text:
The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (ΔIami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of γENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a γENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in γENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.
23 Related JoVE Articles!
Assays for the Identification of Novel Antivirals against Bluetongue Virus
Institutions: University of Alabama at Birmingham, Auburn University.
To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e.
50% inhibitory concentration (IC50
) or effective concentration (EC50
), as well as its range of cytotoxicity (CC50
). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.
Immunology, Issue 80, Drug Discovery, Drug Evaluation, Preclinical, Evaluation Studies as Topic, Drug Evaluation, Feasibility Studies, Biological Assay, Technology, Pharmaceutical, High-Throughput Screening Assays, Animal Diseases, Investigative Techniques, Antiviral, Efficacy, Bluetongue Virus, Cytopathic effect, Dose response, Time-of-Addition, Mechanism-of-Action
The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter
Institutions: Wasatch Microfluidics, University of Utah.
The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.
Bioengineering, Issue 86, submerged printing, cell based assay, cell printing, cell microarray, continuous flow microspotter, microfluidics, high-throughput cellular assays, in vitro cytotoxicity, cellular drug screening
A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels
Institutions: University of South Carolina, School of Medicine.
G protein-gated inward rectifier K+
(GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2
. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi
) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+
out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3
. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4
(3)] or HLB 021-152 (Figure 1
). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1
). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+
efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1
). Thus, drugs that modulate K+
efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4
or radiotracer analysis5
, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.
Medicine, Issue 62, G protein-gated inward rectifier K+ (GIRK) channels, clonal cell lines, drug screening, fluorescent dyes, K+ channel modulators, Pharmacology
High Content Screening in Neurodegenerative Diseases
Institutions: VU University Medical Center, Neuroscience Campus Amsterdam.
The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4
. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5
. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7
. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long.
There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8
. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
High-throughput screening (HTS) is an ideal methodology to address these issues9-12
. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14
. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17
. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS.
HCS has many advantages over HTS18, 19
, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility.
Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20
and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21
Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.
Medicine, Issue 59, High-throughput screening, high-content screening, neurodegeneration, automated cell culturing, Parkinson’s disease
Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs
Institutions: Vanderbilt University, Vanderbilt University, Vanderbilt University, Vanderbilt University Medical Center, Vanderbilt University, Vanderbilt University.
Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems.
In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.
Immunology, Issue 73, Cellular Biology, Medicine, Biomedical Engineering, Bioengineering, Cancer Biology, Molecular Biology, Erythrocytes, Endosomes, Small Interfering RNA, Gene Therapy, Nanomedicine, Gene delivery, Nanoparticles, Endosome Escape, Intracellular Trafficking, Cytosolic Drug Delivery, red blood cells, assay
A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl
Institutions: Peter MacCallum Cancer Centre.
The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB’s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.
Chemistry, Issue 93, Granzyme B, serine protease, peptide thioesters, BOC-Ala-Ala-Asp-S-Bzl, colorimetric substrate, hydrolysis, asp-ase activity
High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Institutions: Massachusetts General Hospital.
We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla
luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla
luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.
Cellular Biology, Issue 88, Luciferases, Gene Transfer Techniques, Transfection, High-Throughput Screening Assays, Transfections, Robotics
siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells
Institutions: University of Dundee, University of Dundee.
Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription. To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA “ubiquitome” library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question. Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter. An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested. Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.
Biochemistry, Issue 87, siRNA screening, ubiquitin, UBL, ubiquitome, hypoxia, HIF1A, High-throughput, mammalian cells, luciferase reporter
A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents
Institutions: University of Maryland School of Medicine, University of Maryland School of Medicine, University of Maryland School of Medicine, University of Maryland School of Medicine, University of Maryland School of Medicine.
Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.
Cellular Biology, Issue 78, Transcription Factors, Vitamin D, Drug Discovery, Enzyme-Linked Immunosorbent Assay (ELISA), DNA-binding, transcription factor, drug screening, antibody
Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Institutions: Vanderbilt University Medical Center, Cincinnati Children's Hospital Medical Center, Vanderbilt University School of Medicine.
egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus
egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3
. Herein, a method is described for isolating Xenopus
egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus
egg extract. One method is visually informative ([35
S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus
egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.
Molecular Biology, Issue 88, Xenopus laevis, Xenopus egg extracts, protein degradation, radiolabel, luciferase, autoradiography, high-throughput screening
Use of the Protease Fluorescent Detection Kit to Determine Protease Activity
Institutions: Sigma Aldrich.
The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 μg/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.
Basic Protocols, Issue 30, Protease Fluorescent Detection Kit, Protease Detection, serine proteases, cysteine proteases, metallo-proteases, aspartic proteases
An In vitro FluoroBlok Tumor Invasion Assay
Institutions: Discovery Labware.
The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2
. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro
providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12
(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at >99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.
Cellular Biology, Issue 29, Tumor Invasion Assay, Chemotaxis, Calcein-AM, Matrigel, Falcon, Fluoroblok, Migration, Invasion, Tumor, BD, Matrigel, Boyden chamber, Motility, Haptotaxis
Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays
Institutions: BioSentinel Inc., Madison, WI.
Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro
replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.
Neuroscience, Issue 85, Botulinum, food testing, detection, quantification, complex matrices, BoTest Matrix, Clostridium, potency testing
High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses
Institutions: Institut Pasteur, CNRS UMR3569, Institut Pasteur, CNRS UMR3523, Institut Pasteur.
RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.
Immunology, Issue 87, Viral infections, high-throughput screening assays, broad-spectrum antivirals, chikungunya virus, measles virus, luciferase reporter, chemical libraries
High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels
Institutions: Vanderbilt University School of Medicine, Vanderbilt University School of Medicine, Vanderbilt University School of Medicine.
Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2
. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3
. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5
, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6
. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+
) flux assay of Kir channel function for high-throughput compound screening7,8,9,10
.The assay is based on the permeability of the K+
channel pore to the K+
. A commercially available fluorescent Tl+
reporter dye is used to detect transmembrane flux of Tl+
through the pore. There are at least three commercially available dyes that are suitable for Tl+
flux assays: BTC, FluoZin-2, and FluxOR7,8
. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+
binding. We began working with FluoZin-2 before FluxOR was available7,8
and have continued to do so9,10
. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+
readily permeates most K+
channels, the assay should be adaptable to most K+
Biochemistry, Issue 71, Molecular Biology, Chemistry, Cellular Biology, Chemical Biology, Pharmacology, Molecular Pharmacology, Potassium channels, drug discovery, drug screening, high throughput, small molecules, fluorescence, thallium flux, checkerboard analysis, DMSO, cell lines, screen, assay, assay development
Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications
Institutions: Purdue University, Eli Lilly and Company.
Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson's disease. Acute activation of Gαi/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro
and in vivo
following the chronic activation of several types of Gαi/o-linked receptors including D2
dopamine and μ opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gbγ signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384 well format would facilitate future studies. Using two D2
dopamine receptor cellular models (i.e
), we have converted our 48-well sensitization assay (>20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.
Bioengineering, Issue 83, adenylyl cyclase, cAMP, heterologous sensitization, superactivation, D2 dopamine, μ opioid, siRNA
High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
Institutions: Aix-Marseille Université, Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France.
Escherichia coli (E. coli)
is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli
cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.
Bioengineering, Issue 89, E. coli, expression, recombinant, high throughput (HTP), purification, auto-induction, immobilized metal affinity chromatography (IMAC), tobacco etch virus protease (TEV) cleavage, disulfide bond isomerase C (DsbC) fusion, disulfide bonds, animal venom proteins/peptides
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line
Institutions: University of Mississippi, University of Mississippi.
Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1
. Leishmania donovani
is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2
;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3
. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro
and axenic amastigotes assays5
are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro
screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro
with Leishmania donovani
for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania
amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani
metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania
-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania
amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al.
Infection, Issue 70, Immunology, Infectious Diseases, Molecular Biology, Cellular Biology, Pharmacology, Leishmania donovani, Visceral Leishmaniasis, THP1 cells, Drug Screening, Amastigotes, Antileishmanial drug assay
Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Institutions: KU Leuven.
For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16
construct is co-transfected. Following ligand binding, activation of the Gα16
subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16
, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.
Cellular Biology, Issue 89, G protein-coupled receptor (GPCR), calcium mobilization assay, reverse pharmacology, deorphanization, cellular expression system, HEK293T, Fluo-4, FlexStation
A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
Institutions: Université de Lille.
Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis
) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb
within fluorescently labeled host cells using automated confocal microscopy. This in vitro
assay allows an image based quantification of the colonization process of Mtb
into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb
mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb
within host cells.
Infection, Issue 83, Mycobacterium tuberculosis, High-content/High-throughput screening, chemogenomics, Drug Discovery, siRNA library, automated confocal microscopy, image-based analysis
A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators
Institutions: The Scripps Research Institute, The Scripps Research Institute.
Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.
). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.
Chemistry, Issue 82, BoNT/A, botulinum neurotoxin, high-throughput screening, FRET, inhibitor, FRET peptide substrate, activator
BioMEMS and Cellular Biology: Perspectives and Applications
Institutions: University of Washington.
The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in drug screening, toxicology, and clinical assays, and is increasingly used in regenerative medicine. However, the traditional cell culture methodology essentially consisting of the immersion of a large population of cells in a homogeneous fluid medium and on a homogeneous flat substrate has become increasingly limiting both from a fundamental and practical perspective. Microfabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, and the medium composition, as well as the neighboring cell type in the surrounding cellular microenvironment. Additionally, microtechnology is conceptually well-suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. In this interview, Albert Folch explains these limitations, how they can be overcome with soft lithography and microfluidics, and describes some relevant examples of research in his lab and future directions.
Biomedical Engineering, Issue 8, BioMEMS, Soft Lithography, Microfluidics, Agrin, Axon Guidance, Olfaction, Interview


Company V-Moda known primarily for the release of headphones premium unusual appearance, introduced the worlds first portable Bluetooth-column. Also this is the first device of its kind with a built-in headphone amplifier. The column can be used with headphones with high impedance because the amplifier provides the power for 83 mW per channel. V-Moda Remix has the support of virtual assistants, such as Alexa, Google Assistant and Siri.
With dimensions of 205 x 65 x 68 mm speaker weight is 710 or 850 g, depending on the materials and design chosen. If desired, buyers can customize the appearance of V-Moda Remix using printed on 3D-printer overlays for each of the six sides. There is also a premium version of the 14-carat gold or platinum.
Frequency range V-Moda Remix is 20 – 20 000 Hz, sensitivity: 79 dB ± 3 dB, and the sound meet the two speakers power of 10 watts. In the column there are two headphone jacks and a USB Type-C for charging. Built-in battery, according to the manufacturer, enough for 10 hours of music playback.


Farnley Falcons rugby club has received much-needed planning permission to build a new permanent clubhouse and changing facilities.
The club, which caters for more than 100 youngsters each week, has been operating out of temporary steel containers which act as changing rooms off Butt Lane.
It used to be based at nearby Farnley Sports and Social Club until it closed in late 2014 and was then demolished after an arson attack in 2015 and replaced with houses.
The Falcons successfully applied to Leeds City Council for the demolition of existing buildings and construction of a new single-storey clubhouse and changing facilities for four teams on the green belt site.
They’ve also got the green light for associated car parking and the extension of one of existing pitches at the Butt Lane Recreation Ground in Farnley.
The club’s proposals received the support from dozens of supporters and local residents. A club statement accompanying the application added:
“The volume of visitors to Butt Lane creates huge congestion on training and game days which we would like to resolve with the creation of off-street parking. This has been widely supported during our planning application.
“There are several opportunities which our club wishes to grasp in the near future.
We don’t currently offer Rugby League to the full spectrum of school children, and
we could create two primary school teams to service this gap. Natural progression will
see the number of Girls teams, and players, increase at the club. which will also
place further stresses and challenges upon our facilities.
“The other major opportunity for our club is to establish an Over 35s ‘Masters’ team,
which would expand our offering to the local community beyond that of the very
competitive Men’s Rugby League currently played at Farnley.
“The opportunity to keep parents and players on Butt Lane, using our facilities, and
buying our food and drink will greatly increase the income of the club and the scope
to which we can best provide for both our members and the wider community.”
A planning statement accompanying the application said the new building would be sited in an unobtrusive location currently occupied by the temporary changing rooms.


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Essay social networking sites good
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Essay social networking sites good
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Persuasive speech example – do social networking sites do more harm than good here is an example of a free persuasive speech topic sent in by matteo berto. Free essays on argumentative essays on social networking sites privacy get help with your writing 1 through 30. Social media and social networking seems to play an imperative part of peoples lives around the world essay on social media. In modern times, people are interacting in many different ways, some by telephone, some by e-mail and some by chatting networks every day, people are using the computer to talk with one. Social media sites empower individuals to make social change and do social good on a community level social media shares popularized nine-year old scottish student, martha payne, and her.

domain: sociology


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When it comes to the value of your home, there are several factors to consider beyond the features of the property itself. The location of your home plays a significant role in determining its long-term value. In the Bay Area, where the real estate market is highly competitive, understanding how neighborhood features can influence property values is crucial. In this article, we will explore some key external factors that can impact the value of your Bay Area home. From schools to noise pollution, we’ll discuss the importance of each factor and how it can affect your investment.
1. Proximity to High-Quality Schools
One of the most desirable features for homebuyers in the Bay Area is proximity to excellent schools. Living in a neighborhood with top-quality school districts can significantly boost property values. On the other hand, residing near low-ranking schools can have the opposite effect. According to realtor.com, the median home price in areas with schools that received a low rating is substantially lower compared to homes in areas with highly-rated schools. Thus, homes in good school districts tend to have higher demand and value.
2. Impact of Disruptive Neighbors
Having disruptive neighbors can negatively impact property values. The Appraisal Institute emphasizes that a home’s proximity to bad neighbors can lead to a decline in value. Unkempt yards, unpleasant odors, poorly maintained exteriors, and noisy pets are all factors that can contribute to this decline. Living near a troublesome neighbor can devalue your Bay Area home by as much as 5-10%. Therefore, it’s essential to research the neighborhood before purchasing a property to ensure a peaceful living environment.
3. Noise Pollution from Surrounding Areas
Noise pollution is another significant factor that can affect the value of your Bay Area home. Living near an airport, train tracks, highways, or industrial areas with constant noise can be undesirable for many buyers. The louder and more inconvenient the noise, the more it can impact your home’s resale value. Properties located next to train tracks or major highways tend to have lower values compared to similar homes located farther away. When considering a property near transportation facilities, it’s crucial to consult with a local real estate agent to evaluate the potential impact on market value.
4. Proximity to Power Lines and Power Plants
The presence of power lines and power plants in the neighborhood can impact property prices. Safety concerns associated with power plants often lead to lower property values. Additionally, the unattractive appearance of power lines can deter potential buyers. Some people also worry about the perceived negative health effects of living near power lines. If you’re considering purchasing a home near power lines, it’s advisable to consult with a local real estate agent to assess the potential impact on market value.
5. Living Near a Cemetery
Living near a cemetery can be a polarizing factor for homebuyers. While some may find the quiet and peaceful environment appealing, others may feel uncomfortable or even fearful. Research by realtor.com shows that homes in ZIP codes with a cemetery have a median price approximately 12% lower than similar homes in areas without a graveyard. Witnessing funerals and seeing the road lined with cars of mourners can be distressing for some individuals. Therefore, the proximity of a cemetery can impact the value of your Bay Area home.
6. Considerations Surrounding Shooting Ranges
While some people may enjoy having a shooting range nearby, it can have a negative impact on your home’s value. The noise generated by gunfire, particularly from outdoor gun ranges, can be loud and disruptive. Environmental and safety concerns, such as lead contamination from spent shells, can also be worrisome. If you’re considering a home near a shooting range, it’s important to research the shooting schedule and determine if you can tolerate the occasional sound of gunshots.
7. The Effect of Billboards
Studies have shown that the presence of billboards near residential areas can lower property values. In urban areas, the closer a billboard is to your Bay Area home, the greater its potential impact on its value. As a result, many communities are implementing strict billboard controls or even enforcing a no-billboard policy to protect home values and promote higher median incomes and lower home-vacancy rates.
8. Multiple Foreclosures in the Area
The occurrence of multiple foreclosures in your Bay Area neighborhood can have a detrimental effect on property values. Foreclosures are often seen as indicators of problems within an area, leading to a decline in the average value of homes. Bank-owned properties are typically not well-maintained, resulting in unsightly yards and poorly maintained exteriors. Studies have shown that living within a quarter-mile radius of a foreclosed home can cause a 4% decline in property values. Additionally, areas with foreclosures may experience an increase in property taxes, as highlighted in a report by The Alliance for a Just Society.
9. Other Factors to Consider
While the aforementioned factors play a significant role in determining the value of your Bay Area home, it’s important to consider additional aspects as well. Factors such as crime rates, access to amenities, transportation infrastructure, and local development projects can all influence the value of your property. Consulting with a local real estate agent who is familiar with the Bay Area market can provide valuable insights into these factors and help you make informed decisions.
When it comes to the value of your Bay Area home, it’s essential to understand the impact of external factors within your neighborhood. Proximity to high-quality schools, disruptive neighbors, noise pollution, power lines, cemeteries, shooting ranges, billboards, and foreclosures can all affect your property’s value. By considering these factors and conducting thorough research, you can make informed decisions when buying or selling a home in the Bay Area. Consulting with a local real estate agent who has expertise in the area can provide valuable guidance throughout the process. Remember, the location of your home plays a crucial role in its long-term value, so choose wisely.

domain: geography


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Last Updated: Tuesday, July 15, 2014, 13:17
It`s nursing season in Europe`s biggest marine sanctuary and schools of whales have brought their young to Italian shores - but environmentalists warn they are at risk from toxins leaked by the wrecked Costa Concordia cruise ship.
Last Updated: Friday, July 11, 2014, 19:21
In a discovery that offers new ways to protect crops from insects, researchers have found that insecticides can be designed to target specific pests without harming beneficial species like bees.
Last Updated: Monday, July 07, 2014, 17:37
In what can alter the course of cancer treatment in the near future, researchers have found a compound that appears to pinpoint all of the malignant cells in a patient`s body.
Last Updated: Monday, July 22, 2013, 19:46
The pesticide that killed 23 schoolchildren last week is a nerve poison banned by many countries because of what the World Health Organisation (WHO) describes as its "high acute toxicity".
Last Updated: Tuesday, December 27, 2011, 21:37
A cancer-causing toxin was found in milk produced by a leading company.
Last Updated: Sunday, March 13, 2011, 09:41
High levels of domoic acid were found in sardines, which may have distressed them off LA coast.
more videos >>

domain: news


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Bluefield, Va Farmers Market
This is the location for the Bluefield, Va Farmers Market. Please check out all the information about Bluefield, Va Farmers Market including the season dates/times and items sold for this farmers' market in Bluefield, Virginia.
Bluefield, Va Farmers Market Season Dates and Times
Types of items sold at this Farmers' Market
Bluefield, Va Farmers Market is located in Bluefield, VA in Tazewell county. They do sell organic products at this market. Make sure you check the date and the times before you head out the door. This farmers market has many products to offer. Search all farmers markets in near Bluefield, Va Farmers Market as well. If the Bluefield, Va Farmers Market needs to be updated and you would like to help, please contact us.
Other Markets in Tazewell
|Warrenton Farmers Market located in Warrenton, VA||View Details|
|Del Ray Farmers' Market located in Alexandria, VA||View Details|
|The Carytown Farmers Market located in Richmond, VA||View Details|
|Lynchburg Community Market located in Lynchburg, VA||View Details|
|Woodstock Farmers' Market located in Woodstock, VA||View Details|

domain: food


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01 December 2016 Current Affairs: The Indian Space Research Organisation (ISRO) is using drones to map disasters in north-eastern States by collecting land details and add it to data from remote sensing satellites.
In this regard, ISRO’s Shillong-based North-Eastern Space Applications Centre (NE-SAC) has tested unmanned aerial vehicles (UAVs) to map various problems and disasters.
NE-SAC has taken the initiative for design and assembling of UAVs for various applications to assess several regional problems in the northeast region.
UAVs can perform efficient surveys for disaster-prone or physically inaccessible areas. It can undertake quick damage assessment of floods, landslides and earthquakes and enable timely relief measures. These drones providing ground-based details which are generally combined with data from ISRO’s remote sensing satellites.
Recently they were used for to map the area affected by landslides along NH40, Meghalaya’s life line. It also gave the extent of damage caused to pest-infested paddy fields in Naramari village of Assam.
North-Eastern Space Applications Centre (NE-SAC) : NE-SAC is a joint initiative of Department of Space (DoS) and North Eastern Council. It was started in the year 2000. It is located at Umiam (near Shillong), Meghalaya. It aims to provide developmental support to the North Eastern region using Space technology-based communication and technology.
Its mandate is to develop high technology infrastructure support to enable NE states to adopt space technology for their development. NE-SAC provides developmental support by undertaking specific application projects using remote sensing, satellite communication, GIS and conducts space science research.
FreshersLive - No.1 Job site in India. Here you can find latest 2018 government as well as private job recruitment notifications for different posts vacancies in India. Get top company jobs for both fresher and experienced. Job Seekers can get useful interview tips, resume services & interview Question and answer. Practice online test free which is helpful for interview preparation. Register with us to get latest employment news/rojgar samachar notifications. Also get latest free govt and other sarkari naukri job alerts daily through E-mail.


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This remarkably inventive novella, published originally in the February 1943 issue of Astounding Science Fiction, sculpts a complicated feudal society based on decadence and warfare, a tragic warrior hero and a brilliantly imagined Venus within its compass of 25,000 words. Clash By Night foreshadows the full-length novel, Fury, which appeared in Astounding three years later. The novella is centered on Brian Scott, a so-called "Free Companion" who is a warrior for one of the competing undersea civilizations of Venus. The novel portrays what Scott intends to be his last mercenary battle. A decadent humanity lives on Venus under the planet's seas. They are the descendants of survivors exiled from Earth after the destructive atomic wars. Life for most of these inhabitants of the Keeps is pleasant though corrupt and is so detailed by Kuttner with great precision and force through the eyes of Scott, whose mercenary services as a warrior are available to the highest bidder. These wars among the undersea Keeps give point to what would otherwise be an aimless, luxurious existence in these shells under the fierce Venusian seas. As Brian Scott, in the employ of the besieged Montana Keep, makes alliances with other mercenaries of the Doones to fight a war for property, he comes to a truer understanding of the nature of his life and of his fate.About the Author
Henry Kuttner (1914-1958) wrote alone as well as in collaboration with his wife, the great science fiction and fantasy writer, C.L. Moore. Moore is one of the four or five most important writers of the 1940s and the writer whose work went furthest in its sociological and psychological insight into making science fiction human as well as technological literature.
Kuttner was an important influence upon every contemporary science fiction writer who succeeded him. In the early 1940s, and under many pseudonyms, Kuttner and Moore published very widely through a range of science fiction and fantasy pulp markets. The Kuttners wrote well-regarded science fiction novels, among them, Fury and Mutant. These, along with over fifty shorter works, remain consistently in print, keeping the Kuttner name front and center.
About Henry KuttnerSee more books from this Author

domain: library


3 Popular Israeli Gun Innovations
Israel is a relatively young nation, with few natural resources.
So, they capitalize on what they do best, which is weapons.
It’s no secret how good Israeli weapons are.
In fact, Israel is one of the world’s top weapons exporters, with approximately $6.5 billion in annual arms sales.
Despite Israel’s small size, it spends about 4.5 percent of GDP on research and development.
Of that amount, about 30% goes to products of a military nature.
By comparison, only 17% of U.S. R&D is for the military.
A big reason Israelis are such successful weapon innovators is their willingness to take more risks than other countries.
This stems from their required military service.
While many of their Western counterparts live in the safety of college dormitories.
Many Israeli 19-year-olds are conducting highly dangerous operations behind enemy lines.
Plus, Israel has been in a perpetual state of conflict.
They’ve fought a war almost every decade.
When you’re constantly under attack, you either innovate or die.
So today, I want to share 3 of the most popular weapons designed or manufactured in Israel.
The Uzi is one of the most distributed weapons in history.
More than ten million manufactured and shipped to over 90 countries.
While it’s been in production and use since the early 1950s, there have been several variants.
The Uzi was first designed by Captain Uziel Gal of the Israeli Defense Forces.
The original models had a wooden stock, before they went to the iconic folding metal one in later models.
The Uzi came in different variations including ammo capacity of 20-50 rounds.
Along with a rate of fire of 1,000 to 1,150 rounds per minute.
Older versions came in 9mm, but .22, and 45 ACP models were also made.
The Uzi was sold to more military and law enforcement forces than any submachine gun ever made.
Its popularity stemmed from its compact size, large ammo capacity, and ability to churn out a high rate of fire.
The Desert Eagle.
Another iconic Israeli gun is the Desert Eagle (AKA “The Desert Eagle Hand Cannon”).
The pistol was designed from 1979-1982, and manufactured in Israel until 1995.
Today it is no longer an Israeli gun, but it was originally manufactured in Israel.
The Desert Eagle is a massive pistol.
It’s designed to withstand enormous amounts of pressure from the large calibers it fires.
It was originally chambered in .357 magnum.
Before the Desert Eagle came along, revolvers were the only handguns that could withstand the pressures of the round.
And while most semi-auto pistols have free-floating barrels…
To compensate for the force of shooting, the Desert Eagle has a fixed barrel.
The bolt face utilizes teeth to lock into battery, much like an AR-15 rifle.
Typically, pistols use the energy from the round fired to cycle the action.
But this would be impractical and unsafe on the Desert Eagle.
Instead, they derived a gas piston system like the one in the Ruger Mini-14.
This system takes the excess gas from the gunpowder to push a piston, and cycle the action.
This diverts some of the recoil from going straight into the operator’s wrists.
There are three main variants to the Desert Eagle:
The most recent edition has a 10” barrel, and a total length of 14.75”.
It comes in .357 Magnum, .41 Magnum, .44 Magnum, or .50 Action Express.
While it never has seen much practical use, it’s been used in film and television.
And it’s remained one of the most popular sidearms in modern gun culture.
The Jericho 941.
The Jericho 941 is a no-nonsense, no-frills, but highly reliable semi-automatic sidearm.
It’s based upon the proven Czech CZ-75 pistol design.
And the Jericho 941 comes in 9mm, .40 S&W, and .45 ACP.
The gun incorporates a low bore axis to minimize recoil and muzzle rise.
It comes in both single and double action configurations.
The weapon has been imported into several countries, including the U.S.
There are several variations and sizes, and both steel and polymer configurations.
The steel frame Jericho is much heavier than the polymer models.
This can lead to a more manageable recoil while shooting.
While not ideal for self-defense carry, it’d still be a good choice for home defense.
It’s desired by operators around the world who work in harsh environments.
These are three of the most popular weapons Israel has produced…
Perhaps you may wish to add one or two of them to your personal arsenal.
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Do not get Left Behind, Learn This article On Vitamins Now
On the subject of good well being, one must study all they’ll to ensure they stay on top of things. That means knowing all you’ll be able to about vitamins and minerals. With a purpose to learn extra, check out the great ideas and tricks discovered within the content we have presented below.
Many people who find themselves in need of supplemental vitamins and minerals merely struggle bodily with swallowing the pills as they can be massive and, in all honesty, horrible tasting. Try drinking water that has vitamins and minerals consists of with some kind of fruit taste added. This lets you get some of the vitamins you require as well as including to your daily water intake.
When shopping for fats-soluble vitamins, like omega-3, it’s a good idea to retailer them within the refrigerator. Since they comprise fats, improper store can cause them to develop into rancid. Additionally, make sure that to keep them away from heat and light because this may break them down and make them ineffective.
Thiamine, or vitamin B1, deficiency may cause neurological and psychiatric signs. These signs can include weight loss, weakness, irregular heart price and psychosis. A vitamin B advanced can make sure you obtain the vitamin B1 you want, or you possibly can eat pork, brown rice, liver, oatmeal, leafy vegetables, eggs and potatoes.
In case you are planning on taking any vitamins and minerals, you need to attempt to schedule them round mealtimes. Once you eat and digest your meals, stomach acid is produced. This can help break down any vitamins you’re consuming, which means that they will be too much simpler.
Vitamin C is one of the crucial helpful vitamins which you could take. It promotes tissue development, reduces most cancers dangers, helps with blood cell formation and quickens the healing course of. Broccoli, peppers, oranges, tomatoes, and a large number of other foods are all rich in vitamin C. You too can take a complement that incorporates vitamin C.
If focus factor https://thebrilliantfactor.com are attempting to grow out your hair or nails, consider including a Biotin supplement. Whereas it will not make your hair or nails grow quicker, it will make them stronger and more resistant to breakage. relevant site is, Biotin is usually really useful to chemotherapy patients to assist them regrow the hair that they misplaced during therapy.
Some vitamins are stored in your physique, and others usually are not. Water-soluable vitamins dissolve within the fluids in your body and are flushed out usually. These vitamins embody B vitamins and vitamin C. Because these vitamins are usually not stored in your physique, it is important to devour these vitamins every day.
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Ask your doctor to advocate a good supplement for you. You can search the online for the very best sorts. Supplements will not be regulated by the FDA, which suggests you might want to analysis them.
Have you ever instructed your self you do not need vitamins and minerals since you never get sick? Nicely you actually nonetheless need them as they not only help fight off infection, they also alleviate signs of stress which we all endure at some point. Vitamin C is the surprise vitamin because it aides in not only well being and reducing stress however even wholesome eyes only for an instance.
Vitamin B6 deficiencies could cause anemia, coronary heart disease and high cholesterol. This highly effective vitamin might be present in cereals, liver, beans, eggs, vegetables and pink meat. Vitamin B6 is used to in patients affected by ADHD, diabetes, autism, Down’s syndrome, sickle cell anemia, migraine complications, asthma and macular degeneration.
All the time learn the instructions earlier than ingesting any type of vitamin. You want to take a few of these when you haven’t had anything to eat, and some have for use right after you eat. You also need to make certain that you don’t take an excessive amount of of any vitamin.
When taking vitamin supplements, at all times read the label to find out the quantity of the vitamin included in each capsule or tablet.Your best option will normally be an quantity that’s close to the usual recommended daily dose. Carefully consider the potential risk of excessive dose vitamins to the potential advantages before you purchase.
In case you have a wheat intolerance, which complement you purchase turns into extra essential. Some pills embody wheat, so you need to examine the label earlier than you purchase something. There are a lot of non-wheat choices for folks such as you, so you will not be denied the supplement you need just due to your medical condition.
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domain: medical


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Sony/ATV Music Publishing has announced a series of "groundbreaking" upgrades to how it reports and pays royalties to its songwriters.
Over the course of the next year, the publisher will roll out new initiatives that will speed up how quickly songwriter earnings are processed, allowing songwriters to get paid faster than ever before.
Sony/ATV is introducing real-time inter-company processing for all foreign earnings from every territory in which the company operates. As a result, foreign earnings will now be reported and paid in the same period in which they are collected.
It will also offer a new Cash Out service, which will allow its songwriters to request some or all of their current royalty balance to be paid immediately, instead of having to wait until the next distribution. This service will be offered to the company’s songwriters without a fee. Once they have made any withdrawals, they will then receive the remainder of their balance during the next regular distribution.
We can now give our songwriters even greater control over their royalties and ensure that they get paid quicker than ever before
Sony/ATV chairman and CEO Jon Platt (pictured) said: “I am proud to announce these ambitious new initiatives that will help to deliver our strategy to be the best service company in the world for songwriters. Powered by our industry-leading administration operations and best-in-class technology, we can now give our songwriters even greater control over their royalties and ensure that they get paid quicker than ever before.”
Real-time inter-company processing and the Cash Out service will start to be made available over the next year to all Sony/ATV songwriters who are signed up to the company’s royalty portal and app, SCORE, which gives songwriters and their teams real-time access to their current royalty account and historic earnings.

domain: finance


The deadly ET-Plus guardrail system, known for jamming and piercing through vehicles, will undergo yet another round of testing, this time conducted by the state of Virginia. Langdon & Emison, a nationwide personal injury firm, represents clients nationwide in cases involving the ET-Plus.
A series of eight crash tests, overseen by the federal government, began in December after a federal jury found Trinity Highway Products, the manufacturer of the ET-Plus, liable for defrauding the Federal Highway Administration by not disclosing design changes it made in 2005.
In March, the federal government announced that the ET-Plus had passed all eight crash tests, but Virginia officials have deemed the tests inadequate, according to a report by The New York Times. State officials are proposing an additional six tests set to begin this month, the Times reported.
“The new tests will include having vehicles hit Trinity’s ET-Plus guardrails at slight angles – said to be representative of many car accidents and generally recommended under newer federal safety standards,” wrote The Times.
Lawmakers had criticized the previous crash tests for not requiring lower-angle testing to address the system’s known defects and to determine whether the ET-Plus is safe. In most accidents, the striking vehicles have struck the ET-Plus from shallow angles, yet one of the initial tests involved crashing a pickup into an ET-Plus at a 15-degree angle.
“Conducting a test in which the vehicle strikes the ET-Plus at 5 degrees is the optimal angle for determining the worst possible result from a crash,” said Langdon & Emison attorney Adam Graves. “At 15 degrees, the striking vehicle typically does not fully engage with the end terminal and instead races past the guardrail on impact, which doesn’t test the system’s actual performance.”
Virginia was one of the first states to suspend further use of the ET-Plus and has been considering removing the guardrail system for more than a year.
About Langdon & Emison
Langdon & Emison represents families in a number of cases involving defective Trinity guardrails and is reviewing a number of potential cases. For a free case evaluation or to work with us, contact our firm at 1-866-931-2115 or click here.


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- Taylor and Francis 2013; US$ 39.95
Previously published as a special issue of Environmental Education Research , this collection includes some of the most influential and important articles contributed to the field over the last decade. Drawing out the best articles from volumes one to ten, the editors highlight six major themes: EE and ESD: tension or transition? locating... more...
- Taylor and Francis 2013; US$ 35.95
In this book, Paul Clarke argues that in order to live sustainably we need to learn how to live and flourish in our environment in a manner that uses finite resources with ecologically informed discretion. Education is perfectly placed to create the conditions for innovative and imaginative solutions and to provide the formulas that ensure that everyone... more...
- Taylor and Francis 2013; US$ 48.95
The links between education and sustainable development are deepening, although subject to much controversy and debate. The success of the sustainability discourse depends both on the pedagogic and research functions of higher education. Similarly, for higher education itself to remain relevant and engaged it faces pressure not only to integrate the... more...
- Taylor and Francis 2013; US$ 119.95
The environment and contested notions of sustainability are increasingly topics of public interest, political debate, and legislation across the world. Environmental education journals now publish research from a wide variety of methodological traditions that show linkages between the environment, health, development, and education. The growth in... more...
- Springer 2013; US$ 69.95
The Nature of Transformation: Environmental Adult Education is based on 15 years of educating for social-environmental change around the world. It is for adult and community educators, trainers, literacy and health care practitioners, social activists, community artists and animators, labour educators, and professors in higher education interested... more...
- Hodder Education 2012; US$ 7.98
Build confidence and understanding throughout the year with hundreds of additional practice questions. This Workbook supports our bestselling Checkpoint series, with exercises specifically matched to the Cambridge Progression tests and the Checkpoint tests. - Develops understanding and builds confidence ahead of assessment with exercises matched to... more...
- Andrews UK 2012; US$ 11.99
Plan for six weeks of learning covering all six areas of learning and development of the EYFS through the topic of the environment. The Planning for Learning series is a series of topic books written around the Early Years Foundation Stage designed to make planning easy. This book takes you through six weeks of activities on the theme of the environment.... more...
- Taylor and Francis 2012; US$ 67.95
Essential Environmental Science brings together within a single volume the vast range of techniques, methods and basic tools necessary for the study of the environment. Environmental science has a massive area of operation, utilising the tools from a plethora of traditional sciences and social sciences. This practical manual draws on contributions... more...
- Diplomica Verlag 2012; US$ 10.36
Hauptbeschreibung Bildung für eine nachhaltige Entwicklung ist eine anerkannte, wichtige Herausforderung unserer Zeit. Sie erfüllt wesentliche Aufgaben in der Sensibilisierung für Nachhaltigkeitsfragen und -probleme und in der Kompetenzentwicklung zu ihrer Bewältigung. Dabei stellt einerseits die Gewährleistung von Grundbildung eine entscheidende... more...


Service tax evasion at 98 billion rupees in April-Dec: official
NEW DELHI (Reuters) - Tax evaders deprived India's coffers of about 98 billion rupees in service taxes during April to December 2012, a finance ministry official said on Friday.
"Of the 98 billion rupees, about 20 billion rupees have been recovered," Lipika Majumdar Roy Choudhury, member, service tax, said.
The department is making efforts to recover the remainder, she added.
Battling tax evasion has become one of the key agendas of Finance Minister P. Chidambaram as he looks to keep an already bloated fiscal deficit target to 5.3 percent for the full year. (Reporting by Manoj Kumar; editing by Malini Menon)
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Having agreed this week to formally adopt inflation targeting as a guiding star for monetary policy, India's government and central bank remain at odds over how crucial decisions are made. Full Article
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peace, love, cross sign
This cute peace, love, cross has leopard print, splatter paint and glitter. I can also add a name for personalization. It is made from 1/4 baltic birch and hand painted. Sealed to protect from fading. Bow may vary depending on ribbon availability. It measures about 20 inches.
All items are handmade by a female veteran owned small business. Thank you for supporting a veteran!
Follow me on Facebook. Www.facebook.com/tiffsdecorandmore
and Instagram www.instagram.com/tiffsdecorandmore

domain: design


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Description Usage Arguments Value
Read a historical climate data CSV file
A path to an (unmodified) CSV file downloaded from the EC service
A tibble with an attribute "flag_info", containing the flag information.
Add the following code to your website.
For more information on customizing the embed code, read Embedding Snippets.


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On June 26, 2013, the U. S. Supreme Court issued an opinion on the case United States v Windsor. They ruled that Section 3 of the Defense of Marriage Act, which said the 'marriage' and 'spouse' only applied to heterosexual unions, was unconstitutional.
According the USA.gov, in response to this ruling, President Obama directed the Attorney General to "work with other members of my Cabinet to review all relevant federal statues to ensure this decision, including its implications for Federal benefits and obligations, is implemented swiftly and smoothly." A number of federal agencies have released information about changes to federal programs and benefits as a result of the Supreme Court's decision.
Taxes - The Dept. of Justice and Internal Revenue Service ruled that "same-sex couples, legally married in jurisdictions that recognize their marriages, will be treated as married for federal tax purposes. The ruling applies regardless of whether the couple lives in a jurisdiction that recognizes same-sex marriage or a jurisdiction that does not recognize same-sex marriage."
Social Security - The Social Security Administration now recognizes same-sex marriages for purposes of determining benefits.
Medicare: - All beneficiaries in private Medicare plans now have access to equal coverage when it comes to care in a nursing home where their spouse lives.
U. S. Visas for Same-Sex Spouses - U. S. embassies and consulates will process visa applications for same-sex marriages the same as for opposite gender spouses.
Benefits for Uniformed Servicemembers - The Department of Defense will extend benefits to same-sex spouses of uniformed service members and civilian employees.
Benefits for Federal Employees - The Office of Personnel Management is now able to provide benefits to legally married same-sex spouses of federal employees and annuitants.
For the most up-to-date information on other government programs that will change as a result of the ruling, visit the agency website. If you don't know the agency, you can browse by program.
For more information on same-sex marriage, visit our page on the topic.


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Native Orchids from the Million Orchid Project to be
Planted on Coral Gables Trees
Thousands of native orchid seedlings, as part of the Million Orchid Project, are being planted on Coral Gables City trees.
In 2014, the Coral Gables City Commission voted to fund $30,000 to reintroduce the orchids that were once plucked from the trees to be shipped north as potted plants. In the late 1800s, masses of orchids were hauled away from South Florida’s forest to be sold as disposable houseplants. At first, there seemed to be an endless supply, but orchid populations eventually dwindled to catastrophically low levels.
“This project is well-matched to George Merrick’s dream of creating ‘a City that is a garden’,” said Coral Gables Mayor Jim Cason. “Our already beautiful green landscape will be simply stunning when the orchids are in full bloom.”
The Coral Gables orchids will be planted in publicly accessible landscapes to be enjoyed by the public. The Million Orchid Project was launched in 2012 by Fairchild Tropical Botanic Garden with the goal to reestablish one million native orchid plants into South Florida’s urban landscapes. Fairchild is growing South Florida native orchid plants by the thousands in its indoor laboratory with the help of volunteers. Four orchid species that were once common in our region are being planted: Florida butterfly orchids, Dollar orchids, Cockleshell orchids and Cigar/Cowhorn orchids. Orchid seedlings will flower in 1-3 years after installation, making our trees even more beautiful!
On Earth Day 2014, the first orchids of the Million Orchid Project were planted at Merrick Park, across from City Hall. Thanks to the partnership between the City of Coral Gables and Fairchild Tropical Botanic Garden, approximately 250,000 native orchids will be installed in various public green spaces , parks and roadway swales.

domain: biology


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Dothan Police Chief Greg Benton recognized some officers at Tuesday's city commission meeting.
Officer Keith Cook is now 'Officer of the Year.'
'Police Supervisor of the Year' went to Lieutenant Scott Heath.
Detention Officer Eric Morris got 'Civilian Employee of the Year.'
Pattie Poole was recognized as 'Telecommunicator 2 Civilian Supervisor of the Year.'
The officers were chosen through peer and management nominations.
|7:00pm||The Big Bang Theory|
|8:00pm||The Crazy Ones|
|8:30pm||Two and a Half Men|
|10:00pm||WTVY News 4 at Ten|
|9:00pm||Law & Order: SVU|
|7:00pm||The Vampire Diaries|
|9:00pm||Primetime News at 9|

domain: law