Objective To search for a good method for generation of hematopoietic stem cells (HSC) by embryonic stem cells (ESC),and to investigate the potential of HSC derived from ESC to reconstruct hematopoiesis in vivo.Methods Using a three-step method to induce a mice ESC line, E14.1-into HSC.And identifying HSC by flow cytometry analysis cell markers with CD34 +/Sca-1+, then HSC (1×109L-1)from ESC differentiation were injected into severe combined immunodefieency (SCID) mice for observing teratoma formation.To validate function of HSC by colonngenic cells assay and to reconstitute the hematopoiesis in lethally irradiated mice.Results Combining to use more hematopoietic stimulating factor to availably promote the El4.1 cell into embryoid body (EBs) with abundant hematopoietic progenitors.EBs were induced after 14 day to fast differentiate for HSC with peak percentages of CD34+/Sca-1+ cells reached to (13.72±2.07)%.To harvested ceils from EBs by day 14 for second -step HSC differentiation, percent of CD34+/Sca-1+ cells rise to(24.62±2.50) % after day 16 induction.Cloning forming units (CFU) analysis showed that more Erythro -myeloid cloning generation was observed at this period.Cells obtained in the second step are subsequently plated on monolayer of mice bone marrow stromal cells, in the presence of TPO, FLt3 ligand and superoatant of mice fetal liver stromal cells, cultured for additional 15 days, followed fast expansion of CD34+/Sca-1+ to maximally (58.64±4.20 ) % with more CFU-E, CFU-GM and CFU-GEMM population.Wright-Giemsa stain showed that its had the character of hematopoietic progenitors.Cells from the third-step were injected into SCID mice, but no teratomas were recovered in 2 mices after 6 weeks.Positive selection of CD34+/Sca-1+ cells by magnetic sorting from the third - step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 days after transplantation, with 71.4% successful engraftment rate.And 3 recipients showed that the cell population of the peripheral blood leukocytes,red cells, hemoglobin approached normal index at 40 days after transplant, hut followed relative'slow renew in platelet count.Survival rate of transplant group is 43.0%, compared to 100% mortality in control mice.Karyotyping assays confirmed female mice with XY.Conclusions The results showed that the three-step differentiation and the culture conditions described here good support differentiation of ESC into HSC.HSC derived from ESC were safe without teratomas formation in body, and its can reconstruct hematopoiesis.

Objective To investigate the 16S rRNA methylase genes and Aminoglycoside modifying enzymes(AMEs)genes in Enterobacter cloacae isolated from the People's Liberation Army 98th Hospital,Huzhou district,Zhejiang province,China.Methods 40 strains of Enterobacter cloacae were isolated from the inpatients between September,2003 and November,2004.5 kinds of 16S rRNA methylase gene (including armA,rmtA,rmtB,rmtC and rmtD)and 9 kinds of AMEs gene[including aac(3)-Ⅰ,aac(3)-Ⅱ,aac(3)-Ⅲ,aac(3)-Ⅳ,aac(6')-Ⅰ b,aac(6')-Ⅱ,ant(3'')-Ⅰ,ant(2'')-Ⅰ and aph(3')-Ⅵ]were analyzed by PCR and verificated by DNA sequencing.Results In 40 strains of Enterobacter cloacae,the positive rates of genes of rmtB,aac(3)-Ⅱ,aac(6')-Ⅰ b,ant(3'')-Ⅰ,ant(2'')-Ⅰ and aph(3')-Ⅵ were 12.5%(5/40),27.5%(11/40),72.5%(29/40),32.5%(13/40),5.0%(2/40)and 5.0%(2/40),respectively.8 kinds of the rest of genes were all tested negative.The total positive rate of AMEs gene was 85.0%(34/40).Among 29 strains of Enterobacter cloacae that the aac(6')-Ⅰ b gene was positive,through PCR and verification by DNA sequencing,7 strains(24.1%)were confirmed to take the aac(6')-Ⅰ b-cr(the GenBank register number:EF375620,EU159121)alone,18 strains(62.1%)were confirmed to take the aac(6')-Ⅰ b-Suzhou(EU085533)alone,3 strains(10.3%)were confirmed to take both aac (6')-Ⅰ b-Suzhou and aac(6')-Ⅰ b-cr while only 1(3.4%)was aac(6')-Ⅰ b(the classical type).Conclusion There was lower positive rate of 16S rRNA methylase gene but very high AMEs genotypes in Enterobacter cloacae isolated from inpatients and the finding of rmtB gene was reported for the first time in the world.At least 5 kinds of AMEs gene existed in Enterobacter cloacae were isolated and they were the new host of both gene of aac(6')-Ⅰ b-cr and aac(6')-Ⅰ b-Suzhou,with aac(6')-Ⅰ b-Suzhou gene was the predominance subtype in aac(6')-Ⅰ b.

In July 1997, a strain (GB30) of virus was isolated from 60 samples of brain tissues of Murina aurata (Chiroptera: Vespertilionidae) co llec ted in Gengma county, Yunnan province, China. Isolation of virus was negative fr om 4 samples of brain tissues of Rousettus leschenaulti (Chiroptera: Pteropo did ae) collected in Gengma. GB30 virus strain could regularly cause illness and dea th in suckling mice, produced evident CPE in BHK21 cells. It agglutinated red b lood cells of dove at pH5.75～7.4. This virus has been identified serologically by hemagglutination inhibition and immunofluorescent tests using Japanese enceph alitis (JE), dengue (DEN) type 1,2,3,4, and chikungunya (CHIK) viruses monoclona l antibodies, and JE and sindbis (SIN) viruses immune sera. It showed specific r eaction to JE virus only and no reaction with DEN 1～4, CHIK and SIN viruses. Th erefore it can be identified as JE virus. This is the first report on the isolat ion of JE virus from Murina aurata. The results showed that bats are conside red as the reservoir and amplifier host of JE virus transmission in nature.

OBJECTIVETo explore the relationship between exon 3 mutation in the methyl CpG-binding protein 2 (MeCP2-E3) gene and Hirschsprung disease (HSCR) and anorectal malformations (ARMs).

METHODSPCR and DNA sequencing were used to detect the mutation of MeCP2-E3 in 120 healthy controls, 120 HSCR, and 50 ARMs.

RESULTSOn sequencing, 45(37.5%) children with HSCR had basic replacement in MeCP2-E3, 12(10.0%) of them were homozygous mutation. Fourteen(28.0%) children with ARMs had basic replacement in MeCP2-E3, 4(8%) of them were homozygous mutation. There were no mutation in the control group.

CONCLUSIONSMutation of MeCP2-E3 is present in the peripheral blood of children with HSCR or ARMs, which may contribute to the development of Hirschsprung disease or anorectal malformations.

Anorectal Malformations ; Anus, Imperforate ; genetics ; Case-Control Studies ; Child, Preschool ; Exons ; Female ; Hirschsprung Disease ; genetics ; Humans ; Male ; Methyl-CpG-Binding Protein 2 ; genetics ; Mutation ; Phenotype

Adult ; Burkitt Lymphoma ; metabolism ; pathology ; CD3 Complex ; metabolism ; DNA Nucleotidylexotransferase ; metabolism ; Diagnosis, Differential ; Female ; Granulosa Cell Tumor ; metabolism ; pathology ; Humans ; Ki-67 Antigen ; metabolism ; Leukocyte Common Antigens ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; surgery ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; surgery ; Sarcoma, Myeloid ; metabolism ; pathology

A model for screening the yeast which can ferment xylose to produce the ethanol was constructed.An ethanol yeast was obtained using the lignocellulose as substrate production the ethanol.By malt extract medium pre-culturing,soil samples use the plate with xylose as sole carbon source as the primary screening,then finally screen by the potassium dichromate color-displaying method.A strain named Y2-3 was screened from the soil.Phenotypic analysis including morphology and physiology and biochemical characteristics and 26D1/D2 sequence analysis were carried out.Based on taxonomy results,the Y2-3 was identified as Pichia caribbica.The strain Y2-3 ferments using xylose as sole carbon source: biomass 23.5 g/L,xylose utilization rate 94.7 %,ethanol final yield 4.57 g/L;using mixture sugar:biomass 28.6 g/L,xylose utilization rate 94.2 %,glucose utilization rate 95.6%,ethanol final yield 20.6 g/L.Pichia caribbica is a yeast which can utilize xylose and mixture sugar as substrate.It established the foundation for further research fermentation of ethanol by yeast using lignocellulose.

Objective To investigate the status of genotype of the KPC(Klebsiella pneumoniae carbapenemase)-encoding genes in Pan-resistant K. Pneumoniae, isolated from the 98th Hospital of People' s Liberation Army, Huzhou district, Zhejiang province, China. Methods 19 strains of Pan-resistant K. Pneumoniae were isolated from the inpatients between November, 2008 and July,2009. Phenotypic confirmatory test for suspected carbapenemases production were carried out by Modified Hodge test. Carbapenemase gene of blaKPC was analyzed by PCR and verified by DNA sequencing. Results In 19 strains of K. Pneumoniae, the positive rates of Modified Hodge test and gene of blaKPC were both 100.0%. These genes all belonged to blaKPC-2 subtype confirmed by nucleotide sequence analysis. Among them, the blaKPC-2 gene sequence of the HZ001 strain (its original serial number was HZ9871 ) had been registered in GenBank (GenBank Accession Number: GU086225).Conclusion All of the Pan-resistant K. Pneumoniae isolated from the inpatients harbored blaKPC-2 type carbapenemases gene and causing an outbreak in a hospital. Carbapenemases that producing type KPC-2 might be the major reason which causing the resistance to Carbapenems antibiotics.

Objective To investigate the presence and genetic background of 16S rRNA methylase gene and Aminoglycoside modifying enzymes(AMEs) genes in Klebsiella pneumoniae isolated from the People's Liberation Army 98th HospitaI,Huzhou district,Zhejiang province,China.Methods 25 strains of Klebsiella pneumoniae were isolated from the inpatienta between September,2005 and April,2006.6 kinds of 16S rRNA methylase gene (including armA,rmtA,rmtB,rmtC,rmtD and npmA ),6 kinds of AMEs genes [ including aac (3)-Ⅰ,sac (3)-Ⅱ,sac (6')-Ⅰ,aac (6')-Ⅱ,ant (3")-Ⅰand ant(2")-Ⅰ],intI1,intI2,intI3,mercuric reductase gene merA (merA gene were the collective genetic markers of transposona of Tn21 and Tn501 ) and tnpA ( tnpA gene were the collective genetic markers of transposons of Tn1,Tn2,Tn3 and Tn1000) were analyzed by PCR and verificated by DNA sequencing.Results In 25 strains of Klebsiella pneumoniae,the positive rate of genes of rmtB,sac (3)-Ⅱ,sac (6')-Ⅰ,ant(3")-Ⅰ and intI1 were 60.0%(15/25),4.0%(1/25),48.0%(12/25),60.0%(15/25) and 96.0%(24/25),respectively.The rest 12 kinds of genes were all tested negative.The total positive rate of 6 kinds of AMEs gene was 84.0%(21/25).Conclusion There were very high positive rate on both genes of rmtB and AMEs genotypes in Klebsiella pneumoniae isolated from inpatients,and this was the first report of the emergence of 16S rRNA methylase gene rmtB in Klebsiella pneumoniae identified in Zhejiang province,China.

OBJECTIVETo analyse the plasmid-mediated SHV type beta-lactamases-encoding genes sequence and to identify its subtype of multiple-drug-resistant acinetobacter baumannii isolated from Huzhou district, Zhejiang province, China.

METHODSSixty strains of acinetobacter baumannii were isolated from hospitalized patients between Jul, 2000 and Dec, 2002. Susceptibility of antimicrobial agents and confirmatory tests for Extended-spectrum beta-lactamases (ESBLs) were tested by microdilute method. SHV type beta-lactamases-encoding genes were tested by polymerase chain reaction (PCR). SHV sequences of acinetobacter baumannii HZ02 and HZ10 strains were detected by ABI automated sequencer and were analysed to compare with SHV genes that had been published in GenBank.

RESULTSEighteen (30.0%) strains of acinetobacter baumannii isolated between Jun, 2001 and Jan, 2002 were carrying SHV beta-lactamases resistant gene of plasmids. Detected SHV sequences of acinetobacter baumannii HZ02 strain and HZ10 strain had 825 and 833 nucleotides respectively and had the same gene sequence as the gene encoding SHV-12 subtype of ESBLs discovered in Switzerland.

CONCLUSIONSThirty percentage of the clinically isolated acinetobacter baumannii were carrying SHV type (extended-spectrum) beta-lactamases resistant gene of plasmids and causing an outbreak in hospital and was discovered to have carried the strains of SHV-12 subtype producing ESBLs gene in acinetobacter baumannii which was the first reported case in the world.

Acinetobacter Infections ; epidemiology ; microbiology ; Acinetobacter baumannii ; enzymology ; genetics ; isolation & purification ; Amino Acid Sequence ; Base Sequence ; China ; epidemiology ; DNA, Bacterial ; genetics ; isolation & purification ; Drug Resistance, Multiple ; Humans ; Molecular Epidemiology ; Molecular Sequence Data ; Sequence Analysis, DNA ; beta-Lactamases ; classification ; genetics

Acinetobacter baumannii ; drug effects ; genetics ; metabolism ; Amino Acid Sequence ; Base Sequence ; Carbonyl Cyanide m-Chlorophenyl Hydrazone ; pharmacology ; DNA Gyrase ; genetics ; DNA Topoisomerase IV ; genetics ; Drug Resistance, Bacterial ; Fluoroquinolones ; pharmacology ; Reserpine ; pharmacology