Proteomic Analysis of Heloderma horridum horridum Venom: Assessment to Its Transcriptome and Newfound Proteins.

Heloderma horridum horridum, a venomous reptile native to America, has a venom with potential applications in treating type II diabetes. In this work, H. h. horridum venom was extracted, lyophilized, and characterized using enzymatic assays for hyaluronidase, phospholipase, and protease. Proteomic analysis of the venom was conducted employing bottom-up/shotgun approaches, SDS-PAGE, high-pH reversed-phase chromatography, and fractionation of tryptic peptides using nano-LC-MS/MS. The proteins found in H. h. horridum venom were reviewed according to the classification of the transcriptome previously reported. The proteomic approach identified 101 enzymes, 36 other proteins, 15 protein inhibitors, 11 host defense proteins, and 1 toxin, including novel venom components such as calcium-binding proteins, phospholipase A2 inhibitors, serpins, cathepsin, subtilases, carboxypeptidase-like, aminopeptidases, glycoside hydrolases, thioredoxin transferases, acid ceramidase-like, enolase, multicopper oxidases, phosphoglucose isomerase (PGI), fructose-1,6-bisphosphatase class 1, pentraxin-related, peptidylglycine α-hydroxylating monooxygenase/peptidyl-hydroxyglycine α-amidating lyase, carbonic anhydrase, acetylcholinesterase, dipeptidylpeptidase, and lysozymes. These findings contribute to understanding the venomous nature of H. h. horridum and highlight its potential as a source of bioactive compounds. Data are available via PRoteomeXchange with the identifier PXD052417.

The Clot Thickens: Differential Coagulotoxic and Cardiotoxic Activities of Anguimorpha Lizard Venoms.

Despite their evolutionary novelty, lizard venoms are much less studied in comparison to the intense research on snake venoms. While the venoms of helodermatid lizards have long been assumed to be for defensive purposes, there is increasing evidence of toxic activities more useful for predation than defence (such as paralytic neurotoxicity). This study aimed to ascertain the effects of Heloderma, Lanthanotus, and Varanus lizard venoms on the coagulation and cardiovascular systems. Anticoagulant toxicity was demonstrated for the Varanus species studied, with the venoms prolonging clotting times in human and bird plasma due to the destructive cleavage of fibrinogen. In contrast, thromboelastographic analyses on human and bird plasmas in this study demonstrated a procoagulant bioactivity for Heloderma venoms. A previous study on Heloderma venom using factor-depleted plasmas as a proxy model suggested a procoagulant factor was present that activated either Factor XI or Factor XII, but could not ascertain the precise target. Our activation studies using purified zymogens confirmed FXII activation. Comparisons of neonate and adult H. exasperatum, revealed the neonates to be more potent in the ability to activate FXII, being more similar to the venom of the smaller species H. suspectum than the adult H. exasperatum. This suggests potent FXII activation a basal trait in the genus, present in the small bodied last common ancestor. This also indicates an ontogenetic difference in prey preferences in the larger Heloderma species paralleing the change in venom biochemistry. In addition, as birds lack Factor XII, the ability to clot avian plasma suggested an additional procoagulant site of action, which was revealed to be the activation of Factor VII, with H. horridum being the most potent. This study also examined the effects upon the cardiovascular system, including the liberation of kinins from kininogen, which contributes to hypotension induction. This form of toxicity was previously described for Heloderma venoms, and was revealed in this study was to also be a pathophysiological effect of Lanthanotus and Varanus venoms. This suggests that this toxic activity was present in the venom of the last common ancestor of the anguimorph lizards, which is consistent with kallikrein enzymes being a shared toxin trait. This study therefore uncovered novel actions of anguimorph lizard venoms, not only contributing to the evolutionary biology body of knowledge but also revealing novel activities to mine for drug design lead compounds.

Exendin-4: A potential therapeutic strategy for Alzheimer's disease and Parkinson's disease.

Neurodegenerative disorders, which affect millions worldwide, are marked by a steady decline of neurons that are selectively susceptible. Due to the complex pathological processes underlying neurodegeneration, at present, there is no viable therapy available for neurodegenerative disorders. Consequently, the establishment of a novel therapeutic approach for such conditions is a clinical void that remains. The potential significance of various peptides as neuroprotective interventions for neurodegenerative disorders is gaining increasing attention. In the past few years, there has been growing scientific interest in glucagon-like peptide-1 receptor agonists due to their claimed neuroprotective effects. Exendin-4 is a glucagon-like peptide-1 receptor agonist that is known to possess anti-diabetic effects and does not degrade for hours, making it a superior candidate for such disorders. Moreover, exendin-4's neuroprotective effects have been reported in several preclinical studies. Exendin-4's diverse therapeutic targets suggest its potential therapeutic uses in neurodegenerative ailments like Alzheimer's disease and Parkinson's disease and have garnered an increasing amount of attention. Given the substantial body of evidence supporting the neuroprotective potential of exendin-4 in various research models, this article is dedicated to exploring the promising role of exendin-4 as a therapeutic agent for the treatment and management of Alzheimer's disease and Parkinson's disease. This review draws insights from the findings of numerous preclinical and clinical studies to highlight the collective neuroprotective advantages of exendin-4 and the potential mechanisms that underlie its neuroprotective effects.

Predicting daily activity time through ecological niche modelling and microclimatic data.

Climate temporality is a phenomenon that affects species activity and distribution patterns across spatial and temporal scales. Despite the global availability of microclimatic data, their use to predict activity patterns and distributions remains scarce, particularly at fine temporal scales (e.g. < month). Predicting activity patterns based on climatic data may allow us to foresee some of the consequences of climate change, particularly for ectothermic vertebrates. The Gila monster exhibits marked daily and seasonal activity patterns linked to physiology and reproduction. Here we evaluate whether ecological niche models fitted using microclimate data can predict temporal activity patterns using the Gila monster Heloderma suspectum as a study system. Furthermore, we identified whether the activity patterns are related to physiological constraints. We used dated occurrences from museum specimens and human observations to generate and test ecological niche models using minimum volume ellipsoids. We generated hourly microclimatic data for each occurrence site for 10 years using the NicheMapR package. For ecological niche modelling, we compared the traditional seasonal approach versus a daily activity pattern strategy for model construction. We tested both using the omission rate of independent observations (citizen science data). Finally, we tested whether unimodal and bimodal activity patterns for each season could be recreated through ecological niche modelling and whether these patterns followed known physiological constraints. The unimodal and bimodal activity patterns previously reported directly from tracking individuals across the year were recovered using niche modelling and microclimate across the species' geographical range. We found that upper thermal tolerances can explain the daily activity patterns of this species. We conclude that ecological niche models trained with microclimatic data can be used to predict activity patterns at high temporal resolutions, particularly on ectotherm species of arid zones coping with rapid climate modifications. Furthermore, the use of high temporal resolution variables can lead to a better niche delimitation, enhancing the results of any research objective that uses correlative models.

La estacionalidad climática es un fenómeno que afecta la actividad de las especies y los patrones de distribución a diferentes escalas espaciales y temporales. A pesar de la disponibilidad global de datos microclimáticos para estudiar dichos patrones, su uso sigue siendo escaso, particularmente en escalas temporales finas (e.g., < mes). La predicción de patrones de actividad basados en datos climáticos puede permitirnos prever algunas de las potenciales consecuencias del cambio climático, particularmente para los vertebrados ectotérmicos. El monstruo de Gila (Heloderma suspectum) exhibe marcados patrones de actividad diarios y estacionales vinculados a la fisiología y la reproducción. En este trabajo evaluamos cómo los modelos de nichos ecológicos ajustados con datos de microclima, pueden predecir patrones de actividad temporal, utilizando al monstruo de Gila como sistema de estudio. Además, identificamos si los patrones de actividad están relacionados con restricciones fisiológicas. Usamos registros de presencia provenientes de colecciones científicas y de ciencia ciudadana para generar y probar modelos de nichos ecológicos usando elipsoides de volumen mínimo. Generamos datos microclimáticos para cada hora en cada sitio de presencia durante diez años utilizando el paquete NicheMapR. Para el modelado de nichos ecológicos, comparamos el enfoque estacional tradicional con una estrategia de patrón de actividad diaria para la construcción del nicho. Ambos enfoques fueron probados utilizando la tasa de omisión de observaciones independientes (provenientes de datos de ciencia ciudadana). Finalmente, probamos si los patrones de actividad unimodales y bimodales para cada estación podían recrearse a través de modelos de nichos ecológicos y si estos patrones seguían restricciones fisiológicas conocidas. Los patrones de actividad unimodal y bimodal previamente informados directamente del seguimiento de individuos a lo largo del año, sí se recuperaron mediante el uso de modelos de nicho y microclimas en todo el rango geográfico de la especie. Encontramos también que las tolerancias térmicas superiores pueden explicar los patrones de actividad diaria de esta especie. Concluimos que los modelos de nichos ecológicos entrenados con datos microclimáticos pueden usarse para predecir patrones de actividad en altas resoluciones temporales, particularmente en especies ectotermas de zonas áridas que se enfrentan a modificaciones climáticas rápidas. Además, consideramos que el uso de variables con alta resolución temporal puede conducir a una mejor delimitación de nichos, mejorando los resultados de cualquier objetivo de investigación que utilice estos modelos correlativos.

The Dragon's Paralysing Spell: Evidence of Sodium and Calcium Ion Channel Binding Neurotoxins in Helodermatid and Varanid Lizard Venoms.

Bites from helodermatid lizards can cause pain, paresthesia, paralysis, and tachycardia, as well as other symptoms consistent with neurotoxicity. Furthermore, in vitro studies have shown that Heloderma horridum venom inhibits ion flux and blocks the electrical stimulation of skeletal muscles. Helodermatids have long been considered the only venomous lizards, but a large body of robust evidence has demonstrated venom to be a basal trait of Anguimorpha. This clade includes varanid lizards, whose bites have been reported to cause anticoagulation, pain, and occasionally paralysis and tachycardia. Despite the evolutionary novelty of these lizard venoms, their neuromuscular targets have yet to be identified, even for the iconic helodermatid lizards. Therefore, to fill this knowledge gap, the venoms of three Heloderma species (H. exasperatum, H. horridum and H. suspectum) and two Varanus species (V. salvadorii and V. varius) were investigated using Gallus gallus chick biventer cervicis nerve-muscle preparations and biolayer interferometry assays for binding to mammalian ion channels. Incubation with Heloderma venoms caused the reduction in nerve-mediated muscle twitches post initial response of avian skeletal muscle tissue preparation assays suggesting voltage-gated sodium (NaV) channel binding. Congruent with the flaccid paralysis inducing blockage of electrical stimulation in the skeletal muscle preparations, the biolayer interferometry tests with Heloderma suspectum venom revealed binding to the S3-S4 loop within voltage-sensing domain IV of the skeletal muscle channel subtype, NaV1.4. Consistent with tachycardia reported in clinical cases, the venom also bound to voltage-sensing domain IV of the cardiac smooth muscle calcium channel, CaV1.2. While Varanus varius venom did not have discernable effects in the avian tissue preparation assay at the concentration tested, in the biointerferometry assay both V. varius and V. salvadorii bound to voltage-sensing domain IV of both NaV1.4 and CaV1.2, similar to H. suspectum venom. The ability of varanid venoms to bind to mammalian ion channels but not to the avian tissue preparation suggests prey-selective actions, as did the differential potency within the Heloderma venoms for avian versus mammalian pathophysiological targets. This study thus presents the detailed characterization of Heloderma venom ion channel neurotoxicity and offers the first evidence of varanid lizard venom neurotoxicity. In addition, the data not only provide information useful to understanding the clinical effects produced by envenomations, but also reveal their utility as physiological probes, and underscore the potential utility of neglected venomous lineages in the drug design and development pipeline.

Report of a severe Heloderma suspectum envenomation.

BACKGROUND: Heloderma bites are rare and generally mild, but a few cases can be life threatening. CASE REPORT: We report a case of Heloderma suspectum envenomation in a healthy 39-year-old herpetologist. The patient rapidly developed tongue and lip swelling associated with stridor. On arrival at ICU, he was hypotensive, and in shock with atrial fibrillation requiring electrical cardioversion. Blood tests showed hypokalemia (2 mmol·L-1), associated with moderate low blood electrolytes which were corrected rapidly. In addition, he presented hematological abnormalities (INR = 1.34 and fibrinogen levels at 80 mg·dL-1) without active bleeding. All clinical and biological signs normalized without specific intervention and was discharged 4 days post-bite. The patient discharged 3 days after hospital presentation and fully recovered in 2 months. DISCUSSION/CONCLUSION: The case presented here showed the three severe complications described after Heloderma bite: a) angioedema, b) fluid loss associated with hypokalemia and metabolic acidosis, and c) cardiac disorders simulating ischemia.

Severe Heloderma spp. envenomation: a review of the literature.

Context: Heloderma bites are rare and generally mild, but a few cases can be life threatening. Methods: Description of Heloderma bite was searched in medical literature. Discussion: We present a synthesis of clinical and biomedical effects of envenomation by Heloderma sp. based on 22 well identified cases described in medical literature. Three life-threatening syndromes, concomitant or not, may be involved: (a) angioedema which can lead to respiratory tract obstruction, (b) significant fluid losses due to diarrhea, vomiting and sweating, associated with hypokalemia and sometimes metabolic acidosis, and (c) atrioventricular conduction disorders simulating cardiac ischemia. Conclusion: Heloderma bite are quite rare and generally mild. However, few severe cases may require emergency resuscitation. There is no antivenom, and the treatment is only symptomatic and supportive.

Venom gland transcriptome from Heloderma horridum horridum by high-throughput sequencing.

Lizards of the Helodermatidae (Anguimorpha) family consist of at least two well recognized species: Heloderma horridum horridum and Heloderma suspectum suspectum. They contain specialized glands in their jaws that produce venomous secretions that causes envenoming symptoms to bitten animals. One way to study proteins from such secretions is by RNA-seq; a powerful molecular tool to characterize the transcriptome of such specialized gland, and its protein secretions. The total RNA from venom gland tissues of H. horridum horridum was extracted and a cDNA library was constructed and sequenced. Overall, 114,172 transcripts were found, and 199 were annotated based on sequence similarities to previously described peptides/proteins. Transcripts coding for putative exendins, defensins, natriuretics and serine protease inhibitors were the most highly expressed. Transcripts that code for several putative serine proteases, phospholipases, metalloproteases, lipases, L-amino oxidase and nucleases were also found. Some of the novel identified transcripts were translationally controlled tumor proteins, venom factors, vespryns, waprins, lectins, cystatins and serine protease inhibitors. All these new protein structures may contribute to a better understanding of the venomous secretions of the Helodermatidae family.

Hypoxia by Altitude and Welfare of Captive Beaded Lizards (Heloderma Horridum) in Mexico: Hematological Approaches.

Heloderma horridum is one of the few known venomous lizards in the world. Their populations are in decline due to habitat destruction and capture for the pet trade. In México, many zoos have decided to take care of this species, most of them at altitudes greater than the natural altitudinal distribution. However, we know little about the capacity of the reptiles to face high-altitude environments. The objective of this study was to compare hematological traits of H. horridum in captivity in high and low altitude environments. Our findings show that H. horridum does not respond to hypoxic environments, at least in blood traits, and that the organisms appear to be in homeostasis. Although we cannot know if individual H. horridum housed in high-altitude environments are completely comfortable, it appears hypoxia can be avoid without modifications of blood parameters. We suggest that future work should address changes in metabolic rates and in behavioral aspects to understand how to maintain the health and comfort of the reptiles native to low altitude when they are housed in high-altitude environments.

Varanid Lizard Venoms Disrupt the Clotting Ability of Human Fibrinogen through Destructive Cleavage.

The functional activities of Anguimorpha lizard venoms have received less attention compared to serpent lineages. Bite victims of varanid lizards often report persistent bleeding exceeding that expected for the mechanical damage of the bite. Research to date has identified the blockage of platelet aggregation as one bleeding-inducing activity, and destructive cleavage of fibrinogen as another. However, the ability of the venoms to prevent clot formation has not been directly investigated. Using a thromboelastograph (TEG5000), clot strength was measured after incubating human fibrinogen with Heloderma and Varanus lizard venoms. Clot strengths were found to be highly variable, with the most potent effects produced by incubation with Varanus venoms from the Odatria and Euprepriosaurus clades. The most fibrinogenolytically active venoms belonged to arboreal species and therefore prey escape potential is likely a strong evolutionary selection pressure. The results are also consistent with reports of profusive bleeding from bites from other notably fibrinogenolytic species, such as V. giganteus. Our results provide evidence in favour of the predatory role of venom in varanid lizards, thus shedding light on the evolution of venom in reptiles and revealing potential new sources of bioactive molecules useful as lead compounds in drug design and development.

SURGICAL TREATMENT OF PULMONARY MELANOPHOROMA IN A BEADED LIZARD (HELODERMA HORRIDUM EXASPERATUM).

An adult male Rio Fuerte beaded lizard (Heloderma horridum exasperatum) was examined because of a history of anorexia and lethargy of one week duration. Diagnostic tests included a physical exam, complete blood cell count, plasma biochemistries, whole-body radiographs, and ultrasonography. The physical exam revealed the presence of a large mass in the midcoelomic cavity. Radiographs confirmed the presence of the midcoelomic mass and showed a smaller mass in the right cranial lung field. The ultrasonogram showed a homogeneous mass with soft tissue echogenicity. A fine-needle aspirate was collected, and the cytology results were suggestive of a melanophoroma. Exploratory surgery revealed a large mass (10×6 cm) within the right lung, with extensive adhesions to the caudolateral margin of the right liver lobe. The smaller mass (2×3 cm) was within the cranial aspect of the right lung. A right pulmonectomy and partial hepatectomy were performed to remove the tumors. The animal died 3.5-yr postsurgery, and histopathologic evaluation did not show evidence of melanophoroma in any of the tissues evaluated.

Gila monster (Heloderma suspectum) envenomation: Descriptive analysis of calls to United States Poison Centers with focus on Arizona cases.

BACKGROUND: The Gila monster (Heloderma suspectum) is a venomous lizard native to the deserts of southwestern United States (US) and northern Mexico. The purpose of this study was to describe human exposures to Gila monsters reported to US poison control centers (PCCs) with a focus on Arizona cases. METHODS: The American Association of Poison Control Centers' National Poison Data System (NPDS) was used to access and retrospectively review all calls to US PCCs, concerning Gila monsters between January 1, 2000 and October 31, 2011. In addition, detailed records from the two Arizona PCCs were reviewed for the same time period. RESULTS: A total of 319 calls regarding Gila monsters were identified in the NPDS. Of these, 105 (33%) were human exposures; most (79%) occurred in males. A total of 71 (68%) of these 105 cases were referred to a health care facility (HCF); 30 (29%) were managed on-site. Of the 71 HCF referrals, 36 (51%) were discharged home and 17 (24%) were admitted. Most (65%) admissions were to an intensive care unit (ICU). Arizona's PCCs received 70 unique reports of Gila monster bite. Most (77%) of the bites in Arizona involved an upper extremity. Eight (11%) involved patients under the age of 18 years. Eleven (16%) Arizona cases were work-related. Twenty-eight (40%) of the 70 bites in Arizona were evaluated in a HCF, but not admitted. Eleven (16%) were admitted, of which five were to an ICU. Six patients had edema of airway structures; three required emergent airway management, one by cricothyrotomy. There were no deaths. CONCLUSION: Gila monster bites are uncommon. Many cases did not require hospitalization. Edema of airway structures is an infrequent, but life-threatening complication.

Problems with mitigation translocation of herpetofauna.

Mitigation translocation of nuisance animals is a commonly used management practice aimed at resolution of human-animal conflict by removal and release of an individual animal. Long considered a reasonable undertaking, especially by the general public, it is now known that translocated subjects are negatively affected by the practice. Mitigation translocation is typically undertaken with individual adult organisms and has a much lower success rate than the more widely practiced conservation translocation of threatened and endangered species. Nonetheless, the public and many conservation practitioners believe that because population-level conservation translocations have been successful that mitigation translocation can be satisfactorily applied to a wide variety of human-wildlife conflict situations. We reviewed mitigation translocations of reptiles, including our own work with 3 long-lived species (Gila monsters [Heloderma suspectum], Sonoran desert tortoises [Gopherus morafkai], and western diamond-backed rattlesnakes [Crotalus atrox]). Overall, mitigation translocation had a low success rate when judged either by effects on individuals (in all studies reviewed they exhibited increased movement or increased mortality) or by the success of the resolution of the human-animal conflict (translocated individuals often returned to the capture site). Careful planning and identification of knowledge gaps are critical to increasing success rates in mitigation translocations in the face of increasing pressure to find solutions for species threatened by diverse anthropogenic factors, including climate change and exurban and energy development.