Objective To establish a convenient,accurate and practical method for detection of adefovir dipivoxil resistance-as- sociated mutation in hepatitis B virus:rtA181V/T/S and rtN236T mutations.Methods According to HBV complete sequences in GenBank,two pairs of primers were designed to amplify the region of HBV reverse transcriptase in order to introduce a BglI restriction site upon PCR product of wild type (wt) and a BseDI restriction site upon PCR product of rt236 mutant type.After amplification,the PCR products were digested with BglI and BseDI separately.We used this method to detect wild,rt181 mu- tant,rt236 mutant plasmids and 3 chronic hepatitis B patients' serum with obvious ADV resistance-associated mutations.We also tested the sensitivity of this method by mixing the wild and mutant plasmids in different proportions.Results The method could detect rt181 and rt236 mutations simultaneously.The result of RFLP analysis was in accordance with that of DNA se- quencing and cloning analysis.This method could detect the mutants even when they comprised only 10% of the total virus population.Conclusions The PCR-RFLP method with high sensitivity can detect rt181 and rt236 mutations simultaneously.It can be used for early detection of ADV resistance-associated mutation in hepatitis B virus.

To synthesize salbutamol immunogen and develop an enzyme immunoassay (ELISA), a new salbutamol immunogen was synthesized using 4-aminobenzoic acid as a linker to connect hapten with carrier protein. An enzyme immunoassay based on the antibody prepared was developed and applied to detect salbutamol residue spiked in swine liver. An unusual coating antigen, clenbuterol-ovalbumin (OVA) conjugate instead of salbutamol-OVA conjugate, was used in the immunoassay and the results were discussed based on the structures of related compounds. The antibodies showed high sensitivity in the heterologous assay when using clenbuterol-OVA as a coating antigen, with an IC50 value of 8.97 ng mL(-1) toward salbutamol. The antibodies prepared showed high cross-reactivity with clenbuterol (107%) and were promising for the simultaneous determination of salbutamol and clenbuterol residues in food and food products. Recovery rates from the salbutamol-spiked swine liver samples were in the range of 70%-99%, while the intra-assay and inter-assay coefficients of variation were <13.3% and <14.3%, respectively. In summary, the antibodies of salbutamol have been successfully prepared. Sensitive and stable analysis for the detection of salbutamol residues in swine liver was obtained based on the competitive ELISA methods developed in this study.
4-Aminobenzoic Acid ; chemistry ; Adrenergic beta-Agonists ; analysis ; immunology ; Albuterol ; analysis ; immunology ; Animals ; Antibodies ; immunology ; Antibody Specificity ; Clenbuterol ; analysis ; immunology ; Drug Residues ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Food Contamination ; Haptens ; immunology ; Immunization ; Liver ; chemistry ; Male ; Ovalbumin ; chemistry ; immunology ; Rabbits ; Serum Albumin, Bovine ; chemistry ; immunology ; Swine

OBJECTIVETo investigate the risk factors of fungal rhinosinusitis.

METHODSThe preoperative clinical data of 57 patients with a diagnosis of fungal rhinosinusitis confirmed pathologically using Gomori methenamine silver staining were analyzed statistically against the data of 57 age- and gender-matched control patients with chronic rhinosinusitis.

RESULTSCompared with chronic rhinosinusitis, fungal rhinosinusitis was characterized by a significantly shorter mean disease course (37.31 months vs 130.84 months, t = 5.59, P = 0.000). The factors related to fungal rhinosinusitis included nasal mucus, purulent nasal discharge, unilateral/bilateral sinus lesion and calcified plaque in CT scan , with odds ratios of 0.17 (0.04-0.62), 0.35 (0.15-0.80), 41 (12.50-100.00) and 91 (24.01-344.95), respectively. Conditional logistic regression identified calcified plaque in CT scan as the high-risk factor of fungal rhinosinusitis.

CONCLUSIONThe presence of calcified plaque in CT scan indicates high risk of fungal rhinosinusitis and may serve as an important evidence for diagnosis of this disease.

Case-Control Studies ; China ; epidemiology ; Female ; Fungi ; Humans ; Male ; Mycoses ; epidemiology ; Rhinitis ; epidemiology ; microbiology ; Risk Factors ; Sinusitis ; epidemiology ; microbiology

OBJECTIVETo investigate the efficacy of oral sweet solutions in relieving pain caused by vaccination in infants aged 1 to 12 months.

METHODSRelated databases were searched to find related randomized control trails (RCTs). The quality of these RCTs was evaluated. The Meta analysis was performed using RevMan 5.3.

RESULTSA total of 20 RCTs involving 2 376 infants were included, and quality assessment showed that 6 RCTs had grade A quality and 14 had grade B quality. The Meta analysis showed that compared with sterile water, 25%-75% oral sweet solution significantly reduced crying time (WMD=-21.16, 95%CI -39.66 to -2.77, P<0.05) and the proportion of crying time (the duration of crying /3-minute periods after the injection) (WMD=-13.83, 95%CI -20.88 to -6.78, P<0.01), while the crying time showed no significant difference between the group treated with oral administration of 12% sucrose solution and non-intervention group. Co

ONCLUSIONSOral sweet solution (25%-75%; 2 mL) given 2 minutes before vaccination can effectively relieve the pain caused by vaccination in infants aged 1-12 months.

Crying ; Humans ; Infant ; Pain ; prevention & control ; Solutions ; Sucrose ; administration & dosage ; Vaccination ; adverse effects

OBJECTIVETo study the relationship between BRAF mutation and clinicopathological features in papillary thyroid carcinoma (PTC).

METHODSFresh samples were examined for the presence of BRAF mutations in 43 patients with PTC and 20 patients with non-PTC thyroid disease and 40 normal thyroid tissues by polymerase chain reaction (PCR) and direct sequencing. The relationship between BRAF mutation and clinicopathological features was studied.

RESULTSBRAF mutation was detected in 39.5% (17/43) of PTC samples, in 0 of non-PTC thyroid disease samples and normal thyroid tissues. Significant association was seen between BRAF mutation and both extrathyroidal invasion and cervical lymph node metastasis (P<0.05, P<0.05). There was no significant relationship between BRAF mutation and gender, age at the time of diagnosis, tumor size and distant metastasis.

CONCLUSIONBRAF mutation is associated with extrathyroidal invasion and lymph node metastasis. It may increase the ability of invasion and metastasis of PTC and have influence on prognosis.

Adenocarcinoma, Papillary ; diagnosis ; genetics ; Adult ; Aged ; Carcinoma, Papillary ; diagnosis ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Lymphatic Metastasis ; genetics ; Male ; Middle Aged ; Neoplasm Invasiveness ; diagnosis ; pathology ; Neoplasm Recurrence, Local ; diagnosis ; pathology ; Prognosis ; Proto-Oncogene Proteins B-raf ; genetics ; Thyroid Neoplasms ; diagnosis ; genetics ; Young Adult

Naringenin (1) was transformed to three metabolites (2-4) by Mucor sp. Based on LCMS(n)-IT-TOF and NMR spectroscopic data, 2-4 were identified as naringenin-7-O-sulphate, naringenin-4'-O-sulphate, and naringenin-5-O-sulphate, respectively. These results might provide hints to the mammalian/human metabolism of naringenin.
Biotransformation ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Flavanones ; chemistry ; metabolism ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Mucor ; metabolism ; Sulfates ; metabolism

Objective To investigate the effect of 2-methoxyestradiol(2ME2)on the expressions of hypoxia-inducible factor 1α and apoptosis-related genes(RTP801 and caspase-3)in the hippocampus of rats following global cerebral ischemia.Methods A total of 168 adult male SD rats were randomized into 2 groups,global cerebral isehemia group(GI group,n=84)and global cerebral ischemia+2ME2 treatment group(GI+2ME2 group,n=84).In GI and GI+2ME2 groups,4 vessel occlusion(4-vo)global ischemia was induced,and the rats were sacrificed at 6,12,24,48,96h,and 5 and 7 days after the reperfusion.Nissl staining was used for quantitative analysis of the hippocampal neurons,and immunohistochemistry and RT-PCR were performed to detect the expressions of HIF-1α protein,caspase-3 protein and RTP801 mRNA.Results At 48,96h and 5 and 7 days after the reperfusion,the numbers of hippocampal neurons in GI+2ME2 group were 37.09±3.52,26.93±3.10,22.22±3.091,and 6.98±3.07,respectively,significantly higher than those in GI group(P＜0.05).2ME2 significantly suppressed the expression levels of HIF-1α and reduced the numbers of the cells positive for HIF-1α protein to 1 1.47±1.9,20.27±2.07,3.12±0.89,1.07±0.83 at these time points(P＜0.05).The expressions of caspase-3 protein were decreased significantly in GI+2ME2 group,with the numbers of positive cells of 12.39±1.67,20.65±2.01,15.61±1.26,and 6.57±1.12 at the time points.The absorbance of RTP801 mRNA expression at the time points from 12 h to 5 days in GI+2ME2 group was 0.750±0.078,1.008±0.090,0.717±0.072,0.43 1±0.047,and 0.23 1±0.028,respectively,significantly lower than that in GI group(P＜0.05).Conclusion 2ME2 offers brain protection in rats with global cerebral ischemia and suppresses the elevation in the expressions of hypoxia-inducible factor 1α,RTP801 and caspase-3.

OBJECTIVETo study the resistant rate of hepatitis B virus (HBV) to ADV and the dynamic evolution of HBV in lamivudine (Lam)-resistant chronic hepatitis B (CHB) patients.

METHODSTwenty-three Lam-resistant CHB patients were assigned to a 10mg/d ADV monotherapy for 68-116 weeks. The baseline and different time point blood samples after ADV monotherapy were analyzed for ADV-resistant mutations using direct sequencing of PCR products; the evolution of HBV mutations was examined by clonal analysis of serial samples from one patient infected with ADV-associated resistant HBV strains.

RESULTSThe cumulative incidence of genotypic ADV resistance at weeks 48 and 96 was 4.3% and 10.5% respectively respectively. The evolution analysis of HBV mutant strains in an ADV-resistant CHB patient showed that the proportion of YMDD mutants gradually decreased with rtA181S mutants increasing over time after ADV monotherapy, and that rtA181S+N236T mutants became the predominant strains during prolonged ADV monotherapy. The addition of Lam to the ongoing ADV treatment had poorer antiviral response in the patient with rtA181S or rtA181S+N236T mutant infection; one clone with multi-drug resistant mutations was selected during Lam and ADV combination therapy.

CONCLUSIONIncreased risk of adefovir resistance and selection of multi-drug resistant mutations are associated with long-term ADV monotherapy in patients with Lam-resistant chronic hepatitis B.

Adenine ; analogs & derivatives ; therapeutic use ; Adult ; Antiviral Agents ; therapeutic use ; Drug Resistance, Viral ; Evolution, Molecular ; Female ; Hepatitis B virus ; classification ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; Male ; Middle Aged ; Organophosphonates ; therapeutic use

BACKGROUNDCervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS). However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro.

METHODSNormal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity.

RESULTSOur results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%.

CONCLUSIONA novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.

Cell Culture Techniques ; methods ; Cervix Uteri ; cytology ; Epithelial Cells ; cytology ; Female ; Humans ; Keratinocytes ; cytology

The purpose of this study was to investigate the effect of simvastatin (SIM) on proliferation and apoptosis of acute monocytic leukemia cell line SHI-1 and its mechanism. Experiments were divided into control and test groups (5 µmol/L, 10 µmol/L, 20 µmol/L SIM groups). The growth inhibitory rate of SHI-1 cells was detected using methyl thiazolyl tetrazolium (MTT) method. The cell cycle distribution and apoptotic rate were measured by using flow cytometry. The expression of BCL-2, caspase-3 mRNA were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of BCL-2, caspase-3 protein levels were analyzed by Western blot. The results demonstrated that SIM inhibited the growth of SHI-1 cells in time- and does-dependent manners. Cell cycle analysis showed that SHI-1 cells significantly arrested in S phase (p < 0.05) after treating with SIM for 48 hours, as compared with control group. 5 µmol/L SIM in test group significantly blocked cell cycle progression, but can not induce apoptosis. The expressions of BCL-2 mRNA and protein were down-regulated and caspase-3 mRNA and protein were up-regulated along with the increase of SIM concentration (p < 0.05). It is concluded that SIM is able to inhibit proliferation and induce apoptosis of SHI-1 cells, the mechanism may be associated with downregulating the expression of apoptosis-related gene BCL-2, upregulating the expression of caspase-3.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Monocytic, Acute ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Simvastatin ; pharmacology