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

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

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

Thermoelectric Limitations of Graphene Nanodevices at Ultrahigh Current Densities

Graphene is atomically thin, possesses excellent thermal conductivity, and is able to withstand high current densities, making it attractive for many nanoscale applications such as field-effect transistors, interconnects, and thermal management layers. Enabling integration of graphene into such devices requires nanostructuring, which can have a drastic impact on the self-heating properties, in particular at high current densities. Here, we use a combination of scanning thermal microscopy, finite element thermal analysis, and operando scanning transmission electron microscopy techniques to observe prototype graphene devices in operation and gain a deeper understanding of the role of geometry and interfaces during high current density operation. We find that Peltier effects significantly influence the operational limit due to local electrical and thermal interfacial effects, causing asymmetric temperature distribution in the device. Thus, our results indicate that a proper understanding and design of graphene devices must include consideration of the surrounding materials, interfaces, and geometry. Leveraging these aspects provides opportunities for engineered extreme operation devices

Interferon beta-1a sc at 25 years: a mainstay in the treatment of multiple sclerosis over the period of one generation.

INTRODUCTION Interferon beta (IFN beta) preparations are an established group of drugs used for immunomodulation in patients with multiple sclerosis (MS). Subcutaneously (sc) applied interferon beta-1a (IFN beta-1a sc) has been in continuous clinical use for 25 years as a disease-modifying treatment. AREAS COVERED Based on data published since 2018, we discuss recent insights from analyses of the pivotal trial PRISMS and its long-term extension as well as from newer randomized studies with IFN beta-1a sc as the reference treatment, the use of IFN beta-1a sc across the patient life span and as a bridging therapy, recent data regarding the mechanisms of action, and potential benefits of IFN beta-1a sc regarding vaccine responses. EXPERT OPINION IFN beta-1a sc paved the way to effective immunomodulatory treatment of MS, enabled meaningful insights into the disease process, and remains a valid therapeutic option in selected vulnerable MS patient groups

Transient hypoxia and downregulation of circulating prohepcidin concentrations in healthy young men

To determine the impact of acute hypoxia on prohepcidin concentrations in humans, we measured concentrations of this peptide in serum collected during and after a transient period (30 min) of hypoxia and during normoxia. Prohepcidin concentrations were significantly lower 150 min after the end of hypoxia than after normoxia (p=0.028)