical effects consisted of local erythema, mild pain, transient bleeding and edema. Two victims experienced progressive local edema that distressed the victims. The medical team's unfamiliarity with snakebites contributed to the incorrect clinical management of one victim including the contraindicated, ineffective provision of antivenom. These cases provide further documentation about local envenoming caused by these species, and also emphasize the need for regional medical personnel to receive increased training in order to improve familiarity with the local snake fauna and evidence-based snakebite management.

Global Protected Areas as refuges for amphibians and reptiles under climate change.

Protected Areas (PAs) are the cornerstone of biodiversity conservation. Here, we collated distributional data for >14,000 (~70% of) species of amphibians and reptiles (herpetofauna) to perform a global assessment of the conservation effectiveness of PAs using species distribution models. Our analyses reveal that >91% of herpetofauna species are currently distributed in PAs, and that this proportion will remain unaltered under future climate change. Indeed, loss of species' distributional ranges will be lower inside PAs than outside them. Therefore, the proportion of effectively protected species is predicted to increase. However, over 7.8% of species currently occur outside PAs, and large spatial conservation gaps remain, mainly across tropical and subtropical moist broadleaf forests, and across non-high-income countries. We also predict that more than 300 amphibian and 500 reptile species may go extinct under climate change over the course of the ongoing century. Our study highlights the importance of PAs in providing herpetofauna with refuge from climate change, and suggests ways to optimize PAs to better conserve biodiversity worldwide.

A case of envenoming by a Persian false-horned viper Pseudocerastes persicus (Duméril, Bibron & Duméril, 1854) (Serpentes: Viperidae) in Southeastern Iran.

Despite the wide distribution of the Persian false-horned viper (Pseudocerastes persicus) in the Middle East, few identified bites have been reported. A 33-year-old herpetologist bitten on the hand by Pseudocerastes persicus in Kerman Province, Southeastern Iran, developed local pain and extensive swelling with mild non-specific systemic symptoms and minimal laboratory evidence of systemic envenoming.

Comparative Venom Proteomics of Iranian, Macrovipera lebetina cernovi, and Cypriot, Macrovipera lebetina lebetina, Giant Vipers.

Envenoming by Macrovipera lebetina subspecies causes severe life-threatening difficulties for people living in North Africa and the Middle East. To better understand the pathophysiology of envenoming and improve patient management, knowledge about the venom components of the subspecies is essential. Here, the venom proteomes of Macrovipera lebetina lebetina from Cyprus and Macrovipera lebetina cernovi from Iran were characterized using RP-HPLC separation of the crude venom proteins, SDS-PAGE of fractionated proteins, and LC-MS/MS of peptides obtained from in-gel tryptic digestion of protein bands. Moreover, we also used high-resolution shot-gun proteomics to gain more reliable identification, where the whole venom proteomes were subjected directly to in-solution digestion before LC-HR-MS/MS. The data revealed that both venoms consisted of at least 18 protein families, of which snake venom Zn2+-dependent metalloprotease (SVMP), serine protease, disintegrin, phospholipase A2, C-type lectin-like, and L-amino acid oxidase, together accounted for more than 80% of the venoms' protein contents. Although the two viper venoms shared mostly similar protein classes, the relative occurrences of these toxins were different in each snake subspecies. For instance, P-I class of SVMP toxins were found to be more abundant than P-III class in the venoms of M. l. cernovi compared to M. l. lebetina, which gives hints at a more potent myonecrotic effect and minor systemic hemorrhage following envenoming by M. l. cernovi than M. l. lebetina. Moreover, single-shot proteomics also revealed many proteins with low abundance (<1%) within the venoms, such as aminopeptidase, hyaluronidase, glutaminyl-peptide cyclotransferase, cystatin, phospholipase B, and vascular endothelial growth factor. Our study extends the in-depth understanding of the venom complexity of M. lebetina subspecies, particularly regarding toxin families associated with envenoming pathogenesis and those hard-detected protein classes expressed in trace amounts.

Case Report: Recent Case Reports of Levant Blunt-Nosed Viper Macrovipera lebetina obtusa Snakebites in Iran.

Envenomation and death resulting from snakebites represent a significant public health problem worldwide, particularly in tropical and subtropical regions. The WHO has defined snakebite as a neglected tropical health concern. Bites from Macrovipera lebetina obtusa usually cause life-threatening systemic hemodynamic disturbances, reduced functionality of the kidneys, and other serious symptoms, including hypotension shock, edema, and tissue necrosis, at the bite site. Herein, we highlight five cases of M. l. obtusa envenomation that presented with wide-ranging manifestations. Many recovered cases were left with long-term musculoskeletal disabilities. In a particular case, a 15-year-old male patient was envenomed in his palm by an 80-cm M. l. obtusa. Within 12 hours, swelling extended to near the shoulder. Fasciotomy was performed on the forearm and part of the upper arm of this patient. Symptoms of severe localized pain and swelling, dizziness, weakness, low blood pressure, and itching around the bite area were documented. The patient remained in the hospital for 13 days.

Integrating Top-Down and Bottom-Up Mass Spectrometric Strategies for Proteomic Profiling of Iranian Saw-Scaled Viper, Echis carinatus sochureki, Venom.

Saw-scaled or carpet vipers (genus Echis) are considered to cause a higher global snakebite mortality than any other snake. Echis carinatus sochureki (ECS) is a widely distributed snake species, also found across the thirteen provinces of Iran, where it is assumed to be responsible for the most snakebite envenomings. Here, we collected the Iranian specimens of ECS from three different geographically distinct populations, investigated food habits, and performed toxicity assessment and venom proteome profiling to better understand saw-scaled viper life. Our results show that the prey items most commonly found in all populations were arthropods, with scorpions from the family Buthidae particularly well represented. LD50 (median lethal dose) values of the crude venom demonstrate highly comparable venom toxicities in mammals. Consistent with this finding, venom characterization via top-down and bottom-up proteomics, applied to both crude venoms and size-exclusion chromatographic fractions, revealed highly comparable venom compositions among the different populations. By combining all proteomics data, we identified 22 protein families from 102 liquid chromatography and tandem mass spectrometry (LC-MS/MS) raw files, including the most abundant snake venom metalloproteinases (SVMPs, 29-34%); phospholipase A2 (PLA2s, 26-31%); snake venom serine proteinases (SVSPs, 11-12%); l-amino acid oxidases (LAOs, 8-11%), C-type lectins/lectin-like (CTLs, 7-9%) protein families, and many newly detected ones, e.g., renin-like aspartic proteases (RLAPs), fibroblast growth factors (FGFs), peptidyl-prolyl cis-trans isomerases (PPIs), and venom vasodilator peptides (VVPs). Furthermore, we identified and characterized methylated, acetylated, and oxidized proteoforms relating to the PLA2 and disintegrin toxin families and the site of their modifications. It thus seems that post-translational modifications (PTMs) of toxins, particularly target lysine residues, may play an essential role in the structural and functional properties of venom proteins and might be able to influence the therapeutic response of antivenoms, to be investigated in future studies.

Venoms of Iranian Scorpions (Arachnida, Scorpiones) and Their Potential for Drug Discovery.

Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda, Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus, in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.

A perspective view of top-down proteomics in snake venom research.

The venom produced by snakes contains complex mixtures of pharmacologically active proteins and peptides which play a crucial role in the pathophysiology of snakebite diseases. The deep understanding of venom proteomes can help to improve the treatment of this "neglected tropical disease" (as expressed by the World Health Organization [WHO]) and to develop new drugs. The most widely used technique for venom analysis is liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based bottom-up (BU) proteomics. Considering the fact that multiple multi-locus gene families encode snake venom proteins, the major challenge for the BU proteomics is the limited sequence coverage and also the "protein inference problem" which result in a loss of information for the identification and characterization of toxin proteoforms (genetic variation, alternative mRNA splicing, single nucleotide polymorphism [SNP] and post-translational modifications [PTMs]). In contrast, intact protein measurements with top-down (TD) MS strategies cover almost complete protein sequences, and prove the ability to identify venom proteoforms and to localize their modifications and sequence variations.