OBJECTIVETo construct the delta-pIRES2-EGFP plasmid and investigate its expression in HEK293 cells.

METHODSFull length cDNA of rat delta opioid receptor gene amplified from rat brain tissues using reverse transcription and nested PCR was cloned into pMD20 T vector. The delta cDNA was inserted into pIRES2-EGFP plasmid to construct the recombinant eukaryotic plasmid delta-pIRES2-EGFP, which was transfected into HEK293 cells via Lipofectamine2000. The expression of delta was examined under fluorescence microscope.

RESULTSThe recombinant delta-pIRES2-EGFP plasmid was successfully constructed, and high expression of delta was detected in HEK293 cells transfected by the plasmid.

CONCLUSIONdelta-pIRES2-EGFP has been successfully cloned, which shows high expression of delta in HEK293 cells.

Animals ; DNA, Complementary ; genetics ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HEK293 Cells ; Humans ; Plasmids ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, delta ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Objective: To explore the predictors in patients of paroxysmal atrial fibrillation (AF) recurrence after radiofrequency catheter ablation (RFCA). Methods: A total of 142 PAF patients received RFCA in our hospital from 2013-03 to 2016-03 were studied. The patients were divided into 2 groups: Recurrence group, n=46 and Non-recurrence group, n=96. Clinical data was compared between 2 groups and AF recurrent predictors were studied by single and multivariate Logistic regression analysis. Based on quartiles of uric acid (UA) level, the patients were categorized in another set of 4 groups: Q1 group, UA<259 μmol/L, n=33, Q2 group, UA 259-320 μmol/L, n=37, Q3 group, UA 321-380 μmol/L, n=37 and Q4 group, UA>380 μmol/L, n=35. The influence of UA on AF recurrence was measured by Kaplan-Meier test, the predictive value of UA combining metabolic syndrome (UA+MS) on AF recurrence was studied by ROC curve analysis. Results: The BMI, diabetes, MS, AF duration, CHADS2 score, creatinine, UA and BNP were different between Recurrence group and Non-recurrence group, all P<0.05. Logistic regression analysis indicated that AF duration (OR=1.02,95% CI 1.01-1.03, P=0.002), UA level (OR=1.01, 95% CI 1.00-1.01, P=0.046) and MS (OR=4.73, 95% CI 1.36-16.45, P=0.014) were the independent predictors for AF recurrence. UA quartile analysis indicated that gender, BMI, MS, creatinine, LVEF and the incidence of AF recurrence had signifcant discrepancy by different UA levels, all P<0.05. ROC curve showed that the predictive values for UA+MS in AF recurrence had the sensitivity at 80.4%, specificity at 74.1% (AUC 0.79±0.04, 95% CI 0.71-0.89, P=0.0001), for UA in AF recurrence had the sensitivity at 73.9%, specificity at 57.2% (AUC 0.66, 95% CI 0.56-0.76, P=0.02); UA+MS had the higher predictive value than UA alone, P<0.05. Conclusion: Both UA and MS were related to AF recurrence, high UA level combining MS had certain predictive value for AF recurrence in PAF patients after RFCA.

Background:Reduced expression of tripartite motif-containing 3 (TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly reduced in human primary hepatocellular carcinoma (HCC) tissues and that low TRIM3 expression was associated with short survival of HCC patients.However,the role of TRIM3 in liver cancer remains unknown.This study aimed to investigate the function of TRIM3 in liver cancer cells.Methods:The protein levels of TRIM3 in five liver cancer cell lines (SK-Hep1,Hep3B,Huh7,HepG2,Bel-7402) and one normal liver cell line (L02) were detected with Western blotting.HepG2 and Bel-7402 cells with IowTRIM3 expression were infected with recombinant lentiviruses overexpressing TRIM3 (LV-TRIM3),whereas Huh7 and Hep3B cells with high TRIM3 expression were transfected with TRIM3-targeted small interfering RNA (siTRIM3).The functions of TRIM3 in the proliferation,colony formation,cell cycle,migration,invasion,and apoptosis of the above cell lines were examined.The effect of TRIM3 on tumor growth and metastases in nude mice was also investigated.Results:TRIM3 was overexpressed in HepG2 and Bel-7402 cells with LV-TRIM3 infection,which further reduced proliferation,colony formation,migration,and invasion of both cell lines.Cell cycle analysis showed thatTRIM3 overexpression induced G0/G1 phase arrest in HepG2 and Bel-7402 cells.Moreover,apoptosis was not increased in HepG2 or Bel-7402 cells overexpressing TRIM3.Contrarily,silencing TRIM3 expression in Huh7 and Hep3B cells by siTRIM3 led to significantly decreased percentages of both cells in the G0/G1 phase and promoted cell proliferation,colony formation,migration,and invasion.In vivo experiment results confirmed thatTRIM3 overexpression suppressed tumor growth and metastasis.Conclusions:TRIM3 plays a tumor-suppressing role in the regulation of liver cancer development by reducing cell proliferation through cell cycle arrest at the G0/G1 phase.

Objective To establish a specific and sensitive Enzyme-linked immunosorbent Assay(ELISA)kit for detection of HIV-1 antibody in urine using Escherichia coli expression products as coating antigen. Methods The truncated HIV-1 gp41 gene fragment of major antigenic epitopes was inserted into the plasmid pET22b to obtain expression plasmid pET22b-mgp41. The recombinant antigen was expressed in BL21 (DE3) strains of Escherichia coli and was purified by immobilized metal chelation and gel filtration chromatography. Using this antigen as coating antigen, a HIV-1 urine antibody ELISA kit was developed. In order to examine the clinical utility of the kit, 5437 urine samples were assayed, which consisted of 641 urine samples from HIV infected patients and 4796 samples from normal subjects. Results The purity of purified antigen is up to 95%. Anti-HIV antibodies were detected in all the urine samples from HIV infected patients, and the diagnostic sensitivity for HIV-1 infection was 100%. In healthy control group, 71 cases showed false positive, the specificity was 98.52%.Conclusion The HIV-1 urine antibody kit can be used in screening and diagnosing for HIV-1 infection.

OBJECTIVETo study the characteristics of lymphocyte nuclear factor kappa B (NF-κB) signal transduction kinase-related molecular mRNA differential expressions at various month age segments in aging process and the intervening effect of Epimedium flavonoids (EF) on it.

METHODSSixty SD rats were divided into six groups, according to animals' age, i.e., the 3 days (d) group, the 4 months (m) group, the 10 m group, the 18 m group, the 27 m group, and the 27 m+EF group. RNA was extracted from separated splenic lymphocytes. Adopting NF-κB signal path functional genome oligonucleotide gene-chip (128 related genes), the integral characteristics and differences of NF-κB signal transduction kinase-related mRNA expressions were determined, and the intervening effect of EF was examined.

RESULTSThe mean level of the NF-κB signal transduction kinase-related mRNA expressions in rats' splenic lymphocytes lowered with aging; the highest expression was presented at 3 d after birth, and then, it lowered gradually, with the lowest level at 18 m or 27 m. After EF intervention, the expression level was raised to the 10-18 m level in the aged rats.

CONCLUSIONThe changing rules of lymphocyte NF-κB-signal-transduction-kinase-related mRNA expressions in various stages of aging are helpful for selecting the well time for preventing and intervening aging, and will also give a hint to the molecular index for assessment of senility retarding researches.

Aging ; drug effects ; genetics ; Animals ; Epimedium ; chemistry ; Flavonoids ; pharmacology ; Gene Expression Regulation ; drug effects ; I-kappa B Kinase ; metabolism ; Lymphocytes ; enzymology ; Male ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; genetics

OBJECTIVETo screen for potential mutations in an ethnic Han Chinese family from Shanxi with hereditary multiple exostoses.

METHODSPolymerase chain reaction and DNA sequencing were used to screen potential mutations in EXT1 and EXT2 genes.

RESULTSFor EXT1 gene, two synonymous mutations (P477P and E587E), three intronic mutations (c.1537 -48A>G, c.1721 +203A>G and c.1722 -103C>G) were detected. For EXT2 gene, five intronic mutations (c.-29 -148A>T, c.1080 -18T>A, c.1336 -93C>T, c.1526 -166C>T, and c.1526 -195C>T) were identified. Among these, EXT1 P477P, EXT1 E587E and EXT2 c.1080 -18T>A are polymorphisms listed by Multiple Osteochondroma Mutation Database, whilst the other 7 sites have not been reported.

CONCLUSIONNo mutations have been found among all exons of the EXT1 and EXT2 genes in this family. Linkage analysis is necessary for identifying the cause of this disease.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Child, Preschool ; China ; Exons ; Exostoses, Multiple Hereditary ; diagnosis ; genetics ; Female ; Genotype ; Humans ; Introns ; Male ; Middle Aged ; Mutation ; N-Acetylglucosaminyltransferases ; genetics ; Young Adult

Adolescent ; Exostoses, Multiple Hereditary ; genetics ; Humans ; Male ; Pedigree

OBJECTIVEAlpha-thalassemia is one of the most common monogene disorders in the world. Most frequently, it is caused by deletions of alpha-globin gene (-alpha or --), and less commonly resulted from the non-deletional mutation (alpha(T)alpha). Hemoglobin H (HbH) disease is the most severe type among survivors of alpha-thalassemia. The clinical presentation of children with the disease was highly heterogeneous. The aim of this study was to investigate the effect of alpha-globin genotypes in the children with HbH disease on predicting the phenotypic severity and to define the factors involved in the disease progress.

METHODSForty-three children with the disease in Zhuhai area of Guangdong, China were examined by using established techniques to detect genotypes of alpha-globin and to determine all hematological parameters. All detailed clinical data of the cases were recorded. Then clinical and hematological findings, and the correlation with genotypes were evaluated.

RESULTSSix alpha-thalassemia mutations were detected and interacted to produce 5 HbH disease genotypes. Of these genotypes, -alpha(3.7)/--(SEA)(60%), -alpha(4.2)/--(SEA) (19%) and alpha(CS)alpha/--(SEA) (12%) HbH diseases were prevalent in the area. Compared with -alpha(3.7)/--(SEA) HbH disease, significantly lower red blood cell (RBC) count, hemoglobin (Hb), mean corpuscular hemoglobin (MCHC) and HbA(2) (P < 0.05, 0.01, 0.01 and 0.01, respectively), and significantly higher mean corpuscular hemoglobin volume (MCV) and HbH levels (both P < 0.01), and more severe clinical phenotypes were found in the HbH disease with alpha(T)alpha/--(SEA) genotype. While the differences were much more significant when compared with -alpha(3.7)/--(SEA) then compared with -alpha(4.2)/--(SEA) not only in the hematological parameters, but also in the severity of clinical phenotypes. In addition, HbH levels showed anegatively correlation with the RBC count (r = -0.39, P < 0.01).

CONCLUSIONThe phenotypes of HbH disease may be mainly related to the underlying genotypes. The children with alpha(T)alpha/--(SEA) genotype presented with more severe hematological and clinical phenotypes followed by the -alpha(4.2)/--(SEA) and then -alpha(3.7)/--(SEA) genotypes. But phenotypic severity was not simply related to the degree of alpha-globin deficiency. HbH levels were found to exacerbate anemia. These data might provide comprehensive and very valuable and basic information for the management of HbH disease, genetic counseling and prenatal diagnosis.

Child ; China ; Disease Progression ; Genotype ; Hemoglobin H ; genetics ; Humans ; Phenotype ; alpha-Globins ; genetics

OBJECTIVEBy means of Delphi method and expert panel consultations, to choose suitable indicators and improve the score table for classifying the hygienic condition of hotels so that it can be widely used at nationwide.

METHODSA two-round Delphi consultation was held to choose suitable indicators among 78 experts from 18 provinces, municipalities and autonomous regions. The suitable indicators were selected according to the importance recognized by experts.

RESULTSThe average length of service in public health of the experts was (21.08 +/- 5.78) years and the average coefficient of experts' authorities C(r) was 0.89 +/- 0.07. The response rates of the two-round consultation were 98.72% (77/78) and 100.00% (77/77). The average feedback time were (8.49 +/- 4.48) d, (5.86 +/- 2.28) d, and the difference between two rounds was statistically significant (t = 4.60, P < 0.01). Kendall's coefficient were 0.26 (chi(2) = 723.63, P < 0.01), 0.32 (chi(2) = 635.65, P < 0.01) and opinions among experts became consistent. The score table for the hygienic quantifying and classification of hotels was composed of three first-class indicators (hygienic management, hygienic facilities and hygienic practices) and 36 second-class indicators. The weight coefficients of the three first-class indicators were 0.35, 0.34, 0.31.

CONCLUSIONDelphi method might be used in a large-scale consultation among experts and be propitious to improve the score table for the hygienic quantifying and classification.

Delphi Technique ; Housing ; classification ; standards ; Hygiene ; Outcome Assessment (Health Care) ; Public Health Administration ; methods

OBJECTIVETo investigate the effect of Chaiqin Chengqi Decoction (,CQCQD) on cholecystokinin receptor 1 (CCKR1)-mediated signal transduction of pancreatic acinar cell in rats with acute necrotic pancreatitis (ANP).

METHODSTwenty-seven Sprague-Dawley rats were randomized into three groups: the control group, the ANP group, and the CQCQD group (9 in each group). ANP rats were induced by two intraperitoneal injections of 8% L-arginine (pH=7.0, 4.4 g/kg) over a 2-h period. Rats were treated with 1.5 mL/100 g body weight of CQCQD (CQCQD group) or physiological saline (control and ANP groups) at 2 h interval. And 6 h after induction, pancreatic tissues were collected for histopathological examination. Pancreatic acinar cells were isolated for determination of CCKR1 mRNA and protein expression, phospholipase C (PLC) and inositol-1,4,5-triphosphate (IP3), and determination of fluorescence intensity (FI) as a measure of intracellular calcium ion concentration [Ca(2+)]i.

RESULTSThe pancreatic histopathological score (6.2 ± 1.1) and the levels of PLC (1,187.2 ± 228.2 μg/mL) and IP3 (872.2 ± 88.4 μg/mL) of acinar cells in the ANP group were higher than those in the control (2.8 ± 0.4, 682.5 ± 121.8 μg/mL, 518.4 ± 115.8 μg/mL) and the CQCQD (3.8 ± 0.8, 905.3 ± 78.5 μg/mL, 611.0 ± 42.5 μg/mL) groups (P<0.05). [Ca(2+)]i FI for the ANP group (34.8±27.0) was higher than that in the control (5.1 ± 2.2) and CQCQD (12.6 ± 2.5) groups (P<0.05). The expression of pancreatic acinar cell CCKR1 mRNA in the ANP group was up-regulated (expression ratio=1.761; P=0.024) compared with the control group. The expression of pancreatic acinar cell CCKR1 mRNA in the CQCQD group was down-regulated (expression ratio=0.311; P=0.035) compared with the ANP group. The ratio of gray values of the CCKR1 and β-actin in the ANP group (1.43 ± 0.17) was higher than those in the control (0.70 ± 0.15) and CQCQD (0.79 ± 0.11) groups (P<0.05).

CONCLUSIONSPancreatic acinar cell calcium overload of ANP induced by L-arginine was related to the up-regulated expressions of pancreatic acinar cell CCKR1 mRNA and protein. CQCQD can down-regulate expressions of pancreatic acinar cell CCKR1 mRNA and protein to reduce the PLC and IP3 of pancreatic acinar cells, relieving the calcium overload and reducing the pathological changes in rats with ANP.

Acinar Cells ; drug effects ; metabolism ; Animals ; Blotting, Western ; Calcium ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fluorescence ; Gene Expression Regulation ; drug effects ; Inositol 1,4,5-Trisphosphate ; metabolism ; Pancreas ; pathology ; Pancreatitis, Acute Necrotizing ; drug therapy ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Receptors, Cholecystokinin ; genetics ; metabolism ; Signal Transduction ; drug effects ; Type C Phospholipases ; metabolism