Metacercariae of Clinostomum complanatum (Trematoda: Digenea) in European newts Triturus carnifex and Lissotriton vulgaris (Caudata: Salamandridae).

Adults of Clinostomum spp. are digenetic trematodes found in fish-eating birds, reptiles and occasionally mammals, including humans. Freshwater snails serve as first intermediate hosts and many fish species and amphibians as second intermediate hosts. To date, amphibian hosts of Clinostomum metacercariae include members of urodele and anuran families in North America, but no data are available on infections of European amphibians, including newts. In this study, we characterize infections of Clinostomum complanatum metacercariae in four smooth (Lissotriton vulgaris) and 18 Italian crested newts (Triturus carnifex) from an artificial pond located in a protected area in Tuscany, Italy. Parasites were surgically removed from the infected newts and identified both morphologically and using sequences of a mitochondrial gene, cytochrome c oxidase I, and the ribosomal markers, internal transcribed spacers. This is the first record of C. complanatum in European newts and, more generally, in amphibians in Europe.

Circulating tumour cells: insights into tumour heterogeneity.

Tumour heterogeneity is a major barrier to cure breast cancer. It can exist between patients with different intrinsic subtypes of breast cancer or within an individual patient with breast cancer. In the latter case, heterogeneity has been observed between different metastatic sites, between metastatic sites and the original primary tumour, and even within a single tumour at either a metastatic or a primary site. Tumour heterogeneity is a function of two separate, although linked, processes. First, genetic instability is a hallmark of malignancy, and results in 'fixed' genetic changes that are almost certainly carried forward through progression of the cancer over time, with increasingly complex additional genetic changes in new metastases as they arise. The second type of heterogeneity is due to differential but 'plastic' expression of various genes important in the biology and response to various therapies. Together, these processes result in highly variable cancers with differential response, and resistance, to both targeted (e.g. endocrine or anti-human epithelial growth receptor type 2 (HER2) agents) and nontargeted therapies (e.g. chemotherapy). Ideally, tumour heterogeneity would be monitored over time, especially in relation to therapeutic strategies. However, biopsies of metastases require invasive and costly procedures, and biopsies of multiple metastases, or serially over time, are impractical. Circulating tumour cells (CTCs) represent a potential surrogate for tissue-based cancer and therefore might provide the opportunity to monitor serial changes in tumour biology. Recent advances have enabled accurate and reliable quantification and molecular characterization of CTCs with regard to a number of important biomarkers including oestrogen receptor alpha and HER2. Preliminary data have demonstrated that expression of these markers between CTCs in individual patients with metastatic breast cancer reflects the heterogeneity of the underlying tumours. Future studies are designed to determine the clinical utility of these novel technologies in either research or routine clinical settings.

MicroRNA-Mediated Direct Reprogramming of Human Adult Fibroblasts Toward Cardiac Phenotype.

Modulation of microRNA expression holds the promise to achieve direct reprogramming of fibroblasts into cardiomyocyte-like cells as a new strategy for myocardial regeneration after ischemic heart disease. Previous reports have shown that murine fibroblasts can be directly reprogrammed into induced cardiomyocytes (iCMs) by transient transfection with four microRNA mimics (miR-1, 133, 208, and 499, termed "miRcombo"). Hence, study on the effect of miRcombo transfection on adult human cardiac fibroblasts (AHCFs) deserves attention in the perspective of a future clinical translation of the approach. In this brief report, we studied for the first time whether miRcombo transient transfection of AHCFs by non-viral vectors might trigger direct reprogramming of AHCFs into cardiomyocyte-like cells. Initially, efficient miRNA delivery to cells was demonstrated through the use of a commercially available transfection agent (DharmaFECT1). Transient transfection of AHCFs with miRcombo was found to upregulate early cardiac transcription factors after 7 days post-transfection and cardiomyocyte specific marker cTnT after 15 days post-transfection, and to downregulate the expression of fibroblast markers at 15 days post-transfection. The percentage of cTnT-positive cells after 15 days from miRcombo transfection was ∼11%, as evaluated by flow cytometry. Furthermore, a relevant percentage of miRcombo-transfected AHCFs (∼38%) displayed spontaneous calcium transients at 30 days post-transfection. Results evidenced the role of miRcombo transfection on triggering the trans differentiation of AHCFs into iCMs. Although further investigations are needed to achieve iCM maturation, early findings from this study pave the way toward new advanced therapies for human cardiac regeneration.

Lactobacillus rhamnosus GG inhibits invasion of cultured human respiratory cells by prtF1-positive macrolide-resistant group A streptococci.

AIMS: This study was designed to determine whether the probiotic strain Lactobacillus GG, which is extensively used in the treatment and prevention of intestinal disorders, is able to inhibit invasion of cultured human respiratory cells by macrolide-resistant group A streptococci (GAS) carrying the prtF1 gene, which encodes the fibronectin (Fn)-binding invasin F1. METHODS AND RESULTS: Eight prtF1-positive erythromycin-resistant GAS strains were used to infect A549 monolayers in competition and displacement assays with Lactobacillus GG. Live (L-LGG) and heat-killed (HK-LGG) lactobacilli and their spent culture supernatant (SCS) significantly reduced (P < 0.001) GAS invasion efficiency in both assays. No antibacterial activity of Lactobacillus GG against GAS was detected. Both L-LGG and HK-LGG and all prtF1-positive GAS induced a strong agglutination reaction using Fn-coated particles. CONCLUSIONS: Lactobacillus GG exerts an antagonistic action against GAS by inhibiting cell invasion. Competitive binding of Lactobacillus GG and GAS to Fn might be involved in the inhibition process. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding that Lactobacillus GG can prevent in vitro invasion of respiratory cells by GAS suggests new applications for this probiotic strain and warrants further studies of its capacity to prevent GAS throat infections.

Temperature and pH sensors based on graphenic materials.

Point-of-care applications and patients' real-time monitoring outside a clinical setting would require disposable and durable sensors to provide better therapies and quality of life for patients. This paper describes the fabrication and performances of a temperature and a pH sensor on a biocompatible and wearable board for healthcare applications. The temperature sensor was based on a reduced graphene oxide (rGO) layer that changed its electrical resistivity with the temperature. When tested in a human serum sample between 25 and 43°C, the sensor had a sensitivity of 110±10Ω/°C and an error of 0.4±0.1°C compared with the reference value set in a thermostatic bath. The pH sensor, based on a graphene oxide (GO) sensitive layer, had a sensitivity of 40±4mV/pH in the pH range between 4 and 10. Five sensor prototypes were tested in a human serum sample over one week and the maximum deviation of the average response from reference values obtained by a glass electrode was 0.2pH units. For biological applications, the temperature and pH sensors were successfully tested for in vitro cytotoxicity with human fibroblast cells (MRC-5) over 24h.

Influence of inducers of monooxygenases on cytotoxic efficiency of ellipticine on leukemia L1210 cells.

The effects of some inducers of microsomal cytochrome P450-dependent monooxygenases on the metabolic bioactivation and the cytotoxicity of the antitumoral drug ellipticine (ELPT) were studied. Rate of growth of leukemia L1210 cells was measured in vitro in the absence and presence of ELPT or measured when the ELPT was metabolically transformed by noninbred Sprague-Dawley rat liver microsomes. The animals used were either untreated or pretreated by various inducers such as phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin, Aroclor 1254, or ELPT. The transformation of ELPT into its two main metabolites, 9-hydroxyellipticine (9-OHE) and 7-hydroxyellipticine, was studied and measured by high-pressure liquid chromatography in conjunction with the determination of cytotoxic activity. A large variability was observed in the bioactivation and cytotoxic efficiency of ELPT mediated by the different microsomal preparations: The more P448 and/or P1-450 forms of cytochrome were induced, the more the 9-OHE was produced and the more the cytotoxicity toward L1210 cells was enhanced. These features were compared with those elicited by the activation of cyclophosphamide, which was transformed into cytotoxic metabolites by the cytochrome P450 form specifically induced by phenobarbital-type inducers.

Uptake, cytofluorescence, and cytotoxicity of oxazolopyridocarbazoles (amino acid-ellipticine conjugates) in murine sarcoma cells.

The uptake, cytofluorescence, and cytotoxicity of elliptinium (NMHE) and a series of fluorescent oxazolopyridocarbazoles [amino acid-ellipticine conjugates (AA-NMHE)] were studied in murine sarcoma cells. For all these drugs, the uptake was rapid, directly proportional to the drug concentration, and unaffected by metabolic inhibitors which is consistent with a diffusion mechanism. By 4 h, the intracellular concentration of NMHE exceeded the external drug concentration by about 100 times; this suggests that the toxicity of NMHE is not, as previously assumed, limited by its transport across tumor cell membranes. Conjugation of NMHE with aliphatic amino acids increased the cellular uptake 5- to 7-fold. Cellular exposure to AA-NMHE conjugates resulted in the appearance of granular cytoplasmic fluorescence which was readily translocated to the nucleus upon continued exposure to fluorescent light. The cytotoxicity of the AA-NMHE conjugates (drug concentration required to reduce colony formation by 63% on the exponential part of the survival curve = 3-14 microM) was less than of NMHE (drug concentration required to reduce colony formation by 63% on the exponential part of the survival curve = 0.7 microM) as shown by colony formation following 4 h drug exposure. In contrast, the isoleucine-NMHE conjugate was the most cytotoxic compound (drug concentration required to reduce colony formation by 63% on the exponential part of the survival curve = 0.045 microM) when the drug exposure period was extended to 8 days. The general lower toxicity of the AA-NMHE conjugates is likely due to loss of the phenolic character of the NMHE moiety; therefore, attempts to link NMHE to amino acids remain attractive but will have to be done without affecting the 9-hydroxy group of NMHE.

Mutagenicity and cytotoxicity of five antitumor ellipticines in mammalian cells and their structure-activity relationships in Salmonella.

The mutagenicity and cytotoxicity of five antitumor compounds (ellipticines) were investigated in the Chinese hamster ovary cell hypoxanthine-guanine phosphoribosyltransferase assay and in six strains of Salmonella. All five compounds (ellipticine, 9-methoxyellipticine, 9-hydroxyellipticine, 9-aminoellipticine, and 2-methyl-9-hydroxyellipticinium) were cytotoxic and mutagenic in the Chinese hamster ovary cell hypoxanthine-guanine phosphoribosyltransferase assay in the presence or absence of Aroclor 1254-induced rat liver S9, and all except the last compound were mutagenic in Salmonella. Based on the reversion pattern obtained in various frame-shift and DNA repair-proficient (uvrB+) or -deficient (uvrB) strains of Salmonella in the presence or absence of S9, the first three compounds appear to cause frame-shift mutations by both intercalation and covalent bonding with DNA; thus, these are classified as reactive intercalators. However, 9-aminoellipticine intercalates only weakly and may instead exert its mutagenic activity primarily (or exclusively) by forming a covalent adduct with DNA. Compared to the published antitumor data obtained in mice, the results in Salmonella and Chinese hamster ovary cells suggest that the ability of ellipticine, 9-methoxyellipticine, and 9-hydroxyellipticine to intercalate with DNA, induce frame-shift mutations, and cause cell killing is associated with and may be the basis for their antitumor activity. The observation that the ellipticines are mutagenic in mammalian cells suggests that these antitumor agents may be carcinogenic.

Antitumor activity of 9-hydroxyellipticine (NSC 210717) ON L1210 mouse leukemia and the effect of route of injection.

The antitumor activity of a new derivative of ellipticine, 9-hydroxyellipticine (NSC 210717), was studied using L1210 mouse leukemia. Low doses of this drug have a high antileukemic activity, whereas high doses have less activity than expected because of a leveling off in the antitumor activity-dose relationship, as if a few cells were resistant to the treatment. The possible causes of this apparent resistance were investigated. It is suggested that this apparent resistance is related to the sequestration of a small number of cells in compartments inaccessible to the drug. A model was developed which takes into account the distribution of cells in various compartments and their drug sensitivity therein. It was predicted and observed that the activity of drugs acting on cells in the small compartments can be observed only in conjunction with the presence of drugs acting on the cells in the major compartment. The importance of this observation in the screening procedures of new drugs, the clinical trial of new chemotherapeutic agents, and the association of anticancer drugs are discussed within this context. 9-Hydroxyellipticine is of interest because it acts on leukemic cells in the brain.

Physicochemical and pharmacological properties of the antitumor ellipticine derivative 2-(diethylamino-2-ethyl)9-hydroxy ellipticinium-chloride, HCl.

2-(Diethylamino-2-ethyl)9-hydroxyellipticinium-chloride, HCl (DHE), a new congener of the antitumor agent elliptinium acetate (Celiptium) (NMHE), has recently been selected for phase I clinical trials. NMHE has a methyl group at nitrogen 2 on the ellipticine ring while DHE possesses a basic diethylaminoethyl chain at this position. Compared to NMHE, the presence of the diethylaminoethyl side chain results in the following: a significant increase in the lipophilicity of the drug; no significant modification in either the binding constant values to DNA or the ability to intercalate between DNA base pairs; a marked decrease in the unwinding angle value of supercoiled DNA; and no significant change in the alteration of the catalytic activity of topoisomerase II in vitro. DHE appears to act as a simple reversible intercalating agent as shown by the selective mutagenic effect on Salmonella TA 1977 tester strain and by its inability to induce the SOS functions in a sfiA lac fusion containing Escherichia coli strain. From a pharmacological point of view, the presence of the diethylaminoethyl chain results in a 2-fold increase in the cytotoxicity to L1210 cultured cells, a strong increase in the antitumor efficiency on experimental murine tumors such as L1210 and P388 leukemia, B16 melanoma, M 5076 reticulosarcoma, and colon 38 adenocarcinoma, and finally an objective decrease in the acute and subacute toxicity in mice, rat, and macaque. The absence of significant differences in the interaction of NMHE and DHE with their potential targets in vitro leads to the hypothesis that the superiority of DHE in terms of cytotoxicity and antitumor efficiency may be due to an increase in the diffusion across cellular membrane and a more favorable biodistribution in vivo.

A graphene oxide pH sensor for wound monitoring.

This article describes the fabrication and characterization of a pH sensor for monitoring the wound status. The pH sensitive layer consists of a graphene oxide (GO) layer obtained by drop-casting 5 µÎ of GO dispersion onto the working electrode of a screen-printed substrate. Sensitivity was 31.8 mV/pH with an accuracy of 0.3 unit of pH. Open-circuit potentiometry was carried out to measure pH in an exudate sample. The GO pH sensor proved to be reliable as the comparison with results obtained from a standard glass electrode pH-meter showed negligible differences (<; 0.09 pH units in the worst case) for measurements performed over a period of 4 days.

Risk of melanoma following adulthood cancer: a case-control study.

Melanoma is a severe skin cancer related to sun exposure. Whether this malignancy is linked to exposure to ionising radiation during adulthood is still controversial. This case-control study examined the risk of melanoma following treatment for an adulthood first malignant neoplasm (FMN). Cases were patients who presented with cutaneous melanoma after a first cancer in adulthood. Controls (3 per case) were patients free of melanoma, matched for age, duration of follow-up since the FMN, type of FMN, and followed in the same institution. A total of 57 cases and 171 controls were included. In the final multivariate analysis, no risk of melanoma was associated with radiotherapy (odds ratio (OR) for 1 Gy = 1.01, 95% confidence interval (95%CI) 0.96-1.07) nor hormonotherapy, whereas chemotherapy use (OR = 2.3, 95%CI 0.93-5.6) and having a history of familial cancer (OR = 2.8, 95%CI 1.3-5.9) exhibited a nearly significant risk. In conclusion, unlike the evidence for risk of exposure to ionising radiation during childhood, we did not substantiate a risk for association of melanoma with exposure to ionising radiation during adulthood. The risk associated with chemotherapy should justify the implementation of skin surveillance for early detection of melanoma in these patients.

The localization of topoisomerase II cleavage sites on DNA in the presence of antitumor drugs.

Type II topoisomerase are enzymes that break and religate DNA phosphodiester bonds while crossing over DNA strands and altering DNA topology. They also are structural proteins that play a role in the spatial organization of chromatin and are involved in several crucial biological functions, such as DNA replication and transcription, chromosome segregation and recombination. Many drugs interfere with type II topoisomerases and can be assigned to two groups. Coumarin derivatives and synthetic quinolones act at the level of ATP binding or hydrolysis and are used for controlling bacterial infections. Drugs belonging to the second group produce DNA lesions by trapping a "cleavable complex" consisting of the normal transient topoisomerase II-DNA reaction intermediate in which the enzyme and the DNA are joined by two covalent bonds. There are four main categories of antitumour drugs that form cleavable complexes in eukaryotes: acridines, anthracyclines, ellipticines and epipodophyllotoxins. These drugs are cytotoxic and many--but not all--are endowed with antitumoral properties. The mechanisms of this pharmacological activity are not understood. Topoisomerase II-induced DNA breaks generated from cleavable complexes display different levels of cytotoxicity depending on their localization on DNA. The primary structure of DNA is not the only parameter that determines this localization. The spatial organization of the enzyme-DNA complex and both the topology and the structure of the underlying chromatin fiber constitute additional critical factors. It, therefore, may be unrealistic to expect that the actual pharmacological potency of antitumor drugs that act on type II topoisomerases can be accurately predicted solely on the basis of simple in vitro test tube experiments carried out using pure enzymes and naked DNA.

Alpha-anomeric DNA: beta-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase.

Nuclease-resistant alpha-anomeric DNA:beta-RNA hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H. RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A)12:d[G-G-(T)12-G-G], or by acid-solubility techniques, using a long substrate, poly(A):poly(dT). Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange. Our results suggest that RNase H, for which DNA:RNA duplexes are the natural substrates, binds to non-physiological alpha-DNA:RNA hybrids and is consequently inhibited. These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities. However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded alpha-DNA itself. Unexpectedly, polymerase activity is highly stimulated by alpha-oligos, analogous in their sequence to the beta primer used at a concentration unable to generate a detectable synthesis. These results suggest that the inhibition of reverse transcriptase activity with the alpha:beta may occur at different levels.

Histochemical evidence of altered development of cholinergic fibers in the rat dentate gyrus following lesions. II. Effects of partial entorhinal and simultaneous multiple lesions.

It has been concluded previously that the septohippocampal fibers which project to the rat dentate gyrus extend or branch in the denervated area of the molecular layer following a complete ipsilateral entorhinal lesion. The septohippocampal fibers thus appear to replace some of the perforant fibers which degenerate as a result of the lesion. The reactive fibers eventually become localized to a much smaller and more superficial area after lesions of immature rats than after lesions made in adulthood. To determine whether this difference in the response results from a selective reaction to loss of the lateral perforant path in the immature rat, various portions of the entorhinal cortex were removed at the age of 11 days, and the cholinergic septohippocampal fibers were visualized by acetylcholinesterase histochemistry. An alternative possibility, that the difference between immature and adult rats is attributable to an interaction with other reactive afferents, was tested by removing other sources of input (the contralateral entorhinal cortex, contralateral hippocampal formation or both) along with the ipsilateral entorhinal cortex at the age of 11 days and then demonstrating the septohippocampal fibers histochemically. Lesions of the lateral part of the ipsilateral entorhinal cortex (source of the lateral perforant path) at 11 days of age evoked a septohippocampal reaction along the outer edge of the molecular layer, where the lateral perforant path fibers normally terminate. This result matched that produced by a complete entorhinal lesion. Lesions of the medial entorhinal cortex evoked no obvious reaction. In contrast, the septohippocampal fibers in adult rats proliferated in the denervated area of the molecular layer after lesions of either part of the entorhinal cortex. Combining lesions of other sources of innervation to the dentate gyrus with an ipsilateral entorhinal lesion at 11 days of age did not alter the response of septohippocampal fibers, as determined histochemically. Neither did the septohippocampal fibers react to removal of commissural afferents alone. The response at any age was unaffected by prior or subsequent removal of the contralateral entorhinal cortex. These results indicate that in immature rats the septohippocampal fibers respond only to loss of the lateral perforant path, but these same fibers can later react to loss of any part of the perforant path. They are regarded as support for the hypothesis that the reactive septohippocampal fibers preferentially interact with dendritic growth cones. Our results do not support explanations based on a hypothetical attraction between septohippocampal and crossed perforant path fibers (which react in the same area) or on competition with commissural fibers (which reinnervate an adjacent area). We suggest further that proximity to the degenerating elements does not in itself determine the pattern of reinnervation after lesions of the central nervous system.

Effects of CYP2D6 activity and tobacco on larynx cancer risk.

The genetically determined capacity of the cytochrome P450 CYP2D6 is suspected to be involved in the activation of tobacco carcinogens. From a multicentric case-control study carried out to analyze the interaction between host and environmental factors on tobacco-related cancers, we reported recently that the effect of tobacco on lung cancer risk rose with increasing CYP2D6 activity, and the effect of CYP2D6 activity rose with increasing tobacco consumption. The aim of the present report was to investigate whether results on lung cancer could be observed for larynx cancer, from a study on 140 cases and 157 controls. A weak interaction between increasing levels of both CYP2D6 activity and average daily consumption of tobacco was found (P = 0.12). The only significant interaction between these two factors was observed when CYP2D6 activity was considered with the two conventional phenotypes (P < 0.05). A dose-response effect of tobacco on larynx cancer risk was found only among one-third of the smokers with the highest level of CYP2D6 activity, and CYP2D6 was a risk factor only among heavy smokers. The highest risk for larynx cancer was then observed among smokers having both the highest levels of CYP2D6 activity and daily consumption of tobacco. The interaction between CYP2D6 activity and tobacco was weaker for larynx cancer than that reported previously for lung cancer. However, the similarities in the results found for these two cancers, i.e., a greater effect of tobacco among smokers with the highest CYP2D6 activity, reinforce the hypothesis that this activity could modify the effect of tobacco on cancer risk.

Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses.

In cell culture the cytotoxicity of some anticancer drugs, especially bleomycin, can be greatly enhanced by exposing cells to non-cytotoxic electric pulses. Nude or conventional mice bearing subcutaneous transplanted tumours were treated with intramuscular doses of bleomycin followed by local delivery of electric pulses similar to those used in vitro. Tumors were reduced and even eradicated after this electrochemotherapy. Thus the antitumour effects of bleomycin in mice can be considerably potentiated by local electric pulses.

Electrochemotherapy of spontaneous mammary tumours in mice.

Electrochemotherapy delivers external electric pulses to the tumour site to induce local potentiation of the antitumour activity of intramuscular injections of bleomycin. C3H/Bi mice with spontaneous mammary carcinomas received weekly injections of 50 micrograms bleomycin followed by electric pulses 30 min later. All the 38 tumours treated exhibited at least a partial regression. 23 complete remissions were observed, 3 of which were cures. One difficulty in assessing the cure rate in this model is that frequent parallel or sequential tumours cause early death. Electrochemotherapy appears similarly efficient in spontaneous tumours as in previously studied transplanted tumours.

Familial relative risk of colorectal cancer: a population-based study.

This study aimed to assess the familial relative risk for colorectal cancer (CRC) and its variation according to age and gender. A population-based family study was carried out in France, from 1993 to 1998, including 761 families. Familial CRC risks were estimated from a cohort analysis of the relatives. No obvious decrease in CRC risk was found with increasing age, except when either the proband, or the relative, were in the youngest age class. The effect of the relatives' and probands' ages on the CRC risk differed according to their gender. The cumulative risk of CRC increased at an earlier age in male relatives of probands younger than 60 years of age, than in female relatives. This result suggests that mechanisms specific to females, possibly interacting with genetic factors, explain the difference in the cumulative risks between families with male and female probands.

Does cruciform DNA provide a recognition signal for DNA-topoisomerase II?

Topoisomerase II displays higher affinity for supercoiled DNA compared to the same relaxed DNA. Moreover, cruciform structures are formed in topologically constrained DNA. Here we report that, using S1 nuclease experiments on supercoiled DNA, hairpin structures are located close to numerous topoisomerase II cleavage sites on the BPV I genome. Therefore, DNA secondary structure may play a role in the recognition mechanism of DNA by topoisomerase II.