1.Rapid screening of katG gene mutation in isoniazid-resistant Mycobacterium tuberculosis
Journal of Zhejiang University. Medical sciences 2002;31(3):178-180
OBJECTIVE: To evaluate the relationship between katG gene mutation and isoniazid (INH) resistance and to develop a rapid screening method of point mutation in the katG gene associated with MTB resistance. METHODS: Twenty-four clinical isolates of MTB with 8 INH resigtance isolates and 16 INH-sensitive isolates were analyzed by PCR-RFLP, with the H(37)Rv reference strain as the control. RESULTS: G-->C point mutations were detected in 7 of 8 isoniazid-resistant strains and no gene mutation was shown in 16 isoniazid-sensitive isolates. The sensitivity and specificity were 87.5 % and 100 % respectively. No katG gene sequence deletion was observed in any specimen. CONCLUSION: Our results suggest katG gene mutation is one of the most important mechanisms of INH-resistant TB. PCR-RFLP may be useful in detection of katG gene mutation.
2.Construction of the mutants of rice nonspecific lipid transfer protein and expression comparison in two kinds of thioredoxin fusion expression vectors.
Xiao-Chun GE ; Ji-Chao CHEN ; Wen-Yi WANG ; Kai-Ming CAO ; Chong-Rong SUN
Chinese Journal of Biotechnology 2002;18(2):167-171
Five structural important residues of rice nonspecific lipid transfer protein LTP110 were mutated by site-directed mutagenesis. Sequence results showed that they were all mutated successfully. After trying various E. coli expression systems, thioredoxin fusion expression system was found to be a proper system to express wild type and mutant LTP110. cDNA sequences encoding wild type LTP110 and the mutants Y17A, P72L, R46A, D43A, C50A were cloned into two kinds of thioredoxin fusion expression vectors. The expression results were compared. In pTrxFus/GI724 expression system, wild type LTP110 and the mutants Y17A, P72L, R46A could be expressed at low level while D43A and C50A could not be expressed normally; in pET32a(+)/BL21 (DE3) trxB- expression system, wild type LTP110 and all mutant proteins could be expressed very well and the levels were higher than that in pTrxFus/GI724 system. LTP110 fusion protein expressed in pET32a(+) vector was purified and its activity was checked by fluorescence labeled fatty acid. Results indicated that the recombinant LTP110 fusion protein has lipid binding activity. This work provides good basis for the further study.
Amino Acid Sequence
;
Carrier Proteins
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genetics
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isolation & purification
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metabolism
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Gene Expression
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Genetic Engineering
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Genetic Vectors
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Oryza
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genetics
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Plant Proteins
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genetics
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isolation & purification
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metabolism
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Recombinant Fusion Proteins
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genetics
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isolation & purification
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metabolism
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Thioredoxins
;
genetics
3.Introduce Tagsk1 into salt-sensitive callus to improve the capacity of salt-tolerance by micropartical bombardment.
Tao XU ; Bao-Cun ZHAO ; Rong-Chao GE ; Yin-Zhu SHEN ; Zhan-Jing HUANG
Chinese Journal of Biotechnology 2006;22(2):211-214
The Tagsk1 (Triticum asetium L. glycogen synthase kinase 1) gene derived from the genome of wheat salt-tolerance mutant RH8706-49 was cloned by PCR. The special primers designed according to full length cDNA sequence of Tagsk1 (AF525086). A binary expression vector pBI121-gsk1 containing Gus and Tagsk1 was constructed. And pBI121-gsk1 was introduced into the callus induced from mature embryos of salt-sensitive wheat H8706-34 and cv. China Spring by particle bombardment. The transformed callus were screened by Kanamycin and 0.5% NaCl. The salt-tolerance callus were obtained, which showed higher ability of salt-tolerance and could diffirentiate roots and buds on the medium containing 0.5% NaCl.
Adaptation, Physiological
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Biolistics
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DNA, Plant
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genetics
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Glycogen Synthase Kinases
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genetics
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Mutation
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Plant Proteins
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genetics
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Plants, Genetically Modified
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Salt-Tolerant Plants
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genetics
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Seeds
;
genetics
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Sodium Chloride
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metabolism
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Transformation, Genetic
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Triticum
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enzymology
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genetics
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physiology
4.Development of Integrated Atomic Force Microscopy and Fluorescence Microscopy for Single-Molecule Analysis in Living Cells
Ge Ge QIN ; Hui Wen LI ; Chao Jia XU ; Long Xiao KOU ; Rong ZHAO ; Fang LUO ; Hong Xiao FANG
Chinese Journal of Analytical Chemistry 2017;45(12):1813-1823
Atomic force microscope ( AFM) and fluorescence microscope ( FM) have been emerging as two most commonly used tools for single-molecule study in living cells. Combining the advantages of two microscopes, the development of the integrated AFM-FM technique with high spatiotemporal resolution and multi-function has attracted increasing interest. In this review, the principles of AFM single-molecular force spectroscopy and single-molecule fluorescence imaging were briefly discussed, and the recent advances in the integrated AFM-FM instrumentation were summarized. Subsequently based on our own research in the investigation of ligand-receptors interactions with the integrated AFM-FM technique, its applications in live-cell single-molecule imaging and characterization were introduced.
5.Prognostic analysis of 76 cases with adenoid cystic carcinoma in salivary gland.
Ming-hua GE ; Jia-feng WANG ; Qing-min XIA ; Zhuo TAN ; Jia-jie XU ; Chao CHEN ; Ke-jing WANG ; Xiang-rong TAN
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2012;47(3):202-206
OBJECTIVETo investigate the prognosis of adenoid cystic carcinoma (ACC) in salivary gland and its influencing factors.
METHODSClinical and following-up data of 76 patients with ACC in salivary glands were reviewed. Major gland tumors represented 35.5% whereas minor gland tumors comprised 64.5% of the cohort, with 8 cases (10.5%) in stage I, 23 (30.3%) in stage II, 18 (23.7%) in stage III and 27(35.5%) in stage IV. Survival rates were calculated by Kaplan-Merier method. Cumulative survival curves were evaluated using the Log-rank test. Multivariate analysis was performed by Cox proportional hazard model.
RESULTSThe regional recurrence rate was 28.9% and distant metastasis rate was 21.1%. The overall 5-year survival rate, tumor-free survival rate and tumor-related survival rate were 73.7%, 61.8% and 74.9% respectively. The overall 10-year survival rate, tumor-free survival rate and tumor-related survival rate were 48.2%, 39.8% and 56.2% respectively. Univariate survival analysis showed pathological type, clinical stage and perineural invasion were relevant to the prognosis of ACC and multivariate analysis showed they were the independent prognostic factors of ACC in salivary gland.
CONCLUSIONSClinical stage, pathological type and perineural invasion were the independent prognostic factors for adenoid cystic carcinoma in salivary gland. Surgery was the first choice for the treatment of adenoid cystic carcinoma in salivary gland, and postoperative radiotherapy may prolong the tumor-free survival time of patients in stage III and IV.
Adult ; Aged ; Carcinoma, Adenoid Cystic ; diagnosis ; Female ; Humans ; Male ; Middle Aged ; Multivariate Analysis ; Prognosis ; Retrospective Studies ; Salivary Gland Neoplasms ; diagnosis ; Survival Rate ; Young Adult
6.The analysis on influencing factors and its clinical application of cupping spot.
Quanxi GE ; Rong MA ; Chao WANG ; Xiangwen MENG
Chinese Acupuncture & Moxibustion 2018;38(12):1359-1362
We retrieved and analyzed the articles with the subject term words or key words of the "cupping spot" from January 1, 1990 to May 20, 2018. The negative pressure, cup-retaining time, temperature and the other factors about cupping spot were compared. There was a single stimulus for the negative pressure and cupping time. Pressure and time, pressure and temperature could be considered interactively, and the best group was on research. The color standard of canister spot diagnosis had not been unified, which could be analyzed from its color characteristic and color spectrum. At present, there is no agreement on the measurement of pressure and temperature, selection criteria of subjects, etc. The experimental researchmethods on the spot are becoming more and more diverse, with modern instruments for cupping spot on clinical efficacy and biological mechanism research.
Acupuncture Therapy
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Medicine, Chinese Traditional
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Pressure
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Temperature
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Treatment Outcome
7.Detection and clinical features of MLL gene rearrangement in adult patients with acute leukemia.
Ping LIU ; Run ZHANG ; Zheng GE ; Zhong-Kun LIN ; Juan LIU ; Si-Xuan QIAN ; Su-Jiang ZHANG ; Hua LU ; Han-Xin WU ; Hong-Xia QIU ; Peng LIU ; Wei XU ; Li-Juan CHEN ; Chao LU ; Bin-Bin LU ; Chun QIAO ; Hai-Rong QIU ; Guang-Rong ZHU ; Jian-Fu ZHANG ; Yu-Jie WU ; Jian-Yong LI
Journal of Experimental Hematology 2012;20(5):1110-1116
This study was purposed to investigate the incidence of mixed lineage leukemia (MLL) gene rearrangement and partner gene types as well as the clinical features and prognosis of acute leukemia (AL) with this rearrangement through detection in adult AL using combination of 3 techniques, and to evaluate the clinical value of this combination detection. The MLL gene rearrangement in 183 cases of adult AL was detected by combination of conventional cytogenetics, split signal FISH and multiplex nested PCR. The results showed that the incidence of MLL rearrangements in adult patients with AL was low (8.2%), and MLL-AF4 fusion gene was most common and predominant in acute lymphoblastic leukemia (ALL), while the MLL-AF6 and MLL-AF9 were most frequent in acute myeloid leukemia (AML). Extramedullary involvements were found in 40% of MLL-rearranged AL patients, and 33.3% of patients with MLL-rearranged AL reached to complete remission within 30 days during induction chemotherapy. In addition, in this cohort of MLL-rearranged adult AL patients, the 3-month relapse rate and 6-month overall survival rate were 50.0% and 50.0% respectively. It is concluded that the rate of missed diagnosis of CC technique for patients with MLL-rearranged AL reached to 60% in this study, while the combination of CC, FISH and multiplex nested PCR has been confirmed to have important significance for evaluating prognosis and conducting clinical therapy of patients with MLL-rearranged AL.
Adolescent
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Adult
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Aged
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Female
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Gene Rearrangement
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Humans
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Leukemia, Myeloid, Acute
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genetics
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Male
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Middle Aged
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Myeloid-Lymphoid Leukemia Protein
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genetics
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Oncogene Proteins, Fusion
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genetics
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Young Adult
8.Construction and expression of multi-gene recombinant plasmid pEG-FP-N1-HBsAg-ROP2
Rong MA ; Ting XIAO ; Jin LI ; Hui SUN ; Chao XU ; Bing-Cheng HUANG ; Kun YIN ; Gui-Hua ZHAO ; Yong CUI ; Song ZHU ; Gong-Zhen LIU ; Ge YAN ; Qing-Kuan WEI
Chinese Journal of Schistosomiasis Control 2018;30(2):184-188
Objective To construct pEGFP-N1-HBsAg-ROP2 recombinant expression plasmid and transfect HEK293T cells for expression,and pay a way for Toxoplasma gondii nucleic acid vaccine development. Methods According to the HBsAg gene sequence and pcDNA3-p30-ROP2 recombinant plasmid restriction sites,the HBsAg gene was amplified by PCR.The HB-sAg gene was cloned into the pcDNA3-p30-ROP2 and instead of p30 gene.The HBsAg-ROP2 fragment was amplified by PCR and digested with HindⅢand KpnⅠto clone into the pEGFP-N1 eukaryotic expression vector and construct the recombinant pEGFP-N1-HBsAg-ROP2.The expression vector was transfected into HEK293T cells based on the identification of PCR amplifi-cation,restriction endonucleases and sequencing.Results The PCR product of HBsAg was about 700 bp,which was consis-tent with the theoretical value.Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pcDNA3-HBsAg-ROP2 recombinant plasmid.The recombinant plasmid pEGFP-N1-HBsAg-ROP2 was double-digested to generate an empty vector fragment of about 4.7 kb and a band of about 1.9 kb of HBsAg-ROP2 fragment.The results of sequencing showed that the sequence was 99.84% identical with the published sequence in GenBank.The target plasmid was successfully transfect-ed into HEK293T cells,and the expression was correct,the protein concentration was 3.08 mg/ml.Conclusion The recombi-nant plasmid pEGFP-N1-HBsAg-ROP2 is successfully constructed and expressed efficiently.
9.Impact of sinonasal anatomic changes after endoscopic anterior skull base surgery on nasal airflow and air conditioning: a computational fluid dynamics study.
Dong DONG ; Yu Lin ZHAO ; Chao WANG ; Jia Song TIAN ; Yu Dong ZHANG ; Rong Han WEI ; Xin Jie QIAO ; Ge GUO ; Tong Nan YIN ; Hao Jie HU
Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2023;58(5):445-451
Objective: To analyze the impact of the sinonasal anatomic changes after endonasal endoscopic anterior skull base surgery on the nasal airflow and heating and humidification by computational fluid dynamics (CFD), and to explore the correlation between the postoperative CFD parameters and the subjective symptoms of the patients. Methods: The clinical data in the Rhinology Department of the First Affiliated Hospital of Zhengzhou University from 2016 to 2021 were retrospectively analyzed. The patients received the endoscopic resection of the anterior skull base tumor were selected as the case group, and the adults whose CT scans had no sinonasal abnormalities were chosen as the control group. The CFD simulation was performed on the sinonasal models after reconstructed from the patients' sinus CT images during the post-surgical follow-up. All the patients were asked to complete the Empty Nose Syndrome 6-Item Questionnaire (ENS6Q) to assess the subjective symptoms. The comparison between two independent groups and the correlation analysis were carried out by using the Mann-Whitney U test and the Spearman correlation test in the SPSS 26.0 software. Results: Nineteen patients (including 8 males and 11 females, from 22 to 67 years old) in the case group and 2 patients (a male of 38 years old and a female of 45 years old) in the control group were enrolled in this study. After the anterior skull base surgery, the high-speed airflow moved to the upper part of the nasal cavity, and the lowest temperature shifted upwards on the choana. Comparing with the control group, the ratio of nasal mucosal surface area to nasal ventilation volume in the case group decreased [0.41 (0.40, 0.41) mm-1 vs 0.32 (0.30, 0.38) mm-1; Z=-2.04, P=0.041], the air flow in the upper and middle part of the nasal cavity increased [61.14 (59.78, 62.51)% vs 78.07 (76.22, 94.43)%; Z=-2.28, P=0.023], the nasal resistance decreased [0.024 (0.022, 0.026) Pa·s/ml vs 0.016 (0.009, 0.018) Pa·s/ml; Z=-2.29, P=0.022], the lowest temperature in the middle of the nasal cavity decreased [28.29 (27.23, 29.35)℃ vs 25.06 (24.07, 25.50)℃; Z=-2.28, P=0.023], the nasal heating efficiency decreased [98.74 (97.95, 99.52)% vs 82.16 (80.24, 86.91)%; Z=-2.28, P=0.023], the lowest relative humidity decreased [(79.62 (76.55, 82.69)% vs 73.28 (71.27, 75.05)%; Z=-2.28, P=0.023], and the nasal humidification efficiency decreased [99.50 (97.69, 101.30)% vs 86.09 (79.33, 87.16)%; Z=-2.28, P=0.023]. The ENS6Q total scores of all patients in the case group were less than 11 points. There was a moderate negative correlation between the proportion of the inferior airflow in the post-surgical nasal cavity negatively and the ENS6Q total scores (rs=-0.50, P=0.029). Conclusions: The sinonasal anatomic changes after the endoscopic anterior skull base surgery alter the nasal airflow patterns, reducing the efficiency of nasal heating and humidification. However, the post-surgical occurrence tendency of the empty nose syndrome is weak.
Adult
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Humans
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Male
;
Female
;
Young Adult
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Middle Aged
;
Aged
;
Retrospective Studies
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Hydrodynamics
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Air Conditioning
;
Nose
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Nasal Cavity
;
Skull Base/surgery*
10.Comprehensive profiling and characterization of the absorbed components and metabolites in mice serum and tissues following oral administration of Qing-Fei-Pai-Du decoction by UHPLC-Q-Exactive-Orbitrap HRMS.
Wei LIU ; Jian HUANG ; Feng ZHANG ; Cong-Cong ZHANG ; Rong-Sheng LI ; Yong-Li WANG ; Chao-Ran WANG ; Xin-Miao LIANG ; Wei-Dong ZHANG ; Ling YANG ; Ping LIU ; Guang-Bo GE
Chinese Journal of Natural Medicines (English Ed.) 2021;19(4):305-320
Qing-Fei-Pai-Du decoction (QFPDD) is a Chinese medicine compound formula recommended for combating corona virus disease 2019 (COVID-19) by National Health Commission of the People's Republic of China. The latest clinical study showed that early treatment with QFPDD was associated with favorable outcomes for patient recovery, viral shedding, hospital stay, and course of the disease. However, the effective constituents of QFPDD remain unclear. In this study, an UHPLC-Q-Orbitrap HRMS based method was developed to identify the chemical constituents in QFPDD and the absorbed prototypes as well as the metabolites in mice serum and tissues following oral administration of QFPDD. A total of 405 chemicals, including 40 kinds of alkaloids, 162 kinds of flavonoids, 44 kinds of organic acids, 71 kinds of triterpene saponins and 88 kinds of other compounds in the water extract of QFPDD were tentatively identified via comparison with the retention times and MS/MS spectra of the standards or refereed by literature. With the help of the standards and in vitro metabolites, 195 chemical components (including 104 prototypes and 91 metabolites) were identified in mice serum after oral administration of QFPDD. In addition, 165, 177, 112, 120, 44, 53 constituents were identified in the lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. These findings provided key information and guidance for further investigation on the pharmacologically active substances and clinical applications of QFPDD.
Administration, Oral
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Alkaloids/analysis*
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Animals
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COVID-19
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Chromatography, High Pressure Liquid
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Drugs, Chinese Herbal/pharmacokinetics*
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Flavonoids/analysis*
;
Mice
;
SARS-CoV-2
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Saponins/analysis*
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Triterpenes/analysis*