Objective To explore the method of repairing wound defects with exposed tibia. Methods 32 patients with soft tissue defects with exposed tibia were treated with three kinds of flaps pedicled with interior,lateral and posterior vascular perforating branches in the leg,respectively.Re- suits One flap with distal part necrotic was treated with change of dressings and got one-stage healing. One case had delayed union for muscular infection under the flap.Other flaps all were successful and sur- vived.No ostemyelitis was found.Conclusion The flap pedicled with vascular perforating branches of leg has abundant blood supply and is a good method for repairing small or middle wound defect with ex- posed tibia.

Objective To study the exact function of human gene 5 transactivated by pre-S1 protein of hepatitis B virus(PS1TP5)by investigating the gene expression of PS1TP5 in yeast cells. Methods Reverse transcription-polymerase chain reaction(RT-PCR)was performed to amplify the gene of PS1TP5 using the mRNA of HepG2 cells as template and the gene was cloned into pGEM-T vector.The gene of PS1TP5 was cut from pGEM-T-PS1TP5 vector and cloned into yeast expressive plasmid pGBKT7,then pGBKT7-PS1TP5 was transformed into yeast cell AH109.The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western hybridization.Results PS1TP5 gene was successfully amplified and identified by DNA sequencing.The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109.The results of SDS-PAGE and Western assay showed that the relative molecular weight of the expressed product was about 36 950,and PS1TP5 protein existed in yeast cells.Conclusion The findings suggest that PS1TP5 can be successfully expressed in yeast cell.

OBJECTIVETo develop and investigate GLL-37, a substitution analogue of the human antimicrobial peptide LL-37 with anti-enzymatic degradation activity and improved efficacy.

METHODSThe bactericidal activities of LL-37 and newly developed GLL-37 against 6 Gram-negative and -positive bacteria were determined by Broth microdilution assays. The minimum inhibitory concentrations of LL-37 and GLL-37 against E.coli ATCC 25922 in different NaCl concentration medium were also detected. Both peptides were co-incubated with elastase, and then analyzed by PAGE electrophoresis and bactericidal activity determination.

RESULTGLL-37 showed a stronger elastase resistance ability than LL-37, and was significantly more effective than LL-37 under high-salt condition.

CONCLUSIONThe antimicrobial peptide GLL-37 derived form LL-37 has the potential as a new therapeutic agent for bacterial infections.

Animals ; Anti-Bacterial Agents ; pharmacology ; therapeutic use ; Antimicrobial Cationic Peptides ; pharmacology ; therapeutic use ; Blood Bactericidal Activity ; drug effects ; Cathelicidins ; Cell Membrane Permeability ; drug effects ; Escherichia coli ; drug effects ; Female ; Humans ; Membrane Proteins ; metabolism ; Monocytes ; drug effects ; Pseudomonas Infections ; drug therapy

BACKGROUNDThe hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein.

METHODSThe reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for alpha-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.

RESULTSForty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2 receptor gamma chain, PALM2-AKAP2 protein (PALM2-AKAP2), eukaryotic translation initiation factor 4A, beta-2-microglobin, solute carrier family 9 (sodium/hydrogen exchanger), calreticulin, asialoglycoprotein receptor 1 (ASGR1), MHC class II lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86 (LY86) and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank (accession number: DQ471327).

CONCLUSIONSGenes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S1 protein.

Amino Acid Sequence ; Base Sequence ; DNA, Complementary ; chemistry ; Gene Library ; Hepatitis B Surface Antigens ; genetics ; physiology ; Humans ; Leukocytes ; metabolism ; Molecular Sequence Data ; Plasmids ; Protein Interaction Mapping ; Protein Precursors ; genetics ; physiology ; Recombinant Fusion Proteins ; biosynthesis ; Transcriptional Activation ; Two-Hybrid System Techniques ; Yeasts ; genetics

OBJECTIVETo screen proteins in leukocytes interacting with PS1TP2 by yeast-two hybrid and to view their subcellular localization in HepG2 cells.

METHODSThe function and structure of PS1TP2 were studied by bioinformatic analysis. PS1TP2 gene was amplified and cloned into plasmid pET32a (+) and pGBKT7 to construct recombinant expression vectors pET32a (+)-PS1TP2 and pGBKT7-PS1TP2. They were transduced into E. coli Rosetta strain and yeast AH109. The transformed yeast mated with yeast Y187 containing leukocyte cDNA library plasmid in a 2xYPDA medium. Diploid yeast cells were plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) for selecting twice and then screening. Then a green fluorescent protein (GFP) expression vector pEGFP-C1-PS1TP2 was established, transduced into HepG2, and its subcellular localization was studied by fluorescence microscopy and confocal microscopy.

RESULTSBioinformatic analysis showed that the PS1TP2 gene was located at 6q24.1, the protein was unstable and the aliphatic index was very high. After transformation of the E. coli and yeast AH109, the expression protein showed: (1) the molecular weight of the expressed product was about 41000 Da, and (2) PS1TP2 existed within the cells. Diploid yeast cells were plated on the synthetic dropout nutrient medium containing X-a-gal for selecting twice and then screening. Twenty-six colonies from blue colonies were sequenced, pEGFP-C1-PS1TP2 was successfully expressed in the HepG2 cells, and PS1TP2 was located in the cell plasma.

CONCLUSIONA prokaryotic expression vector pET32a(+)-PS1TP2 was constructed successfully and the PS1TP2 was successfully expressed in the yeast system. Genes of PS1TP2 interact with leukocyte proteins. These results bring some new clues for studying the biological functions of HBV.

Base Sequence ; Cloning, Molecular ; Dipeptides ; Gene Expression ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Humans ; Protein Precursors ; metabolism ; Proteins ; genetics ; metabolism ; Two-Hybrid System Techniques

OBJECTIVETo reveal the allelic frequencies and haplotype frequencies of fourteen Y-chromosome short tandem repeat (STR) loci in a Tibetan population.

METHODSThe Y-chromosomal STR loci were analyzed from 126 healthy unrelated autochthonous male individuals of Chinese Tibetan using a multiplex PCR system. Allele and haplotype frequencies for these loci were determined by the AmpFISTR Y filer PCR Amplification kit.

RESULTSOne hundred and twenty-one alleles were detected from the 14 STR loci. The allele diversity values (DP) for each locus ranged from 0.4104 (DYS391) to 0.9489 (DYS385a, b), the DP value of these loci were higher than 0.5 except for that of DYS391. A total of 105 haplotypes were identified in the Y-STR loci, among which 103 were unique, while two occurred more than once. The overall haplotype diversity for the Y-STR loci was 0.9998, and the discrimination capacity was 0.9898.

CONCLUSIONThe 14 STR loci above belong to loci of high discriminating ability, the haplotypes are highly polymorphic.

Alleles ; Chromosomes, Human, Y ; genetics ; Gene Frequency ; Haplotypes ; genetics ; Humans ; Male ; Microsatellite Repeats ; genetics ; Polymorphism, Genetic ; genetics ; Tibet

Adult ; Aged ; Biomechanical Phenomena ; Bone Screws ; Female ; Humans ; Joint Instability ; physiopathology ; surgery ; Lumbar Vertebrae ; physiopathology ; surgery ; Male ; Middle Aged ; Spinal Diseases ; physiopathology ; surgery ; Spinal Fusion ; instrumentation

Objective To study the biological significance of HBV gene mutation at nucleotide (nt) 1862. Methods The EB virus eukarotic expression vector for HBV pre-C/C gene was constructed using molecular biological method, HBV pre-C/C gene mutation at nt 1862 was induced by way of site-specific mutation technique, and identified by PCR-RFLP and sequencing analysis. The resulted recombinant plasmid containing the HBV variant was subsequently transfected into Cos7 cell line mediated by lipofectin, to observe the expression of HBeAg. The cells transfected with the recombinant plasmid con- taining wild HBV pre-C/C gene fragment served as control. Results HBeAg expression was detected in the cells transfected with wild recombinant plasmid but not in those with HBV variant transfection. Conclusion The success in the construction of eukarotic expression vector for HBV pre-C/C gene mutation at nt 1862 may pave the way for further studying a series of biological changes of HBV resulted from the mutation addressed in this study.

Objective To study the biological significance of HBV gene mutation at nucleotide (nt) 1862. Methods The EB virus eukarotic expression vector for HBV pre-C/C gene was constructed using molecular biological method, HBV pre-C/C gene mutation at nt 1862 was induced by way of site-specific mutation technique, and identified by PCR-RFLP and sequencing analysis. The resulted recombinant plasmid containing the HBV variant was subsequently transfected into Cos7 cell line mediated by lipofectin, to observe the expression of HBeAg. The cells transfected with the recombinant plasmid con- taining wild HBV pre-C/C gene fragment served as control. Results HBeAg expression was detected in the cells transfected with wild recombinant plasmid but not in those with HBV variant transfection. Conclusion The success in the construction of eukarotic expression vector for HBV pre-C/C gene mutation at nt 1862 may pave the way for further studying a series of biological changes of HBV resulted from the mutation addressed in this study.

Objective To compare the effectiveness of TiRobot assisted F screw technique and inverted triangle parallel nail internal fixation in the treatment of unstable femoral neck fractures.Methods A retrospective analysis was conducted on 72 patients with unstable femoral neck fractures who were treated with percutaneous cannulated screw fixation assisted with TiRobot Orthopaedic robot from December 2019 to April 2021.Among them,37 patients were treated with F screw internal fixa-tion,including 16 males and 21 females,aged47 to 64years old with an average of(53.87±5.28)years old;According to Pauwels classification,there were 1 case of type Ⅰ,19 cases of type Ⅱ,17 cases of type Ⅲ;8 cases of combined medical diseases;17 cases of falling,8 cases of traffic accident and 12 cases of falling from height;The time from injury to operation was 29 to 49 hours with average of(35.00±7.34)hours.Another 35 cases used internal fixation with an inverted triangle parallel nail,including 13 males and 22 females with an average age of 46 to 63 years old(52.36±5.05)years old;According to the Pauwels injury classifi-cation:there were 2 cases of type Ⅰ,21 cases of type Ⅱ,12 cases of type Ⅲ;6 cases of medical diseases,15 cases of falling in-jury,9 cases of traffic accident,11 cases of falling injury;The time from injury to operation was 30 to 45 hours with an average of(33.00±6.83)h.The intraoperative blood loss,operation time,intraoperative fluoroscopy times,follow-up time,fracture healing time,postoperative complications were observed and compared between the two groups.The hip joint function was e-valuated by Harris score at 6 months and 12 months after operation.Results There was no significant difference in operation time,intraoperative blood loss,intraoperative fluoroscopy times and other intraoperative data between two groups(P＞0.05).Both groups were followed up regularly,and the follow-up time was 12 to 16 months.The fracture healing time and Harris score of the F screw internal fixation group were better than those of the inverted triangle parallel nail internal fixation group(P＜0.05).There was 1 case of femoral neck shortening in the F screw internal fixation group,1 case of nonunion,1 case of nail withdrawal,and 1 case of lower extremity deep vein thrombosis in the inverted triangle internal fixation group.The incidence of complications in the F screw internal fixation group was lower than that in the inverted triangle parallel nail internal fixation group(P＜0.05).Conclusion Percutaneous cannulated F screw technique using Tirobot navigation positioning system is a safe and effective treatment for patients with unstable femoral neck fractures.It can significantly shorten the fracture healing time,reduce the incidence of postoperative complications,significantly improve hip joint function,and improve the quality of life.