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Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na + ion channels of type beta-toxin (β-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom.

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na + channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (β-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide β-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

The first toxic component identified against mammals in the venom from Centruroides tecomanus scorpion from Colima, Mexico was Ct1a toxin, which was neutralized by human single chain variable fragment (scFv) RAS27. Venom characterization from these scorpions collected on the Pacific coast of Colima, enabled the identification of a second component of medical importance named Ct71 toxin. Amino acid sequence of Ct71 shares a high identity with Chui5 toxin from C. huichol scorpion, which was neutralized by scFv HV. For this reason, the kinetic parameters of interaction between Ct71 toxin and scFv HV were determined by surface plasmon resonance. Results showed a significantly higher affinity for Ct71 as compared to Chui5. As expected, this toxin was neutralized by scFv HV. The injection of a mixture of scFvs HV and RAS27, resulted in the neutralization of C. tecomanus venom, corroborating that human recombinant antibody fragments can efficiently contribute to the neutralization of medically important toxins and their respective venoms from Mexican scorpions., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Scorpions are a group of arthropods that strike fear in many people due to their severe medical symptoms, even death, caused by their venomous stings. Even so, not all scorpion species contain harmful venoms against humans but still have valuable bioactive molecules, which could be used in developing new pharmaceutical leads for treating important diseases. This work conducted a comprehensive analysis of the venom from the scorpion Thorellius intrepidus. The venom of T. intrepidus was separated by size exclusion chromatography, and four main fractions were obtained. Fraction IV (FIV) contained small molecules representing over 90% of the total absorbance at 280 nm. Analysis of fraction FIV by RP-HPLC indicated the presence of three main molecules (FIV.1, FIV.2, and FIV.3) with similar UV absorbance spectra profiles. The molecular masses of FIV.1, FIV.2, and FIV.3 were determined, resulting in 175.99, 190.07, and 218.16 Da, respectively. Further confirmation through 1 H-NMR and 13 C-NMR analyses revealed that these molecules were serotonin, N-methylserotonin, and bufotenidine. These intriguing compounds are speculated to play a pivotal role in self-defense and increasing venom toxicity and could also offer promising biotechnological applications as small bioactive molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Seven new peptides denominated CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito and their primary structures were determined. The molecular weights ranged between 3760.4 Da and 4357.9 Da, containing 32 to 39 amino acid residues with three putative disulfide bridges. The comparison of amino acid sequences with known potassium scorpion toxins (KTx) and phylogenetic analysis revealed that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to the α-KTx 10.x subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), and CboK7 (α-KTx 2.24) bear > 95% amino acid similarity with members of the α-KTx 2.x subfamily, and CboK5 is identical to Ce3 toxin (α-KTx 2.10). Electrophysiological assays demonstrated that except CboK1, all six other peptides blocked the Kv1.2 channel with Kd values in the picomolar range (24-763 pM) and inhibited the Kv1.3 channel with comparatively less potency (Kd values between 20-171 nM). CboK3 and CboK4 inhibited less than 10% and CboK7 inhibited about 42% of Kv1.1 currents at 100 nM concentration. Among all, CboK7 showed out-standing affinity for Kv1.2 (Kd = 24 pM), as well as high selectivity over Kv1.3 (850-fold) and Kv1.1 (~6000-fold). These characteristics of CboK7 may provide a framework for developing tools to treat Kv1.2-related channelopathies.

Centruroides possanii is a recently discovered species of "striped scorpion" found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD 50 to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii . Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field.

In recent years, morbidity caused by scorpion sting of the species Tityus championi has increased in Panama. Therefore, the LD 50 was determined by intravenous injection in 2.9 mg/kg and the venom of T. championi was separated using a HPLC system and their fractions were tested for biological activities in mice to identify the most toxic fractions to mammals. In addition, the venom fractions were also tested against invertebrates to look for insect-specific toxin peptides. The most toxic fractions were analyzed by MS/MS spectrometry. The primary structures of T. championi venom peptides with the most relevant activity were obtained, and the primary structure of one of most neurotoxic peptides was found at least in other four species of Tityus from Panama. This neurotoxin is quite important to be used as a protein target to be neutralized if developing antivenoms against the sting of this Panamanian scorpion or other relevant species of genera Tityus in the country., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Previously, it was demonstrated that from the single chain fragment variable (scFv) 3F it is possible to generate variants capable of neutralizing the Cn2 and Css2 toxins, as well as their respective venoms (Centruroides noxius and Centruroides suffusus). Despite this success, it has not been easy to modify the recognition of this family of scFvs toward other dangerous scorpion toxins. The analysis of toxin-scFv interactions and in vitro maturation strategies allowed us to propose a new maturation pathway for scFv 3F to broaden recognition toward other Mexican scorpion toxins. From maturation processes against toxins CeII9 from C. elegans and Ct1a from C. tecomanus, the scFv RAS27 was developed. This scFv showed an increased affinity and cross-reactivity for at least 9 different toxins while maintaining recognition for its original target, the Cn2 toxin. In addition, it was confirmed that it can neutralize at least three different toxins. These results constitute an important advance since it was possible to improve the cross-reactivity and neutralizing capacity of the scFv 3F family of antibodies., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Baltazar Becerril Lujan has patent COMPOSITION OF HUMAN RECOMBINANT ANTIBODY FRAGMENTS THAT COMPLETELY NEUTRALIZE THE VENOM OF THE SCORPION CENTRUROIDES SCULPTURATUS pending to 17/937,666., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Scorpine is an antimicrobial and antimalarial peptide isolated from Pandinus imperator scorpion venom. As there are few functional and structural studies reported on scorpine-like peptides, we investigated the recombinant truncated N- and C-terminal domains as well as complete scorpine using biological assays and determined the N- and C-terminal structures using solution nuclear magnetic resonance. The study was conducted using recombinant N- and C-terminal peptides and complete scorpine expressed in Escherichia coli. The results showed that N-scorpine presented a random coil structure in water and adopted α-helical folding in the presence of 50% trifluoroethanol (TFE). C-scorpine contains three disulfide bonds with two structural domains: an unstructured N-terminal domain in water that can form a typical secondary alpha-helix structure in 50% TFE and a C-terminal domain with the CS-αβ motif. Our findings demonstrate cytolytic activity associated with C-scorpine, N-scorpine, and scorpine, as well as channel blocking activity associated with the C-scorpine domain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A novel peptide, Cm39, was identified in the venom of the scorpion Centruroides margaritatus . Its primary structure was determined. It consists of 37 amino acid residues with a MW of 3980.2 Da. The full chemical synthesis and proper folding of Cm39 was obtained. Based on amino acid sequence alignment with different K + channel inhibitor scorpion toxin (KTx) families and phylogenetic analysis, Cm39 belongs to the α-KTx 4 family and was registered with the systematic number of α-KTx 4.8. Synthetic Cm39 inhibits the voltage-gated K + channel hK V 1.2 with high affinity (K d = 65 nM). The conductance-voltage relationship of K V 1.2 was not altered in the presence of Cm39, and the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel; therefore, the pore blocking mechanism is proposed for the toxin-channel interaction. Cm39 also inhibits the Ca 2+ -activated K Ca 2.2 and K Ca 3.1 channels, with K d = 502 nM, and K d = 58 nM, respectively. However, the peptide does not inhibit hK V 1.1, hK V 1.3, hK V 1.4, hK V 1.5, hK V 1.6, hK V 11.1, mK Ca 1.1 K + channels or the hNa V 1.5 and hNa V 1.4 Na + channels at 1 μM concentrations. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels.

Horses have played a prominent role in shaping our modern world, with important effects on health. Unfortunately, better characterization of the horse immune system is still needed. In this report, using flow cytometry techniques, four monoclonal antibodies against horse CD11c integrin were characterized and described for their ability to provide a positive recognition signal in peripheral blood mononuclear cells. Further immune cell phenotype experiments were performed using MHC-II, CD14, TLR4 and the specific anti-horse CD11c monoclonal antibody (1C4). With this staining panel, it was possible to detect a cell population defined by CD11c + MHC-II + TLR4 + CD14 low , which could be considered as putative dendritic cells. This manuscript shows that a new monoclonal antibody (1C4) can be used for the characterization of dendritic cells and their different lineages, opening the possibility of better understanding the mechanisms of immunity in horses., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The Cm28 in the venom of Centruroides margaritatus is a short peptide consisting of 27 amino acid residues with a mol wt of 2,820 D. Cm28 has <40% similarity with other known α-KTx from scorpions and lacks the typical functional dyad (lysine-tyrosine) required to block KV channels. However, its unique sequence contains the three disulfide-bond traits of the α-KTx scorpion toxin family. We propose that Cm28 is the first example of a new subfamily of α-KTxs, registered with the systematic number α-KTx32.1. Cm28 inhibited voltage-gated K+ channels KV1.2 and KV1.3 with Kd values of 0.96 and 1.3 nM, respectively. There was no significant shift in the conductance-voltage (G-V) relationship for any of the channels in the presence of toxin. Toxin binding kinetics showed that the association and dissociation rates are consistent with a bimolecular interaction between the peptide and the channel. Based on these, we conclude that Cm28 is not a gating modifier but rather a pore blocker. In a selectivity assay, Cm28 at 150 nM concentration (>100× Kd value for KV1.3) did not inhibit KV1.5, KV11.1, KCa1.1, and KCa3.1 K+ channels; NaV1.5 and NaV1.4 Na+ channels; or the hHV1 H+ channel but blocked ∼27% of the KV1.1 current. In a biological functional assay, Cm28 strongly inhibited the expression of the activation markers interleukin-2 receptor and CD40 ligand in anti-CD3-activated human CD4+ effector memory T lymphocytes. Cm28, due to its unique structure, may serve as a template for the generation of novel peptides targeting KV1.3 in autoimmune diseases., (© 2022 Naseem et al.)

Ca 2+ channel blockers (CCBs) are commonly used to treat different cardiovascular conditions. These drugs disrupt the intracellular Ca 2+ signaling network, inhibiting numerous cellular functions in different cells, including T lymphocytes. We explored the effect of the CCB verapamil on normal human peripheral blood T cell activation, proliferation, and cytokine production. Cells were activated by ligating CD3 or CD3/CD28 in the presence or absence of verapamil, and the expression of activation-induced cell surface molecules (CD25, CD40L, CD69, PD-1, and OX40), cell proliferation, and cytokine release were assessed by flow cytometry. Verapamil exerted a dose-dependent inhibitory effect on the expression of all the activation-induced cell surface molecules tested. In addition, verapamil diminished T cell proliferation induced in response to CD3/CD28 stimulation. Likewise, the production of Th1/Th17 and Th2 cytokines was also reduced by verapamil. Our data substantiate a potent in vitro suppressive effect of verapamil on T lymphocytes, a fact that might be relevant in patients receiving CCBs.

Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD 50 ) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD 50 of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.

Kv1.3 K + channels play a central role in the regulation of T cell activation and Ca 2+ signaling under physiological and pathophysiological conditions. Peptide toxins targeting Kv1.3 have a significant therapeutic potential in the treatment of autoimmune diseases; thus, the discovery of new toxins is highly motivated. Based on the transcriptome analysis of the venom gland of V. mexicanus smithi a novel synthetic peptide, sVmKTx was generated, containing 36 amino acid residues. sVmKTx shows high sequence similarity to Vm24, a previously characterized peptide from the same species, but contains a Glu at position 32 as opposed to Lys32 in Vm24. Vm24 inhibits Kv1.3 with high affinity (K d  = 2.9 pM). However, it has limited selectivity (~1,500-fold) for Kv1.3 over hKv1.2, hKCa3.1, and mKv1.1. sVmKTx displays reduced Kv1.3 affinity (K d  = 770 pM) but increased selectivity for Kv1.3 over hKv1.2 (~9,000-fold) as compared to Vm24, other channels tested in the panel (hKCa3.1, hKv1.1, hKv1.4, hKv1.5, rKv2.1, hKv11.1, hKCa1.1, hNav1.5) were practically insensitive to the toxin at 2.5 μM. Molecular dynamics simulations showed that introduction of a Glu instead of Lys at position 32 led to a decreased structural fluctuation of the N-terminal segment of sVmKTx, which may explain its increased selectivity for Kv1.3. sVmKTx at 100 nM concentration decreased the expression level of the Ca 2+ -dependent T cell activation marker, CD40 ligand. The high affinity block of Kv1.3 and increased selectivity over the natural peptide makes sVmKTx a potential candidate for Kv1.3 blockade-mediated treatment of autoimmune diseases., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Enzymes are an integral part of animal venoms. Unlike snakes, in which enzymes play a primary role in envenomation, in scorpions, their function appears to be ancillary in most species. Due to this, studies on the diversity of scorpion venom components have focused primarily on the peptides responsible for envenomation (toxins) and a few others (e.g., antimicrobials), while enzymes have been overlooked. In this work, a comprehensive study on enzyme diversity in scorpion venoms was performed by transcriptomic and proteomic techniques. Enzymes of 63 different EC types were found, belonging to 330 orthogroups. Of them, 24 ECs conform the scorpion venom enzymatic core, since they were determined to be present in all the studied scorpion species. Transferases and lyases are reported for the first time. Novel enzymes, which can play different roles in the venom, including direct toxicity, as venom spreading factors, activators of venom components, venom preservatives, or in prey pre-digestion, were described and annotated. The expression profile for transcripts coding for venom enzymes was analyzed, and shown to be similar among the studied species, while being significantly different from their expression pattern outside the telson.

The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD 50 determined for Ct-IT1 is 3.81 μg/100 mg of cricket weight, but low amounts of peptides (0.8 μg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant β-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: The development of more effective antivenoms remains a necessity for countries where scorpionism is a public health problem. Also, the regionalization of antivenoms may be important for some countries with special scorpionism characteristics., Objective: Production of antibodies capable of neutralizing the lethal effect of the venom of three scorpion species from Panama., Methods: The primary structures of two neurotoxins from T. pachyurus, one from T. cerroazul and another from C. bicolor were elucidated using N-terminal amino acid degradation and Sanger gene cloned sequencing. The obtained mRNA transcripts were cloned and expressed using E. coli vectors. Different bacterial expression conditions were tested and the best culture conditions for each expressed protein is reported. The expressed scorpion toxins were purified by chromatographic methods and used as immunogens in rabbits., Results: The antibodies produced under the reported immunization scheme show better neutralization (ED 50 ) than other reported commercial antivenoms used to neutralize similar species scorpion venoms under similar LD 50 conditions., Conclusion: The information reported here shows the proof of concept for selecting recombinant immunogens with the ability to produce antibodies for neutralizing the lethal effects of the most important medical species of scorpions in Panama., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10 -9 and 6.1 × 10 -11 M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.

The peptide, denominated Ct1a, is a β-toxin of 66 amino acids, isolated from venom of the scorpion, Centruroides tecomanus, collected in Colima, Mexico. This toxin was purified using size exclusion, cationic exchange, and reverse phase chromatography. It is the most abundant toxin, representing 1.7% of the soluble venom. Its molecular mass of 7588.9 Da was determined by mass spectrometry. The amino acid sequence was determined by Edman degradation and confirmed by transcriptomic analysis. Since neurons of the suprachiasmatic nucleus (SCN) maintain a spontaneous firing rate (SFR), we evaluated the physiological effects of toxin Ct1a on these neurons. The SFR exhibited a bimodal concentration-dependent response: 100 nM of Ct1a increased the SFR by 223%, whereas 500 nM and 1000 nM reduced it to 42% and 7%, respectively. Control experiments, consisting of recordings of the SFR during a time similar to that used in Ct1a testing, showed stability throughout the trials. Experiments carried out with denatured Ct1a toxin (500 nM) caused no variation in SFR recordings. Action potentials of SCN neurons, before and after Ct1a (100 nM) showed changes in the time constants of depolarization and repolarization phases, amplitude, and half-time. Finally, recordings of hNav1.6 sodium currents indicated that Ct1a shifts the channel activation to a more negative potential and reduces the amplitude of the peak current. These results all demonstrate that toxin Ct1a affects the SFR of SCN neurons by acting upon sodium channels of sub-type 1.6, implicating them in regulation of the SFR of SCN neurons., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Six peptides, belonging to the NDBP-4 family of scorpion antimicrobial peptides were structurally and functionally characterized. The sequence of the mature peptides VpCT1, VpCT2, VpCT3 and VpCT4 was inferred by transcriptomic analysis of the venom gland of the scorpion Mesomexovis variegatus. Analysis of their amino acid sequences revealed patterns that are also present in previously reported peptides that show differences in their hemolytic and antimicrobial activities in vitro. Two other variants, VpCT3W and VpCTConsensus were designed to evaluate the effect of sequence changes of interest on their structure and activity. The synthesized peptides were evaluated by circular dichroism to confirm their α-helical conformation in a folding promoting medium. The peptides were assayed on two Gram-positive and three Gram-negative bacterial strains, and on two yeast strains. They preferentially inhibited the growth of Staphylococcus aureus, were mostly ineffective on Pseudomonas aeruginosa, and moderately inhibited the growth of Candida yeasts. All six peptides exhibited hemolytic activity on human erythrocytes in the range of 4.8-83.7 μM. VpCT3W displayed increased hemolytic and anti-yeast activities, but showed no change in antibacterial activity, relative to its parental peptide, suggesting that Trp6 may potentiate the interaction of VpCT3 with eukaryotic cell membranes. VpCTConsensus showed broader and enhanced antimicrobial activity relative to several of the natural peptides. The results presented here contribute new information on the structure and function of NDBP-4 antimicrobial peptides and provides clues for the design of less hemolytic and more effective antimicrobial peptides., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Colombian scorpion Centruroides margaritatus produces a venom considered of low toxicity. Nevertheless, there are known cases of envenomation resulting in cardiovascular disorders, probably due to venom components that target ion channels. Among them, the human ether-à-go-go-Related gene (hERG1) potassium channels are critical for cardiac action potential repolarization and alteration in its functionality are associated with cardiac disorders. This work describes the purification and electrophysiological characterization of a Centruroides margaritatus venom component acting on hERG1 channels, the CmERG1 toxin. This novel peptide is composed of 42 amino acids with a MW of 4792.88 Da, folded by four disulfide bonds and it is classified as member number 10 of the γ-KTx1 toxin family. CmERG1 inhibits hERG1 currents with an IC 50 of 3.4 ± 0.2 nM. Despite its 90.5% identity with toxin ɣ-KTx1.1, isolated from Centruroides noxius , CmERG1 completely blocks hERG1 current, suggesting a more stable plug of the hERG channel, compared to that formed by other ɣ-KTx.

Epilepsy is one of the most common neurological diseases in the world. The objective of this research was to investigate a new peptide from the venom of the social wasp Chartergellus communis useful to the study or pharmacotherapy of epilepsy. The wasps were collected, and their venom was extracted. Afterward, the steps of fractionation, sequencing, and identification were carried out to obtain four peptides. These molecules were synthesized for behavioral evaluation tests and electroencephalographic assays to determine their antiseizure potential (induction of acute seizures using the chemical compounds, pentylenetetrazole - PTZ, and pilocarpine - PILO) and analysis of neuropharmacological profile (general spontaneous activity and alteration in motor coordination). Chartergellus-CP1 (i.c.v. - 3.0 μg/animal) caused beneficial alterations in some of the parameters evaluated in both models: PTZ (latency and duration of maximum seizures) and PILO (latency and duration of, and protection against, maximum seizures, and reduction of the median of the seizure scores. When evaluated in 3 doses in the seizure model induced by PILO, the dose of 3.0 μg/animal protected the animals against seizures, with an estimated ED 50 of 1.49 μg/animal. Electroencephalographic evaluation of Chartergellus-CP1 showed an improvement in latency, quantity, and percentage of protection against generalized electroencephalographic seizures in the PILO model. Further, Chartergellus-CP1 did not cause adverse effects on general spontaneous activity and motor coordination of animals. This study demonstrated how compounds isolated from wasps' venom may be important resources in the search for new drugs. Such compounds can be considered valuable therapeutic and biotechnological tools for the study and future treatment of epileptic disorders. In this context, a peptide that is potentially useful for epilepsy pharmacotherapy was identified in the venom of C. communis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Peptidase inhibitors (PIs) have been broadly studied due to their wide therapeutic potential for human diseases. A potent trypsin inhibitor from Tityus obscurus scorpion venom was characterized and named ToPI1, with 33 amino acid residues and three disulfide bonds. The X-ray structure of the ToPI1:trypsin complex, in association with the mass spectrometry data, indicate a sequential set of events: the complex formation with the inhibitor Lys 32 in the trypsin S1 pocket, the inhibitor C-terminal residue Ser 33 cleavage, and the cyclization of ToPI1 via a peptide bond between residues Ile 1 and Lys 32 . Kinetic and thermodynamic characterization of the complex was obtained. ToPI1 shares no sequence similarity with other PIs characterized to date and is the first PI with CS-α/β motif described from animal venoms. In its cyclic form, it shares structural similarities with plant cyclotides that also inhibit trypsin. These results bring new insights for studies with venom compounds, PIs, and drug design.

In this communication the isolation, chemical and physiological characterization of three new toxins from the scorpion Centruroides baergi are reported. Their immunoreactive properties with scFvs generated in our group are described. The three new peptides, named Cb1, Cb2 and Cb3 affect voltage-dependent Na + channels in a differential manner. These characteristics, explain the toxicity of this venom. Molecular interactions in real-time among these toxins and the best recombinant antibodies generated in our group, revealed that one of them was able to neutralize the main toxin of this venom (Cb1). These results represent an important advance for the neutralization of this venom and serve as the basis for generating new scFvs that will allow the neutralization of similar toxins from other venoms that have no yet been neutralized., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cl13 is a toxin purified previously from the venom of the Mexican scorpion Centruroides limpidus. This toxin affects the function of voltage gated Na + -channels, human subtypes Nav1.4, Nav1.5 and Nav1.6 in a similar manner as other known β-toxins from scorpion venoms. Here, we report a correction of the primary structure of Cl13, previously published. The peptide does contain 66 amino acids, but residue 58 is a tryptophan and the last C-terminal amino acid is an amidated lysine, instead of arginine. The main contribution of this communication is the determination of the 3D-structure of Cl13, by solution NMR, showing that Cl13 has the classical cysteine-stabilized α/β (CSα/β) folding. It has a triple stranded antiparallel beta sheet commonly present in scorpion sodium channel β-toxins. In addition, we report and discuss a comparison of Cl13 structure with two other toxins (Cn2 and Css2) from scorpions of the same genus Centruroides, which shows important surface similarities with the structure reported here. Finally, the lack of neutralization of Cl13 toxin by two single-chain antibody fragments (scFvs), named LR and 10FG2, which are capable of neutralizing various toxins from Mexican scorpions, is revised. In particular, 10FG2 is capable of neutralizing toxins Cll1 and Cll2 of the same scorpion C. limpidus. The reasons why LR and 10FG2 are unable of neutralizing Cl13 toxin are discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Proteomic characterization of Micrurus browni browni venom showed approximately 41 components belonging to 9 protein families, mainly phospholipases A 2 (PLA 2 s) and three-finger toxins (3FTxs). Venom gland transcriptome yielded 39 venom transcripts belonging to 10 protein families. Functional characterization identified a multimeric toxin, here designated Brownitoxin-1, which comprises at least one PLA 2 and one 3FTx. Its components have no or very low lethality individually but become extremely lethal when combined; both were partially characterized. Other two lethal components were identified: A neurotoxic PLA 2 , and a postsynaptic α-neurotoxin. LD 50 s as well as PLA 2 and nAChR-blocking activities were determined for whole venom and isolated components. Application of venom to murine neuromuscular preparations caused a progressive decrease of twitch force that was irreversible after washing. Inhibition of PLA 2 activity with p-bromophenacyl bromide (pBPB) showed that approximately 90% of toxicity is dependent on this activity. Non-lethal components include diverse 3FTxs, at least three enzymatically active PLA 2 s and the nociceptive toxin MitTx. No evidence of specificity towards prey was observed. This work is one of the most complete characterizations of a coral snake venom so far and its findings highlight the relevance of protein complexes in venom function. SIGNIFICANCE: This study represents a profound analysis of the venom of the coral snake Micrurus browni browni, including a venom proteome, venom gland transcriptomic data and functional studies of whole venom and isolated toxins. It significantly contributes to the understanding of North American coral snake venoms, which are currently largely unknown. It includes characterization of relevant venom components, one of which represents the first description of a lethal multimeric neurotoxin in coral snake venom. This work highlights the importance of protein complexes in coral snake venom and could serve as a basis for the finding of several other multimeric toxins. Finally, we report the absence of taxon specificity, which has been previously reported in the venoms of other snakes of the same genus., Competing Interests: Declaration of competing interest The authors declare no conflict of interest regarding the present work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The study of the effector mechanisms of T helper cells has revealed different phenotypic characteristics that can be manipulated for designing new therapeutic schemes in different pathological scenarios. Ion channels are significant targets in T lymphocyte modulation since they are closely related to their effector activity. Remarkably, some toxins produced by scorpions specifically affect the function of these membrane proteins. For that reason, these toxins are important candidates in the search for new immunomodulators. Here, the effect of two venom fractions of the scorpion Centruroides limpidus was assessed on T lymphocyte proliferation and cytokine production. The venom fractions ClF8 and ClF9 were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and cultured at 25 and 35 µg/ml with murine T lymphocytes. The results indicate that the fraction ClF8 increased both production and secretion levels of IFN-γ, IL-4, IL-17A and IL-10 by CD4 + T cells at 24 h. In contrast, fraction ClF9 only promoted the secretion of IL-17A and IL-10 at its highest concentration (35 µg/ml). Both fractions did not show any effect on T cell proliferation. Subsequent analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed seventeen toxins in the fraction ClF8 and five toxins in the fraction ClF9, most of them with voltage-gated sodium (NaScTx) and potassium (KScTx) channels as molecular targets. These toxins might probably interact with ion channels involved in T lymphocyte activity. Our findings suggest that the difference in composition between the two fractions could be related to the observed effects, and the components identified could be isolated to search for possible immunomodulatory molecules., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest in this work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Centruroides hirsutipalpus (Scorpiones: Buthidae) is related to the "striped scorpion" group inhabiting the western Pacific region of Mexico. Human accidents caused by this species are medically important due to the great number of people stung and the severity of the resulting intoxication. This communication reports an extensive venom characterization using high-throughput proteomic and Illumina transcriptomic sequencing performed with RNA purified from its venom glands. 2,553,529 reads were assembled into 44,579 transcripts. From these transcripts, 23,880 were successfully annoted using Trinotate. Using specialized databases and by performing bioinformatic searches, it was possible to identify 147 putative venom protein transcripts. These include α- and β-type sodium channel toxins (NaScTx), potassium channel toxins (KScTx) (α-, β-, δ-, γ- and λ-types), enzymes (metalloproteases, hyaluronidases, phospholipases, serine proteases, and monooxygenases), protease inhibitors, host defense peptides (HDPs) such as defensins, non-disulfide bridge peptides (NDBPs), anionic peptides, superfamily CAP proteins, insulin growth factor-binding proteins (IGFBPs), orphan peptides, and other venom components (La1 peptides). De novo tandem mass spectrometric sequencing of digested venom identificatied 50 peptides. The venom of C. hirsutipalpus contains the highest reported number (77) of transcripts encoding NaScTxs, which are the components responsible for human fatalities., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The present study evaluated the effect of the change in the orientation of the VH-VL variable domains to VL-VH on the physicochemical and functional properties of two scorpion toxin-neutralizing scFvs. The results showed that the level of expression of proteins obtained from the periplasm of E. coli is the factor mainly affected, either with an increase or decrease in the amount of protein recovered. Likewise, the functional recognition activity in the presence of a denaturing agent showed slight variations in the two orientations. In contrast, recognition and biological activity (neutralizing capacity) are maintained. At the interaction level, the change marginally modified the kinetic association and dissociation constants without significantly modifying the value of the affinity constants. Similarly, it was observed that the thermodynamic stability of the proteins did not show significant variations either. These results contrast with some reports of the effect of changing the orientation of domains, suggesting that it is not possible to predict which orientation of the variable domains of an scFv is more favorable or if they are equivalent, as in the case of scFvs previously matured by directed evolution techniques., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Urotoxin (α-KTx 6), a peptide from venom of the Australian scorpion Urodacus yaschenkoi, is the most potent inhibitor of Kv1.2 described to date (IC 50  = 160 pM). The native peptide also inhibits Kv1.1, Kv1.3 and KCa3.1 with nanomolar affinity but its low abundance in venom precluded further studies of its actions. Here we produced recombinant Urotoxin (rUro) and characterized the molecular determinants of Kv1 channel inhibition. The 3D structure of rUro determined using NMR spectroscopy revealed a canonical cysteine-stabilised α/β (CSα/β) fold. Functional assessment of rUro using patch-clamp electrophysiology revealed the importance of C-terminal amidation for potency against Kv1.1-1.3 and Kv1.5. Neutralization of the putative pore-blocking K25 residue in rUro by mutation to Ala resulted in a major decrease in rUro potency against all Kv channels tested, without perturbing the toxin's structure. Reciprocal mutations in the pore of Uro-sensitive Kv1.2 and Uro-resistant Kv1.5 channels revealed a direct interaction between Urotoxin and the Kv channel pore. Our experimental work supports postulating a mechanism of action in which occlusion of the permeation pathway by the K25 residue in Urotoxin is the basis of its Kv1 inhibitory activity. Docking analysis was consistent with occlusion of the pore by K25 and the requirement of a small, non-charged amino acid in the Kv1 channel vestibule to facilitate toxin-channel interactions. Finally, computational studies revealed key interactions between the amidated C-terminus of Urotoxin and a conserved Asp residue in the turret of Kv1 channels, offering a potential rationale for potency differences between native and recombinant Urotoxin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Every year in Mexico, around 300,000 people suffer from accidents related to scorpion stings. Among the scorpion species dangerous to human is Centruroides ornatus, whose venom characterization is described here. From this venom, a total of 114 components were found using chromatographic separation and mass spectrometry analysis. The most abundant ones have molecular masses between 3000-4000 Da and 6000-8000 Da respectively, similar to other known K + and Na + -channel specific scorpion peptides. Using intraperitoneal injections into CD1 mice, we were able to identify and fully sequenced three new lethal toxins. We propose to name them Co1, Co2 and Co3 toxins, which correspond to toxins 1 to 3 of the abbreviated species name (Co). Electrophysiology analysis of these peptides using heterologously expressed human Na + -channels revealed a typical β-toxin effect. Peptide Co52 (the most abundant peptide in the venom) showed no activity in our in vivo and in vitro model assays. A phylogenetic analysis groups the Co1, Co2 and Co3 among other β-toxins from Centruroides scorpions. Peptide Co52 segregates among peptides of unknown defined functions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction : A few scorpions are dangerous to humans. Their medical relevance was the initial driving force for venom research. By classical biochemistry and molecular cloning, several venom peptides and their coding transcripts were characterized, mainly those related to toxins. The discovery of other components with novel activities and potential applications has revitalized the interest in the field in the last decade and a half. Nontoxic scorpion species have also attracted major interest. Areas covered : Advances in the identification of scorpion venom components via high-throughput venomics (genomics, transcriptomics and proteomics) up to 2019 are summarized. A classification system for venom-related transcripts and proteins, together with an intuitive systematic nomenclature for RNAseq-generated transcripts are proposed. Venom components classified as Na + , K + , Ca 2+ , Cl - and TRP channel toxins, enzymes, protease inhibitors, host defense peptides and other peptidic molecules are briefly reviewed, giving a comprehensive picture of the venom. Expert opinion : Modern high-throughput technologies applied to scorpion venom studies have resulted in a dramatic increase in both, the number and diversity of available sequences, leading to a deeper understanding of the composition of scorpion venoms. Still, many newly-discovered venom constituents remain to be characterized, to complete the puzzle of scorpion venoms.

Growing global viral infections have been a serious public health problem in recent years. This current situation emphasizes the importance of developing more therapeutic antiviral compounds. Hepatitis C virus (HCV) and dengue virus (DENV) belong to the Flaviviridae family and are an increasing global health threat. Our previous study reported that the crude venom of Scorpio maurus palmatus possessed anti-HCV and anti-DENV activities in vitro. We report here the characterization of a natural antiviral peptide (scorpion-like peptide Smp76) that prevents HCV and DENV infection. Smp76 was purified from S. m. palmatus venom and contains 76 amino acids with six residues of cysteine. Smp76 antiviral activity was evaluated using a cell culture technique utilizing Huh7it-1, Vero/SLAM, HCV (JFH1, genotype 2a) and DENV (Trinidad 1751, type 2). A potential antiviral activity of Smp76 was detected in culture cells with an approximate IC 50 of 0.01 μg/ml. Moreover, Smp76 prevents HCV infection and suppresses secondary infection, by inactivating extra-cellular infectious particles without affecting viral replication. Interestingly, Smp76 is neither toxic nor hemolytic in vitro at a concentration 1000-fold higher than that required for antiviral activity. Conclusively, this report highlights novel anti-HCV and anti-DENV activities of Smp76, which may lay the foundation for developing a new therapeutic intervention against these flaviviruses., Competing Interests: Conflict of interestDrs. Hak Hotta, Moustafa M Sarhan, Alaa MH El-Bitar, Lourival D. Possani and Mohamed A. Abdel-Rahman have a patent (anti-virus drug: PCT/JP2017/286) pending containing some of the information described in this work., (© Springer Nature B.V. 2019.)

Scorpion toxins are thought to have originated from ancestral housekeeping genes that underwent diversification and neofunctionalization, as a result of positive selection. Our understanding of the evolutionary origin of these peptides is hindered by the patchiness of existing taxonomic sampling. While recent studies have shown phylogenetic inertia in some scorpion toxins at higher systematic levels, evolutionary dynamics of toxins among closely related taxa remain unexplored. In this study, we used new and previously published transcriptomic resources to assess evolutionary relationships of closely related scorpions from the family Hadruridae and their toxins. In addition, we surveyed the incidence of scorpine-like peptides (SLP, a type of potassium channel toxin), which were previously known from 21 scorpion species. We demonstrate that scorpine-like peptides exhibit gene duplications. Our molecular analyses demonstrate that only eight sites of two SLP copies found in scorpions are evolving under positive selection, with more sites evolving under negative selection, in contrast to previous findings. These results show evolutionary conservation in toxin diversity at shallow taxonomic scale.

The soluble venom of the scorpion Tityus macrochirus was separated by chromatographic procedures and three homogeneous peptides were obtained and their primary structures were determined. They were called: Tma1-Tma3, from the abbreviated name of the scorpion. Tma1 is a peptide containing 65 amino acids with four disulfide linkages and a molecular weight of 7386.2 Da. It is a mammalian toxin, shown to affect human sodium-channels sub-types hNav1.6 and hNav1.4. Tma2 and Tma3 are peptides containing 69 amino acids linked by four disulfide bonds, molecular weights 7819.7 and 7830.0 Da, respectively. They do not affect human sodium-channels but are lethal to insects (crickets). A phylogenic analysis of the three peptides and those of other toxic peptides isolated from the genus Tityus and Centruroides were grouped together and analyzed, permitting to obtain a topology with two main clades, one includes most sodium-channel anti-insect scorpion toxins and others includes mostly sodium-channel scorpion toxins anti-mammalian. Tma1 segregates among a group of well-studied β-class toxins of other Tityus species such as T. discrepans, T. obscurus and T. pachyurus. Tma2 and Tma3 are associated with anti-insect toxins, particularly with one of T. obscurus. This phylogenetic analysis confirms and enforces our experimental results obtained with these three new sodium-channel scorpion toxins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A peptide (Cn29) from the venom of the scorpion Centruroides noxius (about 2% of the soluble venom) was purified and its primary and three-dimensional structures were determined. The peptide contains 27 amino acids with primary sequence: LCLSCRGGDYDCRVKGTCENGKCVCGS. The peptide is tightly packed by three disulfide linkages formed between C2-C23, C5-C18 and C12-C25. Since the native peptide was obtained in limited amounts, the full synthetic peptide was prepared using the standard F-moc-based solid phase synthesis method of Merrifield. The native and synthetic peptides were shown to be identical by sequencing, HPLC separation and mass spectrometry. The solution structure of the peptide solved from NMR data shows that it consists of a well-defined N-terminal region without regular secondary structure extending from Leu 1 to Asp 9, followed by a short helical fragment from Tyr10 to Val14 and two short β strands (Thr17-Glu19 and Lys22-Val24). The primary and tertiary structures of Cn29 are different from all other scorpion peptides described in the literature. Transcriptome analysis of RNA obtained from C. noxius confirmed the expression of a gene coding for Cn29 in its venom gland. Initial experiments were conducted to identify its possible function: lethality tests in mice and insects as well as ion-channel binding using in vitro electrophysiological assays. None of the physiological or biological tests displayed any activity for this peptide, which at present is considered to be another orphan peptide found in scorpion venoms. The peptide is thus the first example of a novel structural component present in scorpion venoms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Many peptides in scorpion venoms are amidated at their C-termini. This post-translational modification is paramount for the correct biological function of ion channel toxins and antimicrobial peptides, among others. The discovery of canonical amidation sequences in transcriptome-derived scorpion proproteins suggests that a conserved enzymatic α-amidation system must be responsible for this modification of scorpion peptides. A transcriptomic approach was employed to identify sequences putatively encoding enzymes of the α-amidation pathway. A dual enzymatic α-amidation system was found, consisting of the membrane-anchored, bifunctional, peptidylglycine α-amidating monooxygenase (PAM) and its paralogs, soluble monofunctional peptidylglycine α-hydroxylating monooxygenase (PHM m ) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL m ). Independent genes encode these three enzymes. Amino acid residues responsible for ion coordination and enzymatic activity are conserved in these sequences, suggesting that the enzymes are functional. Potential endoproteolytic recognition sites for proprotein convertases in the PAM sequence indicate that PAM-derived soluble isoforms may also be expressed. Sequences potentially encoding proprotein convertases (PC1 and PC2), carboxypeptidase E (CPE), and other enzymes of the α-amidation pathway, were also found, confirming the presence of this pathway in scorpions.

Two 1,4-benzoquinone derivatives, found in the venom of the scorpion Diplocentrus melici following exposure to air, have been isolated, characterized, synthesized, and assessed for antimicrobial activities. Initially a white, viscous liquid, the extracted venom colors within minutes under ambient conditions. From this colored mixture, two compounds, one red, the other blue, were isolated and purified using chromatography. After a variety of NMR and mass spectrometry experiments, the red compound was determined to be 3,5- dimethoxy-2-(methylthio)cyclohexa-2,5-diene-1,4-dione, and the blue compound was determined to be 5-methoxy-2,3- bis(methylthio)cyclohexa-2,5-diene-1,4-dione. Because extremely small amounts of these compounds were isolated from the scorpion venom, we developed laboratory syntheses from commercially available precursors, allowing us to produce sufficient quantities for crystallization and biological assays. The red benzoquinone is effective against Staphylococcus aureus [minimum inhibitory concentration (MIC) = 4 µg/mL], while the blue benzoquinone is active against Mycobacterium tuberculosis (MIC = 4 µg/mL) and even against a multidrug-resistant (MDR) strain with nearly equal effectiveness. The bactericidal effects of both benzoquinones show comparable activity to commercially available antibiotics used against these pathogens and were cytotoxic to neoplastic cell lines, suggesting their potential as lead compounds for the development of novel antimicrobial and anticancer drugs. Importantly, the blue benzoquinone was also effective in vivo with mouse models of MDR tuberculosis infection. After treatment for 2 mo, four mice with late-stage active MDR tuberculosis had a significant decrease in pulmonary bacillary loads and tissue damage. Healthy mice served as negative controls and tolerated treatment well, without adverse side effects., Competing Interests: The authors declare no conflict of interest.

Targeting antigens to endocytic receptors on the surface of dendritic cells is a new strategy for increasing the adaptive immune response. The objective of the current study was the construction and bacterial expression of a recombinant antibody single-chain fragment variable (ScFv) directed against chicken DEC 205, an endocytic receptor, for use in the genetic fusion of antigens. In particular, we use as antigen the hemagglutinin-neuraminidase (HN) of Newcastle disease virus. Our results show that inoculation of chickens with HN genetically fused to the ScFv anti-DEC 205 induced an evidently higher immune response against HN, in contrast to inoculation with unconjugated HN. In addition, neutralizing antibodies against Newcastle disease virus were detected only in the serum from chickens immunized with HN fused to ScFv anti-DEC 205. Inoculated fused antigens to ScFv against endocytic receptor DEC 205 resulted in a greater antibody-specific anti-HN production compared with antigens applied alone. The results of this study show that the strategy described here has the potential to be used in the development of more effective vaccines against infectious diseases in chickens., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Venom glands and soluble venom from the Mexican scorpion Centruroides limpidus (Karsch, 1879) were used for transcriptomic and proteomic analyses, respectively. An RNA-seq was performed by high-throughput sequencing with the Illumina platform. Approximately 80 million reads were obtained and assembled into 198,662 putative transcripts, of which 11,058 were annotated by similarity to sequences from available databases. A total of 192 venom-related sequences were identified, including Na + and K + channel-acting toxins, enzymes, host defense peptides, and other venom components. The most diverse transcripts were those potentially coding for ion channel-acting toxins, mainly those active on Na + channels (NaScTx). Sequences corresponding to β- scorpion toxins active of K + channels (KScTx) and λ-KScTx are here reported for the first time for a scorpion of the genus Centruroides . Mass fingerprint corroborated that NaScTx are the most abundant components in this venom. Liquid chromatography coupled to mass spectometry (LC-MS/MS) allowed the identification of 46 peptides matching sequences encoded in the transcriptome, confirming their expression in the venom. This study corroborates that, in the venom of toxic buthid scorpions, the more abundant and diverse components are ion channel-acting toxins, mainly NaScTx, while they lack the HDP diversity previously demonstrated for the non-buthid scorpions. The highly abundant and diverse antareases explain the pancreatitis observed after envenomation by this species.