Objective To analyze the Cyt b gene sequences in Tibet mini-pigs and clarify the differences and genetic relationship with other Chinese pigs.Method The sequence of Cyt b gene was amplified from genome DNA of Tibet mini-pig,Bama miniature pigs,Guizhou xiang pigs and Wuzhishan (WZS) pigs.After sequencing,the base sequences were compared and analysed.The blood relationship tree and evolution position of Tibet mini-pig were established.Result There were 14 mutation sites between domestic pigs in China and pigs from Europe.Besides there was a significant differenee in two nucleotide site:a T→C switch in site 420 and the G→A switch in site 883 at the same time.Conclusion Chinese pigs include Bama miniature pigs,Guizhou xiang pigs and WZS pigs,have a very close blood relationship with some of Tibet mini-pigs.It has been confirmed that there is a certain genetic differentiation in the Tibet mini-pig.

Objective To evaluate the role of β-arrestin-1 in penehyclidine hydrochloride (PHC)-induced inhibition of lipopolysaccharide (LPS)-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells (PMVECs).Methods Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1 × 105 cells/ml and divided into 5 groups (n=15 each) using a random number table:empty plasmid transfection group (group C),LPS plus empty plasmid transfection group (LPS group),PHC plus LPS plus empty plasmid transfection group (P+LPS group),LPS plus β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+shRNA group) and PHC plus LPS plus β-arrestin-1 shRNA transfection group (P+LPS+shRNA group).In LPS and LPS+shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,LPS with the final concentration of 0.1 μg/ml was added at 24 h of incubation,and the cells were then incubated for 1 h.In P+LPS and P+LPS+shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,PHC with the final concentration of 2 μg/ml was added at 24 h of incubation,LPS with the final concentration of 0.1 μg/ml was added at 1 h of incubation,and the cells were then incubated for 1 h.The cell permeability was measured using Transwell chambers.The expression of heat shock protein (HSP27) was detected by immunofluorescence.The expression of β-arrestin-1,p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK (p-p38MAPK) was detected by Western blot.The ratio of pp38MAPK/p38MAPK was calculated.Results Compared with group C,the cell permeability was significantly increased,the expression of HSP27 was up-regulated,p-p38MAPK/p38MAPK ratio was increased,and the expression of β-arrestin-1 was down-regulated in LPS,LPS + shRNA and P + LPS + shRNA groups (P＜0.05),and no significant change was found in the parameters mentioned above in group P+LPS (P＞ 0.05).Compared with group LPS,the cell permeability was significantly decreased,the expression of HSP27 was down-regulated,p-p38MAPK/p38MAPK ratio was decreased,and the expression of β-arrestin1 was up-regulated in group P +LPS,and p-p38MAPK/p38MAPK ratio was significantly increased (P＜0.05),and no significant change was found in the other parameters in group P+LPS+shRNA (P＞0.05).Compared with group P+LPS,the cell permeability was significantly increased,the expression of HSP27 was up-regulated,p-p38MAPK/p38MAPK ratio was increased,and the expression of β-arrestin-1 was down-regulated in group P+LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC inhibits LPS-induced increase in pulmonary microvascular permeability is totally related to β-arrestin-1 in human PMVECs.

Objective To understand the status of knowledge awareness rates and influencing factors of clonorchiasis among the residents in 4 counties(cities)of Hunan Province,so as to provide the references for formulating the prevention and control strategy of clonorchiasis in the province. Methods A total of 1224 subjects were selected by the multi-stage cluster sampling method and investigated with questionnaires and face-to-face interviews. Results The clonorchiasis knowledge awareness rates of the residents were 15.67% in Yongzhou City,9.94% in Yueyang City,13.25% in Wangcheng County,and 21.96% in Tongd-ao County,respectively. In the whole province,the clonorchiasis knowledge awareness rates of the residents were 44.67%, 56.43%,60.95%,and 42.46% in the etiology,clinical manifestations,epidemiological characteristics and daily prevention,re-spectively. The logistic regression showed that the influencing factors of clonorchiasis knowledge awareness rates were the daily dining place,whether or not having the medical personnel in the family,the degree of attention to the relevant news reports, and the history of parasitic diseases. Conclusions The level of clonorchiasis than that in other areas,but the overall level of clonorchiasis knowledge awareness of the residents in Hunan Province is not high. Therefore,the relevant authority should strengthen the health education in clonorchiasis prevention knowledge including personal daily food hygienic knowledge. knowl-edge awareness rate of the residents in the west-southern areas of Hunan Province is higher

Objective To express human HT036 protein in Escherichia coli(E.coli.)and identify it.Methods The cDNA sequence obtained by PCR was cloned into the prokaryotic expression plasmid pET30a(+).The target protein was expressed in E.coli..induced by IPTG and analyzed by Western blotting.Results The interest gene was identified by restriction endonucleases digestion and DNA sequencing.The protein was highly expressed in E.coli..Conclusion We successfully expressed the HT036 protein.

Objective To construct a bait vector containing human Foxp3 gene in yeast two-hybrid system in order to screen the cDNA library of T lymphocyte. Methods RT-PCR was used to amplify the Foxp3 gene fragment from the peripheral blood mononuclear cells (PBMC) with the primers designed in accordance with the sequence in GenBank. The product was inserted into pMD18-T vector. After verified with restriction endonuclease digestion of EcoRⅠ and SalⅠ, the vector was inserted into the “bait plasmid” pGBKT7 (named as pGBKT7-Foxp3). After confirmation with restricted endonuclease digestion and sequence analysis, the plasmid was transformed into the yeast cell AH109, and its toxicity and transcriptional activation was tested by both the phenotype assay and the color assay. Results The amplified product of 1 203 bp was inserted into PMD18-T vector and proven correctly by double restriction enzyme digestion. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame and its expression in yeast was verified. Conclusion The bait plasmid pGBKT7-Foxp3 constructed expresses correctly, and can not activate the transcription of reporter gene alone in yeast two-hybrid system

Objective To explore the interaction between HT036(hypothetical protein HT036)and P311 by co-immunoprecipitation.Methods HA-tagged fusion protein(HA-HT036)expression vector was constructed,identified and transfected into human embryo kidney 293(HEK293)cells alone or with Myc-tagged fusion protein(Myc-P311)expression vector pCMV-Myc-p311.The interaction between P311 and HT036 was detected by co-immunoprecipitation.Results Double restriction enzyme digestion showed that pCMV-HA-HT036 was constructed correctly.When Myc-P311 was immunoprecipitated by anti-Myc antibody,HA-HT036 was identified by Western blotting with anti-HA antibody from immunoprecipitated complex.Conclusion The recombinant vector pCMV-HA-HT036 was constructed successfully.The interaction between HT036 and P311 could be identified by co-immunoprecipitation after co-expression of pCMV-HA-HT036 and pCMV-Myc-p311.The result provides an important basis for further study of the intracellular signal transduction of P311.

Objective To investigate the role of activated cannabinoid receptor 2 (CB2R) in lipopolysaccharide (LPS)-induced secretion of RAW264.7 macrophage inflammatory cytokines and its possible mechanism. Methods Macrophages were seeded in 6-well plates (2 mL/well) at the density of 1×105 cells/mL and randomly divided into four groups (n=6 each group): control group (group C), LPS group (group LPS), LPS plus CB2R agonist HU308 group (group LPS+HU308), and LPS plus HU308 plus 3-Methyladenine group (group LPS+HU308+3-MA). LPS with the final concentration of 1 μg/mL were added in group LPS, group LPS+HU308 and group LPS+HU308+3-MA. After incubation for 15 min, 3-MA with a final concentration of 10 mmol/L was added into group LPS+HU308+3-MA . HU308 with the final concentration of 10 μmol/L was added in group LPS+HU308 and group LPS+HU308+3-MA at 15 min after 3-MA intervention, and the cells were then incubated for 24 h. The concentrations of TNF-α, IL-18 and IL-1β in supernatant serum of each group were determined by ELISA. The expressions of ICAM-1 and NLRP3 mRNA were detected by RT-PCR. The expressions of LC3 and Beclin1 were detected by Western blot, and the ratio of LC3-Ⅱ/LC3-Ⅰ was calculated. LSD-t test was used for sample pairwise comparison, and one way ANOVA for inter-group comparison. A P<0.05 was considered statistically significant. Results Compared with group C, the concentrations of TNF-α [(228.86±10.20) pg/mL vs (140.05±5.54) pg/mL], IL-1β [(363.62±8.14) pg/mL vs (244.82±9.11) pg/mL], and IL-18 [(293.28±13.57) pg/mL vs (202.84±9.54) pg/mL] in supernatant serum were increased (all P<0.05), the expressions of ICAM-1 [(5.88±0.32) vs (1.00±0.03)] and NLRP3 [(8.07±0.93) vs (1.01±0.05)] mRNA were increased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.50±0.03) vs (0.40±0.06)] and Beclin1 [(0.51±0.04) vs (0.16±0.03)] were up-regulated in group LPS (all P<0.05). Compared with group LPS, the concentrations of TNF-α [(165.44±7.07) pg/mL], IL-1β [(272.09±3.35) pg/mL] and IL-18 [(220.41±6.01) pg/mL] in supernatant serum were significantly decreased (all P<0.05), the expressions of ICAM-1 [(3.21±0.35)] and NLRP3 [(1.54±0.30)] mRNA were decreased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.71±0.03)] and Beclin1 [(0.71±0.02)] were up-regulated in group LPS+HU308 (all P<0.05). Compared with group LPS+HU308, the concentrations of TNF-α [(197.06±5.59) pg/mL], IL-1β [(318.98±11.54) pg/mL] and IL-18 [(243.33±8.71) pg/mL] in supernatant serum were significantly increased (all P<0.05), the expressions of ICAM-1 [(4.04±0.21)] and NLRP3 [(5.87±0.77)] mRNA were increased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.44±0.08)] and Beclin1 [(0.32±0.03)] were down-regulated in group LPS+HU308+3-MA (all P<0.05). Conclusions Activation of cannabinoid receptor 2 can alleviate LPS-induced the secretion of RAW264.7 macrophage inflammatory cytokines, and its mechanism may be related to enhanced autophagy.

Objective To evaluate the effect of activating cannabinoid receptor 2 (CB2R) on sepsis-induced acute lung injury and the role of autophagy in mice.Methods Twenty-four SPF male C57BL/6 mice,aged 8-10 weeks,weighing 20-25 g,were divided into 4 groups (n=6 each) using a random number table method:sham operation group (group Sham),sepsis group (group Sep),sepsis plus CB2R agonist HU308 group (group Sep+HU308) and sepsis plus HU308 plus autophagy inhibitor 3-methyladenine group (group Sep+HU308+3-MA).Sepsis was induced by cecal ligation and puncture in anesthetized mice.HU308 2.5 mg/kg was intraperitoneally injected at 15 min after surgery in Sep+HU308 and Sep+ HU308+3-MA groups,and 15 min later 3-MA 10 mg/kg was intraperitoneally injected in group Sep+ HU308+3-MA.Lung tissues were obtained at 12 h after surgery and stained with haematoxylin and eosin for examination of the pathological changes which were scored and for determination of the expression of tumornecrosis factor-alpha (TNF-α),interleukin-18 (IL-18) and IL-1β mRNA (by real-time polymerase chain reaction),expression of autophagy-related protein 5 (Atg5) (by immuno-histochemistry),and expression of microtubule-associated protein 1 light chain 3 (LC3),Beclin-1 and p62 (by Western blot).The ratio of LC3Ⅱ to LC3Ⅰ (LC3Ⅱ/LC3Ⅰ ratio) was calculated.Results Compared with group Sham,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly up-regulated,and LC3 Ⅱ/LC3 Ⅰ ratio and lung injury score were increased in the other three groups,the expression of Beclin-1 was up-regulated,and the expression of p62 was down-regulated in group Sep and group Sep+HU308,and the expression of p62 was significantly up-regulated in group Sep+HU308+3-MA (P＜0.05).Compared with group Sep,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly down-regulated,the expression of Atg5 was up-regulated,and lung injury score was decreased in group Sep+ HU308 and group Sep+ HU308 + 3-MA,LC3Ⅱ/LC3Ⅰ ratio was increased,the expression of Beclin-1 was up-regulated,and the expression of p62 was down-regulated in group Sep+HU308,and the expression of Beclin-1 was down-regulated,and the expression of p62 was up-regulated in group Sep + HU308 + 3-MA (P ＜ 0.05).Compared with group Sep+ HU308,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly up-regulated,the expression of Atg5 and Beclin-1 was down-regulated,LC3Ⅱ/LC3Ⅰ ratio was decreased,the expression of p62 was up-regulated,and lung injury scores were increased in group Sep+HU308+3-MA (P＜0.05).Conclusion Activating CB2R can alleviate acute lung injury in septic mice,and the mechanism may be partially related to enhancing autophagy and reducing inflammatory responses.

Objective To evaluate the effects of day surgery on average days of stay, and to provide scientific basis for shorter average days of stay. Methods Data of average days of stay from November 2011 to December 2016 were extracted from 6 departments, Xiangya Hospital, Central South University. Segmented regression analysis of interrupted time series was used to analyze the trend of average days of stay of pre-and post-day surgery. Results Thanks to the application of day surgery, average days of stay decreased by 0. 071 days per month, and the monthly decline increased by 0. 049 days (P<0. 001) than before. Average days of stay in the department of stomatology, ear-nose-throat, general surgery and hepatobiliary & enteric surgery research center were the most obvious, and the monthly decline increased by 0. 110 days (P<0. 001), 0. 049 days (P=0. 008) , 0. 075 days (P<0. 001), and 0. 057 days (P=0. 003), respectively than before. Conclusions Day surgery could decrease average days of stay, enhancing the utilization of hospital resources.

Objective To investigate and analyze the characteristics of blood physiological and biochemical parameters of Tibetan chickens bred in Guangzhou. Methods Blood samples of Tibetan chickens bred in Guangzhou were collected,and the physiological and biochemical parameters were measured. Results (1)The blood RBC,PLT,PDW, RDW-SD and P-LCR were not significantly different in the males than females(P > 0.05).(2)HCT(P < 0.05), MCHC(P< 0.05),MPV(P< 0.05),HGB(P< 0.01),MCV(P< 0.01)and MCH(P< 0.01)were significantly higher between the males and females.(3)RDW-CV was significantly lower in the blood physiological parameters of males than females.(4)AST,TRIG,ALKP,ALT,Ca,CHOL,CREA,GLU,PHOS and TBIL were not remarkably different in the blood of males than females(P > 0.05).(5)The blood AMYL(P < 0.05)and TP(P < 0.01)were significantly higher in the males than females.(6)The blood ALB(P< 0.01),UREA(P< 0.05), and GLOB(P<0.01)were significantly lower in the males than females. Conclusions The essential data of blood physiological and biochemical indexes of Tibetan chickens bred in Guangzhou are obtained.