text:
Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.
21 Related JoVE Articles!
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)
Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology
Institutions: California Institute of Technology, California Institute of Technology, Massachusetts Institute of Technology, University of Minnesota.
Ideal cell-free expression systems can theoretically emulate an in vivo
cellular environment in a controlled in vitro
This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3
To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo
cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli
based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5
The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli
, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6
Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7
The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8
Accompanying mathematical models are available.9,10
The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."
Cellular Biology, Issue 79, Bioengineering, Synthetic Biology, Chemistry Techniques, Synthetic, Molecular Biology, control theory, TX-TL, cell-free expression, in vitro, transcription-translation, cell-free protein synthesis, synthetic biology, systems biology, Escherichia coli cell extract, biological circuits, biomolecular breadboard
A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay
Institutions: Loyola University Chicago.
Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.
Molecular Biology, Issue 78, Cellular Biology, Biochemistry, Genetics, Biomedical Engineering, Medicine, Cardiology, Heart Diseases, Myocardial Ischemia, Myocardial Infarction, Cardiovascular Diseases, cardiovascular disease, immunoassay, cardiac myosin binding protein-C, cardiac troponin I, creatine kinase MB, electrochemiluminescence, multiplex biomarkers, ELISA, assay
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
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
Magnetic Tweezers for the Measurement of Twist and Torque
Institutions: Delft University of Technology.
Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.
Bioengineering, Issue 87, magnetic tweezers, magnetic torque tweezers, freely-orbiting magnetic tweezers, twist, torque, DNA, single-molecule techniques
Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis
Institutions: National Jewish Health, University of Colorado Denver.
Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes. Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 µl. Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.
Bioengineering, Issue 89, plasma, chemistry techniques, analytical, solid phase extraction, mass spectrometry, metabolomics, fluids and secretions, profiling, small molecules, lipids, liquid chromatography, liquid-liquid extraction, cerebrospinal fluid, bronchoalveolar lavage fluid
Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology
Institutions: DNA Medicine Institute, Harvard Medical School, NASA Glenn Research Center, ZIN Technologies.
Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.
Cellular Biology, Issue 93, Point-of-care, prototype, diagnostics, spaceflight, reduced gravity, parabolic flight, flow cytometry, fluorescence, cell counting, micromixing, spiral-vortex, blood mixing
Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Institutions: University of Exeter.
A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.
Biophysics, Issue 91, differential scanning fluorimetry, dissociation constant, protein-ligand interactions, StepOne, cooperativity, WcbI.
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells
Institutions: ETH Zurich, Switzerland.
We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g.
for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.
Immunology, Issue 80, Microfluidics, proteomics, systems biology, single-cell analysis, Immunoassays, Lab on a chip, chemical analysis
Strategies for Study of Neuroprotection from Cold-preconditioning
Institutions: The University of Chicago Medical Center.
Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro
model that closely reflects their in vivo
counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro
for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-α to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.
Neuroscience, Issue 43, innate immunity, hormesis, microglia, hippocampus, slice culture, immunohistochemistry, neural-immune, gene expression, real-time PCR
Determining Cell Number During Cell Culture using the Scepter Cell Counter
Institutions: Millipore Inc.
Counting cells is often a necessary but tedious step for in vitro
cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1
who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1
. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3
in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.
Cellular Biology, Issue 45, Scepter, cell counting, cell culture, hemocytometer, Coulter, Impedance-based particle detection
Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays
Institutions: University of North Carolina at Chapel Hill.
Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels. One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay. Automated qPCR setup has improved this field by allowing for greater reproducibility. Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment. This method along with internal plate controls also reduces experimental variables common to other techniques.
We recently developed a qPCR assay for profiling of pre-microRNAs (pre-miRNAs) using a set of 186 primer pairs. MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level. These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA). Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs. Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful. There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique. Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs. Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression. However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing. Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.
Biochemistry, Issue 46, pre-microRNAs, qPCR, profiling, Tecan Freedom Evo, robot
Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology
Institutions: BioScale, Inc., BioScale, Inc..
Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.
Bioengineering, Issue 48, Immunoassays, Chinese Hamster Ovary Host Cell Protein, Residual Protein A assay, Assay development, Biomarker detection and quantitation, Phospho-AKT, Gadd34, tissue sample, tumor sample, bioreactor sample
Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)
Institutions: Boston University , Boston University , Boston University , Boston University School of Medicine, Boston University School of Medicine, Istituto di Chimica del Riconoscimento Molecolare.
The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.
Bioengineering, Issue 51, Interferometry, label-free, biosensing, microarray, quantification, real-time detection
Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry
Institutions: Fred Hutchinson Cancer Research Center - FHCRC, University of Victoria, Broad Institute of MIT and Harvard, University of Victoria, Plasma Proteome Institute.
There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1
An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA
(Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2
In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS
) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4
and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7
To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID
) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13
In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.
Molecular Biology, Issue 53, Mass spectrometry, targeted assay, peptide, MRM, SISCAPA, protein quantitation
Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples
Institutions: Institute for Hepatitis and Virus Research, Thomas Jefferson University , Drexel University College of Medicine, Van Andel Research Institute, Serome Biosciences Inc..
In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6
. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12
. In fact, most current cancer biomarkers, such as the L3 fraction of α-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15
, and CA199 for pancreatic cancer 16, 17
are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al.
has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18
. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis
-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4
in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20
. This method has been used successfully in multiple different labs 1, 7, 13, 19-31
. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.
Molecular Biology, Issue 63, Glycoproteins, glycan-binding protein, specific protein glycosylation, multiplexed high-throughput glycan blocked antibody microarray
Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method
Institutions: Luminex Corporation, Luminex Corporation.
The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2
. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5
. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6
. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11
. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.
Molecular Biology, Issue 65, Luminex, xMAP, Multiplex, MAGPIX, MagPlex Low Concentration Microspheres, xMAP Antibody Coupling Kit, ELISA, Immunoassay, Antibody Screening, Optimization, Conversion
Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery
Institutions: Protein Discovery, Inc..
The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free
of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1
Basic Protocols, Cellular Biology, Issue 34, GELFREE, SDS PAGE, gel electrophoresis, protein fractionation, separation, electrophoresis, proteomics, mass spectrometry
Interview: Protein Folding and Studies of Neurodegenerative Diseases
Institutions: MIT - Massachusetts Institute of Technology.
In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.
Neuroscience, issue 17, protein folding, brain, neuron, prion, neurodegenerative disease, yeast, screen, Translational Research


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A three-hour installation performance; a container for questions, nothingness, lineage, and choreography through bodies in relationship to time.
Presented as part of Archive Fever at the University Art Gallery, University of California, San Diego, March 2011
Performed by Anya Cloud, Alicia Peterson Baskel, Julie Burelle, Janet Hayatshahi, Rebecca Salzer and Kyle Sorenson (on Skype)
Sound Design by Adam Tinkle, Julie Burelle (voice).
Photos by Jim Carmody.

domain: artistic


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One week before the official Asia-Europe government meeting (ASEM) gathers in Milan, over 400 people from 42 countries in Europe and Asia gathered at the 10th Asia-Europe Peoples forum (AEPF) to present their demands and recommendations.
At the Asia Europe People’s Forum in Brussels we interviewed civil society activists from across Asia, to find out more about the damaging impacts of free trade agreements on the everyday lives of people in their countries.
Ben Hayes, Praful Bidwai, Susan George, Walden Bello
29 September 2010
Ahead of the Asia Europe People's Forum (AEPF) which coincides with the official ASEM8 summit this year in Brussels, four TNI scholar-activists - Susan George, Praful Bidwai, Ben Hayes and Walden Bello - discuss some of the key struggles facing citizens from both regions.

domain: sociology


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Posted by Dockside Green
Categories: Green, Sustainability, Living
It’s more than a simple garden plot atop a high rise – it’s a small scale farm actually producing food for purchase to the supermarket below. How’s that for locally grown!
Check out the original posting below compliments of www.treehugger.com
Rooftop Supermarket Garden Provides Produce to Shoppers Below
by Sami Grover, Carrboro, NC, USA on 03. 8.11
Image credit: Food from the Sky
Adam Stein once famously derided high-tech vertical farms as pie in the sky, but I've always liked the idea of growing high value produce on the roofs of stores that sell it. After all, who needs to ship those teeny tiny packages of rosemary around the country? One community project has been doing just that—growing food on the rooftop of an urban supermarket, and they've just picked up a prestigious award for their troubles.
Rooftop Garden Grows Food for Store
Food from the Sky rooftop garden—which sits above a Thornton's Budgens supermarket in North London—describes itself as a "world first". Growing vegetables, fruits, mushrooms and herbs using organic methods with children and other members of the community, the project then sells these wares in the supermarket below it every Friday.
Rooftop Farms Proliferate Elsewhere
Of course rooftop farms are proliferating elsewhere too, with some even claiming that rooftop farms could ease Middle East unrest. But whether or not this project really is a world first—or one of a pioneering new trend—hardly seems to matter. The fact is that these folks are growing sustainable, ultra-local produce, and they are providing a much needed connection with nature for inner-city kids.

domain: agronomy


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Volume 1, Issue 1, August 2019
Geo-politics in Indian Ocean and threat to maintenance of Peace in Asia
Author: Nizamuddin Khan, Iftekhar Ahemmed, Mohd Firoz Ahamed
The world’s third largest ocean, Indian Ocean, has been always an important strategic, economic, social and political space for global interaction among world powers. The power and significance of this geographical marine region had been well recognised by Mahan, Mackinder and Spykmen during first half of the 20th century. Colonial era after 15th century was known for Indian Ocean struggles and conflicts among colonisers for controlling the trade and political supremacy. It is a region of several unexploited lands. The sea route is a vital link between the Eastern and the Western part of the world. The region has also tremendous amount of valuable unexploited resources in its beds which attract several littoral countries. The surrounding countries of its rim are of mostly Islamic nations and two giant countries of nuclear power. The increasing influence and intervention of China republic through investment in port development in various countries like Bangladesh, Pakistan, Djibouti and Maldives has presented a challenge and conflicts in peace-making efforts in the region. Silk marine trade route through this ocean is another game of Chinese effort to increase her influence in Africa, West Asia, Pakistan, Sri Lanka and South East Asian countries. 80% of the oil trade through the sea route occurs through this ocean. Keeping in view the high level of strategic and peace maintaining significance in the area , the researchers aimed to explore the status of world power interference in disturbing and balancing geo political and economic power equilibrium in Asia especially in Littoral nations. The excess interference of China, an emerging world economy and its impact on socio economic and political stability especially between India and Pakistan are also explored. The level of relevance of containment policy of USA has been also analysed and searched out. Study is based fully on secondary source of data. Study revealed that the presence of China in Indian Ocean in different forms resulted in geo political unrest in the area. Geo political conflict in South China Sea including Malacca striate, political instability in Maldives, Myanmar conflict and recently Indo Pak tension are some new examples of emergence of conflicts in Indian Ocean region. Rim Land geo strategic model of Spykemen is fully relevant and applicable in contemporary world.
Keywords: Indian Ocean, Silk Road, Resource, Power conflicts

domain: economics


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According to this article there have been five interesting archaeological discoveries in the past decade. First among them, of course, is the finding of Richard III’s remains:- “….When King Richard III was killed at the Battle of Bosworth in 1485, he was buried in the church of the Grey Friars. In 2012, The Richard III Society… Continue reading Five interesting archaeological discoveries….
I found the article below at this site where the numerous posts are Tudor-oriented (Henry VIII), but very interesting and informative. The article is given in full to tempt you into visiting the site to read all the others:- “….On November 25th or 26th, 1533, Henry FitzRoy married Lady Mary Howard. “….Mary Howard… Continue reading Lady Mary Howard married the bastard son of Henry VIII….
The Pharaoh Tutankhamun seems to have been part of our lives forever, so it’s hard to believe that his tomb was found just hundred years ago on 4th November 1922. Even the discoverer, Howard Carter, had no idea what lay within the tomb, only that it didn’t seem to have been got at by… Continue reading Tutankhamun’s tomb was discovered one hundred years ago today….
“….Archaeologists have made an exciting discovery in the Queen Pool at Blenheim Palace prior to dredging work….They believe they have uncovered the remains of a 14th century watermill complex….” So reads the opening of this article. A new discovery at somewhere as historic as Blenheim is very exciting. But maybe it was its Woodstock Palace… Continue reading A new discovery at what was once Woodstock Palace….
The monarch in question is Robert I (Bruce) and the investigation, as part of the Foundation for Mediaeval Genealogy’s Declaration of Arbroath Family History Project, is being carried out by the University of Strathclyde: Graham Holton has reported good progress in this press release: Genetic marker discovered for descendants of Bruce clan, January 2022.A distinct… Continue reading Identifying another King
This excellent Channel Four programme has returned for a third series soon after the second, perhaps because the pandemic interrupted some of the earlier filming. The first episode features Odiham Place in Hampshire, looking for the home of Sir Francis Walsingham, although it was actually built for Henry VIII and was smaller than a 1739… Continue reading The Great British Dig – History in Your Garden (3)
Well, I had heard before that medieval horses were much smaller than we imagine, and now it seems proof may may have been found. It’s a fact that for journeys medieval folk who could afford more than Shanks’ pony used small horses that could keep up a fast trot for a l-o-n-g time. They… Continue reading How big were the horses of medieval knights….?
I can’t find an illustration of the intended development. The above photograph came with this article. It seems a 63-home housing development has been turned down again. For good this time. It would have been close to Bosworth Field.
As you will observe from their appearance on Diana Rubino’s blog , The Legendary Ten Seconds now have a book featuring information on some of their best-known songs about Richard III, his time and Devon, of course. My Review of The Legendary Ten Seconds for the Ricardian Register (magazine of the American branch) As a longtime… Continue reading Diana Rubino on the Legendary Ten Seconds
I also received this from Anglia Water about the “Rutland Sea Dragon”, an ichtyosaur found near Rutland Water. It featured in episode four of Digging for Britain, the latest series of which was shown over two weeks in January. As ever, Alice Roberts was the main presenter, alongside Dr. Onyeka Nubia and Dr. Cat Jarman.… Continue reading Not what you expect with a water bill!

domain: history


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Waterfront Contemporary Craftsman. Escape from the hustle and bustle of the city to this stunning home overlooking the Grand Canal. This finely finished home exudes quality and grace. Every detail was carefully considered through the artful blending of high end modern with ageless architectural aesthetics. Privately set on the closest canal to the ocean, this warm and stunning home is convenient to all that Venice and The Marina have to offer in the way of casual beach living.
|09/16/2019||Listed For Sale||$3,795,000||$1,405.56||Keller Williams Realty Santa Barbara|
Westminster Avenue Elementary
|0.7 miles away||KG - 05|
Coeur D'alene Avenue Elementary
|0.9 miles away||KG - 05|
|1.2 miles away||KG - 06|


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Amerian-based Songstar, o9echi comes through with a new hit single titled “Melanin“.
This single is from his debut EP, “Admission Day” which featured top artists like Bella Shmurda & Duncan Mighty.
“Melanin” is currently garnishing a lot of streams worldwide.
“Melanin” represents a celebration of African Beauty. Enjoy!!!
Watch the Video below:-
What do you think about this Video?
Drop your comments.
RECOMMENDED VIDEOS FOR YOU 👇Pheelz Ft. BNXN – Finesse BNXN (Buju) Ft. Zinoleesky – Kilometer (Remix) Fave – Mr Man Boy Spyce – Nobody T.I Blaze Ft. Blxckie, Rasaqi NFG – Basic Fave – Mr Man DJ Neptune Ft. Omah Lay, Joeboy – Abeg Masterkraft Ft. Chike – Hosanna Shatta Wale – Rising Youth
Follow NL on Social Media

domain: celebrity


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17th August 2023
Bright Futures – Girls Standing Up Campaign
The LGA Foundation have supported Bright Futures over the past six years, and we feel that there is something incredibly unique about the work that they have been doing. We wanted to highlight a particular element of their wide-ranging charity and are keen to promote it in order to increase the support that they are asking for.
Bright Futures NE have launched a petition to give students a legal right to access school toilets when they have their period.
This petition has been launched to run alongside their ‘Girls Standing Up!’ project, which has been running for the past eighteen months. Since the ‘Girls Standing Up! campaign’s inception, they have worked with over 1000 young people to deliver an educational program covering sexual harassment, domestic abuse, healthy relationships, consent, and sexual assault, amongst other topics.
Running parallel to this project, Bright Futures NE has recruited ‘Fearless Female Ambassadors’ to lead on a number of social action projects; covering issues that are important and meaningful to them, as young women and members of the community.
The Fearless Female Ambassadors have proposed that young people should have a legal right to access school toilets when they are on their period. Young people, who are on their period throughout school hours, are sometimes denied the right to use the toilet. This can cause shame, embarrassment and preventing them from changing period products. This is inconsistent with the right to dignity and basic human rights – as per the UN Convention on the Rights of the Child. Bright Futures NE propose young people with periods, by law be given a right to access school toilets.
The LGA Foundation spoke with two members from Bright Futures, who are helping to lead the petition. Bright Futures have gone to great lengths to speak with young people across the community to ascertain how best to ensure that they are allowed to use the bathroom when it is necessary. The Ambassadors have presented the idea of ‘period passes’ to be used within schools and have hosted pop up events, where they distributed period products, whilst promoting their campaign. They plan to host more events throughout the summer; hosting pop-ups and outreach in town centres.
It is their hope that the petition will gain enough signatures and attention to bring this issue to Parliament and for a real discussion to be had and a law to be passed. The campaign is being supported by Emma Lewill-Buck, Labour MP for South Shields.
Quote from Dianne MacKay:
“From the minute we met the team at Bright Futures NE, we wanted to support the important work they were doing with young women within their communities. No issue is too big or too small for this project. Knowledge is a huge part of enabling our young people to identify issues, support each other and speak out and they do it in such a fun and inclusive way. The founders are such inspirational women, this flows throughout the team and the girls attending the groups are so passionate about making a positive impact for themselves, their peers and the wider community. Encouraging young women to find their voices and be heard, lobbying parliament with petitions to make our streets safer for girls and women and tackling period poverty to demand access to toilets in school are just some of the important issues they are addressing.”
We invite followers of the LGA Foundation to sign the petition and provide ample opportunity for this issue to be progressed to Parliament.
To sign the petition, please click the link below or scan the QR code:
Bright Futures are a registered charity (No. 1157578) working with young women up to the age of 25 to give them a brighter future by helping them to tackle some of life’s biggest challenges including substance misuse, domestic abuse, unemployment, poor health and wellbeing, sexual abuse and exploitation.

domain: law


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A set of simple cards for children to match the process to the description; suitable mixture and then match to the picture of the process.separatng mixtures
Separating mixtures matching cards
Subject Science — Physical Science
Grade Level Grades 6-8
Resource Type Handout
Wow, thanks! This are ecellent; just what i need
About to do this topic! -such a helpful resource. Better than anything I've seen in books for doing this.

domain: library


text: If you love to go out fishing then you should check out the top fishing apparel by Malo’o. One of the best fishing apparel our customers love is the Malo’o performance fishing shirt which is made of fast-drying material and is SPF 50+ to protect you on hot days. It is also, stain-resistant and anti-microbial and has a tighter weave to protect you against the cold winds. You can also check out Malo’o fishing equipment such as a fishing rod rack, fishing pole holder, etc. that can help you to make your fishing easier and fun. These products are made of the best quality material and go through top testing before selling. Malo’o fishing rod racks can be used to put all your fishing rods properly in place so they don’t get damaged or broken during traveling. You can shop all your desired product through Malo’o’s official website at the best prices.

domain: hobby


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Related SubjectsNo related subjects.
Tudor, history, --fiction (4)
historical fiction, richard iii (4)
historical fiction, wars of the roses (4)
Richard III, historical fiction (4)
Tudor, history, royalty (3)
England, Tudor, -fiction, -historical novel (3)
Tudor, biography, history (3)
biography, espionage (3)
Tudors, history (3)
British Royalty, biography (3)
Subject: Pretenders to the throne
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cibet's albums beáta judit bérczy, dr
I am a doctor, psychotherapeutist. I have been taking photos from childhood with pleasure. On my photos I try to reveal of the feelings I had at the moment when I was taking them. Although the photography is my passion, I spend more and more time on it. Since I have sold my pictures, I consider it as my second job. I have had several exhibitions in Hungary, some of my photos appeared in magazines or decorate homes, schools, offices, public facilities, both inland and abroad.
Canon 5D Mk II, EF 50mm f/1.4 USM, EF 85mm f/1.8 USM, EF 24-105mm f/4 USM L IS,EF 70-200mm f/4 USM L IS
Sep 20, 2009


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Washington7 hours ago
The American rapper campaigned in South Carolina on Sunday.
- In his speech, Kanye West made several revelations, saying that his father wanted his mother to have an abortion
- She also targeted human rights activist Harriet Tubman, one of America’s most respected figures in the 19th century.
American rapper Kanye West launched the campaign for the presidential election in South Carolina on Sunday. He had announced to contest on July 4. This is their first rally after that. He wept during his speech on Sunday. Kanye was talking about international trade and abortion. He said that Kim Kardashiniya (his wife) and they did not want their first child.
In his speech, he made many controversial comments. He spoke at length on abortion. He claimed that he and his then girlfriend Kim Kardashian thought of ending their first child. He said that women who become mothers for the first time should be given one million dollars.
43-year-old Kanye West was in bulletproof vest during the campaign. He spoke without a microphone on stage. They took their first child and said that she (Kim Kardashian) kept crying for months. We did not want this child. He had puppies in his hand. After much thought we were ready for the baby. Their child’s name is North.
Mother saved my life
West claimed that his father also wanted his mother to have an abortion. They said crying – My mother saved my life. There is no Kanye West, because my father was very busy. My mother gave me life.
Civil right activist also accused Tubman
During his speech, he also targeted human rights activist Harriet Tubman. He said that he always worked for the whites. He never freed slaves. Tubman was one of America’s most respected figures of the 19th century.
The Politico News report says West has missed the deadline to qualify for elections in many states. However, he qualified for Oklahoma last week. By state rule, West needed 10,000 signatures to qualify from South Carolina by Monday afternoon.
Elon Musk supported
It was only after Kanye West announced that he would contest the presidential election that Tusla CEO Elon Musk extended his support. West has won the Grammy 21 times.
read this also
West said – I will contest the presidential election this time, Tesla CEO Musk said – I fully support you

domain: news


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Join up with this Multi Blogger Giveaway and get more TWITTER followers!!
Its time for another Dnbuster and Pals giveaway!!
This giveaway will be for a $50 Amazon gift card.
Hoping to "Pal up" with 35+ Bloggers!
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The group is where the Raffle Copter code will be shared and all questions will be answered:)
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Go here to enter I did!!!!


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The Big East will not go gentle into the good night of college football irrelevance. And the conference’s thrashing bid to stay afloat could be what finally gets Boise State University to the BCS championship game if things keep breaking right for the Broncos.
Several outlets are reporting that the Big East will extend six invitations to schools in an effort to maintain its automatic BCS bid in the wake of departures by Syracuse, Pittsburgh, West Virginia and Texas Christian. Boise State, Navy and Air Force are set to join for football only and Southern Methodist, Houston and Central Florida for all sports. By far the glitziest name being courted is Boise State, the college football arrivistes whose two undefeated seasons since 2006 (with a possible third happening this year) have allowed them to get no closer to the national championship than, well, the mediocre Big East.
Weak schedule and conference aside, Boise has made a case that it should be a part of the title game conversation. But as a member of the Mountain West Conference, their prospects continue to look dim, barring a dramatic collapse from the likes of LSU, Alabama and Stanford. By as soon as Friday, though, Boise could have an invitation to an automatic-bid conference, and provided that they continue to replicate the outstanding success of their last few years, prospects for the Broncos could turn for the better. Don’t call it a comeback quite yet, however. That automatic bid has been called into question by all of this maneuvering, and the likes of Rutgers, Louisville and South Florida don’t exactly scream football nirvana. But there’s a silver lining. “[Big East commissioner John] Marinatto might have botched realignment and expansion, but he could be right about one point he has stressed for months: Being the last league to negotiate a TV deal will work to the Big East’s advantage,” Ralph Russo writes.
Jumping ship to the Big East would cost Boise State considerably in other lost revenue, especially next year, when the school would stand to lose $21 million from an early departure from the MWC, according to the Idaho Statesman. Joining in 2013 would be more affordable. For the five other schools, various obstacles stand in the way: Air Force’s interest appears heavily dependent on Navy’s decision and Conference USA squads UCF, SMU and Houston would each owe a $500,000 exit fee before they could join the Big East in 2013. Still, the Big East’s lucrative TV contract will likely prove too alluring for schools to resist, and reports indicate that, should all six teams accept, the Big East will be split into western and eastern divisions to accommodate universities like Houston and Boise State, which are by no stretch of the imagination in the east. That’s right – get ready for the Big East West.
Speaking of which—Utah’s very own BYU could also be in play, though this hypothetical 12th spot could go to a smaller school like Temple, which could also be called in to replace Navy or Air Force should either decline. It’s a muddled, abstract sketch at this point, but if the Big 12 and Big Ten can contain neither 12 nor 10 schools, then the idea of a Big East featuring a team from Idaho doesn’t sound quite as strange.
* * *
After staining his team with the fallout from a messy divorce and then filing for Chapter 11 bankruptcy, Frank McCourt has finally agreed to sell the Los Angeles Dodgers. The MLB assumed control of the Dodgers in April, and the court-supervised sale of the team will include Dodger Stadium and surrounding parking lots. With its historical cache and large, wealthy market, the team, which was purchased by McCourt in 2004 for $421 million, will surely attract buyers. Mark Cuban told the Los Angeles Times that he looked into making a bid, and the Dodgers offered to sell for between $1 billion and $1.2 billion. But at that price point, Cuban, owner of the NBA’s Dallas Mavericks, said he would pass. (A spokesman for the Dodgers denied ever talking a sale with Cuban.)
Most of the Dodgers community won’t be sad to see him go. “McCourt neither apologized nor expressed remorse for what he had done and how it all ended,” Ramona Shelburne writes for ESPN. “He simply left and walked away. Though he will never be forgotten in these parts, all these awful memories will fade quickly. The pain of these last two years was always going to be temporary. The Dodgers were in purgatory, not hell. As soon as McCourt moved on, Los Angeles would, too.”
* * *
The NCAA basketball regular season begins Monday, and as writers publish their first projections, UNC looks to be in poll position. The Tar Heels, who return every meaningful player from a squad that tied for the ACC regular-season championship and reached the NCAA Tournament’s Elite 8, boast a player-of-the-year contender in Harrison Barnes, plus John Henson and Tyler Zeller in the front-court, and breakout point guard Kendall Marshall. Sports Illustrated’s Luke Winn is one prognosticator coloring this season’s champion Carolina blue. “As a fan of unpredictability, I would be overjoyed if the next champ emerged from outside this top 16, but I fear that my preseason No. 1 has wire-to-wire potential, he writes. Winn’s top five rounded out with the University of Kentucky, Ohio State, Duke and Syracuse, with national champions UConn coming in at sixth.
* * *
With a dearth of marquee stars, long-distance running can often seem like a cavalcade of times and nothing else. Not at the New York Marathon. Scott Cacciola writes for the Journal about the marathon’s unique competitive environment, in which pacemakers—runners paid to blaze a world-record tempo for one or two top stars a race—are banned, and tactical running is stressed above time. “New York is a different experience,” Cacciola wrote. “Runners are constantly jockeying for position and varying tempo. Mahon likened the race to a boxing match: Competitors take turns throwing punches, speeding up and slowing down—Kryptonite for anyone who hopes to break a record.” Such running favors certain athletes who, though they might not post record times, seem well suited to outmaneuvering other runners. This includes Meb Keflezighi, who in 2009 became the first American to win the New York Marathon in 27 years—with a time that was five-and-a-half minutes slower than the world record.
Found a good column from the world of sports? Don’t keep it to yourself — write to us at firstname.lastname@example.org and we’ll consider your find for inclusion in the Daily Fix. You can email Kevin at email@example.com.


The sales of tickets for Hangzhou Asian Games will start in October this year.
Beijing Red Horse Media (Damai), as the main ticketing carrier, will provide professional ticketing service of Hangzhou Asian Games and Asian Para Games for spectators.
The ticketing work will, sticking to the idea of “green, smart, economic, ethical” and “sunny, harmonious, self-improvement, shared”, make full use of the comprehensive advantages, including big data, blockchain, face recognition and other AI technologies, and Smart Hangzhou 2022, to improve the public’s games-watching experience of “Smart Asian Games” from a high-tech, digitalized perspective.
As to the design and pricing of the tickets, the public will be provided with more convenient ways to purchase the tickets and watch the competitions. More young people will be encouraged to engage in the competitions and activities, which will for sure play a positive role in their healthy growth.
“Ticket+” is another highlight. Tickets will serve as a bond connecting multiple steps, including on-the-spot games watching, franchised products, urban trip, food, accommodation, tourism, public benefit, theme activities, and interaction with sponsors, so as to offer “all-in-one” games-watching experience. Meanwhile, the official ticketing website will chain up all tickets to realize digitalized ticketing service using blockchain technology.
Tickets for Hangzhou Asian Games and Asian Para Games will be divided into two categories – big customer ticket and public customer ticket – according to the overall arrangement of the ticketing work. You can buy the tickets on the official website of Hangzhou Asian Games or offline.


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Review about Europcar Car Rental from Singapore, Singapore
Appalling customer relations and lousy after sales experience with Europcar.
Firstly, it took Europcar more than a month to give me a response on my authorisation charge inquiry, despite me calling and emailing on 3 separate occasions. I had to chase Europcar for an answer.
Secondly, Europcar's process of placing an authorisation charge on customers' credit card is ridiculous. When I picked up the car at Perth Airport on 18 Aug 15, a charge of AUD1,410.74 was made to my credit card. This was the deposit plus GPS and additional driver charges. But, the very next day on 19 Aug 15, the same amount was debited back into my credit card account! When I returned the car on 31 Aug 15, a charge of AUD1,409.74 (-$1 deposit) was made on my credit card. Becoz of the transactions on 18 and 19 Aug 15, i lost SGD98.69 due to the exchange rate. Why is there a need to debit back the amount into my account? Why can't Europcar hold the amount until I return the car then debit back the deposit? Renee from Customer Relations Australia was defensive and stubborn by saying this is company policy and that I agreed to the terms & conditions when i signed the rental agreement. This was not what was written in the T&C:
[General Rental Terms, Section 6.2 Terms of Payment]: "When payment is made by means of a credit card, an authorisation will be requested prior to the start of the rental... This amount is not debited. It is held on cardholder's bank account until the final rental charge amount is debited."
[Prepayment Terms and Conditions]: "An authorisation will be obtained at time of rental and only on return of the vehicle will the corresponding charges be posted to the credit card."
Lastly, another charge of AUD512.38 was made on my credit card on 1 Oct 15. I did not receive any invoices, statement, or document to explain what this charge was for. When I called to get an answer, it turned out that they had keyed in my email address wrongly thus the invoice didn't reach me. And the charge was for a small glass chip on the windscreen! When i finally received the invoice and damage report, they had spelt my name and email address incorrectly again! I have requested and have yet to receive the updated invoice and damage report as of now. This administrative lapses is outrageous and unacceptable. It is affecting my insurance claim.
I've never been more disappointed and upset with a car rental company like this one.
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Home / Shop / Basket Patterns
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A PDF digital pattern, available for instant download - A simple little Market style basket. Add some beads and shells and you are ready to decorate the beach house or collect some shells.
Dimensions 6” x 6” x 4” high (8 ½” with handle)
Early Intermediate Weaving Level


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A man in a northeast Georgia jail on a child molestation charge faces a similar charge in another county, officials said.
Isaac Joseph Sgro, 22, of Toccoa, was in the Habersham County Jail on charges of rape, aggravated child molestation, aggravated sexual battery, sexual battery and first-degree cruelty to children when Hall County authorities arrested him on charges of aggravated child molestation and child molestation on July 24.
The initial charges stem from “an act of child molestation against a female juvenile that occurred within the city of Cornelia,” Cornelia police Chief Chad Smith said last month in a news release. Jonathan Edward Lewicki, 31, was also arrested in connection with that case.
The latest charges are related to a case involving the same girl, sheriff’s spokesman Lt. Scott Ware said Friday. The child was molested between June 30, 2013 and June 30, 2018 in east Hall County, according to Ware.
Sgro is being held in the Hall County Jail without bond.
Both cases remain under investigation.
In other news:
Support real journalism. Support local journalism. Subscribe to The Atlanta Journal-Constitution today. See offers.
Your subscription to the Atlanta Journal-Constitution funds in-depth reporting and investigations that keep you informed. Thank you for supporting real journalism.


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With the mybpaid application of bpost you can anytime & anywhere:
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• Consult useful information relating to the use of your bpaid card (loss, theft, FAQ ...)
With the prepaid bpaid card of bpost you don’t need a credit card to make online purchases. You reload your card whenever you want and you set the amount depending on your budget.
• Buy online without credit card
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The bpaid card is available in every Belgian post office.
- Fix infinite loading in transactions list.
Ratings and Reviews
Pourquoi une limitation de 8000 eur??
À recommander à qui le souhaite
Facile , efficace , usage mondial . Très pratique
Bon produit à prix réduit
Carte pratique. Excellent porte-monnaie électronique à usage mondial.
Up to six family members will be able to use this app with Family Sharing enabled.


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ENC Ele-Max Nutrient Concentrate is a combination of chelated micronutrients and is compatible with most insecticides, fungicides and herbicides. It contains a formula of 11-8-5 NPK, boron, manganese, iron, zinc, molybdenum and cobalt. This plant nutrient effectively helps in improving plant conditions. It may be applied to many crops (including row crops) since all plants are capable of absorbing nutrients through the leaves. The use of ELE-MAX Nutrient Concentrate for foliar feeding is particularly advantageous on crops that have fast and heavy foliar growth.
|Active Ingredient:||Total Nitrogen - 11.00%
Available Phosphate - 8.00%
Soluble Potash - 5.00%
Boron - 0.02%, Cobalt - 0.0005%, Iron - 0.10%, Manganese - 0.05%, Molybdenum - 0.0005%, Zinc - 0.05%
|Target pests:||Vegetable plants, potatoes, sugar beets, grapes, hops, cotton, citrus fruits, coffee, tobacco and others
* See label for complete list
|For use in:||Commercial Turf-grass, Nursery, Greenhouse Ornamental Plants and Commercial Farming
* See label for complete list
|Application:||1 - 4 qt. per 100 gallons of water per acre
* See label for complete application instructions
|Pet safe:||Yes, if used as directed on label|
|NOT FOR SALE TO:||AK|
|Special Features:||Derived from potassium hydroxide, ammoinium phosphate, urea, sodium tetraborate, cobalt sulfate, ammonium molybdate, iron EDTA, copper EDTA, manganese EDTA, and zinc EDTA|
|Shipping Weight:||26.28 lbs|
|Manufactured By:||Helena Chemical (Mfg. Number: ELEMAX360)|
This fertilizer is approved for use in the following states:
AL; AR; AZ; CA; CO; CT; DE; FL; GA; HI; IA; ID; IL; IN; KS; KY; LA; MA; MD; ME; MI; MN; MO; MS; MT; NC; ND; NE; NH; NJ; NM; NV; NY; OH; OK; OR; PA; RI; SC; SD; TN; TX; UT; VA; VT; WA; WI; WV; WY.
ENC Ele-Max Nutrient Concentrate may be applied to many crops (including row crops) since all plants are capable of absorbing nutrients through the leaves. The use of Ele-Max Nutrient Concentrate for foliar feeding is particularly advantageous on crops that have fast and heavy foliar growth, including vegetable plants, potatoes, sugar beets, grapes, hops, cotton, citrus, fruits, coffee, tobacco, etc. The application of Ele-Max Nutrient Concentrate is also recommended in combination with herbicidal sprays in many cereals and row crops.
- Should be used in all crop protection sprays with minimum volumes of 5 gallons/A by ground and 2 gallons/A by air, however, the concentration of Ele-Max Nutrient Concentrate should not exceed 1 quart in 5 gallons of solution. (5% by volume).
- The following table is a guide for the use of Ele-Max Nutrient Concentrate as a plant nutrient. Rates and frequency of application will depend on individual management programs. Use of Ele-Max Nutrient Concentrate on any crops not listed on this table must be as a result of recommendations from your local agricultural authority.
Have a question about this product?
Ask our product experts...
Ask a Question
07/29/2015 Brian from Salem, Indiana
QWhat is the mix rate of ENC Ele-Max Nutrient Concentrate for vegetable gardens?
A The amount of ENC Ele-Max Nutrient Concentrate you use depends on the particular crop you are treating. You can view the application rates by following the link below to the product label. http://www.domyownpestcontrol.com/msds/ELEMAXNUTRIENTLABEL.pdf
The amount of ENC Ele-Max Nutrient Concentrate you use depends on the particular crop you are treating. You can view the application rates by following the link below to the product label.
06/12/2015 Denise from Canby, Ca,
QHow much of the Ele-Max nutrient do I add for 1 gallon of water?
I use this product to fertilizer my petunias


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event marked the beginning of the modern era of photography.
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Muybridge went on to develop systems and techniques to photograph motion of
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Wilderness Timeline 1850-1900: Shows small pictures that illustrate the following
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