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Supplemental material, sj-pdf-1-jbf-10.1177_2280800020963471 for Fabrication of pH-sensitive graphene oxide–Benazepril carrier as biosafety controlled release systems by Tianxiong Liu, Yujiao Li, Xianzhe Chen, Xiaoming Zhao, Jianfang Wang and Dongyi Zhang in Journal of Applied Biomaterials & Functional Materials

Although ultraviolet (UV) light illumination has been widely used to increase the sensitivity of semiconductor gas sensors, its underlying mechanism is still blurred and controversial. In this work, the influence of UV light illumination on the sensitivity of ZnO nanofilm gas sensors is explored experimentally and simulated based on a modified Wolkenstein's model. The influential factors on sensitivity are determined respectively: the surface band bending and Fermi level are measured by Kelvin probe force microscopy, the binding energy and extrinsic surface state are calculated by density functional theory, and the depletion of the whole semiconductor caused by the finite size is illustrated by the transfer characteristics of a field effect transistor. With all these factors taken into consideration, the surface state densities of adsorbed O

Rapid nondestructive determination of the moisture content of tea leaves is the basis of intelligent control in tea processing. Visible and near infrared (Vis/NIR) spectroscopy can detect the fundamental vibrations of hydrogen group (O–H) in the organic molecules, and has become one of the most commonly used methods for rapid determination of the moisture content of tea leaves. However, the established spectral model often fails to predict new samples of different varieties or batches, and then it will limit the wide application of this technology. Hence, improvement in the model and removing the impact of these samples variation on the determination model become a key issue for quality control during tea processing. In this study, tea samples of nine batches from three varieties were adopted to verify the performance of model transfer in improving the generalization of spectral models, it is worth noting that the moisture content distribution of different varieties samples is obviously different. It can be found that spectral profile difference of the three batches of tea samples for each variety was effectively eliminated by model transfer. The prediction ability of the PLSR model was improved by correcting the spectra of the tested batches and varieties of tea leaves to solve the problem induced by sample differences. The determination model developed based on the variety of “Longjing” was successfully transferred to the other two varieties of “Wuniuzao” and “Yingshuang”, and the R p 2 were improved from 0.4343 to 0.2066 to 0.7595 and 0.6376, respectively by using DS algorithm comparing with un-transferred models. This study demonstrated that the proposed model transfer strategy by correcting the spectral data could be a robust technique for the rapid detection of the moisture content in different types of tea leaves and lay a basis for industrial application.

Wilms' tumors are one of the most common malignant, solid intra-abdominal tumors observed in children. Although potential tumor markers have been found, inflammatory cytokines interfere with the process of specific protein identification. The present study was undertaken to identify post-traumatic stress-related factors of Wilms' tumors and to verify the accuracy of early-stage tumor-specific serum protein markers. Serum samples were screened for differentially-expressed proteins using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). Potential markers were isolated and purified using solid-phase extraction (SPE) and SDS-PAGE. Following enzymatic digestion of the protein samples, the peptide fragments were detected with high performance liquid chromatography-mass spectrometry. The obtained peptide mass fingerprint was searched in the Swiss-Prot protein sequence database via the Mascot search engine. Differentially-expressed proteins were verified using western blot analysis. Differentially-expressed proteins with a mass/charge of 5,816 were screened out using SELDI-TOF-MS, and significant differences between the tumor and control groups, and the trauma and control groups were observed. Target proteins were isolated and purified using SPE and SDS-PAGE. Thioredoxin 1 (Trx1) was found to be differentially expressed. In the serum of children with Wilms' tumors, there was an increase in the level of the post-traumatic stress-related inflammatory factor, Trx1, as compared with the normal control group. Thus, the results of this study indicate that Trx1 presents a potential post-traumatic stress-related factor of Wilms' tumors.

As one of the main participant in the electricity market, load serving entities (LSE) purchase electricity in the wholesale market, and provide different types of electricity package to users in the retail market. LSE’s profit mainly comes from the difference between the cost of purchasing electricity and the benefit of selling electricity. To reduce the cost, LSE can optimize electricity consumption characteristics of various types of users through real-time price adjustment, which can help to improve the shape of electricity purchase curve and implement demand side response. In this paper, a real-time pricing strategy based on synchronous perturbation stochastic approximation algorithm (SPSA) is proposed. This method considers the difference between diverse users synthetically. Depending on the historical electricity consumption and historical price data, the user side response function is constructed, and the real-time electricity price is formulated with the objective of minimizing load curve’s peak-valley difference. Finally, the effectiveness and advantages of the proposed method are verified by comparing the load curve and the power purchase cost before and after the price adjustment.

A novel graphene oxide (GO)-based carrier was fabricated for the controlled release of Benazepril (BENA). Freeze dried samples of GO–BENA carrier were prepared for controlled drug release at different pHs (pH = 2, 7, and 10) and release kinetics indicate BENA desorption from GO is by Fickian diffusion. The BENA yield from the carrier amounted to ~55% of the adsorbed material in a strongly acidic medium after 50 h. Binding fractions of BENA to 10 mg/L GO was determined for different solution concentrations of the drug. In vitro assays of cell proliferation (WST-1 kit), cell structural integrity (LDH kit) and flow cytometric indicators of necrosis in three different cell lines (CACO-2, SGC-7901, and primary mouse hepatic fibroblast) all demonstrated that the GO carrier had a good biocompatibility. The pH-dependent release sensitivity of the GO-based carrier suggests that it is a potential candidate for use in the controlled release of drugs in the acidic environment of the stomach.

Beijing is severely affected by land subsidence, and rapid urbanisation and building construction might accelerate the land subsidence process. Based on 39 Envisat Advanced Synthetic Aperture Radar (ASAR) images acquired between 2003–2010, 55 TerraSAR-X images acquired between 2010–2016, and urban building information, we analysed the relationship between land subsidence and buildings at the regional, block, and building scales. The results show that the surface displacement rate in the Beijing urban area ranged from −109 mm/year to +13 mm/year between 2003–2010, and from −151 mm/year to +19 mm/year between 2010–2016; two subsidence bowls were mainly distributed in the eastern part of the Chaoyang District. The displacement rate agreed well with the levelling measurements, with an average bias of less than six mm/year. At the regional scale, the spatial pattern of land subsidence was mainly controlled by groundwater extraction, compressible layer thickness, and geological faults. Subsidence centres were located in the area around ground water funnels with a compressible layer depth of 50–70 m. The block-scale analysis demonstrated a clear correlation between the block construction age and the spatial unevenness of subsidence. The blocks constructed between 1998–2005 and after 2005 showed considerably more subsidence unevenness and temporal instability than the blocks constructed before 1998 during both time periods. The examination of the new blocks showed that the spatial unevenness increased with building volume variability. For the 16 blocks with a high building volume, variability, and subsidence unevenness, the building-scale analysis showed a positive relationship between building volume and settlement in most blocks, although the R2 was lower than 0.5. The results indicate that intense building construction in urban areas could cause differential settlement at the block scale in Beijing, while the settlement of single buildings could be influenced by the integrated effects of building volume, foundation structures, and the hydrogeological background.

In recent years, with the development of remote sensing techniques, image classification based on remote sensing imagery with high spatial resolution has played a significant role. At present, there are mainly two ways to classify the imagery of high resolution: pixel-based method and object-based method. Because of the wide differences of spectral features in pixel-based method, 'salt-and-pepper' effect appears in the classification result, which decreases the accuracy. And the object-based method which is widely used in image classification has a low accuracy of classification about objects with similar spectral features and shape features. Each of the two methods has its advantages and disadvantages. It is generally agreed that object-based classification has an advantage over pixel-based classification. However, the previous study found that the results of object-based classification was deeply influenced by reference sampling method So it is necessary to have a comparison between object and pixel-based method under different reference sampling schemes. In this study, we studied pixel-based method and object-based method, selected samples using 'select samples randomly' method and 'select samples separated by objects' method respectively, and built classification models using SVM and RF classifiers respectively. We compared different classification methods and analyzed the impacts of selection of samples and classifiers on classification results. The research showed that the accuracy of classification depends on the distribution of sample points. When sample points were selected randomly(Rand), object-based method got a higher accuracy; when sample points were selected by reference objects separately(Sep), pixel-based method got a higher accuracy.

Hepatocellular carcinoma (HCC) is one of the most widespread malignant human tumors worldwide. Treatment options include radiotherapy, surgical intervention and chemotherapy; however, drug resistance is an ongoing treatment concern. In the present study, the effects of a microRNA (miR/miRNA), miR-9, on the sensitivity of HCC cell lines to the epidermal growth factor receptor inhibitor, cetuximab, were examined. miR-9 has been proposed to serve a role in tumorigenesis and tumor progression. In the present study, bioinformatics analyses identified the eukaryotic translation initiation factor 5A2 (eIF-5A-2) as a target of miR-9. The expression levels of miR-9 and eIF-5A-2 were examined by reverse transcription-quantitative polymerase chain reaction and HCC cell lines were transfected with miR-9 mimics and inhibitors to determine the effects of the miRNA on cell proliferation and viability. The miR-9 mimic was revealed to significantly increase the sensitivity of epithelial phenotype HCC cells (Hep3B and Huh7) to cetuximab, while the miR-9 inhibitor triggered the opposite effect. There were no significant differences in sensitivity to cetuximab observed in mesenchymal phenotype HCC cells (SNU387 and SNU449). Cells lines displaying high expression levels of eIF-5A-2 were more resistant to cetuximab. Transfection of cells with a miR-9 mimic resulted in downregulation of the expression of eIF-5A-2 mRNA, while an miR-9 inhibitor increased expression. When expression of eIF-5A-2 was knocked down with siRNA, the effects of miR-9 on cetuximab sensitivity were no longer observed. Taken together, these data support a role for miR-9 in enhancing the sensitivity of epithelial phenotype HCC cells to cetuximab through regulation of eIF-5A-2.

Fei Xue,1 Yanhui Liu,2 Hongwei Zhang,1 Yu Wen,1 Lei Yan,1 Qiang Tang,1 Erhui Xiao,1 Dongyi Zhang1 1Department of Hepatobiliary and Pancreatic Surgery, Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital, Zhengzhou, People’s Republic of China; 2Department of Hematology, Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital, Zhengzhou, People’s Republic of China Background: Let-7 miRNAs are reported to play an inhibitory role in carcinogenesis, tumor progression, recurrence, and pluripotency of cancer. However, few studies have reported the relationship between let-7 and drug sensitivity, especially for let-7a (a subtype of let-7). This study aimed to investigate the function of let-7a in regulating the sensitivity of hepatocellular carcinoma (HCC) cell lines to cetuximab. Methods: The cytotoxicity of cetuximab on HCC cell lines (Huh7, Hep3B, HepG2, SNU449, and SNU387) was evaluated using a cell viability assay (the Cell Counting Kit-8 assay) and a cell proliferation assay (the Click-iT EdU Imaging Kit) in the presence of a control, a let-7a mimic, and a let-7a inhibitor. Small interfering RNA to knockdown the expression of signal transducer and activator of transcription 3 (STAT3) were employed. Protein and mRNA expression levels were determined using quantitative polymerase chain reaction and Western blot analysis. Results: It was found that let-7a enhances the sensitivity of HCC cells with an epithelial phenotype (Huh7, Hep3B, and HepG2) to cetuximab, but has no effect on cells with the mesenchymal phenotype (SNU449 and SNU387). It was determined that STAT3 was a target mRNA of let-7a using TargetScan. Expression of STAT3 and let-7a mRNA were negatively correlated in HCC cell lines. Moreover, let-7a altered the protein and mRNA expression of STAT3. Furthermore, STAT3 knockdown enhanced the function of cetuximab on HCC cell lines with epithelial phenotypes, but not on HCC cell lines with mesenchymal phenotypes. Finally, a rescue experiment confirmed that let-7a affected the sensitivity of HCC cell lines to cetuximab by interacting with STAT3. Conclusions: There is a functional link between let-7a and STAT3 in enhancing the sensitivity of HCC cells with an epithelial phenotype to cetuximab. Our results provide novel insight into new methodologies for combating HCC drug resistance. Keywords: hepatocellular carcinoma, microRNA, STAT3, let-7a

Applications of Battery Energy Storage Systems (BESS) in the power grids will be expanded in the coming years due to factors such as decreasing costs of such systems. In a modern power network, BESS systems can be optimally controlled for peak-load shaving and other ancillary services such as volt-var control, system balancing and loss reduction. However, all these “system-level” controls will depend on the capability of each individual BESS to independently control the active and reactive power injection/absorption at its connected bus, and to maintain the voltage of the DC link between its AC/DC and DC/DC converters. As a result, understanding the behavior and control requirements for the operation of the BESS is necessary. In this paper, an active AC/DC rectifier and a bidirectional DC/DC converter have been modeled and analyzed for charging and discharging of the batteries in a BESS system. The models used in these analyses have been developed based on an integrated state-space equation set for the converters and the battery. This type of modeling is proper for demonstration of the effects of massively integrated storage systems in the bulk power grids.

Inorganic lead (Pb) is one of the most common environmental pollutants. Much evidence indicates that Pb exposure could directly affect fish growth and development. In this study, we investigated the cytotoxic effects of Pb on cytoskeletal protein stability at both protein and mRNA level in crucian carp Carassius auratus. Pb(NO3)2 treatment in concentration of 100 μmol/L resulted in decreased expression of both α- and β-tubulin but γ-tubulin as assayed with SDS-PAGE, Western Blot, and ELISA. In vivo and in vitro analyses on protein expression of tubulins are consistent. The effect of Pb on mRNA expression varied among different tissues. Our results suggest that cytotoxicity of Pb at protein translation level is stronger than at mRNA expression level.

As genome sequences of many organisms - including humans - are being decoded, there is a great need for genetic tools to analyze newly discovered genes/proteins. A 'unigenic evolution' approach has been previously proposed for dissecting protein domains, which is based on the assumption that functionally important regions of a protein may tolerate missense mutations less well than other regions. We describe a unigenic evolution analysis of general transcription factor IIB (TFIIB) - a protein that is well characterized both structurally and functionally - to better understand the molecular basis of this genetic approach. The overall distribution profile of hypomutable regions within yeast TFIIB correlates extremely well with the known compact structural domains, suggesting that the unigenic evolution approach can help reveal structural properties of a protein. We further show that a small region located immediately carboxyl-terminal to the zinc ribbon motif is functionally important despite its strong hypermutability. Our study further demonstrates the usefulness of the unigenic evolution approach in dissecting protein domains, but suggests that the mutability of different regions of a protein in such a test is determined primarily by their structural properties.

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.

The spider Ornithoctonus huwena is a venomous spider found in southern China. Its venom is a complex mixture of numerous biologically active components. In this study, 41 novel unique transcripts encoding cellular proteins or other possible venom components were generated from the previously constructed cDNA library. These proteins were also annotated by KOG (eukaryotic orthologous group) and GO (gene ontology) terms. A novel cellular transcript contig encoding an EF-hand protein (named HWEFHP1) was found, which might be involved in the secretion of toxins in the venom glands. In order to have an overview of the molecular diversity of the O. huwena venom, the datasets of all the transcripts, peptides and proteins known so far were analyzed. A comparison of the data obtained through a proteomic versus a transcriptomic approach, revealed that only 15 putative cystine knot toxins (CKTs) were identified by both approaches, 29 transcripts coding for CKTs were found in the transcriptome but not as translated peptides in the venom proteome. However, no cellular protein with identical molecular weight was identified by both approaches. Our data may contribute to a deeper understanding of the biology and ecology of O. huwena and the relationship between structure and function of individual toxins.

Jingzhaotoxin-34 (JZTX-34) is a 35-residue polypeptide from the venom of Chinese tarantula Chilobrachys jingzhao . Our previous work reported its full-length cDNA sequence encoding a precursor with 87 residues. In this study we report the protein expression and biological function characterization. The toxin was efficiently expressed by the secretary pathway in yeast. Under whole-cell patch-clamp mode, the expressed JZTX-34 was able to inhibit tetrodotoxin-sensitive (TTX-S) sodium currents (IC 50 ∼ 85 nM) while having no significant effects on tetrodotoxin-resistant (TTX-R) sodium currents on rat dorsal root ganglion neurons. The inhibition of TTX-S sodium channels was completely reversed by strong depolarization (+120 mV). Toxin treatment altered neither channel activation and inactivation kinetics nor recovery rate from inactivation. However, it is interesting to note that in contrast to huwentoxin-IV, a recently identified receptor site-4 toxin from Ornithoctonus huwena venom, 100 nM JZTX-34 caused a negative shift of steady-state inactivation curve of TTX-S sodium channels by approximately 10 mV. The results indicated that JZTX-34 might inhibit mammalian sensory neuronal sodium channels through a mechanism similar to HWTX-IV by trapping the IIS4 voltage sensor in the resting conformation, but their binding sites should not overlay completely.

By sequencing random clones from the venom gland cDNA library of the spider Ornithoctonus huwena, a transcript, named SHL-Ib1, encoding a lectin-like peptide was cloned. The amino acid sequence of the putative mature peptide of SHL-Ib1 is identical, except for seven different residues, with that of SHL-I, a lectin found in the venom of O. huwena. The mature peptides of SHL-Ib1b and SHL-Ib1c are the mutants of SHL-Ib1 with two or three amino acid residues truncated at the C-terminal. The recombinant SHL-Ib1b and SHL-Ib1c were expressed successfully by the yeast expression system and purified by using a combination of ion-exchange and reverse phase high performance liquid chromatography (HPLC). The molecular masses of the two expressed peptides were identified by mass spectrometry, indicating that the C-terminals of the two peptides were not amidated. The two peptides can agglutinate human erythrocytes at minimal concentrations of 0.75 and 1.475mg/ml, respectively. Structure modeling of SHL-Ib1 has given a clue to the low agglutination bioactivities of these recombinant toxins. These lectin-like peptides, due to the small molecular sizes, may have the advantage to investigate the binding mechanism of the lectin and have the potential to be the carrier for drug delivery.

This paper presents an approach to utilize a Chinese machine-readable dictionary to acquire a conceptual network with five semantic relations; and then, the application of this conceptual resource is discussed text retrieval. Because of the low accuracy of current Chinese parser, our method is different from other traditional dictionary processing that need parsing the explanations of words when constructing this noun-based conceptual network. This construction is designed to be a new three-step process by statistical learning mechanism. This method acquires a relatively satisfying conceptual network based on Chinese nouns. In its application, this network is available for providing linkage-based knowledge for text retrieval.

In this paper we present a new similarity of text on the basis of combining cosine measure with the quantified conceptual relations by linear interpolation for text clustering. These relations derive from the entries and the words in their definitions in a dictionary, which are quantified under the assumption that the entries and their definitions are equivalent in meaning. This kind of relations is regarded as "knowledge" for text clustering. Under the framework of k-means algorithm, the new interpolated similarity improves the performance of clustering system significantly in terms of optimizing hard and soft criterion functions. Our results show that introducing the conceptual knowledge from the un-structured dictionary into the similarity measure tends to provide potential contributions for text clustering in future.

Formation of the gastrula organizer requires suppression of ventralizing signals and, in fish and frog, the need to counteract the effect of ubiquitously present maternal factors that activate the expression of Bmps. How the balance between dorsalizing and ventralizing factors is shifted towards organizer establishment at late blastula stages is not well understood. Mutations in zebrafish bozozok (boz) cause severe defects in axial mesoderm and anterior neurectoderm and affect organizer formation. The boz gene encodes the homeodomain protein Bozozok/Dharma and its expression in the region of the organizer is activated through β-catenin signaling. Here, we investigate the molecular mechanism by which boz contributes to the establishment of the organizer. We demonstrate that the homeodomain protein Boz acts as a transcriptional repressor in zebrafish: overexpression of an En-Boz fusion protein can rescue the boz phenotype, whereas a VP16-Boz fusion protein acts as an antimorph. Expression analysis of bmp2b indicates that Boz negatively regulates bmp2b in the prospective organizer. We demonstrate that this Boz activity is independent of that of other zygotic genes, because it also occurs when translation of zygotic genes is suppressed by cycloheximide(CHX). We identify two high-affinity binding sites for Boz within the first intron of the bmp2b gene. Deletion of these control elements abolishes Boz-dependent repression of bmp2b in the early blastula. Thus, Boz directly represses bmp2b by binding to control elements in the bmp2b locus. We propose that early transcriptional repression of bmp2b by Boz is one of the first steps toward formation of a stable organizer, whereas the later-acting Bmp antagonists (e.g. Chordin, Noggin)modulate Bmp activity in the gastrula to induce patterning along the dorsoventral axis. Thus, similar to Drosophila Dpp, asymmetry of Bmp expression in zebrafish is initiated at the transcriptional level, and the shape of the gradient and its function as a morphogen are later modulated by post-transcriptional mechanisms.

The Drosophila morphogenetic protein Bicoid (Bcd) is a homeodomain-containing activator that stimulates the expression of target genes during early embryonic development. We demonstrate that a small domain of Bcd located immediately N-terminally of the homeodomain represses its own activity in Drosophila cells. This domain, referred to as a self-inhibitory domain, works as an independent module that does not rely on any other sequences of Bcd and can repress the activity of heterologous activators. We further show that this domain of Bcd does not affect its properties of DNA binding or subcellular distribution. A Bcd derivative with point mutations in the self-inhibitory domain severely affects pattern formation and target gene expression in Drosophila embryos. We also provide evidence to suggest that the action of the self-inhibitory domain requires a Drosophila co-factor(s), other than CtBP or dSAP18. Our results suggest that proper action of Bcd as a transcriptional activator and molecular morphogen during embryonic development is dependent on the downregulation of its own activity through an interaction with a novel co-repressor(s) or complex(es).

The positions of the disulfide bonds of huwentoxin-I, a neurotoxin from the spider Selenocosmia huwena, have been determined. The existence of three disulfide bonds in the native toxin was demonstrated by mass spectroscopy and the lack of reactivity with a thiol reagent. The assignment procedure involved a combination of tryptic digestion of the native toxin and sequence analysis of both intact and in situ S-carboxymethylated toxin. In situ carboxymethylation is shown to be a useful procedure in sequencing of cysteine- and cystine-containing peptides. Sequence analysis of the intact, cross-linked toxin indicated that no amino acid phenylthiohydantoin (PTH) derivative is seen for the first half-cystine in a cross-linked pair, but that the PTH of dehydroalanine, which can be detected at 313 nm, is seen at the position of the second half-cystine. By sequencing disulfide cross-linked tryptic fragments, the three disulfide linkages in huwentoxin-I could be assigned as Cys2-Cys17, Cys9-Cys22, and Cys16-Cys29.