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Background: Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are then detected by quantitative polymerase chain reaction (qPCR). However, 3C-qPCR assays are often complicated by the necessity of normalization controls to correct for amplification biases. In addition, qPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Recently, digital PCR (dPCR) technology has become available, which allows for highly sensitive nucleic acid quantification. Main advantage of dPCR is its high precision of absolute nucleic acid quantification without requirement of normalization controls. Results: To demonstrate the utility of dPCR in quantifying chromatin interactions, we examined two prostate cancer risk loci at 8q24 and 2p11.2 for their interaction target genes MYC and CAPG in LNCaP cell line. We designed anchor and testing primers at known regulatory element fragments and target gene regions, respectively. dPCR results showed that interaction frequency between the regulatory element and MYC gene promoter was 0.7 (95% CI 0.40-1.10) copies per 1000 genome copies while other regions showed relatively low ligation frequencies. The dPCR results also showed that the ligation frequencies between the regulatory element and two EcoRI fragments containing CAPG gene promoter were 1.9 copies (95% CI 1.41-2.47) and 1.3 copies per 1000 genome copies (95% CI 0.76-1.92), respectively, while the interaction signals were reduced on either side of the promoter region of CAPG gene. Additionally, we observed comparable results from 3C-dPCR and 3C-qPCR at 2p11.2 in another cell line (DU145). Conclusions: Compared to traditional 3C-qPCR, our results show that 3C-dPCR is much simpler and more sensitive to detect weak chromatin interactions. It may eliminate multiple and complex normalization controls and provide accurate calculation of proximity-based fragment ligation frequency. Therefore, we recommend 3C-dPCR as a preferred method for sensitive detection of low frequency chromatin interactions. [ABSTRACT FROM AUTHOR]

Ethidium monoazide and propidium monoazide (EMA and PMA) have been used in combination with PCR for more than a decade to facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving lower limits of detection and quantification of targeted live cells, fewer laborious procedures, lower costs, and potentially higherthroughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry costs that are 3 times higher because of the requirement for much larger amounts for LD discrimination than palladium compounds. Palladium compounds can penetrate dead (compromised) but not live bacteria and can be chelated primarily by chromosomal DNA and cell wall transmembrane proteins, with small amounts of DNA-binding proteins in vivo. The new mechanism for palladium compounds is obviously different from that of platinum compounds, which primarily target DNA. Combining palladium compounds with PCR (Pd-PCR) in water resulted in discrimination between live and dead Enterobacteriaceae bacteria that was much clearer than that seen with the PMA method. Pd-PCR correlated with reference plating or with the currently used PMA-PCR method for pasteurized milk, based on EN ISO 16140:2003 validation. Pd-PCR enabled us to specifically detect and assay viable Enterobacteriaceae cells at concentrations of 5 to 10 CFU/ml in milk while following U.S./EU regulations after a 4.5-h process in a typical laboratory exposed to natural or electric light, as specified by U.S./EU regulations. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma (RCC) is resistant to standard radiotherapy. Ubenimex, an aminopeptidase N inhibitor, is widely used as an adjunct therapy after surgery to enhance the function of immunocompetent cells and confer antitumor effects. Our previous study demonstrated that ubenimex induces autophagic cell death in RCC cells. Recently, the molecular mechanism of autophagy induction has been associated with radiosensitivity in RCC cells. In the present study, the ability of ubenimex to enhance RCC cell sensitivity to radiation via the induction of autophagic cell death was determined, and the mechanism of action of this effect was investigated. The 786-O and OS-RC-2 human RCC cell lines were treated with 0.5 mg/ml ubenimex and different doses of irradiation (IR). The cell viability was measured using a colony-formation assay and flow cytometry. Acridine orange (AO)-ethidium bromide (EB) staining was assessed by fluorescence microscopy as an indicator of autophagic cell death. Protein expression was assessed by western blotting. Autophagosomes were evaluated using transmission electron microscopy. RCC cells were used to evaluate the sensitivity to radiation using clonogenic survival and lactate dehydrogenase assays. Furthermore, these parameters were also tested at physiological oxygen levels. The AO-EB staining and flow cytometry of the OS-RC-2 cells indicated that the combined treatment significantly enhanced autophagic cell death compared with ubenimex or IR alone. Therefore, treatment with ubenimex did not significantly alter cell cycle progression but increased cell death when combined with radiation. An Akt agonist could significantly weaken this effect, indicating that ubenimex may act as an Akt inhibitor. Furthermore, the western blot analysis indicated that the combined treatment inhibited the Akt signaling pathway compared with ubenimex treatment or IR alone. Ubenimex may enhance RCC cell sensitivity to radiation by inducing cell autophagy. This induction changes the role of autophagy from protective to lethal in vitro, and this switch is associated with the inhibition of the Akt signaling pathway. [ABSTRACT FROM AUTHOR]

Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand. [ABSTRACT FROM AUTHOR]

Introduction: The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. Methodology: Two immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis. Results: The results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic capacities were lower. Conclusion: Among the drugs and conditions tested, the use of d4t was the best option for producing Rho-0 cells from hMSCs. Rho-0 cells are useful for studying the role of mitochondria in hMSC differentiation. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial dysfunction is one of the leading causes of neurological disorders in humans. Mitochondrial perturbations lead to adaptive mechanisms that include HIF-1 stabilization, though the consequences of increased levels of HIF-1 following mitochondrial stress remain poorly understood. Results: Using Caenorhabditis elegans, we show that a hif-1 loss-of-function mutation confers resistance towards the mitochondrial toxin ethidium bromide (EtBr) and suppresses EtBr-induced production of ROS. In mammals, the PD-related gene DJ-1 is known to act as a redox sensor to confer protection against antioxidants and mitochondrial inhibitors. A deletion mutant of the C. elegans homolog djr-1.1 also showed increased resistance to EtBr. Furthermore, our data implicates p38 MAP kinase as an indispensable factor for survival against mitochondrial stress in both hif-1 and djr-1.1 mutants. Conclusions: We propose that EtBr-induced HIF-1 activates pathways that are antagonistic in conferring protection against EtBr toxicity and that blocking HIF-1 activity may promote survival in cells with compromised mitochondrial function. [ABSTRACT FROM AUTHOR]

The graphene-based materials with unique, versatile, and tunable properties have brought new opportunities for the leading edge of advanced nanobiotechnology. In this regard, the use of graphene in gene delivery applications is still at early stages. In this study, we successfully designed a new complex of carboxylated-graphene (G-COOH) with ethidium bromide (EtBr) and used it as a nanovector for efficient gene delivery into the AGS cells. G-COOH, with carboxyl functions on its surface, in the presence of EtBr, formaldehyde, and cyclohexylisocyanide were participated in Ugi four component reaction to fabricate a stable amphiphilic graphene-EtBr (AG-EtBr) composite. The coupling reaction was confirmed by further analyses with FT-IR, AFM, UV-vis, Raman, photoluminescence, EDS, and XPS. The AG-EtBr nanocomposite was able to interact with a plasmid DNA (pDNA). This nanocomposite has been applied for transfection of cultured mammalian cells successfully. Moreover, the AG-EtBr composites showed a remarkable decreased cytotoxicity in compared to EtBr. Interestingly, the advantages of AG-EtBr in cell transfection are more dramatic (3-fold higher) than Lipofectamine2000 as a commercial nonviral vector. To the best of our knowledge, this is the first report in which EtBr is used as an intercalating agent along with graphene to serve as a new vehicle for gene delivery application. [ABSTRACT FROM AUTHOR]

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Multiple sclerosis, an autoimmune inflammatory disease of the central nervous system, is characterized by excessive demyelination. The study aimed to investigate the possible protective effect of ozone (O) therapy in ethidium bromide (EB)-induced demyelination in rats either alone or in combination with corticosteroids in order to decrease the dose of steroid therapy. Rats were divided into Group (1) normal control rats received saline, Group (2) Sham-operated rats received saline, Group (3) Sham-operated rats received vehicle (oxygen), Group (4) EB-treated rats received EB, Group (5) EB-treated rats received O, Group (6) EB-treated rats received methylprednisolone (MP), and Group (7) EB-treated rats received half the dose of MP concomitant with O. EB-treated rats showed a significant increase in the number of footfalls in the grid walk test, decreased brain GSH, and paraoxonase-1 enzyme activity, whereas brain MDA, TNF-α, IL-1β, INF-γ, Cox-2 immunoreactivity, and p53 protein levels were increased. A significant decline in brain serotonin, dopamine, norepinephrine, and MBP immunoreactivity was also reported. Significant improvement of the above-mentioned parameters was demonstrated with the administration of either MP or O, whereas best amelioration was achieved by combining half the dose of MP with ozone. [ABSTRACT FROM AUTHOR]

The mitochondrial unfolded protein response (UPRmt) is a surveillance pathway that defends proteostasis in the “powerhouse” of the cell. Activation of the UPRmt protects against stresses imposed by reactive oxygen species, respiratory chain deficits, and pathologic bacteria. Consistent with the UPRmt’s role in adaption, we found that either its pharmacological or genetic activation by ethidium bromide (EtBr) or RNAi of the mitochondrial AAA-protease spg-7 was sufficient to reduce death in an anoxia-based Caenorhabditis elegans model of ischemia-reperfusion injury. The UPRmt-specific transcription factor atfs-1 was necessary for protection and atfs-1 gain-of-function (gf) mutants were endogenously protected from both death and dysfunction. Neurons exhibited less axonal degeneration following non-lethal anoxia-reperfusion (A-R) when the UPRmt was pre-activated, and consistent with the concept of mitochondrial stress leading to cell non-autonomous (ie. “remote”) effects, we found that restricted activation of the UPRmt in neurons decreased A-R death. However, expression of the atfs-1(gf) mutant in neurons, which resulted in a robust activation of a neuronal UPRmt, did not upregulate the UPRmt in distal tissues, nor did it protect the worms from A-R toxicity. These findings suggest that remote signaling requires additional component(s) acting downstream of de facto mitochondrial stress. [ABSTRACT FROM AUTHOR]

Compared to standard treatments for various diseases, photochemotherapy and photo-dynamic therapy are less invasive approaches, in which DNA photocleavers represent promising tools for novel "on demand" chemotherapeutics. A series of p-nitrobenzoyl and p-pyridoyl ester conjugated aldoximes, amidoximes and ethanone oximes were subjected to UV irradiation at 312 nm with supercoiled circular plasmid DNA. The compounds which possessed appropriate properties were additionally subjected to UVA irradiation at 365 nm. The ability of most of the compounds to photocleave DNA was high at 312 nm, whereas higher concentrations were required at 365 nm as a result of their lower UV absorption. The affinity of selected compounds to calf-thymus (CT) DNA was studied by UV spectroscopy, viscosity experiments and competitive studies with ethidium bromide (EB) revealing that all compounds interacted with CT DNA. The fluorescence emission spectra of the pre-treated EB-DNA exhibited a moderate to significant quenching in the presence of the compounds indicating the binding of the compounds to CT DNA via intercalation as concluded also by DNA-viscosity experiments. For the oxime esters the DNA photocleavage and affinity studies aimed to clarify the role of the oxime nature (aldoxime, ketoxime, amidoxime) and the role of the pyridine and p-nitrophenyl moieties both as oxime substituents and ester conjugates. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (> 48 h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity. [ABSTRACT FROM AUTHOR]

In this study, the potential of natural pumice (NP) and iron-coated pumice stone (Fe-CP) as novel low-cost adsorbents to remove ethidium bromide (EtBr) from aqueous solutions was investigated. The operational parameters affecting removal efficiency and adsorption capacity such as adsorbent dose, initial EtBr concentration, pH, and contact time were studied in order to maximize EtBr removal. The maximum amount of adsorbed EtBr (qm) using NP and Fe-CP was 40.25 and 45.08 mg g‒1, respectively. It was found that EtBr adsorption followed the Freundlich isotherm model and fitted the pseudo-second-order kinetics equation for both adsorbents. In addition, the experimental system could be easily modeled by artificial neural network calculations. [ABSTRACT FROM AUTHOR]

Piscirickettsia salmonis is a fastidious intracellular pathogen responsible for high mortality rates in farmed salmonids, with serious economic consequences for the Chilean aquaculture industry. Oxytetracycline and florfenicol are the most frequently used antibiotics against P. salmonis, but routine use could contribute to drug resistance. This study identified differentiated florfenicol susceptibilities in two P. salmonis strains, LF-89 and AUSTRAL-005. The less susceptible isolate, AUSTRAL-005, also showed a high ethidium bromide efflux rate, indicating a higher activity of general efflux pump genes than LF-89. The P. salmonis genome presented resistance nodulation division (RND) family members, a family containing typical multidrug resistance-related efflux pumps in Gram-negative bacteria. Additionally, efflux pump acrAB genes were overexpressed in AUSTRAL-005 following exposure to the tolerated maximal concentration of florfenicol, in contrast to LF-89. These results indicate that tolerated maximum concentrations of florfenicol can modulate RND gene expression and increase efflux pump activity. We propose that the acrAB efflux pump is essential for P. salmonis survival at critical florfenicol concentrations and for the generation of antibiotic-resistant bacterial strains. [ABSTRACT FROM AUTHOR]

Mimicking proton conduction mechanism of Nafion to construct novel proton-conducting materials with low cost and high proton conductivity is of wide interest. Herein, we have designed and synthesized a cationic covalent organic framework with high thermal and chemical stability by combining a cationic monomer, ethidium bromide (EB) (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide), with 1,3,5-triformylphloroglucinol (TFP) in Schiff base reactions. This is the first time that the stable cationic crystalline frameworks allowed for the fabrication of a series of charged COFs (EB-COF:X, X = F, Cl, Br, I) through ion exchange processes. Exchange of the extra framework ions can finely modulate the COFs' porosity and pore sizes at nanoscale. More importantly, by introducing PW12O403- into this porous cationic framework, we can greatly enhance the proton conductivity of ionic COF-based material. To the best of our knowledge, EB-COF:PW12 shows the best proton conductivity at room temperature among ever reported porous organic materials. [ABSTRACT FROM AUTHOR]

Ionic liquids (ILs) are salts with poor ionic coordination, resultantly remaining in liquid state below 100 °C and some may retain liquid state even at room temperature. ILs are known to provide a conducive environment for many biological enzymatic reactions, but their interaction with biomacromolecules are poorly understood. In the present study, we investigate the effect of various ionic liquids on DNA-small molecule interaction using calf thymus DNA (ctDNA)-ethidium bromide (EB) as a model system. The effect of various ionic liquids on these interactions is studied by an array of techniques such as circular dichroism (CD), UV melting, fluorescence exclusion and isothermal titration calorimetry. Interestingly, we observed that presence of IL increased the stability of ctDNA without altering its structure. The binding affinities Kbs for EB binding to ctDNA in the presence of 300 mM ILs are about half order of magnitude smaller than the Kbs in absence of ILs and correspond to a less favorable free energy. We noted that, when adjusted to corresponding buffer condition, the unfavorable shift in ΔG of ctDNA-EB interaction is attributed to decreased entropy in the case of ILs, whereas the same effect by NaCl was due to increased enthalpy. [ABSTRACT FROM AUTHOR]

In this work we focus on the use of novel homo and block copolymers based on poly(vinyl benzyl trimethylammonium chloride) as gene delivery vectors. The homopolymers and block copolymers were synthesized by RAFT polymerization schemes and molecularly characterized. DNA/polymer complexes (polyplexes) in a wide range of N/P (amino-to-phosphate groups) ratios were prepared. The ability of the novel polymers to form complexes with linear DNA was investigated by light scattering, zeta potential, and ethidium bromide fluorescence quenching measurements. The resulting polyplexes were in the size range of 80-300 nm and their surface potential changed from negative to positive depending on the N/P ratio. The stability of polyplexes was monitored by changes in their hydrodynamic parameters in the presence of salt. The novel vector systems were visualized by transmission electron microscopy. The influence of factors such as molar mass, content, and chemical structure of the polycationic moieties as well as presence of a hydrophilic poly[oligo(ethylene glycol) methacrylate] block on the structure and stability of the polyplexes, kinetics of their formation, and effectiveness of the (co)polymers to shrink and pack DNA was discussed. [ABSTRACT FROM AUTHOR]

A new strategy for the synthesis of 6 H-benzo[ c]chromen-6-ones through judicious use of Garratt-Braverman (GB) Cyclization and Minisci acyloxylation reactions in moderate to good yield is reported. The uniqueness of the GB reaction is exemplified in providing the required biaryl esters, which could be successfully converted into the target skeleton by means of Minisci reaction on the free carboxylates. Ethidium bromide displacement and UV-based assay on the final molecules established them as DNA intercalators with binding constants in the range of about 104. [ABSTRACT FROM AUTHOR]

In the present work, isopropyl-6-amino-4-(3,5-bis(trifluoromethyl)phenyl)-5-cyano-2-methyl-4 H -pyran-3-carboxylate (4 H -pyran analog) has been synthesized by a three component reaction catalyzed by CsOH/γ-Al 2 O 3 and characterized. The interaction of 4 H -pyran analog with herring sperm DNA (hs DNA) under physiological conditions (phosphate buffer of pH 7.2) was investigated by UV absorption, FT-IR, fluorescence, 31 P NMR and circular dichroism (CD) spectroscopy. Fluorescence quenching results reveal that static quenching mechanism is involved in binding between 4 H -pyran analog and hs DNA. The calculated thermodynamic parameters (Δ H ° and Δ S °) indicate that hydrogen bonding plays a major role in binding between them. UV absorption and fluorescence shows the binding mode of 4 H -pyran analog with hs DNA as non-intercalative. According to the IR spectroscopy, 4 H -pyran analog binds to guanine, thymine, adenine bases of hs DNA but not to phosphate backbone of hs DNA which is also in good agreement with 31 P NMR results. CD and competitive binding experiment results confirms the minor groove binding of 4 H -pyran analog to hs DNA. [ABSTRACT FROM AUTHOR]

Efflux inhibition is proven bacterial machinery responsible for removal of bacterial wastage including antibiotics. Recently, efflux inhibitors (EI) have been tested with encouraging results as an adjuvant therapy for treatment of tuberculosis (TB). Although, EI have emerged as innovative approach of treatment for multi drug resistant (MDR) & extensively drug resistant tuberculosis (XDR-TB), toxicity profile limits their wider use. To address this issue, we have attempted synthesizing hybrid molecules those results by combining known EI and triazole. This synthesis was aimed to arrive at structure that possesses pharmacophore from known EI. Synthesized molecules were evaluated as growth inhibitors (GI) and Efflux inhibitor of TB initially against Mycobacterium smegmatis mc 2 155. Pharmacologically active compounds were then tested for their cytotoxicity to further narrow down search. Most active compounds 144, 145, 154 and 163 were then tested for their GEI action against Mycobacterium tuberculosis (Mtb). Synthesized compounds were also tested for their synergistic action with first line and second line anti-TB drugs and ethidium bromide (EtBr). We arrived at compound 135 as most potent dual inhibitor of tuberculosis. [ABSTRACT FROM AUTHOR]

In the present paper, the removal of ethidium bromide (EtBr) from aqueous solutions in a batch system using natural (NP) and aluminium-coated pumice (ACP) as alternative low-cost adsorbents was investigated. The maximum adsorption capacity, q m (mg/g) was 58.82 and 76.92 mg/g for NP and ACP, respectively, operating at an initial pH of 8, an adsorbent dose of 8 g/L, a contact time of 210 min and an initial EtBr concentration of 30 mg/L. The equilibrium data of both adsorbents fitted the Freundlich isotherm model, indicating the heterogeneity of the adsorbent surface. In addition, the adsorption rate of both adsorbents was well described by the pseudo-second-order kinetics model. This indicated chemisorption was the rate-controlling step of the adsorption process which occurred by ion exchange. Within the performed study, a three-layer artificial neural network (ANN) model was also developed to predict the efficiency of EtBr removal. Computational results clearly demonstrated that the ANN model was able to predict the combined effect of initial pH, adsorbent dose, contact time and initial EtBr concentration on the adsorption efficiency with a very high determination coefficient ( R 2 = 0.998) and a low relative error (RE = 0.037). [ABSTRACT FROM AUTHOR]

Introduction: Memory and cognitive impairments are some of devastating outcomes of Multiple Sclerosis (MS) plaques in hippocampus, the gray matter part of the brain. The present study aimed to evaluate the intrahippocampal injection of Ethidium Bromide (EB) as a simple and focal model to assess cognition and gray matter demyelination. Methods: Thirty Wistar rats were divided into three groups: control group, which received saline, as solvent of EB, into the hippocampus; and two experimental groups, which received 3 µL of EB into the hippocampus, and then, were evaluated 7 and 28 days after EB injection (n=10 in each group), using a 5-day protocol of Morris Water Maze (MWM) task as well as Transmission Electron Microscopy (TEM) assay. Results: Seven days after EB injection, the behavioral study revealed a significance increase in travelled distance for platform finding in the experimental group compared to the control group. In addition, the nucleus of oligodendrocyte showed the typical clumped chromatin, probably attributed to apoptosis, and the myelin sheaths of some axons were unwrapped and disintegrated. Twenty-eight days after EB injection, the traveled distance and the time spent in target quadrant significantly decreased and increased, respectively in experimental groups compared to the control group. Also, TEM micrographs revealed a thin layer of remyelination around the axons in 28 days lesion group. Discussion: While intracerebral or intraventricular injection of EB is disseminated in different parts of the brain and can affect the other motor and sensory systems, this model is confined locally and facilitates behavioral study. Also, this project could show improvement of memory function subsequent to the physiological repair of the gray matter of the hippocampus. [ABSTRACT FROM AUTHOR]

Quantitative studies of the binding of various DNA-binding antibiotics with dsDNA are useful for drug design, not only for effective antibiotics, but also for antitumor drugs. We studied the binding kinetics, association and dissociation rate constants, and association constants ( k on , k off , and K a , respectively) of intercalators and groove binders, including various antibiotics, to double-stranded DNA (dA 30 ·dT 30 and dG 30 ·dC 30 ) immobilized on a highly sensitive 27 MHz quartz-crystal microbalance (QCM) in aqueous solution. Although a simple ethidium bromide intercalator bound to both dA 30 ·dT 30 and dG 30 ·dC 30 , antibiotics that are side-binding intercalators, such as daunomycin, aclacinomycin A, and actinomycin D, with sugar or peptide moieties on the intercalator parts selectively bound to dG 30 ·dC 30 with high K a and small k off values. Nogalamycin, a dumbbell-shaped penetrating intercalator, showed low k on and k off values owing to slow duplex unwinding during the penetration process. Groove binders (Hoechst 33258, distamycin A, and mithramycin) had high K a values owing to the high k on values. Kinetic parameters depended largely on molecular shapes and DNA-binding molecule binding modes. [ABSTRACT FROM AUTHOR]

Many image acquisition methods of nucleic acids still depend on UV illumination, especially after the electrophoresis when determining the size of the target DNA. Therefore the quality of the UV illuminator in the gel documentation system, and the comparison of the fluorescence detected are crucial. This paper presents a fluorescence standard reference plate using quantum dots compared to the conventional method where an agarose gel containing ethidium bromide is loaded with standard samples. The fluorescence standard reference plate consists of chambers filled with commercially available quantum dots such as phosphor dots. The chamber is made by thermally attaching nylon and polyester, the former on the inside and the latter on the outside, for increased stability. The images of the proposed reference plate were captured more than 2 months in regular intervals. The intensity analysis of the images shows that the proposed reference plate delivers stable fluorescence over a long term period. The proposed reference plate can be utilized to compare the performance of various UV illuminators, or to set a standard fluorescence point for certain analyses. [ABSTRACT FROM AUTHOR]

DNA aptamers are synthetic, single-stranded DNA oligonucleotides selectedby SELEX methods for their binding with specific ligands. Here we present ethidiumbinding results for three related DNA aptamers (PDB code: 1OLD, 1DB6, and 2ARG)that bind L-argininamide (L-Arm). The ligand bound form of each aptamer's structurehas been reported and each are found to be composed primarily of two domainsconsisting of a stem helical region and a loop domain that forms a binding pocket for thecognate ligand. Previous thermodynamic experiments demonstrated that the DNAaptamer 1OLD undergoes a large conformational ordering upon binding to L-Arm. Herewe extend those linkage binding studies by examining the binding of the heterocyclicintercalator ethidium to each of the three aptamers by fluorescence and absorptionspectrophotometric titrations. Our results reveal that ethidium binds to each aptamer with∆Go's in the range of -8.7 to -9.4 kcal/mol. The stoichiometry of binding is 2:1 for eachaptamer and is quantitatively diminished in the presence of L-Arm as is the overallfluorescence intensity of ethidium. Together, these results demonstrate that a portion ofthe bound ethidium is excluded from the aptamer in the presence of a saturating amountof L-Arm. These results demonstrate the utility of ethidium and related compounds forthe probing of non-conventional DNA structures and reveal an interesting fundamentalthermodynamic linkage in DNA aptamers. Results are discussed in the context of thethermodynamic stability and structure of each of the aptamers examined.

A novel protocol to reuse agarose following agarose gel electrophoresis was established in this study. By repeated freeze-and-thaw of the agarose gel, ethidium bromide and other buffer components in the gel were safely removed from the gel without generation of any toxic fume. The agarose recovered using this method can be used for further electrophoretic experiments without any issues. [ABSTRACT FROM AUTHOR]

Plasmid borne antibiotics resistance is the global threat to healthcare facilities. Such antibiotics resistance is inherited stably within the same bacterial generations and transmitted horizontally to other species of bacteria. The elimination of such resistance plasmid is of great importance to contain dispersal of antibiotics resistance. E. coli strains were identified, screened for the presence of antibiotics resistance by disc diffusion method, and cured by sub-lethal concentrations of Ethidium bromide and Acridine orange. After curing, again antibiotic resistance was determined. Before and after curing, plasmids were extracted by column spin Kit and subjected to 1% agarose gel electrophoresis and antibiotic resistance genes were identified by PCR. The Ethidium bromide was more effective than Acridine orange in eliminating antibiotics resistance and resistance genes bearing plasmids (4, 5, 6, 8, 9, 10 and <10kb). The most frequently eliminated antibiotic resistance was against Imipenem and Meropenem followed by Cefoperazone-sulbactam, Amikacin and cephalosporins in sequence. The loss of antibiotic resistance was associated with the elimination of plasmid-borne antibiotic resistance genes; bla-TEM, bla-SHV, bla-CTX-M, qnrA, qnrB, qnrC and qnrD. Some E. coli strains did not show the removal of antibiotics resistance and plasmids, suggesting the presence of resistance genes on main chromosome and or non-curable plasmids.

We propose a simple and fast method for detecting arbitrary DNA mutations. Single-stranded DNA probes labeled with fluorescein amidite (FAM-ssDNA), ethidium bromide (EB), and graphene oxide (GO) are employed in the sensing system. The detection is achieved in two steps. In the first step, the sensing system contains FAM-ssDNA probes and EB molecules. It exhibits different fluorescence emissions in the presence of perfectly matched, mismatched, and random DNA sequences. With the addition of GO in the second step, the fluorescence signal for perfectly matched and random DNA does not vary greatly, which, however, experiences a significant change for mismatched DNA targets. The signal ratio before and after the addition of GO can clearly distinguish mutations from normal and random DNA sequences. The detection method works well regardless of the mutation positions and only requires "mix-and-detect" steps, which are completed within 15 min. [ABSTRACT FROM AUTHOR]

The study of EtBr and H33258 interaction as well as EtBr and MB interaction with DNA has been carried out. It was revealed that, at joint interaction, the effect of two ligands on the change of melting thermodynamic parameters of EtBr-DNA-H33258 or EtBr-DNA-MB complexes is not an addition of separate interaction influences. It was shown that, at joint, binding of EtBr and MB with DNA competition occurs, while in the case of EtBr and H33258, the mutual strengthening of stabilizing effect of each of them on DNA double-stranded structure mainly takes place. [ABSTRACT FROM AUTHOR]

A rapid and efficient method for quantification and discrimination of Salmonella enterica ser. Enteritidis between viable and dead cells killed by heat was developed using ethidium bromide monoazide (EMA) in combination with a real-time loop amplified (Rti-LAMP) DNA assay. The use of 8.0 μg/ml or less of EMA did not inhibit DNA amplification in Rti-LAMP assays derived from viable cells. However, 8.0 μg/ml of EMA notably inhibited DNA amplification and significantly increased the Tp values with dead cells. When the DNA from 2000 viable CFU was subjected to EMA-Rti-LAMP the resulting Tp value was 13 min. In contrast, the DNA from 2000 CFU completely heat destroyed CFU still yielded a Tp value, which was greatly increased to 33.1 min. When the DNA from viable plus heat killed CFU at a ratio of 7:1993 was subjected to EMA-Rti-LAMP, the resulting Tp value was 19.3 min, which was statistically identical (P < 0.05) to the Tp value of 19.9 min. obtained with the DNA from only 7 viable CFU. These results indicate that even though 2000 dead cells yielded a Tp value of 33.1 min., low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tp values. In addition, propidium monoazide (PMA) was found to be ineffective in distinguishing between low numbers of viable and heat killed cells of S. enterica . [ABSTRACT FROM AUTHOR]

Ethidium is a cationic dye with fluorescence that is enhanced ∼9-fold upon binding DNA. In order to better understand how the local environment modulates the behavior of this dye, we measured the photophysical properties of gaseous ethidium ions, using a quadrupole ion trap mass spectrometer that has been modified for fluorescence spectroscopy. The photodissociation maximum of gaseous ethidium measured through action spectroscopy is 485 nm and the emission maximum is 548 nm. The Stokes shift (2370 cm −1 ) of gaseous ethidium is marginally larger than that of ethidium in non-polar solvents, and significantly less than that in polar solvents. Time-resolved fluorescence measurements of gaseous ethidium ions show two components with lifetimes of 21.4 ± 1.5 and 5.1 ± 0.7 ns, which suggest the presence of multiple conformations in the gas phase. Both lifetimes are significantly longer than that of aqueous ethidium, while the longer of the two lifetimes is remarkably similar to that of ethidium in complex with double-stranded DNA in solution. In line with this, the estimated quantum yield of gaseous ethidium is ∼30% lower than that of ethidium in complex with DNA in solution, and ∼10-fold higher than that of aqueous ethidium. These benchmark results provide a reference from which to better understand the factors that modulate the fluorescence of phenanthridine-based dyes by the local environment. [ABSTRACT FROM AUTHOR]

Understanding the interaction of small molecules with DNA has become an active research area at the interface between biology and chemistry. In the present work, we investigated the mode of interaction of nelfinavir (NFV) with herring sperm DNA (hs DNA) under physiological conditions using various biophysical techniques. Analysis of UV-absorption and fluorescence spectra indicates the formation of complex between NFV and hs DNA. According to the fluorescence results, the binding constant ( K ) between NFV and hs DNA was found to be 3.30 × 10 4 LM −1 . The calculated thermodynamic parameters (Δ H ° and Δ S °) suggested that hydrogen bonding plays a major role in binding between them. Phosphate group binding studies revealed that there was no electrostatic interactions occurred between NFV and hs DNA. Circular dichroism (CD) and DNA melting curve were employed to measure the conformational change of hs DNA in the presence of NFV, which verified the minor groove binding mode. These results were further supported by viscosity measurements and competitive displacement assay study using Hoechst 33258. According to the sequence specificity experiments, NFV binds to A–T rich region of hs DNA. [ABSTRACT FROM AUTHOR]

Rifamycin is a broad-spectrum antimicrobial drug produced commercially by submerged fermentation where the yields are far less in comparison to its demand in human drug therapy. Addressing the need, sequential mutational strain improvement was carried using UV and EtBr that resulted in improved strain yielding rifamycin SV up to 4.32 g/L. Further optimization of six important fermentation factors was followed which include temperature, agitation, inoculum level, period of fermentation, inorganic nitrogen source and amino acids. For the first time, we report a maximum yield of 5.32 g/L of rifamycin SV. Among the amino acids, proline known for its slowest assimilation by Amycolatopsis mediterranei produced the highest improvement in antibiotic yields. Following mutational strain improvement and process optimization, a total of 3.8-fold increase in antibiotic titre was achieved. Following a conventional procedure of mutational strain improvement, highest yield of rifamycin SV was reported by optimizing submerged fermentation process. [ABSTRACT FROM AUTHOR]

Sodium dodecyl sulfate (SDS) is widely utilized in biomolecules separation, but high residue SDS in biomolecules samples interfere mass analysis. Ethidium bromide (EtdBr) interacts with SDS, and the formation of EtdBr-SDS complex at low SDS concentration (0-0.1 %) results a large red shift of the n→π* transition of EtdBr from 480 nm to 530 nm. The ion pairs become non-emissive and cause low emission intensity. While the concentration of SDS is above 0.1 %, SDS starts aggregating to form micelle. Micelle formation destabilizes the complex and the absorption maxima shift back to 513 nm while emission intensity increases. Based on the change of absorption and emission of EtdBr, a SDS concentration assay was developed. If absorption maximizes at 480 nm, the concentration of SDS of the sample is below 0.005 %. If absorption maximum is at longer wavelength than 480 nm, a second parameter, the ratio of absorbance at 513 nm and 550 nm is introduced. If the ratio is smaller than 1.5, the concentration of SDS is between 0.01-0.1 %. If the ratio is larger than 1.5, the concentration of SDS is above 0.15 %. Despite the suitable range is small, the lower limit is around the range of no mass interference. [ABSTRACT FROM AUTHOR]