The excavation of novel toxin-resistance proteins against trichothecenes toxins in Paramyrothecium roridum.

Trichothecene toxins cause serious hazard towards human health and economical crops. However, there are no sufficient molecular strategies to reduce the hazard of trichothecene toxins. Thus it is urgent to exploit novel approaches to control the hazard of trichothecenes. In this study, four trichothecene toxin-resistance genes including mfs1, GNAT1, TRP1 and tri12 in Paramyrothecium roridum were excavated based on genome sequencing results, and then expressed in toxin-sensitive Saccharomyces cerevisiae BJ5464, the toxin resistance genes pdr5, pdr10 and pdr15 of which were firstly knocked out simultaneously by the introduction of TAA stop codon employing CRISPR/Cas9 system. Therefore, three novel hazardous toxin-resistance genes mfs1, GNAT1, TRP1 in P. roridum were firstly excavated by the co-incubation of DON toxin and toxin resistant genes-containing BJ5464 strains. The in vitro function and properties of novel toxin-resistance genes coding proteins including GNAT1, MFS1 and TRP1 were identified by heterologous expression and cellular location analysis as well as in vitro biochemical reaction. The excavation of novel trichothecene toxin-resistance genes provide novel molecular clues for controlling the harm of trichothecenes, meanwhile, this study will also pave a new way for the yield improvement of trichothecenes by heterologous expression to facilitate the development of trichothecenes as anti-tumor lead compounds.

Viperin, an IFN-Stimulated Protein, Delays Rotavirus Release by Inhibiting Non-Structural Protein 4 (NSP4)-Induced Intrinsic Apoptosis.

Viral infections lead to expeditious activation of the host's innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin's antiviral action in addition to the intricacy of viperin-virus interaction.

Report of the 1st International Electronic Conference on Toxins (IECT2021), 16-31 January 2021.

The 1st International Electronic Conference on Toxins (IECT2021) was successfully held online by https://sciforum [...].

Ubiquitous Selfish Toxin-Antidote Elements in Caenorhabditis Species.

Toxin-antidote elements (TAs) are selfish genetic dyads that spread in populations by selectively killing non-carriers. TAs are common in prokaryotes, but very few examples are known in animals. Here, we report the discovery of maternal-effect TAs in both C. tropicalis and C. briggsae, two distant relatives of C. elegans. In C. tropicalis, multiple TAs combine to cause a striking degree of intraspecific incompatibility: five elements reduce the fitness of >70% of the F2 hybrid progeny of two Caribbean isolates. We identified the genes underlying one of the novel TAs, slow-1/grow-1, and found that its toxin, slow-1, is homologous to nuclear hormone receptors. Remarkably, although previously known TAs act during embryonic development, maternal loading of slow-1 in oocytes specifically slows down larval development, delaying the onset of reproduction by several days. Finally, we found that balancing selection acting on linked, conflicting TAs hampers their ability to spread in populations, leading to more stable genetic incompatibilities. Our findings indicate that TAs are widespread in Caenorhabditis species and target a wide range of developmental processes and that antagonism between them may cause lasting incompatibilities in natural populations. We expect that similar phenomena exist in other animal species.

Snake antivenom: Challenges and alternate approaches.

Snake envenomation is still a serious threat to many countries in the world. The only mainstay treatment depends on the administration of animal derived immunoglobulin based antivenom. Significant limitations to these antivenoms are a challenge in the treatment of snake envenomation. Many alternate approaches have been explored to overcome the limitations of antivenom. Exploring alternate approaches like use of bioactive components from plant sources, use of peptide and small molecule inhibitors are some aspects taken towards improving the current limitations of antivenom therapy. However, all these alternate approaches also have many drawbacks which should be improved by more in vitro and in vivo experiments. Here, we review some of the limitations of current antivenom therapy and developments as well as drawbacks of these alternate treatment strategies.

Targeting Uremic Toxins to Prevent Peripheral Vascular Complications in Chronic Kidney Disease.

Chronic kidney disease (CKD) exhibits progressive kidney dysfunction and leads to disturbed homeostasis, including accumulation of uremic toxins, activated renin-angiotensin system, and increased oxidative stress and proinflammatory cytokines. Patients with CKD are prone to developing the peripheral vascular disease (PVD), leading to poorer outcomes than those without CKD. Cumulative evidence has showed that the synergy of uremic milieu and PVD could exaggerate vascular complications such as limb ischemia, amputation, stenosis, or thrombosis of a dialysis vascular access, and increase mortality risk. The role of uremic toxins in the pathogenesis of vascular dysfunction in CKD has been investigated. Moreover, growing evidence has shown the promising role of uremic toxins as a therapeutic target for PVD in CKD. This review focused on uremic toxins in the pathophysiology, in vitro and animal models, and current novel clinical approaches in reducing the uremic toxin to prevent peripheral vascular complications in CKD patients.

Review: Southern African medicinal plants used as blood purifiers.

ETHNOPHARMALOGICAL RELEVANCE: Blood purification practices, also referred to as blood cleansing or detoxification, is an ancient concept which is widespread amongst African traditional medicine, but for which no modern scientific basis exists. There prevails considerable ambiguity in defining what a blood purifier is. AIM OF THE STUDY: The purpose of this review is to firstly define what a blood purifier is in the context of African traditional medicine and compare to other cultural and westernized interpretations. Thereafter, this study identifies traditionally used medicinal plants used as blood purifiers in southern Africa and correlates these species to scientific studies, which may support evidence for these "blood purifying plant species". MATERIALS AND METHODS: Ethnobotanical books and review articles were used to identify medicinal plants used for blood purification. Databases such as Scopus, ScienceDirect, PubMed and Google Scholar were used to source scientific articles. An evaluation was made to try correlate traditional use to scientific value of the plant species. RESULTS: One hundred and fifty nine plant species have been documented as traditional remedies for blood purification. Most of the plant species have some pharmacological activity, however, very little link to the traditional use for blood purification. There has been some justification of the link between blood purification and the use as an antimicrobial and this has been explored in many of the plant species identified as blood purifiers. Other pharmacological studies specifically pertaining to the blood require further attention. CONCLUSION: Irrespective of the ambiguity of interpretation, medicinal plants used to "cleanse the blood", play an important holistic role in traditional medicine and this review with recommendations for further study provides some value of exploring this theme in the future.

In Silico Molecular Studies of Antiophidic Properties of the Amazonian Tree Cordia nodosa Lam.

We carried out surveys on the use of Cordia nodosa Lam. in the jungles of Bobonaza (Ecuador). We documented this knowledge to prevent its loss under the Framework of the Convention on Biological Diversity and the Nagoya Protocol. We conducted bibliographic research and identified quercetrin as a significant bioactive molecule. We studied its in silico biological activity. The selected methodology was virtual docking experiments with the proteins responsible for the venomous action of snakes. The molecular structures of quercetrin and 21 selected toxins underwent corresponding tests with SwissDock and Chimera software. The results point to support its antiophidic use. They show reasonable geometries and a binding free energy of -7 to -10.03 kcal/mol. The most favorable values were obtained for the venom of the Asian snake Naja atra (5Z2G, -10.03 kcal/mol). Good results were also obtained from the venom of the Latin American Bothrops pirajai (3CYL, -9.71 kcal/mol) and that of Ecuadorian Bothrops asper snakes (5TFV, -9.47 kcal/mol) and Bothrops atrox (5TS5, -9.49 kcal/mol). In the 5Z2G and 5TS5 L-amino acid oxidases, quercetrin binds in a pocket adjacent to the FAD cofactor, while in the myotoxic homologues of PLA2, 3CYL and 5TFV, it joins in the hydrophobic channel formed when oligomerizing, in the first one similar to α-tocopherol. This study presents a case demonstration of the potential of bioinformatic tools in the validation process of ethnobotanical phytopharmaceuticals and how in silico methods are becoming increasingly useful for sustainable drug discovery.

Structural basis for phospholipase A2-like toxin inhibition by the synthetic compound Varespladib (LY315920).

The World Health Organization recently listed snakebite envenoming as a Neglected Tropical Disease, proposing strategies to significantly reduce the global burden of this complex pathology by 2030. In this context, effective adjuvant treatments to complement conventional antivenom therapy based on inhibitory molecules for specific venom toxins have gained renewed interest. Varespladib (LY315920) is a synthetic molecule clinically tested to block inflammatory cascades of several diseases associated with elevated levels of secreted phospholipase A2 (sPLA2). Most recently, Varespladib was tested against several whole snake venoms and isolated PLA2 toxins, demonstrating potent inhibitory activity. Herein, we describe the first structural and functional study of the complex between Varespladib and a PLA2-like snake venom toxin (MjTX-II). In vitro and in vivo experiments showed this compound's capacity to inhibit the cytotoxic and myotoxic effects of MjTX-II from the medically important South American snake, Bothrops moojeni. Crystallographic and bioinformatics analyses revealed interactions of Varespladib with two specific regions of the toxin, suggesting inhibition occurs by physical blockage of its allosteric activation, preventing the alignment of its functional sites and, consequently, impairing its ability to disrupt membranes. Furthermore, based on the analysis of several crystallographic structures, a distinction between toxin activators and inhibitors is proposed.

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

Coevolution takes the sting out of it: Evolutionary biology and mechanisms of toxin resistance in animals.

Understanding how biotic interactions shape the genomes of the interacting species is a long-sought goal of evolutionary biology that has been hampered by the scarcity of tractable systems in which specific genomic features can be linked to complex phenotypes involved in interspecific interactions. In this review we present the compelling case of evolved resistance to the toxic challenge of venomous or poisonous animals as one such system. Animal venoms and poisons can be comprised of few or of many individual toxins. Here we show that resistance to animal toxins has evolved multiple times across metazoans, although it has been documented more often in phyla that feed on chemically-armed animals than in prey of venomous predators. We review three types of gene-product based resistance: 1) toxin scavenging, where molecules produced by the envenomed organism bind and inactivate the toxins; 2) target-site insensitivity, including landmark cases of convergent changes that make the molecules normally targeted by animal toxins refractory, and; 3) off-target repurposing, where envenomed organisms overcome toxicity by exploiting the function of toxins to alter their physiological effect. We finish by discussing the evolutionary processes that likely played a role in the origin and maintenance of toxin resistance. We conclude that antagonistic interactions involving poisonous or venomous animals are unparalleled models for investigating microevolutionary processes involved in coevolution and linking them to macroevolutionary patterns.

Efficient functional neutralization of lethal peptide toxins in vivo by oligonucleotides.

Medical means to save the life of human patients affected by drug abuse, envenomation or critical poisoning are currently limited. While the compounds at risks are most often well identified, particularly for bioterrorism, chemical intervention to counteract the toxic effects of the ingested/injected compound(s) is restricted to the use of antibodies. Herein, we illustrate that DNA aptamers, targeted to block the pharmacophore of a poisonous compound, represent a fast-acting and reliable method of neutralization in vivo that possesses efficient and long-lasting life-saving properties. For this proof of concept, we used one putative bioweapon, αC-conotoxin PrXA, a marine snail ultrafast-killing paralytic toxin, to identify peptide-binding DNA aptamers. We illustrate that they can efficiently neutralize the toxin-induced (i) displacement of [125I]-α-bungarotoxin binding onto nicotinic receptors, (ii) inhibition of diaphragm muscle contraction, and (iii) lethality in mice. Our results demonstrate the preclinical value of DNA aptamers as fast-acting, safe and cheap antidotes to lethal toxins at risk of misuse in bioterrorism and offer hope for an alternative method than donor sera to treat cases of envenomation.

Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach.

DM64 is a glycosylated protein with antivenom activity isolated from the serum of the opossum Didelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity column with immobilized DM64 as bait was used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 column were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Although venom fractions bound to the column were mainly composed of basic PLA2, a few spots corresponding to acidic PLA2 were also observed. Some unexpected protein spots were also identified: C-type lectins and CRISP may represent putative new targets for DM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies. BIOLOGICAL SIGNIFICANCE: Local tissue damage induced by myotoxic PLA2 remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problem largely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this important antivenom protein.

Vitamin D3 supplementation alleviates rotavirus infection in pigs and IPEC-J2 cells via regulating the autophagy signaling pathway.

Vitamin D had an anti-infection effect and benefited to the intestinal health. Autophagy signaling pathway was regulated by vitamin D3 to inhibit the infection of human immunodeficiency virus type-1. Rotavirus (RV) was a major cause of the severe diarrheal disease in young children and young animals. Although evidence suggested that vitamin D3 attenuates the negative effects of RV infection via the retinoic acid-inducible gene I signaling pathway, little is known of its antiviral effect whether through the regulation of autophagy. The present study was performed to investigate whether vitamin D3 alleviates RV infection in pig and porcine small intestinal epithelial cell line (IPEC-J2) models via regulating the autophagy signaling pathway. RV administration increased the Beclin 1 mRNA abundance in porcine jejunum and ileum. 5000 IU/kg dietary vitamin D3 supplementation greatly up-regulated LC3-II/LC3-I ratios and PR-39 mRNA expression under the condition of RV challenged. The viability of IPEC-J2 was significantly inhibited by RV infection. Incubation with 25-hydroxyvitamin D3 significantly decreased the concentrations of RV antigen and non-structural protein 4 (NSP4), and up-regulated the mRNA expression of Beclin 1 and PR-39 in the RV-infected IPEC-J2 cells. And then, based on the 25-hydroxyvitamin D3 treatment and RV infection, LC3-II mRNA expression in cells was inhibited by an autophagy inhibitor 3-methyladenine (3-MA). Bafilomycin A1 (Baf A1, a class of inhibitors of membrane ATPases, inhibits maturation of autophagic vacuoles) treatment numerically enhanced the LC3-II mRNA abundance, but had no effect on NSP4 concentration. Furthermore, 25-hydroxyvitamin D3 decreased the p62 mRNA expression and increased porcine cathelicidins (PMAP23, PG1-5 and PR-39) mRNA expression in the RV-infected cells. Taken together, these results indicated that vitamin D3 attenuates RV infection through regulating autophagic maturation and porcine cathelicidin genes expression.

Studies on Unprocessed and Acid-Treated Arabinogalactan from Larch as an Inhibitor of Glycan Binding of a Plant Toxin and Biomedically Relevant Human Lectins.

The increasing evidence for the physiological significance of glycan-protein (lectin) interactions prompts considerations for respective bioactivity of plant polysaccharides. Arabinogalactan from larch, a polysaccharide with a ß1,3-linked galactose core and branches at the 6'-hydroxyl, was thus tested, together with two processed forms treated either with oxalic or trifluoroacetic acid. Hydrolysis by acid reduced the arabinose contents without backbone degradation. The three preparations were tested as an inhibitor of lectin binding in solid-phase and cell-based assays, using the toxin from Viscum album and a panel of seven human lectins (six galectins and a C-type lectin). Increasing potency correlating with the molecular contents of galactose was seen for the plant toxin. In general, relatively weak or no inhibitory capacity was detected for the three preparations, when binding of the human galectins and avian orthologues used as controls was measured. Acid-treated polysaccharides also weakly interfered with binding of the galactose-specific C-type lectin of human macrophages. Larch arabinogalactan, tested as a model, will thus most likely not impair (ga)lectin functionality physiologically.

Predicting the Uncertain Future of Aptamer-Based Diagnostics and Therapeutics.

Despite the great promise of nucleic acid aptamers in the areas of diagnostics and therapeutics for their facile in vitro development, lack of immunogenicity and other desirable properties, few truly successful aptamer-based products exist in the clinical or other markets. Core reasons for these commercial deficiencies probably stem from industrial commitment to antibodies including a huge financial investment in humanized monoclonal antibodies and a general ignorance about aptamers and their performance among the research and development community. Given the early failures of some strong commercial efforts to gain government approval and bring aptamer-based products to market, it may seem that aptamers are doomed to take a backseat to antibodies forever. However, the key advantages of aptamers over antibodies coupled with niche market needs that only aptamers can fill and more recent published data still point to a bright commercial future for aptamers in areas such as infectious disease and cancer diagnostics and therapeutics. As more researchers and entrepreneurs become familiar with aptamers, it seems inevitable that aptamers will at least be considered for expanded roles in diagnostics and therapeutics. This review also examines new aptamer modifications and attempts to predict new aptamer applications that could revolutionize biomedical technology in the future and lead to marketed products.

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

Conditional Activation of Toxin-Antitoxin Systems: Postsegregational Killing and Beyond.

Toxin-antitoxin (TA) systems are small genetic modules formed by a stable toxin and an unstable antitoxin that are widely present in plasmids and in chromosomes of Bacteria and Archaea. Toxins can interfere with cell growth or viability, targeting a variety of key processes. Antitoxin inhibits expression of the toxin, interacts with it, and neutralizes its effect. In a plasmid context, toxins are kept silent by the continuous synthesis of the unstable antitoxins; in plasmid-free cells (segregants), toxins can be activated owing to the faster decay of the antitoxin, and this results in the elimination of these cells from the population (postsegregational killing [PSK]) and in an increase of plasmid-containing cells in a growing culture. Chromosomal TA systems can also be activated in particular circumstances, and the interference with cell growth and viability that ensues contributes in different ways to the physiology of the cell. In this article, we review the conditional activation of TAs in selected plasmidic and chromosomal TA pairs and the implications of this activation. On the whole, the analysis underscores TA interactions involved in PSK and points to the effective contribution of TA systems to the physiology of the cell.

Novel class of potential therapeutics that target ricin retrograde translocation.

Ricin toxin, an A-B toxin from Ricinus communis, induces cell death through the inhibition of protein synthesis. The toxin binds to the cell surface via its B chain (RTB) followed by its retrograde trafficking through intracellular compartments to the ER where the A chain (RTA) is transported across the membrane and into the cytosol. Ricin A chain is transported across the ER membrane utilizing cellular proteins involved in the disposal of aberrant ER proteins by a process referred to as retrograde translocation. Given the current lack of therapeutics against ricin intoxication, we developed a high-content screen using an enzymatically attenuated RTA chimera engineered with a carboxy-terminal enhanced green fluorescent protein (RTA(E177Q)egfp) to identify compounds that target RTA retrograde translocation. Stabilizing RTA(E177Q)egfp through the inclusion of proteasome inhibitor produced fluorescent peri-nuclear granules. Quantitative analysis of the fluorescent granules provided the basis to discover compounds from a small chemical library (2080 compounds) with known bioactive properties. Strikingly, the screen found compounds that stabilized RTA molecules within the cell and several compounds limited the ability of wild type RTA to suppress protein synthesis. Collectively, a robust high-content screen was developed to discover novel compounds that stabilize intracellular ricin and limit ricin intoxication.