Study on the Analgesic Activity of Peptide from Conus achates.

BACKGROUND: As a peptide originally discovered from Conus achates by mass spectrometry and cDNA sequencing, Ac6.4 contains 25 amino acid residues and three disulfide bridges. Our previous study found that this peptide possesses 80% similarity to MVIIA by BLAST and that MVIIA is a potent and selective blocker of N-type voltage-sensitive calcium channels in neurons. OBJECTIVE: To recognize the target protein and analgesic activity of Ac6.4 from Conus achates. METHODS: In the present study, we synthesized Ac6.4, expressed the Trx-Ac6.4 fusion protein, tested Ac6.4 for its inhibitory activity against Cav2.2 in CHO cells and investigated Ac6.4 and Trx-Ac6.4 for their analgesic activities in mice. RESULTS: Data revealed that Ac6.4 had strong inhibitory activity against Cav2.2 (IC50 = 43.6 nM). After intracranial administration of Ac6.4 (5, 10, 20 µg/kg) and Trx-Ac6.4 (20, 40, 80 µg/kg), significant analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent. CONCLUSION: This study expands our knowledge of the peptide Ac6.4 and provides new possibilities for developing Cav2.2 inhibitors and analgesic drugs.

[Highly toxic type â ¡ ribosome-inactivating proteins ricin and abrin and their detection methods: a review].

Type Ⅱ ribosome-inactivating proteins (RIPs) are an important class of protein toxins that consist of A and B chains linked by an interchain disulfide bond. The B-chain with lectin-like activity is responsible for binding to the galactose-containing receptors on eukaryotic cell surfaces, which is essential for A-chain internalization by endocytosis. The A-chain has N-glycosidase activity that irreversibly depurinates a specific adenine from 28S ribosomal RNA (28S rRNA) and terminates protein synthesis. The synergistic effect of the A-B chain inactivates the ribosome, inhibits protein synthesis, and exhibits high cytotoxicity. Ricin and abrin that are expressed by the plants Ricinus communis and Abrus precatorius, respectively, are typical type Ⅱ RIPs. The toxicity of ricin and abrin are 385 times and 2885 times, respectively, more that of the nerve agent VX. Owing to their ease of preparation, wide availability, and potential use as a bioterrorism agent, type Ⅱ RIPs have garnered increasing attention in recent years. Ricin is listed as a prohibited substance under schedule 1A of the Chemical Weapons Convention (CWC). The occurrence of ricin-related bioterrorism incidents in recent years has promoted the development of accurate, sensitive, and rapid detection and identification technology for type Ⅱ RIPs. Significant progress has been made in the study of toxicity mechanisms and detection methods of type Ⅱ RIPs, which primarily involve qualitative and quantitative analysis methods including immunological assays, mass spectrometry analysis methods, and toxin activity detection methods based on depurination and cytotoxicity. Immunoassays generally involve the specific recognition of antigens and antibodies, which is based on oligonucleotide molecular recognition elements called aptamers. These methods are fast and highly sensitive, but for highly homologous proteins in complex samples, they provide false positive results. With the rapid development of biological mass spectrometry detection technology, techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are widely used in the identification of proteins. These methods not only provide accurate information on molecular weight and structure of proteins, but also demonstrate accurate quantification. Enzyme digestion combined with mass spectrometry is the predominantly used detection method. Accurate identification of protein toxins can be achieved by fingerprint analysis of enzymatically digested peptides. For analysis of protein toxins in complex samples, abundant peptide markers are obtained using a multi-enzyme digestion strategy. Targeted mass spectrometry analysis of peptide markers is used to obtain accurate qualitative and quantitative information, which effectively improves the accuracy and sensitivity of the identification of type Ⅱ RIP toxins. Although immunoassay and mass spectrometry detection methods can provide accurate identification of type Ⅱ RIPs, they cannot determine whether the toxins will retain potency. The widely used detection methods for activity analysis of type Ⅱ RIPs include depurination assay based on N-glycosidase activity and cytotoxicity assay. Both the methods provide simple, rapid, and sensitive analysis of type Ⅱ RIP toxicity, and complement other detection methods. Owing to the importance of type Ⅱ RIP toxins, the Organization for the Prohibition of Chemical Weapons (OPCW) has proposed clear technical requirements for the identification and analysis of relevant samples. We herein reviewed the structural characteristics, mechanism of action, and the development and application of type Ⅱ RIP detection methods; nearly 70 studies on type Ⅱ RIP toxins and their detection methods have been cited. In addition to the technical requirements of OPCW for the unambiguous identification of biotoxins, the trend of future development of type Ⅱ RIP-based detection technology has been explored.

[Determination of cyanogen chloride in organic and water matrices by gas chromatography-mass spectrometry based on thiol derivatization].

Cyanogen chloride (ClCN) has been widely used in industrial production. ClCN is also listed in the Schedule of the Chemical Weapons Convention (CWC). The use of traditional colorimetric analysis or gas chromatography for the detection of ClCN has been characterized by low efficiency and poor sensitivity. In this study, a method was established for the qualitative analysis and quantitative detection of ClCN in organic and water matrices by gas chromatography-mass spectrometry (GC-MS) based on thiol derivatization. 1-Butylthiol was selected as the optimal derivatization reagent. The optimal temperature for thiol derivatization in the organic matrices was 40 ℃ and the reaction time was 10 min. The pH for derivatization was approximately 9. The ClCN in the organic matrices was directly analyzed by GC-MS after derivatization. The conditions of ClCN derivatization in the water matrices were the same as those in the organic matrices. After the derivatization of ClCN, headspace-solid phase microextraction (HS-SPME) was employed during sample preparation for water matrices. Different temperatures for HS-SPME were explored, and the optimal temperature was found to be 55 ℃. The product of thiol derivatization was confirmed as butyl thiocyanate. The main fragmentation patterns and mass spectrometric cleavage pathway were investigated by GC-MS/MS. The quantitative determination of ClCN in organic and water matrices was conducted via the internal standard and external standard methods, respectively. ClCN showed good linearity in the corresponding ranges in the organic and water matrices. The correlation coefficients for both matrices were greater than 0.99. The linearities of ClCN in the organic and water matrices were in the range of 20-2000 µg/L and 20-1200 µg/L, respectively. An organic sample and water samples from different substrates were selected to verify the accuracy and precision of the method at three spiked levels. The average spiked recoveries of ClCN in the organic sample and water samples were 87.3%-98.8% and 97.6%-102.2%, respectively. The corresponding relative standard deviations (RSDs, n=6) were 2.1%-4.7% and 2.8%-4.2%. The derivatization method established in this study showed good reaction specificity. The method was successfully applied in the analysis of samples obtained from the Organisation for the Prohibition of Chemical Weapons (OPCW). The method established in this study for the detection of ClCN showed high sensitivity and precision, and could aid in the analysis and detection of ClCN in the environment.

Folic acid modified copper nanoclusters for fluorescent imaging of cancer cells with over-expressed folate receptor.

Water-soluble and functional copper nanoclusters (CuNCs) were prepared by using folic acid (FA) that serves both as a reducing reagent and a stabilizer. FA also acts as a functional ligand on the surface of the CuNCs, and this can be exploited to target the folate receptor which is over-expressed on the surface of HeLa cells. The FA-modified CuNCs nanoclusters have an average size of ca. 0.9 nm and are stable in aqueous medium for 30 days. Under photoexcitation at λex 270 and 350 nm, the FA-CuNCs display strong blue fluorescence with an emission peak at 440 nm. The FA-CuNCs exhibit low cytotoxicity and favorable biocompatibility as demonstrated by an MTT assay. A cell viability of >80% is found when incubating HeLa cells for 20 h with FA-CuNCs at levels of up to 200 µg mL-1. The targeting capability of the FA-CuNCs is demonstrated by live cell imaging. It is shown that HeLa cells with over-expressed folate receptor are much brighter than A549 cells where the receptor is not over-expressed. This is further corroborated by the fact that the copper content in HeLa cells (1.5 pg/cell) is 6.5-fold higher than that of A549 cells (0.23 pg/cell), both measured after the same incubation time of 3 h. If free FA is introduced into the cell culture medium, the folate receptors will be preoccupied with FA, and this results in a significant decrease in the cellular uptake of the FA-CuNCs by HeLa cells. Graphical Abstract Biocompatible copper nanoclusters (CuNCs) coated with folic acid (FA) were prepared and are shown to be viable probes for the differentiation between FR-positive HeLa cells and FR-negative A549 cells.

Protein-Stabilized Gadolinium Oxide-Gold Nanoclusters Hybrid for Multimodal Imaging and Drug Delivery.

A protein-stabilized multifunctional theranostic nanoplatform, gadolinium oxide-gold nanoclusters hybrid (Gd2O3-AuNCs), is constructed for multimodal imaging and drug delivery. The Gd2O3-AuNCs nanohybrid is developed by integrating Gd2O3 nanocrystals and gold nanoclusters into bovine serum albumin scaffold as a stabilizer. The nanohybrid exhibits favorable biocompatibility and is capable of enhancing the contrast in magnetic resonance and X-ray computed tomography imaging. Meanwhile, the integrated AuNCs component not only endows the nanohybrid to produce red fluorescence, but also sensitizes the generation of singlet oxygen (1O2) upon near-infrared laser stimulation at 808 nm. Bovine serum albumin surrounding the nanoparticles makes Gd2O3-AuNCs a brilliant carrier for the delivery of indocyanine green (ICG). ICG loading endows the Gd2O3-AuNCs-ICG nanocomposite with a near-infrared fluorescence imaging capability, and improves its photodynamic property and photothermal capability. Ultimately, further experiments have demonstrated that Gd2O3-AuNCs-ICG nanocomposite is a promising theranostic agent for image guided cancer therapy.

Hydrophobic Carbon Nanodots with Rapid Cell Penetrability and Tunable Photoluminescence Behavior for in Vitro and in Vivo Imaging.

Tunable fluorescent emission and applications in both in vitro and in vivo imaging of hydrophobic carbon nanodots (CNDs) with rapid penetration capability are reported. The hydrophobic CNDs are prepared via hydrothermal treatment of ionic liquid 1-ethyl-3-methylimidazolium bromide and exhibit excitation-dependent photoluminescence behavior along with a red-shift in the excitation/emission maxima with concentration. The quantum yields of the as-prepared CNDs are in the range of 2.5-4.8% at an excitation wavelength of 300-600 nm. The rapid penetration behavior (within 1 min) of CNDs into the cell membrane significantly reduces the sample treatment time and avoids potential fluorescence quenching induced by the interaction between CNDs and samples. A co-location study reveals that the hydrophobic CNDs are distributed mainly in the lysosome. The potentials of the hydrophobic CNDs as fluorescent probe in in vitro and in vivo imaging are well demonstrated by the labeling of HeLa cells, MCF-7 cells, A549 cells, and Kunming mice.