Objective To investigate the calcium/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)signaling pathway and expression of brain-derived neurotrophic factor(BDNF)isoforms in mouse model of cognitive dysfunction secondary to repetitive mild traumatic brain injury(rmTBI).Methods A total of 63 male C57BL/6J mice(5 weeksd old,werghing 16～17 g)were divided into model group(n=50)and control group(n=13).The mice of the model group received modified weight drop and repeated head impacts to establish a model of rmTBI,and the mice from the control group were only given anesthetic treatment.From the 30th day after the end of modeling,Morris water maze test was applied to evaluate the spatial learning and memory abilities,and the relative data was evaluate for correlation analysis.According to the taxonomy of cognitive levels,the model group was further stratified into high-,medium-and low-performance groups.After the mice with medium-performance group were eliminated,and the indicators of water maze test were compared among the high-performance,low-performance,and control groups.After the behavioral experiment,Western blotting was conducted to detect the expression of CaMKII,α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor(AMPAR)subunits(GluR1,p-GluR1,GluR2),and different isoforms of BDNF(mBDNF,proBDNF)in the prefrontal cortex and hippocampus.The expression levels were compared among the groups.Results ① There were significant correlations among the water maze-related indicators(P<0.05).The mice in the model group were divided into 3 groups of high-(n=31),medium-(n=11)and low-performance(n=8)levels through hierarchical clustering,and there were differences in cognitive levels in the high-performance,low-performance and control groups.② The phosphorylation level of GluR1 in the hippocampus was significantly decreased in the high-performance group than the control group and the low-performance group(P<0.05).When compared with the control group,the expression of GluR1 and GluR2 in the prefrontal cortex of the low-performance group was increased(P<0.05),and the expression of GluR2 and CaMKⅡ in the prefrontal cortex of the high-performance group was increased(P<0.05).The low-performance group obtained obviously lower GluR1/GluR2 ratio than the high-performance group(P<0.05).③ When compared with the control group,the low-performance group exhibited notably elevated expression of proBDNF and mBDNF(P<0.05)and mBDNF/proBDNF(P<0.05)in the prefrontal cortex and hippocampus,while the high-performance group had enhanced expression of mBDNF in the prefrontal cortex(P<0.05).Conclusion In the chronic phase of rmTBI,prognosis of good cognitive function is accompanied by up-regulation of CaMKⅡ signaling pathway,and the difference in cognitive function is accompanied by the change in proportions of different AMPAR subunits on the postsynaptic membrane.In the cognitive impairment group,there is an overexpression of 2 types of BDNF isoforms and an increase in the conversion rate of mBDNF.

To carry out pathologic diagnoses and whole-genome sequence analyses of the Jaagsiekte sheep retrovirus (JSRV) in Xinjiang, China, we first observed sheep suspected to have the JSRV. Then, the extracted virus suspension was observed by transmission electron microscopy (TEM). Total RNAs from lungs of JSRV-infected sheep were extracted and reverse-transcribed using a cDNA synthesis kit. Six pairs of primers were designed according to the exogenous reference virus strain (AF105220). Reverse transcription-polymerase chain reaction was carried out from JSRV-infected tissue, and the whole genome of the JSRV sequenced. Our results showed: flow of nasal fluid ("wheelbarrow test"); different sizes of adenoma lesions in the lungs; papillary hyperplasia of alveolar epithelial cells; alveolar cavity filled with macrophages; dissolute nuclei in central lesions. TEM revealed JSRV particles with a diameter of 88 nm to 125. 4 nm. The full-length of the viral genome sequence was 7456 bp. BLAST analyses showed nucleotide homology of 96% and 95% compared with that of the representative strain from the USA (AF105220) and UK (AF357971). Nucleotide homology was 89.8% and 89.9% compared with the endogenous Jaagsiekte sheep retrovirus, Inner Mongolia strain (DQ838493) and USA strain (EF680300). The specific pathogenic amino-acid sequence "YXXM" was found in the TM district, similar to the exogenous JSRV: this gene has been reported to be oncogenic. This is the first report of the complete genomic sequence of the exogenous JSRV from Xinjiang, and could lay the foundation for study of the biological characteristics and pathogenic mechanisms of the pulmonary adenomatosis virus in sheep.
Amino Acid Sequence ; Animals ; China ; Genome, Viral ; Jaagsiekte sheep retrovirus ; classification ; genetics ; isolation & purification ; pathogenicity ; Lung ; pathology ; virology ; Molecular Sequence Data ; Phylogeny ; Pulmonary Adenomatosis, Ovine ; pathology ; virology ; Sheep ; Viral Proteins ; chemistry ; genetics ; Virulence

Objective To investigate the effects of microRNA-294 (miR-294) on tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6) and high mobility group box 1 (HMGB 1) secretion in sepsis by targeting triggering receptor expressed on myeloid cell-1 (TREM-1).Methods miRNA-294 was predicted to regulate TREM-1 specially through bioinformatics analysis.Mice macrophage cell lines RAW264.7 were cultured in vitro,the cells were divided into non-inflammatory stage and inflammatory stage,and the cells in the two stages were subdivided into five groups as follows:normal control (NC),NC mimic transfection (NCm),NC inhibitor transfection (NCi),miR-294 mimic transfection (miR-294m) and miR-294 inhibitor transfection (miR-294i) groups.The ability of miR-294 was confirmed with dual-luciferase activity assay.At non-inflammatory stage,the cells were transfected with mimic or inhibitor of miR-294 or NC using TurboFectTM siRNA Transfection Reagent for 48 hours,mRNA expression of TREM-1 was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR).At inflammatory stage,6 hours after stimulation by lipopolysaccharide (LPS,1 mg/L),the concentrations of TNF-α,IL-6 and HMGB1 were determined by enzyme linked immunosorbent assay (ELISA),the protein expression of TREM-1 was determined by Western Blot.Results ① Dual-luciferase activity assay demonstrated that TREM-1 was the target of miR-294.② In non-inflammatory stage,the expression of TREM-1 mRNA (2-ΔΔ~) in miR-294m group was significantly lower than that of the NC and NCm groups (0.673 ± 0.049 vs.1.000 ± 0.003,0.915 ± 0.039,t1=2.184,t2=5.421,both P＜0.001),the expression of TREM-1 protein (gray scale) was (50.00 ± 1.19)％ of NCm group (t=41.586,P＜0.001).③ In inflammatory stage,the concentrations of TNF-α (ng/L) in miR-294m group was significantly lower than that of the NC group (1 547.18 ±47.18 vs.2 702.11 ± 327.20,t=4.212,P=0.010),the concentrations of IL-6 (ng/L) was significantly lower than that of the NC and NCm groups (505.28 ± 33.33 vs.837.66 ± 69.43,918.72 ± 119.39,t1 =4.382,P1=0.015; t2=5.451,P2=0.021),the level of TREM-1 protein (gray scale) was (51.33 ±0.88)％ of NCm group (t=63.368,P＜0.001).Conclusion miR-294 reduce TNF-α and IL-6 secretion in LPS-induced RAW264.7 through inhibiting the expression of TREM-1 specifically.