Similar neurotoxin expression profiles of traditional Chinese scorpion medicine material between juvenile and adult Mesobuthus martensii scorpions revealed by multiple strategic proteomics.

ETHNOPHARMACOLOGICAL RELEVANCE: The Mesobuthus martensii scorpions, called as "Quanxie", are known Chinese medicinal material base on the "Combat poison with poison" strategy for more than one thousand years, and still widely used to treat various diseases according to the the Pharmacopoeia of the People's Republic of China nowadays. AIM OF STUDY: The study aims to investigate the similarity of scorpion neurotoxins at the protein level between the juvenile and adult Mesobuthus martensii scorpions as Chinese medicine materials. MATERIALS AND METHODS: The second-, third- and fourth-instar, and adult Mesobuthus martensii scorpions were collected for the characterization of neurotoxin expression through multiple strategic proteomics, including undigested scorpion venom, endopeptidase-digested, and undigested scorpion telson extract for the sample analysis. RESULTS: Based on the known 107 scorpion neurotoxins from the genomic and transcriptomic analysis of adult Mesobuthus martensii scorpions, the multiple strategic proteomics first revealed that neurotoxins exhibited more stability in telson extract than secreted venom. In the reported transcripts of scorpion neurotoxins, approximately 53%, 56%, 66% and 78% of neurotoxins were detected through undigested scorpion venom, the endopeptidase Arg-C-, Lys-C-digested telson extract, and undigested telson extract strategies, respectively. Nearly 79% of scorpion neurotoxins detected in third-instar Mesobuthus martensii scorpions represent the largest number of scorpion neurotoxins from proteomic analysis to date. Moreover, a total of 84% of scorpion neurotoxins were successfully identified at the protein level, and similar neurotoxin expression profiles in second-, third- and fourth-instar, and adult Mesobuthus martensii scorpions were first revealed by the multiple strategic proteomics. CONCLUSION: These findings for the first time demonstrate the similar neurotoxin expression profiles between the juvenile and adult Mesobuthus martensii scorpions as Chinese medicinal material, which would serve as a paradigm for further toxin analysis from different venomous animals.

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.

Distribution, sources, and eco-risk of Current-Use Pesticides (CUPs) in the coastal waters of the northern Shandong Peninsula, China.

This study investigated the spatial distributions and seasonal variations of 19 CUPs in the coastal areas of the Shandong Peninsula and its surrounding rivers and assessed their ecological risk. In freshwater and seawater, insecticides (chlorpyrifos, methoxychlor, and pyridaben), as well as fungicides (fenarimol) and herbicides (dichlobenil) were the main pollutants (Detection Frequency: 100 %). Spatially, during winter, the regional pollution levels of Σ19CUPs in seawater showed a trend of Laizhou Bay (LZB, mean:4.13 ng L-1) > Yellow River Estuary (YRE, mean:2.57 ngL-1) > Bohai Bay (BHB, mean:2.21 ng L-1) > Yanwei Area (YWA, mean:1.94 ng L-1). The similarities of major substances between rivers and the marine environment suggest that river discharge is the main source of CUPs pollution in coastal areas. In summer, CUPs in rivers posed a high risk. In winter, the risk significantly decreased, indicating a moderate overall risk. Seawater exhibited a low risk in winter.

Scorpion venom peptides: Molecular diversity, structural characteristics, and therapeutic use from channelopathies to viral infections and cancers.

Animal venom is an important evolutionary innovation in nature. As one of the most representative animal venoms, scorpion venom contains an extremely diverse set of bioactive peptides. Scorpion venom peptides not only are 'poisons' that immobilize, paralyze, kill, or dissolve preys but also become important candidates for drug development and design. Here, the review focuses on the molecular diversity of scorpion venom peptides, their typical structural characteristics, and their multiple therapeutic or pharmaceutical applications in channelopathies, viral infections and cancers. Especially, the group of scorpion toxin TRPTx targeting transient receptor potential (TRP) channels is systematically summarized and worthy of attention because TRP channels play a crucial role in the regulation of homeostasis and the occurrence of diseases in human. We also further establish the potential relationship between the molecular characteristics and functional applications of scorpion venom peptides to provide a research basis for modern drug development and clinical utilization of scorpion venom resources.

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.

Mechanism of agonist-induced activation of the human itch receptor MRGPRX1.

Mas-related G-protein-coupled receptors X1-X4 (MRGPRX1-X4) are 4 primate-specific receptors that are recently reported to be responsible for many biological processes, including itch sensation, pain transmission, and inflammatory reactions. MRGPRX1 is the first identified human MRGPR, and its expression is restricted to primary sensory neurons. Due to its dual roles in itch and pain signaling pathways, MRGPRX1 has been regarded as a promising target for itch remission and pain inhibition. Here, we reported a cryo-electron microscopy (cryo-EM) structure of Gq-coupled MRGPRX1 in complex with a synthetic agonist compound 16 in an active conformation at an overall resolution of 3.0 Å via a NanoBiT tethering strategy. Compound 16 is a new pain-relieving compound with high potency and selectivity to MRGPRX1 over other MRGPRXs and opioid receptor. MRGPRX1 was revealed to share common structural features of the Gq-mediated receptor activation mechanism of MRGPRX family members, but the variable residues in orthosteric pocket of MRGPRX1 exhibit the unique agonist recognition pattern, potentially facilitating to design MRGPRX1-specific modulators. Together with receptor activation and itch behavior evaluation assays, our study provides a structural snapshot to modify therapeutic molecules for itch relieving and analgesia targeting MRGPRX1.

The tick saliva peptide HIDfsin2 promotes the tick-borne virus SFTSV replication in vitro by enhancing p38 signal pathway.

Pathogens co-evolved with ticks to facilitate blood collection and pathogen transmission. Although tick saliva was recently found to be rich in bioactive peptides, it is still elusive which saliva peptide promotes virus transmission and which pathways are invovled. Here, we used a saliva peptide HIDfsin2 and a severe fever with thrombocytopenia syndrome virus (SFTSV) both carried by the tick Haemaphysalis longicornis to elucidate the relationship between tick saliva components and tick-borne viruses. HIDfsin2 was found to promote the replication of SFTSV in a dose-dependent manner in vitro. HIDfsin2 was further revealed to MKK3/6-dependently magnify the activation of p38 MAPK. The overexpression, knockdown and phosphorylation site mutation of p38α indicated that p38 MAPK activation facilitated SFTSV infection in A549 cells. Moreover, the blockade of p38 MAPK activation significantly suppressed SFTSV replication. Differently, HIDfsin2 or pharmacological inhibition of p38 MAPK activation had no effect on a mosquito-borne Zika virus (ZIKV). All these results showed that HIDfsin2 specifically promoted SFTSV replication through the MKK3/6-dependent enhancement of p38 MAPK activation. Our study provides a new perspective on the transmission of tick-borne viruses under natural conditions, and supports that the blockade of p38 MAPK activation can be a promising strategy against the mortal tick-borne virus SFTSV.

The efficacy and cerebral mechanism of intradermal acupuncture for major depressive disorder: a study protocol for a randomized controlled trial.

Background: Major depressive disorder (MDD) has emerged as the fifth leading cause of years lived with disability, with a high prevalent, affecting nearly 4% of the global population. While available evidence suggests that intradermal acupuncture may enhance the effectiveness of antidepressants, whether its efficacy is a specific therapeutic effect or a placebo effect has not been reported. Moreover, the cerebral mechanism of intradermal acupuncture as a superficial acupuncture (usually subcutaneous needling to a depth of 1-2 mm) for MDD remains unclear. Methods: A total of 120 participants with MDD will be enrolled and randomized to the waiting list group, sham intradermal acupuncture group and active intradermal acupuncture group. All 3 groups will receive a 6-week intervention and a 4-week follow-up. The primary outcome will be measured by the Hamilton Depression Rating Scale-17 and the secondary outcome measures will be the Self-Rating depression scale and Pittsburgh sleep quality index. Assessments will be conducted at baseline, 3 weeks, 6 weeks, and during the follow-up period. In addition, 20 eligible participants in each group will be randomly selected to undergo head magnetic resonance imaging before and after the intervention to explore the effects of intradermal acupuncture on brain activity in MDD patients. Discussion: If the intradermal acupuncture is beneficial, it is promising to be included in the routine treatment of MDD. Clinical Trial Registration: Clinicaltrials.gov, NCT05720637.

Discovery and analysis of a novel antimicrobial peptide B1AW from the skin secretion of Amolops wuyiensis and improving the membrane-binding affinity through the construction of the lysine-introduced analogue.

In the development and study of antimicrobial peptides (AMPs), researchers have kept a watchful eye on peptides from the brevinin family because of their extensive antimicrobial activities and anticancer potency. In this study, a novel brevinin peptide was isolated from the skin secretions of the Wuyi torrent frog, Amolops wuyiensis (A. wuyiensisi), named B1AW (FLPLLAGLAANFLPQIICKIARKC). B1AW displayed anti-bacterial activity against Gram-positive bacteria Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA), and Enterococcus faecalis (E. faecalis). B1AW-K was designed to broaden the antimicrobial spectrum of B1AW. The introduction of a lysine residue generated an AMP with enhanced broad-spectrum antibacterial activity. It also displayed the ability to inhibit the growth of human prostatic cancer PC-3, non-small lung cancer H838, and glioblastoma cancer U251MG cell lines. In molecular dynamic (MD) simulations, B1AW-K had a faster approach and adsorption to the anionic membrane than B1AW. Therefore, B1AW-K was considered a drug prototype with a dual effect, which deserves further clinical investigation and validation.

Functional Characterization of a New Degradation Peptide BmTX4-P1 from Traditional Chinese Scorpion Medicinal Material.

Thermally processed Buthus martensii Karsch scorpion is an important traditional Chinese medical material that has been widely used to treat various diseases in China for over one thousand years. Our recent work showed that thermally processed Buthus martensii Karsch scorpions contain many degraded peptides; however, the pharmacological activities of these peptides remain to be studied. Here, a new degraded peptide, BmTX4-P1, was identified from processed Buthus martensii Karsch scorpions. Compared with the venom-derived wild-type toxin peptide BmTX4, BmTX4-P1 missed some amino acids at the N-terminal and C-terminal regions, while containing six conserved cysteine residues, which could be used to form disulfide bond-stabilized α-helical and ß-sheet motifs. Two methods (chemical synthesis and recombinant expression) were used to obtain the BmTX4-P1 peptide, named sBmTX4-P1 and rBmTX4-P1. Electrophysiological experimental results showed that sBmTX4-P1 and rBmTX4-P1 exhibited similar activities to inhibit the currents of hKv1.2 and hKv1.3 channels. In addition, the experimental electrophysiological results of recombinant mutant peptides of BmTX4-P1 indicated that the two residues of BmTX4-P1 (Lys22 and Tyr31) were the key residues for its potassium channel inhibitory activity. In addition to identifying a new degraded peptide, BmTX4-P1, from traditional Chinese scorpion medicinal material with high inhibitory activities against the hKv1.2 and hKv1.3 channels, this study also provided a useful method to obtain the detailed degraded peptides from processed Buthus martensii Karsch scorpions. Thus, the study laid a solid foundation for further research on the medicinal function of these degraded peptides.

A study on the correlation of the asymmetric regulation between the periaqueductal gray and the bilateral trigeminal nucleus caudalis in migraine male rats.

BACKGROUND: The study was designed to explore the correlation of the asymmetric regulation between periaqueductal gray (PAG) and bilateral trigeminal nucleus caudalis (TNC) in migraine rats through studying the changes of metabolites in pain regulatory pathway of acute migraine attack. METHODS: Thirty male Sprague-Dawley (SD) rats were randomly divided into three groups: blank, control, model groups. Then, blank group was intraperitoneally injected with ultrapure water, while control group injected with saline and model group injected with Glyceryl Trinitrate (GTN). Two hours later, PAG and bilateral TNC were removed respectively, and metabolite concentrations of PAG, Left-TNC, Right-TNC were obtained. Lastly, the differences of metabolite among three brain tissues were compared. RESULTS: The relative concentrations of rNAA, rGlu, rGln, rTau, rMI in PAG or bilateral TNC had interaction effects between groups and sites. The concentration of rLac of three brain tissues increased in migraine rats, however, the rLac of LTNC and RTNC increased more than that of PAG. Besides, the concentrations of rNAA and rGln increased in RTNC, while rGABA decreased in RTNC. CONCLUSIONS: There is correlation between PAG, LTNC and RTNC in regulation of pain during acute migraine attack, and the regulation of LTNC and RTNC on pain is asymmetric.

Altered brain activity and functional connectivity in migraine without aura during and outside attack.

BACKGROUND: Migraine is commonly seen as a cyclic disorder with variable cortical excitability at different phases. Herein, we investigated the cortical excitability in migraine without aura patients during an attack (MWoA-DA) and interictal period (MWoA-DI) and further explored the functional connectivity (FC) in brain regions with cortical excitability abnormalities in patients. METHODS: Seven MWoA-DA patients, twenty-seven MWoA-DI patients, and twenty-nine healthy controls (HC) underwent resting-state functional magnetic resonance imaging (rs-fMRI) scan. The amplitude of low-frequency fluctuations (ALFF) was assessed to identify spontaneous brain activity. Then, brain regions showing significant differences across groups were identified as regions of interest (ROI) in FC analysis. RESULTS: Compared with MWoA-DI patients and HC, the ALFF in the trigeminocervical complex (TCC) was higher in the MWoA-DA patients. Decreased FC in MWoA-DA patients was found between TCC and left postcentral gyrus compared with MWoA-DI patients. Compared with HC, ALFF was lower in the right cuneus but higher in the right rolandic operculum of MWoA-DI patients. Additionally, the ALFF in the right cuneus was negatively correlated with the Migraine Disability Assessment Scale (MIDAS) in MWoA-DI patients. CONCLUSIONS: The trigeminovascular system and impairments in descending pain modulatory pathways participate in the pathophysiology of migraine during the ictal period. The defense effect exists in the interictal phase, and the dysfunction in the cuneus may be related to the disease severity. This dynamic change in different brain regions could deepen our understanding of the physiopathology underlying migraine.

Key domains and residues of the receptor MRGPRX1 recognizing the peptide ligand BAM8-22.

Mas-related G protein-coupled receptors (Mrgprs) are a newly discovered class of G protein-coupled receptors consisting of more than 50 members in recent years. MRGPRX1 can be activated by bovine adrenal medulla peptide 8-22 (BAM8-22), triggering Ca2+ influx and then causing pain and itch. It is very important for the discovery of analgesic and antipruritic drugs to elucidate the molecular mechanism of MRGPRX1 recognizing BAM8-22. Here, we identified the functional domains and residues of the receptor MRGPRX1 activating BAM8-22 through molecular model, mutation and living cell calcium imaging. The molecular docking predicted that BAM8-22 interacted with N-terminal, TM4, TM5, TM6 and ECL3 of MRGPRX1. Both ECL3 and TM6 domains were further revealed to play a critical role in the BAM8-22-induced MRGPRX1 activation, whereas TM3 region performed a secondary function. Moreover, the mutation F237A of MRGPRX1 completely lost the activation ability of BAM8-22. These results were consistent with the cryogenic electron microscopy (cryo-EM) structure of MRGPRX1-Gαq in complex with BAM8-22 reported most recently. Taken together, our work shows insights into the molecular mechanism of the interaction between the receptor MRGPRX1 and the peptide agonist BAM8-22, and will also provide some valuable clues for the design of analgesic and antipruritic drugs targeting MRGPRX1.

Impaired fertility in 4930590J08Rik mutant male mice is associated with defective sperm energy metabolism.

Testis-specifically expressed genes are important for male reproduction according to their unique expression patterns. However, the functions of most of these genes in reproduction are unclear. Here, we showed that mouse 4930590J08Rik was a testis-specifically expressed gene. 4930590J08Rik knockout mice exhibited a delay in the first wave of spermatogenesis and a reduction of cauda epididymal sperm. Furthermore, knockout spermatozoa exhibited defective acrosome reactions and decreased progressive motility, which led to impaired in vivo fertilization. Transcriptome analysis of testes revealed that most of the differentially expressed genes in knockout testes were associated with metabolic processes. 4930590J08Rik knockout sperm exhibited oxidative phosphorylation deficiency and were highly dependent on increased anaerobic glycolysis to compensate for ATP demands. Taken together, the 4930590J08Rik-disrupted mouse partially mimics the phenotypes of human asthenospermia and oligozoospermia, which provides a new model for further understanding the pathogenesis of idiopathic male infertility.

Norbisabolane-type sesquiterpenoid derivatives, benzofuran lignans and a phenolic glycoside from the roots of Glochidion wilsonii Hutch.

Three highly oxygenated norbisabolane sesquiterpenoid glycosides (glochiwilsonosides A-C), five benzofuran lignans (glochiwilsonises A-E) and a phenolic glycoside (glochiwilsophe-noside), together with forty-one known compounds, were isolated from the roots of Glochidion wilsonii Hutch. The chemical structures of the compounds were identified by spectroscopic methods and previous literature data. Glochiwilsonoside A displayed anti-proliferative activity on A-549 and RAW 264.7 cell lines with an IC50 value of 34.5 ± 0.9 µM and CC50 value of 16.0 ± 0.9 µM, respectively. Twenty-three known compounds were reported from the genus Glochidion for the first time, and the chemotaxonomic characteristics of the isolated compounds were also summarized. The bisabolane/norbisabolane-type sesqui-terpenoid derivatives could be used as chemotaxonomic markers for G. wilsonii.