Objective To explore the optimal experiment conditions of CCK-8 and MTS for cell proliferation assays in human amniotic epithelial cells and to evaluate the cytotoxicity of these reagents. Methods Human amniotic epithelial cells (hAECs) in logarithm growth stages were prepared in different cell concentrations with DMEM/F12 and 10% FBS. The sensitivity and optimal wavelengths was determined based on the optical density (OD) measured at 450 nm and 492 nm. The optimal time was determined under the conditions of the same cell concentration and defined OD values. HAECs were treated with DMSO, CCK-8 and MTS for 1 h, 2 h, 3 h, and 4 h, respectively. 24 h later, cytotoxicity of the CCK-8 and MTS was evaluated by determination of cell proliferation and Trypan Blue staining. Results The optimal detection wavelength was 450 nm for CCK-8, and 492 nm for MTS. The sensitivity of CCK-8 was slightly lower then that of MTS. The optimal time for incubation hAECs with CCK-8 was 4 h within 1~4 h. The inhibitory on cell proliferation and cytotoxicity of CCK-8 were weaker then those of MTS. Conclusion CCK-8 is a convenient reagent with low cytotoxicity for detection of the proliferation of hAECs.

OBJECTIVETo prepare rabbit anti-mouse zona pellucida 2 (mZP2) polyclonal antibodies and test their immunoactivity.

METHODSRecombinant proteins of mZP2 expressed in Rosetta transformant was separated by SDS-PAGE, and the gel strips containing the recombinant mZP2 were cut out and emulsified to immunize New Zealand white rabbits. The antibody response of the antiserum was detected by ELISA, and the specificity of the antiserum was verified by immunohistochemical assay. The effect of the antiserum on the binding of oocytes with acrosomal reacted sperm was tested by sperm-egg binding assay.

RESULTSELISA results showed that the immunized rabbit produced anti-mZP2 antiserum. The antiserum reacted specifically with the zona pellucida of mouse ovarian sections. Sperm-egg binding assay showed that treatment of the oocytes with the anti-mZP2 antiserum caused decreased binding of zona pellucida with the acrosomal reacted sperm by 43.7%.

CONCLUSIONWe obtained rabbit anti-mouse ZP2 polyclonal antibodies that can inhibit the binding of oocytes with acrosomal reacted sperm.

Animals ; Antibodies ; immunology ; Antibody Formation ; Egg Proteins ; immunology ; Female ; Immune Sera ; Male ; Membrane Glycoproteins ; immunology ; Mice ; Oocytes ; Rabbits ; Receptors, Cell Surface ; immunology ; Recombinant Proteins ; immunology ; Sperm-Ovum Interactions ; Spermatozoa ; Zona Pellucida Glycoproteins

OBJECTIVETo optimize pre-coated multiple-probe fluorescence in situ hybridization (FISH) to improve its efficiency in cytogenetic diagnosis of acute leukemia.

METHODSThe original multiple-probe FISH techniques were optimized by adjusting the cell density and adding a process of protease digestion. Cytogenetic anomalies were detected in 141 patients with acute lymphocytic leukemia (ALL) or acute myeloid leukemia/ myelodysplastic syndromes (AML/MDS) using the modified technique, and 35 of the patients were also examined using the original technique. The successful detection rate and positive site detection rate were compared between the modified and original techniques.

RESULTSModification of the pre-coated multiple-probe FISH technique resulted in an significant increase of the successful detection rate (from 85.3% to 100%) and the positive site detection rate (from 5.1% to 8.6%) in ALL patients; in AML/MDS patients, the successful detection rate was significantly improved from 67.4% to 99.8% and the positive site detection rate from 3.5% to 6.0% (P<0.01).

CONCLUSIONThe modified pre-coated multiple-probe FISH technique can significantly increase the diagnostic efficiency of cytogenetic abnormalities in leukemic patients.

Chromosome Aberrations ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotyping ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Myelodysplastic Syndromes ; diagnosis ; genetics

Objective To evaluate the clinical efficacy and safety of endoscopic selective varices devascularization.Methods From November 2015 to July 2017,at the First Affiliated Hospital of Anhui Medical University,282 patients with liver cirrhosis complicated with gastroesophageal varices who underwent endoscopic treatment were selected and divided into traditional treatment group (n=102) and selective treatment group (n=180).The patients of traditional treatment group were treated with tissue glue "sandwich method" (lipiodol-tissue glue-lipiodol),and the patients of selective treatment group were treated with modified " sandwich method" (lauromacrogol-tissue glue-0.9％ sodium chloride solution).After operation,all the patients were followed up for three months.The rates of remarkable efficacy,efficacy,improvement,rebleeding and complications were compared between the two groups respectively.T test,chi-square test and rank sum test were performed for groups comparison.Results One month after operation,the rates of remarkable efficacy and efficacy of the traditional treatment group and the selective treatment group were 46.1％ (47/102) and 20.6％ (21/102),and 67.8％ (122/180) and 30.0％ (54/180),respectively.The results of rank sum test indicated that the differences in rates of remarkable efficacy and efficacy between two groups were statistically significant (Z=-5.428,bilateral P＜0.05).The improvement rate of the selective treatment group was higher than that of the traditional treatment group (97.8％,176/180 vs.66.7％,68/102),and the difference was statistically significant (x2-54.048,P＜ 0.05).At two weeks and three months after operation,the rebleeding rate of traditional treatment group was higher than that of selective treatment group (10.8 ％,11/102 vs.3.3 ％,6/180;21.6％,22/102 vs.7.2％,13/180),and the difference was statistically significant (x2 =6.380 and 12.327,both P＜0.05).No serious complications occurred in both groups.There was no statistically significant difference in overall complication rate between selective treatment group and traditional treatment group (37.8％,68/180 vs.30.4％,31/102;x2 =1.559,P-0.212).Conclusion The treatment with endoscopic selective varices devascularization shows good efficacy and safety,and is worth further study.

OBJECTIVESTo study the correlation between positive rates of RNA in clinical confirmed severe acute respiratory syndrome (SARS) patients and its appearance in relation to the development of the disease in order to provide scientific basis for early diagnosis, effective prevention and treatment of the disease.

METHODSOne-step reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the SARS RNA in the clinical specimens from different courses of the disease. The representative amplicons were then sequenced. Chi-square for trend test was performed to study the correlation between positive rates of RT-PCR and at different periods after the onset of the disease.

RESULTSThe fragments amplified from the sputum specimens of SARS patients were shown to share 100% homology with the published SARS-associated coronavirus. Of the different clinical specimens, positive rate in the stools appeared to be the highest (21.55%). Chi-square for trend test revealed that the positive rates of stools and sputa of SARS patients decreased with the development of the disease (chi(2) for trend = 12.55 and 16.408, P = 0.0004 and P = 0.000 05 respectively).

CONCLUSIONOne-step RT-PCR proved to be an effective method for the detection of SARS-associated coronavirus from clinical specimens. Data as indicated that the positive rates of SARS coronavirus were decreasing in SARS patients along with the disease progression.

Adolescent ; Adult ; Aged ; Chi-Square Distribution ; China ; Disease Progression ; Feces ; virology ; Female ; Humans ; Male ; Middle Aged ; Mucus ; virology ; RNA, Viral ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; isolation & purification ; Severe Acute Respiratory Syndrome ; pathology ; virology

OBJECTIVEIn order to find out the factors related to hemorrhagic fever with renal syndrome (HFRS) infection, and to evaluate the probability of ecdemic hantaviruses (HV) infection in rodents in Beijing areas.

METHODSRodents were collected in a large-scale railway station and a produce market with 'trap nights' method from April to May, 2004. The IgG reacting sera to HV antigen were detected using ELISA. The partial M and S segment of HV from captured rodent lung samples were amplified with RT-PCR. The PCR products were purified and sequenced. BLAST program was then used to perform on nucleotide pairwise alignment with all available sequence in GenBank. The alignment of the multiply nucleotide and the deduced amino acid sequences, together with phylogenetic analysis were completed with DNASTAR software.

RESULTSThe average population density was 3.49% (24/690). The overall seroprevalence of HV infection was 8.3% (2/24). RT-PCR positive rates were 8.3% (2/24). The nucleotide sequences of 356 bp region (1958 - 2313) of M segment obtained from 2 samples were all identified to Seoul virus (SEOV), with 7.6% heterogeneity. The dc501 strain from railway station was closely related to SD227 and Hebei4 from Shandong and Hebei provinces respectively. BjFT01 strain from the farm product market had more special nucleotide transitional mutations than other known SEOV from Beijing in GenBank. This strain, together with known HN71 from Hainan province, K24-E7 from Zhejiang province, L99 from Jiangxi province and R22 from Henan province, represented a monophylogentic linkage.

CONCLUSIONThe higher HV prevalence of rodents in transportation center was the potential and important risk for HFRS epidemic in Beijing. The increasing prevalence of M. musculus should call for attention. It was possible that SEOV in Beijing was imported by infected rodents through vehicles from other provinces.

Animals ; Antigens, Viral ; immunology ; China ; epidemiology ; Enzyme-Linked Immunosorbent Assay ; Hantavirus ; classification ; genetics ; isolation & purification ; Hantavirus Infections ; epidemiology ; immunology ; Hemorrhagic Fever with Renal Syndrome ; epidemiology ; Immunoglobulin G ; blood ; Lung ; virology ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Rodent Diseases ; epidemiology ; virology ; Rodentia ; Seroepidemiologic Studies

Objective:To assess the practical value of visual scores of magnetic resonance imaging(MRI)features in the diagnosis and classification of dementia with Lewy bodies(DLB).Methods:In this study, 102 DLB patients were prospectively recruited, with 102 cognitively normal elderly people as the normal control group(NC).All included subjects underwent MRI examinations and neuropsychological assessments.Based on the clinical dementia rating(CDR)scale, DLB patients were divided into a mild(CDR=1.0), a moderate(CDR=2.0)and a severe(CDR=3.0)group.The results of MRI were scored visually and the rating scales included medial temporal lobe atrophy(MTA), global cortical atrophy-frontal subscale(GCA-F), posterior cortical atrophy(PCA), white matter lesions(the Fazekas scale), cerebral microbleeds(CMBs), and the Evans Index(EI).Statistical differences were compared between the DLB and NC groups and between DLB patients with different degrees of cognitive impairment.Results:In terms of neuropsychology, the Mini-Mental State Examination(MMSE) score of the DLB group[16.0(11.0, 21.0)]was statistically significantly lower than that of the NC group[29.0(28.0, 30.0)]( Z=-12.31, P<0.001), the Montreal Cognitive Assessment(MoCA)score of the DLB group[9.5(6.0, 15.0)]was statistically significantly lower than that of the NC group[28.0(27.0, 29.0)]( Z=-12.40, P<0.001), and the Activities of Daily Living(ADL)score of the DLB group[32.0(23.8, 40.0)]was statistically significantly higher than that of the NC group[20.0(20.0, 20.0)]( Z=-11.98, P<0.001).The scores of all MRI visual assessment scales in DLB patients were statistically significantly higher than those in the NC group( P<0.001).There were significant differences in MTA scores between DLB patients with different degrees of cognitive impairment( P0<0.001).The MTA score of the mild group[1.0(1.0, 1.0)]was statistically significantly lower than that of the moderate group[2.0(1.0, 2.0)]( P1<0.001, P2<0.001); The MTA score of the moderate group[2.0(1.0, 2.0)]was statistically significantly lower than that of the severe group[2.0(2.0, 3.0)]( P1=0.003, P2=0.010). Conclusions:This study has for the first time after comprehensively evaluated the value of various visual scores in DLB diagnosis, MTA can be used to help diagnose DLB and distinguish the severity of DLB, providing a new supplemental tool for clinical diagnosis.

BACKGROUNDGenetic association studies on populations of European origin have identified the DCDC2 gene as a susceptibility locus for developmental dyslexia. Here, we sought to investigate the association of DCDC2 polymorphisms with developmental dyslexia in children of Han Chinese origin.

METHODSWe undertook a case-control genetic association study on 76 dyslexic children and 79 non-dyslexic matched controls. We isolated DNA from oral mucosal cell samples and genotyped two DCDC2 coding-sequence single nucleotide polymorphisms, rs2274305 and rs6456593, in each sample using SNaPshot single nucleotide extension. We compared the allele and genotype frequencies between the groups using the χ(2) test and analyzed the relationship between dyslexia and the polymorphism at both loci using unconditional logistic regression. We also predicted haplotypes and compared their frequencies between the two groups.

RESULTSThe differences in the genotype distribution and the allelic genes of the two single nucleotide luci of the DCDC2 gene, rs2274305 and rs6456593, between the two dyslexic and non-dyslexic groups were statistically meaningless (P > 0.05). The differences in the haplotype distributions of the DCDC2 gene between the dyslexic and normal group were statistically meaningless (P > 0.05).

CONCLUSIONThe DCDC2 gene may not be a susceptibility factor for developmental dyslexia among the Han Chinese. However, methodological issues may have prevented the detection of positive associations.

Asian Continental Ancestry Group ; Child ; Dyslexia ; genetics ; Female ; Genetic Predisposition to Disease ; genetics ; Genotype ; Haplotypes ; genetics ; Humans ; Male ; Microtubule-Associated Proteins ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; genetics

The industry of germ-free animals has been a hot spot in research along with the rapid development of studies on the relationship between microbiota and host diseases. Because it is pathogen?free, and the high degree of simi?larity in anatomy, physiology, pathogenesis to humans, germ?free pig is considered a clinical relevant model to be widely used in life science research. Based on the current state of research of germ?free pig cultivation at home and abroad and the experimental studies carried out in our laboratory as well, this article gives a simple discussion on germ?free technique of domestic pigs.

OBJECTIVETo investigate hantanvirus infection of captured rodents in Haidian district and Changping district of Beijing and to type hantavirus using molecular technique.

METHODSThe captured mice were classified and the density of distribution was calculated. Reverse transcription-polymerase chain reaction (RT-PCR) technique was used to amplify the partial M fragnments of hantaviruse. Several representative positive samples were sequenced and analysed by ClustalX (5.0) and DNAClub software.

RESULTSA total of 414 animals were captured, among which Battus norvegicus was the dominant group. In Haidian district, the median infection rates with hantavirus were 13.14% in Battus norvegicus and 0 in Mus musculus Linnaeus. In Changping district, the average infection rates were 17.46% in Battus norvegicus and 3.57% in Mus musculus Linnaeus. Nucleotide sequences analysis showed that the virus detected all belonged to SEO-type. They clustered with Z37 virus and could be branched into 2 different subclades.

CONCLUSIONThe major hosts of hantavirus in Haidian and Changping district were Battus norvegicus and the epidemic strains in the two districts of Beijing were genotyped as SEO-type. Nucleotide sequence and deduced amino acid sequence from different rodents were highly homologous, while nucleotide mutation had also been observed. Further studies are required to explore the possible virus sequence mutation.

Animals ; China ; epidemiology ; DNA, Viral ; genetics ; Disease Reservoirs ; Fluorescent Antibody Technique ; Hantavirus ; classification ; genetics ; isolation & purification ; Hantavirus Infections ; epidemiology ; veterinary ; virology ; Hemorrhagic Fever with Renal Syndrome ; epidemiology ; veterinary ; virology ; Mice ; Molecular Epidemiology ; Phylogeny ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Rodent Diseases ; epidemiology ; virology