Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu"-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu"-ts18(cd) were indistinguishable from the reported ts mutant Wu"-ts 18. Then we cultured the ts mutant Wu"-ts 18(cd) at non-permissive temperature 39.5℃, which "forced" the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu"-ts 18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

OBJECTIVE: One multiplex genotyping system was developed in using silver staining with allelic ladders for three Y-chromosome STR markers (DYS434, DYS443, and DYS456), with a view towards the application of rapid and simple genotyping assay methods for DNA profiling. The distributions of haplotypes for three Y-STR loci(DYS434, DYS443, and DYS456) was investigated in a Tibetan ethnic group of Chinese population. METHODS: Allele and haplotype frequencies at these Y-STRs loci(DYS434, DYS443, and DYS456) were analysed by PCR amplification using Y-STR multiplexes, followed by horizontal non-denaturing polyacrylamide gelelec-trophoresis in 101 unrelated males of Tibetan ethnic group in Lasa of China. RESULTS: A total of 31 different haplotypes were found, 16 of them being unique. The haplotype diversity value (which is the same as the discrimination index) calculated from all three loci combined was 0.9481, which is informative. CONCLUSION The Y-STR multiplexes provide useful information for forensic analysis and paternity tests and can also be of great benefit for providing information not normally available from autosomal DNA systems.
Alleles ; China/ethnology* ; Chromosomes, Human, Y/genetics* ; Female ; Forensic Medicine ; Gene Frequency ; Genetic Markers ; Genetics, Population ; Genotype ; Haplotypes ; Humans ; Male ; Microsatellite Repeats/genetics* ; Paternity ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Silver Staining

OBJECTIVETo evaluate the replication and encapsidation of HBV mutants with the truncated C gene.

METHODSThe HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome.

RESULTSThe C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay.

CONCLUSIONThe C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.

Cell Line, Tumor ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; genetics ; physiology ; Humans ; Mutation ; Plasmids ; genetics ; Transfection ; Virus Replication

In order to investigate the effect of N-tosyl-L-phenylalnylchloromethyl ketone (TPCK) and dexamethasone (Dex) on expression of nuclear transcription factor-kappaB (NF-kappaB) in childhood acute lymphoblastic leukemia (ALL) and its significance, so as to provide the experimental basis for corresponding clinical treatment of ALL, in which NF-kappaB is taken as a target. The biotin-streptavidin method was used to detect the expression of NF-kappaB P65 protein and the effects of TPCK and Dex at clinically relevant dosage on activity of NF-kappaB P65 protein in 20 childhood ALL patients. The results indicated that the expression of NF-kappaB P65 protein was strongly diminished and reached to negative level at 2 hours by treatment with 40 micromol/L TPCK, the positive expression of NF-kappaB P65 protein was (2.5 +/- 1.6)%. TPCK had a time-dependent inhibitory effect on ALL cells cultured in vitro. The expression of NF-kappaB P65 protein in ALL cells was strongly inhibited by clinically relevant concentration of dexamethasone 5.0 microg/ml for 24 hours in vitro. The positive expression was (25.0 +/- 3.0)%, there was significant difference, as compared with untreated ALL cells (T=55, P<0.01). It is concluded that TPCK and Dex can inhibit NF-kappaB activity. Inhibition of NF-kappaB activity may be one of the effect mechanism of dexamethasone on ALL cells. Inhibition of NF-kappaB conduction pathway may have a significant value in childhood ALL treatment.
Bone Marrow Cells ; pathology ; Cells, Cultured ; Child ; Child, Preschool ; Dexamethasone ; pharmacology ; Female ; Humans ; Infant ; Leukocytes, Mononuclear ; pathology ; Male ; NF-kappa B ; biosynthesis ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; Protein Synthesis Inhibitors ; pharmacology ; Tosylphenylalanyl Chloromethyl Ketone ; pharmacology ; Tumor Cells, Cultured

The study was aimed to observe the effect of recombinant human granulocyte-colony stimulating factors (rhG-CSF) in low dose on peripheral blood stem cell (PBSC) mobilization in unrelated healthy normal donors. G-CSF was administered at 5 microg/(kg x d) subcutaneously for successive 5 or 6 days to 56 unrelated donors. Stem cells were harvested on the fourth and fifth days or on the fifth and sixth days. The numbers of mononuclear cells (MNC), CD34(+) cells and Hb, Plt, and CD3(+), CD4(+), CD8(+) and CD20(+) cells were determined during the mobilization. The results showed that most common adverse events were bone pain (17.9%, 10/56), agrypnia (8.9%, 5/56) and lassitude (4.5%, 3/56) during rhG-CSF mobilization, but all donors were suffered less than grade II according to the WHO criteria, and did not need to stop the mobilization and not need to give special treatment. In harvest on day 4 - 5 and 5 - 6, MNC count was (5.95 +/- 1.52) x 10(8)/kg and (7.19 +/- 2.12) x 10(8)/kg; CD34(+) cells count was (3.03 +/- 1.09) x 10(6)/kg and (7.92 +/- 2.50) x 10(6)/kg. There were no significant differences in hemoglobin level and platelet count, the percentage of CD3(+) cells, CD4(+) cells, CD8(+) cells and CD20(+) cells between pre-mobilization and post-mobilization of rhG-CSF. It is concluded that the low dose of rhG-CSF 5 microg/(kg x d) for peripheral blood stem cell mobilization in unrelated healthy normal donors is safe and effective.
Adolescent ; Adult ; Blood Donors ; Female ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; Recombinant Proteins

Objective To investigate the metabolic alterations in the brain of neonates with HIE and correlate those alterations with clinical grading and prognosis of HIE.Methods Fourty-six eases of full-term neonates diagnosed as HIE clinically were performed MRI and 1~H-MRS,9 healthy neonates without the evidence of asphyxia were studied as controls,1~H-MRS techniques included single voxel proton MRS and two dimensional muhi-voxel chemical shift spectroscopy imaging,point resolved spectroscopy sequence was used for 1~H-MRS.Metabolic changes in the spectroscopy were analyzed in neonates with HIE,and study the relationgship between MRS findings and prognosis.Results(1)The typical 1~H-MRS manifestations of full- term neonates suffering from HIE were as follows:the peaks of Lac were elevated,GLx-? were elevated and NAA were decreased.(2)GLx-?/Cr ratio in control,mild,moderate and severe HIE group was 0.16, 0.21,0.64,and 1.31,respectively.Lac/Cr ratio in control,mild,moderate and severe HIE group was 0.12,0.14,0.19,and 0.26,respectively.There was a significant difference in the ratio of GLx-? and Lac/Cr between HIE group and control group(t=5.01,P

Objective:To construct HIV-1 pseudovirus containing enhanced green fluorescent protein ( EGFP ) gene. To understand the interaction between the virus and the cells. Methods: HIV-1 pseudovirus containing EGFP gene was constructed by lentiviral packaging systems, and its EGFP gene was amplified using RT-PCR. The level of genomic integration and transcription of HIV-1 pseudovirus containing EGFP gene were detected on iDCs infected with HIV-1 pseudovirus. At the same time, research on expression of the EGFP gene in iDCs infected with HIV-1 pseudovirus was performed. Results:The EGFP gene of HIV-1 pseudovirus was detected through RT-PCR. The EGFP gene was identified in iDCs infected with HIV-1 pseudovirus through PCR and RT-PCR. The EGFP was observed in iDCs infected with HIV-1 pseudovirus under fluorescence microscopy. Conclusion: HIV-1 pseudovirus containing EGFP gene has been successfully produced. The HIV-1 pseudovirus that we constructed can infect iDCs,then its RNA can integrate into the genome of iDCs in the way of reverse transcription,and the EGFP gene could express in the iDCs after infected with HIV-1 pseudovirus.

OBJECTIVE Paeoniflorin (PF) and albiflorin (AF) are the major active components of total peony glucosides(TPG)from Paeonia lactiflora Pal,which have many biological activities such as anti-inflammatory, antioxidation and anti-hypertension effects. The drug-drug pharmacokinetic interaction among PF,AF and TPG,the pharmacokinetic comparisons of AF between hypoxia and normoxia,the transport of AF cross the blood-brain barrier cell model and the transport of AF/PF/TPG cross Caco-2 cell model were investigated.METHODS A highly sensitive and rapid UPLC-MS method with multiple-reaction monitoring(MRM)scanning via electrospray ionization(ESI)source operating both in the positive and negative ionization mode was successfully developed and validated for simultaneous quantitation of PF and AF in rat plasma after an oral administration of PF,AF and TPG. RESULTS The validated and developed UPLC-MS/MS method was successfully applied to simultaneously determine the AF and PF concentration in rat plasma and investigate pharmacokinetic interactions after a single intragastrical ad-ministration of PF,AF,co-administration of PF with AF and TPG,respectively.The elimination of both PF co-administered with AF and PF in TPG were slower than those for PF alone and the distribution in the tissues was wider.The combination of PF with AF or TPG could significantly increase the values of the AUC, MRT and t1/2of the drug PF, and reduce the values of CL of PF. From a comparison of the main pharmacokinetic parameters among AF alone, AF combined with PF and AF in TPG, the values of the MRT and t1/2of AF in TPG were greater than that of AF alone,and there were statistically signifi-cant differences in these parameters(P<0.05,P<0.01).It was also noticed that AUC and Cmaxof PF in hypoxia rats were significantly decreased compared with that of normaxia rats, suggesting that there was a decreased exposure of PF in rats under hypoxia. The multiple active components in TPG may lead to DDIs between some P-gp substrates. CONCLUSION The clinical performance of total peony glucosides would be better than that of single constitute. The outcomes of the study are expected to serve as a basis for development of clinical guidelines on total peony glucosides usage.

OBJECTIVETo evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy.

METHODSA fragment containing the small envelope gene of HBV was replaced with the reporter gene green fluorescent protein (GFP) to construct the recombinant HBV vector, which was transfected into HepG2 cells with liposome. The expression of GFP was observed with fluorescence microscope. The HBV cccDNA was testified using semi-nest PCR. The viral particles of the recombinant HBV in culture medium were detected by PCR as well as Southern blot.

RESULTSThe HBV vector carrying the interesting gene of GFP could express the functional protein in the transfected hepatocytes. However, the recombinant HBV vector was replication-deficient, which could not be packed and replicated in the hepatocytes to secrete mature recombinant HBV particles carrying the interesting gene of GFP when transfected solely but could when cotransfected with the recombinant and helper construct which lacked part of 5'-proximal HBV RNA packaging signal epsilon.

CONCLUSIONIt is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.

Cell Transformation, Viral ; Cells, Cultured ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; physiology ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; cytology ; virology ; Humans ; Liver ; cytology ; virology ; Recombinant Proteins ; genetics ; Transfection ; Virus Replication

OBJECTIVETo explore the clinical effect of talar neck fractures treated by open reduction and internal fixation with screws.

METHODSAmong 28 cases in the study, 20 cases were males and 8 cases were female. The age ranged from 22 to 72 years with an average of 38 years. Based on the Hawkins classification,there were 6 cases of type I,18 of type I and 4 of type II. They were treated by open reduction and internal fixation with screws.

RESULTSTwenty-eight cases were followed up for 1 to 7 years(mean 2.8 years). The evaluation of the results by Hawkins functional rating scale revealed excellent in 14 cases,good in 9 cases, fair in 3 cases and poor in 2 cases. The excellent and good rate were 83.2%. Osteonecrosis occurred in 5 cases with 3 of type II and 2 of type III fractures. Two of 3 cases with talar displacement were found with osteonecrosis or painful arthritis. The subtalar arthritis occurred in 6 cases, 3 of which were associated with ankle arthritis. Two cases underwent arthrodesis because of painful arthritis of the subtalar joint or osteonecrosis of the talar body. Wound infection and anteromedial skin necrosis of the ankle were not found.

CONCLUSIONTreatment of talar neck fractures could obtain satisfactory clinical results through open reduction and internal fixation with screws. Protection of the residual blood supply, anatomically reduction and stable fixation are essential for successful treatment of talar neck fractures.

Adult ; Aged ; Bone Screws ; Female ; Fracture Fixation, Internal ; methods ; Fractures, Bone ; diagnostic imaging ; surgery ; Humans ; Male ; Middle Aged ; Radiography ; Talus ; injuries ; surgery