To establish a simple, sensitive and effective technique for the identification of six common dermatophytes, polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene were used. The DNA of 6 common dermatophytes was amplified by primer dPsD1 and then primers dPsD2. The products generated by dPsD2 were digested with restriction enzyme Hinc II and Hinf I separately. A DNA fragment of about 3390 bp was amplified by using primer dPsD1 from the genomic DNA of each dermatophyte species. The product of dPsD2 was 2380 bp and the restriction profiles of Hinc II and Hinf I were between 58-1670 bp. By using PCR-RFLP, all of the 6 dermatophytoses were diagnosed to species level and no obvious difference identification between Hinc II and Hinf I. It is concluded that the PCR-RFLP identification of dermatophytes by Hinc II or Hinf I is efficient and rapid in clinical practice.
Arthrodermataceae/*classification ; Arthrodermataceae/genetics ; Arthrodermataceae/*isolation & purification ; DNA Topoisomerases, Type II/genetics ; DNA, Fungal/analysis ; DNA, Fungal/genetics ; Dermatomycoses/*microbiology ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

Antigens, Neoplasm ; genetics ; metabolism ; Breast Neoplasms ; enzymology ; genetics ; metabolism ; DNA Topoisomerases, Type II ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; physiology ; Humans

Among 155 clinical respiratory isolates of Haemophilus influenzae in Korea, 6 (3.9%) isolates had reduced levofloxacin susceptibility (MICs > or = 0.5 microg/mL). These six isolates had no significant quinolone resistance-determining region (QRDR) mutations in gyrA, gyrB, parC, or parE. This phenomenon suggests that neither evolution nor spread of any significant QRDRs mutations in clinical isolates occurred in Korea. Therefore, continued surveillance is necessary to observe the evolution of antibiotic-resistance and take measures to avoid the spread of drug-resistant clones.
Anti-Bacterial Agents/*pharmacology ; DNA Gyrase/*genetics ; DNA Topoisomerases, Type II/*genetics ; Haemophilus influenzae/*drug effects/pathogenicity ; Korea ; Microbial Sensitivity Tests ; Mutation ; Ofloxacin/*pharmacology

Among 155 clinical respiratory isolates of Haemophilus influenzae in Korea, 6 (3.9%) isolates had reduced levofloxacin susceptibility (MICs > or = 0.5 microg/mL). These six isolates had no significant quinolone resistance-determining region (QRDR) mutations in gyrA, gyrB, parC, or parE. This phenomenon suggests that neither evolution nor spread of any significant QRDRs mutations in clinical isolates occurred in Korea. Therefore, continued surveillance is necessary to observe the evolution of antibiotic-resistance and take measures to avoid the spread of drug-resistant clones.
Anti-Bacterial Agents/*pharmacology ; DNA Gyrase/*genetics ; DNA Topoisomerases, Type II/*genetics ; Haemophilus influenzae/*drug effects/pathogenicity ; Korea ; Microbial Sensitivity Tests ; Mutation ; Ofloxacin/*pharmacology

OBJECTIVETo study the mechanisms of dystrophin gene deletion, the junction fragment with exons 45-54 deletion were cloned and sequenced.

METHODSA Duchenne muscular dystrophy (DMD) patient with exons 45-54 deletion has been substantiated by PCR amplification of the exons. Then we used a PCR-based genome-walking method for localizing the breakpoints in introns 44 and 54. At last, the deletion-junction fragment was directly amplified by PCR approach with forward and reverse primers annealing to a DNA sequence as close as possible to the breakpoints in introns 45 and 54. The sequencing result of the deletion-junction fragment was compared with the normal intronic sequences.

RESULTSA total of 2716 bp sequence containing the junction fragment was obtained. The 5' breakpoint was located in LINE/L1 element of intron 44 and close to a matrix attachment region (MAR). The 3' breakpoint was located in the minor potential MAR with topoisomerase II cleavage sites around. Beside the 3' breakpoint there was a 6 bp palindromic sequence. A 4 bp microhomologous sequence (AGAG) was in the joint of the deletion-junction fragment.

CONCLUSIONThe nonhomologous recombination caused by L1 repeated element, topoisomerase II cleavage sites, MARs and the nonhomologous end joining of microhomologous sequence may be the important factors in this huge gene fragment deletion.

Base Sequence ; Child ; Cloning, Molecular ; DNA Topoisomerases, Type II ; metabolism ; Dystrophin ; genetics ; Exons ; genetics ; Humans ; Introns ; genetics ; Male ; Muscular Dystrophy, Duchenne ; genetics ; Mutation ; Polymerase Chain Reaction ; Sequence Deletion

Objective To investigate the expression of topoisomeraseⅡα (TOP2α) in hepatocellular carcinoma (HCC) and its role in predicting prognosis of HCC patients. Methods We used HCC-related datasets in UALCAN, HCCDB, and cBioPortal databases to analyze the expression and mutation of TOP2α and its co-expressed genes in HCC tissues. GO function and KEGG pathway enrichment of TOP2α and its co-expressed genes were identified. The TIMER database was used to analyze infiltration levels of immune cells in HCC. The impacts of TOP2α and its co-expression genes and the infiltrated immune cells on the survival of HCC patients were assayed by Kaplan-Meier plotter analysis. Results TOP2α and its co-expression genes were highly expressed in HCC (P< 0.001) and detrimental to overall survival of HCC patients (P< 0.001). TOP2α and its co-expression genes were mainly involved in cell mitosis and proliferation, and cell cycle pathway (ID: hsa04110, P = 0.001945). TOP2α and its co-expression genes were mutated in HCC and the mutations were significantly detrimental to overall survival (P = 0.0247) and disease-free survival (P = 0.0265) of HCC patients. High TOP2α expression was positively correlated with the infiltration of B cell (r = 0.459, P< 0.01), CD8⁺ T cell (r = 0.312, P< 0.01), CD4⁺ T cell (r = 0.370, P< 0.01), macrophage (r = 0.459, P< 0.01), neutrophil (r = 0.405, P< 0.01), and dendritic cell (r = 0.473, P< 0.01) in HCC. The CD8⁺ T cell infiltration significantly prolonged the 3- and 5-year survival of HCC patients (all P< 0.05), and CD4⁺ T cell infiltration significantly shortened the 3-, 5-, and 10-year survival of HCC patients (all P< 0.05). ConclusionTOP2α may be an oncogene, which was associated with poor prognosis of HCC patients and could be used as a biomarker for the prognostic prediction of HCC.
Humans ; Biomarkers, Tumor/genetics* ; Carcinoma, Hepatocellular/genetics* ; CD8-Positive T-Lymphocytes ; Computational Biology ; Liver Neoplasms/genetics* ; Prognosis ; DNA Topoisomerases, Type II/genetics*

OBJECTIVE: To investigate the expression and significance of muscle segment homeobox2 (Msx2) and topo II-alpha in sinonasal inverted papilloma (SNIP), and the relationship in the process of malignant transformation of SNIP. METHOD: Immunohistochemical method was used to detect the expression of Msx2 and topo II-alpha in 32 cases of SNIP, 30 cases of inflammatory nasal polyp (INP) and 30 cases of SNIP with carcinoma. According to the pathology results, SNIP were divided into mild atypical hyperplasia, moderate atypical hyperplasia and severe atypical hyperplasia. RESULT: The mean optical density of Msx2 in SNIP and SNIP with carcinoma tissues were 0.2183 +/- 0.0598 and 0.2521 +/- 0.0761,which were significantly higher than 0.1878 +/- 0. 0372 in the INP tissue (P<0.05 or 0.01). The mean optical density of topo II-alpha in SNIP and SNIP with carcinoma tissues were 0.2303 +/- 0.0397 and 0.2666 +/- 0.0483, which were significantly higher than 0.1978 +/- 0.0388 in the NIP tissue (P<0.01). There were significant difference of Msx2 and topo II-alpha in SNIP between any two of the three groups divided according to pathological morphology (P<0.01 or 0.05). The expression of Msx2 and topo II-alpha in SNIP were positively correlated (P<0.05). CONCLUSION Msx2 and topo II-alpha may play an important role in the occurrence and development of SNIP. So it can be used as new therapeutic targets.
Antigens, Neoplasm ; genetics ; metabolism ; DNA Topoisomerases, Type II ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Male ; Middle Aged ; Nose Neoplasms ; genetics ; metabolism ; pathology ; Papilloma, Inverted ; genetics ; metabolism ; pathology

Alu Elements ; physiology ; Chromosome Aberrations ; DNA Topoisomerases, Type II ; metabolism ; DNA, Neoplasm ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Humans ; Sarcoma ; genetics ; metabolism ; Soft Tissue Neoplasms ; genetics ; metabolism ; Translocation, Genetic

To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene. The DNA of 7 dermatophytes, along with Candida albicans, Aspergillus terreus and Aspergillus flavus were amplified by consensus primer dPsD1. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc II. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect for T. mentagrophytes and T. tonsurans. From the restriction profiles of Hinc II, 6 of the 7 dermatophytoses were diagnosed to species level including T. mentagrophytes and T. tonsurans. By combining the results of the PCR and PCR-RFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase II gene is rapid and efficient.
Arthrodermataceae ; classification ; isolation & purification ; Aspergillus ; isolation & purification ; Candida albicans ; isolation & purification ; DNA Topoisomerases, Type II ; genetics ; Dermatomycoses ; microbiology ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Trichophyton ; isolation & purification

To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene. The DNA of 7 dermatophytes, along with Candida albicans, Aspergillus terreus and Aspergillus flavus were amplified by consensus primer dPsD1. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc II. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect for T. mentagrophytes and T. tonsurans. From the restriction profiles of Hinc II, 6 of the 7 dermatophytoses were diagnosed to species level including T. mentagrophytes and T. tonsurans. By combining the results of the PCR and PCR-RFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase II gene is rapid and efficient.
Arthrodermataceae/classification ; Arthrodermataceae/*isolation & purification ; Aspergillus/*isolation & purification ; Candida albicans/isolation & purification ; DNA Topoisomerases, Type II/genetics ; Dermatomycoses/microbiology ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Trichophyton/*isolation & purification