Comprehensive assessment of 12 commercial DNA-binding dyes as alternatives to ethidium bromide for agarose gel electrophoresis.

Staining and visualization of the nucleic acid bands on agarose gels using ethidium bromide (EB) has been a widely used technique in molecular biology. Although it is an efficient dye for this purpose, EB is known to be mutagenic and genotoxic in humans. This led to the emergence of various alternative dyes, which were claimed to be safer and more efficient than EB. However, these dyes portray varied sensitivity and interference with the electrophoretic mobility of nucleic acids. This work aimed at assessing ten nucleic acid-binding dyes and two prestained dyes for these properties by three staining techniques, such as precasting, preloading, and poststaining. Of these, preloading was not suitable for any of the dye while poststaining worked optimal for most of them. Precasting was suitable for only four dyes viz. DNA Stain G, SYBR™ safe, EZ-Vision® in-gel, and LabSafe™. Poststaining was, in general, a costlier method than precasting. The work gives a comprehensive understanding of the performance of nucleic acid-binding dyes for routine molecular biology experiments.

Deep mutational scan of a drug efflux pump reveals its structure-function landscape.

Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational scanning (DMS) on the bacterial ABC transporter EfrCD to determine the drug efflux activity profile of more than 1,430 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity toward positively charged ethidium, whereas additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryogenic electron microscopy structure of EfrCD uncovered a high-affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert EfrCD into a drug-specific ABC importer.

Mode of interaction of altretamine with calf thymus DNA: biophysical insights.

Insights into drug-DNA interactions have importance in medicinal chemistry as it has a major role in the evolution of new therapeutic drugs. Therefore, binding studies of small molecules with DNA are of significant interest. Spectroscopy, coupled with measurements of viscosity and molecular docking studies were employed to obtain mechanistic insights into the binding of altretamine with calf thymus DNA (CT-DNA). The UV-visible spectroscopic measurements study confirmed altretamine-CT-DNA complex formation with affinity constant ([15.68 ± 0.04] × 103 M-1), a value associated with groove binding phenomenon. The associated thermodynamic signatures suggest enthalpically driven interactions. The values of standard molar free energy change (ΔGmo) -(23.93 ± 0.23) kJ mol-1, enthalpy change (ΔvHHmo) -(50.84 ± 0.19) kJ mol-1 and entropy change (ΔSmo) -(90.29 ± 0.12) JK-1 mol-1 indicate the binding is thermodynamically favorable and an important role of the hydrogen bonds and Van der Waals interactions in the binding of altretamine with CT-DNA. Circular dichroism spectroscopy indicated insignificant conformational changes in the DNA backbone upon interaction with altretamine suggesting no distortion and/or unstacking of the base pairs in the DNA helix. UV-melting study suggested that the thermal stability of the DNA backbone is not affected by the binding of the drug. Competitive displacement assays with ethidium bromide, Hoechst-33258 and DAPI established the binding of altretamine with CT-DNA in the minor groove. The mode of binding was further confirmed by viscosity and molecular docking studies. Molecular docking further ascertained binding of altretamine in the minor groove of the CT-DNA, preferably with the A-T rich sequences.[Formula: see text]HighlightsAltretamine binds CT-DNA which is enthalpically driven with Ka of the order of 103Insignificant conformational change is observed due to DNA-altretamine complexationAltretamine binds favorably with A-T rich sequences in the minor groove of CT-DNAMechanistic insights obtained based on thermodynamic signaturesCommunicated by Ramaswamy H. Sarma.

Adsorption and removal of ethidium bromide from aqueous solution using optimized biogenic catalytically active antibacterial palladium nanoparticles.

Due to being low cost and eco-friendly, biological nanomaterial synthesis and development have made broad spectral progress. This study aimed to optimize the phytomediated synthesis of catalytically active, antibacterial palladium nanoparticles (PdNPs) for adsorption-based removal of ethidium bromide (EtBr) from an aqueous solution. Optimization of synthesis demonstrated that a precursor to extract ratio of 4:1, pH 3, and incubation at 80 °C for 60 min were the optimum conditions that led to the synthesis of negatively charged, highly stable, polycrystalline, spherical, and monodispersed PdNPs of 5-10 nm. When tested as catalysts, PdNPs successfully catalyzed Suzuki-Miyaura cross-coupling between aryl halides and arylboronic acids resulting in the synthesis of 4-acetylbiphenyl. Furthermore, the antibacterial activity test demonstrated that biogenic PdNPs were most effective and potent against Staphylococcus aureus and Proteus vulgaris followed by Escherichia coli, Bacillus subtilis, and Bacillus cereus. In addition, PdNPs were found as an excellent adsorbent for adsorption of EtBr from water as the adsorption reaction obeyed pseudo-second-order kinetics with a linear regression coefficient (R2 > 0.995). The adsorption reaction fitted well with the Freundlich and Temkin isotherm models, indicating multi-layer adsorption. Estimating thermodynamic parameters resulted in a positive value of ΔH0 and ΔG0, demonstrating adsorption was non-spontaneous and endothermic.

Application of the SwitchSense Technique for the Study of Small Molecules' (Ethidium Bromide and Selected Sulfonamide Derivatives) Affinity to DNA in Real Time.

The discovery and introduction of the switchSense technique in the chemical laboratory have drawn well-deserved interest owing to its wide range of applications. Namely, it can be used to determine the diameter of proteins, alterations in their tertiary structures (folding), and many other conformational changes that are important from a biological point of view. The essence of this technique is based on its ability to study of the interactions between an analyte and a ligand in real time (in a buffer flow). Its simplicity, on the other hand, is based on the use of a signaling system that provides information about the ongoing interactions based on the changes in the fluorescence intensity. This technique can be extremely advantageous in the study of new pharmaceuticals. The design of compounds with biological activity, as well as the determination of their molecular targets and modes of interactions, is crucial in the search for new drugs and the fight against drug resistance. This article presents another possible application of the switchSense technique for the study of the binding kinetics of small model molecules such as ethidium bromide (EB) and selected sulfonamide derivatives with DNA in the static and dynamic modes at three different temperatures (15, 25, and 37 °C) each. The experimental results remain in very good agreement with the molecular dynamics docking ones. These physicochemical insights and applications obtained from the switchSense technique allow for the design of an effective strategy for molecular interaction assessments of small but pharmaceutically important molecules with DNA.

In vitro biological activity of copper(II) complexes with NSAIDs and nicotinamide: Characterization, DNA- and BSA-interaction study and anticancer activity.

Through the reaction of copper(II) acetate with nicotinamide (pyridine-3-carboxylic acid amide, niacinamide) and some derivatives of N-phenylanthranilic acid (fenamates), seven new mixed-ligand copper(II) compounds were isolated: [Cu(tolf-O)(tolf-O,O')nia-N)2(EtOH)] (1), [Cu(tolf-O)(tolf-O,O')(nia-N)2(MeOH)] (2), [Cu(meclf-O)(meclf-O,O')(nia-N)2(EtOH)] (3), [Cu(meclf-O)(meclf-O,O')(nia-N)2(MeOH)] (4), [Cu(meclf-O)(meclf-O,O')(nia-N)2(ACN)] (5), [Cu(mef-O)(mef-O,O')(nia-N)2(EtOH)] (6) and [Cu(mef-O)(mef-O,O')(nia-N)2(ACN)] (7) containing a molecule of relevant solvent as ligand in their primary crystal structure (tolf = tolfenamate, meclf = meclofenamate, mef = mefenamate, nia = nicotinamide, EtOH = ethanol, MeOH = methanol, ACN = acetonitrile). The structures of the complexes were determined by single-crystal X-ray analysis. The intermolecular interactions were studied by Hirshfeld surface analysis. The complexes were characterized by IR, UV-vis and EPR spectroscopy and their redox properties were determined by cyclic voltammetry. The interaction of the complexes with bovine serum albumin was studied by fluorescence emission spectroscopy and the albumin-binding constants of the compounds were calculated. The interaction of the complexes with calf-thymus DNA was monitored by diverse techniques (UV-vis spectroscopy, cyclic voltammetry, viscosity measurements) suggesting intercalation as the most possible mode of binding. DNA-competitive studies of the complexes with ethidium bromide were monitored by fluorescence emission spectroscopy. The cytotoxic effects of copper(II) complexes on lung carcinoma cells and healthy cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric technique.

G-Quadruplex binding of cavity-containing anthraquinonesulfonyl-ß-cyclodextrin conjugate. Effect of encapsulation of ethidium bromide and berberine.

An anthraquinonesulfonyl derivative of ß-cyclodextrin is prepared and characterized employing spectroscopic techniques. The binding interactions of the compound with ethidium bromide, berberine, calf-thymus DNA, quadruplex DNAs viz., kit22, telo24, and myc22 are investigated by ultraviolet-visible, and fluorescence spectroscopic methods. Anthraquinonesulfonyl-ß-cyclodextrin conjugate acts as a host molecule and enhances ethidium bromide and berberine fluorescence due to their encapsulation in cyclodextrin's cavity. The binding constant values are 9.0 × 105 mol-1 dm3 and 5.7 × 104 mol-1 dm3 for the formation of host: guest complexes of the ß-CD derivative with ethidium bromide and berberine respectively. The proximity of the protons of ethidium bromide and berberine protons with those of the internal cavity of ß-CD in the anthraquinonesulfonyl-ß-CD conjugate is confirmed by two-dimensional rotating-frame Overhauser effect spectroscopy. The conjugate displays a quenching of fluorescence selectively to the quadruplexes kit22 and telo24 that is contrast to the spectral behavior with duplex DNA. ctDNA and myc22 exhibit different absorption and emission profiles with ethidium bromide on encapsulation by ß-CD. The encapsulation of berberine leads to a fluorescence enhancement on binding to ctDNA, telo24, and myc22 with binding constants of 5.6 × 105, 3.3 × 105 mol-1 dm3, and 1.5 × 105 mol-1 dm3 respectively. In contrast, kit22 leads to fluorescence quenching on berberine encapsulated-anthraquinonesulfonyl-ß-cyclodextrin conjugate with a Stern-Volmer constant of 3.3 × 105 mol-1 dm3.Communicated by Ramaswamy H. Sarma.

Modulating the chemo-mechanical response of structured DNA assemblies through binding molecules.

Recent advances in DNA nanotechnology led the fabrication and utilization of various DNA assemblies, but the development of a method to control their global shapes and mechanical flexibilities with high efficiency and repeatability is one of the remaining challenges for the realization of the molecular machines with on-demand functionalities. DNA-binding molecules with intercalation and groove binding modes are known to induce the perturbation on the geometrical and mechanical characteristics of DNA at the strand level, which might be effective in structured DNA assemblies as well. Here, we demonstrate that the chemo-mechanical response of DNA strands with binding ligands can change the global shape and stiffness of DNA origami nanostructures, thereby enabling the systematic modulation of them by selecting a proper ligand and its concentration. Multiple DNA-binding drugs and fluorophores were applied to straight and curved DNA origami bundles, which demonstrated a fast, recoverable, and controllable alteration of the bending persistence length and the radius of curvature of DNA nanostructures. This chemo-mechanical modulation of DNA nanostructures would provide a powerful tool for reconfigurable and dynamic actuation of DNA machineries.

Ex vivo imaging and analysis of ROS generation correlated with microglial activation in rat model with acute neuroinflammation induced by intrastriatal injection of LPS.

Neuroinflammation and oxidative stress are hallmarks of neurodegenerative diseases. Microglia, the major important regulators of neuroinflammation, are activated in response to excessive generation of reactive oxygen species (ROS) from damaged cells and resulting in elevated and sustained damages. However, the relationship between microglia and ROS-regulatory system in the early stages of neuroinflammation prior to the appearance of neuronal damages have not been elucidated in detail. In this study, we analyzed the time-dependent changes in ROS generation during acute neuroinflammation in rats that were given an intrastriatal injection of lipopolysaccharide (LPS). We evaluated the effects of minocycline, an anti-inflammatory antibiotic, and N,N'-dimethylthiourea (DMTU), a radical scavenger, to understand the correlation between activated microglia and ROS generation. Ex vivo fluorescence imaging using dihydroethidium (DHE) clearly demonstrated an increased ROS level in the infused side of striatum in the rats treated with LPS. The level of ROS was changed in time-dependent manner, and the highest level of ROS was observed on day 3 after the infusion of LPS. Immunohistochemical studies revealed that time-dependent changes in ROS generation were well correlated to the presence of activated microglia. The inhibition of microglial activation by minocycline remarkably reduced ROS levels in the LPS-injected striatum, which indicated that the increased ROS generation caused by LPS was induced by activated microglia. DMTU decreased ROS generation and resulted in remarkable inhibitory effect on microglial activation. This study demonstrated that ROS generation during acute neuroinflammation induced by LPS was considerably associated with microglial activation, in an intact rat brain. The results provides a basis for understanding the interaction of ROS-regulatory system and activated microglia during neuroinflammation underlying neurodegenerative diseases.

Ethidium Binding to Salmonella enterica ser. Typhimurium Cells and Salmon Sperm DNA.

Bacterial resistance to antibiotics due to increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for efflux analysis are needed. Here, we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et+) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenylalanyl-arginyl-ß-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et+ concentration. The results of our experiments indicated that Et+ fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et+, the intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et+. Measurements of Et+ interaction with the purified DNA demonstrated that the affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et+ considerably decreases in the presence of Mg2+, Polymyxin B or when DNA is incubated in high ionic strength media.

Natural DNA assisted white light generation and stimuli responsive colour tuning.

The unique structure of a natural nucleic acid, calf thymus DNA, which can provide an appropriate scaffold for an efficient cascaded energy transfer among organic chromophores, has been used for the generation of bright and pure white light on UV light excitation. Two most commonly used DNA stains, 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide (EB) have been used as a part of the donor-acceptor pairs. We have judiciously selected 10-anthracene-10-yl-3-methylbenzothiazol-3-ium chloride (AnMBTZ), an ultrafast molecular rotor, to act as a bridge between DNA bound DAPI and EB for the cascaded flow of energy. The unique molecular rotor properties of AnMBTZ and its exceptional binding ability with natural DNA help to form a distinct tri-chromophoric system in DNA template which can produce bright and pure white light on UV excitation. Detailed flow of energy from photoexcited DAPI to EB via AnMBTZ has been explored using steady state and time-resolved emission spectroscopy. Further, unique binding nature of AnMBTZ with DNA molecules has been used to modulate the colour of the emission from the present tri-chromophoric system by external stimuli, like salt and temperature. Such unique stimuli responsive multi-chromophoric system in a bio-template has great potential for different lightening applications.

Stabilization and structural changes of 2D DNA origami by enzymatic ligation.

The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.

Synthesis, electrochemistry, DNA binding and in vitro cytotoxic activity of tripodal ferrocenyl bis-naphthalimide derivatives.

A series of tripodal ferrocenyl bis-naphthalimide derivatives were synthesized and characterized. All of the bis-naphthalimide derivatives exhibited good DNA binding ability which was confirmed by ethidium bromide (EB) displacement experiment and ultraviolet (UV)-visible absorption titration. And the binding mode of these compounds was proved to be a hybrid binding mode by experiments. The cytotoxicity of synthesized compounds against 4 different human cancer cell lines (EC109, BGC823, SGC7901 and HEPG2) was evaluated by thiazolyl blue tetrazolium bromide (MTT) assay. All of the bis-naphthalimide derivatives exhibited good anticancer activity than the positive control drug (amonafide), which was due to the promotion of reactive oxygen species (ROS) level in test cancer cells by the reversible one-electron redox process of ferrocenyl bis-naphthalimide derivatives. Although there was no obvious relationship between the binding constants and the chain length, the structure cytotoxicity relationship revealed that the linker of n = 3, m = 1 was the best choice for the tested tripodol bis-naphthalimide derivatives. SYNOPSIS: A series of tripodal ferrocenyl bis-naphthalimide derivatives were synthesized to study the DNA binding ability and the cytotoxicity induced by reactive oxygen species. All of the compounds exhibited good DNA binding ability. And the structure cytotoxicity relationship revealed that the structure of 5h was the best choice.

Ethidium bromide interactions with DNA: an exploration of a classic DNA-ligand complex with unbiased molecular dynamics simulations.

Visualization of double stranded DNA in gels with the binding of the fluorescent dye ethidium bromide has been a basic experimental technique in any molecular biology laboratory for >40 years. The interaction between ethidium and double stranded DNA has been observed to be an intercalation between base pairs with strong experimental evidence. This presents a unique opportunity for computational chemistry and biomolecular simulation techniques to benchmark and assess their models in order to see if the theory can reproduce experiments and ultimately provide new insights. We present molecular dynamics simulations of the interaction of ethidium with two different double stranded DNA models. The first model system is the classic sequence d(CGCGAATTCGCG)2 also known as the Drew-Dickerson dodecamer. We found that the ethidium ligand binds mainly stacked on, or intercalated between, the terminal base pairs of the DNA with little to no interaction with the inner base pairs. As the intercalation at the terminal CpG steps is relatively rapid, the resultant DNA unwinding, rigidification, and increased stability of the internal base pair steps inhibits further intercalation. In order to reduce these interactions and to provide a larger groove space, a second 18-mer DNA duplex system with the sequence d(GCATGAACGAACGAACGC) was tested. We computed molecular dynamics simulations for 20 independent replicas with this sequence, each with ∼27 µs of sampling time. Results show several spontaneous intercalation and base-pair eversion events that are consistent with experimental observations. The present work suggests that extended MD simulations with modern DNA force fields and optimized simulation codes are allowing the ability to reproduce unbiased intercalation events that we were not able to previously reach due to limits in computing power and the lack of extensively tested force fields and analysis tools.

Mechanisms and pathways of ethidium bromide Fenton-like degradation by reusable magnetic nanocatalysts.

Ethidium bromide (3,8-diamino-6-phenyl-5-ethylphenanthridinium bromide, EtBr) is a carcinogenic compound widely used for staining nucleic acids that is difficult to treat. In this study, magnetic nanocatalysts (MNCs) were synthesized for the heterogeneous Fenton-like degradation of EtBr. The initial pH, MNC content, and H2O2 concentration were the key factors affecting the EtBr degradation performance and dynamics. An EtBr removal efficiency of 98.97% was achieved within 4 h under optimal conditions (initial pH, 3.0; MNC content, 1 g/L; H2O2 concentration, 50 mM), and the degradation followed the ring-open pathway with (2E,4Z,8E)-3-amino-N-ethyl-7,9-dihydroxynona-2,4,8-trienamide as an intermediate, as determined by liquid chromatography and mass spectrometry (LC/MS). Unexpected and satisfactory Fenton-like oxidation of EtBr occurred under basic conditions, which was explained by a novel denitration pathway with 2-[nitro(phenyl)methyl]-(1,1'-biphenyl)-4,4'-diamine as an intermediate. The MNCs retained 62.17% of their degradation efficiency after five consecutive reaction and harvest cycles. Our work elucidated the mechanisms and pathways of EtBr removal in a Fenton-like reaction using MNCs, and comprehensively discussed the optimal reaction conditions and its potential for re-use.

Novel indole-thiazole and indole-thiazolidinone derivatives as DNA groove binders.

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.

Diosgenin Loaded Polymeric Nanoparticles with Potential Anticancer Efficacy.

This study aims to determine the anticancer efficacy of diosgenin encapsulated poly-glycerol malate co-dodecanedioate (PGMD) nanoparticles. Diosgenin loaded PGMD nanoparticles (variants 7:3 and 6:4) were synthesized by the nanoprecipitation method. The synthesis of PGMD nanoparticles was systematically optimized employing the Box-Behnken design and taking into account the influence of various independent variables such as concentrations of each PGMD, diosgenin and PF-68 on the responses such as size and PDI of the particles. Mathematical modeling was done using the Quadratic second order modeling method and response surface analysis was undertaken to elucidate the factor-response relationship. The obtained size of PGMD 7:3 and PGMD 6:4 nanoparticles were 133.6 nm and 121.4 nm, respectively, as measured through dynamic light scattering (DLS). The entrapment efficiency was in the range of 77-83%. The in vitro drug release studies showed diffusion and dissolution controlled drug release pattern following Korsmeyer-Peppas kinetic model. Furthermore, in vitro morphological and cytotoxic studies were performed to evaluate the toxicity of synthesized drug loaded nanoparticles in model cell lines. The IC50 after 48 h was observed to be 27.14 µM, 15.15 µM and 13.91 µM for free diosgenin, PGMD 7:3 and PGMD 6:4 nanoparticles, respectively, when administered in A549 lung carcinoma cell lines.

Polyacrylamide Gel Electrophoresis.

Cross-linked chains of polyacrylamide can be used as electrically neutral gels to separate double-stranded DNA fragments according to size and single-stranded DNAs according to size and conformation. Polyacrylamide gels have the following three major advantages over agarose gels: (1) Their resolving power is so great that they can separate molecules of DNA whose lengths differ by as little as 0.1% (i.e., 1 bp in 1000 bp). (2) They can accommodate much larger quantities of DNA than agarose gels. Up to 10 µg of DNA can be applied to a single slot (1 cm × 1 mm) of a typical polyacrylamide gel without significant loss of resolution. (3) DNA recovered from polyacrylamide gels is extremely pure and can be used for the most demanding purposes (e.g., microinjection of mouse embryos). However, polyacrylamide gels have the disadvantage of being more difficult to prepare and handle than agarose gels. Methods are presented here for preparing and running nondenaturing polyacrylamide gels and for detection of DNA in these gels by staining.

Identification of novel inhibitors of the ABC transporter BmrA.

The resistance of microbes to commonly used antibiotics has become a worldwide health problem. A major underlying mechanism of microbial antibiotic resistance is the export of drugs from bacterial cells. Drug efflux is mediated through the action of multidrug resistance efflux pumps located in the bacterial cell membranes. The critical role of bacterial efflux pumps in antibiotic resistance has directed research efforts to the identification of novel efflux pump inhibitors that can be used alongside antibiotics in clinical settings. Here, we aimed to find potential inhibitors of the archetypical ATP-binding cassette (ABC) efflux pump BmrA of Bacillus subtilis via virtual screening of the Mu.Ta.Lig. Chemotheca small molecule library. Molecular docking calculations targeting the nucleotide-binding domain of BmrA were performed using AutoDock Vina. Following a further drug-likeness filtering step based on Lipinski's Rule of Five, top 25 scorers were identified. These ligands were then clustered into separate groups based on their contact patterns with the BmrA nucleotide-binding domain. Six ligands with distinct contact patterns were used for further in vitro inhibition assays based on intracellular ethidium bromide accumulation. Using this methodology, we identified two novel inhibitors of BmrA from the Chemotheca small molecule library.

Selective Inhibition of Coxiella burnetii Replication by the Steroid Hormone Progesterone.

Coxiella burnetii is a zoonotic bacterial obligate intracellular parasite and the cause of query (Q) fever. During natural infection of female animals, C. burnetii shows tropism for the placenta and is associated with late-term abortion, at which time the pathogen titer in placental tissue can exceed one billion bacteria per gram. During later stages of pregnancy, placental trophoblasts serve as the major source of progesterone, a steroid hormone known to affect the replication of some pathogens. During infection of placenta-derived JEG-3 cells, C. burnetii showed sensitivity to progesterone but not the immediate precursor pregnenolone or estrogen, another major mammalian steroid hormone. Using host cell-free culture, progesterone was determined to have a direct inhibitory effect on C. burnetii replication. Synergy between the inhibitory effect of progesterone and the efflux pump inhibitors verapamil and 1-(1-naphthylmethyl)-piperazine is consistent with a role for efflux pumps in preventing progesterone-mediated inhibition of C. burnetii activity. The sensitivity of C. burnetii to progesterone, but not structurally related molecules, is consistent with the ability of progesterone to influence pathogen replication in progesterone-producing tissues.