OBJECTIVE: To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR). METHODS: By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique. RESULTS: The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected. CONCLUSION A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.
Exosomes ; Gene Expression ; Humans ; Leukemia, Promyelocytic, Acute ; Oncogene Proteins, Fusion ; analysis ; Polymerase Chain Reaction ; Protein Isoforms

OBJECTIVE: To study the expression of Shh and Wnt5a genes in the limb buds of NIPBL fetal rats and the association of these two genes with Cornelia de Lange syndrome (CdLS). METHODS: A total of 72 NIPBL fetal rats were divided into an experimental group and a control group, with 36 rats in each group. The limb buds were collected from 12 fetal rats each on embryonic days 10, 11 and 12 (E10, E11 and E12) respectively. Real-time PCR and Western blot were used to measure the mRNA and protein expression of Shh and Wnt5a. RESULTS: The mRNA and protein expression of Shh and Wnt5a was detected in the limb buds on E10, E11 and E12, and the experimental group had significantly lower expression than the control group (P<0.01). The mRNA and protein expression of Shh and Wnt5a in limb buds was at a low level on E10, followed by an increase on E11 and a reduction on E12, and the expression on E12 was still lower than that on E10 (P<0.01). CONCLUSIONS The mRNA and protein expression of Shh and Wnt5a are consistent. The pathogenesis of CdLS may be associated with the low mRNA and protein expression of Shh and Wnt5a inhibited by the low expression of NIPBL gene.
Animals ; De Lange Syndrome ; Hedgehog Proteins ; Mutation ; Phenotype ; Proteins ; RNA, Messenger ; Rats ; Wnt-5a Protein

BACKGROUNDTrichophyton rubrum is superficial fungi characteristically confined to dead keratinized tissues. These observations suggest that the soluble components released by the fungus could influence the host immune response in a cell in contact-free manner. Therefore, this research aimed to analyze whether the culture supernatant derived from T. rubrum grown in the nail medium could elicit the immune response of keratinocyte effectively.

METHODSThe culture supernatants of two strains (T1a, T XHB ) were compared for the β-glucan concentrations and their capacity to impact the innate immunity of keratinocytes. The β-glucan concentrations in the supernatants were determined with the fungal G-test kit and protein concentrations with bicinchoninic acid protein quantitative method, then HaCaT was stimulated with different concentrations of culture supernatants by adopting morphological method to select a suitable dosage. Expressions of host defense genes were assessed by quantitative polymerase chain reaction after the HaCaT was stimulated with the culture supernatants. Data were analyzed with one-way analysis of variance, followed by the least significant difference test.

RESULTSThe T. rubrum strains (T1a and T XHB ) released β-glucan of 87.530 ± 37.581 pg/ml and 15.747 ± 6.453 pg/ml, respectively into the media. The messenger RNA (mRNA) expressions of toll-like receptor-2 (TLR2), TLR4, and CARD9 were moderately up-regulated in HaCaT within 6-h applications of both supernatants. HaCaT cells were more responsive to T1a than T XHB . The slight increase of dendritic cells-specific intercellular adhesion molecule 3-grabbing nonintegrin expression was faster and stronger, induced by T1a supernatant than T XHB . The moderate decreases of RNase 7, the slight up-regulations of Dectin-1 and interleukin-8 at the mRNA level were detected only in response to T1a rather than T XHB . After a long-time contact, all the elevated defense genes decreased after 24 h.

CONCLUSIONThe culture supernatant of T. rubrum could directly and transiently activate the innate immune response of keratinocytes.

Cell Line, Tumor ; Culture Media, Conditioned ; pharmacology ; Humans ; Immunity, Innate ; drug effects ; Keratinocytes ; drug effects ; metabolism ; Trichophyton ; metabolism ; beta-Glucans ; metabolism

To understand Lophatherum gracile plant community's structural characteristics, a survey of community structure and species diversity was conducted through quadrat sampling in Yongchuan district of Chongqing. The results showed that there were 386 species vascular plants, belonging to 117 families and 229 genera. Based on habitat, community structure and species composition, L. gracile were found in three community types: Pinus massoniana community, banboo community, shurb community. Vertical structure was composed of three layers, including tree layer, shrub layer and herb layer. Species in shrub layer was the richness. P. massoniana is the only dominant species of the community, it can not regenerate naturally, the shrub layer has a greater effect on the community of L. gracile in the future. In addition, the banboo community and shurb community is not stable because of human's activity. Therefore, the community characters of L. gracile should be taken care of conservation when the resources are utilized.
China ; Ecosystem ; Pinus ; physiology ; Plants ; Poaceae ; physiology

To investigate the risk factors of hepatic encephalopathy in patients with liver failure. Nine-hundred-and-seventy-six hepatitis B virus (HBV) patients with liver failure were retrospectively analyzed. Clinical data (sex, age, family history, liver cirrhosis, diabetes, celiac infection, pulmonary infection, liver kidney syndrome, upper gastrointestinal hemorrhage) and laboratory findings (albumin, globulin, total bilirubin, direct bilirubin, alanine aminotransferase, aspartate aminotransferase (AST), gamma-glutamyl transferase, alkaline phosphatase, cholesterol, cholinesterase, K+, Na+, creatinine, international normalized ratio (INR), alpha-fetoprotein, HBV DNA, white blood cell, hemoglobin, platelet) were collected and used to screen the risk factors for hepatic encephalopathy by univariate and multiple regress analyses. Multiple logistic regression analysis indicated that upper gastrointestinal hemorrhage [risk (R) = 0.993, relative hazard (RH) = 2.699, 95% confidence interval (CI): 1.567-4.651], pulmonary infection [R = 1.043, RH = 2.839, 95% CI: 1.680-4.797], INR [R = 0.257, RH = 1.293, 95% CI: 1.220-1.370], AST level [R = 0.001, RH = 1.001, 95% CI: 1.000-1.001], and cirrhosis [R = 0.569, RH = 1.815, 95% CI: 1.112-2.965] were closely correlated with hepatic encephalopathy. HBV-infected patients presenting with upper gastrointestinal haemorrhage, pulmonary infection, prolonged INR, elevated AST, or liver cirrhosis should be carefully monitored for indications of hepatic encephalopathy to initiate timely therapeutic interventions.
Adult ; Female ; Hepatic Encephalopathy ; etiology ; Hepatitis B ; complications ; Humans ; Liver Cirrhosis ; complications ; virology ; Male ; Middle Aged ; Multivariate Analysis ; Prognosis ; Risk Factors

Adult ; Female ; Hepatitis B, Chronic ; diagnosis ; therapy ; Humans ; Liver Failure ; diagnosis ; therapy ; Longitudinal Studies ; Male ; Middle Aged ; Prognosis ; Severity of Illness Index ; Software ; Treatment Outcome

BACKGROUNDLiver cirrhosis is the fatal consequence of chronic hepatitis, making early diagnosis of liver cirrhosis critical. Liver biopsy is still the standard diagnostic method for liver cirrhosis, although its use in a broad population with alcoholism or hepatitis B virus (HBV) infection remains difficult. In this study, we used a metabonomic approach to detect potential biomarkers for early diagnosis of liver cirrhosis.

METHODSSerum specimens were collected prospectively from normal control subjects (n = 22) and patients with alcoholic cirrhosis (n = 18) or HBV-induced cirrhosis (n = 19). The serum metabonome was analyzed using ultraperformance liquid chromatography (LC)/time-of-flight mass spectrometry (MS) integrated with chemometrics. The acquired LC-MS data were normalized and processed using principal component analysis (PCA) and partial least squares discrimination analysis (PLS-DA).

RESULTSSignificant differences in the metabonomics among the three groups were observed. Lysophosphatidyl cholines (LPCs) (LPC C16:0, LPC C18:0, LPC C18:2, LPC C18:3, LPC C20:3, LPC C20:5) were decreased in the serum of patients with hepatic cirrhosis, whereas bile acids (glycocholic acid, glycochenodeoxycholic acid), hypoxanthine, and stearamide were increased in the serum of patients with hepatic cirrhosis. These metabolites are considered "common" biomarkers for hepatic cirrhosis. Oleamide and myristamide were increased in the serum of patients with alcoholic cirrhosis but decreased in those with HBV-induced cirrhosis. These could be specific biomarkers for differential diagnosis between alcohol- and HBV-induced hepatic cirrhosis.

CONCLUSIONSThere are significant metabonomic differences between alcohol- and HBV-induced liver cirrhosis. Metabonomics is a top-down systems biology tool for conducting research on clinical problems.

Adult ; Aged ; Alcohols ; adverse effects ; Chromatography, Liquid ; methods ; Female ; Hepatitis B virus ; pathogenicity ; Humans ; Liver Cirrhosis ; blood ; etiology ; metabolism ; virology ; Male ; Mass Spectrometry ; methods ; Middle Aged ; Principal Component Analysis

OBJECTIVETo investigate the protective effect of Crocin against hypoxia damage of cardiac myocytes of neonatal rats and the regulation of HIF-1 and prolyhydroxylase (PHDs).

METHODSA model of CoCl2 simulated hypoxia damage was established in primary cultural myocardial cell. Expression levels of HIF-1alpha, VEGF, iNOS, as well as PHD1, 2, 3 protein in myocardial cells were detected by Western blot.

RESULTSCompared with CoCl2 group, the viability of myocardial cell was significantly increased after treated 24 h at 10(-5)mol/L Crocin (P < 0.01), HIF-1alpha, VEGF and iNOS were expressed higher than those in Crocin + CoCl2 group (P < 0.01), the expression of PHD2 was significantly increased (P < 0.01), while the expression of PHD3 was remarkably reduced in Crocin + CoCl2 Group (P < 0.01).

CONCLUSIONCrocin has better protective effect on hypoxic damage of myocardial cell. The mechanisms of protective effect of Crocin may be related to the activation of HIF-1-mediated pathway of the hypoxia response. PHDs may be involved in the pathophysiology regulated process of myocardial cells.

Animals ; Carotenoids ; pharmacology ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Homeodomain Proteins ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Hypoxia-Inducible Factor-Proline Dioxygenases ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Procollagen-Proline Dioxygenase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo examine the relationship between prepregnancy body mass index (BMI) and the risk of pregnancy-induced hypertension (PIH) in Chinese population.

METHODSData were collected in 6 counties/cities covered by Perinatal Health Care Surveillance System which was part of the Sino-American cooperative project on neural tube defects prevention established in 1992. The study population consisted of 83 159 women who attended premarital or preconception medical physical examination and delivered single live births with at least 20 gestational weeks from 1995 to 2000 in Jiaxing area. The Chi-square test was employed to test the difference in the rates of PIH among groups with different BMI. Multivariate logistic regression was conducted to examine the association between prepregnancy BMI and the risk of PIH.

RESULTSThe rate of PIH was 11.01% (9153/83 159; 95% CI: 10.79% - 11.22%). The rate of PIH among women with BMI < 18.5 kg/m(2), 18.5 - 22.9 kg/m(2), 23.0 - 24.9 kg/m(2), and > or = 25.0 kg/m(2) were 9.08% (1405/15 472; 95% CI: 8.63% - 9.54%), 10.82% (6389/59 054; 95% CI: 10.57% - 11.07%), 14.63% (943/6444; 95% CI: 13.78% - 15.52%), and 19.00% (416/2189; 95% CI: 17.38% - 20.71%), respectively, the difference was significant (chi(trend)(2) = 261.028, P = 0.000). Taking those with BMI 18.5 - 22.9 kg/m(2) as reference, the unadjusted RR for PIH was 0.82 (95% CI: 0.77 - 0.87) among women with BMI < 18.5 kg/m(2), 1.41 (95% CI: 1.31 - 1.52) among women with BMI 23.0 - 24.9 kg/m(2), and 1.93 (95% CI: 1.73 - 2.16) among women with BMI > or = 25.0 kg/m(2). After controlling for area, maternal age at delivery, educational level, occupation, parity, times of prenatal visit as well as the individual or family history of chronic hypertension, the estimated RR were 0.85 (95% CI: 0.80 - 0.90), 1.37 (95% CI: 1.27 - 1.47) and 1.88 (95% CI: 1.68 - 2.10), respectively.

CONCLUSIONHigh prepregnancy BMI could increase the risk of PIH.

Adult ; Body Mass Index ; Female ; Humans ; Hypertension, Pregnancy-Induced ; epidemiology ; Incidence ; Logistic Models ; Pregnancy ; Rural Population ; Urban Population

OBJECTIVETo investigate the effect of lamivudine, interferon alpha and oxymatrine treatment for surviving hepatic failure patients with hepatitis B.

METHODS200 hepatitis B patients, including 100 subacute or acute-on-chronic hepatic failure survivals (group A), and 100 chronic (group B, n=100) hepatic failure survivals, were enrolled in this study. Patients in group A received interferon alpha (n=35), lamivudine (n=33) , or combinational lamivudine and oxymatrine (n=32) therapy for six months; Patients in group B received lamivudine (n=49), or combinational lamivudine and oxymatrine (n=51) therapy for six months, respectively. After the treatment, all patients were followed-up for six months.

RESULTSAt the end of follow-up, all patients in group A survived, while in group B three patients (6.1%) receiving lamivudine, and four (7.8%, P>0.05) receiving combinational therapy died; more than 90% of all survivals had their HBV DNA loss. The HBeAg/anti-HBe seroconversion rate in patients of group A treated with interferon alpha (9/17, 52.9%) was higher than that in patients treated with combinational lamivudine and matrine (5/16, 31.3%, P<0.05), which was higher than that in the patients treated with lamivudine alone (1/17, 5.9%, P<0.01), and the Knodell histological activity index score in patients treated with lamivudine (7.2+/-0.8, P<0.05) was lower than that in patients treated with interferon alpha (8.2+/-1.3, P<0.05), and the best efficacy was found in receiving combinational therapy (6.9+/-0.7, P<0.01); Lamivudine or lamivudine in combination with matrine significantly inhibited the intrahepatic inflammatory activities, but had no effect on the existing fibrosis in group B patients.

CONCLUSIONLong term nucleotide analogues treatment may delay the progress of fibrosis in hepatitis B-induced hepatic failure survivals, and the administration of matrine in time may further enhance the anti-fibrotic effect of nucleotide analogues.

Adolescent ; Adult ; Alkaloids ; administration & dosage ; therapeutic use ; Antiviral Agents ; administration & dosage ; therapeutic use ; DNA, Viral ; blood ; Drug Therapy, Combination ; Female ; Follow-Up Studies ; Hepatitis B ; complications ; drug therapy ; pathology ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; drug effects ; Humans ; Interferon-alpha ; administration & dosage ; therapeutic use ; Lamivudine ; administration & dosage ; therapeutic use ; Liver Failure ; blood ; drug therapy ; pathology ; Liver Function Tests ; Male ; Middle Aged ; Quinolizines ; administration & dosage ; therapeutic use ; Treatment Outcome ; Young Adult