Objective To optimize the conditions of process for extraction of volatile oil of Rhizoma Zedoariae (RZ) in Weishu Granules and preparation of its ? - cyclodextrin- inclusion complexes. Methods Orthogonal design was adopted. Extraction content of RZ volatile oil was used as the index for the optimizing of extraction process. The amount of inclusion complexes and efficient inclusion rate served as the indexes for the optimizing of inclusion process. Ultraviolet spectrophotometry was applied to detect the content of volatile oil. Results The optimal extraction conditions were: gringding the crude RZ into coarse grains, adding 12- fold water and distilling the mixture for 6 h; the optimal inclusion conditions were: ? - cyclodextrin and volatile oil were mixed in the proportion of 8 :1, 2- fold water was added into the mixture and the mixture was triturated for 60 min. The maximum UV spectrum absorbance of aethylis acetas liquor of valatile oil located at wavelength of 253nm. Conclusion Higher extraction rate of RZ volatile oil can be achieved by this technique; volatile oil is powdered and its stability increased after being included by ? - cyclodextrin.

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.
Amino Acid Sequence ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; Antibody Affinity ; Asthma ; blood ; Base Sequence ; DNA, Complementary ; metabolism ; Escherichia coli ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lymphocytes ; chemistry ; Peptide Library ; RNA, Messenger ; isolation & purification ; Recombination, Genetic ; genetics ; Ribosomes ; chemistry ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; isolation & purification ; Transformation, Genetic

OBJECTIVETo study the clinical characteristics of acute myocardial infarction (AMI) among younger adults and to explore the possible mechanisms of early myocardial infarction, combined with the newly discovered risk factors of coronary heart disease.

METHODSData on comparative analysis to the exposure rates of the risk factors and inducing factors of non-CAD patients with two groups of AMI patients including younger adults group (< or =40 years old) and aged adults group (> or =50 years old). Coronary angiography was applied.

RESULTSThere were differences noticed between the frequencies of risk factors of the two AMI groups. In younger adults group the exposure rates of smoking, hyperlipidemia, positive family history, C-reactive protein (CRP) and fibrinogen were markedly higher, while in elderly group the exposure rates of hypertension, smoking, hyperlipidemia, diabetes, CRP, fibrinogen and homocysteine (HCY) were markedly higher (P < 0.05). Although the clustering status of risk factors of the younger adult group was not higher than that of the elderly group. There were obvious inducing factors before the patients were attacked by AMI and the inducing factors inclined to cluster, which had obvious dose-reaction relationships with the occurrence of AMI in young people.

CONCLUSIONEarly AMI of younger adults might relate to the clustering status of inducing factors. The coexistence of several kinds of inducing factors was resulted in the occurrence of AMI of the atherosclerosis (As) and non-As patients by means of myocardial ischemia accumulation effect.

Adult ; Age Factors ; Aged ; Atherosclerosis ; epidemiology ; China ; epidemiology ; Coronary Angiography ; Humans ; Middle Aged ; Myocardial Infarction ; epidemiology ; pathology ; Myocardial Ischemia ; epidemiology ; Risk Factors

This study aims at trying to establish a novel method of sterility test for injections based on biothermodynamics, in order to overcome the deficiencies of routine sterility tests such as long detecting cycle, low sensitivity and prone to misjudgments. A biothermodynamics method was adopted to rapidly detect the microorganism contamination of injections by monitoring the heat metabolism during the growth of microbe. The growth rate equal to or greater than zero and the heat power difference of P(i) and P(0) with three folds higher than the noise of baseline were chosen as indexes to study the heat change rule of microbe. In this way, the effectiveness of the new method to detect strains required by conventional sterility test or in injection samples was also investigated. Results showed that the Gram-positive bacteria, Gram-negative bacteria and fungi demanded by sterility testing methodology could be detected by biothermodynamics method within 10 hours, with the sensitivity lower than 100 CFU x mL(-1). Meanwhile, this method was successfully applied to the sterility test of Compound Yinchen injection (FFYC), Shuanghuanglian powder injection (SHL) and Compound Triamcinolone injection (TAND) which were sterilized with different degrees. Therefore, the biothermodynamics method, with advantages of fast detection and high sensitivity, could be a complementary solution for conventional sterility tests.
Anti-Inflammatory Agents ; administration & dosage ; chemistry ; Drug Contamination ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Fungi ; isolation & purification ; Gram-Negative Bacteria ; isolation & purification ; Gram-Positive Bacteria ; isolation & purification ; Hot Temperature ; Injections ; Microbiological Techniques ; methods ; Sensitivity and Specificity ; Sterilization ; Triamcinolone ; administration & dosage ; chemistry

OBJECTIVETo investigate the correlation of radiation pneumonitis (RP) with standardized uptake value (SUV) for fluorodeoxyglucose (FDG) positron emission tomography and computed tomography (PET-CT) in lung cancer patients treated with radiation therapy.

METHODSFourty patients with unresectable non-small cell lung cancer (NSCLC) received FDG PET-CT before and after radiotherapy. The average SUV of the lung tissue irradiated with a dose of < or = 5 Gy, 5.1 approximately 15 Gy, 15.1 approximately 35 Gy, 35.1 approximately 60 Gy, >60 Gy were measured. The correlation between SUV and RP was analyzed by comparing the SUV in the patients with RP and without. The SUV ratio of the irradiated lung tissue to that of the non-irradiated lung tissue (L/B) was also calculated.

RESULTSOf the 40 patients, 8 developed RP, including 6 cases of grade 2 and 2 cases of grade 3. The SUV of irradiated lung tissues with a dose of 35.1 approximately 60 Gy was significantly correlated with RP. When SUV > or =1, the RP incidence rate was 41.7% versus 20.0% in the whole group, with a statistically significant difference. (chi2 = 3.96, P < 0.05). The sensitivity and specificity of SUV in predicting RP was 62.5% and 78.1%, respectively. When the value of L/B > or = 2.5, the RP incidence rate was 40.7% in this group versus 20.0% in the whole group, with a statistical significance (chi(2) = 4.92, P < 0.05). If taking L/B > or = 2.5 as a threshold value, the sensitivity and specificity in predicting RP was 72.7% and 90.9%, respectively. No statistically significant difference was found in predicting radiation pneumonitis between SUV > or =1 and L/B > or = 2.5 (chi2 = 0.002, P > 0.05).

CONCLUSIONThe standardized uptake value (SUV) and the SUV ratio of the irradiated lung tissue to that of the non-irradiated lung tissue (L/B) for FDG PET-CT are positively correlated with radiation pneumonitis, and clinicians may use it to predict the occurrence of radiation pneumonitis.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung ; radiotherapy ; Female ; Fluorodeoxyglucose F18 ; pharmacokinetics ; Follow-Up Studies ; Humans ; Lung Neoplasms ; radiotherapy ; Male ; Middle Aged ; Positron-Emission Tomography ; Radiation Dosage ; Radiation Pneumonitis ; diagnosis ; etiology ; metabolism ; Radiopharmaceuticals ; pharmacokinetics ; Radiotherapy, Conformal ; adverse effects ; Tomography, X-Ray Computed

OBJECTIVETo investigate the effects of promoter methylation of p16, death-associated protein kinase (DAPK) and retinoic acid receptor-beta (RAR beta) genes on clinical data in non-small cell lung cancers, and to study the effect of smoking on the risk of gene methylation.

METHODSThe promoter methylation of p16, DAPK and RAR beta genes in 200 primary non-small cell lung cancers and the corresponding nonmalignant lung tissues were determined by methylation-specific PCR.

RESULTSMethylation in the tumor tissues was detected in 51.0% for p16, 60.0% for DAPK, and 58.0% for RAR beta gene, with significant differences (P < 0.05) when compared with those in the corresponding nonmalignant tissues(12.5%, 11.5% and 15.0%) respectively. p16 gene methylation in tumor tissue was associated with age significantly in unconditional logistic regression analysis (P < 0.01) and histologic type (P < 0.05). DAPK gene methylation in tumor tissue was associated significantly with age (P < 0.05), gender (P < 0.05) and clinical type (P < 0.05). RAR beta gene methylation in tumor tissue was associated with clinical type (P < 0.05) and tumor stage (P < 0.05) significantly. The interaction odds ratio (OR) for the gene-gene interaction in tumor tissue between p16 and DAPK was 1.987 (95%CI:1.055-3.743). The results of the gene-smoking analyses revealed that a relationship existed between cigarette smoking and p16 gene methylation (OR = 3.139, 95%CI: 1.046-9.419), the OR for the relationship of DAPK gene methylation and cigarette smoking was 3.585(95%CI: 1.270-10.123) in tumor tissue. The RAR beta gene methylation did not differ based on the smoking status of patients in tumor tissue.

CONCLUSIONThe p16, DAPK and RAR beta genes methylation are strongly associated with clinical data of non-small cell lung cancer, and methylation of p16 and DAPK genes are associated with tobacco smoking.

Apoptosis Regulatory Proteins ; genetics ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; Carcinoma, Non-Small-Cell Lung ; etiology ; genetics ; pathology ; DNA Methylation ; Death-Associated Protein Kinases ; Genes, p16 ; Logistic Models ; Lung Neoplasms ; etiology ; genetics ; pathology ; Neoplasm Staging ; Promoter Regions, Genetic ; Receptors, Retinoic Acid ; genetics ; Smoking ; adverse effects

OBJECTIVETo outline the clinical manifestations and compare the different radiological methods of detecting malformation of cervical segment of internal carotid artery.

METHODSA retrospective analysis of 7 cases with malformation of cervical segment of internal carotid artery between May, 2004 and April, 2011. CT angiography (CTA) and magnetic resonance angiography (MRA) were used to detect the morphology of cervical segment of internal carotid artery.

RESULTSThis disease entity provided no obvious symptoms in five cases, and such complains as pharyngeal foreign body sensation in one and odynophagia in another. Physical examination showed a bulge with pulsation on pharyngeal wall in four cases, and apparent normal pharynges in other three patients, all of which were covered with intact pharyngeal mucosa. Twelve carotid arteries were observed in seven cases, five of which were showed tortuosity and seven kinking. All of the five patients with recorded radiological materials had identified malformations of internal carotid artery, two of which were tortuosity bilaterally and two kinking bilaterally and one tortuosity and kinking respectively. CTA and MRA revealed tortuosity of cervical segment of internal carotid arteries.

CONCLUSIONSNo typical clinical symptoms were shown in the malformation of cervical segment of internal carotid artery. Pharyngeal bulge with pulsation could be encountered. CTA and MRA showed excellent ability to depict the malformation of cervical segment of internal carotid artery and its relationship with surrounding structures, which could protect carotid artery from unintended damage.

Aged ; Angiography ; Carotid Artery Diseases ; diagnostic imaging ; pathology ; Carotid Artery, Internal ; abnormalities ; diagnostic imaging ; pathology ; Female ; Humans ; Magnetic Resonance Angiography ; Male ; Middle Aged ; Retrospective Studies ; Tomography, X-Ray Computed

BACKGROUNDPrevious studies have investigated the technique of linear ablation at the mitral isthmus (MI) in patients with idopathic atrial fibrillation (AF), but MI ablation in patients with prosthetic natural mitral valves (MVs) was not described in detail. Present study sought to summarize our initial experience of ablating MI in patients with prosthetic MVs

METHODSPatients with drug refractory AF and prosthetic MVs were eligible for this study, and the patients with natural MVs but received MI ablation served as control group. Left atrium (LA) mapping and ablation was carried out guided by CARTO system. The anatomy of MI was assessed via computer topography scan.

RESULTSDuring the study period, a consecutive of 19 patients (male/female = 12/7, mean age of (48 ± 6) years) with prosthetic MVs (16 with metal valves, 3 with biologic valves) entered for AF ablation, other 35 patients served as control group. In study group, mapping along MI documented lower voltages ((2.0 ± 1.0) vs. (3.1 ± 1.3) mV, P = 0.002), more fragmented potentials (19/19 vs. 20/15, P < 0.001), and higher impedance ((132 ± 34) vs. (110 ± 20) Ω, P = 0.004). After initial ablation, more residual gaps along the MI lesions were found in study group (2.4 ± 0.4 vs. 1.7 ± 0.3, P < 0.001). The mean length of MI ((6.2 ± 3.3) vs. (7.1 ± 2.3) cm, P = 0.25) was comparable between 2 groups, but the MI in study group was much thicker ((3.1 ± 1.8) vs. (2.1 ± 1.07) cm, P = 0.01) and all were found as pouch type (19/19 vs. 2/35, P < 0.001). The follow-up results were comparable (65.1% vs. 72.3%, P = 0.30).

CONCLUSIONFor patients with prosthetic MVs, linear ablation at MI could be successfully carried out despite anatomical and pathological changes.

Adult ; Atrial Fibrillation ; surgery ; Catheter Ablation ; methods ; Female ; Heart Atria ; surgery ; Heart Valve Prosthesis ; Humans ; Male ; Middle Aged ; Mitral Valve ; surgery

OBJECTIVETo investigate the correlation between RARbeta gene promoter methylation and P53 gene mutations in non-small cell lung cancer (NSCLC).

METHODSPromoter methylation of RARbeta and P53 mutations of exons 5 through 9 in 198 resected primary NSCLC tissues were determined by methylation-specific PCR and direct sequencing.

RESULTSRARbeta gene promoter methylation and P53 mutation were detected in 58.1% and 36.4% of tumors, respectively. Both were higher in males than in females and in smokers than in nonsmokers. A higher prevalence of RARbeta promoter methylation was found in patients with advanced stage tumors than those with TNM stage I. P53 gene mutations were more frequent in squamous cell carcinoma and adeno-squamous carcinoma than adenocarcinoma. All such differences were statistically significant (P< 0.05). Frequencies of P53 mutations, including G:C>T:A mutations, transversions and missense mutations were significantly higher in tumors with RARbeta methylation than in those without (P< 0.05). A significantly higher prevalence of RARbeta methylation was found in tumors with only G:C>T:A mutation in P53 gene than those without P53 mutations (P< 0.05). This difference (OR=3.737, 95%CI: 1.414-9.873) was still statistically significant (P< 0.05) in smokers (OR=4.020, 95%CI: 1.263-12.800), squamous cell carcinomas (OR=5.480, 95%CI: 1.400-21.446) or patients with advanced tumors (OR=3.446, 95%CI: 1.054-11.267) after adjusting for age and sex.

CONCLUSIONRARbeta methylation is associated with G:C>T:A mutations in P53 gene in NSCLC.

Adult ; Aged ; Base Sequence ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; DNA Methylation ; Female ; Genes, p53 ; Genetic Predisposition to Disease ; Humans ; Lung Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Promoter Regions, Genetic ; Receptors, Retinoic Acid ; genetics