OBJECTIVETo screen the proteins interacting with inhibitor of differentiation 1(Id1) using yeast two-hybrid analysis in adult human lung cDNA libraries.

METHODSThe coding sequence of Id1 was amplified by PCR and cloned into the bait plasmid. The recombinant bait vector pHybLex/Zeo-Id1 was verified by restriction endonuclease digestion before transformation into the yeast strain EGY48/pSH18-34, which was tested subsequently for reporter genes Leu2 and LacZ activation. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into the yeast strains and screened to obtain Leu2(+) and Leu2(+)LacZ(+) clones, with the false positive clones excluded using positive and negative controls, and the plasmid of the true positive clone was sequenced and blasted for homological analysis.

RESULTSSuccessful construction of pHybLex/Zeo-Id1 was confirmed by enzyme digestion. After transformation of pHybLex/Zeo-Id1 into EGY48/pSH18-34, no specific reporter genes Leu2 and LacZ activation was found. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into yeast strain, and 198 Leu(+) clones and 19 Leu(+)LacZ(+) double positive clones were obtained. After elimination of the false positive clones, one true positive clone was obtained, whose plasmid analysis by sequencing and blasting indicated high homology (99.5%, 556/559) to AGGF1 (an angiogenic factor with G-patch and FHA domains 1). AGGF1 expression was confirmed in the true positive yeast cells by Western blotting.

CONCLUSIONAGGF1 is confirmed to interact with Id1 by yeast two-hybrid analysis for screening adult human lung cDNA libraries.

Adult ; Angiogenic Proteins ; genetics ; metabolism ; Cell Proliferation ; Endothelial Cells ; cytology ; metabolism ; Gene Library ; Humans ; Inhibitor of Differentiation Protein 1 ; metabolism ; Lung ; cytology ; Plasmids ; genetics ; Protein Binding ; Two-Hybrid System Techniques

PBL teaching method is a new mode of teaching which is originated from the West and implemented into China in recent years with an expectation that it would mainly develop the students’ self-learning ability,and enhance their skills of comprehensive thinking and solving actual problems.The author summarizes the practical experience of using PBL teaching methods in the theory teaching in Department of Medical Microbiology and Human Parasitology,China Medical University in the past three years,and then proved this method is very helpful to improving the students’integrated thinking by analysis of sample.At the same time the results also suggested that the students showed high enthusiasm in discussing the cases.By this way,the students showed great subjective intiative in their studies.

Objective: To observe the effects of acupoint thread-embedding therapy and low-carbohydrate diet therapy on obese patients with food addiction. Methods: Sixty-five eligible patients were randomized into a thread-embedding group of 33 cases and a diet group of 32 cases to respectively receive 12-week treatment. Before treatment, after treatment and at 6-month follow-up, the two groups were observed and compared in terms of body mass (BM), waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), body mass index (BMI), body fat rate (BFR), basal metabolic rate (BMR) and Yale food addiction scale version 2.0 (YFAS 2.0). Results: At the end of treatment, there were no significant differences in the general efficacy, and the improvements in BM, BMI, WC, HC, WHR and BFR between the thread-embedding group and diet group (all P>0.05). At follow-up, the thread-embedding group showed more significant improvements in all the aforementioned indicators compared with the diet group except HC (all P<0.05). At the end of treatment and follow-up, BMR and YFSA 2.0 had more significant improvements in the thread-embedding group than in the diet group (all P<0.05). Conclusion: Acupoint thread-embedding therapy can produce significant efficacy in treating obese patients with food addiction; it can improve the food addiction state and work better in maintaining the efficacy compared with low-carbohydrate diet therapy.

Abdomen ; Hernia ; Humans ; Prostheses and Implants ; Prosthesis Implantation ; Treatment Outcome

OBJECTIVETo investigate the expression of long non-coding RNA HOTAIR in the plasma of breast cancer patients and its value in the diagnosis of breast cancer.

METHODSHOTAIR levels were measured in 24 tumor tissues and 70 plasma samples from breast cancer patients using quantitative real-time PCR. The correlations of plasma HOTAIR level with the clinicopathological features of the patients were analyzed. A multivariate logistic regression model was established to analyze the value of plasma HOTAIR in comparison with plasma CA153 and CEA levels for breast cancer diagnosis. We further detected HOTAIR levels in the plasma and breast cancer tissues of 24 patients before and after operation and investigated their correlation.

RESULTSBreast cancer patients had increased expressions of HOTAIR in the tumor tissues and plasma, and plasma HOTAIR level was significantly correlated with estrogen receptor (ER) level (P=0.004) and lymph node metastasis (P=0.010). Receiver operating characteristic (ROC) curve and the multivariable logistic regression model showed that the area under ROC curve (AUC) of plasma HOTAIR was 0.82 (P<0.001) for breast cancer diagnosis with a diagnostic sensitivity and a specificity of 73.3% and 93.3%, respectively. The diagnostic power and specificity of plasma HOTAIR was much higher than those of CA153 (AUC=0.66, P=0.030) and CEA (AUC=0.52, P=0.001), and the combination of the 3 markers further enhanced the diagnostic power (AUC=0.84) and specificity (96.7%). Plasma HOTAIR level was significantly reduced in the patients after the operation (P<0.0001) and showed a moderate correlation with its expression in tumor tissues (r=0.62, P<0.0001).

CONCLUSIONPlasma HOTAIR may serve as a potential biomarker for breast cancer diagnosis.

Biomarkers, Tumor ; blood ; Breast Neoplasms ; blood ; diagnosis ; Carcinoembryonic Antigen ; blood ; Female ; Humans ; Logistic Models ; Lymphatic Metastasis ; Mucin-1 ; blood ; Prognosis ; RNA, Long Noncoding ; blood ; ROC Curve ; Real-Time Polymerase Chain Reaction ; Receptors, Estrogen ; metabolism ; Sensitivity and Specificity

OBJECTIVETo study the applied valuation of Onodera prognostic nutrition index (Onodera index) in elderly patients with colorectal cancer.

METHODSOnodera indexes of 163 elderly patients with colorectal cancer were calculated and these patients were divided into better-nourished group (Onodera index ≥45) and under-nourished group (Onodera index <45). Correlations of Onodera index with general data, operation type, postoperative complication, recovery of gastrointestinal function, clinicopathological feature and prognosis were analyzed. Cox proportional hazards model was also established to identify the independent prognostic factors for prognosis of elderly patients with colorectal cancer.

RESULTSPatients in better-nourished group had significantly higher radical resection rate [90.9% (70/77) vs. 62.8% (54/86), P<0.01], lower postoperative complication rate [17.1% (12/70) vs. 53.7% (29/54), P<0.01] and earlier postoperative defecation [(3.09±1.14) d vs. (3.43±1.98) d, P<0.05] than those in under-nourished group. Onodera index was found to be related to age, tumor location, tumor size, and operation type (all P<0.05). Better-nourished group had significantly better survival than worse-nourished group (5-year survival rate: 64% vs. 24%, P<0.01). Onodera index was identified as an independent prognostic factor for elderly patients with colorectal cancer (RR=0.888, 95%CI:0.800-0.985, P=0.025).

CONCLUSIONOnodera index is a valuable clinical marker in preoperative estimation as well as prognosis prediction for elderly patients with colorectal cancer.

Aged ; Aged, 80 and over ; Colorectal Neoplasms ; surgery ; Female ; Humans ; Male ; Nutrition Assessment ; Prognosis ; Retrospective Studies

A new dibenzocyclooctadiene lignan, renchangianin E (1) was isolated from the stems of Kadsura renchangiana. Its structure and stereochemistry were elucidated by spectroscopic methods, including 2D-NMR techniques.
Cyclooctanes ; chemistry ; Kadsura ; chemistry ; Lignans ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure

AIMTo study the regular pattern and mechanism of positive inotropic effect after washout of ACh (rebound of myocardial contractile force) in isolated rabbit hearts.

METHODSThe changes of myocardial contractile force after perfusion and washout of ACh were observed in isolated Langendorff perfused rabbit hearts.

RESULTSMaximum rebound rate induced by ACh of 10(-8)-10(-3) mol/L were 2.20% +/- 1.70%, 6.71% +/- 3.40%, 9.18% +/- 3.54%, 14.16% +/- 3.27%, 4.37% +/- 5.86% and 1.03% +/- 6.86%, respectively. Compared with the ACh of 10(-5) mol/L in control group, adrenaline enhanced rebound of myocardial contractile force, maximum rebound rate in adrenaline group was 29.25% +/- 5.83% (P < 0.05), propranolol reduced rebound, and maximum rebound rate in propranolol group was 5.15% +/- 4.45% (P < 0.05), we had not detected rebound of myocardial contractile force in 800 s after addition ACh in verapamil group.

CONCLUSIONIn isolated rabbit heart, positive inotropic effect after washout of ACh has relevance to the activities of calcium current channel and beta-adrenergic receptor. Perhaps there are some different aspects in the mechanism of positive inotropic effect between perfusion of high concentration and after washout of ACh.

Acetylcholine ; pharmacology ; Animals ; Cardiotonic Agents ; pharmacology ; Heart ; drug effects ; In Vitro Techniques ; Myocardial Contraction ; drug effects ; physiology ; Rabbits

OBJECTIVETo observe the effect of nitric oxide (NO) on adhesion, proliferation, and migration of human epidermal stem cells (ESC) in vitro.

METHODSESC were isolated and cultured by the modified method of rapid attachment to type IV collagen. (1) Morphology of cells was observed under inverted phase-contrast microscope. Expression levels of integrin β(1) and cytokeratin 19 (CK19) of cells were determined by Western blotting and immunofluorescence staining. (2) After being treated with scratching, ESC adhered to the wall was respectively treated with nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) in the concentration of 1, 10, 100, 500 µmol/L. ESC without treatment of SNAP was used as control. The migration rate of ESC was detected at post scratching hour (PSH) 12 and 24. The chemotaxis of ESC (treated with SNAP in above-mentioned concentration) was tested by Transwell assay, and the transferred cell number was counted. (3) ESC was respectively treated with SNAP in the concentration of 10, 100, 500 µmol/L for 1 h. ESC without treatment of SNAP was used as control. The adhesion of ESC was detected with adhesion test, and the inhibition rate of adhesion was calculated. The proliferation of ESC (denoted as absorbance value) was determined by microplate reader at post-treatment hour (PTH) 0, 12, 24, 48. Data were processed with one-way analysis of variance and Dunnett t test.

RESULTS(1) Small clone formed on post culture days (PCD) 5 to 9. On PCD 10 to 14, cell proliferation sped up. CK19 and integrin β(1) were detected to be expressed in the isolated cells. The cells were identified as ESC. (2) Compared with that of ESC without treatment of SNAP [(35.7 ± 0.3)%, (45.7 ± 5.0)%], migration of ESC treated with SNAP in the concentration from 1 to 100 µmol/L was promoted at PSH 12 and 24. Migration rates of ESC treated with 100 µmol/L SNAP were the highest [respectively (48.8 ± 2.7)%, (82.1 ± 15.8)%, with t value respectively 8.34, 5.10, P values both below 0.01]. The number of ESC transferred to membrane after being treated with 100 µmol/L SNAP was significantly larger than that of ESC without treatment of SNAP (t = 9.24, P = 0.00). (3) Absorbance values of ESC treated with 100, 500 µmol/L SNAP were obviously higher than that of ESC without treatment of SNAP (with t value respectively 4.30, 4.67, P values both equal to 0.00). Proliferation of ESC treated with 100, 500 µmol/L SNAP was obviously stronger than that of cells without treatment of SNAP at PTH 24, 48 (with t values from 2.84 to 8.17, P values all below 0.05).

CONCLUSIONSExogenous NO in suitable concentration can promote the migration of human ESC. Exogenous NO can inhibit the adhesion and promote the proliferation of human ESC in vitro.

Cell Movement ; drug effects ; Cell Proliferation ; Cells, Cultured ; Epithelial Cells ; cytology ; drug effects ; Humans ; Nitric Oxide ; pharmacology ; Stem Cells ; cytology ; drug effects

In order to visually detect H1, N1 and N2 subtype of avian influenza virus (AIV), three reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed. According to the sequences of AIV gene available in GenBank, three degenerate primer sets specific to HA gene of H1 subtype AIV, NA gene of N1 and N2 subtype AIV were designed, and the reaction conditions were optimized. The results showed that all the assays had no cross-reaction with other subtype AIV and other avian respiratory pathogens, and the detection limit was higher than that of conventional RT-PCR. These assays were performed in water bath within 50 minutes. Without opening tube, the amplification result could be directly determined by inspecting the color change of reaction system as long as these assays were fin-ished. Fourteen specimens of H1N1 subtype and eight specimens of H1N2 subtype of AIV were identified from the 120 clinical samples by RT-LAMP assays developed, which was consistent with that of virus isolation. These results suggested that the three newly developed RT-LAMEP assays were simple, specific and sensitive and had potential for visual detection of H1, N1 and N2 subtype of AIV in field.
Animals ; Chickens ; DNA Primers ; genetics ; Ducks ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H1N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza A virus ; classification ; genetics ; isolation & purification ; Influenza in Birds ; diagnosis ; virology ; Nucleic Acid Amplification Techniques ; methods ; Poultry Diseases ; diagnosis ; virology ; Reverse Transcription ; Turkeys