Toxin Fraction Research Articles

Two Novel Peptides from Buthotus saulcyi Scorpion Venom: Proteomic Analysis and Approaches

Background and Objectives: Venomous scorpions play a crucial role in medicine and public health. Buthotus saulcyi scorpion is known as one of the most populous species in East Asia and Iran, while its venom proteome has still not been fully determined. background: In the present study, we report for the first time a proteomic profile of Buthotus saulcyi scorpion venom with the aim of looking ahead and determining the structural and functional characteristics of these compounds for use as medical tools. Aims: In the current research, the proteomic profile of Buthotus saulcyi scorpion venom to determine the structural and functional characteristics of its compounds used for treatment will be examined for the first time. objective: In the present study, we report for the first time a proteomic profile of Buthotus saulcyi scorpion venom with the aim of looking ahead and determining the structural and functional characteristics of these compounds for use as medical tools. Method:: 2D-PAGE, HPLC, SDS-PAGE, sequencing, and MALDI-TOF MS techniques were used to investigate the properties of these peptides. Result: The 2D-PAGE analysis of crude toxin from B. saulcyi revealed a minimum of 96 protein spots, with isoelectric points ranging from 4 to 9 and molecular weights spanning from 3.6 to 205 kDa. Following this, HPLC was used to isolate 14 fractions of crude toxins, and the protein content of these fractions was measured. SDS-PAGE analysis identified 7 protein bands within the B. saulcyi crude toxin fractions, with molecular weights ranging from 13 to 217 kDa. Further examination of fraction 7 through amino acid sequencing resulted in the identification of two protein bands labeled peptide 3 and peptide 4. Ultimately, these protein bands were extracted, and their molecular mass and amino acid sequences were analyzed using MALDI-TOF MS. result: 2D-PAGE data from the B. saulcyi crude toxin showed at least 96 protein spots in isoelectric point between 4 and 9 and a molecular weight between 3.6 and 205 kDa. Then 14 fractions of crude toxins were isolated using HPLC and their protein content was estimated. SDS-PAGE analysis of fractions of B. saulcyi crude toxin showed 7 protein bands with a molecular weight between 13 and 217 kDa. Subsequently, the amino acid sequencing of Fraction 7 led us to two protein bands designated as Peptide 3 and Peptide 4 peptides. Finally, these protein bands were removed, and molecular mass and amino acid sequence analysis was performed using MALDI-TOF MS. Conclusion:: According to our results, the alignment of P3 and P4 protein sequences revealed the highest similarity to chrysophsin 2 and pheromone-bound protein 2, respectively.

Evaluation of cellular immune response in rabbits after exposure to cobra venom and purified toxin fraction

Snakebite by a cobra is considered neurotoxic as the cause of neuromuscular paralysis mediated by low molecular weight toxins, which are major toxin components of cobra. However, these toxins represent a problem in generating antibodies owing to their low immunogenicity. Developing complementary strategies to improve the antibody response could be a useful approach to creating better therapeutic antivenoms with higher neutralizing potencies. To develop simple immunization strategies for more potent antivenoms by studying the effects of combining crude cobra venom and toxin fraction in a complementary way. The evaluation of specific cell immunology and cytokine mediators for relevant immune responses will be measured in a rabbit model using four simple immunization strategies. Flow cytometry will be used to quantify the number of B and T cells, and qRT-PCR will be used to ascertain the cytokine genes expressed. B cells with anti-CD20 were seen on D14, and a booster dose was insufficient to maximize the antibodies. Conversely, anti-CD5 for T cells decreased periodically but remained stable. Using a mixture of crude cobra venom and its <10 kDa fraction, peak expression of pro-inflammatory cytokine genes was seen in D42 or D58, with a rise of 4 and 6 folds. Similarly, gene expression of pro-inflammatory cytokines was greater than that of anti-inflammatory cytokines (IL-4 and IL-10), which were up-regulated after D42. Thus, immunization with both the crude and its <10 kDa fraction of cobra venom seems to have synergistic effects that boost cytokines, activate the immune system, and cause lymphocyte differentiation.

Proteomic analysis of two novel peptides from the Odontobuthus doriae scorpion venom

The venom of the Odontobuthus doriae scorpion, prevalent in East Asia and Iran, has not been fully characterized. This study provides the first proteomic profile of O. doriae venom to explore its potential as a medical. 2D-PAGE analysis revealed 96 protein spots with isoelectric points from 3 to 9 and molecular weights between 6.6 to 205 kDa. Fourteen toxin fractions were isolated via HPLC, and SDS-PAGE showed seven protein bands ranging from 3.8 to 182 kDa. MALDI-TOF MS identified Peptide 1 and Peptide 2, resembling Hemoglobin beta-2 chain and Chaperonin HSP60 and suggest potential therapeutic applications for P1 and P2.

Exploring the effects of three-finger toxins from Naja ashei venom on neuronal and immunological cancer cell membranes

Three-finger proteins are the most abundant toxins in the venom of Naja ashei, a snake species from the Elapidae family. This research aimed to describe the effects of varying charges of these proteins, isolated from Naja ashei venom using SEC and IEX chromatography. The study examined how differently charged three-finger toxin fractions interact with and affect neuroblastoma (SK-N-SH) and promyeloblast (HL-60) cells, as well as model Langmuir membranes and liposomes designed to mimic cellular lipid composition. Findings revealed that protein surface charges significantly impact cell survival (MTT assay), membrane damage (lactate dehydrogenase release, malondialdehyde formation), and the structural and electrochemical properties of model membranes (Langmuir membranes and zeta potential for liposomes and cancer cell lines). Results indicated that SK-N-SH cells, characterized by a higher negative charge on their cell membranes, interacted more effectively with positively charged toxins than HL-60 cells. However, the mechanism of these electrostatic interactions is complex. The research demonstrated that electrostatic and mechanical membrane modifications induced by venom proteins can significantly affect cell metabolism. Additionally, the total charge of the membrane, influenced by polar lipid components and phospholipid saturation, plays a decisive role in toxin interaction.

Toxicogenomic Effects of Dissolved Saxitoxin on the Early Life Stages of the Longfin Yellowtail (Seriola rivoliana).

Harmful algal blooms (HABs) can produce a variety of noxious effects and, in some cases, the massive mortality of wild and farmed marine organisms. Some HAB species produce toxins that are released into seawater or transferred via food webs (particulate toxin fraction). The objective of the present study was to identify the toxicological effects of subacute exposure to saxitoxin (STX) during embryonic and early larval stages in Seriola rivoliana. Eggs were exposed to dissolved 19 STX (100 μg L-1). The toxic effects of STX were evaluated via the hatching percentage, the activity of three enzymes (protein and alkaline phosphatases and peroxidase), and the expression of four genes (HSF2, Nav1.4b, PPRC1, and DUSP8). A low hatching percentage (less than 5%) was observed in 44 hpf (hours post fertilization) embryos exposed to STX compared to 71% in the unexposed control. At this STX concentration, no oxidative stress in the embryos was evident. However, STX induced the expression of the NaV1.4 channel α-subunit (NaV1.4b), which is the primary target of this toxin. Our results revealed the overexpression of all four candidate genes in STX-intoxicated lecithotrophic larvae, reflecting the activation of diverse cellular processes involved in stress responses (HSF2), lipid metabolism (PPRC1), and MAP kinase signaling pathways associated with cell proliferation and differentiation (DUSP8). The effects of STX were more pronounced in young larvae than in embryos, indicating a stage-specific sensitivity to the toxin.

Bothrops atrox venom: Biochemical properties and cellular phenotypes of three highly toxic classes of toxins

Snake venoms have a complex mixture of compounds that are conserved across species and act synergistically, triggering severe local and systemic effects. Identification of the toxin classes that are most damaging to cell homeostasis would be a powerful approach to focus on the main activities that underpin envenomation. Here, we focus on the venom of Bothrops atrox, snake responsible for most of the accidents in Amazon region of South America. We identified the key cytotoxic toxin fractions from B. atrox venom and mapped their biochemical properties, protein composition and cell damage. Five fractions were obtained by mass exclusion chromatography and contained either a single class of enzymatic activity (i.e., L-amino acid oxidases or Hyaluronidases) or different activities co-distributed in two or more protein fractions (e.g., Metalloproteinases, Serine Proteases, or Phospholipases A2). Only three protein fractions reduced cell viability of primary human cells. Strikingly, such activity is accompanied by disruption of cell attachment to substratum and to neighbouring cells. Such strong perturbation of morphological cell features indicates likely defects in tissue integrity in vivo. Mass spectrometry identified the main classes of toxins that contribute to these phenotypes. We provide here a strategy for the selection of key cytotoxic proteins for targeted investigation of their mechanism of action and potential synergism during snakebite envenomation. Our data highlights putative toxins (or combinations of) that may be the focus of future therapeutic interference.

PREDICTIVE VALUE OF PROTEIN-BOUND UREMIC TOXINS FOR HEART FAILURE EVENTS IN PATIENTS WITH CHRONIC KIDNEY DISEASE

Objective: Evaluate whether the plasma free fraction of four important protein-bound uremic toxins (PBUTs), indoxyl sulfate (IxS), p-cresyl sulfate (pCS), p-cresyl glucuronide (pCG) and hippuric acid (HA), are independently associated with new heart failure (HF) events in patients with chronic kidney disease (CKD), not on dialysis. Design and method: Retrospective, single center analysis. 526 patients with CKD stages G1-G5 (not on dialysis), were included between January 2011 and January 2014. Outcome parameters were monitored until June 2017. Exclusion criteria were pregnancy, age <18 years, active infection, active malignancy, history of transplantation or a cardiovascular event within the past three months. Free fractions of uremic toxins were quantified by ultrahigh-performance liquid chromatography. Kaplan-Meier survival curves were constructed to investigate the univariate association between PBUTs and the composite endpoint of hospitalization or death due to HF. Multivariate Cox regression models were used to study the independent association of PBUTs with outcome. Results: The study population (57.8% male) had a median age of 66 years and varying degrees of CKD: stage G1 (12.2%), 2 (16.3%), 3A (21.1%), 3B (27.8%), 4 (19.2%) and 5 (3.4%). After a median follow up of 5.4 years 43 patients (8.4%) developed the primary endpoint, 18.6% (8/43) were fatal. In Cox regression analysis with adjustment for age, gender, body mass index, diabetes and systolic blood pressure, the free fractions of all four PBUTs remained independent predictors of new heart failure events [IxS: HR 1.46 (95% CI: 1.06 – 2.01; P = 0.019); pCS: HR 1.75 (95% CI: 1.21 - 2.54; P = 0.003); pCG: HR 1.65 (95% CI: 1.17 - 2.32; P = 0.004); HA: HR 1.43 (95% CI: 1.05 - 1.95; P = 0.022)]. Univariate survival analyses are shown in Figure 1. Conclusions: Free fraction of the PBUTs indoxyl sulfate, p-cresyl sulfate, p-cresyl glucuronide and hippuric acid were independently associated with hospitalization and mortality due to HF in patients with CKD. The pathophysiological pathways by which these toxins may contribute to heart failure remain to be elucidated.

Snake Venomics and Antivenomics of Cape Cobra (Naja nivea) from South Africa: Insights into Venom Toxicity and Cross-Neutralization Activity.

Naja nivea (Cape Cobra) is endemic to southern Africa. Envenoming by N. nivea is neurotoxic, resulting in fatal paralysis. Its venom composition, however, has not been studied in depth, and specific antivenoms against it remain limited in supply. Applying a protein decomplexation approach, this study unveiled the venom proteome of N. nivea from South Africa. The major components in the venom are cytotoxins/cardiotoxins (~75.6% of total venom proteins) and alpha-neurotoxins (~7.4%), which belong to the three-finger toxin family. Intriguingly, phospholipase A2 (PLA2) was undetected-this is a unique venom phenotype increasingly recognized in the African cobras of the Uraeus subgenus. The work further showed that VINS African Polyvalent Antivenom (VAPAV) exhibited cross-reactivity toward the venom and immunorecognized its toxin fractions. In mice, VAPAV was moderately efficacious in cross-neutralizing the venom lethality with a potency of 0.51 mg/mL (amount of venom completely neutralized per milliliter of antivenom). In the challenge-rescue model, VAPAV prevented death in 75% of experimentally envenomed mice, with slow recovery from neurotoxicity up to 24 h. The finding suggests the potential para-specific utility of VAPAV for N. nivea envenoming, although a higher dose or repeated administration of the antivenom may be required to fully reverse the neurotoxic effect of the venom.

Paralytic shellfish poisoning toxins in butter clams (Saxidomus gigantea) from the Kodiak Archipelago, Alaska.

Consumption of toxic butter clams (Saxidomus gigantea) is the most frequent cause of paralytic shellfish poisoning (PSP) in Alaskan coastal communities. This study examines seasonal variation in total paralytic shellfish toxin concentrations and congener distribution in tissues of butter clams collected in three communities in the Kodiak Islands, Alaska: the City of Kodiak, Ouzinkie and Old Harbor. In response to questions from local harvesters, the efficacy of removing particular clam tissues on total toxin levels was also assessed. Butter clam samples were collected ∼monthly during 2015–2020 in each community to monitor shellfish toxin levels. Results were combined with clam monitoring data collected previously (2013–2015) to document the seasonal distribution of saxitoxin (STX) and its congeners (neosaxitoxin, gonyautoxin) in clam tissues. Seasonally, paralytic shellfish toxin levels in butter clams were highest in summer, declined in winter, but often remained above regulatory limits throughout the year in the three Kodiak communities. Butter clams collected from Ouzinkie (2013–2020) averaged 165 ± 87 µg STX equivalents (Eq.) 100 g − 1, compared to Kodiak 73 ± 54 µg STX Eq. 100 g − 1 and Old Harbor 143 ± 103 µg STX Eq. 100 g − 1. STX accounted for 59–71% of the total toxin concentration in clams at Ouzinkie, Kodiak, and Old Harbor, while neosaxitoxin (neoSTX) accounted for 12–18%. Gonyautoxins (GTXs) represented 31–60% of the total toxin concentration during the seasonal Alexandrium catenella bloom in June-July, with lower percentages in other months. The fraction of total toxin varied among clam tissues: the siphon tip (2–29%), the neck (3–56%), the gut (3–65%) and the body (6–85%). Removal of the siphon tip reduced total toxin content substantially in some samples but had little effect in others. Saxitoxin congeners varied greatly and somewhat unpredictably among clam tissues, and the results indicate removal of specific tissues was not an effective strategy for reducing paralytic shellfish toxin levels in butter clams for safe consumption.

Production of a Plasma Derived Universal Antivenom against All Elapid Neurotoxic Snake Venoms

Snakebite envenoming has killed about 138000 people and maimed 400,000 victims annually. WHO has designated this medical problem as one of the most neglected tropical diseases for which effective, affordable antivenoms (AVs) are urgently needed. Production of potent AV against neurotoxic venoms was difficult and was thought to be due to the low immunogenicity of the postsynaptic neurotoxins (PSNT) which cause death in the victims. However, it was showed that the use of ineffective adjuvant in the immunization of horse was the root cause. The highly effective Freund adjuvant (FA) causes severe local reactions and could not be used. A novel immunization protocol termed ‘low dose low volume multi-site’ was tested and shown to obliterate the local side effect and allow for the safe use of FA in horse. The immunization protocol led to the production of 7 highly potent monovalent AVs, and 2 potent polyvalent AVs, one against 4 neurotoxic venoms and another against 3 hematotoxic venoms. These AVs allow the treatment of snakebite victims without the need to identify the culprit snakes. Furthermore, we have tested a novel immunization strategy using ‘Diverse toxin repertoire’ of 12 Asian elapid toxin fractions. The resulting antiserum effectively neutralized at least 36 elapid venoms of 28 species encompassing 10 genera and from 20 countries on 4 continents, and most likely all the elapid neurotoxic snake venoms. These results indicate that effective universal antivenom against all elapid neurotoxic venoms of the world can be produced and save numerous lives.

Experimental study on the effect of toxin fractions isolated from hydatid cyst fluid of sheep on the cardiac muscles of mice

The aim of this study was to investigate the effectiveness of hydatid cyst toxin fractions in mice. Fifty male mice were divided into five groups with 10 mice for each group the first group as control the second group was injected with toxin fractions at the concentrations 25Mg/ml PBS, the third group are the mice that injected intraperitoneally with toxin fractions at the concentrations 25Mg/ml PBS and treated with vitamin E at the concentration of 40 mg/ 100 g of feed, the fourth group was injected intraperitoneally with 1 ml of raw fluid and the fifth group was injected intraperitoneally of 1ml of row fluid with the vitamin E at the concentration of 40 mg /100gm feed. The mice were sacrificed after 15 and 30days post injection, specimen of heart are fixed in 10% neutral buffered formalin for histological techniques. The histopathological changes in cardiomyocyte were edema, infiltration of mononuclear cell and thickening of blood vessels wall with congestion in it. The results confirm that the toxin fraction have more effect than the raw fluid and that there is the regeneration effect of vitamin E on hydatid cyst cardiomyocyte.

Proteomics and neutralization of Bungarus multicinctus (Many-banded Krait) venom: Intra-specific comparisons between specimens from China and Taiwan

The Many-banded Krait (Bungarus multicinctus) is a medically important venomous snake in East Asia. This study investigated the venom proteomes of B. multicinctus from Guangdong, southern China (BM-China) and insular Taiwan (BM-Taiwan), and the neutralization activities of two antivenom products (produced separately in China and Taiwan) against the lethal effect of the venoms. The venom proteomes of both specimens contained similar toxin families, notwithstanding small variations in the subtypes and abundances of minor components. More than 90% of the total venom proteins belong to three-finger toxins (3FTx, including alpha-neurotoxins) and phospholipases A2 (PLA2, including beta-bungarotoxins), supporting their key involvement in the pathophysiology of krait envenomation which manifests as pre- and post-synaptic neurotoxicity. The venoms exhibited potent neurotoxic and lethal effects with extremely low i.v. LD50 of 0.027 μg/g (Bm-China) and 0.087 μg/g (Bm-Taiwan), respectively, in mice. Bungarus multicinctus monovalent antivenom (BMMAV) produced in China and Neuro bivalent antivenom (NBAV) produced in Taiwan were immunoreactive toward both venoms and their toxin fractions. The antivenoms neutralized the venom lethality variably, with BMMAV being more efficacious than NBAV by approximately two-fold. Findings suggest that the monovalent antivenom has a higher potency presumably due to its species-specificity toward the krait venom.

A Quest for a Universal Plasma-Derived Antivenom Against All Elapid Neurotoxic Snake Venoms.

This review describes the research aimed at the development of universal antivenom against elapid neurotoxic snake venoms. The antivenoms produced in Thailand in the 1980s were of low potency, especially against the elapid venoms. This was thought to be due to the low immunogenicity of the α-neurotoxins, which are the most lethal toxins in these venoms. Comparisons of various α-neurotoxin conjugates and polymers, and also different immunological adjuvants, showed that the adjuvant used is the major determinant in the antibody response in horses. The potent Freund’s adjuvant was not used due to its severe local side-effect in horses. Therefore, a novel immunization protocol termed ‘low dose, low volume multi-site’ was developed for use in horses. This immunization protocol has led to the production of highly potent monospecific antivenoms against several elapid and viperid venoms, and two potent polyspecific antivenoms, one against 4 neurotoxic and another against 3 hematotoxic venoms. The immunization protocol has also led to other improvements in antivenom production including: several fold increases in antiserum potency, a reduction in the time required to reach therapeutically useful antibody titers, a 90% reduction in the amount of venom used, and 100% of the horses responding to the immunization program. This development is partly responsible for significant decrease in the Thailand’s annual snakebite death toll from a few dozens to mostly nil in recent years. Finally, a simple and novel immunization strategy, using a ‘diverse toxin repertoire’ composed of numerous elapid toxin fractions as immunogen, was proposed and tested. This immunization procedure has resulted in the successful production of a widely paraspecific antiserum against at least 36 neurotoxic venoms of 28 species encompassing 10 genera and from 20 countries on four continents, and possibly against all elapid venoms with α-neurotoxins as the lethal toxins. These results indicate that, with optimizations of the composition of the ‘diverse toxin repertoire’, the immunization scheme and antibody fractionation to increase the antivenom neutralizing potency, an effective universal antivenom against the neurotoxic elapid snakes of the world can be produced.

A pan-specific antiserum produced by a novel immunization strategy shows a high spectrum of neutralization against neurotoxic snake venoms

Snakebite envenomation is a neglected tropical disease of high mortality and morbidity largely due to insufficient supply of effective and affordable antivenoms. Snake antivenoms are mostly effective against the venoms used in their production. It is thus crucial that effective and affordable antivenom(s) with wide para-specificity, capable of neutralizing the venoms of a large number of snakes, be produced. Here we studied the pan-specific antiserum prepared previously by a novel immunization strategy involving the exposure of horses to a ‘diverse toxin repertoire’ consisting of 12 neurotoxic Asian snake toxin fractions/ venoms from six species. This antiserum was previously shown to exhibit wide para-specificity by neutralizing 11 homologous and 16 heterologous venoms from Asia and Africa. We now show that the antiserum can neutralize 9 out of 10 additional neurotoxic venoms. Altogether, 36 snake venoms belonging to 10 genera from 4 continents were neutralized by the antiserum. Toxin profiles previously generated using proteomic techniques of these 36 venoms identified α-neurotoxins, β-neurotoxins, and cytotoxins as predominant toxins presumably neutralized by the antiserum. The bases for the wide para-specificity of the antiserum are discussed. These findings indicate that it is feasible to generate antivenoms of wide para-specificity against elapid neurotoxic venoms from different regions in the world and raises the possibility of a universal neurotoxic antivenom. This should reduce the mortality resulting from neurotoxic snakebite envenomation.

Innovations in dialysis membranes for improved kidney replacement therapy.

Haemodialysis is a life-saving therapy. However, in comparison with the healthy kidney, it removes only a small fraction of the uraemic toxins produced, does not function continuously and cannot replicate biological kidney functions. Innovations in membrane design hold promise to overcome these limitations with potential to improve patient outcomes.

High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches.

Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates. After fraction collection, any solvent present in the wells was removed by means of freeze-drying, after which it was possible to perform a plasma coagulation assay in order to detect coagulopathic activity. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors.

Identification of a native Bacillus thuringiensis strain from Sri Lanka active against Dipel-resistant Plutella xylostella.

BackgroundBiopesticides based on strains of the bacterium Bacillus thuringiensis (Bt) are used globally for effective and environmentally friendly pest control. The most serious threat to the sustainable use of these microbial pesticides is the development of resistance on targeted pests. Populations of Plutella xylostella (diamondback moth) have evolved field resistance to Bt pesticides at diverse locations worldwide. Discovery of novel Bt strains with varied toxin profiles that overcome resistance is one of the strategies to increase sustainability of Bt pesticides against P. xylostella. In this study, we report isolation and characterization of a Bt strain named AB1 from Sri Lanka displaying toxicity towards larvae of P. xylostella resistant to the commercial Bt pesticide Dipel.MethodsStrains of Bt from diverse environments in Sri Lanka were evaluated for protein crystal production through Differential Interference Contrast (DIC) microscopic examination, and for insecticidal activity against P. xylostella in bioassays. The genome of the AB1 strain was sequenced by Hiseq Illumina sequencing to identify the insecticidal genes present in the genome and nano liquid chromatography followed by tandem mass spectrometry (nanoLC/MS/MS) of purified crystal proteins of AB1 was performed to identify the expressed insecticidal proteins. Multilocus sequence typing and Gyrase B gene sequence analyses were performed to identify the phylogenetic origin of the AB1 strain.ResultsThe AB1 strain was identified as producing high levels of bipyramidal crystals and displaying insecticidal activity against susceptible and Dipel-resistant strains of P. xylostella. Multilocus sequence typing and phylogenetic analysis of the Gyrase B gene identified that AB1 belongs to the B. thuringiensis subsp. aizawai serotype. Comparative analysis of genomic and proteomic data showed that among the insecticidal protein coding genes annotated from the AB1 genome (cry1Aa, cry1Ca, cry1Da, cry1Ia, cry2Ab and cry9), Cry1Ca and Cry1Da toxins represented most of the toxin fraction in parasporal crystals from AB1. Overall findings warrant further development of B. thuringiensis subsp. aizawai AB1 strain as a pesticide to control P. xylostella.

Characterization of Functional Effects of Two New Active Fractions Isolated From Scorpion Venom on Neuronal Ca2+ Spikes: A Possible Action on Ca2+-Dependent Dependent K+ Channels

Introduction:It is a long time that natural toxin research is conducted to unlock the medical potential of toxins. Although venoms-toxins cause pathophysiological conditions, they may be effective to treat several diseases. Since toxins including scorpion toxins target voltage-gated ion channels, they may have profound effects on excitable cells. Therefore, elucidating the cellular and electrophysiological impacts of toxins, particularly scorpion toxins would be helpful in future drug development opportunities.Methods:Intracellular recording was made from F1 cells of Helix aspersa in the presence of calcium Ringer solution in which Na+ and K+ channels were blocked. Then, the modulation of channel function in the presence of extracellular application of F4 and F6 toxins and kaliotoxin (KTX; 50 nM and 1 μM) was examined by assessing the electrophysiological characteristics of calcium spikes.Results:The two active toxin fractions, similar to KTX, a known Ca2+-activated K+ channel blocker, reduced the amplitude of AHP, enhanced the firing frequency of calcium spikes and broadened the duration of Ca2+ spikes. Therefore, it might be inferred that these two new fractions induce neuronal hyperexcitability possibly, in part, by blocking calcium-activated potassium channel current. However, this supposition requires further investigation using voltage clamping technique.Conclusion:These toxin fractions may act as blocker of calcium-activated potassium channels.

Role of temperature and nutrients on the growth and toxin production of Prorocentrum hoffmannianum (Dinophyceae) from the Florida Keys

The benthic dinoflagellate Prorocentrum hoffmannianum M.A. Faust is typical of tropical warm waters and produces biotoxins responsible for diarrhetic shellfish poisoning (DSP). In this study, the effect of temperature and nutrient limitation on growth and toxin production of P. hoffmannianum isolated from field samples collected in the Florida Keys was investigated.Batch culture experiments were ran at two temperatures (i.e. 21 ± 0.1 and 27 ± 0.1 °C) and under nitrogen-limited (14.7 μmol L−1 N-NO3- and 18.1 μmol L−1 P-PO43-) and phosphorus-limited (441 μmol L−1 N-NO3- and 0.6 μmol L−1 P-PO43-) levels with respect to control nutrient conditions (441 μmol L−1 N-NO3-and 18.1 μmol L−1 P-PO43-).Both temperature and nutrient conditions significantly affected growth rates and maximum yield of P. hoffmannianum with the maximum values being recorded at the higher temperature and in the replete medium. Production of okadaic acid was induced under all conditions (from 13.5 to 859.8 pg cell−1), with values up to one order of magnitude higher than those observed in other DSP toxin producing species.Toxin production was enhanced under P limitation at 27 °C, corroborating the theory that toxin production is modulated by cell physiological conditions, which are in turn affected by a wide spectrum of factors, including environmental stressors such as nutrient availability. Toxin fraction released in the growth medium was negligible. No okadaic acid esters were detected in this strain of P. hoffmannianum.

Characterization of Functional Effects of Two New Active Fractions Isolated From Scorpion Venom on Neuronal Ca2+ Spikes: A Possible Action on Ca2+-Dependent Dependent K+ Channels.

Introduction:Based on the literature, unihemispheric concurrent dual-site anodal transcranial Direct Current Stimulation (a-tDCSUHCDS) of primary Motor cortex (M1) and Dorsolateral Prefrontal Cortex (DLPFC) would be more efficient than conventional a-tDCS of M1 to induce larger and longer-lasting M1 corticospinal excitability. The main objective of the present study was to compare the effects of a-tDCSUHCDS and conventional M1 a-tDCS on the extent and durability of the motor sequence acquisition in healthy individuals.Methods:In this randomized sham-controlled study, healthy volunteers were randomly divided into three groups: experimental (a-tDCSUHCDS), control (M1 a-tDCS), and sham stimulation groups. The participants practiced serial response time task over three consecutive days when they simultaneously received a-tDCS. Using the skill measure, we assessed motor learning up to 4 weeks after the completion of experimental conditions.Results:Data analysis revealed that all groups exhibited the improved trend over the training course (P<0.001). There were no significant differences in skill acquisition among groups at post-intervention (P>0.05), while a significant improvement was observed between experimental and sham group at the retention time (P<0.05). Moreover, there were no significant differences between the control and two other groups with regard to the retention time (P>0.05).Conclusion:These results revealed a significant increase in the skill acquisition by a-tDCSUHCDS technique with regard to retention issue, which could be a valuable finding in neuro-rehabilitation field.