Imaging FlowCytobot modified for high throughput by in-line acoustic focusing of sample particles

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Limnology and Oceanography: Methods 15 (2017): 867–874, doi:10.1002/lom3.10205.Imaging FlowCytobot, a submersible instrument that measures optical properties and captures images of nano- and microplankton-sized particles, has proved useful in plankton studies, but its sampling rate is limited by the ability of hydrodynamic focusing to accurately position flowing sample particles. We show that IFCB's sampling rate can be increased at least several-fold by implementing in-line acoustic focusing upstream of the flow cell. Particles are forced to the center of flow by acoustic standing waves created by a piezo-electric transducer bonded to the sample capillary and driven at the appropriate frequency. With the particles of interest confined to the center of the sample flow, the increased size of the sample core that accompanies increased sample flow rate no longer degrades image and signal quality as it otherwise would. Temperature affects the optimum frequency (through its effect on the speed of sound in water), so a relationship between sample temperature and optimum frequency for acoustic focusing was determined and utilized to control the transducer. The modified instrument's performance was evaluated through analyses of artificial particles, phytoplankton cultures, and natural seawater samples and through deployments in coastal waters. The results show that large cells, especially dinoflagellates, are acoustically focused extremely effectively (which could enable, for example, > 10-fold increased sampling rate of harmful algal bloom species, if smaller cells are ignored), while for nearly all cell types typically monitored by IFCB, threefold faster data accumulation was achieved without any compromises. Further increases are possible with more sophisticated software and/or a faster camera.NSF Grant Numbers: OCE-1130140 , OCE-113113

The SCEC Southern California Reference Three-Dimensional Seismic Velocity Model Version 2

We describe Version 2 of the three-dimensional (3D) seismic velocity model of southern California developed by the Southern California Earthquake Center and designed to serve as a reference model for multidisciplinary research activities in the area. The model consists of detailed, rule-based representations of the major southern California basins (Los Angeles basin, Ventura basin, San Gabriel Valley, San Fernando Valley, Chino basin, San Bernardino Valley, and the Salton Trough), embedded in a 3D crust over a variable depth Moho. Outside of the basins, the model crust is based on regional tomographic results. The model Moho is represented by a surface with the depths determined by the receiver function technique. Shallow basin sediment velocities are constrained by geotechnical data. The model is implemented in a computer code that generates any specified 3D mesh of seismic velocity and density values. This parameterization is convenient to store, transfer, and update as new information and verification results become available

Membrane Insertion for the Detection of Lipopolysaccharides: Exploring the Dynamics of Amphiphile-in-Lipid Assays

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assays to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. We also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures

Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

detection. by flow cytometry and whole-cell ELISA. strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA. F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies

Hydrogeologic Investigations of Pavement Subsidence in the Cumberland Gap Tunnel

Cumberland Gap Tunnel was constructed under Cumberland Gap National Historical Park in 1996 to improve transportation on a segment of U.S. 25E, connecting Kentucky and Tennessee and restoring Cumberland Gap to its historical appearance. The concrete pavement in the tunnel started to subside in 2001. Ground penetrating radar surveys revealed voids in many areas of the limestone roadbed aggregate beneath the pavement. To investigate possible hydrogeologic processes that may have caused favorable conditions for voids to form in the aggregate, we studied geology, groundwater flow, and groundwater chemistry in the tunnel using a variety of methods, including bore drilling, packer test, dye tracing, groundwater- and surface-flow monitoring, water-chemistry modeling, and an aggregate dissolution experiment. The study revealed that the aggregate receives a large volume of groundwater from much of the bedrock invert, but the flow velocity is too slow to transport small particles out of the aggregate. Calcite saturation indices calculated from water-chemistry data suggest that the groundwater was capable of continuously dissolving calcite, the primary mineral in the limestone aggregate. Water samples taken during different flow conditions indicate that groundwater under low-flow conditions. The dissolution experiment showed that all the limestone aggregate placed beneath the roadbed and in contact with groundwater lost mass; the highest mass loss was 3.4 percent during a 178-day period. The experiment also suggested that water with higher calcite-dissolving potential removed limestone mass quicker than water with low calcite-dissolving potential. We recommend that the limestone aggregate be replaced with noncarbonate aggregate, such as granite, to prevent dissolution and future road subsidence

Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coli

Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx1, stx2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system

Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coil

Citation: Woods, T. A., Mendez, H. M., Ortega, S., Shi, X. R., Marx, D., Bai, J. F., . . . Deshpande, A. (2016). Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coil. Frontiers in Cellular and Infection Microbiology, 6, 12. doi:10.3389/fcimb.2016.00092Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx(1), stx(2)) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system

Three-dimensional anisotropic pressure free boundary equilibria

Free boundary three-dimensional anisotropic pressure magnetohydrodynamic equilibria with nested magnetic flux surfaces are computed through the minimisation of the plasma energy functional $W={\int}_{V}{d^3}x\left[{B^2}/(2\mu_0)+p_{||}/(\Gamma-1)\right]$. The plasma–vacuum interface is varied to guarantee the continuity of the total pressure $\left[{p}_{\perp}+{B^2}/(2\mu_0)\right]$ across it and the vacuum magnetic field must satisfy the Neumann boundary condition that its component normal to this interface surface vanishes. The vacuum magnetic field corresponds to that driven by the plasma current and external coils plus the gradient of a potential function whose solution is obtained using a Green's function method. The energetic particle contributions to the pressure are evaluated analytically from the moments of the variant of a bi-Maxwellian distribution function that satisfies the constraint ${\bf B\cdot\nabla}{\cal F}_h=0$. Applications to demonstrate the versatility and reliability of the numerical method employed have concentrated on high-β off-axis energetic particle deposition with large parallel and perpendicular pressure anisotropies in a 2-field period quasiaxisymmetric stellarator reactor system. For large perpendicular pressure anisotropy, the hot particle component of the pperpendicular distribution localises in the regions where the energetic particles are deposited. For large parallel pressure anisotropy, the pressures are more uniform around the flux surfaces