Seven compounds were isolated from Onychium japonicum by macroporous resin, silica gel, ODS, Sephadex LH-20 column chromatography and semi-preparative HPLC. Their structures were identified by NMR, MS and other spectroscopic methods as onychone A (1), quercetin (2), quercetin-3-O-α-L-rhamnoside (3), kaempferol-7-O-β-D-glucopyranoside (4), kaempferol-3-O-α-L-rhamnopyranoside (5), (-)-prunin (6), and norathyriol (7). Compound 1 is a novel macrocyclic flavonoid, and all the others are reported from this plant for the first time. In vitro cytotoxic activities of compounds 1-7 were evaluated by MTS testing with five cancer cell lines. Compound 7 exhibited weak cytotoxicity against tumor cell lines A549, SMMC-7721, and SW480.

OBJECTIVETo obtain isolated human metapneumovirus (HMPV) strains from clinical specimens collected from infants and children in Beijing and to promote the investigation on this important respiratory pathogen.

METHODClinical specimens including throat swabs from outpatients and nasopharyngeal aspirates from hospitalized children were collected from infants and children visited the affiliated children's hospital for acute respiratory infections during May 2008 to April 2009. HMPV positive specimens identified by RT-PCR and/or direct immunofluorescent assay with monoclonal antibody against HMPV were inoculated to LLC-MK(2) cells and incubated at 37°C and 33°C, respectively. The replication of the virus in the cells was detected by direct immunofluorescent assay followed by RT-PCR. The genotypes of the isolated virus strains were identified by RT-PCR.

RESULTOut of 1092 clinical specimens, 81 were HMPV positive by RT-PCR, the positive rate was 7.4% (81/1092). Among these positive specimens, 33 were inoculated to LLC-MK(2) cells and the replication of HMPV was revealed by antigen detection and RT-PCR from 5 out of these 33 inoculates. These isolated viruses could be passed in LLC-MK(2) cells and were not cross-reacted with other common respiratory viruses, such as ADV, RSV and Parainfluenza viruses 1/2/3 by monoclonal antibodies against these viruses in direct immunofluorescent assay. The HMPV was more likely to be isolated from fresh specimens within 24 hours after the collection of specimens which were not frozen. Four of the 5 isolated strains were identified as genotype A and 1 as genotype B. Unlike other respiratory viruses, these isolated HMPV did not show specific CPE in cell culture and the replication of the virus was identified by antigen detection and RT-PCR.

CONCLUSIONHMPV of both genotypes were isolated from infants and children with acute respiratory infections in Beijing which will accelerate the investigation of this important virus.

Acute Disease ; Child ; China ; Genes, Viral ; genetics ; Genotype ; Humans ; Infant ; Metapneumovirus ; isolation & purification ; Respiratory Tract Infections ; virology

OBJECTIVETo identify pathogenic mutations of ANTXR2 gene in a patient with juvenile hyaline fibromatosis.

METHODSGenomic DNA was extracted from peripheral venous blood sample from the patient. All coding exons (exons 1-17) and splicing sites of the ANTXR2 gene were amplified with PCR. Potential mutations were detected with direct sequencing of the PCR products. 100 unrelated healthy subjects were used as the controls. CLUSTALX (1.81) was employed to analyze cross-species conservation of the mutant amino acid. Impact of the mutations was analyzed with software including SIFT, PolyPhen-2 and MutationTaster.

RESULTSA compound heterozygous mutation c.1074delT/c.1153G>C, was identified, among which c.1153G>C has not been reported previously and was predicted to be probably damaging. Both mutations were not found among the 100 healthy controls.

CONCLUSIONThe patient's condition may be attributed to the compound heterozygous mutations of c.1074delT and c.1153G>C of the ANTXR2 gene. Above results has facilitated molecular diagnosis for this patient.

Child, Preschool ; Female ; Heterozygote ; Humans ; Hyalinosis, Systemic ; diagnosis ; genetics ; Mutation ; Receptors, Peptide ; genetics

Objective To investigate the clinical characteristics of incomplete Kawasaki disease(KD).Methods Clinical data includi-ng test results,therapeutic methods were analyzed retrospectively in 579 patients with Kawasaki disease.They were divided into classic KD and incomplete KD and made a compared analysis.Results There were no significant differences in gender,age,symptom and laboratory examination between classic and incomplete KD.But the rate of coronary artery lesions was higher in incomplete KD(18.4%) than that of classic KD(11%).Conclusion The rate of coronary artery lesions was higher in incomplete KD,and it should be paid more attention to earlier diagnosis and earlier treatment.

Objective To explore the causes of twisted or tailed DNA bands in agarose gels after electro-phoresis .Methods Prestained and poststained methods were employed to exam 3 kinds of DNA marker bands in agarose gel with GeneGreen and Ethidium Bromide ,respectively .Results Three kinds of DNA marker bands in agarose gel with 1:10000 and 1:5000 concentration of GeneGreen showed obvious distortions and trailing phenomena compared with the same concentration of Ethidium Bromide w hen the prestained method was used for electrophoresis ,which were improved when the poststained method was used in agarose gel with GeneGreen and Ethidium Bromide ,respectively .Conclusion Nucleic acid dye GeneGreen can affect the quali-ty of DNA bands in agarose gel electrophoresis .When the quality of DNA itself ,agarose gel quality ,electro-phoretic fluid quality and voltage are excluded ,the quality of nucleic acid dye should be taken into considera-tion if the bands twisting or tailing occurs when the DNA nucleic acid electrophoresis is carried out by pres-tained method .The quality of DNA electrophoresis bands can be improved by using poststained method or re-placing nucleic acid dye .

Objective To investigate the prevalence of unintended pregnancy (UP) and exploring the risk factors of UP for married women of child-bearing age from Qingshan district,Wuhan.Methods A cross-sectional study was adopted in this study.Cluster sampling method was used with 3256 women recruited,in 2010.Information on history and risks related to social-demographic factors of UP were collected,using a self-administered questionnaire.Results Of the 3256 participants,over half of them (53.8％) reorted ever having had the history of UP and 9.1％ reported UP in the past year.Rate of UP in the past year for different age cohorts (18-30,31-40,41-49 years) were 31.8％,10.5％ and 1.8％ respectively.The most frequently reported reason for UP across all the age cohorts was "Did not use any contraceptive methods",with proportions on the reason that reported by women at 18-30,31-40 and 41-49 year-olds,were 69.7％,51.1％ and 42.4％ respectively.The second frequently reported reasons for UP were "Failure of traditional contraception" for younger cohort ( 18-30 years:13.0％ ) and "IUD dropped or pregnancy with IUD" for older-age cohorts (23.4％ at 31-40 year-olds and 37.0％ at the 41-49 year-oplds).The most frequently cited reason for "Did not use any contraceptive methods" was "Believe we were lucky so far,not to get pregnant" (59.6％).The risk factors of UP were being at older age,experiencing sex debut at younger age and got married at younger age.Conclusion The prevalence of lifetime UP history was high among women at child-bearing age from Qingshan district,Wuhan.Reproductive health services and interventions should be taken according to the needs from different age cohorts of women.Younger cohort of women should receive more attention.

BACKGROUNDHuman metapneumovirus (hMPV) was discovered by scientists in the Netherlands as a novel respiratory virus in 2001 and had been found in children with acute respiratory tract infections (ARTI) in China. The objective of this study was to determine the importance of hMPV infection in children in Beijing and the genotypes of the circulating virus by the surveillance during a four-consecutive-year period.

METHODSClinical specimens collected from children with ARTI from January 2006 to December 2009 were tested for hMPV by RT-PCR using primers targeting the matrix (M) gene, followed by genotyping of hMPV directly from positive samples by diplex PCR with primers for glycoprotein (G) genes. Sequence analysis was used for genotyping of those un-typable samples. Common respiratory viruses in these clinical specimens were tested by virus isolation and antigen detection, in addition to hMPV detection.

RESULTSOf 4730 tested specimens, 191 (4.0%) were positive for hMPV and 62.8% of 191 were identified as genotype A. The positive rate of hMPV from hospitalized patients was higher than that from outpatients each year. Most of hMPV positive children were under five years old. The peak of hMPV activity mostly occurred in late spring and overlapped with or followed that of respiratory syncytial virus (RSV) and followed by parainfluenza virus 3. Of hMPV infected cases, 68.6% were lower respiratory tract infection, among which 79.4% were hospitalized, and upper respiratory tract infection was diagnosed for 31.4% of hMPV infected children. The 9.4% of hMPV positive samples were found to co-exist with other respiratory viruses.

CONCLUSIONShMPV was an important pathogen for ARTI in pediatric patients, especially those under five years old. Both genotypes A and B circulated simultaneously in Beijing.

Adolescent ; China ; Female ; Genotype ; Humans ; Male ; Metapneumovirus ; genetics ; pathogenicity ; Respiratory Tract Infections ; virology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo assess the value of whole-genome high-resolution chromosome microarray analysis (CMA) for the investigation of fetuses with ultrasound abnormalities.

METHODSWhole genome high-resolution CytoScanHD array from Affymetrix was employed to investigate 651 fetuses with structural abnormalities detected by ultrasound, for whom standard G-banded chromosome analysis has revealed a normal karyotype. The fetuses were divided into a single malformation group (n=264) and a multiple malformations group (n=387). In total there were 130 chorionic villus samples, 192 amniotic fluid samples and 329 cord blood samples. Extraction of fetal DNA and CMA experiment have followed the standard guidelines from the manufacturers. All copy number variations (CNVs) detected by CMA were confirmed by fluorescence in situ hybridization (FISH) or real-time polymerase chain reaction (RT-PCR).

RESULTSCMA analysis has detected genomic CNVs in 475 (73%) cases. Clinically significant CNVs were found in 11.5% (75/651) of fetuses, including two uniparental disomies (UPD) and two cryptic mosaicisms. Variations of unknown significance (VOUS) was found in 2.0% (13/651) of tested fetuses.

CONCLUSIONAbove results have suggested that whole-genome and high-resolution CMA is valuable for the analysis of fetuses with structural abnormalities detected by ultrasound, which can increase the detection rate by approximately 11%. CMA using single nucleotide polymorphism (SNP) array has the ability to detect UPD and low-level mosaicisms. Sufficient communication between technicians and genetic counselors, parental testing and comparison the results with in-house and relevant online databases can significantly reduce the rate of VOUS.

Chromosome Aberrations ; Chromosomes, Human ; genetics ; DNA Copy Number Variations ; Female ; Fetal Diseases ; diagnosis ; diagnostic imaging ; genetics ; Genome, Human ; Humans ; Karyotyping ; Male ; Oligonucleotide Array Sequence Analysis ; methods ; Pregnancy ; Prenatal Diagnosis ; methods ; Ultrasonography

OBJECTIVEAdenovirus (ADV) is one of the most common causes of acute respiratory infections in infants and children. The objective of this study was to investigate the prevalence of adenovirus infection among pediatric patients with acute respiratory infections in Beijing and the types of the adenoviruses circulating in Beijing on the molecular bases.

METHODClinical specimens including throat swabs from outpatients and nasopharyngeal aspirates from hospitalized patients were collected from patients with acute respiratory infections in a consecutive period of 6 years from Jan 2003 to Dec 2008. Adenoviruses were identified from the collected clinical specimens by tissue culture and/or immunofluorescence assay and typed by nested-PCR based on the sequence of the encoding gene of hexon. Primers were designed for PCR amplification using hexon gene of adenovirus as target. One primer pair was designed as universal primers for amplifying a 1278 bp gene fragment located at the hexon gene of adenovirus types 3, 7, 11 and 21. Four primer pairs with the sequences located within the region of this 1278 bp fragment were designed specifically for amplifying adenoviruses types 3, 7, 11 or 21, respectively, which were used for a multiplex nest-PCR in a single tube. The products from this multiplex nest-PCR were 502 bp (for type 3), 311 bp (for type 7), 880 bp (for type 11) and 237 bp (for type 21), respectively, and the type of the adenovirus tested can be determined after agarose electrophoresis analysis of the PCR products. For those strains which could not be typed by the multiplex nest-PCR, the gene fragment was amplified by a universal primer pair for all adenovirus types from group A to F and the PCR products were sequenced directly.

RESULTOut of 17 941 clinical specimens collected, including 4378 throat swabs from outpatients and 13 563 nasopharyngeal aspirates from hospitalized patients, 304 were adenovirus positive by tissue culture and/or immunofluorescence assay, the overall positive rate was 1.69% (304/179 41). Among these 304 adenovirus positive specimens, 184 were by virus isolation and 184 by immunofluorescence assay, among which 64 were positive by both methods. The types of the adenoviruses were tested for 285 patients including 174 viral isolates and 111 clinical specimens. By using the multiplex nest-PCR, 272 were typable, including 174 (61.1%, 174/285) for ADV3, 92 (32.3%, 92/285) for ADV7, 6 for ADV11 (2.1%, 6/285) and no adenovirus type 21 was detected. Sequence analysis for those 13 nontypable specimens by the multiplex nest-PCR showed that 9 were ADV2 (3.2%, 9/285), 2 were ADV6 (0.7%, 2/285), 1 was ADV1 (0.4%, 1/285) and 1 was ADV5 (0.4%, 1/285). Most of the patients positive for adenovirus were under 5 years of age and 64.4% were from patients with lower respiratory infections, such as bronchiolitis and pneumonia. All the 5 cases of severe pneumonia with pulmonary failure were caused by ADV7 infection.

CONCLUSIONAdenovirus is still an important pathogen for acute respiratory infection in infants and young children and most of the adenoviruses associated with acute respiratory infections in children in Beijing from 2003 to 2008 were ADV3 and ADV7. ADV7 could cause severe lower respiratory infections.

Acute Disease ; Adenoviridae ; classification ; isolation & purification ; Adenoviridae Infections ; epidemiology ; prevention & control ; Adolescent ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Prevalence ; Respiratory Tract Infections ; epidemiology ; prevention & control ; virology

OBJECTIVETo investigate over-expression of wild-type alpha-synuclein inducing the aberrant aggregation of alpha-synuclein in HEK293 cell in vitro.

METHODSThe cDNA encoding the human alpha-synuclein without the stop code was cloned into PGEM T-easy vector. Using enzyme map and DNA sequencing analyzed and determined the recombinant plasmid, and then sub-clone the alpha-synuclein cDNA fragment into pEGFP-N1 vector. The recombinant plasmids alpha-synuclein-pEGFP were transfected into HEK293 cells by lipofectamin 2000. The aberrant aggregation of alpha-synuclein was measured by EGFP fluorescence, anti-alpha-synuclein immunocytochemistry. The inclusions in the cultured cells were identified with HE staining.

RESULTSThe restriction enzyme map suggested that eukaryotic expression vector for human wild-type alpha-synuclein gene was constructed successfully. By EGFP fluorescence, anti-alpha-synuclein immunocytochemistry, it could be observed that the alpha-synuclein protein could aggregate in cytoplasm and the Lewy body-like inclusions found in cytoplasm of cultured cells.

CONCLUSIONThe over-expression of wild-type alpha-synuclein can induce protein aberrant aggregation and Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro.

Cells, Cultured ; Gene Expression ; Humans ; Immunohistochemistry ; Inclusion Bodies ; metabolism ; Lewy Bodies ; metabolism ; Parkinson Disease ; genetics ; metabolism ; alpha-Synuclein ; genetics ; metabolism