Objective To discover novel conopeptides which are the antagonists of neuronal nicotinic acetylcholine receptors (nAChRs) in order to contribute to the development of novel analgesic drugs and neuropharmacological probes.Methods Based on the conserved untranslated region and intron of A-superfamily conotoxins,a novel α-conotoxin Lt1.1 was cloned from Conus litteratus.The peptide-resin was synthesized using the solid-phased method and was cleaved.The resulting linear peptide was oxidized by air to give the product containing disulfide bridges.The folding product was finally purified by HPLC.The disulfide bond connectivity was determined using the two-step oxidative folding methods.The cRNA of rat nAChRs was expressed on the membrane of Xenopus oocyte.Membrane currents were recorded using the two electrode voltage-clamp technique.Results A novel α-conotoxin designated as Lt1.1(GCCSHPACNVNNPDIC-NH2) was cloned and its disulfide connectivity was C1-C3,C2-C4.Lt1.1 selectively inhibited the α3β2 and α3β4 nAChRs with an IC50 of 166.76 and 190.00 nmol/L,respectively.Conclusion Lt1.1 is a novel 4/7 α-conotoxin that selectively targets α3β2 and α3β4 nAChRs.

Objective To clone a new conotoxin Bt14.10 from Conus betulinus derived from the South China Sea, synthesize the peptide , and to determine linkage of its disulfide bridges .Methods The genomic DNA was extracted from C.betulinus venom duct while the Bt14.10 sequence was cloned using primers designed based on the untranslated region and intron.The peptide was then synthesized using solid-phase method and folded into the target product whose disulfide bridge connection was further determined by two-step oxidative folding .Results A novel conotoxin designated as Bt 14.10 (CAHSVPGMHPCKCNNTC-NH2) was obtained,the disulfide connectivity of which was C1-C3,C2-C4.Conclusion Bt14.10 is a new A-superfamily conotoxin and has a distinct loop spacing pattern between cysteines in A-superfamily conotoxins.

Objective To study the imaging distribution feature and diagnostic value of high resolution computed tomography(HRCT)in peripheral lung cancer(PLC).Methods The feature of imaging distribution was analysed in 37 patients with PLC by pathological proved,which compared with those in 23 cases with lung benign nodules by selected randomly.A double blind method was taken on the manifestations of HRCT about lung nodules tumor-lung interface in near heart side and far heart side.①cloudy or/and shaggy②spiculate③smooth.To search and define the correlation between its distributing feature;manifestations of 3 kinds HRCT;alteration of segment level bronchus and lung benign malignancy nodules.Results Cloudy,or shaggy,spiculalte departing from heart side in lung-tumor interface by HRCT were observed in peripheral lung cancer(79%) and benign nodules(22%);smooth was observed in peripheral lung cancer(14%) and benign nodules(74%).Some cases possed simultaneously two or more than two kinds HRCT's signs.Incidence rate of emphraxis and stenosis signs of segment level bronchus in PLC was higher than that in benign nodules.Conclusion Asymmetry apo-tip dominant position distribution of cloudy or shaggy,and spiculate change of tumor-lung interface by HRCT played an important role in qualitative diagnosis of peripheral lung cancer(≤3.5 cm).The appearance reason relates with the bronchial ventilation that the lesion results in occlusion.

Objective:To explore the key pathways and genes involved in microglia inflammation through transcriptome sequencing and bioinformatics analysis.Methods:BV2 cells were stimulated by lipopolysaccharide to establish microglia inflammation model. The levels of IL-6 and TNF-α were detected by ELISA and RT-qPCR. The established microglia inflammation model was sequenced by transcriptome sequencing, and the differentially expressed genes were screened by bioinformatics method. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes were performed. The protein-protein interaction network of differentially expressed genes was constructed by using string database, and the protein-protein interaction network was visualized by using Cytoscape software. The protein interaction network module was extracted by using MCODE app. The hub gene was extracted by using cytohubba app and was verified through RT-qPCR. We conducted enrichment analysis of hub genes, predicted their targeted miRNAs and interacting drugs.Results:The microglia inflammation model was successfully established and verified by ELISA and RT-qPCR. We screened 434 differentially expressed genes by bioinformatics analysis of transcriptome sequencing results. GO analysis showed that these differentially expressed genes were mainly concentrated in cellular response to cytokine stimulus, inflammatory response, regulation of response to external stimulation. KEGG analysis showed that these differentially expressed genes were mainly concentrated in Chemokine signaling pathway, TNF signaling pathway, IL-17 signaling pathway. We constructed the protein interaction network of these differentially expressed genes, and carried out module analysis and extraction of hub genes. Most of hub genes are located in module 1, and the seed gene of module 1 is S1pr1. Hub genes include S1pr1, Cxcr4, Cx3cl1, Cx3cr1, Cxcl10, Cxcl2, Ccl4, Ccl5, Ccl9, Fpr1. RT-qPCR results showed that compared with the culture medium group, the mRNA expressions of S1pr1, Cxcr4, Cx3cl1 and Cx3cr1 were down-regulated, and the mRNA expressions of Cxcl10, Cxcl2, Ccl4, Ccl5, Ccl9 and Fpr1 were up-regulated in the LPS group. The enrichment analysis of hub genes mainly focused on chemokine-mediated signaling pathway, Class A/1 (Rhodopsin-like receptors), cell chemotaxis and so on. Drugs and miRNAs that may interact with hub genes were predicted. Conclusion:Through transcriptome sequencing and bioinformatics analysis of microglia inflammation model, differentially expressed genes were screened, hub genes and seed genes were extracted, which will help us further understand the molecular mechanism of microglia inflammation and provide potential targets for the treatment of related diseases.

ObjectiveTo explore the establishment and evaluation methods of the rat model of acute myocardial infarction （AMI） in coronary heart disease with the syndrome of Qi and Yin deficiency by sleep deprivation （SD） combined with isoproterenol （ISO） and preliminarily explore its biological basis. MethodForty SD rats were assigned into normal （no treatment）， SD （treatment in modified multi-platform water environment for 96 h）， ISO （subcutaneous injection of ISO at 100 mg·kg^-1 once every other day for a total of 2 times）， and SD+ISO （injection of 100 mg·kg^-1 ISO after SD for 72 h and 96 h） groups. The cardiac function was detected by small animal echocardiography. The serum levels of creatine kinase （CK）， creatine kinase isoenzyme （CK-MB）， lactate dehydrogenase （LDH）， and cardiac troponin T （cTnT） were measured by biochemical methods. The pathological changes of the myocardial tissue were observed by hematoxylin-eosin staining. The general state， body weight， grip strength， body temperature， behaviors in open field test， serum levels of cyclic adenosine monophosphate （cAMP）， cyclic guanosine monophosphate （cGMP）， cAMP/cGMP ratio， red （R）， green （G）， blue （B） values of the tongue surface， and pulse amplitude were observed and measured to evaluate the modeling results. Enzyme-linked immunosorbent assay was employed to determine the serum levels of interleukin-18 （IL-18）， tumor necrosis factor-α （TNF-α）， superoxide dismutase （SOD）， malondialdehyde （MDA）， corticotropin-releasing factor （CRF）， adrenocorticotropic hormone （ACTH）， triiodothyronine （T3）， tetraiodothyronine （T4）， cluster of differentiation 4 （CD4）， and cluster of differentiation 8 （CD8）. ResultIn terms of disease indicators， the ISO and SD+ISO groups had lower cardiac function indicators than the normal group （P<0.01）. The levels of CK， CM-MB， LDH and cTnT elevated in each model group compared with the normal group （P<0.01）. The pathological changes of myocardial tissue were obvious in the ISO and SD+ISO groups. In terms of syndrome indicators， compared with the normal group， the SD and SD+ISO groups showed decreased body weight at each time point （P<0.01）， and the ISO group showed decreased body weight at the time points of 48 h and 72 h （P<0.05， P<0.01）. The paw temperature and rectal temperature increased in the SD group （P<0.01）. The model groups showed weakened grasp strength， lowered R， G， and B values of the tongue surface （P<0.01）， prolonged immobility time （P<0.01）， reduced total distance and number of entering the central area （P<0.01）， decreased average speed （P<0.05， P<0.01）， and increased cAMP and cGMP （P<0.05， P<0.01）. The cAMP/cGMP ratio was increased in the SD+ISO group （P<0.01）， and the pulse amplitude was decreased in the SD and SD+ISO groups （P<0.01）. In terms of serological indicators，compared with the normal group， the levels of IL-18， TNF-α， SOD and MDA were significantly increased in the ISO and SD+ISO groups （P<0.01）， the CRF， ACTH， CORT， T3， T4， CD4 and CD8 in the model groups were increased （P<0.05， P<0.01）. ConclusionSleep deprivation for 96 h combined with high-dose ISO can successfully establish a rat model of acute myocardial infarction in coronary heart disease with the syndrome of Qi and Yin deficiency. The model evaluation system can be built with disease indicators of western medicine， histopathological indicators， macroscopic indicators of traditional Chinese medicine， and serological indicators.