Concentrations Of Ethidium Bromide Research Articles

Chronic Mitochondrial Dysfunction Renders Acute Myeloid Leukemia Resistant to Multipronged Inhibition of Mitochondrial Metabolism

Acute Myeloid Leukemia (AML) is a genetically diverse and aggressive myeloid neoplasm that awaits novel therapeutics to improve patient survival. Targeting AML metabolic aberrations as a genotype-agnostic strategy has shown promise, evidenced by the clinical success of venetoclax and the rapid pre-clinical development of other mitochondrial (mt) metabolism targeted therapies. However, the mechanism of how AML develops resistance to drugs targeting key mt metabolic pathways is not fully understood. We previously identified sirtuin 5 (SIRT5), a deacylase that regulates several pathways of mt metabolism in AML including oxidative phosphorylation and glutamine metabolism. In AML, it functions as a master metabolic regulator that is necessary for leukemic stem cell survival. Genetic or pharmacological inhibition of SIRT5 leads to rapid AML cell apoptosis in vitro and disease regression in multiple AML mouse models. However, a subset of AML cell lines and primary samples can survive SIRT5 inhibition, indicating an unusual intrinsic resistance to mt dysfunction. To provide a mechanistic explanation, we first compared the molecular features between SIRT5-dependent and independent cases. Among the 25 primary AML samples we tested, NPM1c mutation, but not FLT3-ITD, TP53, WT1, NRAS, or IDH1 mutations, is significantly enriched in SIRT5-independent samples. Accordingly, among the panel of 22 AML cell lines we tested, the only NPM1 mutant cell line (OCI-AML3) sits at the most SIRT5-independent extreme of the spectrum. NPM1 mutant AML cells have increased cytoplasmic mitochondrial DNA (mtDNA) and fragmented mt morphology, indicating chronic mt damage (Wu H et al. Cancer Discovery. 2021). Because SIRT5 exerts its metabolic maintenance functions within the mt, we hypothesize that AML cells that can adapt to chronic, sublethal mt dysfunction will be capable of proliferation without SIRT5. To test this, we took three orthogonal approaches: (1) introducing NPM1c mutation, (2) using low concentrations of ethidium bromide (EtBr) to selectively block mtDNA replication, or (3) genetically inducing chronic mt dysfunction by silencing the essential mt transcription factor TFAM in 3 AML cell lines that are heavily dependent on SIRT5 - OCI-AML2, CMK, and SKM-1. SIRT5-dependent cells expressing ectopic NPM1c exhibit mt dysfunction as evidenced by decreased oxygen consumption rate (OCR) measured using the Seahorse metabolic flux assay. Also, the lethal effect of SIRT5 depletion is rescued in these cells. Pharmacologically blocking NPM1c nuclear export with selinexor in the typically SIRT5-independent OCI-AML3 cells sensitizes them to the lethal effect of SIRT5 depletion. After culturing in 100 ng/mL EtBr for 3, 5, or 11 days, cells had significantly decreased mt abundance as measured by mtDNA content relative to nuclear DNA and had significantly decreased OCR and extracellular acidification rate (ECAR). SIRT5-dependent cells cultured in EtBr survived shRNA-induced SIRT5 depletion, in contrast to the rapid lethality of cells not cultured in EtBr. Additionally, cells initially cultured in EtBr and allowed to recover in the absence of EtBr assume their original phenotype of SIRT5 dependence. However, inhibiting specific subunits of the mt using potent complex I and III inhibitors (rotenone and antimycin A) shows no effect on SIRT5 dependence. Together these data suggest that mt damage, rather than loss of function in one pathway, is needed to convert SIRT5-dependent cells to SIRT5 independence. TFAM, a nuclearly coded transcription factor for mtDNA, is necessary for the replication and transcription of mt genes. We used cas13d-mediated knockdown of TFAM to deplete mtDNA and induce mt dysfunction. Experiments that investigate how this method of inducing mt dysfunction affects OCR, ECAR, and SIRT5-dependent cell line proliferation are currently underway and will be presented at the ASH conference. In summary, we present a mechanism of how NPM1 mutation-associated or experimentally induced chronic sublethal mt dysfunction renders AML resistant to therapies targeting vital mt metabolic pathways, such as SIRT5 inhibitors, demonstrating AML's metabolic flexibility. Experiments are scheduled to validate these findings in primary samples and additional metabolomic and transcriptional studies will be done to deduce the mechanism for SIRT5 dependence in AML.

Protective Effect of Biflavones from Platonia insignis Mart. against Ethidium Bromide-Induced Lethality in Staphylococcus aureus

Brazilian flora are highly diverse and have great potential for research into new medicines of plant origin. Platonia insignis Mart. is a fruit tree from the Amazon region with several potential pharmacological applications already demonstrated. The present study aimed to investigate the protective effect of a mixture of the biflavones volkensiflavone and moreloflavone obtained from P. insignis flowers against the lethality induced by ethidium bromide in efflux pump overproducer strains of Staphylococcus aureus. Microdilution tests were performed to determine the minimum inhibitory concentration of ethidium bromide in the absence or presence of biflavones. The addition of biflavones to the growth medium increased the minimum inhibitory concentration of ethidium bromide against all strains tested, indicating that biflavones protected the bacterial cells against cell damage induced by this biocide agent. Comparative analysis of the infrared absorption spectra of each isolated compound, as well as of the associated compounds, demonstrated the complexation reaction, indicating that there was a preferential interaction between the H2N- groups of primary amines of ethidium bromide with the carbonyls of biflavones that formed imines -C=NH. Such complexation could reduce the amount of free ethidium bromide molecules available for DNA intercalation, resulting in the observed protective effect. These results suggest a possible technological application of the biflavones from P. insignis as protective agents against cell damage induced by ethidium bromide.

Simple, rapid, and reproducible capillary gel electrophoresis separation and laser-induced fluorescence detection of DNA topoisomers with unmodified fused silica separation capillaries.

The topology of DNA is a critical quality attribute for plasmid-based pharmaceuticals, making quantification of trace levels of plasmid topoisomers an important analytical priority. An automated and cost-effective method based on capillary gel electrophoresis laser-induced fluorescence detection is described. The method outlined in this report is significant because it is easily implemented by any laboratory for which routine analyses of plasmid topology are critical for the development of new plasmid-based therapies as well as for quality control of gene therapies utilizing supercoiled DNA. Detection of topoisomers was achieved by incorporating ethidium bromide in the separation medium. The detector response was improved by 3 orders of magnitude by utilizing a 605-nm optical filter with a 15-nm bandwidth. Separations of linear, open circle, supercoiled, and multimer DNAplasmids ranging from 4.2 to 10.5kbp were accomplished in under 6min using an unmodified fused silica capillary (50-μm internal diameter). The background electrolyte was comprised of 0.5% gel, which was hydroxypropylmethyl cellulose, 1mM ethylenediaminetetraacetic acid, and 50mMN-(2-acetamido)-2-aminoethanesulfonic acid (pH of 6.25). The separations, which balanced the bulk electroosmotic flow, the electrophoretic mobility of the DNA, and gel sieving were dependent upon the pH of the electrolyte and the gel concentration. Reproducibility was dependent upon the procedure used to prepare the gel as well as other factors including the ethidium bromide concentration and capillary conditioning. A single unmodified capillary operated for more than 150 runs had an across-day migration time precision of 1% relative standard deviation and percent area precision of 10% relative standard deviation.

Expression Levels of Efflux pump mexR and norA Genes in Multi-Drug Resistant in Some Bacteria by Using Quantitative RT-PCR Under Stress of Effect Efflux Pump Inhibitors.

The study was suggested because of the major role of multidrug-resistant Pseudomonas aeruginosa andStaphylococcus aureus in a wide range of clinical infections besides increasing a high resistance against thecommonly used difference antibiotic. The efflux pumps have a vital role in multidrug resistance for extrudingmany toxic materials and antibiotics. This study aimed to measure the expression level of efflux pump genesin MDR Pseudomonas aeruginosa and Staphylococcus aureus using RT-qPCR technique. In this study, onehundredclinical isolates of MDRP. aeruginosa and S. aureus isolated from wounds were examined, then20 isolates selected based on their ability as MDR and wereexposed to different concentrations of EthidiumBromide (Cartwheel method) to determine the presence of efflux pumps activity. Efflux pump genesmexR,and norA were screened by PCR. The results demonstrated the presence of mexR and norA genesin all MDRisolates. RT-qPCR assay was used for investigating the efflux pump genes expression. The difference in geneexpression between active and nonactive efflux pumps was determined when exposed to the antibiotics andefflux pump inhibitors.

Fluorescent reversible regulation of cysteamine-capped ZnSe quantum dots successively induced by photoinduced electron transfer of herring sperm DNA and intercalation binding of ethidium bromide

A fluorescent reversible regulation was studied by fluorescence spectra, ultraviolet–visible spectra in the combination of molecular docking, which based on the photoinduced electron transfer(PET) from hsDNA (herring sperm DNA) to CA (cysteamine)-capped ZnSe QDs (quantum dots) and intercalation of ethidium bromide (EB) into hsDNA. It was proven that the QDs bound with the adding hsDNA by electrostatic force and formed 1:1 hsDNA-QDs complexes, leading to the PET from hsDNA to QDs, and consequently the fluorescence quenching of the QDs; with EB being added in the complex solution, it bound with hsDNA by intercalation interaction and caused hsDNA releasing from hsDNA-QDs complex with forming 2.5:1 EB-hsDNA complex, leading to the recovery of fluorescence, based on the greater binding constant (1.74 × 106 L·mol−1) of hsDNA with the embedded EB comparing to that of QDs with the captured hsDNA (4.25 × 104 L·mol−1). A good linear relationship existed between the fluorescence recovery yield and the EB concentrations under the range of 1.0—12.0 × 10−6 mol·L−1 with bare interference of related substances. This work provided some useful insights into the study of binding mechanism between DNAs with their intercalators and fluorescence bi-direction regulation, and showed great potential for the determination of trace EB.

Improved biosurfactant production from Aspergillus niger through chemical mutagenesis: characterization and RSM optimization

Biosurfactants are surface-active natural polymers produced within several microorganisms and are secreted outside the cellular environment. They are the focus of modern researches due to their eco-friendly nature and high production capability using low-cost agro-industrial wastes. In this research, we have evaluated Aspergillus niger (A. niger) for its biosurfactant production potential during solid state fermentation of banana stalks powder. The native strain of A. niger produced 2.3 g/L of biosurfactant with 49.74 cm2 oil displacement, 57% emulsification index and an emulsification activity of 1.024 (OD540). Sequential mutagenesis was induced in the native strain of A. niger by exposing the strain with different concentrations of ethidium bromide (EtBr), for different time periods. Significantly higher amount of biosurfactant (3.3 g/L) was obtained from the mutant strain A. niger M2 exposed to 50 µg/10 mL of EtBr for 60 min. The screening tests revealed the improvement in oil displacement (59.81 cm2), emulsification index (62.3%) and emulsification activity (OD540, 1.262) of biosurfactant. FTIR analysis confirmed the presence of amine, amide, fatty acids and triglycerides functional groups. The maximum biosurfactant synthesizing mutant (A. niger M2) was further optimized using RSM under CCD. After optimization, the highest biosurfactant (5.50 g/L) was obtained at 35 °C temperature, 7 pH, 5.75 g substrate concentration and 168 h of overall incubation period. In conclusion, the cost-effective production of biosurfactant, along with novel structural and multifunctional characteristics, this study may be useful for different industrial and biotechnological applications.

Antimicrobial activity and inhibition of the NorA efflux pump of Staphylococcus aureus by extract and isolated compounds from Arrabidaea brachypoda

Arrabidaea brachypoda is a native shrub of the Brazilian Cerrado widely used in the folk medicine for treatment of renal diseases and articular pains. This study aimed to, first, evaluate the antimicrobial activity of both extracts and isolated molecules Brachydins BR-A and BR-B obtained from the flowers of A. brachypoda against Staphylococcus aureus, Escherchia coli and Candida albicans species. A second objective was to investigate if these natural products were able to potentiate the Norfloxacin activity against the strain Staphylococcus aureus SA1199-B that overexpress the norA gene encoding the NorA efflux pump. Extracts and isolated compounds were analyzed by HPLC-PDA and LC-ESI-MS respectively. Minimal inhibitory concentrations of Norfloxacin or Ethidium Bromide (EtBr) were determined in the presence or absence of ethanolic extract, dichloromethane fraction, as well as BR-A or BR-B by microdilution method. Only BR-B showed activity against Candida albicans. Addition of ethanolic extract, dichloromethane fraction or BR-B to the growth media at sub-inhibitory concentrations enhanced the activity of both Norfloxacin and EtBr against S. aureus SA1199-B, indicating that these natural products and its isolated compound BR-B were able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. Moreover, BR-B inhibited the EtBr efflux in the SA1199-B strain confirming that it is a NorA inhibitor. Isolated BR-B was able to inhibit an important mechanism of multidrug-resistance very prevalent in S. aureus strains, thus its use in combination with Norfloxacin could be considered as an alternative for the treatment of infections caused by S. aureus strains overexpressing norA.

Phenotypic and genotypic methods for identification of slime layer production, efflux pump activity, and antimicrobial resistance genes as potential causes of the antimicrobial resistance of some mastitis pathogens from farms in Menoufia, Egypt.

Mastitis caused by multi- or pan-drug resistant bacteria is a growing health concern. A total of 110 milk samples were collected: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecalis, and Escherichia coli were present in 54/110 (49.09%), 37/110 (33.63%), 25/110 (22.72%), 7/110 (6.36%), and 50/110 (45.45%) samples, respectively. A total of 20 methicillin-resistant S. aureus (MRSA) isolates, 19 Streptococcus sp. isolates, and 15 E. coli isolates were selected, and 100% were positive for (coagulase and hemolysins), streptokinase, and hemolytic activity, respectively. A number of 11 E. coli isolates were serotyped, and the serotypes were: O26, O55, O111, O119, O124, O125, O127, and O158. The antimicrobial resistance index ranges for MRSA, Streptococcus sp., and E. coli were 0.49-0.83, 0.39-0.83, and 0.56-1, respectively. The most effective antimicrobials on Gram-positive isolates were cephradine, ciprofloxacin, doxycycline, norfloxacin, and vancomycin, while doxycycline and norfloxacin were effective on E. coli serotypes. All of the selected isolates exhibited slime layer production. The efflux pumps of the 12 MRSA, 12 Streptococcus sp., and 11 E. coli isolates exhibited activity with ethidium bromide concentrations of 1, 1.5, and 0.5µg/ml, respectively. There was a simultaneous antimicrobial activity of the efflux pump inhibitor chlorpromazine with amoxicillin/clavulanic acid, erythromycin, and oxacillin, to which the isolates were resistant. The 12 MRSA isolates harboured the methicillin resistance genes mec(A,A1, and A2), mecA1, and mecC at frequencies of 9/12 (75%), 9/12 (75%), and 8/12 (66.7%), respectively, and the penicillin resistance gene BlaZ was present at a frequency of 5/12 (41.7%). The distributions of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 8/12 (66.7%), 5/12 (41.7%), 12/12 (100%), 2/12 (16.7%), 0/12 (0.0%), and 8/12 (66.7%), respectively. The 12 Streptococcus sp. isolates harboured mec(A, A1, and A2), mecA1, mecC, and blaZ at rates of 4/12 (33.33%), 4/12 (33.33%), 5/12 (41.7%), and 4/12 (33.33%), respectively. The frequencies of erm(A) and erm(F) were 4/12 (33.33%), and 9/12 (75%), respectively. The 11 E. coli isolates harboured the extended-spectrum β-lactamases integrase1, integrase2, blaCTX-M, blaCTX-M-1, and blaTEM at frequencies of 10/11 (90.90%), 11/11 (100%), 9/11 (81.81%), 6/11 (54.54%), and 10/11 (90.90%), respectively. Moreover, the frequencies of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 7/11 (63.63%), 4/11 (36.36%), 4/11 (36.36%), 5/11 (45.45%), 10/11 (90.90%), and 10/11 (90.90%), respectively. Our results demonstrated the high antimicrobial resistance of the investigated isolates and confirmed the existence of multiple mechanisms underlying multidrug resistance.

<p>A novel mechanism of action of ketoconazole: inhibition of the <em>NorA</em> efflux pump system and biofilm formation in multidrug-resistant <em>Staphylococcus aureus</em></p>

Background: The rapid emergence of antimicrobial resistance among Gram-positive organisms, especially staphylococci, has become a serious clinical challenge. Efflux machinery and biofilm formation are considered two of the main causes of antimicrobial resistance and therapy failure.Aim: Our study aims to evaluate the antibiofilm and efflux pump inhibitory activity of the antifungal ketoconazole against multidrug-resistant (MDR) Staphylococcus aureus.Methods: Ketoconazole was tested for its effect on the following: minimum inhibitory concentrations (MICs) of ciprofloxacin, norfloxacin, levofloxacin, and ethidium bromide (EtBr) by the broth microdilution method, the efflux of EtBr by NorA-positive MDR S. aureus, and the relative expression of NorA, NorB, and NorC efflux pump genes. Docking studies of ketoconazole were performed using 1PW4 (glycerol-3-phosphate transporter from Escherichia coli which was the representative structure from the major facilitator superfamily).Results: Ketoconazole significantly decreased the MICs of levofloxacin, ciprofloxacin, norfloxacin, and EtBr (a substrate for efflux pump) by 8 to 1024-fold (P<0.01) and decreased the efflux of EtBr. Furthermore, a time-kill assay revealed that combinations of levofloxacin with ketoconazole or carbonyl cyanide m-chlorophenylhydrazone showed no growth for the tested strains after 24 h in comparison to the effect of levofloxacin alone. Docking studies and the ability of ketoconazole to diminish the relative expression of NorA gene in comparison to control (untreated strains) confirmed its action as an efflux pump inhibitor. Conclusion: The findings showed that the antifungal ketoconazole has no antibacterial activity but can potentiate the activity of the fluroquinolones against MDR S. aureus via inhibiting efflux pump and biofilm formation in vitro.

Mutagen-induced phytotoxicity in maize seed germination is dependent on ROS scavenging capacity

Ethidium bromide (EB) and acridine orange (AO) bind to nucleic acids and are thus considered as potential mutagens. In this study, the effects of EB and AO on the germination behaviours of white, yellow, red, and purple maize seeds were investigated. The results indicate that low concentrations of EB (50 μg mL−1) and AO (500 μg mL−1) promote germination, particularly for the white and yellow seeds. However, high concentrations of EB (0.5 mg mL−1) and AO (5 mg mL−1) significantly inhibit germination, with the level of inhibition decreasing in the following order: white > yellow > red > purple. In addition, EB and AO induce H2O2 production in a concentration-dependent manner. The effects of these mutagens on seed germination were partly reversed by dimethyl thiourea, a scavenger of reactive oxygen species (ROS), and diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, while the effects were enhanced by treatment with H2O2 and 3-amino-1,2,4-triazole, a specific inhibitor of catalase. In addition, AO and EB profoundly increased NADPH oxidase activities in germinating seeds. The treatment of seeds with EB and AO did not affect the growth or drought tolerance of the resultant seedlings. The findings suggest that the mechanism of mutagen toxicity is related to the induction of ROS production.

Heteroassociation of Caffeine and a Phenanthridine Dye in Aqueous Solution

Association of ethidium bromide and caffeine in aqueous solution was studied using spectrophotometry. The results were analyzed using a molecular dimer heteroassociation model. The molecular association equilibrium constants were determined. The experimental absorption spectrum of a mixture of heterocyclic molecules was deconvoluted into individual components using a regularization method. Quantitative information on the structure of the heterocomplex was obtained using data for the individual spectral components. The distance and angle between point transition dipole moments of the heterogeneous molecules were 0.3 nm and 16o. A calculation of the relative contents of different types of associates in a mixed solution showed that heteroassociation of the molecules decreased the effective concentration of ethidium bromide and, accordingly, the mutagenic activity of the dye.

MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features.

Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process. Failure in completing that process caused lethal consequences. The mtDNA depleted (MtDP) PC3 cells could be steadily maintained in the special medium in slow cycling status. The MtDP PC3 cells contained immature mitochondria and exhibited Warburg effect. Furthermore, the MtDP PC3 cells were resistant to therapeutic treatments and contained greater cancer stem cell-like subpopulations: CD44+, ABCG2+, side-population and ALDHbright. In conclusion, these results highlight the association of mtDNA content, mitochondrial function and cancer cell stemness features.

Efflux pump inhibitory activity of flavonoids isolated from Alpinia calcarata against methicillin-resistant Staphylococcus aureus

AbstractMethicillin-resistant

Efflux-pump inhibitory activity of a gallotannin from Terminalia chebula fruit against multidrug-resistant uropathogenic Escherichia coli

This study was carried out to evaluate the possible efflux-pump inhibitory activity of a gallotannin 1,2,6-tri-O-galloyl-β-d-glucopyranose isolated from Terminalia chebula fruit against multidrug-resistant (MDR) uropathogenic Escherichia coli. Susceptibility tests of antibiotics and ethidium bromide in the presence and absence of isolated gallotannin and phenylalanine-arginine β-naphthylamide (PAβN) were conducted by using the microbroth dilution assay. Ethidium bromide accumulation and efflux assays were performed spectrofluorometrically to elucidate the possible resistance-modifying activity of isolated gallotanin, if any. Minimum inhibitory concentrations of test antibiotics and ethidium bromide were reduced in the presence of isolated gallotannin and PAβN. The isolated gallotannin also demonstrated efflux-pump inhibitory activity against the studied bacteria as evidenced from ethidium bromide accumulation and efflux assays. The isolated gallotannin, 1,2,6-tri-O-galloyl-β-d-glucopyranose exhibited efflux-pump inhibitory activity which may be one of the possible mechanisms of its antibacterial action against MDR uropathogenic E. coli.

A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

Interference of intrinsic curvature of DNA by DNA-intercalating agents

It has been demonstrated in our studies that the intrinsic curvature of DNA can be easily interrupted by low concentrations of chloroquine and ethidium bromide. In addition, the changes of DNA curvature caused by varying the concentration of these two DNA intercalators can be readily verified through using an atomic force microscope.

An oligonucleotide-based label-free fluorescent sensor: highly sensitive and selective detection of Hg2+ in aqueous samples

A highly sensitive and selective detection method for mercury(II) in aqueous samples was developed based on the use of an oligonucleotide-based label-free fluorescent probe. The novel design was composed of two oligonucleotides with partially complementary sequences and the label-free dye ethidium bromide (EB). The fluorescence signal was very low when EB was free in water solution. However, the fluorescence signal increased by nearly 15 times when the DNA duplex was present. This dsDNA–EB system was used as a fluorescent sensor for Hg2+. The presence of Hg2+ led to the release of EB from the grooves of dsDNA and the fluorescence intensity was quenched. In this investigation, the optimal concentrations of dsDNA and EB were 0.285 μM and 0.9 μM, respectively. The calibration curve was linear in the range of 30 to 120 nM for Hg2+ (R2 = 0.992), with a detection limit of 9.5 nM. The method was simple and convenient, and this sensor also provided potential applications for developing aptasensors.

An assay of DNA by resonance light scattering technique and its application in screening anticancer drugs

In this contribution, a novel assay has been developed to detect the interaction between DNA and drugs using ethidium bromide (EB) as a probe based on the decreased resonance light scattering (RLS) technique. It was found that, in pH 2.0 Britton–Robinson buffer solution, EB underwent dramatic enhancement of RLS signal when binding to DNA. Additionally, the strong RLS signal of the EB–DNA system was remarkably decreased after the addition of DNA-targeted anticancer drugs. The results showed a linear relationship between the enhanced RLS intensity of the EB–DNA system and concentration of DNA in the range of 0.015–40.8 μg mL−1. The linear regression equation is represented as follows: ΔIRLS = 4.15 + 21.14c with regression coefficient r = 0.9970 (n = 10). The detection limit was 0.3 ng mL−1. Only 2.0 × 10−8 mol L−1 was selected as the optimum EB concentration in this assay. Further study demonstrated that the anticancer efficacy of anticancer drugs varied inversely with RLS intensity of the EB–DNA-drug system. So, it is intuitive to see the sequence of antitumor efficiency of anticancer drugs without data processing by RLS screening spectra. Compared with the traditional cell-based screening methods, the proposed method was more convenient, rapid and intuitive. The reaction mechanism is discussed.

Fluorescence Detection of Hg2+ Based on Hg2+-induced Formation of dsDNA

Abstract A simple and rapid fluorescence method was developed for Hg2+ detection on the basis of the specific thymine-Hg2+-thymine (T-Hg2+-T) structure and ethidium bromide (EB). Duplex DNA with thymine-thymine (T-T) mismatches was used. The melting temperature of double-stranded DNA (dsDNA) is lower than room temperature because there are several T-T mismatches in its structure. A single strand is retained in the absence of Hg2+ at room temperature. However, in the presence of Hg2+, the coordination chemistry of T-Hg2+-T leads to the formation of DNA duplexes. EB can intercalate into dsDNA, accompanied by the enhancement of EB fluorescence. The dsDNA and EB concentrations were optimized, and the detection was completed 1 min after the addition of Hg2+. Under optimal conditions, the change in fluorescence was proportional to the Hg2+ concentration in the range 1.0 × 10−8–9.0 × 10−7 M, and the detection limit was 3.0 × 10−9 M. The interferences from selected inorganic ions were also investigated. The sensor showed good selectivity for Hg2+, Ca2+ and Mg2+, but not for other metal ions. Therefore, the proposed method was simple and selective.

Analysis of DNA looped domains organization duringTriturus cristatusspermatogenesis

Chromatin from eukaryotes is organized in DNA loops with sequential attachments to a nucleoskeleton named nuclear matrix. This organization plays major roles in replication, transcription, recombination, DNA repair, chromosome condensation and segregation. During spermatogenesis, chromatin undergoes several dynamic transitions, which are often associated with important changes not only in its physical conformation but even in its compositions and structure. To understand the periodical change in the functional organization of chromatin during spermatogenesis, the higher order organization of chromatin in different testicular cell types (pachytene spermatocytes, round spermatids) and the epididymal sperm of Triturus cristatus have been investigated. The expansion and the contraction of nucleoid DNA were measured with a fluorescence microscope following exposure of nucleoids to increasing concentrations of ethidium bromide (EtBr) (2.5-200 μg/ml) as an intercalating dye to induce DNA-positive supercoils. Nucleoids from all studied cell types exhibited a biphasic change (condensed-relaxed-condensed) in size as a consequence of exposure to increasing concentrations of EtBr, indicating that they contained negatively supercoiled DNA. At higher EtBr concentrations, maximum positive supercoiling occurred in pachytene DNA loops. Our data suggest that pachytene DNA is the most open chromatin conformation in terms of EtBr intercalation.