Conopeptides (Conotoxin) derived from tropical marine gastropod, cone snail ( Conus ) venoms have been an useful tool agent in neuro investigation and new drug source. Recently, new method of Conotoxin genes cloning from Conus genomic DNA has been established. So to get Conus genomic DNAs rapidly is the basis for diverse Conotoxin gene isolation, gene bank construction and the medical source exploitation. In this report, different DNA extractions from different tissue and organs of six cone snail species were performed in order to optimize the DNA isolation methods. The optimized brief phenol SDS method was established which fit for cone snails genomic DNA extraction.

Two methods of different RNA extractions from Conus venom tube and toxin gland of seven cone snail species were performed. ThermoScript~ TM RNase H~ - Transcriptase and AMV Transcriptase were used to synthesize cDNA separately. The cDNAs were used as templates to amplify conotoxin genes by PCR.The optimized RNA isolation and cDNA synthesis methods were obtained according to RT-PCR results, which would be the basis for cloning of new conotoxin genes.

Objective To isolate and clone novel O-superfamily conotoxin genes of Conus capitaneus Linnaeus collected from Hainan,which would provide the initial drug leads to investigate and develop Conus capitaneus conotoxins.Methods 3'-RACE(Rapid Amplification of cDNA Ends) was used for cloning the novel O-superfamily conotoxins.The specific amplified cDNA fragments were sequenced and analyzed,as well as the genetic diversity of the O-superfamily conotoxins.Results The full-length cDNA(CaHr91N) of a new O-superfamily conotoxin(CaHr91) was cloned and sequenced from Conus capitaneus Linnaeus.The novel conotoxin precursor CaHr91P with 77 amino acids(aa) encoded by the cDNA consists of three typical regions of signal with 21aa, pro-peptide with 22aa and mature peptide with 34aa.Predicted sequence of the toxin region CaHr91M is "ECREQSQGC TNTSPPCC SGLRC SGQSQGGVC ISN" with a common O-superfamily cysteine pattern C-C-CC-C-C.Percent identities between CaHr91P and other published homologue O-superfamily sequences were compared systematically,as well as research status on conopeptides from C.capitaneus.Conlusion The elucidated cDNA of the novel CaHr91P conotoxin will be the basis for a better understanding of its bioactivity and application,as well as finding more novel conotoxins from C.capitaneus.

AIM:To compared the differential sensitivity of nicotinic acetycholine receptors (nAChRs) consisting of α and β subunits with different ratios.METHODS:The cRNA of α and β subunits was obtained by in vitro transcription.The α3β2 and α3β4 nAChR subtypes were expressed in Xenopus laevis oocytes by microinjection of cRNA coding α and β subunits at α∶β ratios of 1∶10, 1∶1 and 10∶1.The pharmacological activities of nAChRs to agonist acetycholine (ACh) and antagonist α-conotoxin (CTx) RegⅡA were investigated by two-electrode voltage-clamp techniques.RESULTS:For α3β2 nAChR expressed at the ratios of 1∶10, 1∶1 and 10∶1, the EC50 values of ACh were 91.2 μmol/L, 104.4 μmol/L and 130.6 μmol/L, respectively, while the IC50 values of α-CTx RegⅡA were 40.2 nmol/L, 36.4 nmol/L and 42.3 nmol/L, respectively.For α3β4 nAChR at the ratios of 1∶10, 1∶1 and 10∶1, the EC50 values of ACh were 44.0 μmol/L, 110.0 μmol/L and 230.0 μmol/L, respectively, while the IC50 values of α-CTx RegⅡA were 226.8 nmol/L, 71.5 nmol/L and 49.4 nmol/L, respectively.CONCLUSION:The results imply that the α3 and β4 subunit stoichiometry can change the structure and pharmacological activity of α3β4 nAChR, but the stoichiometry of α3 and β2 subunits has no effect on α3β2 nAChR.

Objective To investigate the prevalence of diabetes and prediabetes in northeast Sichuan area.Methods A total of 8 053 adult residents(over 18 years old)from the hospital who had underwent physical examination were recruited in the study.The concentrations of Fasting plasma glucose(FPG),glycated hemoglobin A1c(HbA1C)and blood lipids were measured.Diabetes and prediabetes were defined according to the 2013 American Diabetes Association criteria.Results The FPG and HbA1C concentra-tions increased with age.The overall prevalence of diabetes and prediabetes were estimated to be 8.5% and 39.6% in the adult pop-ulation in northeast sichuan area respectively.The prevalence of diabetes and prediabetes in men were significantly higher than those in women(10.7% vs.5.7%,41.5% vs.37.2%,P <0.05,respectively).Furthermore,the prevalence of diabetes increased with age,and the prevalence of prediabetes had a trend of first enhancement followed by a decline.Conclusion The prevalence of diabe-tes and prediabetes are relatively high in northeast Sichuan area.

To investigate activities of three isomers of α-conotoxin TxID on human α3β4 and α6/α3β4 nicotinic acetylcholine receptors(nAChRs). The three isomers of α-conotoxin TxID were synthesized using solid phase Fmoc chemistry and fully folded by two-step oxidations. Human α3β4 and α6/α3β4 nAChRs were expressed in oocytes of Xenopus laevis, which were used for bioassay of the three isomers, including inhibition and washout reversibility. There were obvious differences between the inhibition potency of each isomers at human α3β4 and α6/α3β4 nAChRs. The blocking was reversible and washout rapidly. The most potent isomer is the globular form with an IC50 of 9. 3 nmol/L on human α3β4 and α6/α3β4 nAChRs respectively. The 2nd potent isomer was the ribbon form with much less potency, which had an IC50 of > 5 μmol/L. The bead isomer had little or no block on human α3β4 and α6/α3β4 nAChRs with an IC50 of > 10 μmol/L. The three isomers of α-conotoxin TxID were synthesized successfully with two pairs of desired disulfide bond. Inhibition activities of the 3 isomers on human α3β4 and α6/α3β4 nAChRs were obtained respectively, which would be basis for new marine drug development of α-conotoxin TxID.

Malignant tumor has become an urgent threat to global public healthcare. Because of the heterogeneity of tumor, single therapy presents great limitations while synergistic therapy is arousing much attention, which shows desperate need of intelligent carrier for co-delivery. A core‒shell dual metal-organic frameworks (MOFs) system was delicately designed in this study, which not only possessed the unique properties of both materials, but also provided two individual specific functional zones for co-drug delivery. Photosensitizer indocyanine green (ICG) and chemotherapeutic agent doxorubicin (DOX) were stepwisely encapsulated into the nanopores of MIL-88 core and ZIF-8 shell to construct a synergistic photothermal/photodynamic/chemotherapy nanoplatform. Except for efficient drug delivery, the MIL-88 could be functioned as a nanomotor to convert the excessive hydrogen peroxide at tumor microenvironment into adequate oxygen for photodynamic therapy. The DOX release from MIL-88-ICG@ZIF-8-DOX nanoparticles was triggered at tumor acidic microenvironment and further accelerated by near-infrared (NIR) light irradiation. The

Pulmonary drug delivery has attracted increasing attention in biomedicine, and porous particles can effectively enhance the aerosolization performance and bioavailability of drugs. However, the existing methods for preparing porous particles using porogens have several drawbacks, such as the inhomogeneous and uncontrollable pores, drug leakage, and high risk of fragmentation. In this study, a series of cyclodextrin-based metal-organic framework (CD-MOF) particles containing homogenous nanopores were delicately engineered without porogens. Compared with commercial inhalation carrier, CD-MOF showed excellent aerosolization performance because of the homogenous nanoporous structure. The great biocompatibility of CD-MOF in pulmonary delivery was also confirmed by a series of experiments, including cytotoxicity assay, hemolysis ratio test, lung function evaluation,

In the original publication of the article the keywords are incorrectly online published. The correct keywords should read as α-Conotoxin; Nicotinc acetylcholine receptor; Acetylcholine binding protein; X-ray crystallography".

The α3* nAChRs, which are considered to be promising drug targets for problems such as pain, addiction, cardiovascular function, cognitive disorders etc., are found throughout the central and peripheral nervous system. The α-conotoxin (α-CTx) LvIA has been identified as the most selective inhibitor of α3β2 nAChRs known to date, and it can distinguish the α3β2 nAChR subtype from the α6/α3β2β3 and α3β4 nAChR subtypes. However, the mechanism of its selectivity towards α3β2, α6/α3β2β3, and α3β4 nAChRs remains elusive. Here we report the co-crystal structure of LvIA in complex with Aplysia californica acetylcholine binding protein (Ac-AChBP) at a resolution of 3.4 Å. Based on the structure of this complex, together with homology modeling based on other nAChR subtypes and binding affinity assays, we conclude that Asp-11 of LvIA plays an important role in the selectivity of LvIA towards α3β2 and α3/α6β2β3 nAChRs by making a salt bridge with Lys-155 of the rat α3 subunit. Asn-9 lies within a hydrophobic pocket that is formed by Met-36, Thr-59, and Phe-119 of the rat β2 subunit in the α3β2 nAChR model, revealing the reason for its more potent selectivity towards the α3β2 nAChR subtype. These results provide molecular insights that can be used to design ligands that selectively target α3β2 nAChRs, with significant implications for the design of new therapeutic α-CTxs.
Animals ; Aplysia ; Binding Sites ; Conotoxins ; chemistry ; Crystallography, X-Ray ; Humans ; Protein Structure, Quaternary ; Receptors, Nicotinic ; chemistry