Journal of Venomous Animals and Toxins Including Tropical Diseases最新文献

Background: Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately seven million individuals worldwide, with the highest number of cases in Latin America. CD has two phases, of which the chronic phase is characterized by reduced efficacy in drug therapies. This and other factors make developing new strategies that aim to identify molecules capable of becoming alternatives to or complement current chemotherapy vitally important.

Methods: Cruzain and AsCystatin were obtained recombinantly through expression in E. coli. Bioinformatic assays were conducted with both molecules, followed by in vitro enzyme inhibition assays. Subsequently, in silico studies allowed for the design of peptides, which were then assessed for molecular interactions with cruzain. The designed peptides were synthesized, and their inhibitory potential on cruzain and their trypanocidal and cytotoxic effects in vitro were finally assessed.

Results: AsCystatin, a potential inhibitor of cysteine proteases, was identified from previously published scientific literature. In silico assays suggested that AsCystatin interacts with key regions of cruzain, and was subsequently produced through heterologous expression, obtaining a protein with a high degree of purity. Next, the inhibition of AsCystatin on the activity of cruzain was assessed, observing that approximately 20 µM of cystatin could inhibit 50% of the catalytic activity of the recombinant enzyme. Based on the in-silico analysis performed previously, original, and modified peptides were designed and tested, which allowed for identifying four peptides with inhibitory capacity on the enzymatic activity of cruzain. Finally, three of these peptides showed trypanocidal activity on epimastigote forms of T. cruzi in in vitro models.

Conclusion: It was possible to identify AsCystatin and four peptides derived from this protein with inhibitory activity on cruzain, highlighting the trypanocidal effect of these peptides observed in in vitro assays.

Background: Temperature regulation is essentially important for survival of poikilotherms such as snakes. Body temperature is regulated by snakes through behavioral and physiological responses. The global-warming crisis, combined with the need to house large population of snakes in limited spaces, increases the likelihood of exposing snakes to high ambient temperature (HTa), requiring it reliance on physiological responses. This study aimed to study the effect of HTa exposure on physiological responses and venom production, which have rarely been studied.

Methods: Eleven adult monocled cobras (Naja kaouthia Lesson, 1831) were divided into two groups. The concurrent control group was housed in a temperature-controlled room, and the heat exposed group was housed in the same room with gradually increasing temperatures (25°C-35°C) for 4 h on four consecutive days. Data were collected 3 days before the experiment as the baseline and then compared with day 1 and day 4 after HTa exposure data representing immediate and prolonged effects. Body temperature, body weight, water intake, heart rate, hematology, plasma biochemistry, body-fluid compartments, hormonal response, heat shock protein expression and venom production were measured.

Results: In response to HTa exposure, body temperature and heart rate increased, plasma volume significantly decreased, but water intake increased. Hematocrit and plasma protein progressively decreased in the latter stages of experimentation, but HTa diminished this effect. HTa only increased plasma corticosterone on day 1. Exposure to HTa increased venom protein concentration on day 4 and diminished the decreased proportion effect of frequent venom collection on phospholipase A₂ component.

Conclusion: Increased heart rate and fluid shift from the intravascular compartment appeared to be the underlying mechanism for heat dissipation during HTa exposure. Under the study condition, HTa caused heat stress, but the snake could adapt after continued exposure. Additionally, HTa increased venom protein concentration in N. kaouthia, particularly phospholipase A₂ component.

Background: Non-small cell lung cancers (NSCLC) represent the primary cause of cancer-related deaths worldwide. Rhopalurus junceus venom has been shown to exert cytotoxic effects against a panel of epithelial cancer cells in vitro and suggested that NSCLC was the subtype most susceptible to the treatment.

Methods: This study evaluated the effect of Rhopalurus junceus scorpion venom on cell viability, in non-cancerous (MRC-5, lung; CHO-K1, ovary) and NSCLC (A549; NCI-H460) cell lines. The effects on cell cycle, apoptosis, and cell signaling-related proteins were determined by flow cytometry and WB. Protein fractions responsible for the observed effect were identified using HPLC.

Results: Scorpion venom was more effective against NSCLC than non-cancerous cells. E_max values were 20.0 ± 5.8% and 22.47 ± 6.02% in A549 and NCI-H460 cancer cells, respectively, as compared to 50 ± 8.1% in MRC-5 and 54.99 ± 7.39% in CHO-K1 cells. It arrested NSCLC cells in the G2/M phase, while non-cancerous cells were arrested in the S (MRC-5) or G0/G1 (CHO-K1) phases. No changes were observed in the Bax/Bcl-2 or the cleaved-caspase 3/Total caspase 3 ratios in cells treated with venom. Likewise, the scorpion venom treatment did not affect p-ERK, p-AKT, or p-38MAPK protein levels. In contrast, scorpion venom treatment increased the cytosolic apoptosis-inducing factor (AIF) in A549 cells, indicating caspase-independent apoptosis. Additionally, combined etoposide/venom exposure provoked G2/M arrest and apoptosis in NSCLC more strongly than either substance alone. Furthermore, upon crude venom fractioning through RP-HPLC, we found two soluble fractions with high cytotoxic effects.

Conclusion: The present study concludes that a specific fraction of Rhopalurus junceus venom reduces cell viability of NSCLC cells. The AIF protein plays a key role in mediating caspase-independent apoptotic cell death. These findings suggest that Rhopalurus junceus venom enhances the anticancer effect of etoposide in vitro by causing cell cycle arrest and caspase-independent apoptosis.

Background: This study examines the impact of Phα1β, a spider peptide derived from the venom of Phoneutria nigriventer, on the Kv11.1 potassium channel in HEK293 cells transfected with the human ERG potassium channel. Phα1β inhibits high-voltage calcium channels and acts as an antagonist of the TRPA1 receptor, both of which play crucial roles in pain transduction pathways. Over the past 15 years, our research has demonstrated the potential of Phα1β, in both its native and recombinant forms, as a promising analgesic drug through preclinical tests conducted on rodent pain models. Regulatory agencies require the evaluation of new drugs on human ERG channels.

Methods: To assess hERG potassium channel inhibition, we utilized the FLIPR® Potassium Assay, a commercially available kit. The assay involved testing the effects of Phα1β alongside the well-established hERG potassium channel blocker dofetilide, which served as a positive control. The viability of HEK-293 cells was assessed using the colorimetric MTT reduction test (3-(4, dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereby viable cells reduce the MTT salt, forming a formazan complex within their mitochondria, as previously described.

Results: Phα1β was tested at concentrations of 56, 225, 450, and 900 pMol, resulting in a discreet inhibition of hERG potassium channel activity at higher concentrations, approximately 13.47%, with an IC₅₀ value exceeding 900 pMol. Dofetilide, administered at concentrations ranging from 0.0001 to 10 µM, displayed a concentration-dependent inhibition of the hERG potassium channel, with a mean IC₅₀ value of 0.1642 µM (0.1189-0.2282 µM). To evaluate cytotoxicity, HEK293-hERG cells were exposed to Phα1β concentrations of 56/900 pMol for 24 hours, resulting in no significant alteration in cell viability.

Conclusion: Our findings indicate that even at high concentrations, Phα1β does not impede the functionality of the hERG potassium channel nor affect cell viability.

Background: Fish venoms have been poorly characterized and the available information about their composition suggests they are uncomplicated secretions that, combined with epidermal mucus, could induce an inflammatory reaction, excruciating pain, and, in some cases, local tissue injuries.

Methods: In this study, we characterized the 24-hour histopathological effects of lionfish venom in a mouse experimental model by testing the main fractions obtained by size exclusion-HPLC. By partial proteomics analysis, we also correlated these in vivo effects with the presence of some potentially toxic venom components.

Results: We observed a strong lesion on the skin and evident necrosis in the skeletal muscle. None of the tissue-damaging effects were induced by the fraction containing cytolysins, membrane pore-forming toxins ubiquitously present in species of scorpionfish, stonefish, and lionfish, among others. On the contrary, injuries were associated with the presence of other components, which have remained practically ignored so far. This is the case of an abundant protein, present in venom, with homology to a Golgi-associated plant pathogenic protein 1-like (GAPR1), which belongs to the same protein superfamily as venom CRISPs and insect allergens.

Conclusion: This GAPR1-like protein and the hyaluronidase are probably responsible for the hemostasis impairment and hemorrhagic lesions observed in mouse skin, whereas muscle injuries can be indirectly caused by a combination of inflammatory and hemorrhagic events. More information is required to establish the components accountable for the myonecrotic effect.

Envenomation by aquatic species is an under-investigated source of human morbidity and mortality. Increasing population density along marine and freshwater coastlines increases these incidents. Specific occupational groups - including commercial fishery workers, fisherfolk, marine tourism workers, and researchers - rely on aquatic resources for their livelihood. While diverse venomous aquatic species exhibit a broad array of habitats worldwide, they are most abundant in the tropics. Specific tropical regions present historic "hot spot" areas of concern for occupational groups with heightened risk of aquatic envenomation. Towards the overall objective of characterizing the health burden of aquatic envenomations, this review seeks to define (1) vulnerable, high-risk populations and (2) geographic hot-spot regions. To formally assess these metrics, a systematic literature review was performed where inclusion criteria requirements were peer-reviewed, published, epidemiological studies with defined denominators from January 1, 2000, to July 31, 2024, on the topic of human envenomation by aquatic species. Fifty-three articles met the inclusion criteria. Excluded articles were comprised of case reports, news and magazine articles, and those in languages aside from English, French, Portuguese, and Spanish. Most of the included articles examined emergency department and poison-control datasets that reported few overall envenomations (< 1%) from populations with physical and financial access to medical care. In contrast, datasets surveying beachgoers or fisherfolk directly, and life-guard incident reports, demonstrated that aquatic envenomation is an important source of injury for these groups and settings (envenomation frequency mean: 71%, median: 80%). Reports on additional high-risk groups, including marine and aquatic biologists, military personnel etc., and in key high-risk geographic regions including Thailand, Indonesia, and other Indo-Pacific countries were missing from the reviewed literature. Socio-demographic data were also largely missing from the literature. This systematic review highlights critical gaps where further research is needed, especially in under-represented regions and vulnerable populations.

Background: Micrurus mipartitus is a coral snake of public health concern in Colombia. Its venom is mainly composed of three-finger toxins (3FTxs), Mipartoxin-1 being the most abundant protein partially responsible for its lethal effect. In this work, we present the production of Mipartoxin-1 in a recombinant form and evaluate its immunogenic potential. Methods: A genetic construct HisrMipartoxin-1 was cloned into the pET28a vector and heterologous expression was obtained in E. coli BL21 (DE3). The recombinant HisrMipartoxin-1 protein was extracted from inclusion bodies, refolded in vitro, and isolated by affinity and RP-HPLC chromatography. The lethal effect of HisrMipartoxin-1 was tested, and antibodies against HisrMipartoxin-1 were produced by immunization in rabbits. The antibody titers were monitored by an ELISA test. The neutralizing ability of the antibodies, against the lethal effect of native toxins and M. mipartitus venom, was also assessed. Results: HisrMipartoxin-1 was detected on SDS-PAGE, with a molecular mass of around 11 kDa. The retention time was 16.0 minutes. HisrMipartoxin-1 did not exhibit lethality in mice; however, antibodies against HisrMipartoxin-1 recognized the native toxin, the whole venom of M. mipartitus, and a 3FTx from another species within the Micrurus genus. Furthermore, antibodies against HisrMipartoxin-1 completely neutralized the lethal effect of native Mipartoxin-1 in mice but not M. mipartitus whole venom. Conclusion: These findings indicate that HisrMipartoxin-1 might be used as an immunogen to develop anticoral antivenoms or complement them. This work is the first report of the heterologous expression of 3FTx from M. mipartitus.

Background: Medications currently used to treat pain are frequently associated with serious adverse effects and rapid development of tolerance. Thus, there is a need to develop more effective, and safer medicines for the population. Blocking NMDA receptors (NMDAR) has shown to be a promising target for the development of new drugs. That statement is due to NMDAR activation and glutamate release in the spinal cord which affects chronic pain modulation. Therefore, the aim of this study was to evaluate the possible spinal antinociceptive activity of PnTx4(5-5) toxin. The peptide is purified from the venom of the spider P. nigriventer and its affinity for NMDAR and sodium channels Nav1.2-1.6 has already been established.

Methods: We compared its effect and safety with MK-801 (NMDAR antagonist) and evaluated its influence on glutamate and reactive oxygen species (ROS) levels in CSF. PnTx4(5-5) was administered intrathecally in the Formalin test and co-administered with NMDA in the Spontaneous pain test. After three minutes of observation, mice cerebrospinal fluid was collected to measure glutamate and ROS levels.

Results: The spider peptide inhibited nociception as post-treatment in the inflammatory phase of the Formalin test. Furthermore, it inhibited spontaneous nociception induced by NMDA, being more potent and effective than MK-801 in both models tested. A glutamate rise level in the CSF of mice was significantly reduced by the toxin, but ROS increase was not affected. The animals' motor skills were not affected by the tested doses of NMDAR inhibitors.

Conclusion: In conclusion, the results suggest PnTx4(5-5) may mediate its antinociceptive effect in the spinal cord not only by inhibiting postsynaptic receptors but probably also by acting on autoreceptors. This effect does not affect the motricity of mice at the highest dose tested, which suggests that it has therapeutic potential and safety for use as a painkiller.

Background: Sea anemones are well known to contain multiple peptide toxins. However, of more than 1100 species of sea anemones distributed worldwide, only a little over 50 have been studied for peptide toxins. Therefore, innumerable unique and novel peptide toxins remain to be discovered in unstudied sea anemones.

Methods: Isolation of peptide toxins in the sea anemone Heteractis aurora was attempted by gel filtration and reverse-phase high performance liquid chromatography, using the toxicity to crabs as an index. The amino acid sequences of the isolated four toxins (Hau I-IV) and their precursors were determined using a combination of protein sequencing and cDNA cloning.

Results: Hau I and IV were potently lethal to crabs, whereas Hau II and III were only paralytic. The precursor proteins of the four toxins were commonly composed of a signal peptide, a propart, and the remaining region including a mature peptide. Interestingly, four and two copies of the mature peptide were present in the precursor proteins of Hau II and III, respectively. Homology searches revealed that Hau I (30 amino acid residues) is a novel peptide toxin, although it has the same cysteine pattern CXXC-C-C as the boundless β-hairpin (BBH) family. Hau II (27 amino acid residues) and III (28 amino acid residues) were homologous with the BBH family, whereas Hau IV (49 amino acid residues) was a new member of the well-known type 1 sodium channel toxin family.

Conclusion: This study showed that a novel class of toxin (Hau I), two BBH family toxins (Hau II and III), and a type 1 sodium channel toxin (Hau IV) are present in the toxin of the sea anemone H. aurora.

Visceral leishmaniasis (VL) is a neglected disease that is typical of tropical and subtropical parts of the world and is caused by the trypanosomatid Leishmania donovani complex. This disease is a multifactorial condition that involves parasitic, environmental, and immunogenetic characteristics. Genetic changes in genes encoding cytokines may be associated with changes in their expression and, consequently, with the development of clinical resistance or susceptibility to the disease. This systematic review and meta-analysis aimed to assess whether single nucleotide polymorphisms (SNPs) in interleukin genes influence the clinical consequences of visceral leishmaniasis infection. To this end, we carried out a systematic review and meta-analysis with structured searches in the EMBASE, PubMed, Scopus, SciELO, and Web of Science databases without time restrictions. Two independent reviewers examined the studies, performed data extraction, and assessed quality by assigning scores. If there were any discrepancies, a third reviewer with more experience was consulted. After the screening process, 28 articles were included in the systematic review and 9 in the final analysis of the meta-analysis. Statistical analyses were carried out using various genetic models. The odds ratio (OR) and corresponding 95% confidence intervals (CIs) were calculated to estimate the associations. Overall, the main clinical outcomes were classified as not associated or associated when they presented susceptibility, resistance, risk, or protective factors for the development of the disease. Associations between IFN-γ +874T/A polymorphisms in the dominant model (OR 1.64, 95% CI 1.13-2.38, I² = 0%, p < 0.01) and heterozygous model (OR 1.72, 95% CI 1.15-2.57, I² = 0%, p < 0.01) and IL-18 -137G/C in the recessive model (OR 1.33, 95% CI 1.02-1.71, I² = 9%, p = 0.03) and VL were observed. For the IL-10 gene SNPs, there was no significant association. Our findings suggest that SNPs in the IFN-γ and IL-18 genes may be associated with the risk of developing VL.