Reversible DNA i-motif to hairpin switching induced by copper(II) cations

i-Motif DNA structures have previously been utilised for many different nanotechnological applications, but all have used changes in pH to fold the DNA. Herein we describe how copper(ii) cations can alter the conformation of i-motif DNA into an alternative hairpin structure which is reversible by chelation with EDTA

Redox-dependent control of i-Motif DNA structure using copper cations

Previous computational studies have shown that Cu+ can act as a substitute for H+ to support formation of cytosine (C) dimers with similar conformation to the hemi-protonated base pair found in i-motif DNA. Through a range of biophysical methods, we provide experimental evidence to support the hypothesis that Cu+ can mediate C–C base pairing in i-motif DNA and preserve i-motif structure. These effects can be reversed using a metal chelator, or exposure to ambient oxygen in the air that drives oxidation of Cu+ to Cu2+, a comparatively weak ligand. Herein, we present a dynamic and redox-sensitive system for conformational control of an i-motif forming DNA sequence in response to copper cations

Capture of Escherichia coli O157:H7 Using Immunomagnetic Beads of Different Size and Antibody Conjugating Chemistry

Immunomagnetic beads (IMB) were synthesized using anti-Escherichia coli O157 antibodies and magnetic beads of two different sizes (1 μm and 2.6 to 2.8 μm) that contained a streptavidin coating, activated carboxyl groups or tosylated surfaces. The synthesized IMB, together with a commercially available IMB, were used to capture different strains of E. coli O157:H7 and E. coli O157:NM. The E. coli capture was measured by the time resolved fluorescence (TRF) intensity using a sandwich assay which we have previously demonstrated of having a sensitivity of 1 CFU/g after 4.5 hour enrichment [1]. The analyses of measured TRF intensity and determined antibody surface concentration indicated that larger beads provided higher response signals than smaller beads and were more effective in capturing the target of interest in pure culture and ground beef. In addition, while each type of IMB showed different favorable capture of E. coli O157:H7, streptavidin-coated IMB elicited the highest response, on average. Streptavidin-coated IMB also provided an economic benefit, costing less than $0.50 per assay. The results could be used to guide the proper choice of IMB for applications in developing detection processes for E. coli O157:H7

Advances in Foodborne Pathogen Analysis

As the world population has grown, new demands on the production of foods have been met by increased efficiencies in production, from planting and harvesting to processing, packaging and distribution to retail locations. These efficiencies enable rapid intranational and global dissemination of foods, providing longer “face time” for products on retail shelves and allowing consumers to make healthy dietary choices year-round. However, our food production capabilities have outpaced the capacity of traditional detection methods to ensure our foods are safe. Traditional methods for culture-based detection and characterization of microorganisms are time-, labor- and, in some instances, space- and infrastructure-intensive, and are therefore not compatible with current (or future) production and processing realities. New and versatile detection methods requiring fewer overall resources (time, labor, space, equipment, cost, etc.) are needed to transform the throughput and safety dimensions of the food industry. Access to new, user-friendly, and point-of-care testing technologies may help expand the use and ease of testing, allowing stakeholders to leverage the data obtained to reduce their operating risk and health risks to the public. The papers in this Special Issue on “Advances in Foodborne Pathogen Analysis” address critical issues in rapid pathogen analysis, including preanalytical sample preparation, portable and field-capable test methods, the prevalence of antibiotic resistance in zoonotic pathogens and non-bacterial pathogens, such as viruses and protozoa

Apparent Thixotropic Properties of Saline/Glycerol Drops with Biotinylated Antibodies on Streptavidin-Coated Glass Slides: Implications for Bacterial Capture on Antibody Microarrays

The thixotropic-like properties of saline/glycerol drops, containing biotinylated capture antibodies, on streptavidin-coated glass slides have been investigated, along with their implications for bacterial detection in a fluorescent microarray immunoassay. The thixotropic-like nature of 60:40 saline-glycerol semisolid droplets (with differing amounts of antibodies) was observed when bacteria were captured, and their presence detected using a fluorescently-labeled antibody. Semisolid, gel-like drops of biotinylated capture antibody became liquefied and moved, and then returned to semisolid state, during the normal immunoassay procedures for bacterial capture and detection. Streaking patterns were observed that indicated thixotropic-like characteristics, and this appeared to have allowed excess biotinylated capture antibody to participate in bacterial capture and detection. When developing a microarray for bacterial detection, this must be considered for optimization. For example, with the appropriate concentration of antibody (in this study, 0.125 ng/nL), spots with increased diameter at the point of contact printing (and almost no streaking) were produced, resulting in a maximal signal. With capture antibody concentrations greater than 0.125 ng/nL, the excess biotinylated capture antibody (i.e., that which was residing in the three-dimensional, semisolid droplet space above the surface) was utilized to capture more bacteria. Similarly, when the immunoassay was performed within a hydrophobic barrier (i.e., without a coverslip), brighter spots with increased signal were observed. In addition, when higher concentrations of cells (∼108 cells/mL) were available for capture, the importance of unbound capture antibody in the semisolid droplets became apparent because washing off the excess, unbound biotinylated capture antibody before the immunoassay was performed reduced the signal intensity by nearly 50%. This reduction in signal was not observed with lower concentrations of cells (∼106 cells/mL). With increased volumes of capture antibody, abnormal spots were visualized, along with decreased signal intensity, after bacterial detection, indicating that the increased droplet volume detrimentally affected the immunoassay

Study on the mechanism of antibacterial action of magnesium oxide nanoparticles against foodborne pathogens

BACKGROUND: Magnesium oxide nanoparticles (MgO nanoparticles, with average size of 20 nm) have considerable potential as antimicrobial agents in food safety applications due to their structure, surface properties, and stability. The aim of this work was to investigate the antibacterial effects and mechanism of action of MgO nanoparticles against several important foodborne pathogens. RESULTS: Resazurin (a redox sensitive dye) microplate assay was used for measuring growth inhibition of bacteria treated with MgO nanoparticles. The minimal inhibitory concentrations of MgO nanoparticles to 10(4) colony-forming unit/ml (CFU/ml) of Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella Enteritidis were determined to be 0.5, 1 and 1 mg/ml, respectively. To completely inactivate 10(8−9) CFU/ml bacterial cells in 4 h, a minimal concentration of 2 mg/ml MgO nanoparticles was required for C. jejuni whereas E. coli O157:H7 and Salmonella Enteritidis required at least 8 mg/ml nanoparticles. Scanning electron microscopy examination revealed clear morphological changes and membrane structural damage in the cells treated with MgO nanoparticles. A quantitative real-time PCR combined with ethidium monoazide pretreatment confirmed cell membrane permeability was increased after exposure to the nanoparticles. In a cell free assay, a low level (1.1 μM) of H(2)O(2) was detected in the nanoparticle suspensions. Consistently, MgO nanoparticles greatly induced the gene expression of KatA, a sole catalase in C. jejuni for breaking down H(2)O(2) to H(2)O and O(2). CONCLUSIONS: MgO nanoparticles have strong antibacterial activity against three important foodborne pathogens. The interaction of nanoparticles with bacterial cells causes cell membrane leakage, induces oxidative stress, and ultimately leads to cell death

Antimicrobial activity of spherical silver nanoparticles prepared using a biocompatible macromolecular capping agent: evidence for induction of a greatly prolonged bacterial lag phase

<p>Abstract</p> <p>Background</p> <p>We have evaluated the antimicrobial properties of Ag-based nanoparticles (<it>Np</it>s) using two solid phase bioassays and found that 10-20 μL of 0.3-3 μM keratin-stabilized <it>Np</it>s (depending on the starting bacterial concentration = <it>C</it>_I) completely inhibited the growth of an equivalent volume of <it>ca</it>. 10³to 10⁴colony forming units per mL (CFU mL^-1) <it>Staphylococcus aureus</it>, <it>Salmonella </it>Typhimurium, or <it>Escherichia coli </it>O157:H7 on solid surfaces. Even after one week at 37°C on solid media, no growth was observed. At lower <it>Np </it>concentrations (= [<it>Np</it>]s), visible colonies were observed but they eventually ceased growing.</p> <p>Results</p> <p>To further study the physiology of this growth inhibition, we repeated these experiments in liquid phase by observing microbial growth via optical density at 590 nm (OD) at 37°C in the presence of a [<it>Np</it>] = 0 to 10^-6M. To extract various growth parameters we fit all OD[t] data to a common sigmoidal function which provides measures of the beginning and final OD values, a first-order rate constant (<it>k</it>), as well as the time to calculated 1/2-maximal OD (<it>t</it>_m) which is a function of <it>C</it>_I, <it>k</it>, as well as the microbiological lag time (<it>T</it>).</p> <p>Performing such experiments using a 96-well microtitre plate reader, we found that growth <it>always </it>occurred in solution but <it>t</it>_mvaried between 7 (controls; <it>C</it>_I= 8 × 10³CFU mL^-1) and > 20 hrs using either the citrate-([<it>Np</it>] ~ 3 × 10^-7M) or keratin-based ([<it>Np</it>] ~ 10^-6M) <it>Np</it>s and observed that {∂<it>t</it>_m/∂ [<it>Np</it>]}_citrate~ 5 × 10⁷and {∂<it>t</it>_m/∂ [<it>Np</it>]}_keratin~ 10⁷hr·L mol^-1. We also found that there was little effect of <it>Np</it>s on <it>S. aureus </it>growth rates which varied only between <it>k </it>= 1.0 and 1.2 hr^-1(1.1 ± 0.075 hr^-1). To test the idea that the <it>Np</it>s were changing the initial concentration (<it>C</it>_I) of bacteria (<it>i.e</it>., cell death), we performed probabilistic calculations assuming that the perturbations in <it>t</it>_mwere due to <it>C</it>_Ialone. We found that such large perturbations in <it>t</it>_mcould only come about at a <it>C</it>_Iwhere the probability of any growth at all was small. This result indicates that much of the <it>Np</it>-induced change in <it>t</it>_mwas due to a greatly increased <it>T </it>(<it>e.g</it>., from <it>ca</it>. 1 to 15-20 hrs). For the solid phase assays we hypothesize that the bacteria eventually became non-culturable since they were inhibited from undergoing further cell division (<it>T </it>> many days).</p> <p>Conclusion</p> <p>We propose that the difference between the solid and liquid system relates to the obvious difference in the exposure, or residence, time of the <it>Np</it>s with respect to the bacterial cell membrane inasmuch as when small, <it>Np</it>-inhibited colonies were selected and streaked on fresh (<it>i.e</it>., no <it>Np</it>s present) media, growth proceeded normally: <it>e.g</it>., a small, growth-inhibited colony resulted in a plateful of typical <it>S. aureus </it>colonies when streaked on fresh, solid media.</p

Evolution of the CDKN1C-KCNQ1 imprinted domain

<p>Abstract</p> <p>Background</p> <p>Genomic imprinting occurs in both marsupial and eutherian mammals. The <it>CDKN1C </it>and <it>IGF2 </it>genes are both imprinted and syntenic in the mouse and human, but in marsupials only <it>IGF2 </it>is imprinted. This study examines the evolution of features that, in eutherians, regulate <it>CDKN1C </it>imprinting.</p> <p>Results</p> <p>Despite the absence of imprinting, CDKN1C protein was present in the tammar wallaby placenta. Genomic analysis of the tammar region confirmed that <it>CDKN1C </it>is syntenic with <it>IGF2</it>. However, there are fewer LTR and DNA elements in the region and in intron 9 of <it>KCNQ1</it>. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of <it>KCNQ1 </it>and promoter elements could not be detected, the antisense transcript <it>KCNQ1OT1 </it>that regulates <it>CDKN1C </it>imprinting in human and mouse is still expressed.</p> <p>Conclusion</p> <p>CDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. The expression of <it>KCNQ1OT1 </it>in the absence of CDKN1C imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. These findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that <it>CDKN1C </it>imprinting cannot be due to its synteny with <it>IGF2 </it>or with its placental expression in mammals.</p