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The efficient removal of Ethidium Bromide (EB), a toxic yet widely used chemical in biochemical industries and laboratories, is crucial to avoid its detrimental effect on human health, aquatic life, and the environment. One effective method is adsorption onto inert surfaces, but the choice of adsorbents is critical for the success of the removal protocols, primarily because of the limitations such as low adsorption capacity, high cost, limited availability, and a lack of understanding of the adsorption mechanism. In this work, Pyrophyllite Nanoclay (PNC), a naturally abundant aluminosilicate is explored as a promising adsorbent for EB. The adsorption process follows the Freundlich isotherm and displays pseudo-second-order kinetics. Under the optimized condition, PNC demonstrates nearly quantitative adsorption of EB and the maximum adsorption efficiency obtained from experiment was 6.53 mg/g (0.017 mmol/g) at room temperature under optimized condition. Through thermodynamic, kinetic analysis coupled with characterization techniques, the mechanism of adsorption is proposed. The adsorbent proves to be reusable, maintaining its performance over three cycles. The utility of the process is further validated and optimized using Response Surface Methodology (RSM). The present work focuses on the performance of PNC as adsorbent and elucidates distinctive surface adsorption mechanism (in comparison to other nanoclays), thus emphasizing the significance of structural, experimental, mechanistic and validation studies in choosing the right adsorbent for the removal toxic chemicals. [Display omitted] • Adsorption of Ethidium bromide on Pyrophyllite nanoclay (PNC) is investigated for the first time. • Kinetic and thermodynamic studies supported by characterization techniques reveal surface adsorption mechanism. • PNC is recyclable for at least three times. • Response Surface Methodology (RSM) optimization is conducted to validate the process. [ABSTRACT FROM AUTHOR]

Ethidium bromide is extensively used in many biochemical laboratories. Due to its high toxicity, its removal from the wastewater before being disposed becoming an emerging issue. Swelling clay minerals, due to their inexpensive material cost, high efficiency, and vast reserves, have been evaluated for the removal of different types of contaminants. In this study, the removal of ethidium bromide, particularly its mechanism of removal was assessed, using Ca-montmorillonite (SAz-1) as a representative swelling clay minerals. The ethidium bromide removal could reach equilibrium within 1 h and a capacity of 1,276 mmol kg-1, same magnitude as the cation exchange capacity of SAz-1. The total amount of metal cations desorbed from SAz-1 to the amount of ethidium removed was linearly correlated with a slope of 0.7, suggesting cation exchange as the dominant mechanism. In addition, the less-than-unity desorption/adsorption ratio could be attributed to the removal of ethidium dimers bridged with bromide counterions. An increase of the basal spacing to 18.3 Å for SAz-1 after adsorption implied that intercalation of flat-lying ethidium dimers into the clay interlayer, which increased its thermal stability, and active participation of the amine groups of ethidium molecules in the adsorptive interaction was evidenced from Fourier transform infrared band shifts. The results suggested that Ca-montmorillonite is an excellent candidate for fast and effective removal of ethidium bromide from aqueous solution. As such, it could serve as an ideal packing material for kits to be manufactured and used for ethidium bromide removal in case of spill. [ABSTRACT FROM AUTHOR]

• The polyoxometalate compounds, stabilized by the biologically relevant ethidium counter cations. • The binding studies of Keggin-Ethidium hybrid with CT-DNA (calf thymus DNA). • The single crystal X-ray structure of Keggin-Ethidium hybrid compounds. • The supramolecular interactions between Keggin polyanion and ethidium cation. We report the synthesis and photoluminescence (PL) properties of three compounds [C 21 H 20 N 3 ] 2 [HPMo 12 O 40 ] (1), [C 21 H 20 N 3 ] 3 [PW 12 O 40 ]∙4C 3 H 7 NO (2) and [C 21 H 20 N 3 ] 4 [SiW 12 O 40 ] (3) consisting of ethidium cation (3,8-diamino-5-ethyl-6-phenylphenanthridinium) and Keggin type polyoxometalates. Compounds 1, 2 and 3 have been characterized unambiguously by the single crystal X-ray diffraction studies. The binding studies of inorganic-organic hybrid compounds 1, 2 and 3 with CT-DNA (calf thymus DNA) have been carried out and monitored by UV–Vis- and emission-spectroscopy. The compounds 1 and 2 show fluorescence enhancement where as compound 3 with silicotungstate polyanion shows fluorescence quenching. The photoluminescence properties of three hybrid compounds arise due to the interaction of ethidium cation with DNA. We have demonstrated that the interaction between ethidium cation and CT-DNA is influenced by a particular type of polyoxometalate (POM) Keggin anion. The crystal structures show supramolecular interactions between the Keggin anion and ethidium cation. Compounds 1, 2 and 3 are further characterized by routine spectral analysis. [Display omitted] [ABSTRACT FROM AUTHOR]

• Therapeutic potential of Embelin against Ethidium Bromide-Induced Multiple Sclerosis in Rats. • Role of Embelin against the motor deficit and biochemical abnormalities in rats. • Role of Embelin against neurochemical, neuroinflammatory, and histological alterations in rats. Multiple sclerosis (MS) is a debilitating inflammatory disease characterized by demyelination, varied remyelination conservation, and partial axonal retention in central nervous system (CNS) lesions. The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in the pathophysiology of MS. Embelin (EMB), derived from the Embelia ribes plant, possesses diverse biological activities, including anti-inflammatory properties. This study aimed to investigate the neuroprotective effects of EMB in an ethidium bromide (EB)-induced model of MS in Wistar rats. Wistar rats were randomly divided into five groups (n = 8). MS-like manifestations were induced by injecting EB (0.1 %/10 µl) into the intracerebropeduncle (ICP) region of the rat brain for seven consecutive days. EMB was administered at doses of 1.25, 2.5, and 5 mg/kg. Behavioral assessments, neuroinflammatory cytokine analysis like tumor necrosis factor-α, interleukin-1-β, interleukin-6 (TNF-α, IL-1β, IL-6), oxidative stress marker measurements malondialdehyde, reduced glutathione, superoxide dismutase (MDA, GSH, SOD), and nitrite (NO), Acetylcholinesterase enzyme (AchE), and neurotransmitter level analysis, dopamine, serotonin, and norepinephrine (DA, 5-HT, and NE) were conducted. The study assessed behavioral, neurochemical, biochemical, and neuroinflammatory parameters, along with the modulation of p38 MAPK signaling. EMB administration significantly ameliorated neurological consequences induced by EB, improving motor coordination and gait abnormalities in rats. Furthermore, EMB effectively reduced neuroinflammatory cytokines (TNF-α, IL-1β, IL-6) and oxidative stress markers (AchE, SOD, MDA, GSH, nitrite). Notably, EMB exhibited a modulatory effect on neurotransmitter levels, increasing GABA, DA, and 5-HT, while reducing glutamate in EB-treated groups. This study demonstrates the neuroprotective potential of EMB against the EB-induced model of MS in rats. EMB administration mitigated neurological impairments, attenuated neuroinflammation, alleviated oxidative stress, and restored neurotransmitter balance. These findings highlight the promise of EMB as a therapeutic candidate for MS treatment, providing insights into its potential mechanism of action involving the modulation of p38 MAPK signaling. [ABSTRACT FROM AUTHOR]

In this study, we report the synthesis of eight novel indole-thiazole and indole-thiazolidinone derivatives, as well as their ability to interact with DNA, analysed through the UV–vis absorption, fluorescence, circular dichroism (CD), viscosity techniques and molecular docking. The ctDNA interaction analysis demonstrated different spectroscopic effects and the affinity constants (Kb) calculated by the UV–vis absorption method were between 2.08 × 105 and 6.99 × 106 M−1, whereas in the fluorescence suppression constants (Ksv) ranged between 0.38 and 0.77 × 104 M−1 and 0.60–7.59 × 104 M−1 using Ethidium Bromide (EB) and 4′,6-Diamidino-2-phenylindole (DAPI) as fluorescent probes, respectively. Most derivatives did not alter significantly the secondary structure of the ctDNA according to the CD results. None of the compounds was able to change the relative viscosity of the ctDNA. These results prove that compounds interact with ctDNA via groove binding, which was confirmed by A-T rich oligonucleotide sequence assay with compound JF-252 , suggesting the importance of both the phenyl ring coupled to C-4 thiazole ring and the bromo-unsubstituted indole nucleus. • Unsubstituted and 5-bromo-indole with thiazole and thiazolidinone were synthetized. • Thiazole and thiazolidinone rings bear a naphthalene and lateral substituents. • DNA interaction was performed by different techniques with the eight derivatives. • Bromo-unsubstituted indole and phenyl linked to C4 thiazole ring favor DNA bond. • The mode of interaction for all derivative was proven to be by DNA groove. [ABSTRACT FROM AUTHOR]

Abstract A new Dichlorotetra(4-hydroxy-N′-(pyridin-4-ylmethylene)benzohydrazone)Ru(II) complex 1 has been synthesized and characterized using spectroscopic techniques. The structure of complex 1 has been optimized through ORCA computational programme package using B3LYP functionals. The complex binds efficiently with calf thymus DNA (CT-DNA) as monitored by UV–visible titrations (K b = 4.8 × 105), ethidium bromide displacement studies (K sv = 1.39) as well as Circular Dichroism (CD) titrations. The complex intercalates with DNA base pairs. It shows cleavage of supercoiled (SC) DNA into nicked circular (NC) DNA efficiently via oxidative pathway. Complex 1 also inhibits Topoisomerase I (Topo I) relaxation activity at concentration < 20 μM. The molecular docking studies support that Topo I inhibition occur via blocking religation of G11 hydroxyl group. Graphical Abstract A new ruthenium(II) hydrazone complex cis , fac -[RuCl 2 (Py-CH=N-NH-CO-phenyl-OH) 4 ] 1 has been synthesized, characterized and optimized for DNA binding and self-nuclease activity of DNA via oxidative pathway. Complex 1 inhibited Topo I activity. The mechanism of DNA binding and Topo I inhibition activity was supported by molecular docking studies. Unlabelled Image Highlights • Ruthenium(II)-hydrazone complex cis , fac -[RuCl 2 (Py-CH=N-NH-CO-phenyl-OH) 4 ] 1 is synthesized and characterized. • The structure of complex 1 has optimized by ORCA computational programme package using B3LYP functionals. • The complex interacts with calf thymus DNA (CT-DNA) and binding of the complex is supported by molecular docking studies. • Complex 1 inhibits topoisomerase I with IC 50 values of 15 μM via blocking religation of G11 hydroxyl group. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Synthesis of amino acid conjugates of 3-(1-chloro-3,4-dihydronaphthalen-2-yl) acrylic acid. • Screening for NorA inhibitors against Staphylococcus aureus. • The potent inhibitors displayed MEC of 1.56 μg/mL against NorA overexpressing strain SA-1199B. • Mechanism for NorA specificity elucidated by ethidium bromide efflux. Abstract Inhibitors for NorA efflux pump of Staphylococcus aureus have attracted the attention of many researchers towards the discovery and development of novel efflux pump inhibitors (EPIs). In an attempt to find specific potent inhibitors of NorA efflux pump of S. aureus , a total of 15 amino acid conjugates of 3-(1-chloro-3,4-dihydronaphthalen-2-yl)acrylic acid (4 – 18) were synthesized using a simple convenient synthetic approach and bioevaluated against NorA efflux pump. Two compounds 7 and 8 (each having MEC of 1.56 µg/mL) were found to restore the activity of ciprofloxacin through reduction of the MIC elucidated by comparing the ethidium bromide efflux in dose dependent manner in addition to ethidium bromide efflux inhibition and accumulation study using NorA overexpressing strain SA-1199B. Most potent compounds among these were able to restore the antibacterial activity of ciprofloxacin completely against SA-1199B. Structure activity relationship (SAR) studies and docking study of potent compounds 7 and 8 could elucidate the structural requirements necessary for interaction with the NorA efflux pumps. On the whole, compounds 7 and 8 have ability to reverse the NorA efflux mediated resistance and could be further optimized for development of potent efflux pump inhibitors. [ABSTRACT FROM AUTHOR]

We have set-up an electrochemical advanced oxidation process for ethidium bromide (1), based on the Eu-doped MnWO 4 (Eu:MnWO 4), obtained through a template-driven synthesis, along with developing a suitable monitoring method. Under galvanostatic conditions, Eu:MnWO 4 -coated graphite electrode serves as anode, applicable for removal of 1. To go further and augment the catalytic method, we have applied a modified carbon paste electrode for the monitoring of 1 with the limit of detection (LOD) of 54 nM. Enhancement of the hydrogen evolution reaction is an indication of electrocatalytic properties of the material, whereby developed method emerges as a candidate for straightforward application in electrochemical advanced oxidation processes (EAOPs). We have enriched experimental data with theoretical insights, provided by Density Functional Theory (DFT), and proposed oxidation mechanism of 1. Based on obtained results, we propose the new nanomaterial as a potent electrochemical modifier, suitable for catalytic treatment and process monitoring of the 1 -polluted waters. [Display omitted] • Eu:MnWO 4 -based nanomaterial for ethidium bromide sensing and control. • A novel analytical approach for both ethidium bromide degradation and concentration monitoring. • Ethidium bromide degradation mechanism was studied theoretically. • The practical feasibility of this system was tested by monitoring the treated water parameters. [ABSTRACT FROM AUTHOR]

This study was carried out to test the essential oil from C. ambrosioides leaves and its main constituent, α-Terpinene, in an antibacterial activity assay. As well, it was evaluated ability reduce resistance to norfloxacin and ethidium bromide was compared the Staphylococcus aureus 1199B whith 1199 wild type strain. The MIC of the C. ambrosioides essential oil and α-Terpinene were determined by microdilution method. The MIC of the essential oil and α-Terpinene presented a value ≥ 1024 μg/mL. However, when associated with antibacterials, the essential oil from C. ambrosioides leaves significantly reduced the MIC of antibiotics and ethidium bromide, characterizing an efflux pump inhibition. The C. ambrosioides essential oil, despite having no direct antibacterial activity against the S. aureus 1199B strain, showed a potentiating action when associated with antibacterial agents, this being attributed to an inhibition of efflux pumps. [ABSTRACT FROM AUTHOR]

A new zinc(II) complex, [Zn(L)](ClO 4 ) with hexadentate N 4 S 2 donor azo-thioether ligand (HL) was synthesized and characterized by several spectroscopic techniques. The structure was confirmed by single crystal X-ray analysis. The interaction of the complex with CT DNA was investigated by UV–vis method and binding constant is found to be 6.6 × 10 4  M −1 . Competitive binding titration with ethidium bromide (EB) by fluorescence titration method reveals that the complex efficiently displaces EB from EB-DNA system and the Stern-Volmer dynamic quenching constant, K sv is found to be 2.6 × 10 4  M −1 . DFT and TDDFT calculations were carried out to interpret the electronic structure and electronic spectra of the complex. [ABSTRACT FROM AUTHOR]

An improved microwave-induced synthesis of five ethidium derivatives (Ethidium derivatives, 2a–d ) is presented. As the derivatives 2a–d have been proposed previously to be telomerase inhibitors, the binding interactions of these ethidium derivatives with G-quadruplex DNA were evaluated by means of photometric and fluorimetric titration, thermal DNA denaturation, CD and 1 H NMR spectroscopy. In particular, the compound bearing 3,8-bis(pyrrolidin-1-yl)propanamido substituent 2a exhibits high selectivity for G-quadruplex DNA relative to duplex DNA. [ABSTRACT FROM AUTHOR]

PCR-based methods for enumerating bacteria cells could lead to an overestimation of viable cells due to the amplification of DNA from dead cells. To overcome this disadvantage, DNA intercalating dyes such as Ethidium monoazide (EMA) or Propidium monoazide (PMA) combined with qPCR have been considered promising alternative methods to discriminate between viable and nonviable cells. The drawback of those DNA intercalating dyes, however, could not assess the physiological states of cells. Thus, the objective of this study was to develop a novel molecular viability assay to selectively detect only the cells that exhibit metabolic activity. This study's results showed that DyeTox13 Green C-2 Azide dye coupled with qPCR can be used effectively to distinguish between active and nonactive gram-negative bacteria ( P. aeruginosa PAO1) and gram-positive bacteria ( E. faecalis v583). In this study, we evaluated the usefulness of the DyeTox13 Green C-2 Azide-qPCR assay for selectively amplifying nucleic acids of microorganisms that have metabolic activities. We conclude that the use of DyeTox13 Green C-2 Azide-qPCR is a promising alternative for discriminating active cells from nonactive cells but the dye's performance might be dependent on the kind of bacterial species. [ABSTRACT FROM AUTHOR]

The interaction between lomefloxacin (LMF) and a ctDNA-H 1 complex was investigated by various spectroscopies, as well as viscometry and molecular modeling techniques. The RLS results pointed at different intensities of the ctDNA, H 1 and ctDNA-H 1 complex systems in the presence of LMF. The fluorescence intensity measurements of the ctDNA, H 1 and ctDNA-H 1 complex in the presence of LMF as binary and ternary systems at three temperatures exhibited regular quenching of the fluorescence emission curves signifying a static behavior of the binary and ternary systems. Stern-Volmer constant values of ctDNA-LMF complex formation were 7.94 × 10 7 , 4.94 × 10 7 and 3.54 × 10 7 M −1 at 298, 303 and 308 K respectively. In the presence of H1, the Stern-Volmer constant values had been changed to 1.41 × 10 8 , 0.84 × 10 8 and 0.21 × 10 8 M −1 at 298, 303 and 308 K respectively. The Stern-Volmer constant values of binary and ternary systems clearly revealed static quenching behavior of ctDNA upon interaction with LMF in absence and presence of H1. Thermodynamic parameters obtained from Van't Hoff plots revealed the different essence of interaction between LMF with ctDNA, H 1 and ctDNA complex systems. The interaction between LMF with ctDNA and the ctDNA-H 1 complex in the presence of ethidium bromide (EB) and acridine orange (AO) as intercalator probes showed that a competitive ness occurred between EB and AO with LMF, indicating that the LMF bound to ctDNA as an intercalator. Circular dichroism (CD) curves in far UV-CD were used to determine the enhancement of ellipticity in ctDNA upon interaction with LMF in the absence and presence of H 1 . The specific viscosity of ctDNA in the absence and presence of H 1 increased with an enhancement of the LMF concentration which was another reason for LMF binding to ctDNA as an intercalator. Molecular modeling data confirmed the experimental results pertaining to the interaction behavior in the ctDNA-LMF complex in the absence and presence of H 1 . [ABSTRACT FROM AUTHOR]

Although qPCR is indisputably very powerful tool for identification and quantification of various microorganisms, one of its main disadvantages is the inability to distinguish between signals originating from different sources (live, dead or compromised cells). Sample pre-treatment with DNA intercalating dyes such as ethidium monoazide (EMA) or propidium monoazide (PMA) is a widely used solution which helps to reduce this issue. Efficacy of this pre-treatment, however, depends on membrane permeability. Therefore in this work, a killing assay was performed, evaluating the impact of several cell inactivation techniques with various modes of action on EMA/PMA-qPCR results. Three main thermotolerant Campylobacter spp. ( C. jejuni , C. coli and C. lari ) in mixed suspension were inactivated by a panel of eight lethal and one sub-lethal procedures, which bacteria can encounter during their life span (e.g. food processing and storage, surface disinfection or laboratory maintenance in terms of sterilization or aseptic work), and afterwards were quantified using previously designed multiplex EMA/PMA-qPCR. It was shown that not only various inactivation mechanisms affect the efficacy of qPCR, but species-specific differences were apparent as well. C. lari was the most resistant species to EMA/PMA treatment, while C. coli seemed to be the most sensitive. Results of this killing assay might be generally helpful in every research where EMA/PMA-qPCR is used, as they clearly show that this pre-treatment technique cannot be blindly used for every sample without at least considering the way of the bacterial inactivation. [ABSTRACT FROM AUTHOR]

Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis ( 4a – c , 7 and 8 ; H37Rv-MIC 99 ≤1.25 µM, R5401-MIC 99 ≤2.5 µM, X_61-MIC 99 ≤5 µM) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a – c and 3b (MIC 90 ≤13.42 µM) and inhibition of EB efflux demonstrated by these compounds are encouraging. [ABSTRACT FROM AUTHOR]

Guggulsterone, a sterol found in plants is used as an ayurvedic medicine for many diseases such as obesity, internal tumors, ulcers etc. E and Z are two isoforms of guggulsterone, wherein guggulsterone-E (GUGE) has also been shown to have anticancer potential. Most of the anticancer drugs target nucleic acids. Therefore, we studied the mode of interaction between ctDNA and GUGE using UV–Vis, fluorescence and CD spectroscopy, isothermal calorimetry along with molecular docking studies. Hoechst 3325, ethidium bromide and rhodamine-B displacement experiments confirms that GUGE binds in the minor groove of DNA. ITC results further suggest these interactions to be feasible and spontaneous with hydrogen bond formation and van der waals interactions. Lastly, molecular docking also suggests GUGE to be a minor groove binder interacting through a single hydrogen bond formation between OH group of GUGE and nitrogen (N3) of adenosine (A6). [ABSTRACT FROM AUTHOR]

Two new derivatives of pyrimido[4′,5′;4,5]thieno(2,3-b)quinoline (PTQ), 9-hydroxy-4-(3-diethylaminopropylamino)pyrimido[4′,5′;4,5]thieno(2,3-b)quinoline (Hydroxy-DPTQ) and 8-methoxy-4-(3-diethylaminopropylamino)pyrimido[4′,5′;4,5]thieno(2,3-b)quinoline (Methoxy-DPTQ) were synthesized and their DNA binding ability was analyzed using spectroscopy (UV–visible, fluorescence and circular dichroism), ethidium bromide dye displacement assay, melting temperature ( T m ) analysis and computational docking studies. The hypochromism in UV–visible spectrum and increased fluorescence emission of Hydroxy-DPTQ and Methoxy-DPTQ in the presence of DNA suggested the molecule-DNA interaction. The association constants calculated from UV–visible and spectral titrations were of the order 10 4 to 10 6 M −1 . Circular dichroism studies corroborated the induced conformational changes in DNA upon addition of molecules. The change in the ellipticity was observed both in negative and positive peak of DNA, thus, suggesting the intercalation of molecules. The observed displacement of ethidium bromide from the DNA and increased T m , upon addition of DNA confirmed the intercalative mode of binding. This was further validated by computational docking, which showed clear intercalation of molecules into the d(GpC)-d(CpG) site of the receptor DNA. Anticancer activities of these molecules are evaluated by using MTT assay. Both molecules showed antiproliferative activity against all the three cancer cells studied, with Hydroxy-DPTQ being more potential molecule among the two. IC 50 value of Hydroxy-DPTQ and Methoxy-DPTQ were in the range of 3–5 μM and 130–250 μM, respectively. [ABSTRACT FROM AUTHOR]

This study described a novel multiplex qualitative detection method using pyrosequencing. Based on the principle of the universal primer-multiplex-PCR, only one sequencing primer was employed to realize the detection of the multiple targets. Samples containing three genetically modified (GM) crops in different proportions were used to validate the method. The dNTP dispensing order was designed based on the product sequences. Only 12 rounds (ATCTGATCGACT) of dNTPs addition and, often, as few as three rounds (CAT) under ideal conditions, were required to detect the GM events qualitatively, and sensitivity was as low as 1% of a mixture. However, when considering a mixture, calculating signal values allowed the proportion of each GM to be estimated. Based on these results, we concluded that our novel method not only realized detection but also allowed semi-quantitative detection of individual events. [ABSTRACT FROM AUTHOR]

• The incidence of clinical signs of MS was reduced in rats with chronic toxoplasmosis. • Weight loss was lower in the MS group with chronic toxoplasmosis. • There was no significant difference in the occurrence of clinical symptoms in MS with acute toxoplasmosis group compared to the control group. • In histological examination, higher cell density, less spongy tissue, more viable cells and less pyknotic cells were observed. • TNF-α and INF-γ levels were decreased in MS with chronic toxoplasmosis compared to the MS group. Multiple sclerosis (MS) is an autoimmune neurodegenerative disease. Since the modulation of the immune system by parasites has been proven, and there have been reports of a reduction in the clinical symptoms of MS in people with toxoplasmosis, this study aimed to investigate the effect of toxoplasmosis on MS in an animal model. MS model was induced by the ethidium bromide injection in the areas specified in the Rat's brain in the stereotaxic device and Toxoplasma gondii RH strain injection of the rat's peritoneal for creation of toxoplasmosis. The effect of acute and chronic toxoplasmosis on the MS model was evaluated by examining the development of clinical symptoms of MS, body weight, changes in the levels of inflammatory cytokines, inflammatory cell infiltration, cell density, and spongy tissue in the brain. The body weight in the acute toxoplasmosis with MS was the same as the MS group, and a significant decrease was observed, but no weight loss was observed in the chronic toxoplasmosis with MS. In the chronic toxoplasmosis, the progress of clinical signs such as Immobility of limbs, including tail, hands, and feet, was observed less compared to other groups. The histology results in the group of chronic toxoplasmosis showed high cell density and inhibition of spongy tissue formation, and the infiltration of inflammatory cells in this group was less. TNF-α and INF-γ decreased in MS with chronic toxoplasmosis compared to the MS group. Our findings showed that chronic toxoplasmosis with inhibition of spongy tissue formation and prevention of cell infiltration and. As a result, the reduction of inflammatory cytokines could reduce clinical symptoms in MS in the animal model. [ABSTRACT FROM AUTHOR]

The use of natural products is crucial to suppress the development of these micro-organisms and to reduce the concentration necessary to inhibit these microrganisms, reducing the toxicity risks also. In this study, the essential oil from Chenopodium ambrosioides Leaves and its main constituent α-Terpinene were used in the antibacterial and potentiating activity of antibiotics and ethidium bromide assays, against the bacterial strains Staphylococcus aureus IS-58, carriers of efflux pumps. The Minimum Inhibitory Concentration (MIC) was determined using a microdilution method. The capacity of the aforementioned was also tested in combination with tetracycline and ethidium bromide, with the aim of improving the activity of the antibacterials. The MIC of the C. ambrosioides L. essential oil and of α-Terpinene were above 1024 μg/mL, comprising a clinically irrelevant value. However, when associated with the antibiotics, the C. ambrosioides L. essential oil, significantly decreased the MIC of tetracycline and ethidium bromide. The efflux pump is the only mechanism the bacteria possesses to reduce the toxicity of ethidium bromide, and thus this reduction in the MIC demonstrates that the C. ambrosioides L. essential oil is an effective option in the inhibition of the efflux pump present in these micro-organisms. [ABSTRACT FROM AUTHOR]

Introduction Few, if any, radiotracers are available for the in vivo imaging of reactive oxygen species (ROS) in the central nervous system. ROS play a critical role in normal cell processes such as signaling and homeostasis but overproduction of ROS is implicated in several disorders. We describe here the radiosynthesis and initial ex vivo and in vivo evaluation of [ 11 C]hydromethidine ([ 11 C]HM) as a radiotracer to image ROS using positron emission tomography (PET). Methods [ 11 C]HM and its deuterated isotopologue [ 11 C]( 4 ) were produced using [ 11 C]methyl triflate in a one-pot, two-step reaction and purified by high performance liquid chromatography. Ex vivo biodistribution studies were performed after tail vein injections of both radiotracers. To demonstrate sensitivity of uptake to ROS, [ 11 C]HM was administered to rats treated systemically with lipopolysaccharide (LPS). In addition, ex vivo autoradiography and in vivo PET imaging were performed using [ 11 C]HM on rats which had been microinjected with sodium nitroprusside (SNP) to induce ROS. Results [ 11 C]HM and [ 11 C]( 4 ) radiosyntheses were reliable and produced the radiotracers at high specific activities and radiochemical purities. Both radiotracers demonstrated good brain uptake and fast washout of radioactivity, but [ 11 C]( 4 ) washout was faster. Pretreatment with LPS resulted in a significant increase in brain retention of radioactivity. Ex vivo autoradiography and PET imaging of rats unilaterally treated with microinjections of SNP demonstrated increased retention of radioactivity in the treated side of the brain. Conclusions [ 11 C]HM has the attributes of a radiotracer for PET imaging of ROS in the brain including good brain penetration and increased retention of radioactivity in animal models of oxidative stress. [ABSTRACT FROM AUTHOR]

Continuous increases in the rates of tumor diseases have highlighted the need for identification of novel and inexpensive antitumor agents from natural sources. In this study, we investigated the effects of enriched fraction from hydroalcoholic Brazilian red propolis extract against Hep-2 cancer cell line. Initially 201 fractions were arranged in 12 groups according to their chromatographic characteristics (A–L). After an in vitro cell viability screening, J and L were further selected as promising enriched fractions for this study. The chemical characterization was performed and Biochanin A, Formononetin, and Liquiritigenin compounds were quantified. Through MTT viability assay and morphological changes observed by Giemsa and DAPI staining, the results showed that red propolis inhibited cancer cells growth. Flow cytometry results indicated effects that were partly mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA cleaved assay, increase at SUB G1-G0 phase in cell cycle analysis and loss of mitochondrial membrane potential. In conclusion, our results demonstrated that red propolis enriched fractions promoted apoptotic effects in human cancer cells through the mechanisms involving mitochondrial perturbation. Therefore, red propolis fractions contain candidate agents for adjuvant cancer treatment, which further studies should elucidate the comprehensive mechanistic pathways. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphism (SNP) genotyping is an important aspect in understanding genetic variations. Here, we discriminate SNPs using toe-hold mediated displacement reactions. The biological target is an 80 nucleotide long double-stranded–DNA from the mtDNA HV1 region, associated with maternal ancestry. This target has been specially designed with a pendant toehold and a cationic fluorophore, ATTO 647N, as a reporter, produced in a polymerase chain reaction. Rates of reaction for the toehold-polymerase chain reaction products (TPPs) with their corresponding complementary displacing sequences, labelled with a Black Hole Quencher 1, followed the order TPP–Cytosine > TPP–Thymine > TPP–Adenine ≥ TPP–Guanine. Non-complementary rates were the slowest with mismatches involving cytosine. These reactions, operating in a static/or contact mode, gave averaged readouts between SNPs within 15 min (with 80–90% quenching), compared to 25–30 min in previous studies involving fluorescence resonance energy transfer. Addition of an intercalating agent, ethidium bromide, retarded the rate of reaction in which cytosine was involved, presumably through stabilization of the base pairing, which resulted in markedly improved discrimination of cytosine containing SNPs. [ABSTRACT FROM AUTHOR]

In the absence of activating anionic lipids and cholesterol, the nicotinic acetylcholine receptor (nAChR) from Torpedo adopts an uncoupled conformation that does not usually gate open in response to agonist. The uncoupled conformation binds both agonists and non-competitive channel blockers with a lower affinity than the desensitized state, consistent with both the extracellular agonist-binding and transmembrane channel-gating domains individually adopting resting-state like conformations. To test this hypothesis, we characterized the binding of the agonist, acetylcholine, and two fluorescent channel blockers, ethidium and crystal violet, to resting, desensitized and uncoupled nAChRs in reconstituted membranes. The measured K d for acetylcholine binding to the uncoupled nAChR is similar to that for the resting state, confirming that the agonist binding site adopts a resting-state like conformation. Although both ethidium and crystal violet bind to the resting and desensitized channel pores with distinct affinities, no binding of either probe was detected to the uncoupled nAChR. Our data suggest that the transmembrane domain of the uncoupled nAChR adopts a conformation distinct from that of the resting and desensitized states. The lack of binding is consistent with a more constricted channel pore, possibly along the lines of what is observed in crystal structures of the prokaryotic homolog, ELIC. [ABSTRACT FROM AUTHOR]

Maximising the number of cells arrested at metaphase and their resolution is fundamentally important for molecular cytogenetic investigations, particularly in fish, which typically yield low mitotic index and have highly condensed chromosomes. To overcome these limitations, fish were injected with a mitotic stimulator (the yeast, Saccharomyces cerevisiae) to improve the mitotic index, and the intercalating agent ethidium bromide to produce elongated chromosomes. Specifically, adults were injected with activated yeast and then Colcemid (0.025 µg/µl solution, 10 µl per 1 g of body weight) at 24–96 h post yeast injections, followed by chromosome preparations from multiple tissues. Results showed that gill tissue had the highest number of dividing cells at 72 h post yeast exposure with no significant (p > 0.05) differences between the sexes. Nonetheless, sex-specific differences in the mitotic index were observed in spleen, kidney, and liver, which may be attributed to sex-specific differences in immune responses. For elongation of mitotic chromosomes, individuals (both sexes) were first injected with activated yeast and after 48 h with ethidium bromide (2 or 4 µg/ml) and Colcemid (0.05 µg/µl solution, 10 µl per 1 g of body weight). Following which, animals were sampled at three time points (1, 4 and 8 h) for chromosome preparations. The results show that the optimum elongation of metaphase chromosomes of males and females was achieved by using 2 µg/ml and 4 µg/ml, respectively, for 1 h. Interestingly, the average mitotic chromosome length (μm) of males and females post-ethidium bromide exposure was significantly different (p < 0.05) for both concentrations, except at 1 h exposure for 2 µg/ml EtBr. Such differences can be attributed to overall chromosomal condensation differences between sexes. Regardless, the increased mitotic index and chromosome resolution could benefit cytogenetic studies in other fish species. • The use of yeast and ethidium bromide boosts mitotic index and chromosome resolution for cytogenetic analysis. • The sex-specific mitotic indexsuggests sexual dimorphisms in immunological responses from haemopoietic organs in G. holbrooki. • Sexual dimorphism in chromosome elongation indicates differences in their genetic content and/or epigenetic modifications. [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]

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]

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]

Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol–chloroform–isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations. [ABSTRACT FROM AUTHOR]

Human norovirus (HuNoV) is the most common cause of gastroenteritis worldwide. The lack of a virus culture system makes it difficult to determine the viability of norovirus by only reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative RT-PCR (qRT-PCR). The aim of this study was to investigate the detection of viable murine norovirus (MNV) by combining propidium monoazide (PMA) or ethidium monoazide (EMA) with qRT-PCR. MNV (5.21 log 10 PFU/mL) was subjected to heat treatment at room temperature, 65, 70, 75, 80, 85, or 90 °C in a water bath for 1 min. The plaque assay, qRT-PCR, PMA-combined qRT-PCR, and EMA-combined qRT-PCR were then performed with heat exposed MNV samples. The MNV titer was reduced by 0.38, 1.34, and 3.71 log 10 PFU/mL at temperatures of 65, 70, and 75 °C, respectively. MNV was reduced >4.21 log 10 PFU/mL at 80, 85, and 90 °C heat inactivation. PMA (EMA) value equation for the interpretation of the viability of MNV was derived as follows: PMA (EMA) value = −log R N − log R P ( R N : the relative quantity value of the not-treated sample, and R P : the relative quantity value of the PMA- or EMA-treated sample as determined by qRT-PCR). By PMA-combined qRT-PCR, the viable PMA value was 0.32, 0.83, and 2.62 for the 65, 70, and 75 °C preheated MNVs, respectively. The viable PMA values for the viruses heated at 80, 85, and 90 °C were all greater than 3.0, which was the cutoff value for discriminating between live and dead MNVs. The results of EMA-combined qRT-PCR were similar to those of qRT-PCR. Thus, PMA-combined qRT-PCR correlated well with the plaque assay in detecting viable MNVs. [ABSTRACT FROM AUTHOR]

A series of 2,3,7-trisubstituted phenazines were synthesized using an easy, efficient, and straightforward condensation method. These compounds were tested in vitro on human pancreatic (MiaPaCa-2) cell lines and dose response curves proved that these compounds appeared to be more cytotoxic than standard gemcitabine. Standard ethidium bromide assay showed that these phenazine derivatives were not intercalating agents suggesting a new mode of action. Finally, in vivo test on mice subcutaneously implanted with MiaPaca-2 cells with phenazine 11 showed an activity similar to that of gemcitabine (Gemzar®) standard drug at a 10 times lower concentration (1 mg/kg vs 10 mg/kg for gemcitabine); this result confirms the potential of this new class of phenazines. [ABSTRACT FROM AUTHOR]

The interaction of DNA with anthragallol (Ant) was investigated using ethidium bromide (EB) as a fluorescence probe, and the binding mechanism of Ant with DNA was researched via viscosity measurements. The results indicate that there is a complex of Ant and DNA, as confirmed by Ultraviolet visible absorption spectroscopy (UV–vis), Fluorescent and Resonance Light Scattering spectrum (RLS) and viscosity measurements. Ant molecules could intercalate with the base pairs of DNA as evidenced by the hyperchromic effect of absorption spectra, the relative viscosity of DNA and significant increases in the melting temperature. The binding constants of Ant and DNA were obtained by the fluorescence quenching technique. Furthermore, the binding mechanisms of the reaction of Ant with DNA were also investigated. The RLS assay successfully evaluated the saturated value and measured the potential toxicity of Ant. Adriamycin, chrysophanol, rhein, and alizarin can be used as references to build a method based on the mechanism of interactions with DNA and the DNA-saturation binding value to rapidly evaluate the potential toxicity of Ant. [ABSTRACT FROM AUTHOR]

Periprosthetic joint infection (PJI) is one of the most dreading complications that hinder the merits of an arthroplasty. A prerequisite for treatment of the above procedure is rapid detection of live bacteria to prevent its recurrence and proper choice of antibiotics. Conventional culture methods are time-consuming and associated with a high false negative rate. Amplification of bacterial genetic materials requires a tedious process but is associated with a high false positive rate. An integrated microfluidic system capable of molecular diagnosis for detecting live bacteria was reported in our previous work. However, the system could not provide detailed information about infectious bacteria for the subsequent antibiotic choices. Furthermore, it took at least 55 min to finish the entire process. In this work, a microfluidic platform using ethidium monoazide (EMA) which can only penetrate into dead bacteria is presented for live bacteria detection and typing within a short period of time (30 min for the detection of live bacteria and another 40 min for the typing of bacteria strains). We tested the proposed system by using human joint fluid samples and found its limit of detection for bacterial detection equal to 10 2 CFU (colony formation unit) for live bacteria detection with gold nanoparticle probes and 10 2 –10 4 CFU for typing bacteria by an on-chip polymerase chain reaction. The whole procedure of the integrated microfluidic system is automated with little human intervention. Moreover, this is the first time that sequential live bacteria detection and typing are demonstrated on the same microfluidic platform. Based on the promising results, the proposed system may become in the near future an auxiliary tool for immediate medical decision and choice of antibiotics in routine arthroplasties or PJI's. [ABSTRACT FROM AUTHOR]

Mercaptopropionic acid-capped Mn-doped ZnS quantum dots/ethidium bromide (EB) nanohybrids were constructed for photoinduced electron transfer (PIET) and then used as a room-temperature phosphorescence (RTP) probe for DNA detection. EB could quench the RTP of Mn-doped ZnS QDs by PIET, thereby forming Mn-doped ZnS QDs/EB nanohybrids and storing RTP. Meanwhile, EB could be inserted into DNA and EB could be competitively desorbed from the surface of Mn-doped ZnS QDs by DNA, thereby releasing the RTP of Mn-doped ZnS QDs. Based on this mechanism, a RTP sensor for DNA detection was developed. Under optimal conditions, the detection limit for DNA was 0.045 mg L −1 , the relative standard deviation was 1.7%, and the method linear ranged from 0.2 to 20 mg L −1 . The proposed method was applied to biological fluids, in which satisfactory results were obtained. [ABSTRACT FROM AUTHOR]

Human spermatozoa are compromised by production of reactive oxygen species (ROS), and detection of ROS in spermatozoa is important for the diagnosis of male infertility. The probes 2′,7′-dichlorohydrofluorescein diacetate (DCFH), dihydroethidium (DHE), and MitoSOX red (MSR) are commonly used for detecting ROS by flow cytometry; however, these probes lack sensitivity to hydrogen peroxide (H 2 O 2 ), which is particularly damaging to mammalian sperm cells. This study reports the synthesis and use of three aryl boronate probes, peroxyfluor-1 (PF1), carboxyperoxyfluor-1, and a novel probe, 2-(2-ethoxyethoxy)ethoxyperoxyfluor-1 (EEPF1), in human spermatozoa. PF1 and EEPF1 were effective at detecting H 2 O 2 and peroxynitrite (ONOO − ) produced by spermatozoa when stimulated with menadione or 4-hydroxynonenal. EEPF1 was more effective at detection of ROS in spermatozoa than DCFH, DHE, or MSR; furthermore it distinguished poorly motile sperm as shown by greater ROS production. EEPF1 should therefore have a significant role in the diagnosis of oxidative stress in male infertility, cryopreservation, age, lifestyle, and exposure to environmental toxicants. [ABSTRACT FROM AUTHOR]

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli. [ABSTRACT FROM AUTHOR]

Abstract: The present study investigated the changes of bacterial community composition including bacterial pathogens along a biogas plant, i.e. from the influent, to the biogas reactor and to the post-digester. The effects of post-digestion temperature and time on the changes of bacterial community composition and bacterial pathogens were also studied. Microbial analysis was made by Ion Torrent sequencing of the PCR amplicons from ethidium monoazide treated samples, and ethidium monoazide was used to cleave DNA from dead cells and exclude it from PCR amplification. Both similarity and taxonomic analysis showed that the bacterial community composition in the influent was changed after anaerobic digestion. Firmicutes were dominant in all the samples, while Proteobacteria decreased in the biogas reactor compared with the influent. Variations of bacterial community composition in the biogas reactor with time were also observed. This could be attributed to varying composition of the influent. Batch experiments showed that the methane recovery from the digested residues (obtained from biogas reactor) was mainly related with post-digestion temperature. However, post-digestion time rather than temperature had a significant effect on the changes of bacterial community composition. The changes of bacterial community composition were also reflected in the changes of relative abundance of bacterial pathogens. The richness and relative abundance of bacterial pathogens were reduced after anaerobic digestion in the biogas reactor. It was found in batch experiments that bacterial pathogens showed the highest relative abundance and richness after 30 days' post-digestion. Streptococcus bovis was found in all the samples. Our results showed that special attention should be paid to the post-digestion since the increase in relative abundance of bacterial pathogens after post-digestion might reflect regrowth of bacterial pathogens and limit biosolids disposal vectors. [Copyright &y& Elsevier]

Highlights: [•] New application of partial filling affinity capillary electrophoresis was developed. [•] Strength of DNA–ligand interactions was quantitatively evaluated. [•] Binding constants of DNA–ligand complexes were determined. [•] DNA–EtBr complex was found to be stronger than DNA–oligophenylenes complexes. [Copyright &y& Elsevier]

Abstract: Four series of cassiarin A derivatives with alkanoyl (3a–3d), aroyl (4a–4d), hydroxy/amino-substituted ethylene glycol (5a–5c) and selenium-containing (6a) side chains were synthesized. Their antitumor activities were evaluated against BT474, CHAGO, HepG2, KATO-III, SW620 and CaSki cancer cell lines. Preliminary results demonstrated that 5b had moderate activities against HepG2 and KATO-III cell lines, while 5c showed moderate to high cytotoxicity against most tested cell lines. In addition, 6a exhibited moderate cytotoxicity against cervical cells, CaSki. DNA-binding and ethidium bromide displacement experiments suggested that 5c and 5b binds to DNA via an intercalative mode, whereas 6a did not. However, the selenium-containing cassiarin A derivative 6a inhibited topoisomerase II with more than 95% inhibition at the concentration of 50μM. These cassiarin A derivatives showed lower toxicity to normal cells, WI-38 than amonafide therefore they are potential lead compounds to be further developed as new anticancer agents. [Copyright &y& Elsevier]

Abstract: Aim: The purpose of this study was to investigate whether the flavonoid quercetin can prevent alterations in the behavioral tests and of cholinergic neurotransmission in rats submitted to the ethidium bromide (EB) experimental demyelination model during events of demyelination and remyelination. Main methods: Wistar rats were randomly distributed into four groups (20 animals per group): Control (pontine saline injection and treatment with ethanol), Querc (pontine saline injection and treatment with quercetin), EB (pontine 0.1% EB injection and treatment with ethanol), and EB+Querc (pontine 0.1% EB injection and treatment with quercetin). The groups Querc and Querc+EB were treated once daily with quercetin (50mg/kg) diluted in 25% ethanol solution (1ml/kg) and the animals of the control and EB groups were treated once daily with 25% ethanol solution (1ml/kg). Two stages were observed: phase of demyelination (peak on day 7) and phase of remyelination (peak on day 21 post-injection). Behavioral tests (beam walking, foot fault and inclined plane test), acetylcholinesterase (AChE) activity and lipid peroxidation in pons, cerebellum, hippocampus, hypothalamus, striatum and cerebral cortex were measured. Key findings: The quercetin promoted earlier locomotor recovery, suggesting that there was demyelination prevention or further remyelination velocity as well as it was able to prevent the inhibition of AChE activity and the increase of lipidic peroxidation, suggesting that this compound can protect cholinergic neurotransmission. Significance: These results may contribute to a better understanding of the neuroprotective role of quercetin and the importance of an antioxidant diet in humans to provide benefits in neurodegenerative diseases such as MS. [Copyright &y& Elsevier]

Highlights: [•] Spectroscopic and molecular modeling methods were used. [•] The quenching of DNA–EB system by 5-HMF was a static quenching. [•] The binding of 5-HMF to DNA was a non- intercalative binding. [•] Hydrophobic force and hydrogen bonds both played a major role. [ABSTRACT FROM AUTHOR]

Abstract: Recently, D.J. Hall et al. reported that ethidium (E+) is formed as a major product of hydroethidine (HE) or dihydroethidium reaction with superoxide (O2 −) in intact animals with low tissue oxygen levels (J. Cereb. Blood Flow Metab. 32:23–32, 2012). The authors concluded that measurement of E+ is an indicator of O2 − formation in intact brains of animals. This finding is in stark contrast to previous reports using in vitro systems showing that 2-hydroxyethidium, not ethidium, is formed from the reaction between O2 − and HE. Published in vivo results support the in vitro findings. In this study, we performed additional experiments in which HE oxidation products were monitored under different fluxes of O2 −. Results from these experiments further reaffirm our earlier findings (H. Zhao et al., Free Radic. Biol. Med. 34:1359, 2003). We conclude that whether in vitro or in vivo, E+ measured by HPLC or by fluorescence lifetime imaging is not a diagnostic marker product for O2 − reaction with HE. [Copyright &y& Elsevier]

Abstract: Background: Nearly ten years ago, we demonstrated that superoxide radical anion ( ) reacts with the hydroethidine dye (HE, also known as dihydroethidium, DHE) to form a diagnostic marker product, 2-hydroxyethidium (2-OH-E+). This particular product is not derived from reacting HE with other biologically relevant oxidants (hydrogen peroxide, hydroxyl radical, or peroxynitrite). This discovery negated the longstanding view that reacts with HE to form the other oxidation product, ethidium (E+). It became clear that due to the overlapping fluorescence spectra of E+ and 2-OH-E+, fluorescence-based techniques using the “red fluorescence” are not suitable for detecting and measuring in cells using HE or other structurally analogous fluorogenic probes (MitoSOXTM Red or hydropropidine). However, using HPLC-based assays, 2-OH-E+ and analogous hydroxylated products can be easily detected and quickly separated from other oxidation products. Scope of review: The principles discussed in this chapter are generally applicable in free radical biology and medicine, redox biology, and clinical and translational research. The assays developed here could be used to discover new and targeted inhibitors for various superoxide-producing enzymes, including NADPH oxidase (NOX) isoforms. Major conclusions: HPLC-based approaches using site-specific HE-based fluorogenic probes are eminently suitable for monitoring in intra- and extracellular compartments and in mitochondria. The use of fluorescence-microscopic methods should be avoided because of spectral overlapping characteristics of -derived marker product and other, non-specific oxidized fluorescent products formed from these probes. General significance: Methodologies and site-specific fluorescent probes described in this review can be suitably employed to delineate oxy radical dependent mechanisms in cells under physiological and pathological conditions. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. [Copyright &y& Elsevier]

Highlights: [•] Two dyes ethidium bromide and propidium iodide bind to single stranded polyriboadenylic acid. [•] Binding affinity is greater for propidium iodide than ethidium bromide. [•] Binding are characterized by both negative enthalpy and entropy changes. [•] Ionic strength dependence of binding revealed lesser electrolytic contribution to the binding process. [Copyright &y& Elsevier]

Highlights: [•] Using a SWNT-based approach 1.8nM synthetic S. typhi target DNA demonstrated the configuration. [•] The approach was highly selective and could distinguish between sequences of high homology (1- or 3-nt). [•] Using PCR, S. typhi trpS gene prepared from culture could be detected. [Copyright &y& Elsevier]

Abstract: The development of rapid and sensitive methods for the detection and quantification of Legionella viable cells is essential for monitoring water quality and preventing legionellosis. The aim of this study was to verify the applicability of a quantitative PCR (qPCR) method used in combination with ethidium monoazide (EMA) to the quantification of Legionella spp. in samples collected from swimming pools, water recirculation systems and hot water systems in two fitness clubs. This molecular technique (EMA–qPCR) allows the amplification of target DNA from culturable and viable cells, but prevents the amplification of DNA from non-viable cells. The effectiveness of this new method able to detect alive legionellae was also compared with conventional qPCR and culture method. Our results confirm that EMA–qPCR allows to discriminate the non-viable cells from those viable and that it is particularly indicated for monitoring the effectiveness of thermal treatments for the Legionella contamination control in water environments, also providing information about the presence of Viable But Non-Culturable (VBNC) cells. Other Gram-negative bacteria typically associated with biofilm were identified in samples taken from swimming pools and balance tanks, suggesting that also the presence of biofilm should be monitored for a more general view of water contamination. [Copyright &y& Elsevier]