text:
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
28 Related JoVE Articles!
Quantification of γH2AX Foci in Response to Ionising Radiation
Institutions: The Alfred Medical Research and Education Precinct, The University of Melbourne, The Alfred Medical Research and Education Precinct.
DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX1
. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2
. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3
. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete γH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2
. The loss of γH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8
. The disappearence of γH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6
. Further, removal of γH2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8
. Importantly, the quantitative analysis of γH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of γH2AX foci in γ-irradiated adherent human keratinocytes9
Medicine, Issue 38, H2AX, DNA double-strand break, DNA damage, chromatin modification, repair, ionising radiation
Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c
Institutions: University of North Carolina , University of North Carolina .
Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1
. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c
from the mitochondrial intermembrane space to the cytoplasm2
. The release of cytochrome c
from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c
release from mitochondria5
. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c
(heme-attached) protein into cells6-9
. Cytochrome c
is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11
. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c
protein instead of its cDNA, because while the expression of cytochrome c
from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c
protein is a useful tool to mimic mitochondrial cytochrome c
release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c
protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c
for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.
Cellular Biology, Issue 52, Microinjection, apoptosis, cytochrome c, fibroblasts, neurons
Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Institutions: Loyola University Chicago.
Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria.
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2
DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2
DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2
. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions.
Neuroscience, Issue 51, Mitochondrial membrane potential, reactive oxygen species, neuroscience, cortical neurons
The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures
Institutions: University of Basel.
Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3
. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11
are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4
. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.
Neuroscience, Issue 61, dendritic development, dendritic branching, cerebellum, Purkinje cells
Assessment of Vascular Function in Patients With Chronic Kidney Disease
Institutions: University of Colorado, Denver, University of Colorado, Boulder.
Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA
) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA
, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA
, aPWV, and vascular endothelial cell protein expression.
Medicine, Issue 88, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets
Institutions: University of Alabama at Birmingham.
Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.
Immunology, Issue 85, bioenergetics, translational, mitochondria, oxidative stress, reserve capacity, leukocytes
Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response
Institutions: University of Alabama.
Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster.
Here, we have used the conserved innate immune response in Drosophila
to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster,
and this assay can be modified to be applicable to other Drosophila
tissues or even other small, whole-organism inflammation models.
Immunology, Issue 82, biology (general), environmental effects (biological, animal and plant), immunology, animal models, Immune System Diseases, Pathological Conditions, Signs and Symptoms, Life Sciences (General), Neuroinflammation, inflammation, nitric oxide, nitric oxide synthase, Drosophila, neurodegeneration, brain, Griess assay, nitrite detection, innate immunity, Parkinson disease, tissue culture
Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy
Institutions: École Polytechnique Fédérale de Lausanne, Oregon Health & Science University.
Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.
Bioengineering, Issue 86, Intravital imaging, epifluorescence, two-photon imaging, Tumor matrix, Matrix remodeling
Visualization of G3BP Stress Granules Dynamics in Live Primary Cells
Institutions: Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535.
SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer's disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.
Cellular Biology, Issue 87, Stress granule (SG), G3BP, primary cells, neurons
Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions
Institutions: University of Florence, University of Oxford, University of Florence, University of Florence, National Institute of Optics-National Research Council, Italy, International Center of Computational Neurophotonics.
The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.
Bioengineering, Issue 90, Single molecule biophysics, Optical tweezers, fluorescence microscopy, DNA binding proteins, lactose repressor, microfluidics
Purification of Transcripts and Metabolites from Drosophila Heads
Institutions: University of Florida , University of Florida , University of Florida , University of Florida .
For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila
as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila
are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.
Genetics, Issue 73, Biochemistry, Molecular Biology, Neurobiology, Neuroscience, Bioengineering, Cellular Biology, Anatomy, Neurodegenerative Diseases, Biological Assay, Drosophila, fruit fly, head separation, purification, mRNA, RNA, cDNA, DNA, transcripts, metabolites, replicates, SCA3, neurodegeneration, NMR, gene expression, animal model
Infinium Assay for Large-scale SNP Genotyping Applications
Institutions: Oklahoma Medical Research Foundation.
Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina's panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.
Basic Protocol, Issue 81, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina
Quantitation of γH2AX Foci in Tissue Samples
Institutions: The Alfred Medical Research and Education Precinct, The Alfred Medical Research and Education Precinct, The University of Melbourne, Royal Children's Hospital, The University of Melbourne.
DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks1
. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2
. Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2
. Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3
. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo
and in vivo4,5
. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.
Cellular Biology, Issue 40, immunofluorescence, DNA double-strand breaks, histone variant, H2AX, DNA damage, ionising radiation, reactive oxygen species
Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae
Institutions: University of Maine.
Disseminated candidiasis caused by the pathogen Candida albicans
is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6
. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9
. Very little is known about the dynamics of C. albicans
interaction with innate immune cells in vivo.
Extensive in vitro
studies have established that outside of the host C. albicans
germinates inside of macrophages, and is quickly destroyed by neutrophils10-14
. In vitro
studies, though useful, cannot recapitulate the complex in vivo
environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18
. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host.
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21
, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24
. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5
. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color.
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25
. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26
, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25
zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro
studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5
Immunology, Issue 65, Infection, Molecular Biology, Developmental Biology, Candida albicans, candidiasis, zebrafish larvae, Danio rerio, microinjection, confocal imaging
Viability Assays for Cells in Culture
Institutions: Duquesne University.
Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.
Cellular Biology, Issue 83, In-cell Western, DRAQ5, Sapphire, Cell Titer Glo, ATP, primary cortical neurons, toxicity, protection, N-acetyl cysteine, hormesis
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining
Institutions: CNRS UMR 5534, Université de Lyon 1, LabEX DEVweCAN, CNRS UPR 3296, CNRS UMR 5286.
Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ
in animal models. We describe a DNA-fluorescent in situ
hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.
Neuroscience, Issue 83, Life Sciences (General), Virology, Herpes Simplex Virus (HSV), Latency, In situ hybridization, Nuclear organization, Gene expression, Microscopy
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
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
Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease
Institutions: Louisiana State University Health Sciences Center.
There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson’s disease.
Behavior, Issue 90, Exercise, locomotor, Parkinson’s disease, aging, treadmill, bradykinesia, Parkinsonism
Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE
Institutions: University of Cambridge, UK, University of Cambridge, UK.
Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via
functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v
.) or intracerebroventricular (i.c.v.
) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.
) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo
Here we describe the methods that we have developed for the i.v
. and i.c.v.
delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.
Immunology, Issue 86, Somatic neural stem/precursor cells, neurodegenerative disorders, regenerative medicine, multiple sclerosis, experimental autoimmune encephalomyelitis, systemic delivery, intravenous, intracerebroventricular
Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease
Institutions: SRI International, University of California-Santa Cruz.
Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer's disease, but its effects in Parkinson's disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone. To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e.
5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder. Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD. The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates. This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.
Medicine, Issue 83, MPTP, dopamine, Iba1, TH, GFAP, lipoxygenase, transgenic, gene-environment interactions, mouse, Parkinson's disease, neurodegeneration, neuroinflammation
Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways
Institutions: University of Colorado School of Medicine, Oregon Health & Science University, University of Colorado School of Medicine.
Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.
Neuroscience, Issue 82, male, female, sex, neuronal culture, ischemia, cell death, neuroprotection
Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice
Institutions: Cornell University.
Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/-
consistently develop thymic lymphoma early in life 1,2
, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3
. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4
. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/-
mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.
Immunology, Issue 50, mouse, thymic lymphoma, Atm, p53, T-cell lines
Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model
Institutions: Minneapolis Veterans Affairs Health Care System, University of Minnesota, University of Minnesota, University of Minnesota.
The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro
hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.
Neuroscience, Issue 86, apoptosis, obesity, caspase, resazurin, DEVD, palmitic acid, hypothalamic model
In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts
Institutions: Emory University.
Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.
Cellular Biology, Issue 19, Current Protocols Wiley, Xenopus Egg Extracts, Nuclear Assembly, Nuclear Membrane
Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection
Institutions: The Salk Institute for Biological Studies, The Salk Institute for Biological Studies.
Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2
and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al.
for the preparation of the slices and from Gogolla et al.
for the staining procedure 3,4
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro
, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.
Neuroscience, Issue 51, Cerebellum, Organotypic, Fas, Apoptosis, Purkinje cell
BioMEMS: Forging New Collaborations Between Biologists and Engineers
Institutions: University of California, Irvine (UCI).
This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons. This device is compatible with live-cell optical microscopy (DIC and phase contrast), as well as confocal and two photon microscopy approaches. This method uses precision-molded polymer parts to create miniature multi-compartment cell culture with fluidic isolation. The compartments are made of tiny channels with dimensions that are large enough to culture neurons in well-controlled fluidic microenvironments. Neurons can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration.
Neuroscience, Issue 9, Microfluidics, Bioengineering, Neuron

domain: medical


text:
Dainty monogram signet rings make the perfect gift for her, made in solid sterling silver with a modern matte finish.
Signet rings are back and these delicate and feminine statement rings are perfect to freshen up your jewellery collection.
Available in a choice of shapes, the diamond has bold faceted edges, whilst the oval is a little softer with its curved edges. Go for the rectangle if you like uncomplicated and easy to wear luxury.
Popular pinkie signet rings or as a simple yet delicate sovereign ring for women, and ideal gift for her if she loves geometric jewellery.
Each design was originally hand carved in wax before being cast into solid silver, inspired by my love for art deco design they are a touch of luxury that can be worn every day.
The initial is laser engraved giving a strikingly crisp monogram that will not fade. Engraved to order which can take a little longer, but that is so worth the wait for a truly unique piece of heirloom jewellery.
Choose to have your initial oxidised black to make the letter pop, or go for the subtle look with the letter left silver.
These geometric rings are the perfect gift for her, a simple design with sharp lines and smooth facets.
Made to your chosen ring size using solid sterling silver.
Presented in a gorgeous jewellery gift box.
Handmade in solid sterling silver and hallmarked for your quality guarantee.
Diamond measures 5mm x 11mm. Thinnest part of ring measures 1.5mm x 1.5mm.
Rectangle measures 6mm x 13mm. Thinnest part of ring measures 1.5mm x 1.5mm.
Oval measures 5mm x 13mm. Thinnest part of ring measures 1.5mm x 1.5mm.
This item is made to order, please allow 11 -12 working days for delivery.
Delivery is FREE within the UK. A signature may be required upon arrival.
International delivery to the rest of the world is available, the cost is dependent on your location. Please add your item to the basket where the cost will be calculated during checkout
If you require your order sooner please do get in touch to find out if I can help.
What to expect when you order arrives...
I want you to experience joy and excitement when you receive your jewellery - whether its a gift for a loved one or a gift for yourself!
All orders come in a branded gift box suitable for your chosen piece, along side a care card on how to look after your jewellery, a polishing cloth, and a gift card to write a note to the receipt. If you are sending directly I can gift wrap and add a gift note for you, free of charge, taking the hassle out of Christmas!

domain: artistic


text:
Six-Part Documentary Series Premiering Sept. 5, 2019, Will Drive Change for the Most Pressing Global Issues Impacting Humanity.
Submitted by P&G
National Geographic announced that ACTIVATE: THE GLOBAL CITIZEN MOVEMENT, the six-part documentary series from National Geographic and Procter & Gamble, co-produced by Global Citizen and RadicalMedia, will premiere Sept. 5 at 9/8c in the U.S. and will air globally on National Geographic in 172 countries and 43 languages.
ACTIVATE is a multiplatform storytelling partnership and six-part documentary series — bolstered by custom content distributed across National Geographic’s unrivaled social, digital, linear and print channels. It will raise awareness about extreme poverty, inequality and sustainability issues to mobilize Global Citizens to drive meaningful and lasting change on the Global Citizen platform.
Each episode delves into a different issue connected to the root causes of extreme poverty, following a specific Global Citizen campaign and the organizers, amplifiers, grassroots activists, action takers and people whose lives are being changed. Episode topics include eradicating extreme poverty, ending cash bail, breaking down barriers to girls’ education, funding education for displaced children, ending plastic pollution and helping solve the water crisis.
ACTIVATE will take audiences from the Global Citizen Festival stages to the front lines of change around the world while spotlighting real-time efforts to address issues related to extreme poverty. Through the eyes of activists including Hugh Jackman, Priyanka Chopra Jonas, Becky G, Usher, Common, Rachel Brosnahan, Gayle King, Bonang Matheba, Pharrell Williams, Darren Criss and Uzo Aduba, and told through the iconic storytelling lens of National Geographic, viewers will hear the stories, meet the people and witness firsthand the work that is being done to combat extreme poverty and inspire a global movement.
The series has been filmed internationally in Mexico, Nigeria, Peru, the Philippines and South Africa, and across the United States in California, New York, Tennessee and Washington, D.C.
The ACTIVATE partnership is the result of a shared commitment National Geographic, P&G and Global Citizen have made to leverage their collective influence, audience and scale to both raise awareness and inspire a global community to take action.
For more than 10 years, Global Citizen has been working to defeat extreme poverty through advocacy and citizen engagement, motivating businesses, organizations and governments to make financial commitments and policy changes to achieve the United Nations Sustainable Development Goals and eradicate extreme poverty by 2030. At the same time, P&G has a rich history of leveraging its brands and its people as a force for good to make the world a better place by helping solve critical issues related to girls’ education, clean drinking water, disaster relief, sustainability and inequality. And for more than 130 years, National Geographic has utilized its powerful imagery and impact storytelling to help audiences around the globe understand the world around them so that, in turn, they care more about it.
The multiplatform ACTIVATE storytelling partnership will expand beyond the documentary series to include a 10-page custom print section for ACTIVATE in the September issue of National Geographicmagazine as well as custom story stretches aligned to each episode, custom digital articles and social media moments highlighting various issues within ACTIVATE.
The partnership will also include digital hubs on NatGeo.com and GlobalCitizen.org for viewers who would like to dive deeper into the six critical issues highlighted in the series. Viewers will be able to take action on Global Citizen campaigns, earn rewards and learn more about P&G’s brand impact efforts led by Tide, Always, Head & Shoulders, Charmin and more.
The ACTIVATE series will illustrate experimentation with new content and distribution models, and new ways to mobilize viewers across the world.
ACTIVATE: Eradicating Extreme Poverty
Premieres: Thursday, Sept. 5, 2019
Hugh Jackman, Priyanka Chopra Jonas and Becky G join Global Citizen in campaigns to push world leaders into enacting policy changes that would end extreme poverty around the globe. As Jackman prepares for the organization’s massive Central Park festival, Becky G travels to Mexico to amplify Global Citizen’s campaign to push companies to support women-owned businesses. Experts describe the state of extreme poverty around the world, the enormous progress that’s been made in the past 20 years and innovative solutions being implemented across the globe, including P&G and its Charmin brand’s supply chain sourcing efforts.
ACTIVATE: Ending Cash Bail
Premieres: Thursday, Sept. 12, 2019
Artists and activists Common and Usher team up with Global Citizen and grassroots organizers as they attempt to achieve historic criminal justice reform by ending the use of cash bail in New York state.
ACTIVATE: Education Cannot Wait
Premieres: Thursday, Sept. 19, 2019
Emmy Award-winning actor Rachel Brosnahan teams up with Global Citizen and travels to the border of Peru to see what happens to children’s education during conflicts and natural disasters. After an emotional experience with displaced families, Brosnahan and Global Citizen return to the U.S., where they urge Ireland, the U.K. and the U.S. to pledge millions to Education Cannot Wait, a global fund for education during crises. Experts describe the disproportionate impact that natural disasters and conflict have on developing countries and ways people and brands around the world, like Tide, are providing effective relief during emergencies.
ACTIVATE: Keeping Girls in School
Premieres: Thursday, Sept. 26, 2019
Priyanka Chopra Jonas joins Global Citizen and activists around the globe as they campaign to break down barriers to girls’ education. Meanwhile, Gayle King and Bonang Matheba rally tens of thousands of people to call on the South African government to commit $58 million toward ending “period poverty” and providing girls with the menstrual education and resources they need to stay in school and stay confident. Experts describe the broad range of issues that keep girls from getting an education in developing countries, and the enormous progress that’s being made around the world, like the work by feminine care product brand Always to provide free educational resources.
ACTIVATE: Ending Plastic Pollution
Premieres: Thursday, Oct. 3, 2019
Pharrell Williams joins Global Citizen’s push to get governments, companies and individuals to solve the ocean plastic pollution crisis. Meanwhile, Darren Criss travels to the Philippines to witness plastic’s impact on people living in extreme poverty and calls on global citizens to urge their mayors to commit their cities to zero-waste futures. Experts describe the disproportionate impact of plastic pollution on people in developing countries and the ways people and brands, like Head & Shoulders, are solving the problem around the world.
ACTIVATE: Clean Water
Premieres: Thursday, Oct. 10, 2019
“Orange Is the New Black” star Uzo Aduba joins Global Citizen as they rally millions around the world to push for clean drinking water and proper sanitation for the world’s most vulnerable people. They travel to Aduba’s parents’ homeland of Nigeria, where they urge governors to commit state funds to eradicate the contaminated water and open defecation crises. Experts describe the effects of contaminated drinking water on people in developing countries and solutions being implemented around the world, including the P&G Children’s Safe Drinking Water Program.
About National Geographic
National Geographic Partners LLC (NGP), a joint venture between the National Geographic Society and Disney, is committed to bringing the world premium science, adventure and exploration content across an unrivaled portfolio of media assets. NGP combines the global National Geographic television channels (National Geographic Channel, Nat Geo WILD, Nat Geo MUNDO, Nat Geo PEOPLE) with National Geographic’s media and consumer-oriented assets, including National Geographic magazines; National Geographic studios; related digital and social media platforms; books; maps; children’s media; and ancillary activities that include travel, global experiences and events, archival sales, licensing and e-commerce businesses. Furthering knowledge and understanding of our world has been the core purpose of National Geographic for 131 years, and now we are committed to going deeper, pushing boundaries, going further for our consumers … and reaching millions of people around the world in 172 countries and 43 languages every month as we do it. NGP returns 27 percent of our proceeds to the nonprofit National Geographic Society to fund work in the areas of science, exploration, conservation and education. For more information visit natgeotv.com or nationalgeographic.com, or find us on Facebook, Twitter, Instagram, YouTube, LinkedIn and Pinterest.
About Procter & Gamble
P&G serves consumers around the world with one of the strongest portfolios of trusted, quality, leadership brands, including Always®, Ambi Pur®, Ariel®, Bounty®, Charmin®, Crest®, Dawn®, Downy®, Fairy®, Febreze®, Gain®, Gillette®, Head & Shoulders®, Lenor®, Olay®, Oral-B®, Pampers®, Pantene®, SK-II®, Tide®, Vicks® and Whisper®. The P&G community includes operations in approximately 70 countries worldwide. Please visit http://www.pg.com for the latest news and information about P&G and its brands.
About Global Citizen
Since the first Global Citizen Festival in New York in 2012, Global Citizen has grown into one of the largest, most visible platforms for young people around the world, calling on world leaders to honor their responsibilities in achieving the United Nations Sustainable Development Goals and ending extreme poverty by 2030. Global Citizens have generated commitments and policy announcements from leaders valued at over $37.9 billion that are set to affect the lives of more than 2.25 billion people. The organization has taken its action-based model to South Africa, Australia, India, the United Kingdom, Germany, Belgium and Canada, and has created a platform for activists to learn about the issues they care most about, take action and earn rewards for doing so.
RadicalMedia produces premium content across all media platforms. With offices in New York, Los Angeles, London, Berlin and Shanghai, RadicalMedia is a global intradisciplinary media company that creates and produces film, television, music programming, commercials, brand partnerships, interactive design, and digital and immersive experiences for all distribution platforms. RadicalMedia is fluent in every discipline of storytelling, with accolades that include Oscar, Emmy, Peabody and Grammy awards. Embracing the future and pushing innovation, RadicalMedia is aptly referred to as Never Established. Check out its latest work on Instagram and on www.radicalmedia.com.
P&G defines our commitment to sustainable development as "ensuring a better quality of life for everyone, now and for generations to come." We have a long heritage as a sustainability leader and we remain committed to improving consumers' lives through P&G brands and by contributing to the sustainability of our planet and the communities in which we live and work.
More from P&G

domain: news


text:
The Finance Ministry says capital investment by Japanese corporations fell three percent in the first quarter, compared with the same quarter of 2002.
The survey also shows that average corporate profits rose 10 percent in the first quarter compared with a year earlier. But that is actually a much smaller increase than the 23 percent jump in the previous quarter.
Analysts say the statistics suggest companies are only managing to improve profitability by cutting costs and trimming capacity, but are not making new investments in equipment or plants.
Japan Research Institute senior economist Hisashi Yamada does not see capital spending increasing any time soon. Mr. Yamada says further deregulation and incentives are needed to help existing businesses and to create a better environment for entrepreneurs.
The Finance Ministry also reports large Japanese companies are more pessimistic about the outlook for the economy than they were in the first quarter of the year. The bleaker mood reflects worries about the strengthening yen, which hurts exports, and the effects of SARS on the region.
The outbreak of Severe Acute Respiratory Syndrome, as well as the effects of the U.S. led war in Iraq, has weakened Japan Airlines. Asia's largest carrier says it will seek 17,000 volunteers to take a month's leave without pay. It is the first time a major Japanese company has called for such across-the-board furloughs.
Europe's top automaker is launching a new passenger car in Japan. Volkswagen, which sold 60,000 autos in Japan last fiscal year, says it will soon release the convertible version of its Beetle.
Volkswagen Japan President Tsutomu Umeno told TV Tokyo that his company wants to increase its market share in the competitive Japanese market. Volkswagen holds the biggest share of the market for imported cars in Japan and expects to capture about 24 percent of the market this year.
Intel, the world's largest chipmaker, is investing $100 million in Elpida Memory. The deal gives Intel a stake in the struggling chip maker, but the size of the stake has not been reported.
Elpida is a joint venture between Hitachi and NEC and is Japan's last big maker of standard computer memory chips. The Japanese partners also say they will inject more money into the unprofitable company, which has promised to turn a profit by 2005.


The bank, which spent £12 million in 2005, according to Nielsen Media Research, uses Media Planning Group to handle planning and buying.
MPG has held the account since ING Direct launched in 2003, backed with £25 million in marketing support.
The AAR is handling the review process and MPG will repitch for the account.
Gina Fusco, the sales and marketing director at ING Direct, said: "Now that we have passed the important one million-customer milestone and new products are on the horizon, it is appropriate and best practice to review our current media handling arrangements."
Marc Mendoza, the MPG managing partner, said: "We are looking forward to continuing a successful relationship and fully understand that a financial services company that has now reached this size would want to undertake a benchmarking exercise."
ING Direct's creative account will continue to be handled by VCCP.
ING Direct, a division of the Dutch banking group ING, has 15 million customers worldwide.
- Media Perspective, page 10.


The circular economy at the core of Enel X
The future of the economy is circular. Enel X has placed this model at the centre of its strategy with the aim of accelerating and multiplying the benefits for clients and suppliers. The advantages do not only concern the environment, but also economic growth and new business opportunities
Published on 26 February 2019
The current economic system, based on the linear model of “produce, consume, dispose” is no longer sustainable. By 2050 the global population will reach 9.8 billion, the raw material needs of urban centres – which already use two thirds of global energy and are responsible for 70% of greenhouse gas emissions – will climb from 40 to 90 billion tonnes per year and the amount of waste produced will rise from 1.3 to 2.2 billion tonnes by as early as 2025. Pollution levels will increase, the scarcity of resources will make it hard to satisfy everyone’s needs; nations, companies and citizens will have to bear increasingly high costs. How can we reverse this trend?
The new paradigm: circularity
The circular economy is a sustainable systemic solution for most of these problems. For this reason Enel X has placed it at the heart of its strategy with the aim of becoming an accelerator and amplifier of the beneficial effects of this paradigm within the vast ecosystem of our suppliers and clients. The advantages are not confined to reducing the impact on the environment, but include economic growth, and new business opportunities.
What does circular economy mean? Imagine taking the straight line of the current economic system and bending it until it is transformed into a circle. Now you’ll have a system that operates in a similar way to natural cycles, an economy where used materials are regenerated. Products are designed along modular lines for a longer life, to be easily dismantled and regenerated. The amount of waste produced is cut to a minimum, and this waste is given value through transformation into resources for producing other goods, prolonging the lifecycle into infinity. Exclusive ownership of goods is replaced by use as a service or through sharing, like the car-sharing model. Thanks to digitalisation, decentralisation and electrification, energy consumption is more efficient, mobility will trend towards zero impact, the use of renewable sources will be maximised and every user will also be transformed into a potential supplier, as is the case with the Vehicle-to-Grid (V2G) technology applied to e-vehicles by Enel X.
Our Circular Innovation
Enel X was the first service provider to offer a practical application in the context of the circular economy, with, for example, its Pole Station e-vehicle charging service. To achieve this we looked at the roots of the problem of sustainability, rethinking the entire value production chain on the basis of the five keystones of the circular economy, which can be expressed through five business models. The Pole Station and the new-generation gamma Juice stations are the result of applying the “product as a service” and “sharing” models that offer clients access to the assets without transferring ownership. The “circular supply chain” model based on renewable energy and organically-derived materials underpins technologies like the adaptive public lighting systems, which use AI to regulate lighting levels automatically according to weather, traffic and light conditions, leading to significant savings for the public authorities. The modular design of most of our solutions, and regeneration wherever possible, facilitates the replacement of all the parts and makes it possible to recycle modules no longer in use: this approach is based on two mainstays of the circular economy, “Extending product lifecycles” and “Recovering the value of what is no longer in use”.
These are just a few examples of the many solutions that Enel X has already developed to increase the circularity and sustainability of our economy. We have an ecosystem that enables us to collaborate with startups, research centres, universities and clients, opening up our product innovation to different realities. This hyper-connected system relies on the horizontal sharing of ideas, requirements and solutions that can then be trialled and rolled out on an international scale, a process of constant innovation that we have also incorporated into our corporate approach.
Measuring circularity: an economic and environmental value
To promote the new paradigm Enel X has rethought the entire value generation chain, from the supply of materials to the final phases of the product or service, taking into account the economic, social and environmental impacts. As a result we have created a “circularity scale”, the Enel X Score, which measures how sustainable our solutions are. Its purpose is to create a means of providing transparency for our clients and suppliers, making them aware of the fact that adopting circular models offers advantages in terms of sustainability, greater savings and added value.
The “circularity scale”, which ranges from a minimum level of 1 to a maximum of 5, assesses the degree to which our solutions reflect the cornerstones of circularity and the five key dimensions identified by Enel X. The use of recyclable or biodegradable materials, renewable resources and types of use that encourage sharing, efficiency and asset reuse and the recovery of materials at the end of their life are vital elements in this assessment. Applied to our solutions, this measurement technique is reflected by a RINA-certified sustainability level label that enhances clients’ environmental awareness and our efforts to constantly improve on our goods and services as well as our level of circularity.
We are also replicating this virtual model abroad. Enel X offers an innovative report that measures the level of energy circularity of companies and cities, already successfully tested on some industrial clients and public administrations. It is available to all – Enel X will produce a detailed report on the level of circularity of their business with a guided, structured road map for improvement.
Choosing Enel X means changing your life and business so that respect for the environment takes on an even more important role.
Read all the stories of Enel X and discover how the world can become ever more smart and sustainable
The combined photovoltaic, storage and fuel cell system developed by Demand Energy is the first microgrid in New York City to ensure both peak load reduction and an autonomous backup power supply through self-generated power
Major sports competitions for electric cars and motorcycles are based on Enel X technology, and the related innovations are also having positive impacts on the development of road going vehicles
The energy retrofitting designed by Enel X offers its customers green lighting solutions which reduce energy consumption by 50%


text: Dr. Kathleen Puntillo is Professor of Nursing, Emerita, at University of California, San Francisco. She has a long-standing program of research on pain in critically ill and injured patients. Currently she is completing a European Society of Intensive Care Medicine-funded international study of procedural pain in ICU patients and recently completed an NIH-funded study of thirst in ICU patients. She publishes and lectures extensively on the topics of pain and other symptoms, ICU communication, and ICU palliative care. Dr Puntillo is a Fellow in the American Academy of Nursing and a Fellow in Critical Care Medicine.


text:
The double exposure technique using alternating phase shift mask (alt-PSM) has been proposed and it is well
used for the gate layer of the high performance logic devices as strong resolution enhancement technology (RET). This
technique has advantage that the fine resist profile is obtained on wafer with extensive process margin. However, this
double exposure technique is very expensive because of the alt-PSM cost. This time, the new double exposure technique
without alt-PSM is developed for gate layer of 45 nm node logic devices. In this new double exposure method,
attenuated phase shift mask (att-PSM) or binary mask (BIM) is used with dipole illumination. It is thought that this new
double exposure method is effective for random logic devices which have various pattern pitches by the optimization of
dipole illumination condition and pattern placement. Firstly, the optical contrast and depth of focus (DOF) is calculated.
From these results, dipole illumination condition is optimized. It is found that DOF of new double exposure method is
wider than that of conventional method. In addition, mask pattern is optimized to obtain wide process margin. For dense
pattern, mask biasing is effective and optimization of shifter width is effective for isolated pattern. Furthermore, it is
found that assist pattern is very effective for isolated pattern. From experimental results, it is proved that new double
exposure method have wider process margin than that of conventional one. The strong design for manufacturing (DFM)
rule that required the severe line width control is placed at single direction is proposed to realize the new double
exposure method. Finally, it is found that the lithographic performance of new double exposure method has same level as
conventional method with alt-PSM for gate layer of 45 nm logic devices.

domain: literature


Jonas Birgersson, a 28-year-old Swedish Internet entrepreneur, doesn't look to Americans such as Amazon.com's Jeff Bezos for role models. He takes his cues from the military strategists of the 19th century Prussian army. A military history buff, Birgersson runs Framfab, an Internet consultancy with a market cap of $1.7 billion, according to lessons learned by the Prussian military in their battles against Napoleon. That approach should come in handy as Birgersson girds himself for war on two fronts: in Europe, where he is challenging the Continent's dominant phone companies by offering cheap high-speed Internet access, and in the U.S. market, where he is competing for the digital services business of multinationals.
Just as the Prussians learned from Napoleon's army that troops divided into small corps are more effective and respond better to rapid change than large groupings, Birgersson has split Framfab into a network of small, independent cells. This structure has served the firm well. Founded by Birgersson in 1995, the company, which offers strategic consulting, employs 1,700 people and operates in eight countries. It counts multinationals like Volvo, Kellogg's, Commerzbank, Bosch, Ikea and Nike among its clients. Describing Framfab's strategy in the U.S. market as a "flank attack," Birgersson clearly views the marketplace as a battlefield. "Only idiots compete on equal terms," he says. "This is war."
For Birgersson, who made his first million at age 26 yet still rides a bicycle to work, the battle is not about money or personal clout, but about empowering consumers. "We want to give ordinary people more for less," he says. Enter another of his businesses: B2 or Bredbandsbolaget, which means broadband network in Swedish, a high-speed Internet service provider that offers Web access to people across Europe at cut-rate prices. But unlike most high-tech business plans, this one starts by targeting the bottom of the personal income pyramid instead of the middle or top.
The idea behind B2, which is 25% owned by U.K.-based cable operator NTL and also backed by Intel, is to install in apartment buildings the local area networks (lans) used in offices to provide high-speed connections for PCs. Typically, Internet surfers use 56 kilobits per second modems and pay a per minute charge. The lan does away with the need for modems. In fact, B2's advertised speed is around 20 times faster for downloading data from the Web and around 80 times faster for uploads than the average cable modem or digital subscriber line consumer offer, says Sam Paltridge, an Internet expert at the Paris-based o.e.c.d. "The telcos' dsl and cable modem offers make a big thing of the fact that they are x times faster than ordinary modems," says Paltridge. "Yet B2 is offering something that is less expensive than many of the telcos' narrow band offerings over the public switched telephone network, but at a speed around 180 times a 56 kbit/sec modem."
A recent o.e.c.d. study on the pricing of broadband services for consumers in the U.S., Japan and many European countries shows the average monthly cost for digital subscriber line connections is $60.80, and $44.80 for cable modem service—anywhere from two to three times Bredsbolaget's charge of $20. B2's 10 megabits per second connection can transmit and receive services such as video-on-demand, radio, television and telephony. It also allows users to create their own portals or Web TV channels. The monthly fee even includes free phone calls to other users of B2's network. And, for an as yet unspecified additional charge, B2 plans within the next few months to introduce flat rate local and national telephone calls over the traditional phone network, using a converter box which will allow consumers to use their normal telephones.
The goal, says Birgersson, is to give consumers all of these services for the same amount as—or even less than—they currently pay the phone company for regular phone service and slow Internet access. Small wonder that the offer is proving appealing to the residents of government-run public housing. Last August HSB, the largest Swedish organization of tenant-owned housing, signed a contract with B2 covering 350,000 households. Birgersson says phone companies could have long ago introduced the kind of high-speed lan service B2 is offering consumers, but didn't because it would cut their profits from existing services. B2 filed a complaint last November to the European Commission's Directorate alleging that Sweden's former monopoly, Telia, has reduced its prices 90% for high speed lan services in some of the places where B2 has introduced competition. B2 charges that Telia is abusing its dominant position by using predatory and discriminatory pricing. The case is still pending.
Birgersson has no use for any of Europe's major telecommunication firms and goes so far as to claim that they are "absolutely worthless" because their copper wire networks are too time-consuming and expensive to upgrade to handle the next generation of high-speed services. That is why B2 is building its own fiber optic network from scratch, one that allows faster and cheaper connections to the Internet—without the need for modems.
Framfab is working with the Swedish government on a plan to link 70 Swedish cities through a high-speed fiber optic network that would allow companies to rent "raw" fiber optics—fiber which is not connected to a traditional phone company network—and run any type of service over it. Eventually, the national network being built by the government will link up with the local loop being built by B2. "It's a paradox that it takes government infrastructure to get competition to start in broadband," Birgersson remarks. By the end of this year, B2 plans to connect 100,000 housing units in Sweden. The eventual goal will be to connect 50 million households in Europe over the next five to seven years, primarily by striking deals with local new entrants which have fiber optic networks.
A key part of Framfab's global strategy is to entice U.S. companies to test their Web commerce offerings in Sweden first, since it will be one of the first countries in the world to have full national broadband coverage. "We want to create the world's greatest lab in Sweden," Birgersson says. "It's a small market, so if it blows up, who cares. But if it works, you can deploy worldwide." Birgersson, who says he has ideas for other companies in the works, is convinced his projects will be winners in the long run. "We are judged by history," he says. "It's okay if in our lifetime nobody understands what we are doing. Inspiring and motivating people in the future will be our greatest achievement." Spoken like a true warrior.


text:
Although the baroque music is unquestionably beautiful, there’s a problem with the plot of Handel’s opera Rinaldo.
Simply put, the storyline is wacko.
It’s even wacko by the usual standards of opera, a bizarro world in which wrong babies are mistakenly tossed onto fires and heroines fling themselves off parapets at the slightest provocation. In Rinaldo’s mish-mash of a plot, dragons cavort, sorceresses unleash crazed minions, Christian magicians pop up to do good works and people fall in and out of love within nano-seconds (OK, the latter is actually fairly common).
Thank goodness, then, for stage director Glynis Leyshon, who, for Pacific Opera Victoria’s invigorating revival, has hatched a clever and charming solution to Rinaldo’s kookiness. Repositioning the 18th-century opera in wartime Britain, she resets the shenanigans as a children’s fantasy. After daddy dons his Second World War helmet and goes off to battle, his young son and daughter — clad in pajamas and nightgown — imagine the entire thing, witnessing it all from the sidelines à la Clara in The Nutcracker.
With such a conceit, any zaniness becomes charming and even amusing. And for the modern audience, the approach also serves to make the notion of a romantic male hero with a disconcertingly high-pitched voice more palatable. (In fact, Rinaldo features two good-guy counter-tenors: Rinaldo the bold knight and Goffredo the brave general. The roles are nicely sung by the POV’s Andrey Nemzer and David Trudgen. However, there’s something unintentionally risible about two men stoically trudging through mountains while exchanging falsetto bon mots.)
This is a superb production, beautifully staged, cleverly directed and well-performed. In this opera, there is a lot of lyrical repetition, yet thanks to Leyshon and company, we are unceasingly entertained.
The Victoria Symphony benefits from conductor Timothy Vernon’s obvious love for the material. On Thursday, the tempos were pleasingly brisk and the playing accomplished and vivacious. Harpsichordist Tatiana Vassilieva was a special delight throughout — her extended solo as poor Rinaldo is buffeted by the furies was a virtuoso workout earning its own applause. Handel’s brilliant orchestration is fresh and witty, with the harpsichord and brass echoing the singers’ florid passages enchantingly.
The standout star is Nemzer, the Russian counter-tenor playing Rinaldo. One of this opera’s celebrated arias is Cara sposa, in which Rinaldo laments the disappearance of his love, Almirena. Nemzer, clad in a leather jerkin, sang this heartbreaking song with superb technical control and phrasing. Performing throughout with admirable projection, he revealed a naturally beautiful voice: pure, clear, polished.
As Almirena, soprano Stéphanie Lessard wears a puffy pink Disney princess gown in keeping with the fantasy theme (all the fine costumes are by Pam Johnson, who also designed the excellent set).
Lessard was captivating when she sang Rinaldo’s best-known aria, Lascia ch’io pianga (Let me weep), winning over the audience with her buttery delivery and big, rounded tones, the orchestra mirroring each of her sigh-like phrases.
Also notable was soprano Jennifer Taverner as Armida, an evil sorceress who enters to thunder claps and lightning flashes. Taverner sang well, successfully navigating difficult coloratura passages and projecting the powerful stage presence such a pivotal role demands. The effect was boosted by a trio of capering furies (resembling satanic members of ABBA in their fright wigs and wildly flared trousers) and such devices as a giant cage in the shape of a spider, in which Almirena is imprisoned.
The visual tricks in Rinaldo are unrelenting, giving the production a circus-like appeal. Rinaldo ascends skyward in a full-sized boat; a Chinese-style dragon lashes its tail; English rowhouses belching smoke suggest a children’s storybook come to life. One of the design team’s triumphs is a giant moon projection with many incarnations — often featuring a creeping spider representing the sorceress’s diabolical inclinations.
Leyshon’s gift for intelligent humour works wonderfully. Argante, well-sung and acted by baritone Christopher Dunham, arrived in a chariot with two prancing gladiators for horses. One of the horse-men twitched his head in an amusingly equine manner. Elsewhere, a row of roses dropped dramatically from the ceiling like lawn darts and with a thunk impaled a table, causing the woman beside me to exclaim “That’s amazing — what a hoot!” Such sharp attention to detail is the mark of a superior production.
This is one of the best creations I’ve seen from Pacific Opera Victoria. Don’t miss it.
Performances continue today, April 27 and April 29.