Differential fluorescence features and recovery speeds of different scorpion exoskeleton parts during the molting process.

Scorpion fluorescence under ultraviolet light is a well-known phenomenon, but its features under excitation in the UVA, UVB and UVC bands have not been characterized. Systematic fluorescence characterization revealed indistinguishable fluorescence spectra with a peak wavelength of 475 nm for whole exuviae from second-, third- and fifth-instar scorpions under different ultraviolet light ranges. In-depth investigations of the chelae, mesosoma, metasoma and telson of adult scorpions further indicated heterogeneity in the typical fluorescence spectrum within the visible light range and in the newly reported fluorescence spectrum with a peak wavelength of 320 nm within the ultraviolet light range, which both showed excitation wavelength-independent features. Dynamic fluorescence changes during the molting process of third-instar scorpions revealed the fluorescence heterogeneity-dependent recovery speed of scorpion exoskeletons. The typical fluorescence spectra of the molted chelae and telson rapidly recovered approximately 6 h after ecdysis under UVA light and approximately 36 h after ecdysis under UVB and UVC light. However, it took approximately 12 h and 24 h to obtain the typical fluorescence spectra of the molted metasoma and mesosoma, respectively, under UVA irradiation and 72 h to obtain the typical fluorescence spectra under UVB and UVC irradiation. The fluorescence heterogeneity-dependent fluorescence recovery of the scorpion exoskeleton was further confirmed by tissue section analysis of different segments from molting third-instar scorpions. These findings reveal novel scorpion fluorescence features and provide potential clues on the biological function of scorpion fluorescence.

The inhibitory activity of a new scorpion venom-derived antimicrobial peptide Hp1470 against Gram-positive bacteria.

Antimicrobial peptides (AMPs) are a new type of antibiotic and target a variety of microbes, including antibiotic-resistant strains; thus, AMPs have attracted widespread interest. Scorpion venoms contain many bioactive peptides, including AMPs, and have become an important natural resource of peptide-based drugs. Here, the antibacterial peptide gene Hp1470 from the venom of the scorpion Heterometrus petersii was characterized, and its antibacterial activity was determined. The cDNA sequence of Hp1470 is 300 nt in length and contains an open reading frame (ORF) of 207 nt. The ORF was shown to encode 68 amino acid residues, including a signal peptide (23 aa), a mature peptide (13 aa), a C-terminal posttranslational processing signal (3 aa), and a propeptide (29 aa). Multiple sequence alignment results indicated that Hp1470 is an antibacterial peptide. The mature peptide Hp1470, which has a molecular mass of 1564.09 Da, was further chemically synthesized with a purity of greater than 95%. Antimicrobial assays showed that the synthesized Hp1470 exerted an inhibitory effect on Gram-positive bacteria and clinical drug-resistant strains, including PRSA and MRSA, but not Gram-negative bacteria. Hp1470 was further found to protect mice from MRSA infection, suggesting its potential application as an in vivo antimicrobial agent. Interestingly, Hp1470 only inhibited bacterial growth but did not kill bacteria, which was consistent with scanning electron microscopy results showing that Hp1470 did not lyse the cell membrane of Staphylococcus aureus. Our work provides a new direction for developing antibacterial agents with different modes of action from natural scorpion venoms.

Dynamic synthesis and transport of fluorescent substances from moulting scorpions.

Scorpion fluorescence under ultraviolet light is a well-known phenomenon, and its change is also a known biological feature during the scorpion moulting process. However, the synthesis and transport of fluorescent substances during the moulting stage remain unclear. In this study, in-depth investigations on the global fluorescence changes from the exoskeleton, fluorescence layer, coelomic fluid, and abdomen to the digestive glands indicated that the digestive glands, which occupy most of the space in the abdomen of the scorpion mesosoma segment, were responsible for synthesizing the fluorescent substances. More importantly, these fluorescent substances were produced in advance, before the moulting process, which contributed to the recovery of the fluorescent exoskeleton as early as possible. The synthesized fluorescent substances first entered the coelomic fluid, then successively passed through the inherent epithelial cell layer and two new formed endocuticle and exocuticle layers, and ultimately reached and became enriched in the new formed fluorescent layer, which was protected by the new epicuticle layer. These four new layers were the first to illustrate the structural features of the fluorescent exoskeleton. Due to the very soft body and the inability of the newly moulted scorpion to resist attacks from the predator, this special synthesis and transport strategy of the fluorescent substances could guarantee the rapid formation of the integrated fluorescent exoskeleton during the 24 h after ecdysis, which would be a novel biological feature during the scorpion evolution.

ML-SA1 and SN-2 inhibit endocytosed viruses through regulating TRPML channel expression and activity.

Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3), as key channels in the endosomal-lysosomal system, are associated with many different cellular processes, including ion release, membrane trafficking and autophagy. In particular, they can also facilitate viral entry into host cells and enhance viral infection. We previously identified that two selective TRPML agonists, ML-SA1 and SN-2, that showed antiviral activities against dengue virus type 2 (DENV2) and Zika virus (ZIKV) in vitro, but their antiviral mechanisms are still elusive. Here, we reported that ML-SA1 could inhibit DENV2 replication by downregulating the expression of both TRPML2 and TRPML3, while the other TRPML activator, SN-2, suppressed DENV2 infection by reducing only TRPML3 expression. Consistently, the channel activities of both TRPML2 and TRPML3 were also found to be associated with the antiviral activity of ML-SA1 on DENV2 and ZIKV, but SN-2 relied only on TRPML3 channel activity. Further mechanistic experiments revealed that ML-SA1 and SN-2 decreased the expression of the late endosomal marker Rab7, dependent on TRPML2 and TRPML3, indicating that these two compounds likely inhibit viral infection by promoting vesicular trafficking from late endosomes to lysosomes and then accelerating lysosomal degradation of the virus. As expected, neither ML-SA1 nor SN-2 inhibited herpes simplex virus type I (HSV-1), whose entry is independent of the endolysosomal network. Together, our work reveals the antiviral mechanisms of ML-SA1 and SN-2 in targeting TRPML channels, possibly leading to the discovery of new drug candidates to inhibit endocytosed viruses.

The rapid development of the first instar telson with venom secretion highlights the remarkable survival ability of scorpions.

The scorpion venom system plays a critical role in capturing prey and defending against predators. In this study, the rapid developmental process of the first instar telson was first presented. The small amount of venom in the first instar could be stored well by the distorted and blocked venom ducts, which disappeared in the older scorpions. This special developmental process of the first instar telson revealed the notable survival ability of scorpions.

Different pharmacological properties between scorpion toxin BmKcug2 and its degraded analogs highlight the diversity of K+ channel blockers from thermally processed scorpions.

Novel degraded potassium channel-modulatory peptides were recently found in thermally processed scorpions, but their pharmacological properties remain unclear. Here, we identified a full-length scorpion toxin (i.e., BmKcug2) and its four truncated analogs (i.e., BmKcug2-P1, BmKcug2-P2, BmKcug2-P3 and BmKcug2-P4) with three conserved disulfide bonds in processed scorpion medicinal material by mass spectrometry. The pharmacological experiments revealed that the recombinant BmKcug2 and BmKcug2-P1 could selectively inhibit the human Kv1.2 and human Kv1.3 potassium channels, while the other three analogs showed a much weaker inhibitory effect on potassium channels. BmKcug2 inhibited hKv1.2 and hKv1.3 channels, with IC50 values of 45.6 ± 5.8 nM and 215.2 ± 39.7 nM, respectively, and BmKcug2-P1 inhibited hKv1.2 and hKv1.3, with IC50 values of 89.9 ± 9.6 nM and 1142.4 ± 64.5 nM, respectively. The chromatographic analysis and pharmacological properties of BmKcug2 and BmKcug2-P1 boiled in water for different times further strongly supported their good thermal stability. Structural and functional dissection indicated that one amino acid, i.e., Tyr36, determined the differential affinities of BmKcug2 and four BmKcug2 analogs. Altogether, this research investigated the different pharmacological properties of BmKcug2 and its truncated analogs, and the findings highlighted the diversity of K+ channel blockers from various scorpion species through thermal processing.

ML-SA1, a selective TRPML agonist, inhibits DENV2 and ZIKV by promoting lysosomal acidification and protease activity.

Arboviruses, especially Dengue virus (DENV) and Zika virus (ZIKV), have been a severe threat to human health in the last few years due to uncontrollable transmission. There are no approved vaccines or clinical drugs available for use to prevent and treat their infections. Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3) were reported to modulate viral entry, but the antiviral function of these modulators was unknown. Here, we reported that ML-SA1, a TRPML agonist, inhibited DENV2 and ZIKV in vitro in a dose-dependent manner. Time-of-drug-addition experiments showed that ML-SA1 mainly restricted viral entry. Moreover, the selective TRPML3 activator SN-2 was found to share a similar antiviral effect against DENV2 and ZIKV, but the specific TRPML1 agonist MK6-83 was not effective. Although ML-SA1 was further revealed to induce autophagy, its antiviral role was independent of autophagy induction. Finally, ML-SA1 was found to inhibit DENV2 and ZIKV by promoting lysosome acidification and protease activity to cause viral degradation. Together, our study identifies two TRPML agonists, ML-SA1 and SN-2, as potent inhibitors of DENV2 and ZIKV, which may lead to the discovery of new candidates against viruses.

Inhibitory Activity of a Scorpion Defensin BmKDfsin3 against Hepatitis C Virus.

Hepatitis C virus (HCV) infection is a major worldwide health problem which can cause chronic hepatitis, liver fibrosis and hepatocellular carcinoma (HCC). There is still no vaccine to prevent HCV infection. Currently, the clinical treatment of HCV infection mainly relies on the use of direct-acting antivirals (DAAs) which are expensive and have side effects. Here, BmKDfsin3, a scorpion defensin from the venom of Mesobuthus martensii Karsch, is found to dose-dependently inhibit HCV infection at noncytotoxic concentrations and affect viral attachment and post-entry in HCV life cycle. Further experimental results show that BmKDfsin3 not only suppresses p38 mitogen-activated protein kinase (MAPK) activation of HCV-infected Huh7.5.1 cells, but also inhibits p38 activation of Huh7.5.1 cells stimulated by tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) or lipopolysaccharide (LPS). BmKDfsin3 is also revealed to enter into cells. Using an upstream MyD88 dimerization inhibitor ST2345 or kinase IRAK-1/4 inhibitor I, the inhibition of p38 activation represses HCV replication in vitro. Taken together, a scorpion defensin BmKDfsin3 inhibits HCV replication, related to regulated p38 MAPK activation.

Transcriptome analysis of the venom gland of the scorpion Scorpiops jendeki: implication for the evolution of the scorpion venom arsenal.

BACKGROUND: The family Euscorpiidae, which covers Europe, Asia, Africa, and America, is one of the most widely distributed scorpion groups. However, no studies have been conducted on the venom of a Euscorpiidae species yet. In this work, we performed a transcriptomic approach for characterizing the venom components from a Euscorpiidae scorpion, Scorpiops jendeki. RESULTS: There are ten known types of venom peptides and proteins obtained from Scorpiops jendeki. Great diversity is observed in primary sequences of most highly expressed types. The most highly expressed types are cytolytic peptides and serine proteases. Neurotoxins specific for sodium channels, which are major groups of venom components from Buthidae scorpions, are not detected in this study. In addition to those known types of venom peptides and proteins, we also obtain nine atypical types of venom molecules which haven't been observed in any other scorpion species studied to date. CONCLUSION: This work provides the first set of cDNAs from Scorpiops jendeki, and one of the few transcriptomic analyses from a scorpion. This allows the characterization of a large number of venom molecules, belonging to either known or atypical types of scorpion venom peptides and proteins. Besides, our work could provide some clues to the evolution of the scorpion venom arsenal by comparison with venom data from other scorpion lineages.

Imcroporin, a new cationic antimicrobial peptide from the venom of the scorpion Isometrus maculates.

The pace of resistance against antibiotics almost exceeds that of the development of new drugs. As many bacteria have become resistant to conventional antibiotics, new drugs or drug resources are badly needed to combat antibiotic-resistant pathogens, like methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial peptides, rich sources existing in nature, are able to effectively kill multidrug-resistant pathogens. Here, imcroporin, a new antimicrobial peptide, was screened and isolated from the cDNA library of the venomous gland of Isometrus maculates. The MIC of imcroporin against MRSA was 50 microg/ml, 8-fold lower than that of cefotaxime and 40-fold lower than that of penicillin. Imcroporin killed bacteria rapidly in vitro, inhibited bacterial growth, and cured infected mice. These results revealed that imcroporin could be considered a potential anti-infective drug or lead compound, especially for treating antibiotic-resistant pathogens.

Characterization of LmTxLP11 and LmVP1.1 transcripts and genomic organizations: alternative splicing contributing to the diversity of scorpion venom peptides.

Scorpion venoms are rich resources of bioactive peptides with extreme variability. Multiple molecular mechanisms are involved in the diversity of scorpion venom peptides. However, alternative splicing, which plays a major role in the generation of proteomic and functional diversity in metazoan organisms, hasn't been reported in genes coding for scorpion venom peptides. In the EST analysis of venom peptide transcripts from scorpion Lychas mucronatus, we reported an alternative splicing event. Transcripts of LmTxLP11 and LmVP1.1 share identical 5' region. LmVP1.1 is a novel type of scorpion venom peptides constrained by one disulfide bridge, whereas LmTxLP11 is an extended version of LmVP1.1. By transcript alignment with its genomic sequence, it is found that both transcripts are generated from a single gene by alternative poly A site and terminal exon. The gene encoding LmTxLP11 and LmVP1.1 is the first one harboring three introns ever reported from scorpion venoms. This work demonstrates for the first time that alternative splicing is involved in regulating the diversity of scorpion venom peptides.

A HindIII BAC library construction of Mesobuthus martensii Karsch (Scorpiones:Buthidae): an important genetic resource for comparative genomics and phylogenetic analysis.

Scorpions are "living but sophisticated fossils" that have changed little in their morphology since their first appearance over the past 450 million years ago. To provide a genetic resource for understanding the evolution of scorpion genome and the relationships between scorpions and other organisms, we first determined the genome size of the scorpion Mesobuthus martensii Karsch (about 600 Mbp) in the order Scorpiones and constructed a HindIII BAC library of the male scorpion M. martensii Karsch from China. The BAC library consists of a total of 46,080 clones with an average insert size of 100 kb, providing a 7.7-fold coverage of the scorpion haploid genome size of 600 Mbp as revealed in this study. High-density colony hybridization-based library screening was performed using 18S-5.8S-28S rRNA gene that is one of the most commonly used phylogenetic markers. Both library screening and PCR identification results revealed six positive BAC clones which were overlapped, and formed a contig of approximately 120 kb covering the rDNA. BAC DNA sequencing analysis determined the complete sequence of M. martensii Karsch rDNA unit that has a total length of 8779 bp, including 1813 bp 18s rDNA, 157 bp 5.8s rDNA, 3823 bp 28s rDNA, 530 bp ETS, 2168 bp ITS1 and 288 bp ITS2. Interestingly, some tandem repeats are present in the rRNA intergenic sequence (IGS) and ITS1/2 regions. These results demonstrated that the BAC library of the scorpion M. martensii Karsch and the complete sequence of rDNA unit will provide important genetic resources and tools for comparative genomics and phylogenetic analysis.