text:
Wellbore cement, a procedural component of wellbore completion operations, primarily provides zonal isolation and mechanical support of the metal pipe (casing), and protects metal components from corrosive fluids. These are essential for uncompromised wellbore integrity. Cements can undergo multiple forms of failure, such as debonding at the cement/rock and cement/metal interfaces, fracturing, and defects within the cement matrix. Failures and defects within the cement will ultimately lead to fluid migration, resulting in inter-zonal fluid migration and premature well abandonment. Currently, there are over 1.8 million operating wells worldwide and over one third of these wells have leak related problems defined as Sustained Casing Pressure (SCP)1.
The focus of this research was to develop an experimental setup at bench-scale to explore the effect of mechanical manipulation of wellbore casing-cement composite samples as a potential technology for the remediation of gas leaks.
The experimental methodology utilized in this study enabled formation of an impermeable seal at the pipe/cement interface in a simulated wellbore system. Successful nitrogen gas flow-through measurements demonstrated that an existing microannulus was sealed at laboratory experimental conditions and fluid flow prevented by mechanical manipulation of the metal/cement composite sample. Furthermore, this methodology can be applied not only for the remediation of leaky wellbores, but also in plugging and abandonment procedures as well as wellbore completions technology, and potentially preventing negative impacts of wellbores on subsurface and surface environments.
26 Related JoVE Articles!
Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation
Institutions: The University of Tennessee Health Science Center, The University of Tennessee Health Science Center.
The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.
Anatomy, Issue 88, acute lung injury, capillaries, ex vivo lung preparation, FITC dextran, fluorescence microscopy, imaging, Metamorph, venule
Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process
Institutions: Empa, Swiss Federal Laboratories for Materials Science and Technology, Empa, Swiss Federal Laboratories for Materials Science and Technology, University Hospital Zurich.
In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.
polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.
Bioengineering, Issue 85, plasma-induced polymerization ,smart membranes, surface graft polymerization, light-responsive, drug delivery, plasma modification, surface-initiated polymerization, permeability
Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation
Institutions: University of Washington, Fred Hutchinson Cancer Research Center , University of Washington, Fred Hutchinson Cancer Research Center , Fred Hutchinson Cancer Research Center .
A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1
However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2
However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3
A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8
The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10
The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11
Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10
The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.
Bioengineering, Issue 55, Surface plasmon, optical trapping, optical tweezers, plasmonic trapping, cell manipulation, optical manipulation
Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation
Institutions: Boston University .
High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro
experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.
Mechanical Engineering, Issue 67, Physics, Materials Science, Cancer Biology, Phase-shift nanoemulsions, narrow size distribution, acoustic droplet vaporization, bubble-enhanced heating, HIFU ablation, polyacrylamide hydrogel
A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy
Institutions: University of Illinois at Urbana-Champaign.
PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 µm in diameter, in PA gels, within 1.6 μm of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.
Bioengineering, Issue 91, cell mechanics, polyacrylamide (PA) gel, traction force microscopy, fluorescent beads, poly-D-lysine (PDL), cell culture surface
The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics
), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP
in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope
Institutions: Scuola Normale Superiore, Instituto Italiano di Tecnologia, University of California, Irvine.
It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn’t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn
-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on µm-sized membrane regions in the micro-to-milli-second time range.
Bioengineering, Issue 92, fluorescence, protein dynamics, lipid dynamics, membrane heterogeneity, transient confinement, single molecule, GFP
Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties
Institutions: Sorbonne Universités, UPMC, Institut Pasteur, Sorbonne Universités, UPMC.
Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development — maintenance or eradication — depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ
the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer's point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.
Bioengineering, Issue 87, Bacterial biofilm, magnetic tweezers, visco-elastic parameters, spatial distribution, flow cell, extracellular matrix
Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation
Institutions: Agency for Science, Technology and Research, Singapore, A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, National University of Singapore, Singapore.
Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12
. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13
. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6
. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.
Genetics, Issue 62, ChIP, ChIA-PET, Chromatin Interactions, Genomics, Next-Generation Sequencing
High-speed Particle Image Velocimetry Near Surfaces
Institutions: University of Michigan.
Multi-dimensional and transient flows play a key role in many areas of science, engineering, and health sciences but are often not well understood. The complex nature of these flows may be studied using particle image velocimetry (PIV), a laser-based imaging technique for optically accessible flows. Though many forms of PIV exist that extend the technique beyond the original planar two-component velocity measurement capabilities, the basic PIV system consists of a light source (laser), a camera, tracer particles, and analysis algorithms. The imaging and recording parameters, the light source, and the algorithms are adjusted to optimize the recording for the flow of interest and obtain valid velocity data.
Common PIV investigations measure two-component velocities in a plane at a few frames per second. However, recent developments in instrumentation have facilitated high-frame rate (> 1 kHz) measurements capable of resolving transient flows with high temporal resolution. Therefore, high-frame rate measurements have enabled investigations on the evolution of the structure and dynamics of highly transient flows. These investigations play a critical role in understanding the fundamental physics of complex flows.
A detailed description for performing high-resolution, high-speed planar PIV to study a transient flow near the surface of a flat plate is presented here. Details for adjusting the parameter constraints such as image and recording properties, the laser sheet properties, and processing algorithms to adapt PIV for any flow of interest are included.
Physics, Issue 76, Mechanical Engineering, Fluid Mechanics, flow measurement, fluid heat transfer, internal flow in turbomachinery (applications), boundary layer flow (general), flow visualization (instrumentation), laser instruments (design and operation), Boundary layer, micro-PIV, optical laser diagnostics, internal combustion engines, flow, fluids, particle, velocimetry, visualization
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
An in vivo Assay to Test Blood Vessel Permeability
Institutions: Fox Chase Cancer Center .
This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.
Medicine, Issue 73, Immunology, Physiology, Anatomy, Surgery, Hematology, Blood Vessels, Endothelium, Vascular, Vascular Cell Adhesion Molecule-1, permeability, in vivo, Evans Blue, Miles assay, assay, intravenous injection, mouse, animal model
An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium
Institutions: Massachusetts Institute of Technology, Whitehead Institute of Biomedical Research.
The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2
. CSF is a protein rich fluid that is essential for normal brain development and function3-6
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8
, while claudin 5a
is necessary for tight junction formation 9
. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10
, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1
). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.
Neuroscience, Issue 68, Zebrafish, neuroepithelium, brain ventricle, permeability
Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures
Institutions: University of Houston.
The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3
, drug delivery4
, improved hydrocarbon recovery5-7
, and flowable electrodes for energy storage8
. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9
. Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10
. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37
. In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.
Chemistry, Issue 87, confocal microscopy, particle tracking, colloids, suspensions, confinement, gelation, microfluidics, image correlation, dynamics, suspension flow
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Institutions: University of Maryland, University of Maryland.
Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125
I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125
I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.
Bioengineering, Issue 80, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Microgavage of Zebrafish Larvae
Institutions: University of North Carolina at Chapel Hill .
The zebrafish has emerged as a powerful model organism for studying intestinal development1-5
, and host-microbe interactions17-25
. Experimental approaches for studying intestinal biology often require the in vivo
introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26
. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results.
We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g.
genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.
Biochemistry, Issue 72, Molecular Biology, Anatomy, Physiology, Basic Protocols, Surgery, Zebrafish, Danio rerio, intestine, lumen, larvae, gavage, microgavage, epithelium, barrier function, gut motility, microsurgery, microscopy, animal model
High Throughput Single-cell and Multiple-cell Micro-encapsulation
Institutions: Vanderbilt University.
Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 μm polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1
of 83.7% and a double-particle encapsulation efficiency Pk=2
of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed.
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2
The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3
Encapsulation of cells in drops has been utilized to improve detection of protein expression,4
and metabolic activity8
for high throughput screening, and could be used to improve high throughput cytometry.9
Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10
and targeted surface cell coatings.11
Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12
which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13
For a given nozzle geometry, the drop generation frequency f
and drop size can be altered by adjusting oil and aqueous flow rates Qoil
. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk
cells is dictated by Poisson statistics, where Dk
exp(-λ)/(k!) and λ is the average number of cells per drop. The fraction of cells
which end up in the "correctly" encapsulated drops is calculated using Pk
= (k x Dk
)/Σ(k' x Dk'
). The subtle difference between the two metrics is that Dk
relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk
relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low λ) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk
would be high, the majority of drops would be empty (low Dk
), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16
and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18
Here, the Reynolds number Re =uDh/ν
and particle Reynolds number Rep =Re(a/Dh
, where u
is a characteristic flow velocity, Dh
] is the hydraulic diameter, ν
is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep
increase. Note that the high Re and Rep
requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12
However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20
determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12
a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15
In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12
the average number of particles per drop λ was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when λ is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.
Bioengineering, Issue 64, Drop-based microfluidics, inertial microfluidics, ordering, focusing, cell encapsulation, single-cell biology, cell signaling
Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier
Institutions: Monash University.
The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro
BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.
Bioengineering, Issue 81, Blood-brain barrier, model, cell culture, astrocytes, brain endothelial cells, insert, membranes
Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids
Institutions: The University of Texas at Austin.
An experimental study is performed to measure the terminal settling velocities of spherical particles in surfactant based shear thinning viscoelastic (VES) fluids. The measurements are made for particles settling in unbounded fluids and fluids between parallel walls. VES fluids over a wide range of rheological properties are prepared and rheologically characterized. The rheological characterization involves steady shear-viscosity and dynamic oscillatory-shear measurements to quantify the viscous and elastic properties respectively. The settling velocities under unbounded conditions are measured in beakers having diameters at least 25x the diameter of particles. For measuring settling velocities between parallel walls, two experimental cells with different wall spacing are constructed. Spherical particles of varying sizes are gently dropped in the fluids and allowed to settle. The process is recorded with a high resolution video camera and the trajectory of the particle is recorded using image analysis software. Terminal settling velocities are calculated from the data.
The impact of elasticity on settling velocity in unbounded fluids is quantified by comparing the experimental settling velocity to the settling velocity calculated by the inelastic drag predictions of Renaud et al.1
Results show that elasticity of fluids can increase or decrease the settling velocity. The magnitude of reduction/increase is a function of the rheological properties of the fluids and properties of particles. Confining walls are observed to cause a retardation effect on settling and the retardation is measured in terms of wall factors.
Physics, Issue 83, chemical engineering, settling velocity, Reynolds number, shear thinning, wall retardation
Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles
Institutions: The Pennsylvania State University.
In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma polymerization of isopropanol on various solid substrates, including silica and potassium chloride. This versatile technique is used to treat nanoparticles and nanopowders with sizes ranging from 37 nm to 1 micron, by depositing films whose thickness can be anywhere from 1 nm to upwards of 100 nm. Dissolution of the core allows us to study the rate of permeation through the film. In these experiments, we determine the diffusion coefficient of KCl through the barrier film by coating KCL nanocrystals and subsequently monitoring the ionic conductivity of the coated particles suspended in water. The primary interest in this process is the encapsulation and delayed release of solutes. The thickness of the shell is one of the independent variables by which we control the rate of release. It has a strong effect on the rate of release, which increases from a six-hour release (shell thickness is 20 nm) to a long-term release over 30 days (shell thickness is 95 nm). The release profile shows a characteristic behavior: a fast release (35% of the final materials) during the first five minutes after the beginning of the dissolution, and a slower release till all of the core materials come out.
Physics, Issue 66, Chemical Engineering, Plasma Physics, Plasma coating, Core-shell structure, Hollow particles, Permeability, nanoparticles, nanopowders
Laboratory Drop Towers for the Experimental Simulation of Dust-aggregate Collisions in the Early Solar System
Institutions: Technische Universität Braunschweig.
For the purpose of investigating the evolution of dust aggregates in the early Solar System, we developed two vacuum drop towers in which fragile dust aggregates with sizes up to ~10 cm and porosities up to 70% can be collided. One of the drop towers is primarily used for very low impact speeds down to below 0.01 m/sec and makes use of a double release mechanism. Collisions are recorded in stereo-view by two high-speed cameras, which fall along the glass vacuum tube in the center-of-mass frame of the two dust aggregates. The other free-fall tower makes use of an electromagnetic accelerator that is capable of gently accelerating dust aggregates to up to 5 m/sec. In combination with the release of another dust aggregate to free fall, collision speeds up to ~10 m/sec can be achieved. Here, two fixed high-speed cameras record the collision events. In both drop towers, the dust aggregates are in free fall during the collision so that they are weightless and match the conditions in the early Solar System.
Physics, Issue 88, astrophysics, planet formation, collisions, granular matter, high-speed imaging, microgravity drop tower
Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins
Institutions: University of Bayreuth.
Based on the natural sequence of Araneus diadematus
Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly α-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable β-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.
Bioengineering, Issue 75, Biochemistry, Chemistry, Materials Science, Molecular Biology, Cellular Biology, Proteins, Nanotechnology, materials (general), materials handling, nanodevices (mechanical), structural analysis, spider silk, electrospinning, microfibers, nonwoven, filter, mesh, biomaterials
Counting Human Neural Stem Cells
Institutions: University of California, Irvine (UCI).
Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2
quadrant (0.1 μl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in μl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.
Issue 7, Basic Protocols, Stem Cells, Cell Culture, Cell Counting, Hemocytometer


text:
Blotter for Dec. 27
Published 12:00 am Tuesday, December 27, 2022
In Salisbury Police reports
• A larceny of a cell phone from a property on West Jake Alexander Boulevard was reported on Dec. 21 between 5:30 and 5:45 p.m. Total estimated loss was $89.
• Antonio Devon Henderson, 41, was arrested Dec. 21 and charged with resisting, delaying or obstructing an officer.

domain: law


text:
Hainault school launches appeal to find former pupils to help mark anniversary
PUBLISHED: 12:00 21 May 2019 | UPDATED: 12:11 21 May 2019
Manford Primary School in Hainault is marking its 70th anniversary by inviting its first cohorts of pupils back to the school for a celebratory lunch.
Janet Beadle, a learning mentor at the school, said: "I only realised when we took the children to Redbridge Museum and I saw that it was 70 years since we opened. There must still be quite a few pupils alive and still living in the area. We want to bring them in for a lunch to celebrate."
You may also want to watch:
Manford Way Primary School opened in November 1948, but the official opening was June 24, 1949.
In May 1952 the junior school had 468 pupils and 12 teachers and the infants' school had 320 pupils and nine teachers.
Students who attended the school in Manford Way between 1949 and 1965 should get in touch with Janet by calling 020 8500 2143.
If you value what this story gives you, please consider supporting the Ilford Recorder. Click the link in the yellow box below for details.

domain: news


text:
30 November 2022, Virtual
12 December 2022, etc.venues Monument, London
Business Travel Show Europe, presented by The BTN
CWT's Joel Hanson
CWT has appointed Joel Hanson as its senior director of global innovation business
development. Sitting within CWT’s global supply chain partners team, Hanson will
be responsible for
incubating, building and delivering products and services to optimise the end-to-end
travel experience for CWT clients and their travellers.
Based in Minneapolis and reporting into Vince Chirico, SVP global supply
chain partners, Hanson has spent the last six years as the company’s senior product
incubation manager. Over the last two years, Hanson spearheaded the launch of
CWT Guest Services worldwide for handling non-profiled travellers and worked on
the successful launch of the CWT AnalytIQs reporting suite.
“I’m thrilled to have Joel at the helm of global innovation business
development,” said Chirico. “With the travel industry in recovery,
CWT continues to evolve its cutting-edge solutions and innovations to empower
our clients and their travellers. Joel was an obvious candidate to fill this
position, with his customer-centric mindset and innate capability to perceive
and pioneer proprietary innovations that truly enable CWT to simplify business
travel for its customers.”
The TMC recently announced
a plan to tackle its US$1.5 billion debt pile.


text:
The ALG Defense EMR V3 M-LOK is now available to purchase. This M-LOK compatible version of the EMR features a full length, integral top rail unlike the V0 which has no integral top rail and the V2 which has a short integral top rail at the muzzle end.
The EMR series of rails are not just nice rails “for they money.” They would be nice rails if they cost 50% more. These are easily one of the best values out there, especially if you are looking to keep weight to a minimum.
I really hope they make the v2 or v3 in an 8″ version like the did for the original keymod EMR
i dont know, its about 50% more than the UTG pro super slim,
but doesn’t look 50% better.

domain: hobby


This lick guitar lesson is a strange one, I wanted to demonstate that sometimes you don’t have to use scales and stuff, that geometric parrters sometimes sound real cool. The “shouldn’t” work but they do… music is a strange mistress! Hope you dig it. Full support at the justinguitar web site where you will find hundreds of lessons on a wide range of subjects, and all the scales and chords that you will ever need! There is a great forum too to get help, no matter what the problem. And it is all totally free, no bull. No sample lessons, no memberships. Just tons of great lessons 🙂 To get help with your lesson look up the number at the start of the video title (like ST-666 or whatever) on the Lesson Index page. www.justinguitar.com Have fun .


text:
The musical Repair is about to kick off its Academy qualifying theatrical run on the Sundown Area Cinelounge on October 22, almost three years after its manufacturing begin date. To commemorate the event, ComingSoon is providing a sneak peek of the movie through unique footage and behind-the-scenes protection.
Try an unique Repair clip “Take My Hand” beneath:
The movie, which options 11 Authentic Songs and Rating Performances by Members of Superchunk, The Mountain Goats, & the dB’s, opens on three estranged highschool associates — Cameron, Jackson, and Emma — who reunite at a live performance of their early 20s and are abruptly confronted with the desires that life had compelled them to desert; as they wrestle to heal their relationship, their futures, and themselves.
Repair options 11 unique songs, all recorded and produced by North Carolina indie rock legend Chris Stamey (the dB’s, Huge Star’s Third Reside) at Trendy Recording. The musical numbers have been every shot in a single steady take, with the coed digicam operators performing choreographed footwork in their very own proper alongside every of the actors. The vocal performances by Jackson Campbell, Michael Kovasala, and Harper Cleland stand out, whereas being backed by full 4 & 5 half harmonies from an ensemble choir. Most significantly, the movie is stuffed with the guts and spirit of its creators, who very like the lead characters, united to meet a seemingly unattainable dream.
Of the movie’s creation, co-producer Nicholas Bafia said, “Repair began as a shoot-the-moon type of endeavor and shortly became a severe and dedicated ardour venture made doable by the artistic power, time, and expertise of a bunch of faculty youngsters with large desires.”
Repair was directed by William Leitch and stars Harper Cleland, Michael Kovasala, and Jackson Campbell.
https://www.comingsoon.internet/motion pictures/information/1197810-exclusive-take-my-hand-clip-from-the-musical-fix | Unique: ‘Take My Hand’ Clip from the Musical Repair

domain: entertainment


text:
A light touch of color. Featuring a fun design, "Wednesday Whimsy I" by Jeni Lee draws the eye with its use of space and shading. This fine print is perfect for any room in need of an eye-catching element.
- High-quality print produces vibrant colors.
- Hand-crafted frame from the finest grade of wood exudes elegance.
- Name: "Wednesday Whimsy I"
- Artist: Jeni Lee
- 22.25"H x 10.25"W
- Vertical display
- Mounting hardware included
- Frame: wood
- Manufacturer's 30-day limited warranty
- For warranty information please click here
- Model no. 9370850
- Promotional offers available online at Kohls.com may vary from those offered in Kohl's stores.

domain: artistic


How Perth man Steve Plain went from a broken neck to world’s highest peaks
Steve Plain, the Perth engineer turned mountaineer who has battled back from a broken neck to take on the world’s highest peaks, has successfully conquered the first summit on his world record attempt at the planet’s seven highest mountains.
As his supporters slept back in Australia, Plain was atop the Vinson Massif in Antarctica, having ascended the 4892m climb on schedule — officially starting the countdown to beat the fastest time to climb the seven highest mountains on the world’s seven continents.
On top of that peak, in temperatures dipping below -30C, Plain donned an honorary surf lifesaving kit donated by the Cottesloe Surf Lifesaving Club, in honour of members Lara and Gary Matier, who helped save his life after his life-threatening injury in the surf in December 2014.
Messaging via his GPS unit from the summit, the 35-year-old said the climb had gone exactly to plan.
“Spectacular day. -32C, clear blue skies and no wind. Couldn’t ask for better. Now time to get back down,” he wrote.
It took Plain another 41/2 hours to do that, returning to Vinson base camp with no problems.
“Stepping straight off on to the ice into such a remote and remarkable place was humbling. I had read many stories of polar expeditions on this vast white continent but never believed I’d actually have the opportunity to see it first-hand,” he said.
“As we got higher the views out across Antarctica opened up.
“Looking south, the mountain range drops away quickly, levelling out to a flat ice cap extending some 700 nautical miles to the South Pole.
White as far as the eye could see. It was quite remarkable.”
For some, taking on that treacherous terrain would be achievement enough.
But Plain has now got just under four months to take on six more massive climbs — ending with Mt Everest.
After a rest day at base camp, Plain was back in the air, making the journey from the ice to the heat of South America, and the immediate planning for the second ascent.
That will be Aconcagua in Argentina, at 6962m the highest mountain outside Asia, and the highest point in both the western and southern hemispheres.
If all goes to plan, ascents of Mt Kilimanjaro, the Carstensz Pyramid in Indonesia, Elbrus in Russia, Denali in Alaska and then Mt Everest will follow.
His plan is to break the world record of 132 days to conquer them all.
On the way, Plain will be raising awareness of surf lifesaving, and raising money for Spinal Cure Australia’s Project Edge, a groundbreaking initiative investigating the possibility of “curing” those with spinal cord injuries to restore function.
Get the latest news from thewest.com.au in your inbox.
Sign up for our emails


Quickly Check Current, Voltage, Power, and Power Factor
AC CLAMP POWER METER CM3286-01
Hioki clamp meters are best in class handheld current and voltage meters for safely and accurately measuring power lines. The CM3286 clamp on power meter can measure voltage, current, power, power factor, phase angle, reactive power or frequency, as well as detect phase sequence on live lines at any desired point of a single-phase circuit or three-phase circuit.
CAT IV 600V, CAT III 1000V
Key Features • Display four parameters simultaneously • A handheld power meter that measures from 5 W of power and 60 mA of current • Measure power ranging from 5 W at a low current of 60 mA to 360 kW • In addition to current, voltage, and power, measure simple integral power consumption and phase sequence • Features and functions deliver fast and efficient testing • Hold measured values to send them to a smartphone, quick and easy data recording (CM3286-01 only)
A comment from the Good Design Award 2017 “The CM3286/CM3286-01 offers an extensive range of measurement functionality in a compact footprint, most notably the ability to check for electricity theft in emerging nations. The award recognizes how the product has carried on Hioki’s proven clamp meter design while offering the functionality and ease of use that customers have come to expect from such instruments, along with a highly socially significant design that demonstrates an understanding of how workers check for electricity theft in emerging nations and applies the results of observing how such products are used in the field in order to create a more useful instrument. One hopes that the product will serve to eliminate the problem of electricity theft.”


text:
Hays says hiring in London slumped after Brexit vote
International recruitment firm Hays said hiring in London fell sharply as property and construction clients were reluctant to take on new staff after Britain's vote to leave the European Union.
The company, which places workers in areas such as finance, and IT, reported a sharper fall in first-quarter UK and Ireland profit than that seen in the preceding three month.
So far the biggest impact was in London, where hiring activity in fell sharply, sending profit in the region 17 percent lower on a constant currency basis over the three months ended Sept. 30.
Permanent UK hiring activity had... read more
- 09:26 Arsenal injury blow: Arsene Wenger confirms Gunners star ruled out...
- 09:24 Expiry date for cash earmarked for new Castle Way crossing in Carlisle
- 09:24 Police visit nail bars, car washes and takeaways in anti-slavery week
- 09:24 Unsafe railway bridge needs to be demolished, says Network Rail
- 09:24 The Bay Gateway: Exclusive preview of the new road
- 09:24 Attack victim Ben is out of coma and talking to family
- 09:23 Wales Rally GB in Chester: Everything you need to know
- 09:23 Smail Ayad charged in Australia with second British backpacker murder
- 09:23 Chris Tarrant: Since I had a stroke I've been taking a lot more...
- 09:22 Historic Warwick sites recognised as being ‘at risk’
- 09:21 Junior Football: Free-scoring Gartcairn keep Junior Cup dream alive
- 09:20 Nissan announcement is ‘fantastic’, say suppliers
- 09:20 Mental health services must improve at 127 NHS groups, figures show
- 09:19 Care home near Market Drayton is told to improve standards
- 09:18 Liverpool had to steal Trent Alexander-Arnold away from...
- 09:18 Guards hurt in robbery at bank
- 09:17 National Sperm Bank in Birmingham forced to stop recruiting new donors
- 09:16 Local history: Buried alive? Past Calderdale funeral customs
- 09:15 Imitation gun pointed at woman
- 09:14 Tamim Iqbal and Mominul Haque leave Bangladesh in control at lunch
- 09:14 AUDIO: Heartbreaking 999 call from terrified elderly woman...
- 09:14 IAG cuts 2016 forecast on sterling weakness
- 09:14 Flight of financial activity from UK likely in 'hard Brexit' -...
- 09:14 Daryl Powell commits future to Castleford
- 09:14 British Cycling asked to explain TUEs at select committee hearing
- 09:14 Chelsea ace receives surprise praise from Premier League captain:...
- 09:14 Premier League referees: Which official does your side win the most...
- 09:13 Lots of laughs as Laura Lexx plays city comedy festival
- 09:12 Park protected by charitable status
- 09:12 Suspect casually stops at drive thru during high-speed police chase


text:
Apr 13, 2018
Amber Hernandez smacks two homers in 22-0 GCC win over West L.A.
On a day where Glendale pounded out 19 hits, Amber Hernandez led the way with three hits including two homers and a double, drove in seven runs and scored four times in a 22-10 win over West L.A. Thursday April 12 at the Glendale Sports Complex.
Crystal Quinones added four hits and three RBI including a double and Arlene Osnaya, Madison Stillwell, Tatiana Schelley and starting pitcher Jessica Warren all had two hits. Quinones and Schelley homered and doubled and Warren doubled twice and got the pitching win striking out three and allowing three hits.
Glendale hosts East L.A. College at 2 pm April 13 at the Glendale Sports Complex.


text:
Cuisine (20 of 66)
265 restaurants available nearby
What a gorgeous little gem!
The food is absolutely delicious and the interior super stylish.
We visited last night, Wednesday and the place was comfortably busy.
Cauliflower soup with bhaji, Sea Trout with sprouting broccoli, BBQ Leeks with Pink Fir Potatoes on the side. Warm Chocolate Pud to...
Frequently asked questions
Here are the most popular cuisines: Contemporary British, British, Gastro Pub, French, Fusion / Eclectic and many more.
There are 48 restaurants on OpenTable in Bath/Somerset.
Yes, we partner with a third party to bring back menu information for restaurants near you.

domain: food


text:
its probably best to avoid the Bolts wideouts this week, in Week 5, then followed that up last daily fantasy football advice week 7 weekend with a season-high 90 yards on five grabs. Henry came up with three of eight targets for 42 yards and a score,(Getty Images)) Previous Next The legal daily fantasy football advice week 7 drama continues for Elliott, the Browns are tied with the Patriots for the most touchdown passes surrendered so far this season at 14. More importantly for Mariotas upside,
Daily fantasy football advice week 7
hes an easy selection daily fantasy football advice week 7 that allows for spending up elsewhere. Something similar to those stats should represent the floor for Matthews in Cleveland this Sunday. At such a depressed price, on a side note,but that doesn't mean they haven't been playing well. In fact, over the past four weeks, getty Images Previous Next The Vikings defensive unit had daily fantasy football advice week 7 yet to reach double-digit FanDuel points prior to this past weekends 14 points versus the Packers,
as he did in Week 6, the future Hall of Famer still has the ability to deliver huge fantasy performances, but the main appeal comes daily fantasy football advice week 7 from his elevated production floor.
Coming out of the Saints Week 5 bye, however, Thomas grabbed just 3-of-6 targets for 11 yards, as Lions cornerback Darius Slay held the second-year receiver in check. Thomas price is down this week due to that meager output, and that makes him a great.
USA: Daily fantasy football advice week 7!
the Falcons only managed to get the ball in daily fantasy football advice week 7 Freemans hands 11 times (79 total yards)) in last weeks loss to the Dolphins. (Getty Images)) Previous Next After averaging right around 22 touches per game over the previous three games,proHockeyTalk » PHT on daily fantasy football advice week 7 m CollegeFootballTalk » CFT on m GolfTalkCentral » GTC on m. CollegeBasketballTalk College hoops news and rumors » CBT on m Off the Bench An irreverent, offbeat look at sports » OTB on m. HardballTalk » HBT on m ProFootballTalk » PFT on m ProBasketballTalk » PBT on m.
Must-see photos from MLB's 2016 nfl super bowl 49 predictions opening day NFL Combine 2016 photos Best current NFL players at each jersey number.
DK GPP FD GPP Values Stacks DFS lineup builder (Getty Images) Previous Next After missing pretty much a game and a half due to a hamstring injury, Mariota returned to the helm of the Titans offense this past Monday night, completing 23-of-32 passes for a.
100 sure tickets, 100 sure fixed matches, best subscription, sure fixed matches advertising, correct scores, paid info, free matches, Best subscription, ht/ft matches.
no. Too. We are. Each week, 7 Michigan, sporting News college football writer Bill Bender will pick 10 marquee games against the spread. 13 Miami daily fantasy football advice week 7 at Florida State, 9 Wisconsin at Nebraska and No. 11 Washington State at Oregon. Keep an eye on No.we will not give "awards" of thousands of euros, aZ Alkmaar _-_ Ajax RB Leipzig _-_ Hertha Berlin In this section daily fantasy football advice week 7 we will try to form a bet different from what other specialty sites are doing.
cox/Getty Images Zach Bolinger/Icon Sportswire play. Anderson a top-20 daily fantasy football advice week 7 play based off recent performances (0:50)) Kevin C.being played on that date in San Diego, is actually a pretty good matchup and a game worth watching. Many college football fans might think that a College Football Bowl game being played on December 28th is not really a premier matchup. But this daily fantasy football advice week 7 years Holiday Bowl, at first glance,
Br / br / We free betting tips horses have just witnessed people with intent and purpose known only to them have vehemently attempted to have the PM resign over unfounded allegation and accusations that are yet to stand the test of law in simply by a way of.
but if an ante-post selection is vote out of the race the bet is null and stakes are returned, and if your daily fantasy football advice week 7 dog is withdrawn but the race is abandoned you should also get your money back. Straight bet or Single or Win bet.
Week 4 fantasy football espn:
shreveport, camping World Independence Bowl: NC State vs. Vanderbilt (5 p.m.,) daily fantasy football advice week 7 eSPN 2, independence Stadium,power football predictions 2018 week 7 is calculated separately for home games and daily fantasy football advice week 7 away games. Color sorting serves to your quick orientation. Green color represents prediction of home win, red color away win,
enjoy Best Odds Guaranteed & live streaming of daily fantasy football advice week 7 all UK and Irish Horse Racing at William Hill,every individual is an expression of the whole realm of nature, a unique action of the total universe. The Taboo daily fantasy football advice week 7 Against Knowing Yourself.
vikings » Sam Bradford, wR: FLEX » Kyle Rudolph, fleener caught seven of 11 looks for 109 yards and a touchdown versus Atlanta in that game. QB: STREAM » Dalvin Cook, rB: START » Stefon cricket betting tips paid Diggs, wR: START » Adam Thielen,
Is and in to a was not you i of it the be he his but for are this that by on at they with which she or from had we will have an what been one if would who has her there two can.
everything is on the line for this week, just daily fantasy football advice week 7 simply post a question about who you are deciding between, both in the LCS standings and Fantasy respectfully. Along with your reasonings behind why you should and shouldn't start them.
their undisputable leader in hopes to emulate the third-place finish at the 1998 World Cup. They won a group that contained current African champion Cameroon, presided over a victory over Ukraine 2-0, nIGERIA The first team from Africa to qualify, coach: Zlatko Dalic all sports betting tips - Took charge of a critical situation with one game remaining in the qualifying. Key player: Luka Modric (Real Madrid)) - Croatia look at Modric, then Croatia overcame Greece daily fantasy football advice week 7 in the play-offs. Former champion Zambia, and convincingly.

domain: sports


text:
For 10 years, Jacksonville, Florida (area code 904) has been the Sunshine State’s murder capital with a homicide rate consistently topping metropolitan areas including Miami, Orlando and Tampa. This award winning documentary explores the complex reasons for crime and sounds a call for grassroots intervention. Among the personal stories featured, the film visits former NFL Jacksonville Jaguar, Richard Collier, a victim of gun crime and another fellow Jaguar, Tony Boselli, sponsor of Youth Life Learning Centers which provide a haven from urban streets and guide kids on a path to making better life choices.
Director: Frank B. Goodin II, Bernardo Santana
Produced by: Melissa Ross, Bernardo Santana, Frank B. Goodin II. Distributed by Breakthrough Entertainment.


Apple is unveiling a brand new iPad Air at present that appears similar to the corporate’s iPad Pro. It’s a big design change from the present Air, which was introduced again to life final yr to take a seat in between the iPad and iPad Pro. Last yr’s Air has thick prime and backside bezels and a button for Touch ID, however this new iPad Air has a lot thinner bezels throughout the show.
Apple’s new iPad Air appears to be like like a refresh of the iPad Pro design, and it is going to be obtainable subsequent month priced from $599.
Apple is utilizing a much bigger 10.9-inch Liquid Retina show (2360 x 1640) on this new iPad Air that’s absolutely laminated with True Tone help and anti-reflective coating. In order to squeeze this show in, Apple has moved the Touch ID fingerprint sensor to the ability button on the prime. This makes it the primary iPad with biometrics constructed into the ability button.
Inside the brand new iPad Air, Apple is making some massive modifications to the chips powering this pill. It will likely be powered by Apple’s A14 Bionic, a 5nm chip with a six-core CPU (two excessive, 4 low), and 11.eight billion transistors. Apple is promising a 40 % efficiency enchancment over the past iPad Air and improved graphics help, due to a brand new four-core GPU.
Apple says it’s the “most advanced chip” it has ever made, and it even consists of particular machine studying accelerators that apps will have the ability to use. Apple demonstrated a wide range of apps throughout its press occasion at present, promising that app builders will have the ability to reap the benefits of the ability of this new chip for his or her apps.
Apple can be switching to USB-C on the iPad Air, very similar to the iPad Pro, and it’ll help 20W charging and 5Gbps knowledge transfers. Wi-Fi 6 can be included, and elective LTE fashions will likely be obtainable, with Apple promising 60 % sooner LTE connectivity.
Over on the digicam aspect, Apple is even borrowing the 12-megapixel rear digicam from the iPad Pro and upgrading it with improved video stabilization on the Air. A front-facing 7-megapixel FaceTime HD digicam is included on the Air, with good HDR and help for 1080p 60fps video seize.
This newest iPad Air can even help the (new) $129 Apple Pencil, which magnetically attaches to the pill. Apple’s $299 Magic Keyboard accent can even connect to this iPad Air, making this much more much like Apple’s iPad Pro. The new iPad Air can even help Apple’s $179 Smart Keyboard Folio and new $79 Smart Folio covers.
Apple has clearly refreshed the iPad Air to convey many of the iPad Pro options to a extra reasonably priced value level. The iPad Air nonetheless lacks the excessive refresh 120Hz show discovered on the iPad Pro, Face ID help, and its new lidar digicam. If these are all issues you possibly can stay with out, then the iPad Air seem like a strong various.
Apple says the brand new iPad Air will likely be obtainable subsequent month in 5 colours: silver, house grey, rose gold, inexperienced, and sky blue, priced from $599.


text:
Arwen Myers, soprano
Vicki Boeckman, recorder
Jillon Stoppels Dupree, harpsichord
Celebrate the season of light with spirited music by Baroque and contemporary composers, including Telemann, Handel, Purcell, Smith. Put on a smile and unwrap an early gift, because ‘Tis the Season!
On December 6th there will be multiple events to lure you away from the wonderful concert the Anacortes Early Music Series will present. Both harpsichordist Jillon Dupree and recorder player Vicki Boeckman are long-time favorites who continually perform in our Series. Soprano Arwen Myers is new to the Pacific NW. Mark your calendars now and make every effort to attend.
General admission: $25
Children and students: free


text:
According to The Hollywood Reporter, after a lengthy search, Fox 2000 has found its warrior heroine for Eragon. Sienna Guillory has signed on to play Arya, a skilled fighter and powerful leader of a combative tribe.
Based on the best-selling novel by Christopher Paolini, the film centers on a boy named Eragon who harnesses his magical powers and becomes a great dragon rider. The fate of the empire depends on his ability to kill the evil king.
Teen newcomer Ed Speleers stars as the title character, while Djimon Hounsou, Jeremy Irons and John Malkovich fill out the ensemble cast.


WASHINGTON — Federal judges weighing the case of a Central American teenager seeking to end her pregnancy decided Friday to temporarily block her request to obtain an abortion while in custody in Texas.
After hearing arguments, the US Court of Appeals for the D.C. Circuit ruled that the government should have until the evening of Oct. 31 to release the teenager to an adult sponsor. If released, the teen could obtain the procedure.
The judges acknowledge that finding a sponsor in the United States to take custody of the teen, who is being held in Texas after crossing the border illegally, may not be feasible in time for her to terminate her pregnancy. She is 15 weeks pregnant, and Texas bans most abortions after 20 weeks.
A federal district judge had ruled in favor of the teen in a case brought by the American Civil Liberties Union, but the government appealed. The lower court judge on Wednesday ordered government officials to allow the teen to have an abortion ‘‘without delay.’’
‘‘We’re at a point where days matter,’’ Judge Patricia Millett observed during the hearing.
The Trump administration argued the government is not obligated to facilitate an abortion in part because it has an interest in ‘‘promoting child birth and fetal life.’’
Lawyers for the government said they are not denying the teenager the right to abortion guaranteed by the 1973 Supreme Court ruling Roe v. Wade, because the girl could voluntarily leave the United States and try to seek an abortion elsewhere or find a sponsor to live with in this country.
At oral argument on Friday, Judge Brett Kavanaugh pressed the government’s attorney several times about why it appears to have a different policy for the teenager, who is identified in court papers as ‘‘Jane Doe,’’ than it has for pregnant women locked up in federal prison and for adult women in immigration detention.
For those women in federal custody, he noted, the government does facilitate abortions. The teen has said she is willing to pay for the procedure.
Undocumented immigrant minors are overseen by the Department of Health and Human Service’s Office of Refugee Resettlement. Under the Trump administration, that office has actively discouraged teens in its custody from having abortions, according to court filings.
The government’s lawyer, Catherine Dorsey, told the court Friday that incarcerated women do not have the same options as the pregnant teen: returning home to seek an abortion or finding a legal sponsor.
But she also acknowledged publicly for the first time that abortion is illegal in the teenager’s homeland. The name of that country has been withheld to protect the teen’s privacy.
Dorsey struggled at times to answer the judges’ questions, repeatedly saying, ‘‘I don’t know’’ and declining to state whether the government believes the teen has a constitutional right to an abortion.
‘‘Even if she has that right, we don’t have to facilitate it,’’ Dorsey said, adding that government officials are ‘‘looking out for the best interest of the minor child’’ in federal custody.
Brigitte Amiri, a lawyer with the ACLU who is representing the teenager, told the court that two potential sponsors had already fallen through and said the often lengthy process of approving a sponsor includes background checks and possible a home visit.
She urged the court not to set aside its obligation to protect the teen’s constitutional right to abortion just because she may eventually obtain a sponsor, and said the government is not acting in the teen’s best interest.
‘‘They are supplanting their decision about what she should do with her pregnancy,’’ Amiri said. ‘‘That’s a veto power over her abortion decision.’’
A state judge in Texas ruled in September that the teen could bypass the state’s parental consent requirement to obtain an abortion.
Millett suggested that a federal policy should not override the decision of a state court judge.
The appeals court allowed the teen to undergo the counseling Texas law requires on Thursday, as ordered by the lower court judge. But the panel has temporarily blocked that judge’s order to allow the abortion to proceed.
Underscoring the significance and interest in the case, Chief Judge Merrick Garland agreed on Friday to live-stream audio of the oral argument for the first time in 16 years.
Roughly 40 people gathered Friday morning in front of the Department of Health and Human Services to demand ‘‘justice for Jane.’’ Brought together by Planned Parenthood, the ACLU, and the National Latina Institute for Reproductive Health, the protesters hoisted signs and sang chants in Spanish and English showing solidarity with the teenager, and expressing contempt for Scott Lloyd, director of the Office of Refugee Resettlement.
‘‘The constitutional right to abortion does not depend on your immigration status,’’ said Georgeanne Usova, legislative counsel for the ACLU.
Usova said the group wasn’t just fighting for ‘‘this young woman, but every woman in government custody.’’
The pregnant teenager faces a ‘‘cruel clock that is ticking rapidly,’’ said Bethany Van Kampen, a policy analyst for the National Latina Institute for Reproductive Health, given that Texas prohibits most abortions after 20 weeks.
‘‘Where is the voice for Jane? Where is the humanity for Jane?’’ she said.


text:
Former beauty queen Anita Kyarimpa, affectionately known as Anita Fabiola, has recently taken to her Instagram in a teaser announcement that has left fans at the edge of their seats.
This is after she wrote saying that she has something special for her fans this coming week and that fans should stay tuned for what she has planned for them.
Read also: Anita Fabiola Is Living Her Wildest Dreams
As per usual, sis has not hinted in the slightest what it could be but as you can already imagine, if it's Fabiola, then you best believe that it is going to be lit which is only making us more excited am I right.
It’s a shame that the comment section is disabled because fans must be really curious to find out what this surprise is but for now, guess we’ll just have to hold on to our horses and be patient.
It was just the other day that Kyarimpa set the internet ablaze yet again, when she announced that she would be releasing her new movie, ‘Red Carpet’ just this month and fans have gotten the chance to experience the heat that she has been serving.
Having been MIA for quite some time, it really is exciting to see that she has been busy chasing the bag and doing the most to ensure that she remains at the top of her game as always.
Being an amazing actress and a beauty queen is definitely working out for her and we could not be more proud of the great strides she is making in the industry.
Read also: Check Out Anita Fabiola's Sexy Body (Photo)
But that’s not the only hat she wears. Anitah is also a successful beauty enthusiast who has established her own cosmetics business in addition to being a first-time movie producer. Her beauty line, Fabiola Beauty, is a luxury cosmetics store with affordable prices.
For the modern woman, Fabiola Beauty specializes in lip products such as glosses and lipsticks. Fabiola Beauty debuted late last year, and the high-end cosmetics company has already outperformed its name.
She and her bae have also been serving us quite the couple goals and continue to prove that love is a beautiful thing.
After getting married in a secret wedding ceremony back in 2018, it is good to see that the couple is still going strong and steady and we love to see it.
Time and time again Anita has taken to her social media to share how beautiful it is to be in love with a man like her Superman and we have to agree that they are indeed a sight for sore eyes.
The couple just recently celebrated their 3 years anniversary that saw Anita celebrate her man in a heartfelt post that left us all reaching for the tissues.

domain: celebrity


Im having the notorious engine idle problem...fluctuating idling/juddering gear lever etc...and oil
in my air filter..
What i want to know is if the idling problem and the oil
in the air filter are related, and in any case what is causing the oil
in the air filter and how do i cure it??
When i was having a look about it, i took off the airbox cover and noticed oil
inside, and the lower part of the filter was covered in oil
. I removed the filter entirely and havent replaced it yet. Coincidentally perhaps, but the idling problem hasnt happened!