Objective To study the effect of hypothermia on cerebral mitochondrial respiratory function and ultrastructure after traumatic brain injury(TBI). Methods Sprague-Dawley rats were subjected to moderate brain injury by using lateral fluid-percussion(LFP)and randomly divided into sham operation group,normothermic TBI group(rectal temperature for 36-37℃)and hypothermic TBI group(rectal temperature for 31-32℃ lasting for two hours).The ipsilateral brains were dissected and homogenized brain tissues were extracted to obtain mitochondfia by density-centrifugation and speed-centrifugation at 2,24 hours and at days 3 and 7 after TBI.The mitochondrial uhrastructure was studied by electron microscope.The indices of respiratory control rate(RCR)and P/O ratio of mitochondrial respiratory function were measured after oxygen consumption was determined with a Clark-type electrode.Results The mitochondrial uhrastructure of normothermic TBI group was damaged severely while that of hypothermic TBI group kept relatively integrated.The RCR and P/O ratio were markedly decreased two hours after TBI and reached the lowest level at the 24th hour(P＜0.01).At day 7,RCR kept at a lower level compared with sham operation group but P/O ratio recovered to normal.Change of RCR was similar in hypothermie TBI group and normothermic TBI group.However,RCR of the hypothermic TBI group was significantly higher than that of the normothermic TBI group within three days after TBI.In the meantime,P/O ratio recovered to normal three days after TBI. Conclusion Hypothermia can improve cerebral mitochondrial respiratory function and protect the mitochondrial structure after TBI.

Objective: To compare the peri-implant tissue stability between immediate implant and delayed implant in maxillary anterior region after loading 2 years.Methods: In the study, 38 patients with single anterior tooth loss in the Second Clinical Division of Peking University School and Hospital of Stomatology from October 2010 to December 2011 were enrolled , and 43 implants were inserted .The gin-gival contour was induced using implant-supported temporary crowns prior to restoration till permanent prostheses delivered .The gingival papilla height , labial gingival margin level and peri-implant bone level were measured immediately after the permanent restoration and 2 years later .Results: In the study , 16 patients were treated by immediate implant for 17 implants;22 patients were treated by delayed im-plant for 26 implants .The implant stability quotient ( ISQ ) value of the 2 groups showed no significant difference before permanent restoration (P>0.05).In all the cases after loading 2 years, the average mesial gingival papilla height in the implant area of the immediate group and delayed group increased by (0.15 ±0.42) mm and (0.06 ±0.65) mm, respectively;the distal gingival papilla height increased by (0.06 ±0.50) mm and (0.02 ±0.57) mm respectively;while the labial gingival margin level shrinka-ges were (0.15 ±0.23) mm and (0.15 ±0.46) mm, respectively.The peri-implant bone losses in the mesial side were (0.67 ±0.35) mm and (0.69 ±0.49) mm, respectively, while in the distal side were (0.73 ±0.31) mm and (0.75 ±0.48) mm, respectively.All these indicators showed no significant difference between the 2 groups ( P>0 .05 ) .Conclusion:Both the cases obtained optimizer results after loading 2 years, and the soft and hard tissues around the implant were very stable , which means that both the protocols can achieve reliable therapeutic effects .If we can handle the indications , immediate implant for anterior teeth shows similar efficacy with delayed implant in the short term .But immediate implant in terms of shortening the course of treatment is clearly superior to delayed implant .

ObjectiveTo study the relationship between plasma riboflavin levels and esophageal squamous cell carcinoma.Methods We detected and compared plasma concentrations of riboflavin in patients with esophageal squamous cell carcinoma (ESCC) and immigrants of Linzhou living in Changzhi.Plasma riboflavin levels were quantified in 445 ESCC patients,689 healthy control subjects and 347 immigrants of Linzhou living in Changzhi by using enzyme-linked immunosorbent assay.ResultsThe plasma riboflavin levels in patients with ESCC were significantly lower than those in the healthy controls and immigrants of Linzhou living in Changzhi [ (731.69 ± 330.67 ) μg/L vs ( 1090.43 ± 445.08 ) μg/L,(731.69 ± 330.67) μg/L vs ( 897.58 ± 177.78) μg/L,respectively,all P ＜ 0.05 ],and the plasma riboflavin levels of the healthy controls were higher than those in the immigrants of Linzhou living in Changzhi (P ＜ 0.05).ConclusionPatients with ESCC have decreased plasma riboflavin levels as compared with the healthy controls and immigrants of Linzhou living in Changzhi,there exists a lack of riboflavin in ESCC patients,but the specific mechanism needs further study.

Objective To explore the association of C20orf54 gene rs3746804 position single nucleotide polymorphism and susceptibility to esophageal squamous cell carcinoma (ESCC).Methods Purification of genomic DNA from whole blood was used the Maxwell(R) 16 System.rs3746804 in C20ort54 was detected by direct sequencing in 434 ESCC patients from Changzhi (Shanxi province) and Linzhou (Henan province) and 554 healthy controls from Changzhi,Linzhou and including immigrators from Linzhou to Changzhi.Results For rs3746804,the genotypic frequencies of CT(37.5％ vs 51.0％,37.5％ vs 52.0％),CC (44.2％ vs 34.8％,44.2％ vs 33.0％) in Changzhi ESCC patients showed significant differences with healthy Changzhi controls and the healthy immigrator controls (all P ＜ 0.05),and the frequencies of TT(18.3％ vs 4.1％) and CC (44.2％ vs 54.6％) in Changzhi ESCC patients showed significant differences with Linzhou ESCC patients (all P ＜0.05).The genotypic frequencies of TT (4.1％ vs 15.0％),CT (41.2％ vs 52.0％) and CC(54.6％ vs 33.0％) showed significant differences between Linzhou ESCC patients and the healthy immigrator controls (all P ＜ 0.05),and the frequencies of TT (4.1％ vs 14.1％) and CC (54.6％ vs 34.8％) showed significant differences between Linzhou ESCC patients and Changzhi healthy controls (all P ＜ 0.01).Meanwhile,there were significant differences between ESCC patients (including Changzhi and Linzhou ESCC patients) and healthy controls (including the healthy Changzhi,Linzhou and immigrator controls) in genotypic frequencies of CT(39.2％ vs 48.7％) and CC (48.8％ vs 38.2％) (all P ＜ 0.01).CT and CT + TT genotype could decrease the risk of ESCC compared with the CC genotype (OR =0.630,95％ CI0.481-0.826 ; OR =0.654,95％ CI 0.507-0.844).Conclusion There is a closed relationship between SNP rs3746804 in C20orf54 and susceptibility to ESCC.

Objective To analyze the conding region of hantanvirus S gene and predict the structure of nucleoprotein for diagnostic antigen study.Methods RT-PCR was used to amplify the S gene of hantanvirus Hunan03 strain after designing specific primers.The amplification product was cloned into pGM-T vector and then the recombinant vector was transformed into E.coli TOP10,gene sequencing was carried out after blue-white selection and PCR screening for positive clones.The database of NCBI and Swiss-Prot/TrEMBL were used to predict and analyze the structure,biological characteristics and protein structures of S gene.Results The amplification product was about 1290 bp,the pGM-T/S vector was constructed and successfully sequenced,the whole length of the open reading frame (ORF) was composed of 1290 nucleotide residues,among them the GC content was 44.11％ and the AT content was 55.89％,it was composed of 429 amino acids (20 kinds),the accession number of the sequence submitted to GenBank was JN712306,its homology of nucleotides to the 76-118 strain was 83％ and the homology of amino acids was 98％,ten nonspecific variation sites were found.The grand average of hydropathicity was-0.405.There were three transmembrane domains and four non transmembrane domains in the secondary structure of nucleoprotein including 55％ of helix structure,6.1％ of sheet structure and 38.9％ of loop structure.Conclusion The bioinformatics analysis of Hunan03 strain S gene might be important for provide the substructure data to reveal the significance of S gene characteristics on hemorrhagic fever renal syndrome (HFRS) prevention and control.

Objective:To investigate the effects and mechanism of sodium hydrosulfide on rat epidermal cells intervened with serum from burned rat (hereinafter referred to as burn serum).Methods:The experimental research method was used. Ten male Sprague-Dawley (SD) rats aged eight months were taken to prepare normal rat serum (hereinafter referred to as normal serum), 30 male SD rats aged eight months were taken to prepare burn serum after full-thickness burn, and epidermal cells (the third passage)isolated from 10 SD rats born one day were used for the experiments. The cells were divided into normal serum group treated with normal serum and burn serum group treated with burn serum. Cell counting kit 8 method was used to detect cell survival rate after 1, 2, 4, 6, and 8 h of culture, respectively, to screen the subsequent intervention time of burn serum. The cells were divided into burn serum control group treated only with burn serum and 50, 100, 150, 200, 250 μmol/L sodium hydrosulfide groups treated with burn serum+ sodium hydrosulfide at corresponding final molarity. After 30 min of culture following the burn serum intervention, the cell survival rate was detected as above to screen the subsequent intervention concentration of sodium hydrosulfide. The cells were divided into burn serum control group treated with burn serum only and sodium hydrosulfide only group, glibenclamide only group, and sodium hydrosulfide+ glibenclamide group treated with burn serum+ corresponding reagents. After 5, 10, 15 min of culture following the burn serum intervention, the cell survival rate was detected as above to screen the subsequent intervention time of glibenclamide. The cells were divided into burn serum control group treated with burn serum and sodium hydrosulfide only group, glibenclamide only group, and sodium hydrosulfide+ glibenclamide group treated with burn serum+ corresponding reagents. After completing corresponding culture time of each reagent, the mitochondria were extracted to detect cytochrome c oxidase (CCO) activity using a spectrophotometer, and the protein expression level of adenosine triphosphate (ATP)-sensitive potassium channel was detected by Western blotting. Except for the number of samples for ATP-sensitive potassium channel protein detection, which was 3, the number of samples for the other indicators was 10. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference (LSD)- t test, LSD test, and Bonferroni correction. Results:Compared with that of normal serum group, the cell survival rate was significantly decreased in burn serum group after only 4 and 6 h of culture ( t=4.02, 6.42, P<0.05). An overall comparison showed statistically significant differences in cell survival rate among the time points within normal serum group and burn serum group ( F=19.74, 4.48, P<0.05 or P<0.01). Four hours of culture was selected as the subsequent intervention time of burn serum. After 30 min of culture following the burn serum intervention, compared with that of burn serum control group, only 150, 200, 250 μmol/L sodium hydrosulfide groups had a significantly higher cell survival rate ( P<0.01), thus 150 μmol/L was selected as the subsequent intervention concentration of sodium hydrosulfide. Compared with that of burn serum control group, the cell survival rate decreased significantly in glibenclamide only group after 5 and 15 min of culture following burn serum intervention ( P<0.05) and increased significantly in glibenclamide only group after 10 min of culture following the burn serum intervention and sodium hydrosulfide only group at each time point ( P<0.05 or P<0.01). The cell survival rate in sodium hydrosulfide+ glibenclamide group was significantly lower than that of sodium hydrosulfide only group at each time point ( P<0.05). The difference in cell survival rate was statistically significant among the time points within glibenclamide only group ( F=11.81, P<0.01). Five minutes of culture was selected as the subsequent intervention time of glibenclamide. After 35 min of culture following the burn serum intervention, compared with (1.62±0.08) nmol·min -1·mg -1 and 0.682±0.063 in burn serum control group, the CCO activity of cells and the protein expression level of ATP-sensitive potassium channel were significantly increased in sodium hydrosulfide only group ((1.99±0.09) nmol·min -1·mg -1 and 0.932±0.014, P<0.01) and significantly decreased in glibenclamide only group ((1.44±0.09) nmol·min -1·mg -1 and 0.600±0.012, P<0.01); the CCO activity of cells and the protein expression level of ATP-sensitive potassium channel in sodium hydrosulfide+ glibenclamide group ((1.79±0.06) nmol·min -1·mg -1 and 0.744±0.071) was significantly lower than those of sodium hydrosulfide only group ( P<0.05 or P<0.01). Conclusions:Sodium hydrosulfide can improve the survival rate of rat epidermal cells after burn serum intervention, by a mechanism which is related to the alleviation of epidermal cell mitochondrial damage and mediated by ATP-sensitive potassium channel.

OBJECTIVETo investigate the relationship between the molecular characteristics and phylogenetic evolution of rabies N gene.

METHODSSaliva samples were collected from rabies cases, and RT-PCR was used to amplify the N gene of rabies virus with the specific primers. The amplifying product of RT-PCR was cloned to pUCm-T vector and transformed into E.coli XL1-Blue and then the blue-white selection, PCR screening and gene sequencing were carried out to identify the positive clones. Finally, ExPASy and other bioinformatics software were used to analyze and predict the structure and biological characteristics of the N genome.

RESULTSThe amplification product of RT-PCR was 1 353 bp, the recombinant plasmid pUCm-T/N was constructed, the whole length of the N gene open reading frame was composed of 1 353 nucleotide residues to code 450 amino acids (20 kinds), the accession number submitted to the Genbank was HM756692, its sequence homology of nucleotides and amino acids compared with the vaccine strain CTN-1-V was 90% and 99% respectively. The evolutionary analysis showed that the isolated strain belonged to genotype I with certain geographic regionality.

CONCLUSIONThe characteristics investigation and bioinformatics analysis of Hunan0806 N gene will provide fundamental data to reveal the significance of the N gene characteristics for rabies epidemiology and its prevention & control.

Amino Acid Sequence ; Gene Expression Regulation, Viral ; physiology ; Humans ; Models, Molecular ; Molecular Sequence Data ; Nucleocapsid Proteins ; genetics ; metabolism ; Phylogeny ; Protein Conformation ; Rabies ; virology ; Rabies virus ; genetics ; metabolism ; Saliva ; virology

Objective:To confirm the first imported Chikungunya fever (CHIK) epidemic in Hunan province.Methods:Serum samples of patients and colleagues were collected. The nucleic acids of Dengue virus (DENV), Yellow fever virus (YFV), Chikungunya virus (CHIKV) were detected by real- time fluorescent quantitative PCR. The positive PCR products were sequenced. Phylogenetic tree was constructed.Results:The serum samples of the patient and one of the five colleagues were positive for CHIKV. The Blast comparison of gene sequence showed 99% homology with CHIKV sequences. The infected CHIKV belonged to ECSA genotype in the phylogenetic tree.Conclusions:The first imported CHIK epidemic in Hunan province was confirmed through the epidemiological survey and etiologic detection.

Objective To inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).Methods Western blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T.The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry.The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot.The cell growth curve was drawn by MTT test.The Ki-67-positive rate was calculated using immunofluorescence staining.The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.Results Western blot results showed that BTG2 relative expression levels were 0.83±0.12,0.18±0.04, 0.20±0.05 and 0.36±0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively.The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5%(33/40), 77.5%(31/40) and 17.5%(7/40), respectively, with a significant difference between two groups (P<0.05).Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2± 8.0)%, (81.4±9.7)%and (50.1±7.1)%, respectively, showing a significant difference between two groups ( P<0.05 ) .The scratch test results showed that in the control group, empty vector group and BTG2 transfection group , the distance of SW620 cells between two sides was ( 79.27 ±11.24) μm, ( 80.65 ± 12.17) μm and (124.77±19.63) μm, respectively, with a significant difference between two groups ( P<0.05) .Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5±13.1)%, (73.2±12.9)%and (47.4±9.1)%,respectively, showing a significant difference between two groups ( P<0.05) .The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.Conclusion s BTG2 gene is involved in colon cancer cell proliferation and metastasis , and effectively restores the function of BTG2 protein.Therefore, it may be expected to become a new option in gene therapy for colon cancer.

Objective To inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).Methods Western blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T.The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry.The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot.The cell growth curve was drawn by MTT test.The Ki-67-positive rate was calculated using immunofluorescence staining.The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.Results Western blot results showed that BTG2 relative expression levels were 0.83±0.12,0.18±0.04, 0.20±0.05 and 0.36±0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively.The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5%(33/40), 77.5%(31/40) and 17.5%(7/40), respectively, with a significant difference between two groups (P<0.05).Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2± 8.0)%, (81.4±9.7)%and (50.1±7.1)%, respectively, showing a significant difference between two groups ( P<0.05 ) .The scratch test results showed that in the control group, empty vector group and BTG2 transfection group , the distance of SW620 cells between two sides was ( 79.27 ±11.24) μm, ( 80.65 ± 12.17) μm and (124.77±19.63) μm, respectively, with a significant difference between two groups ( P<0.05) .Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5±13.1)%, (73.2±12.9)%and (47.4±9.1)%,respectively, showing a significant difference between two groups ( P<0.05) .The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.Conclusion s BTG2 gene is involved in colon cancer cell proliferation and metastasis , and effectively restores the function of BTG2 protein.Therefore, it may be expected to become a new option in gene therapy for colon cancer.