Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ70RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ70RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RPo formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RPo formation detected by AFM, for a simple tandem gene model containing two λPR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP > Heparin or HepS > DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RPos for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies

Isolation and Characterization of Antimicrobial Compounds in Plant Extracts against Multidrug-Resistant <i>Acinetobacter baumannii</i>

<div><p>The number of fully active antibiotic options that treat nosocomial infections due to multidrug-resistant <i>Acinetobacter baumannii</i> (<i>A. baumannii</i>) is extremely limited. <i>Magnolia officinalis</i>, <i>Mahonia bealei</i>, <i>Rabdosia rubescens</i>, <i>Rosa rugosa</i>, <i>Rubus chingii</i>, <i>Scutellaria baicalensis</i>, and <i>Terminalia chebula</i> plant extracts were previously shown to have growth inhibitory activity against a multidrug-resistant clinical strain of <i>A. baumannii</i>. In this study, the compounds responsible for their antimicrobial activity were identified by fractionating each plant extract using high performance liquid chromatography, and determining the antimicrobial activity of each fraction against <i>A. baumannii</i>. The chemical structures of the fractions inhibiting >40% of the bacterial growth were elucidated by liquid chromatography/mass spectrometry analysis and nuclear magnetic resonance spectroscopy. The six most active compounds were identified as: ellagic acid in <i>Rosa rugosa</i>; norwogonin in <i>Scutellaria baicalensis</i>; and chebulagic acid, chebulinic acid, corilagin, and terchebulin in <i>Terminalia chebula</i>. The most potent compound was identified as norwogonin with a minimum inhibitory concentration of 128 µg/mL, and minimum bactericidal concentration of 256 µg/mL against clinically relevant strains of <i>A. baumannii</i>. Combination studies of norwogonin with ten anti-Gram negative bacterial agents demonstrated that norwogonin did not enhance the antimicrobial activity of the synthetic antibiotics chosen for this study. In conclusion, of all identified antimicrobial compounds, norwogonin was the most potent against multidrug-resistant <i>A. baumannii</i> strains. Further studies are warranted to ascertain the prophylactic and therapeutic potential of norwogonin for infections due to multidrug-resistant <i>A. baumannii</i>.</p></div

Determination of antimicrobial activity of purified compounds from plant extracts against two <i>A.</i> baumannii<i> </i> strains.

<p>Two-fold serially diluted norwogonin (<b>A</b>), terchebulin (<b>B</b>), chebulagic acid (<b>C</b>) and corilagin (<b>D</b>) suspensions were prepared in cation-adjusted Mueller-Hinton broth and mixed with an equal volume of either strain 31P or BAA-1605 suspension (5×10⁵ CFU/mL final). Bacterial growth was measured after a 16 h incubation at 37°C. The final test concentration for each compound ranged from 0.25 to 128 µg/mL for norwogonin (MIC₉₀ = 128 µg/mL), and 7.8 to 1,000 µg/mL for terchebulin (MIC₉₀ = 500 µg/mL), chebulagic acid and corilagin. • 31P and ▴ BAA-1605.</p

Dose response testing of synthetic anti-Gram negative bacterial agents in combination with norwogonin.

<p>The IC₉₀ (µg/mL) of each antibiotic either alone or in combination with 8 or 16 µg/mL norwogonin against strain 31P were determined. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061594#s3" target="_blank">Results</a> are presented as the average IC₉₀ for two experiments each done in duplicate.</p>*<p>IC₉₀ for trimethoprim/sulfamethoxazole could not be determined as maximum inhibition remained below 90% at all concentrations tested.</p

MIC₉₀ determination of purified norwogonin against three clonally distinct strains of <i>A. baumannii</i>.

<p>MIC₉₀ of norwogonin against 31P, 125P and 152P was determined by measuring OD_{600 nm} (<b>A</b>) and was confirmed by resazurin reduction assay (<b>B</b>).</p

Time-kill kinetic analysis of norwogonin against 31P.

<p>The time-kill kinetics of 31P by norwogonin at 1× and 2× MIC was studied over a 24 h incubation. Aliquots were collected at 0, 4, 8 and 24 h, serially diluted in phosphate buffered saline before plating on Mueller-Hinton agar plates. Surviving colonies were enumerated after an 18 h incubation at 37°C. * Estimate: no colonies were observed at the highest concentration plated.</p

Enrichment of (8,4) Single-Walled Carbon Nanotubes Through Coextraction with Heparin

Heparin sodium salt is investigated as a dispersant for dispersing singlewalled carbon nanotubes (SWNTs). Photoluminescence excitation (PLE) spectroscopy is used for identification and abundance estimation of the chiral species. It is found that heparin sodium salt preferentially disperses larger-diameter Hipeo SWNTs. When used to disperse CoMoCAT nanotube samples, heparin has a strong preference for (8,4) tubes, which have larger diameter than the predominant (6,5) in pristine CoMoCA T samples. PLE intensity due to (8,4) tubes increases from 7% to 60% of the total after threefold extractions. Computer modeling verifies that the complex of (8,4) SWNTs and heparin has the lowest binding energy amongst the four semiconducting species present in CoMoCA T. Network field-effect transistors are successfully made with CoMoCAT/heparin and CoMoCAT/sodium dodecylbenzene sulfonate (SDBS)-heparin (x3), confirming the easy removability of heparin.close212