text:
Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms' HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms' frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljanić groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte's structure and - because of cruciforms' charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.
23 Related JoVE Articles!
Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography
Institutions: Imperial College London.
The highly diastereoselective preparation of cis
-Ts-iodoaziridines through reaction of diiodomethyllithium with N
-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 °C
in the dark. These conditions are essential for the stability of the LiCHI2
reagent generated. The subsequent dropwise addition of N
-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 °C promotes cyclization to afford iodoaziridines with exclusive cis
-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1
H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1
H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.
Chemistry, Issue 87, organic chemistry; aziridines, heterocycles, organolithium reagents, chromatography, purification, iodoaziridines
Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization
Institutions: Yale University.
Whole mount in situ
hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ
hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ
hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2
. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3
. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4
. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila
indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5
. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.
Developmental Biology, Issue 25, zebrafish, tyramide signal amplification, in situ hybridization, nuclear labeling
Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna
Institutions: Doshisha University, RIKEN Brain Science Institute, RIKEN Brain Science Institute.
Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.
Neuroscience, Issue 87,
Developmental biology, Drosophila, Whole mount immunolabeling, olfactory receptor neurons, antennae, sensory organ
Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging
Institutions: University of Texas at Arlington .
Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis
plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al.
2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.
Plant Biology, Issue 44, Plant innate immunity, propidium iodide staining, biotic and abiotic stress, leaf microscopy, guard cell, stomatal defense, plant defense, Arabidopsis, Pseudomonas syringae
Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with β-arrestin
Institutions: University of Louisville.
G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2
. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of β-arrestins3
. β-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6
) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4
receptor 1 (BLT1; a high affinity receptor) and LTB4
receptor 2 (BLT2; a low affinity receptor)7-9
. The LTB4
-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17
. The current paper describes the methodologies developed to monitor LTB4
-induced leukocyte migration and the interactions of BLT1 with β-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21
. These cells were tested in live cell imaging methods to demonstrate LTB4
induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and β-arrestin1 tagged with green fluorescent protein (β-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19
. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.
Immunology, Issue 46, Live cell imaging, Chemotaxis, G-protein coupled receptor, receptor internalization, leukotriene B4, leukotriene B4 receptor 1
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry
Institutions: University of Georgia, University of Georgia, Ishikawa Prefectural University.
Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive β-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.
Chemistry, Issue 93, glycoprotein, glycosylation, in-gel reductive β-elimination, O-linked glycan, sulfated glycan, mass spectrometry, protein ID, SDS-PAGE, glycomics, sulfoglycomics
The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures
Institutions: University of Basel, University of Basel.
Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in vitro
models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo
. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.
Neurobiology, Issue 92, blood-brain-barrier, neurovascular remodeling, hippocampus, pyramidal cells, excitotoxic, ischemia
A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays
Institutions: McMaster University .
Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays.
Chemistry, Issue 78, Biochemistry, Chemical Engineering, Molecular Biology, Genetics, Bioengineering, Biomedical Engineering, Chemical Biology, Biocompatible Materials, Siloxanes, Enzymes, Immobilized, chemical analysis techniques, chemistry (general), materials (general), spectroscopic analysis (chemistry), polymer matrix composites, testing of materials (composite materials), Sol-gel, microarray, high-throughput screening, acetylcholinesterase, kinase, drug discovery, assay
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Institutions: Environmental Health Centre.
mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo
male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro
positive selection assay to measure in vivo
mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Genetics, Issue 90, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology
Recapitulation of an Ion Channel IV Curve Using Frequency Components
Institutions: University of Utah.
INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.
METHODS: Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages.
RESULTS AND CONCLUSIONS: In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|<0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.
Biophysics, Issue 48, Ion channel, Kir2.1, impedance spectroscopy, frequency response, voltage clamp, electrophysiology
Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis
Institutions: Institut Nanosciences et Cryogénie, CEA-Grenoble, Université Paris-Sud, Institut de Biologie Structurale.
In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.
Bioengineering, Issue 91, electronic tongue, combinatorial cross-reactive receptor, surface plasmon resonance imaging, pattern recognition, continuous evolution profiles, continuous evolution landscapes, protein analysis
Determination of the Gas-phase Acidities of Oligopeptides
Institutions: University of the Pacific.
Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6
. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8
, the results are often complicated by solvent effects 7
. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10
. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3
CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1
. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2
). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.
Chemistry, Issue 76, Biochemistry, Molecular Biology, Oligopeptide, gas-phase acidity, kinetic method, collision-induced dissociation, triple-quadrupole mass spectrometry, oligopeptides, peptides, mass spectrometry, MS
Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells
Institutions: University of Virginia Health Sciences Center.
Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae
in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.
Microbiology, Issue 79, Immunology, Infection, Cancer Biology, Genetics, Cellular Biology, Molecular Biology, Medicine, Biomedical Engineering, Microscopy, Confocal, Microscopy, Fluorescence, Bacteria, Bacterial Infections and Mycoses, bacteria, infection, viability, fluorescence microscopy, cell, imaging
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Institutions: University College London.
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA
Rs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAA
Rs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.
To elucidate the underlying molecular mechanisms, a novel in vitro
co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAA
R subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAA
R subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro
model system can be used to reproduce, at least in part, the in vivo
conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAA
Rs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line
Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction
Institutions: Lehigh University .
Although initially considered an esoteric reagent, SmI2
has become a common tool for synthetic organic chemists. SmI2
is generated through the addition of molecular iodine to samarium metal in THF.1,2-3
It is a mild and selective single electron reductant and its versatility is a result of its ability to initiate a wide range of reductions including C-C bond-forming and cascade or sequential reactions. SmI2
can reduce a variety of functional groups including sulfoxides and sulfones, phosphine oxides, epoxides, alkyl and aryl halides, carbonyls, and conjugated double bonds.2-12
One of the fascinating features of SmI-2
-mediated reactions is the ability to manipulate the outcome of reactions through the selective use of cosolvents or additives. In most instances, additives are essential in controlling the rate of reduction and the chemo- or stereoselectivity of reactions.13-14
Additives commonly utilized to fine tune the reactivity of SmI2
can be classified into three major groups: (1) Lewis bases (HMPA, other electron-donor ligands, chelating ethers, etc.), (2) proton sources (alcohols, water etc.), and (3) inorganic additives (Ni(acac)2
Understanding the mechanism of SmI2
reactions and the role of the additives enables utilization of the full potential of the reagent in organic synthesis. The Sm-Barbier reaction is chosen to illustrate the synthetic importance and mechanistic role of two common additives: HMPA and Ni(II) in this reaction. The Sm-Barbier reaction is similar to the traditional Grignard reaction with the only difference being that the alkyl halide, carbonyl, and Sm reductant are mixed simultaneously in one pot.1,15
Examples of Sm-mediated Barbier reactions with a range of coupling partners have been reported,1,3,7,10,12
and have been utilized in key steps of the synthesis of large natural products.16,17
Previous studies on the effect of additives on SmI2
reactions have shown that HMPA enhances the reduction potential of SmI2
by coordinating to the samarium metal center, producing a more powerful,13-14,18
sterically encumbered reductant19-21
and in some cases playing an integral role in post electron-transfer steps facilitating subsequent bond-forming events.22
In the Sm-Barbier reaction, HMPA has been shown to additionally activate the alkyl halide by forming a complex in a pre-equilibrium step.23
Ni(II) salts are a catalytic additive used frequently in Sm-mediated transformations.24-27
Though critical for success, the mechanistic role of Ni(II) was not known in these reactions. Recently it has been shown that SmI2
reduces Ni(II) to Ni(0), and the reaction is then carried out through organometallic Ni(0) chemistry.28
These mechanistic studies highlight that although the same Barbier product is obtained, the use of different additives in the SmI2
reaction drastically alters the mechanistic pathway of the reaction. The protocol for running these SmI2
-initiated reactions is described.
Chemistry, Issue 72, Organic Chemistry, Chemical Engineering, Biochemistry, Samarium diiodide, Sml2, Samarium-Barbier Reaction, HMPA, hexamethylphosphoramide, Ni(II), Nickel(II) acetylacetonate, nickel, samarium, iodine, additives, synthesis, catalyst, reaction, synthetic organic chemistry
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ
characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3
(bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ
time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ
Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ
IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
Spatial Separation of Molecular Conformers and Clusters
Institutions: CFEL, DESY, University of Hamburg, University of Hamburg.
Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.
Physics, Issue 83, Chemical Physics, Physical Chemistry, Molecular Physics, Molecular beams, Laser Spectroscopy, Clusters
Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Institutions: The Hebrew University of Jerusalem, The Hebrew University of Jerusalem.
Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.
Chemistry, Issue 82, solution structure determination, NMR, peptide models, copper-binding proteins, copper complexes
Preparation and Use of Photocatalytically Active Segmented Ag|ZnO and Coaxial TiO2-Ag Nanowires Made by Templated Electrodeposition
Institutions: University of Twente.
Photocatalytically active nanostructures require a large specific surface area with the presence of many catalytically active sites for the oxidation and reduction half reactions, and fast electron (hole) diffusion and charge separation. Nanowires present suitable architectures to meet these requirements. Axially segmented Ag|ZnO and radially segmented (coaxial) TiO2
-Ag nanowires with a diameter of 200 nm and a length of 6-20 µm were made by templated electrodeposition within the pores of polycarbonate track-etched (PCTE) or anodized aluminum oxide (AAO) membranes, respectively. In the photocatalytic experiments, the ZnO and TiO2
phases acted as photoanodes, and Ag as cathode. No external circuit is needed to connect both electrodes, which is a key advantage over conventional photo-electrochemical cells. For making segmented Ag|ZnO nanowires, the Ag salt electrolyte was replaced after formation of the Ag segment to form a ZnO segment attached to the Ag segment. For making coaxial TiO2
-Ag nanowires, a TiO2
gel was first formed by the electrochemically induced sol-gel method. Drying and thermal annealing of the as-formed TiO2
gel resulted in the formation of crystalline TiO2
nanotubes. A subsequent Ag electrodeposition step inside the TiO2
nanotubes resulted in formation of coaxial TiO2
-Ag nanowires. Due to the combination of an n
-type semiconductor (ZnO or TiO2
) and a metal (Ag) within the same nanowire, a Schottky barrier was created at the interface between the phases. To demonstrate the photocatalytic activity of these nanowires, the Ag|ZnO nanowires were used in a photocatalytic experiment in which H2
gas was detected upon UV illumination of the nanowires dispersed in a methanol/water mixture. After 17 min of illumination, approximately 0.2 vol% H2
gas was detected from a suspension of ~0.1 g of Ag|ZnO nanowires in a 50 ml 80 vol% aqueous methanol solution.
Physics, Issue 87, Multicomponent nanowires, electrochemistry, sol-gel processes, photocatalysis, photochemistry, H2 evolution


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These cake pops are very easy to make. If you have already been making cake pops like myself, basically you are good to go. You can simply use white Candy Melts and you can have white skeleton, ghost or mummy cake pops. However, I was too forgetful to buy it and was too overwhelming with the other options that all the other colours could provide.
All it takes for these cute little treats is an edible marker. Make sure you buy one that works with candy coating. Typical edible marker tends to drag through or jab into the candy coating. If you write too hard, it will even break your cake pop.
Ingredients (about 45 pops):
1 cup granulated sugar
3/4 cup plus 2 tablespoons all-purpose flour
1/4 cup plus 2 tablespoons unsweetened cocoa powder, sifted
3/4 teaspoon baking powder
3/4 teaspoon baking soda
1/2 teaspoon salt
1 large egg, room temperature
1/2 cup sour cream, room temperature
1/4 cup canola oil
1 tablespoon vanilla extract
1/2 cup boiling water
1 stick unsalted butter, softened
1 cup powdered sugar, sifted
1/2 teaspoon vanilla extract
1 tablespoon heavy cream
1/4 cup unsweetened cocoa powder, sifted
1 bag of dark chocolate Candy Melts
1 bag of orange Candy Melts
1 bag of lavender Candy Melts
1/2 bag of green Candy Melts
black candy decorating pen
To prepare the cake, preheat oven to 350F. Grease and line an 8-inch square cake pan.
In a large bowl, mix together sugar, flour, cocoa powder, baking powder, baking soda and salt.
Crack in the egg, spoon in the sour cream, and pour in the oil and the vanilla. Mix with a wooden spoon until just combined.
Pour in the boiling water, and slowly mix until smooth and liquidy.
Pour batter in the prepared pan, and bake for 20 to 25 minutes. Transfer pan to a wire rack and let cake cool in pan for 15 minutes. Then invert it onto a greased rack. Let cool completely.
To prepare buttercream, beat butter for a few minutes in a standing mixer with the paddle attachment on medium speed. Add powdered sugar and turn the mixer on low, beat until the sugar incorporates with the butter. Add vanilla, heavy cream and cocoa powder, beat on medium speed for another 3 minutes, or until buttercream becomes light and fluffy.
To assemble cake pops, prepare two cookie sheets lined with parchment paper.
Break the cooled cake into a few big trunks by hand, place them into the bowl of an electric mixer fitted with the paddle attachment and mix on medium speed until the big pieces turn into crumbs.
Add frosting gradually into the cake crumbs and blend until the mixture becomes a dough, about 1 to 1 1/2 cups of frosting.
Use a small cookie scoop, roll dough into evenly sized balls and place them on the prepared cookie sheets. Refrigerate for 30 minutes.
Melt small amount of chocolate Candy Melts in the microwave gradually, 10 second intervals and stirring in-between. Dip one end of a lollipop stick in the chocolate and insert into the cake ball. Repeat until you are done with 15 cake pops. Refrigerate again for an hour.
Repeat with orange and lavender Candy Melts. So you will have three group of 15 cake pops in chocolate, orange and lavender.
Remove one group of cake pops from the refrigerator. Melt the corresponding Candy Melts in the microwave. Dip each cake pop in the Candy Melts and gently tap off excess. Place onto a piece of wax paper. Let it dry completely. Repeat with the remaining cake pops.
Repeat with the remaining two groups.
For the pumpkin, draw faces on the orange cake pops with an edible marker. Melt the green Candy Melts in the microwave. Transfer 2 to 3 tablespoons to a piping bag with a small round tip. Pipe the green Candy Melts on the pumpkin tops as leaves.
For the Frankensteins, dip each chocolate cake pop half way in the remaining green Candy Melts and gently tap excess. Place back to the wax paper and let dry completely. Draw faces on the green part.
For the purple skeletons, draw faces on the lavender cake pops with the edible marker.

domain: food


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Posted 02 March 2011 - 12:03 PM
By David A. Bene
A quick review. I bought the book off Amazon and enjoyed reading through it. It's divided into four major sections: (1) history of the league, (2) Statistics for each team and each season, (3) Index, and (4) Linescores for every season.
The last three are self-explanatory and should require no explanation. The first part, the history of the league, is well researched and a good read. My lone complaint is the format. It is double spaced with very large margins around the page so the 103 page length of this section is in reality about half that.
Other than that, it is an enjoyable and informative read and, IMHO, worth the price.
Posted 12 March 2011 - 01:04 PM
A Year-by-Year History, 1926-1965
by Joseph S. Page
Paperback: 232 pages
Publisher: McFarland (December 10, 2010)
Included because it covers the AAFC Championship games. A very nicely written book with a good write-up of each game and the circumstances, if any (such as the 1946 Filchock and Hapes gambling scandal), surrounding it. The write-ups on the games are very much like reading a detailed account of the action in a newspaper. They include game lineups and statistics but no pictures. Overall, a nice book on the subject; well worth the cost.
Posted 12 March 2011 - 01:26 PM
A History of the All-America Football Conference Team, 1946-1949
by Kenneth R. Crippen
Paperback: 327 pages
Publisher: McFarland (November 10, 2009)
Great book on the early Buffalo Bisons/Bills of the AAFC. Good history with schedules, rosters, detailed game summaries and a detailed section on Chapter Notes and Bibliography. It's a great resource for anyone interested in this team (by PFRA's very own, Ken Krippen)!
Posted 12 March 2011 - 01:31 PM
THe 1946-1955 Cleveland Browns
Pro Football's Greatest Dynasty
by Andy Piascik
Paperback: 432 pages
Publisher: Taylor Trade Publishing; Reprint edition (August 16, 2010)
Possibly the best book written on this era of Browns history. It's exceptional from front-to-back and makes an excellent case for the AAFC being on par with the NFL of that time--and the Browns being the best team in either league.
Posted 12 March 2011 - 01:36 PM
by Kevin O'Connell
Paperback: 123 pages
Publisher: Green Road Press
A solid history of the Browns' undefeated 1948 season with photos and detailed player information. Hard to find but worth the search.

domain: library


With the ‘Revolution Radio’ tour whipping fans into a frenzy on a global scale, Californian pop-punk maestros Green Day bring their dominant musical force to Sheffield tonight.
Further to a short UK run back in February, these last few days have seen the band play gigs in Belfast and Dublin and a huge show at London’s Hyde Park as part of the British Summer Time festival. But now – absolutely rammed inside Sheffield Arena – this is where it’s at. With a baying Yorkshire crowd giving it their all along to ‘Bohemian Rhapsody’ and ‘Blitzkrieg Bop’, you just know this is going to be massive.
As ‘The Good, the Bad and the Ugly’ fades out, the blue-haired Tré Cool dances onto stage to screams of delight, before bassist Mike Dirnt and the uncontrollably energetic Billie Joe Armstrong join him. Hitting us up with ‘Know Your Enemy’, it’s during the first track that Billie Joe has a fan pulled up to sing the final verse. The lad does a good job, before being encouraged to run the length of the runway for a stage dive back into the crowd. It’s a great start to what becomes a two-
Along with a trio of touring members, Green Day belt out a set-list which just about covers their quarter century plus album discography. ‘Boulevard of Broken Dreams’ is the first real breather as Billie Joe dedicates it to everybody here – a song “for all us weirdos, freaks and strangers” he says. It’s a starry-eyed moment for plenty of youngsters, although perhaps blurred for many when Billy describes their use of phones as “like cancer” and “as addictive as heroin”. Although he does make a good point.
“Any old school Green Day fans here?” enquires Billy Joe, before welcoming Mike Dirnt during the bass intro into ‘Longview’. It’s during the debut single from ‘Dookie’ that another fan is pulled up to sing the last verse. This guy does really well, a performer already in the making it would appear.
Track-after-track is delivered with captivating buoyancy – the politically charged ‘Holiday’, ‘Youngblood’ one of several tracks from the latest album and ‘Hitchin’ a Ride’ the first single off of 1997’s ‘Nimrod’. There’s even one for “real” old school fans with ‘Paper Lanterns’ off the ‘Slappy’ EP.
Amongst the mesmerising light show, backdrops, flames and explosions, Billy Joe hoses down the crowd and fires tee-shirts the full length of the arena. During ‘When I Come Around’ I swear that every single pair of hands in the 13,500 capacity venue are in the air, and when he tells Sheffield to “jump”, it feels like the entire Steel City is obeying his command. He has this huge crowd eating out of his hand, apart from when they let him know that this is “Yorkshire! Yorkshire! Yorkshire!” – mistakenly and amusingly interpreted by Billy as being called a “cock sucker”.
While the main man has never been known to shy away from sharing the bands political views, it seems to be pretty much on the back burner tonight. Yeah, there’s the mention of our freedom to self-expression and so on, but there’s no ranting or chanting. The 45-year-old displaying an impressive feat of stamina and clearly enjoying himself, during what he describes as one of his “favourite ever shows”.
A circle pit opens up during ‘Welcome to Paradise’, there’s full accordion and harmonica accompaniment to ‘Minority’, before another fan is up onstage to play guitar along to the Operation Ivy cover ‘Knowledge’. This lucky lady – “Eleanora” – not only gets to play with Green Day, but also gets to keep the guitar. Wow! How’s that for a fan participating show?
Even though Green Day have ventured down many paths over the years there’s no song that’s more representative of the band than ‘Basket Case’ – the prime punk-pop track almost tearing the roof off. But still there’s still more to come with ‘She’ and ‘King for a Day’ featuring a fantastic sax solo from Jason Freese, including a tribute to George Michael with the riff to ‘Careless Whisper’.
Of course, there comes the introduction to “the world’s most dangerous drummer” with Tré Cool out from behind his kit doing high kicks before the band lie down and have the crowd in full voice for a medley of covers: ‘Shout’, ‘Teenage Kicks’, ‘Always Look on the Bright Side of Life’, ‘(I Can’t Get No) Satisfaction’ and ‘Hey Jude’.
‘Still Breathing’ and ‘Forever Now’ bring the main set to a close before an encore of ‘American Idiot’ and ‘Jesus of Suburbia’ leave all of those here – young, old and older – totally annihilated with passion, excitement and energy. Billie Joe returns with an acoustic guitar for a calming end-of-show ’21 Guns’ and ‘Good Riddance (Time of Your Life)’ with Mike and Tré joining him at the end.
You can read it anywhere, that Green Day are one of the greatest live bands of all time. But not until you see it, hear it and feel it do you know that for sure. Never has a song lyric been more appropriate as a show finisher. “It’s something unpredictable, but in the end is right, I hope you had the time of your life”. It is, it was, and we did. Cheers Green Day.
Review & Photography: Steve Johnston


“Under the knife” experts iFixit got Apple watch Series 5, which the vendor introduced at the fall presentation. Experts popular resource checked the suitability for repair of the gadget and found due to what the manufacturer has managed to preserve the autonomy of the novelties.
Features Of The Apple Watch Series 5. One of the main differences of a novelty from the predecessor was the capacity of the internal battery. Model 40-millimeter case has been a significant increase in 10%, and for version 44 millimeters figure was a more modest 1.4 per cent, reports.
Visual differences between the two generations hours almost no, but under the “hood” you notice a number of changes. The fifth generation got the display, made by technology LTPO, a new light sensor, and an improved gyroscope, and compass.
In the Apple Watch Series 5, except for the doubled memory, uses a new single-chip system Apple S5. The processor and graphic accelerator new identical model Series 4 is probably the main differences between the devices are as energy efficiency.
Maintainability of hours. Disassembly procedure has not changed from last year: to “opening” hours will need to heat the case, for fixing part of elements of which an adhesive has been used. The battery can be disconnected from the motherboard, only the almost complete disassembly of the gadget. Given the small number of modules maintainability of the Apple Watch Series 5 was estimated at 6 out of 10 possible points on a scale iFixit.
Other news relating to events in the world of technology, gadgets, artificial intelligence, and space, see Techno


By Daniel Katzive
Gale Brewer, New York City Council Member for District 6, toured 72nd Street between Columbus and Amsterdam with Department of Sanitation Manhattan Borough Chief James Leavy and other DSNY officials on Tuesday.
The walk-through comes three months after Brewer met with Sanitation officials on this block to discuss community quality-of-life concerns around sanitation and rats, according to Eddie Amador, Director of Communications for Brewer’s office. Brewer noted significant improvement in today’s walk-through, with just a few areas of concern noted on the north side of the block.
Chief Leavy indicated trash had been accumulating on the sides of the sheds, interfering with drainage. DSNY teams with shovels had done a targeted cleanup and then outreach teams had met with local businesses to reinforce the need to keep these areas clean. He noted his officers had had more success with outreach to local businesses than with summonses.
Chief Leavy said that summonses do remain an important tool in dealing with illegally parked cars which interfere with the ability of sweepers to access the curb. He noted that there have also been issues with improperly used parking placards in this area which DSNY officers have had to address.
While significant improvement was noted by the officials this morning, a few remaining problem areas were noted on the north side of the street. Officials on the walk-through spoke with restaurant staff in one location and a walk back through that area a short time later showed the issue had been remediated.
Deputy Chief Mohammad Aburmeileh noted that business owners are responsible for keeping the street clean out to the limits of their sheds, but sometimes claim staff shortages hinder their capacity to comply and need reminders of this requirement.
Going forward, Council Member Brewer indicated her office would be continuing to research problem areas in the district in order to bring them to the attention of sanitation officials. She noted particular concerns about major cross streets such as 72nd, 86th and 96th Streets.
If you want to report a problem area, call 311 first and get a service request number. Then, call Gale’s office (212-873-0282) and give them the number. Don’t forget to get a number!
Every time I hear that restaurant employees (and more importantly, managers) have to be alerted to a sanitation/appearance/safety/etc. issue DIRECTLY in front of their door, I shudder at the thought what they need to be alerted to when it comes to the kitchen/dishes.
I’m not one to praise local politicians, but I know for a fact that Gale was instrumental in removing that monstrosity of an encampment that was on the corner or Amsterdam and 72nd for months. I cant figure out why that block just remains unsightly, but I would encourage as much cleanup as possible. As much as I enjoy Grays Papaya, I can’t help but think it’s a major part of the problem.
The major part of the problem — in fact, the entire problem – is that UWSers give people in these encampments money. Don’t do that. Give money to organizations that help the mentally ill. That’s what Gale does. I used to give street people money until I found out the woman I was funding had her own apartment (provided by Breaking Ground) in the Bronx.
I’m late to this but have to echo Lisa — as a 72nd St. resident, it’s really frustrating to see UWSers handing cash to people who are camped out for weeks or months on the 72nd St. sidewalks.
I really encourage people to instead donate their spare dollars to the council house on 72nd — it does phenomenal work all year long helping the needy with food, clothing, school supplies, etc.
P.S. The Gray’s Papaya corner has improved quite a bit since the sidewalk shed came down fairly recently. Fingers crossed the sheds above Capital One and Trader Joe’s also come down soon.
Just shocking that almost no one building restaurant sheds considered drainage beneath those structures and ran the plywood walls down to the asphalt. Oh, wait, that flaw was obvious.
Businesses and buildings are responsible for keeping the sidewalk and past the curb clean of debris.
We own a small brownstone and I sweep everyday, even when I didn’t work at home.
It’s nice to have a trash free(ish) street. Also, since putting our trash in a can for the garbage men, it’s cut down on rats a lot! Putting a bag of trash on the street is an invitation for a dinner party! The G men probably don’t like lifting bags out of “cans” but they use cans in other boroughs so, I was told, they have to do it here. Try it, it works!
We also put our garbage out in the cans instead of leaving the black bags (aka “rat buffet”) on the curb and I think it is the single most effective way to help reduce rats (with curbside composting a close second).
Nowif we can get a sweep of the cross streets on the upper west side that would be WONDERFUL.
Staff shortages make it impossible for them to sweep the streets outside their restaurants? There are countless able-bodied people in our neighborhood who are not working and receiving government aid who should be doing these jobs. Go to one of our many shelters and round up some workers and tell them they can keep a portion of the salary, with the rest going to the government to underwrite some of their costs.
It is basic supply and demand – shouldn’t be difficult.
How about adding more trash cans to the block? In the 90’s there used to be a trash can mid-block between Columbus and Amsterdam. And given how infrequently the trash on 72nd/Columbus and 72nd/Amsterdam is picked up, let’s do as Boston does, and put two trash cans side by side so they don’t overflow. So simple. Are you listening Gale.
Not only do street sweepers fail to arrive even once a week these days, often no one even tickets. At this point, most car owners don’t bother to move their cars at all. The streets are filthy.
Why can’t sweeper vehicles be fitted with cameras to record the plate numbers of vehicles that fail to move? And why are vehicles with drivers in them not ticketed? Because “standing” isn’t “parking.” Absurd.
The entire street cleaning system is broken. The city would be cleaner — and revenue higher — with just a small amount of common sense and competence.
Hate to say this, because I am pro-labor. But streetsweepers (DSNY) are Union which means: they will reject anything that could be considered an additional responsibility, because it signals to management that the Union is willing to take on more work for the same pay. So — quite unlikely a simple common-sense change like this is gonna happen.
Hello Neighbors: This is an old story but we’ll repeat anyway. Since the Fairway takeover at 74th/Broadway, that store front curb is a dirty disgraceful eyesore. The empty pallets, broken carts, and rats galore make it impossible to cross the street or get a cab and worst of all, the stores remain unrented for blocks and blocks. Who wants to open a store next to such an eyesore? We’ve called, complained etc. Fairway #718-569-4500-they will tell you they own the curb and sidewalk. There must be a city ordinance or something making this illegal. Mayor Adams please help! The “broken windows” concept has turned our once good neighborhood into blocks of empty stores, homeless encampments and hostile panhandlers.


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At home with minted artist Brandy Brown-Bergquist. A monthly glimpse inside the homes and studio of Minted artists. Get to know the creative force behind Marabou Design. For more interviews go to Domino.
Decorating with bright colors is fun! The trick is how to do it right. When playing with color we 2 Ladies keep it simple. In this vignette simple means sticking to pink and gold as our bright colors. The neutral white fluffy pillow and grey throw from Ho

domain: artistic


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South Africa and Africa in general benefitted immensely from the recent US-Africa Leaders' Summit held in Washington last week, Trade and Industry Minister Rob Davies said on his return.
13 Aug 2014
Sanlam says it has R3bn in discretionary capital that is earmarked for growth opportunities mainly in Africa and South-East Asia.
Phakamisa Ndzamela 6 Jun 2014
Shipping and logistics group Grindrod has been a big spender on infrastructure for some time. Growing its network of port and rail operations allows the group to diversify away from international shipping, a notoriously cyclical business.
19 May 2014
Zimbabwe's new central bank chief said the country's economy was weakening and the bank did not have the tools to fix it.
8 May 2014
Zimbabwe is planning a second auction of its Marange diamonds next February‚ buoyed by the interest in and sale of more than 279‚000 carats of diamonds in Antwerp‚ Belgium. The sale raked in US$10.7m.
Ray Ndlovu 24 Dec 2013
President Jacob Zuma says South Africa and the Democratic Republic of Congo (DRC) must explore ways to stimulate trade and investment between the two countries.
31 Oct 2013
Business leaders from the BRICS group of emerging economies met on Tuesday (20 August) to discuss trade with Africa, insisting that the bloc's planned development bank has not been abandoned.
21 Aug 2013
Growth in SA will continue to lag that of other African countries‚ with the latest African Development Bank African Economic Outlook report released on Wednesday (7 August) listing the country among the 10 that will record the slowest growth this year.
9 Aug 2013

domain: news


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Marbled Clay Ring Dish
How To Grow A Kiwi #Plant From Seed – DIY:
COmo hacer y cuidar un Terrarium
Organizing Your Home: Making the Best Use of Every Space | Decorating Your Small Space
How to Grow Tomatoes Without Seeds #gardeninghacks #vegetablegarden
Homemade DIY Extracts. Easy to make your own at home and fun to gift!
Nisbets Next Day Catering Equipment: Infographic: Know Your Knives
Here's how to make a sweetly whimsical DIY fairy house planter from a terra cotta pot & other inexpensive items. It's really easy, so why not give it a try?

domain: hobby


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Celebrity Big Brother boosts Channel 5 ratings
'Celebrity Big Brother' has proved to be a winner for Channel 5 in its first two weeks, pushing the channel's average audience during the key 9pm and 10pm slots up by 46% and 94% respectively.
Since ‘Celebrity Big Brother’ started (on 18 August), the show has been watched by an average audience of 2.35 million when it has been broadcast at 9pm, and 2.25 million when on air at 10pm.
Compared to the slot averages for the same period (18 August to 1 September) last year, the reality show has increased Channel 5’s audience from 1.61 million between 9pm and 10pm, and from 1.16 million between 10pm and 11pm.
ITV has struggled to retain audiences over the summer and ‘Celebrity Big Brother’ has beaten ITV1 twice in the past five days in the 9pm slot.
Last night, an average audience of 2.10 million people watched former Atomic Kitten and reality star Kerry Katona get cross with 'The X Factor' contestants Jedward, between 10pm and 11pm on Channel 5, a 10.8% share of the available audience.
"Celebrity Big Brother' burst onto Channel 5's schedule a fortnight ago with an audience of 5.08 million, making it the fourth-most-watched show in the channel’s history, and the highest rating show that was not a film or football game.
As the graph below shows, apart from last Saturday (27 August), since launch, the audience of 'Celebrity Big Brother’' has stayed above two million, with many of the shows delivering around 2.5 million viewers, a large audience for the channel.
Despite struggling earlier in the week at 9pm, ITV1 performed well last night with '9/11: Day that Changed the World', which was watched by 5.14 million viewers between 9pm and 10.30pm, a 23.1% share of the TV-viewing audience.
This article was first published on brandrepublic.com
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Geeks & Geezers - AchieveMax® Top Ten Book Review
Geeks & Geezers: How Era, Values, and Defining Moments Shape Leaders by Warren G. Bennis & Robert J. Thomas
Most every organization today employs members of the younger generation just out of college and, in some cases, even high school. In fact, I recently read of a west coast firm that hired several middle school students to do part-time programming after school and on weekends. Those same companies employ their share of A.A.R.P. members nearing retirement. Both age groups, as well as those in between, bring tremendous value to the table in terms of education, experience, creativity, talent, etc. However, consider the cultural, environmental, motivational and personal goal differences of these two diverse employee groups. Then consider the fact that they are being thrown into the same chaotic, rapidly changing business environment and instructed to achieve ever-increasing levels of productivity and profitability! To expect such lofty expectations to be met would, of course, require, at the very least, a basic orientation to members of each group of the vast differences existing in each of the areas mentioned earlier. In fact, education and discussion of these differences and their possible consequences should be on-going. Now think about your own organization. Is this orientation currently being offered? Has it ever been offered? Should it be?
If any organization were to recognize the importance of this education and decide to provide it for their employees, the benefits would be phenomenal! The ideal textbook for such a program would have to be Geeks & Geezers by Bennis & Thomas. In this groundbreaking study, the authors compare and contrast these two disparate groups-affectionately labeled "geeks" (aged 21-34) and "geezers" (aged 70-82). They asked successful geeks to share the secrets of their youthful triumphs and distinguished geezers to tell them how they continue to stay active and engaged despite the changes wrought by age. Today's young leaders grew up in the glow of television and computers; the leaders of their grandparents' generation in the shadow of the Depression and World War II.
The authors, who bring considerable experience to the table (Bennis has written over 30 books on leadership and Thomas is a senior fellow with Accenture's Institute for Strategic Change), interviewed more than 40 leaders who they deem either "geeks" or "geezers" to evaluate the effect of era on values and success. The two groups vary in terms of their ambitions, heroes and family lives, but members of both sets share one common experience: all have "undergone at least one intense, transformational experience," which the authors call a "crucible." In some cases the crucible was an actual hardship, e.g., geezer Sidney Rittenberg spent 16 years in prison in China for speaking out against the government. For others, it was a dramatic experience, such as NYSE pioneer Muriel Siebert's entry into male-dominated Wall Street in 1967 or geek Liz Altman's stint working at a Japanese Sony factory before becoming a Motorola VP.
Among the findings of their research, Bennis and Thomas learned that Geezers and Geeks had quite different concerns when in the age range of 25-30. The Geezers' concerns were making a living, earning a good salary, starting and supporting a family, stability and security, working hard and getting rewarded by the system, listening to their elders, paying "dues" to various organizations, and using retirement to enjoy life. It also reveals the critical traits they share, including adaptability, vision, integrity, unquenchable optimism, and "neoteny"-a youthful curiosity and zest for knowledge.
In contrast, Geeks' concerns (during the same age range) were making history, achieving personal wealth, launching a career, change and impermanence, working hard so they can write their own rules, wondering if their elders "got it wrong," deciding where loyalty should lie, and achieving a balance between work and life. These are significant differences which Bennis and Thomas explain in terms of the different eras in which Geeks lived (at ages 25-30), the societal values of their respective generations, and various "defining moments" such as those associated with the Great Depression, World War II as well as Vietnam and the emergence of the Internet and World Wide Web.
Geeks and Geezers is a book that will forever change how we view not just leadership but the very way we learn and ultimately live our lives. Highlighting the forces that enable any of us to learn and lead not for a time, but for a lifetime, this book is essential reading for geeks, geezers, and everyone in between.
More than 100 business book reviews written by Harry K. Jones are available at http://www.AchieveMax.com/books/.
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Harry K. Jones is a professional speaker and consultant for AchieveMax®, Inc., a firm specializing in custom-designed keynote presentations, seminars, and consulting services. Harry has made presentations ranging from leadership to employee retention and time management to stress management for a number of industries, including education, financial, government, healthcare, hospitality, and manufacturing. He can be reached at 800-886-2MAX or by visiting http://www.AchieveMax.com.
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