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  • 106 Reads
The positive action of resveratrol analogs against Helicobacter pylori
Published: 27 June 2022 by MDPI in Pathogens and Natural Toxins e-Conference section Pathogenic/Toxic Bacteria

Helicobacter pylori is a gastroduodenal pathogen that colonizes the human stomach, thereby inducing inflammation of the gastric mucosa causing gastritis, peptic ulcer and gastric cancer. H. pylori finds multiple ways for its survival in the human host which is performed by various virulence factors, collectively responsible for persistence and colonization. One of the major virulence factors produced by H. pylori is Vacuolating cytotoxin A (VacA) exerting multiple effects on epithelial cells.

Resveratrol is a naturally occuring phytoalexin, found, in particular, in grape and in grapeskin, that inhibits a wide array of bacteria. In particular, its role in effecting the oxidative stress and inflammation in H. pylori-infected mucosa has been described. Although resveratrol possesses antimicrobial benefits, it is noteworthy that its availability from plants is limited, and it has been associated with poor bioavailability. In a recent work, we reported the positive effects of a series of resveratrol analogs against resistant H. pylori clinical strains, evaluating antibacterial (MIC/MBC) and antivirulence effects (biofilm reduction and swarming motility inhibition), alone and combined with levofloxacin.

In this communication we describe the synthesis and antibacterial effect of new resveratrol analogs against H. pylori. New molecules kept unaltered the 4’-hydroxyl group of resveratrol, important for biological activity, while 3,5-hydroxy groups were replaced with a substituent in 4-position. Furthermore, a new substituent was added in 3’-position. We also report a preliminary microbiological evaluation.

  1. Chauhan, A. Chin Yen Tay, B. J. Marshall, U. Jain. Helicobacter pylori VacA, a distinct toxin exerts diverse functionalities in numerous cells: An overview. Helicobacter 2019, 24, e12544
  2. Di Fermo, S. Di Lodovico, R. Amoroso, B. De Filippis, S. D'Ercole, E. Di Campli, L. Cellini, M. Di Giulio. Searching for new tools to counteract the Helicobacter pylori resistance: the positive action of resveratrol derivatives. Antibiotics 2020, 12, 891
  • Open access
  • 40 Reads
Fabrication of Microbicidal Silver Nanoparticles: Green Synthesis and Implications in the Containment of Bacterial Biofilm on Orthodontal Appliances
Published: 27 June 2022 by MDPI in Pathogens and Natural Toxins e-Conference section Pathogenic/Toxic Bacteria

Among various metal-based nanoparticles, silver nanoparticles (AgNPs) manifest superior inhibitory effects against several microorganisms. In fact, the AgNP-based treatment has been reported to inhibit both sensitive and resistant isolates of bacteria and other disease-causing microbes with equal propensity. Keeping this fact into consideration, we executed bio-mediated synthesis of AgNPs employing extract of flower and various other parts (such as bud and leaf) of the Hibiscus rosa-sinensis plant. The physicochemical characterization of as-synthesized AgNPs was executed employing transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, Fourier transform infrared (FTIR) spectroscopy, UV-Vis spectroscopy, etc. The as-synthesized AgNPs demonstrated strong antimicrobial activity against both Gram-positive and Gram-negative bacteria with equal propensity. The as-synthesized AgNPs successfully inhibited Streptococcus mutans (S. mutans), one of the main causative bacteria responsible for dental caries. Considering the fact that orthodontic appliances facilitate infliction of the oral cavity with a range of microbes including S. mutans, we determined the growth inhibitory and anti-adherence activities of AgNPs on orthodontic appliances. We performed microbiological assays employing AgNPs adsorbed onto the surface of nickel-titanium (Ni-Ti) orthodontic wires. Topographic analysis of the decontaminated Ni-Ti orthodontic wires was performed by scanning electron microscopy. In addition to antimicrobial and anti-biofilm activities against oral S. mutans, the as-fabricated AgNPs demonstrated significant inhibitory and anti-biofilm properties against other biofilm-forming bacteria such as Escherichia coli and Listeria monocytogenes.

  • Open access
  • 100 Reads
A Symphony of Destruction- Evolution and Clinical Implications of Coagulotoxicity in Rattlesnakes
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Published: 27 June 2022 by MDPI in Pathogens and Natural Toxins e-Conference section Venomous Animals

How reliable is our knowledge of even the most prominent functional traits in extensively studied venomous animals? This study adopts rattlesnakes as a model group to investigate the evolutionary history of venom coagulotoxicity and its most relevant medical implications. Venom-induced clotting of human plasma and fibrinogen was determined and mapped onto the phylogenetic tree of rattlesnakes to reconstruct the evolution and diversity of coagulotoxic activity across the group. Our results indicate that venom phenotype is often independent of phylogenetic relationships, rather suggesting a relevant influence of diet and other environmental factors on venom evolution. We report the first instance of true procoagulant venom activity in Neotropical rattlesnakes for the species Crotalus culminatus, whose ontogenetic clotting factor-activating pattern emphasized the need for more accurate research on the mechanisms of rattlesnake envenomation. Underscoring this unmet need, one of the mainstream Mexican antivenoms (Antivipmyn®) failed to neutralize the coagulotoxic action of C. culminatus venom, highlighting potential issues in snakebite management for this species. On the other hand, metalloprotease inhibitors were able to effectively counter the procoagulant activity of the venom, not only indicating that this enzyme class is likely responsible for the pathophysiological actions, but also that metalloproatease inhibitors may have therapeutic benefit in treating the envenomed patient. Ultimately, our findings highlight that activity rather than protein composition is the target of selection pressure in venom evolution and underscore the need for further research into rattlesnake venom patterns from a functional perspective. Such remarkable, yet long overlooked variability in venom activity also bears considerable clinical relevance in terms of the impact of rattlesnake envenoming and potential shortfalls of current antivenom treatments.

  • Open access
  • 52 Reads
The simplified synthesis of AFB1-lys/ AFG1-lys to produce analytical standards for human exposure analysis
Published: 27 June 2022 by MDPI in Pathogens and Natural Toxins e-Conference section Toxic Fungi

Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin that can be found contaminating crops such as maize and ground nuts worldwide. It is a carefully monitored for mycotoxin with 99 countries, accounting for 87% of the global population, having in place systems to monitor for AFB1. The most common estimate for human exposure to aflatoxin is by urinary excretion of the hydroxylated AFB1 metabolite AFM1 however it only provides an estimate of recent AFB1 exposure. Chronic AFB1 exposure can be measured by quantifying the AFB1-human serum albumin adduct. This is reported with techniques like ELISA, HPLC, or LC-MS/MS and measures the amount of AFB1-lysine released after proteolysis of serum albumin. Although measuring this toxic is very important from a human health perspective, analytical standards and reference materials are not commercially available. This work goes outlines a simplified synthesis of, unlabelled and isotopically labelled, AFB1-lys without the production of AFB1-lysine epoxide, a potent carcinogen. Additionally, the stability of these standards under long-term storage was determined. We also synthetically linked AFB1 to human serum albumin in order to make reference material.

  • Open access
  • 75 Reads
Differential toxicity of viperid (Macrovipera) venoms and neutralisation by antivenoms and enzyme inhibitors
Published: 27 June 2022 by MDPI in Pathogens and Natural Toxins e-Conference section Venomous Animals

Species within the viperid genus Macrovipera are some of the most dangerous snakes in the Eurasian region, injecting copious amounts of potent venom. Despite their medical importance, the pathophysiological actions of their venoms have been neglected. Particularly poorly known are the coagulotoxic effects and thus the underlying mechanisms of lethal coagulopathy. In order to fill this knowledge gap, we ascertained the effects of venom upon human plasma for Macrovipera lebetina cernovi ,M. l. lebetina, M. l. obtusa, M. l. turanica, and M. schweizeri using diverse coagulation analysing protocols. All five were extremely potent in their ability to promote clotting but varied in their relative activation of Factor X, being equipotent in this study to the venom of the better studied, and lethal, species Daboia russelii. The Insoserp European viper antivenom was shown to be highly effective against all the Macrovipera venoms, but performed poorly against the D. russelii venom. Reciprocally, while Daboia antivenoms performed well against D. russelii venom, they failed against Macrovipera venom. Thus despite the two genera sharing a venom phenotype (Factor X activation) driven by the same toxin type (P- IIId snake venom metalloproteases), the surface biochemistries of the toxins differed significantly enough to impede antivenom cross- neutralization. The differences in venom biochemistry were reflected in coagulation co-factor dependence. While both genera were absolutely dependent upon calcium for the activation of Factor X, dependence upon phospholipid varied. The Macrovipera venoms had low levels of dependence upon phospholipid while the Daboia venom was three times more dependent upon phospholipid for the activation of Factor X. This suggests that the sites on the molecular surface responsible for phospholipid dependence, are the same differential sites that prevent inter- genera antivenom cross- neutralization. Due to cold-chain requirements, antivenoms may not be stocked in rural settings where the need is at the greatest. Thus we tested the efficacy of enzyme inhibitor Prinomastat as a field-deployable treatment to stabilise patients while being transported to antivenom stocks, and showed that it was extremely effective in blocking the Factor X activating pathophysiological actions. Marimastat however was less effective. These results thus not only shed light on the coagulopathic mechanisms of Macrovipera venoms, but also provide data critical for evidence-based design of snakebite management strategies.

  • Open access
  • 59 Reads
Clinical and Evolutionary Implications of Dynamic Coagulotoxicity Divergences in Bothrops (Lancehead Pit Viper) Venoms
Published: 27 June 2022 by MDPI in Pathogens and Natural Toxins e-Conference section Venomous Animals

Despite coagulotoxicity being a primary weapon for prey capture by Bothrops species (lancehead pit vipers) and coagulopathy being a major lethal clinical effect, a genus-wide comparison has not been undertaken. To fill this knowledge gap, we used thromboelastography to compare 37 venoms, from across the full range of geography, taxonomy, and ecology, for their action upon whole plasma and isolated fibrinogen. Potent procoagulant toxicity was shown to be the main venom effect of most of the species tested. However, the most basal species (B. pictus) was strongly anticoagulant; this is consistent with procoagulant toxicity being a novel trait that evolved within Bothrops subsequent to their split from anticoagulant American pit vipers. Intriguingly, two of the arboreal species studied (B. bilineatus and B. taeniatus) lacked procoagulant venom, suggesting differential evolutionary selection pressures. Notably, some terrestrial species have secondarily lost the procoagulant venom trait: the Mogi Mirim, Brazil locality of B. alternatus; San Andres, Mexico locality of B. asper; B. diporus; and the São Roque of B. jararaca. Direct action on fibrinogen was extremely variable; this is consistent with previous hypotheses regarding it being evolutionary decoupled due to procoagulant toxicity being the primary prey-capture weapon. However, human patients live long enough for fibrinogen depletion to be clinically significant. The extreme variability may be reflective of antivenom variability, with these results thereby providing a foundation for such future work of clinical relevance. Similarly, the venom diversification trends relative to ecological niche will also be useful for integration with natural history data, to reconstruct the evolutionary pressures shaping the venoms of these fascinating snakes.

  • Open access
  • 232 Reads
Venomic adaptations of prey specialised spiders
Published: 27 June 2022 by MDPI in Pathogens and Natural Toxins e-Conference section Venomous Animals

Venom represents an important trait of countless animals, including many predators. Predatory venoms are usually very effective against certain prey as the result of evolutionary arms races. Nevertheless, many venomous animals remain understudied, including spiders, one of the most diversified groups of venomous predators. The vast majority of spiders are generalists who prey on a wide range of prey. However, a small proportion of spiders are prey-specialists that preferentially hunt only one prey type, often represented by dangerous prey, such as termites, ants or other spiders. They utilise morphological, behavioural and venomic adaptations to subdue such prey. Despite the current evidence suggesting that prey-specialised spiders immobilise prey with potent venom, their venoms haven’t been investigated in detail. In the research of our lab, we discovered that stenophagous prey-specialised spiders have smaller venom glands and less complex venoms compared to euryphagous generalists, as the venoms of specialists have presumably evolved to target only one prey type. Moreover, our laboratory bioassays revealed that the venoms of specialists are prey-specific. We also investigated the venom composition of one spider-eating specialist, the white-tailed spider (genus Lampona). The proteo-transcriptomic analysis identified more than 200 compounds in the Lampona venom. The main components were Cysteine-rich lampotoxins represented by peptides containing 8 to 10 Cysteine residues. Overall, we confirmed that prey-specialised spiders possess venomic adaptations allowing them to quickly subdue dangerous prey. Many aspects are still not explored, such as the venom composition and the mechanisms of prey-specificity of the majority of specialists, and I aim to investigate these features in my current research.

  • Open access
  • 258 Reads
The real-life invertebrate vampires and how proteins in their saliva enable them to consume blood
Published: 27 June 2022 by MDPI in Pathogens and Natural Toxins e-Conference section Venomous Animals

Haematophagous animals rely on the blood of vertebrates for nutrition, egg development, and survival. To obtain blood meals, these organisms have evolved salivary constituents that potently circumvent the host haemostatic response, which otherwise prevents blood loss through the formation of a blood clot. These salivary proteins can directly affect platelet aggregation, induce vasodilation and inhibit proteinases of the clotting cascade. There are over >15,000 species of blood-feeding invertebrates, and the sialome of several of these animals has been characterised. Within these sialomes, antihaemostatic proteins have been isolated and developed as antithrombotic therapeutics. Consequently, examining the saliva of neglected haematophagous invertebrates could be pharmacologically relevant and provide further insight into evolutionary adaptations to blood-feeding.

Using proteotranscriptomic analyses, I aim to explore the salivary gland venom composition of underexplored haematophagous arthropods, including Australian native horseflies (family Tabanidae), Australian native ticks such as the eastern paralysis tick Ixodes holocyclus, southern paralysis tick Ixodes cornuatus, and ornate kangaroo tick Amblyomma triguttatum, and the only native Australian kissing bug, Triatoma leopoldi. By examining their saliva, I hope to identify coagulotoxic molecules which allow them to overcome host haemostatic systems. Since venom peptides from non-haematophagous animals have also been shown to potently modulate haemostatic systems, I will test a range of peptides from venomous invertebrates for coagulotoxicity against mammalian blood and invertebrate haemolymph. Finally, I will characterise identified molecules both structurally and functionally to gain further knowledge into their role in the saliva of blood-feeding invertebrates. This research will expand the knowledge available on the salivary toxins of blood-feeding invertebrates, and discover new molecules with potential as therapeutics for thromboembolic disorders.

  • Open access
  • 316 Reads
Ostreopsis cf. ovata an emerging threat to human health in the Mediterranean basin: LC-HRMS-based approach for monitoring and risks mitigation strategies

The benthic marine dinoflagellate Ostreopsis cf. ovata, once confined to tropical and subtropical regions, since the end of 1990s has been wide spreading in temperate areas including the Mediterranean basin, with harmful algal blooms (HABs) increasing along Italian coastlines. Particularly, in the summer 2005 a massive proliferation of O. ovata along the Ligurian coasts of Genoa induced a toxic outbreak of alarming proportions with negative impact on marine environment and humans. During that toxic event, people who had spent time on or close to beaches, apparently just breathing marine aerosols, manifested a severe respiratory syndrome with symptoms including fever, respiratory distress, conjunctivitis with, in some cases, needs of hospitalization. The chemical agents responsible for toxicity were identified by liquid chromatography coupled to mass spectrometry (LC-MS) and LC-high-resolution MS (LC-HRMS) studies conducted by MarBioTox research group and were named ovatoxins (OVTXs)1, structural congeners of palytoxin (PLTX), one of the most potent non-protein marine toxins known to date. Since 2005, MarBioTox group has kept on investigating the Ostreospis phenomena that become recurrent during summer-autumn in the Mediterranean area being responsible for numerous human poisonings following inhalation of toxic aerosols, and skin contact, posing serious concerns to human health also related to a possible ingestion of contaminated seafood. Consequently, the European Food Safety Authority (EFSA) in 2009, announced the need to optimize efficient analytical methods for OVTXs and PLTXs detection. Currently, LC-HRMS is the best analytical strategy to detect OVTXs in different matrices (algae, seawater, seafood) and, over the time, it allowed to bring to light different toxin profiles for Mediterranean strains of O. cf. ovata mostly dominated by OVTX-a followed by OVTX-b, OVTX-d and -e isomers, OVTX-c and isobaric PLTX.2

The Italian Ministry of Health solicited the introduction of surveys actions to manage risks associated to O. cf. ovata blooms, defining a three-phase monitoring plan: routine, alert and emergency phases, identified by thresholds of cells concentrations in the water. In the alert phase (cell density 10,000-30,000 cell/L) an assessment of the extension of the affected area and health surveillance are required, while the emergency phase (cell density >30,000 cells/L) represents the favoring conditions for aerosol and spray formation and chemical analysis of toxins in water is requested.3 As a result, new and timely early monitoring methods of alert had to be found. Solid Phase Adsorption Toxin Tracking technology represents a viable and sustainable method that can passively adsorb toxins produced by harmful microalgae and dissolved in water.4 This method offers several advantages over current monitoring techniques: it provides reliable, sensitive, and time-integrated sampling of various aquatic toxins and also has the potential to provide an early warning system for the presence of toxic microalgae and the possible bioaccumulation of these toxins in food.

1 P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, L. Tartaglione, C. Grillo, N. Melchiorre, J. Am. Soc. Spectrom., 2008, 19, 1, 111–120

2 L. Tartaglione, E. Dello Iacovo, A. Mazzeo, S. Casabianca, P. Ciminiello, A. Penna, C. Dell’Aversano, Environ. Sci. Technol., 2017, 51, 13920−13928

3 E. Funari, M. Manganelli, E. Testai, Harmful Algae, 50, 2015, 45–56

4 M. McCarthy, F. N.A.M. van Pelt, V. Bane, J. O'Halloran, A. Furey, (2014), Toxicon, 89, 77-86

  • Open access
  • 85 Reads
Idiosyncratic treatment of motor neuron disease: selective inhibition of voltage-gated sodium channels using spider venom peptide (ProTX-III) to treat motor neuron disease

Amyothropic lateral sclerosis (ALS) commonly known as Motor neuron disease is plaguing humanity for generations, impeding the basic vital functions. Both upper and lower, motor neurons facilitate voluntary and involuntary actions. Suppressing such action’s leads to muscle weakness, atrophy, paralysis and death. This however can be halted upon modulation of hyperexcitability by selectively inhibiting voltage-gated sodium channel subtypes 1.1, 1.2, 1.3, and 1.6 on motor neurons. ProTX-III is one such spider venom peptide which has shown to selectively modulate aforementioned subtypes and partially inhibit 1.4 (off target). This presentation will highlight the rationale behind choosing our drug targets, optimisation of ProTX-III and evaluating the efficacy of the drug. We hope this study will lead to novel drug candidates to treat ALS.

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