Gut microbiota regulates the ALK5/NOX1 axis by altering glutamine metabolism to inhibit ferroptosis of intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (ICC) is a kind of hepatobiliary tumor that is increasing in incidence and mortality. The gut microbiota plays a role in the onset and progression of cancer, however, the specific mechanism by which the gut microbiota acts on ICC remains unclear. In this study, feces and plasma from healthy controls and ICC patients were collected for 16S rRNA sequencing or metabolomics analysis. Gut microbiota analysis showed that gut microbiota abundance and biodiversity were altered in ICC patients compared with controls. Plasma metabolism analysis showed that the metabolite glutamine content of the ICC patient was significantly higher than that of the controls. KEGG pathway analysis showed that glutamine plays a vital role in ICC. In addition, the use of antibiotics in ICC animals further confirmed that changes in gut microbiota affect changes in glutamine. Further experiments showed that supplementation with glutamine inhibited ferroptosis and downregulated ALK5 and NOX1 expression in HuCCT1 cells. ALK5 overexpression or NOX1 overexpression increased NOX1, p53, PTGS2, ACSL4, LPCAT3, ROS, MDA and Fe2+ and decreased FTH1, SLC7A11 and GSH. Knockdown of NOX1 suppressed FIN56-induced ferroptosis. In vivo, supplementation with glutamine promoted tumor growth. Overexpression of ALK5 repressed tumor growth and induced ferroptosis in nude mice, which could be reversed by the addition of glutamine. Our results suggested that the gut microbiota altered glutamine metabolism to inhibit ferroptosis in ICC by regulating the ALK5/NOX1 axis.

Structure and polymerization of liquid sulfur across the λ-transition.

The anomalous λ-transition of liquid sulfur, which is supposed to be related to the transformation of eight-membered sulfur rings into long polymeric chains, has attracted considerable attention. However, a detailed description of the underlying dynamical polymerization process is still missing. Here, we study the structures and the mechanism of the polymerization processes of liquid sulfur across the λ-transition as well as its reverse process of formation of the rings. We do so by performing ab initio-quality molecular dynamics simulations thanks to a combination of machine learning potentials and state-of-the-art enhanced sampling techniques. With our approach, we obtain structural results that are in good agreement with the experiments and we report precious dynamical insights into the mechanisms involved in the process.

Swarming magnetic nanorobots bio-interfaced by heparinoid-polymer brushes for in vivo safe synergistic thrombolysis.

Biocompatible swarming magnetic nanorobots that work in blood vessels for safe and efficient targeted thrombolytic therapy in vivo are demonstrated. This is achieved by using magnetic beads elaborately grafted with heparinoid-polymer brushes (HPBs) upon the application of an alternating magnetic field B(t). Because of the dense surface charges bestowed by HPBs, the swarming nanorobots demonstrate reversible agglomeration-free reconfigurations, low hemolysis, anti-bioadhesion, and self-anticoagulation in high-ionic-strength blood environments. They are confirmed in vitro and in vivo to perform synergistic thrombolysis efficiently by "motile-targeting" drug delivery and mechanical destruction. Moreover, upon the completion of thrombolysis and removal of B(t), the nanorobots disassemble into dispersed particles in blood, allowing them to safely participate in circulation and be phagocytized by immune cells without apparent organ damage or inflammatory lesion. This work provides a rational multifaceted HPB biointerfacing design strategy for biomedical nanorobots and a general motile platform to deliver drugs for targeted therapies.

O1-conotoxin Tx6.7 cloned from the genomic DNA of Conus textile that inhibits calcium currents.

Background: Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. Methods: In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDWCPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Results: Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 µM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Conclusion: Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.

Swarming Responsive Photonic Nanorobots for Motile-Targeting Microenvironmental Mapping and Mapping-Guided Photothermal Treatment.

Micro/nanorobots can propel and navigate in many hard-to-reach biological environments, and thus may bring revolutionary changes to biomedical research and applications. However, current MNRs lack the capability to collectively perceive and report physicochemical changes in unknown microenvironments. Here we propose to develop swarming responsive photonic nanorobots that can map local physicochemical conditions on the fly and further guide localized photothermal treatment. The RPNRs consist of a photonic nanochain of periodically-assembled magnetic Fe3O4 nanoparticles encapsulated in a responsive hydrogel shell, and show multiple integrated functions, including energetic magnetically-driven swarming motions, bright stimuli-responsive structural colors, and photothermal conversion. Thus, they can actively navigate in complex environments utilizing their controllable swarming motions, then visualize unknown targets (e.g., tumor lesion) by collectively mapping out local abnormal physicochemical conditions (e.g., pH, temperature, or glucose concentration) via their responsive structural colors, and further guide external light irradiation to initiate localized photothermal treatment. This work facilitates the development of intelligent motile nanosensors and versatile multifunctional nanotheranostics for cancer and inflammatory diseases.

Ultrasmall Fe2O3 Tubular Nanomotors: The First Example of Swarming Photocatalytic Nanomotors Operating in High-Electrolyte Media.

Self-propelled chemical micro/nanomotors (MNMs) have demonstrated considerable potential in targeted drug delivery, (bio)sensing, and environmental remediation due to their autonomous nature and possible intelligent self-targeting behaviors (e.g., chemotaxis and phototaxis). However, these MNMs are commonly limited by their primary propulsion mechanisms of self-electrophoresis and electrolyte self-diffusiophoresis, making them prone to quenching in high electrolyte environments. Thus, the swarming behaviors of chemical MNMs in high-electrolyte media remain underexplored, despite their potential to enable the execution of complex tasks in high-electrolyte biological media or natural waters. In this study, we develop ultrasmall tubular nanomotors that exhibit ion-tolerant propulsions and collective behaviors. Upon vertical upward UV irradiation, the ultrasmall Fe2O3 tubular nanomotors (Fe2O3 TNMs) demonstrate positive superdiffusive photogravitaxis and can further self-organize into nanoclusters near the substrate in a reversible manner. After self-organization, the Fe2O3 TNMs exhibit a pronounced emergent behavior, allowing them to switch from random superdiffusions to ballistic motions near the substrate. Even at a high electrolyte concentration (Ce), the ultrasmall Fe2O3 TNMs retain a relatively thick electrical double layer (EDL) compared to their size, and the electroosmotic slip flow in their EDL is strong enough to propel them and induce phoretic interactions among them. As a result, the nanomotors can rapidly concentrate near the substrate and then gather into motile nanoclusters in high-electrolyte environments. This work opens a gate for designing swarming ion-tolerant chemical nanomotors and may expedite their applications in biomedicine and environmental remediation.

Lighting up Micro-/Nanorobots with Fluorescence.

Micro-/nanorobots (MNRs) can be autonomously propelled on demand in complex biological environments and thus may bring revolutionary changes to biomedicines. Fluorescence has been widely used in real-time imaging, chemo-/biosensing, and photo-(chemo-) therapy. The integration of MNRs with fluorescence generates fluorescent MNRs with unique advantages of optical trackability, on-the-fly environmental sensitivity, and targeting chemo-/photon-induced cytotoxicity. This review provides an up-to-date overview of fluorescent MNRs. After the highlighted elucidation about MNRs of various propulsion mechanisms and the introductory information on fluorescence with emphasis on the fluorescent mechanisms and materials, we systematically illustrate the design and preparation strategies to integrate MNRs with fluorescent substances and their biomedical applications in imaging-guided drug delivery, intelligent on-the-fly sensing and photo-(chemo-) therapy. In the end, we summarize the main challenges and provide an outlook on the future directions of fluorescent MNRs. This work is expected to attract and inspire researchers from different communities to advance the creation and practical application of fluorescent MNRs on a broad horizon.

Synthesis and evaluation of a novel analgesic conotoxin Lt7b that inhibits calcium currents and increases sodium currents.

Conotoxins are promising neuropharmacological tools and drug candidates due to their high efficiency and specificity in targeting ion channels or neurotransmitter receptors. In this study, a novel O2 -superfamily conotoxin, Lt7b, was synthesized and its pharmacological functions were evaluated. Lt7b with three modified amino acids and three disulfide bonds was successfully synthesized. CD spectra showed that Lt7b had a typical α-helix in the secondary structure. Patch clamp experiments on rat DRG neurons showed that Lt7b could significantly inhibit calcium currents with an IC50 value of 856 ± 95 nM. Meanwhile, 10 µM Lt7b could significantly increase the sodium currents by 77 ± 8%, but it had no obvious effects on the potassium currents in DRG neurons. In addition, patch clamp experiments on ion channel subtypes showed that 10 µM Lt7b could inhibit 7.0 ± 1.2%, 8.0 ± 1.5%, 4.6 ± 3.4%, and 9.5 ± 0.1% of the hCav 1.2, hCav 2.1, hCav 2.2, and hCav 3.2 currents, respectively, while it did not increase the rNav 1.7, rNav 1.8, hNav 1.5, hNav 1.7, and hNav 1.8 currents. Lt7b had no obvious toxicity to HaCaT and ND7/23 cells up to 1 mM and significantly increased the pain threshold at the testing time of 0.5-4 h in a dose-dependent manner in the mouse hotplate assay. This novel conotoxin Lt7b may be a useful tool for ion channel studies and analgesic drug development.

Analysis of complexity and dynamic functional connectivity based on resting-state EEG in early Parkinson's disease patients with mild cognitive impairment.

To explore the abnormal brain activity of early Parkinson's disease with mild cognitive impairment (ePD-MCI) patients, the study analyzed the dynamic fluctuation of electroencephalogram (EEG) signals and the dynamic change of information communication between EEG signals of ePD-MCI patients. In this study, we recorded resting-state EEG signals of 30 ePD-MCI patients and 37 early Parkinson's disease without mild cognitive impairment (ePD-nMCI) patients. First, we analyzed the difference of the complexity of EEG signals between the two groups. And we found that the complexity in the ePD-MCI group was significantly higher than that in the ePD-nMCI group. Then, by analyzing the dynamic functional network (DFN) topology based on the optimal sliding-window, we found that the temporal correlation coefficients of ePD-MCI patients were lower in the delta and theta bands than those in the ePD-nMCI patients. The temporal characteristic path length of ePD-MCI patients in the alpha band was higher than that of ePD-nMCI patients. In the theta and alpha bands, the temporal small world degrees of ePD-MCI patients were lower than that of patients with ePD-nMCI. In addition, the functional connectivity strength of ePD-MCI patients affected by cognitive impairment was weaker than that of ePD-nMCI patients, and the stability of dynamic functional connectivity network was decreased. This finding may serve as a biomarker to identify ePD-MCI and contribute to the early intervention treatment of ePD-MCI.

Synthesis and characterization of αM-conotoxin SIIID, a reversible human α7 nicotinic acetylcholine receptor antagonist.

α-Conotoxins, a group of small marine peptide toxins that target nAChRs with high potency and selectivity, are valuable pharmacological tools and potential drug leads. In this study, we reported the synthesis and physiological functions of a novel αM-superfamily conotoxin SIIID (CCGEGSSCPKYFKNNFICGCC) from a fish-hunting Conus striatus. Three SIIID isomers with different cystine connectivities were synthesized by solid-phase polypeptide synthesis and confirmed by mass spectrometry. Patch clamp experiments on HEK293 cells expressing nAChR subtypes showed that 1 µM SIIID (1-4, 2-5, 3-6) inhibited PNU-120596 and acetylcholine induced human α7 nAChR currents by 48.45%, which was higher than 5.08% of SIIID (1-5, 2-4, 3-6) and 9.57% of SIIID (1-6, 2-4, 3-5). Further study on the most active SIIID isomer showed that 10 µM SIIID inhibited PNU-120596 and acetylcholine induced human α7 nAChR currents by 76.33% but had no obvious effect on acetylcholine induced human α3ß4 nAChR currents. In addition, SIIID inhibited PNU-120596 and acetylcholine induced human α7 nAChR currents with an IC50 value of 880.71 ± 271.91 nM, and this inhibition was reversible. Patch clamp experiments on rat DRG neurons showed that 10 µM SIIID had <15% inhibitory effects on sodium, potassium and calcium currents. Our results suggested that SIIID would be a promising neuropharmacology tool for the study of human α7 nAChR and its related diseases.

Liquid-Liquid Critical Point in Phosphorus.

The study of liquid-liquid phase transitions has attracted considerable attention. One interesting example of this phenomenon is phosphorus, for which the existence of a first-order phase transition between a low density insulating molecular phase and a conducting polymeric phase has been experimentally established. In this Letter, we model this transition by an ab initio quality molecular dynamics simulation and explore a large portion of the liquid section of the phase diagram. We draw the liquid-liquid coexistence curve and determine that it terminates into a second-order critical point. Close to the critical point, large coupled structure and electronic structure fluctuations are observed.

A novel proline-rich M-superfamily conotoxin that can simultaneously affect sodium, potassium and calcium currents.

BACKGROUND: Conotoxins have become a research hotspot in the neuropharmacology field for their high activity and specificity in targeting ion channels and neurotransmitter receptors. There have been reports of a conotoxin acting on two ion channels, but rare reports of a conotoxin acting on three ion channels. METHODS: Vr3a, a proline-rich M-superfamily conotoxin from a worm-hunting Conus varius, was obtained by solid-phase synthesis and identified by mass spectrometry. The effects of synthesized Vr3a on sodium, potassium and calcium currents were tested on rat DRG cells by patch clamp experiments. The further effects of Vr3a on human Cav1.2 and Cav2.2 currents were tested on HEK293 cells. RESULTS: About 10 µM Vr3a has no effects on the peak sodium currents, but can induce a ~10 mV shift in a polarizing direction in the current-voltage relationship. In addition, 10 µM Vr3a can increase 19.61 ± 5.12% of the peak potassium currents and do not induce a shift in the current-voltage relationship. An amount of 10 µM Vr3a can inhibit 31.26% ± 4.53% of the peak calcium currents and do not induce a shift in the current-voltage relationship. The IC50 value of Vr3a on calcium channel currents in rat DRG neurons is 19.28 ± 4.32 µM. Moreover, 10 µM Vr3a can inhibit 15.32% ± 5.41% of the human Cav1.2 currents and 12.86% ± 4.93% of the human Cav2.2 currents. CONCLUSIONS: Vr3a can simultaneously affect sodium, potassium and calcium currents. This novel triple-target conotoxin Vr3a expands understanding of conotoxin functions.

Suppressing ERK Pathway Impairs Glycochenodeoxycholate-Mediated Survival and Drug-Resistance in Hepatocellular Carcinoma Cells.

Glycochenodeoxycholate (GCDA), a toxic component in bile salts, is involved in carcinogenesis of gastrointestinal tumors. The objective of this research was to study the function of ERK1/2 in the GCDA-mediated survival and drug-resistance in hepatocellular carcinoma cells (HCCs). Firstly, extracellular signal-regulated kinase 1/2 (ERK1/2) was detected extensively expressed in liver cancer cells, and silencing ERK1/2 by RNA interference could suppress GCDA-stimulated survival and promote apoptosis. Furthermore, phosphorylation of endogenous ERK1/2 could be potently stimulated by GCDA in combination with enhanced chemoresistance in QGY-7703 hepatocellular carcinoma cells. The GCDA-mediated proliferation and chemoresistance could be impaired by PD98059, which acted as an inhibitor to block the phosphorylation of ERK1/2. Mechanistically, PD98059 was able to potently suppress GCDA-stimulated nuclear aggregation of ERK1/2 and p-ERK1/2, upregulate pro-survival protein Mcl-1 and downregulate pro-apoptotic protein Bim. The results of this study indicated that disruption of ERK1/2 by blocking phosphorylation or nuclear translocation may put forward new methods for solving the problem of GCDA-related proliferation and drug-resistance in liver cancer treatment.

Lactiplantibacillus plantarum AR113 Exhibit Accelerated Liver Regeneration by Regulating Gut Microbiota and Plasma Glycerophospholipid.

Emerging evidence indicates that probiotics have been proved to influence liver injury and regeneration. In the present study, the effects of Lactiplantibacillus plantarum AR113 on the liver regeneration were investigated in 70% partial hepatectomy (PHx) rats. Sprague-Dawley (SD) rats were gavaged with L. plantarum AR113 suspensions (1 × 1010 CFU/mL) both before and after partial hepatectomy. The results showed that L. plantarum AR113 administration 2 weeks before partial hepatectomy can accelerate liver regeneration by increased hepatocyte proliferation and tumor necrosis factor-α (TNF-α), hepatocyte growth factor (HGF), and transforming growth factor-ß (TGF-ß) expression. Probiotic administration enriched Lactobacillus and Bacteroides and depleted Flavonifractor and Acetatifactor in the gut microbiome. Meanwhile, L. plantarum AR113 showed decline of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidyl serine (PS), and lysophosphatidyl choline (LysoPC) levels in the serum of the rats after the L. plantarum AR113 administration. Moreover, L. plantarum AR113 treated rats exhibited higher concentrations of L-leucine, L-isoleucine, mevalonic acid, and lower 7-oxo-8-amino-nonanoic acid in plasma than that in PHx. Spearman correlation analysis revealed a significant correlation between changes in gut microbiota composition and glycerophospholipid. These results indicate that L. plantarum AR113 is promising for accelerating liver regeneration and provide new insights regarding the correlations among the microbiome, the metabolome, and liver regeneration.

A novel proline-rich M-superfamily conotoxin that can simultaneously affect sodium, potassium and calcium currents

Background Conotoxins have become a research hotspot in the neuropharmacology field for their high activity and specificity in targeting ion channels and neurotransmitter receptors. There have been reports of a conotoxin acting on two ion channels, but rare reports of a conotoxin acting on three ion channels. Methods Vr3a, a proline-rich M-superfamily conotoxin from a worm-hunting Conus varius, was obtained by solid-phase synthesis and identified by mass spectrometry. The effects of synthesized Vr3a on sodium, potassium and calcium currents were tested on rat DRG cells by patch clamp experiments. The further effects of Vr3a on human Cav1.2 and Cav2.2 currents were tested on HEK293 cells. Results About 10 μM Vr3a has no effects on the peak sodium currents, but can induce a ~10 mV shift in a polarizing direction in the current-voltage relationship. In addition, 10 μM Vr3a can increase 19.61 ± 5.12% of the peak potassium currents and do not induce a shift in the current-voltage relationship. An amount of 10 μM Vr3a can inhibit 31.26% ± 4.53% of the peak calcium currents and do not induce a shift in the current-voltage relationship. The IC50 value of Vr3a on calcium channel currents in rat DRG neurons is 19.28 ± 4.32 μM. Moreover, 10 μM Vr3a can inhibit 15.32% ± 5.41% of the human Cav1.2 currents and 12.86% ± 4.93% of the human Cav2.2 currents. Conclusions Vr3a can simultaneously affect sodium, potassium and calcium currents. This novel triple-target conotoxin Vr3a expands understanding of conotoxin functions.(AU)

miR-let-7a-5p Inhibits Invasion and Migration of Hepatoma Cells by Regulating BZW2 Expression.

PURPOSE: This study was performed to investigate the effect of miRNA let-7a-5p on the proliferation, invasion, and migration of human hepatoma cells as well as to determine BZW2 expression in these cells. METHODS: Western blotting and real-time quantitative polymerase chain reaction were used to detect changes in the expression of miRNA let-7a-5p and BZW2 protein and gene, respectively. A luciferase reporter gene assay was used to examine whether BZW2 is the target gene of miR-let-7a-5p. The effect of miR-let-7a-5p on the invasion, migration, and proliferation of human hepatoma Bel-7404 and HepG2 cells was determined using the transwell invasion assay, scratch test, and CCK-8 assay, respectively. Flow cytometry was used to assess the effect of miR-let-7a-5p and BZW2 expression on apoptosis of hepatoma cells. RESULTS: The luciferase reporter gene assay identified BZW2 as the target gene of miR-let-7a-5p. Moreover, increased expression of miR-let-7a-5p was found to significantly decrease BZW2 expression; inhibit proliferation, invasion, and migration; and promote apoptosis of hepatoma cells. CONCLUSION: miR-let-7a-5p can inhibit proliferation, invasion, and migration as well as promote apoptosis of hepatoma cells by decreasing BZW2 expression.

µ-conotoxin TsIIIA, a peptide inhibitor of human voltage-gated sodium channel hNav1.8.

TsIIIA, the first µ-conotoxin from Conus tessulatus, can selectively inhibit rat tetrodotoxin-resistant sodium channels. TsIIIA also shows potent analgesic activity in a mice hotplate analgesic assay, but its effect on human sodium channels remains unknown. In this study, eight human sodium channel subtypes, hNav1.1- hNav1.8, were expressed in HEK293 or ND7/23 cells and tested on the chemically synthesized TsIIIA. Patch clamp experiments showed that 10 µM TsIIIA had no effects on the tetrodotoxin-sensitive hNav1.1, hNav1.2, hNav1.3, hNav1.4, hNav1.6 and hNav1.7, as well as tetrodotoxin-resistant hNav1.5. For tetrodotoxin-resistant hNav1.8, concentrations of 1, 5 and 10 µM TsIIIA reduced the hNav1.8 currents to 59.26%, 36.21% and 24.93% respectively. Further detailed dose-effect experiments showed that TsIIIA inhibited hNav1.8 currents with an IC50 value of 2.11 µM. In addition, 2 µM TsIIIA did not induce a shift in the current-voltage relationship of hNav1.8. Taken together, the hNav1.8 peptide inhibitor TsIIIA provides a pharmacological probe for sodium channels and a potential therapeutic agent for pain.

miR-132-5p regulates apoptosis and autophagy in MPTP model of Parkinson's disease by targeting ULK1.

Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by a loss of dopaminergic neurons in the substantia nigra of the brain. Numerous investigations have focused on the underlying mechanism involved in the progression of PD in recent decades. miR-132 is abnormal expression in many diseases including PD. However, the functional role and molecular mechanism of miR-132-5p in PD pathogenesis are still not elucidated. In our study, we found miR-132-5p was upregulated in 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP) model of PD. MTT assay and flow cytometric analysis revealed that inhibition of miR-132-5p increased cell survival ability and reduced MPTP-induced apoptosis of SH-SY5Y cells. Furthermore, inhibition of miR-132-5p could significantly suppressed mRNA and protein expression levels of LC3 and Beclin 1, indicating inhibition of miR-132-5p might restrain autophagy in PD. Subsequently, ULK1 was identified as a target of miR-132-5p and positively regulated by miR-132-5p at both mRNA and protein levels. Additionally, ectopic expression of ULK1 was able to reverse the effects of miR-132-5p inhibition. Taken together, our results demonstrated that miR-132-5p inhibition might exert a protective role in MPTP-treated PD models by targeting ULK1, indicating that miR-132-5p may be a prospective therapeutic target for PD.

Insertions and Deletions Play an Important Role in the Diversity of Conotoxins.

Previous studies have indicated that each conotoxin precursor has a hyperconserved signal region, a rather conserved pro region and a hypervariable mature region, and nucleotide mutations are the main driven factor. However, in this study, we made an in-depth analysis on the M-superfamily conotoxin precursors and found that the diversity of the signal, pro and mature regions are more complicated than previous findings. Different conotoxin precursors can have same signal, pro and/or mature regions, especially different conotoxin precursors with same mature region but different signal and pro regions. In addition, insertions and deletions (indels) were detected in conotoxin precursors. Indels are infrequent in the signal region but frequent in the pro and mature regions. In contrast to deletions that dominate in the pro region, insertions dominate in the mature region. The number of amino acids is crucial for the physiological functions of mature conotoxins, therefore indels, especially insertions in the mature region, play an important role in the sequence and function diversity of conotoxins.