Search

Nanosystems play an important role in many applications. Due to their complexity, it is challenging to accurately characterize their structure and properties. An important means to reach such a goal is computational simulation, which is grounded on ab initio electronic structure calculations. Low scaling and accurate electronic-structure algorithms have been developed in recent years. Especially, the efficiency of hybrid density functional calculations for periodic systems has been significantly improved. With electronic structure information, simulation methods can be developed to directly obtain experimentally comparable data. For example, scanning tunneling microscopy images can be effectively simulated with advanced algorithms. When the system we are interested in is strongly coupled to environment, such as the Kondo effect, solving the hierarchical equations of motion turns out to be an effective way of computational characterization. Furthermore, the first principles simulation on the excited state dynamics rapidly emerges in recent years, and nonadiabatic molecular dynamics method plays an important role. For nanosystem involved chemical processes, such as graphene growth, multiscale simulation methods should be developed to characterize their atomic details. In this review, we review some recent progresses in methodology development for computational characterization of nanosystems. Advanced algorithms and software are essential for us to better understand of the nanoworld.

The epitaxial growth of single-crystal thin films relies on the availability of a single-crystal substrate and a strong interaction between epilayer and substrate. Previous studies have reported the roles of the substrate (

The potential energy surface (PES) is crucial for interpreting a variety of chemical reaction processes. However, predicting accurate PESs with high-level electronic structure methods is a challenging task due to the high computational cost. As an appealing application of quantum computing, we show in this work that variational quantum algorithms can be integrated with machine learning (ML) techniques as a promising scheme for exploring accurate PESs. Different from using a ML model to represent the potential energy, we encode the molecular geometry information into a deep neural network (DNN) to represent parameters of the variational quantum eigensolver (VQE), leaving the PES to the wave function ansatz. Once the DNN model is trained, the variational optimization procedure that hinders the application of the VQE to complex systems is avoided, and thus the evaluation of PESs is significantly accelerated. Numerical results demonstrate that a simple DNN model is able to reproduce accurate PESs for small molecules.

High-temperature reactions widely exist in nature. However, they are difficult to characterize either experimentally or computationally. The minimum energy path (MEP) model routinely used in comput...

Chemical reactions on metal surfaces are important in various processes such as heterogeneous catalysis and nanostructure growth. At moderate or lower temperatures, these reactions generally follow the minimum energy path, and temperature effects can be reasonably described by a harmonic oscillator model. At a high temperature approaching the melting point of the substrate, general behaviors of surface reactions remain elusive. In this study, by taking hydrocarbon species adsorbed on Cu(111) as a model system and performing extensive molecular dynamics simulations powered by machine learning potentials, we identify several important high-temperature effects, including local chemical environment, surface atom mobility, and substrate thermal expansion. They affect different aspects of a high-temperature surface reaction in different ways. These results deepen our understanding of high-temperature reactions.

Spider venoms are known to contain various toxins that are used as an effective means to capture their prey or to defend themselves against predators. An investigation of the properties of Ornithoctonus huwena (O.huwena) crude venom found that the venom can block neuromuscular transmission of isolated mouse phrenic nerve-diaphragm and sciatic nerve-sartorius preparations. However, little is known about its electrophysiological effects on cardiac myocytes. In this study, electrophysiological activities of ventricular myocytes were detected by 100 μg/mL venom of O.huwena, and whole cell patch-clamp technique was used to study the acute effects of the venom on action potential (AP), sodium current (INa), potassium currents (IKr, IKs, Ito1 and IK1) and L-type calcium current (ICaL). The results indicated that the venom prolongs APD90 in a frequency-dependent manner in isolated neonatal rat ventricular myocytes. 100 μg/mL venom inhibited 72.3 ± 3.6% INa current, 58.3 ± 4.2% summit current and 54 ± 6.1% the end current of IKr, and 65 ± 3.3% ICaL current, yet, didn't have obvious effect on IKs, Ito1 and IK1 currents. In conclusion, the O.huwena venom represented a multifaceted pharmacological profile. It contains abundant of cardiac channel antagonists and might be valuable tools for investigation of both channels and anti- arrhythmic therapy development.

The electron-phonon (e-ph) interaction in ${\mathrm{Ca}}_{2}\mathrm{N}$ monolayer, the first electrene material with two-dimensional (2D) electron gas floating in free space, is expected to be very weak, which can be used to design weak-scattering transport channels. Therefore, it is highly desirable to quantitatively evaluate the carrier mobility of this electrene material. In this study, the e-ph interaction in ${\mathrm{Ca}}_{2}\mathrm{N}$ monolayer is investigated by using a precise Wannier interpolation-based first-principles technique. The calculated e-ph coupling matrix elements of ${\mathrm{Ca}}_{2}\mathrm{N}$ monolayer are indeed small compared with other 2D materials such as graphene, which leads to an intrinsic mobility of $189\phantom{\rule{4pt}{0ex}}{\mathrm{cm}}^{2}\phantom{\rule{0.16em}{0ex}}{\mathrm{V}}^{\ensuremath{-}1}\phantom{\rule{0.16em}{0ex}}{\mathrm{s}}^{\ensuremath{-}1}$, which is much higher than those of conventional metals. Other factors affecting mobility are discussed in a comparison with graphene. It is predicted that, based on a momentum mismatch mechanism, mobility of the ${\mathrm{Ca}}_{2}\mathrm{N}$ monolayer can be increased further to above $3000\phantom{\rule{4pt}{0ex}}{\mathrm{cm}}^{2}\phantom{\rule{0.16em}{0ex}}{\mathrm{V}}^{\ensuremath{-}1}\phantom{\rule{0.16em}{0ex}}{\mathrm{s}}^{\ensuremath{-}1}$ via hole doping. Our results confirm that ${\mathrm{Ca}}_{2}\mathrm{N}$ electrene is a promising electronic material.

Kv4 potassium channels are responsible for transient outward K+ currents in the cardiac action potential (AP). Previous experiments by our group demonstrated that Jingzhaotoxin-V (JZTX-V) selectively inhibits A-type potassium channels. However, the specific effects of JZTX-V on the transient outward (Ito) current of cardiomyocytes and underlying mechanism of action remain unclear. In the current study, 100 nM JZTX-V effectively inhibited the Ito current and extended the action potential duration (APD) of neonatal rat ventricular myocytes (NRVM). We further analyzed the effects of JZTX-V on Kv4.2, a cloned channel believed to underlie the Ito current in rat cardiomyocytes. JZTX-V inhibited the Kv4.2 current with a half-maximal inhibitory concentration (IC50) of 13 ± 1.7 nM. To establish the molecular mechanism underlying the inhibitory action of JZTX-V on Kv4.2, we performed alanine scanning mutagenesis of Kv4.2 and JZTX-V and assessed the effects of the mutations on binding activities of the proteins. Interestingly, the Kv4.2 mutations V285A, F289A, and V290A reduced the affinity for JZTX-V while I275A and L277A increased the affinity for JZTX-V. Moreover, mutation of positively charged residues (R20 and K22) of JZTX-V and the hydrophobic patch (formed by W5, M6, and W7) led to a significant reduction in toxin sensitivity, indicating that the hydrophobic patch and electrostatic interactions played key roles in the binding of JZTX-V with Kv4.2. Data from our study have shed light on the specific roles and molecular mechanisms of JZTX-V in the regulation of Ito potassium channels and supported its utility as a potential novel antiarrhythmic drug.

Jingzhaotoxin-34 (JZTX-34) is a selective inhibitor of tetrodotoxin-sensitive (TTX-S) sodium channels. In this study, we found that JZTX-34 selectively acted on Nav1.7 with little effect on other sodium channel subtypes including Nav1.5. If the DIIS3-S4 linker of Nav1.5 is substituted by the correspond linker of Nav1.7, the sensitivity of Nav1.5 to JZTX-34 extremely increases to 1.05 µM. Meanwhile, a mutant D816R in the DIIS3-S4 linker of Nav1.7 decreases binding affinity of Nav1.7 to JZTX-34 about 32-fold. The reverse mutant R800D at the corresponding position in Nav1.5 greatly increased its binding affinity to JZTX-34. This implies that JZTX-34 binds to DIIS3-S4 linker of Nav1.7 and the critical residue of Nav1.7 is D816. Unlike β-scorpion toxin trapping sodium channel in an open state, activity of JZTX-34 requires the sodium channel to be in a resting state. JZTX-34 exhibits an obvious analgesic effect in a rodent pain model. Especially, it shows a longer duration and is more effective than morphine in hot pain models. In a formalin-induced pain model, JZTX-34 at dose of 2 mg/kg is equipotent with morphine (5 mg/kg) in the first phase and several-fold more effective than morphine in second phase. Taken together, our data indicate that JZTX-34 releases pain by selectively binding to the domain II voltage sensor of Nav1.7 in a closed configuration.

In this paper, we demonstrate a Hybrid Manufacturing software-HybridCAM. HybridCAM provides a variety of tool path generation methods for sculpture surfaces. The tool path patterns include contour-parallel pattern, zigzag pattern, and helical pattern. HybridCAM can input multiple format of models, such as IGS, STL, and STP, and can be applied to multi-axis Additive Manufacturing (AM) and multi-axis Subtractive Manufacturing (SM). We present several tool path patterns for different parts and different applications generated by HybridCAM. An actual experiment was carried out to verity the feasibility and practicality of the software.

This paper presents a novel geometric simulation technique for multi-axis Additive Manufacturing (AM). In the proposed methodology, additive swept volume elements, which are represented with Tri-dexel models, are formulated for updating the virtual additive material. Triangular meshes are extracted from the Tri-dexel models using Marching Cube Algorithm for visualization. With the proposed methodology, either three-axis or five-axis AM tool paths of sculpture surfaces can be simulated before actually manufactured. Workpieces after additive manufacturing can be used as blanks for further Subtractive Manufacturing(SM). We developed a geometric simulation software based on the multi-axis AM simulation algorithm, and we carried out an actual three-axis AM experiment to compare with the simulation results. Computer implementation and practical example demonstrate the feasibility of the proposed multi-axis AM simulation method.

Our previous work demonstrated that huwentoxin-IV was an inhibitor cystine knot peptide from Chinese tarantula Ornithoctonus huwena venom that blocked tetrodotoxin-sensitive voltage-gated sodium channels from mammalian sensory neurons [Peng, K., Shu, Q., Liu, Z., Liang, S., 2002. Function and solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider Selenocosmia huwena. J. Biol. Chem. 277(49), 47564-47571]. However, the actions of the neurotoxin on central neuronal sodium channels remain unknown. In this study, we chemically synthesized native huwentoxin-IV and found that sodium channel isoforms from rat hippocampus neurons were also sensitive to native and synthetic toxins, but the toxin-binding affinity (IC(50) approximately 0.4 microM) was 12-fold lower than to peripheral isoforms. The blockade by huwentoxin-IV could be reversed by strong depolarization due to the dissociation of toxin-channel complex as observed for receptor site 3 toxins. Moreover, small unilamellar vesicle-binding assays showed that in contrast to ProTx-II from the tarantula Thrixopelma pruriens, huwentoxin-IV almost lacked the ability to partition into the negatively charged and neutral phospholipid bilayer of artificial membranes. These findings indicated that huwentoxin-IV was a sodium channel antagonist preferentially targeting peripheral isoforms via a mechanism quite different from ProTx-II.

Peptide toxins often have divergent pharmacological functions and are powerful tools for a deep review on the current understanding of the structure-function relationships of voltage-gated sodium channels (VGSCs). However, knowing about the interaction of site 3 toxins from tarantula venoms with VGSCs is not sufficient. In the present study, using whole-cell patch clamp technique, we determined the effects of Jingzhaotoxin-I (JZTX-I) on five VGSC subtypes expressed in HEK293 cells. The results showed that JZTX-I could inhibit the inactivation of rNav1.2, rNav1.3, rNav1.4, hNav1.5 and hNav1.7 channels with the IC50 of 870 ± 8 nM, 845 ± 4 nM, 339 ± 5 nM, 335 ± 9 nM, and 348 ± 6 nM, respectively. The affinity of the toxin interaction with subtypes (rNav1.4, hNav1.5, and hNav1.7) was only 2-fold higher than that for subtypes (rNav1.2 and rNav1.3). The toxin delayed the inactivation of VGSCs without affecting the activation and steady-state inactivation kinetics in the physiological range of voltages. Site-directed mutagenesis indicated that the toxin interacted with site 3 located at the extracellular S3-S4 linker of domain IV, and the acidic residue Asp at the position1609 in hNav1.5 was crucial for JZTX-I activity. Our results provide new insights in single key residue that allows toxins to recognize distinct ion channels with similar potency and enhance our understanding of the structure-function relationships of toxin-channel interactions.

Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity. Background: Spider venom is a complex mixture and contains over 400 venom peptides/species. Results: Highly abundant hypermutation, fragment insertion/deletion, and variable post-translational modifications were observed in venom gland, and the highly diverse toxins exhibit diverse functions. Conclusion: Spider toxins are abundant and variable, factors that work together to generate diverse bioactivities. Significance: Our study reveals the mechanism of structural and functional diversity of peptide toxins from Ornithoctonus hainana.

We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3'-rapid amplification of cDNA ends and 5'-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC(50) value of 0.38 microm in rat cardiac myocytes. Different from scorpion beta-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 microm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion alpha-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.

Intermediate-conductance Ca2+-activated K+ (IK) channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to be potent activators of IK channels. However, the poor selectivity of these molecules limits their therapeutic value. Venom-derived peptides usually block their targets with high specificity. Therefore, we searched for novel peptide activators of IK channels by testing a series of toxins from spiders. Using electrophysiological experiments, we identified hainantoxin-I (HNTX-I) as an IK-channel activator. HNTX-I has little effect on voltage-gated Na+ and Ca2+ channels from rat dorsal root ganglion neurons and on the heterologous expression of voltage-gated rapidly activating delayed rectifier K+ channels (human ether-à-go-go-related gene; human ERG) in HEK293T cells. Only 35.2% ± 0.4% of the currents were activated in SK channels, and there was no effect on BK channels. We demonstrated that HNTX-I was not a phrenic nerve conduction blocker or acutely toxic. This is believed to be the first report of a peptide activator effect on IK channels. Our study suggests that the activity and selectivity of HNTX-I on IK channels make HNTX-I a promising template for designing new drugs for cardiovascular diseases.

Cardiac ion channels are membrane-spanning proteins that allow the passive movement of ions across the cell membrane along its electrochemical gradient, which regulates the resting membrane potential as well as the shape and duration of the cardiac action potential. Additionally, they have been recognized as potential targets for the actions of neurotransmitters, hormones and drugs of cardiac diseases. Spider venoms contain high abundant of toxins that target diverse ion channels and have been considered as a potential resource of new constituents with specific pharmacological properties. However, few peptides from spider venoms were detected as cardiac channel antagonists. In order to explore the effects of the venom of Ornithoctonus hainana on the action potential and ionic currents of neonatal rat ventricular myocytes (NRVMs), whole cell patch clamp technique was used to record action potential duration (APD), sodium current (I Na ), L calcium current (I CaL ), rapidly activating and inactivating transient outward currents (I to1 ), rapid (I Kr ) and slow (I Ks ) components of the delayed rectifier currents and the inward rectifier currents (I K1 ). Our results showed that 100 μg/mL venom obviously prolonged APDs. Significantly, the venom could inhibit I Na and I CaL effectively, while no evident inhibitory effects on cardiac K + channels (I to1 , I ks , I kr and I k1 ) were observed, suggesting that the venom represented a multifaceted pharmacological profile. The effect of venom on Na + and Ca 2+ currents of ventricular myocytes revealed that the hainan venom as a rich resource of cardiac channel antagonists might be valuable tools for the investigation of both channels and drug development.

The voltage-gated sodium channel (VGSC) interacting peptide is of special interest for both basic research and pharmaceutical purposes. In this study, we established a yeast-two-hybrid based strategy to detect the interaction(s) between neurotoxic peptide and the extracellular region of VGSC. Using a previously reported neurotoxin JZTX-III as a model molecule, we demonstrated that the interactions between JZTX-III and the extracellular regions of its target hNav1.5 are detectable and the detected interactions are directly related to its activity. We further applied this strategy to the screening of VGSC interacting peptides. Using the extracellular region of hNav1.5 as the bait, we identified a novel sodium channel inhibitor SSCM-1 from a random peptide library. This peptide selectively inhibits hNav1.5 currents in the whole-cell patch clamp assays. This strategy might be used for the large scale screening for target-specific interacting peptides of VGSCs or other ion channels.

N-type calcium channels play important roles in the control of neurotransmission release and transmission of pain signals to the central nervous system. Their selective inhibitors are believed to be potential drugs for treating chronic pain. In this study, a novel neurotoxin named Huwentoxin-XVI (HWTX-XVI) specific for N-type calcium channels was purified and characterized from the venom of Chinese tarantula Ornithoctonus huwena. HWTX-XVI is composed of 39 amino acid residues including six cysteines that constitute three disulfide bridges. HWTX-XVI could almost completely block the twitch response of rat vas deferens to low-frequency electrical stimulation. Electrophysiological assay indicated that HWTX-XVI specifically inhibited N-type calcium channels in rat dorsal root ganglion cells (IC50 ∼60 nM). The inhibitory effect of HWTX-XVI on N-type calcium channel currents was dose-dependent and similar to that of CTx-GVIA and CTx-MVIIA. However, the three peptides exhibited markedly different degrees of reversibility after block. The toxin had no effect on voltage-gated T-type calcium channels, potassium channels or sodium channels. Intraperitoneal injection of the toxin HWTX-XVI to rats elicited significant analgesic responses to formalin-induced inflammation pain. Toxin treatment also changed withdrawal latency in hot plate tests. Intriguingly, we found that intramuscular injection of the toxin reduced mechanical allodynia induced by incisional injury in Von Frey test. Thus, our findings suggest that the analgesic potency of HWTX-XVI and its greater reversibility could contribute to the design of a novel potential analgesic agent with high potency and low side effects.

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.

With conserved structural scaffold and divergent electrophysiological functions, animal toxins are considered powerful tools for investigating the basic structure-function relationship of voltage-gated sodium channels. Jingzhaotoxin-III (β-TRTX-Cj1α) is a unique sodium channel gating modifier from the tarantula Chilobrachys jingzhao, because the toxin can selectively inhibit the activation of cardiac sodium channel but not neuronal subtypes. However, the molecular basis of JZTX-III interaction with sodium channels remains unknown. In this study, we showed that JZTX-III was efficiently expressed by the secretory pathway in yeast. Alanine-scanning analysis indicated that 2 acidic residues (Asp1, Glu3) and an exposed hydrophobic patch, formed by 4 Trp residues (residues 8, 9, 28 and 30), play important roles in the binding of JZTX-III to Nav1.5. JZTX-III docked to the Nav1.5 DIIS3-S4 linker. Mutations S799A, R800A, and L804A could additively reduce toxin sensitivity of Nav1.5. We also demonstrated that the unique Arg800, not emerging in other sodium channel subtypes, is responsible for JZTX-III selectively interacting with Nav1.5. The reverse mutation D816R in Nav1.7 greatly increased the sensitivity of the neuronal subtype to JZTX-III. Conversely, the mutation R800D in Nav1.5 decreased JZTX-III's IC₅₀ by 72-fold. Therefore, our results indicated that JZTX-III is a site 4 toxin, but does not possess the same critical residues on sodium channels as other site 4 toxins. Our data also revealed the underlying mechanism for JZTX-III to be highly specific for the cardiac sodium channel.

Jingzhaotoxin-V(JZTX-V) isolated from the venom of the spider Chilobrachys jingzhao is a novel potent inhibitor that acts on tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in adult rat dorsal root ganglion(DRG) neurons. It is a 29-residue polypeptide toxin including three disulfide bridges. To investigate the structure-function relationship of the toxin, a mutant of JZTX-V in which Arg20 was substituted by Ala, was synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. The synthetic linear peptide was then purified by reversed-phase high performance liquid chromatography and oxidatively refolded under the optimal conditions. The refolded product was analyzed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF MS) and electrophysiological experiments for its relative molecular weight and prohibitive activity of sodium channels respectively. The present findings show that the prohibitive effect of R20A-JZTX-V on TTX-S sodium channels in DRG neurons is almost the same as that of native JZTX-V, suggesting that Arg20 does not play any important role in inhibiting TTX-S sodium currents in DRG neurons. In contrast, the prohibitive level of R20A-JZTX-V on TTX-R sodium channels is reduced by at last 18.3 times, indicating that Arg20 is a key amino acid residue relative to the bioactivity of JZTX-V. It is presumed that the decrease in activity of R20A-JZTX-V is due to the changes of the property in the binding site in TTX-R sodium channels.

Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide with three disulfide bonds, was a novel spider neurotoxin preferentially inhibiting cardiac sodium channel inactivation. Its activities of phospholipid membrane-binding were studied by a combination of reversed-phase high performance liquid chromatography (HPLC) and fluorescence spectroscopy. Small unilamellar vesicles binding assays showed that the partitioning of JZTX-I into lipid bilayer did not require negatively charged phospholipids. Further, JZTX-I also exhibited a blue shift of 6.4 nm or 4.7 nm as well as red-edge excitation shift of 7.4 nm or 8.0 nm in the presence of 75% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE )/25% 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (POPG) or 100% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles respectively, suggesting that some tryptophan residues on the hydrophobic surface of the toxin were located within a motion restricted membrane interfacial region. Fluorescence quenching experiments suggested that some tryptophan residues of JZTX-I were positioned within the membrane and protected from aqueous quenching agents. These findings should provide further insight into the molecular mechanism of the channel gating of JZTX-I.

An N-terminal tyrosine residue truncate of Jingzhaotoxin-V (Y1-JZTX-V) was synthesized by solid-phase chemical methods using Fmoc-protected amino acids. Reversed-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding of Y1-JZTX-V to find the optimal renaturation conditions of the synthetic linear peptide. When Y1-JZTX-V (0.05 mg/L) was dissolved in 0.1 mol/L Tris-HCl buffer containing 1 mmol/L GSH and 0.1 mmol/L GSSG at pH 7.50 and 4 degrees C, the best renaturation yield of the truncate toxin was obtained. Under the whole-cell patch-clamp mode, Y1-JZTX-V could inhibit tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents in adult rat dorsal root ganglion neurons with IC50 values of 160 nmol/L and 39.6 nmol/L, respectively. The inhibition potentiality of Y1-JZTX-V on TTX-S sodium currents was almost the same as the natural JZTX-V, while that on TTX-R sodium currents was obviously weakened. The IC50 value of Y1-JZTX-V on TTX-R sodium currents was 5.8 times as many as that of natural JZTX-V. Present findings indicated that the first tyrosine residue (Y1) in the N-terminal of JZTX-V was involved in the binding activities of JZTX-V to TTX-R sodium channels.

Hainantoxin-III (HNTX-III) was synthesized using solid-phase chemical method with Fmoc-protected amino acids. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to monitor the oxidative refolding of linear HNTX-III. It is found that the best refolding yield for 0.1 g/L linear HNTX-III could be obtained in double-distilled aqueous solution at pH 7.5 containing 1.0 mol/L L-arginine, 1.0 mmol/L reduced glutathione (GSH), and 0.1 mmol/L oxidized glutathione (GSSG). The relative molecular mass of the refolded HNTX-III was determined to be 3 607.68 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Only one peak was found in the RP-HPLC chromatogram for the mixed sample of native HNTX-III and refolded HNTX-III at ratio of 1 : 1, and the effect of refolded HNTX-III on neuromuscular transmission in isolated mouse phrenic nerve-diaphragm preparations was almost same as that of native HNTX-III. The results indicate that refolded HNTX-III had the same structure and biological activities as the native HNTX-III.

Jingzhaotoxin-XII (JZTX-XII), a 29-residue polypeptide, was purified from the venom of the Chinese tarantula Chilobrachys jingzhao. Electrophysiological recordings carried out in Xenopus laevis oocytes showed that JZTX-XII is specific for Kv4.1 channels, with the IC50 value of 0.363 μM. It interacts with the channels by modifying the gating behavior. JZTX-XII shares 80% sequence identity with phrixotoxin1, a potent inhibitor for Kv4.2 and Kv4.3 channels. Structure analysis indicates that the difference of the charge distribution in the interactive surface perhaps influences the specific pharmacology of the toxins. JZTX-XII should be a valuable tool for the investigation of the Kv4.1 channels.

Jingzhaotoxin-V (JZTX-V), a 29-residue polypeptide, is derived from the venom of the spider Chilobrachys jingzhao. Its cDNA determined by rapid amplification of 3' and 5'-cDNA ends encoded an 83-residue precursor with a pro-region of 16 residues. JZTX-V inhibits tetrodotoxin-resistant and tetrodotoxin-sensitive sodium currents in rat dorsal root ganglion neurons with IC50 values of 27.6 and 30.2 nM, respectively. Moreover, the toxin exhibits high affinity to the resting closed states of the channels. JZTX-V also inhibits Kv4.2 potassium currents expressed in Xenpus Laevis oocytes (IC50=604.2 nM), but has no effects on outward delay-rectified potassium channels expressed in Xenopus laevis oocytes. JZTX-V alters the gating properties of sodium channels by shifting the activation curves to the depolarizing direction and the inactivation curves to the hyperpolarizing direction. Small unilamellar vesicles binding assays show that the partitioning of JZTX-V into lipid bilayer requires negatively charged phospholipids. The phospholipid membrane binding activity of JZTX-V is also verified using intrinsic tryptophan fluorescence analysis as well as acrylamide-quenching assays. Importantly, human multiple sodium channel subtypes are attractive targets for treatment of pain, highlighting the importance of JZTX-V as potential lead for drug development.

The spider Macrothele raveni was recently identified as a new species of Genus Macrothele. The crude venom from M. raveni was found to be neurotoxic to mice and the LD50 of the crude venom in mice was 2.852 mg/kg. Two neurotoxic peptides, raventoxin-I and raventoxin-III, were isolated from the crude venom by ion-exchange and reverse phase high performance liquid chromatography. Raventoxin-I was the most abundant toxic component in the venom, while raventoxin-III was a lower abundant component. Both toxins can kill mice and block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation, but have no effect on cockroaches. The LD50 of raventoxin-I in mice is 0.772 mg/kg. The complete amino acid sequences of raventoxin-I and raventoxin-III were determined and found to consist of 43 and 29 amino acid residues, respectively. It was determined by mass spectrometry that all Cys residues from raventoxin-I and raventoxin-III are involved in disulphide bonds. raventoxin-III showed no significant sequence homology with any presently known neurotoxins in the protein/DNA databases, while raventoxin-I has limited sequence identity with δ-AcTx-Hv1 and δ-AcTx-Ar1, which target both mammalian and insect sodium channels. Both raventoxin-I and raventoxin-III only work on vertebrates, but not on insects. Moreover, raventoxin-I could exert an effect of first exciting and then inhibiting the contraction of mouse diaphragm muscle caused by electrically stimulating the phrenic nerve, but raventoxin-III could not.

Spider venom comprises a mixture of compounds with diverse biological activities, which are used to capture prey and defend against predators. The peptide components bind a broad range of cellular targets with high affinity and selectivity, and appear to have remarkable structural diversity. Although spider venoms have been intensively investigated over the past few decades, venomic strategies to date have generally focused on high-abundance peptides. In addition, the lack of complete spider genomes or representative cDNA libraries has presented significant limitations for researchers interested in molecular diversity and understanding the genetic mechanisms of toxin evolution. In the present study, second-generation sequencing technologies, combined with proteomic analysis, were applied to determine the diverse peptide toxins in venom of the Chinese bird spider Ornithoctonus huwena. In total, 626 toxin precursor sequences were retrieved from transcriptomic data. All toxin precursors clustered into 16 gene superfamilies, which included six novel superfamilies and six novel cysteine patterns. A surprisingly high number of hypermutations and fragment insertions/deletions were detected, which accounted for the majority of toxin gene sequences with low-level expression. These mutations contribute to the formation of diverse cysteine patterns and highly variable isoforms. Furthermore, intraspecific venom variability, in combination with variable transcripts and peptide processing, contributes to the hypervariability of toxins in venoms, and associated rapid and adaptive evolution of toxins for prey capture and defense.