text:
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.
27 Related JoVE Articles!
Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants
Institutions: University of Alabama.
The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.
Neuroscience, Issue 18, epilepsy, seizure, Caenorhabditis elegans, genetics, worm, nematode, aldicarb, pentylenetetrazole, synaptic, GABA
Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
Institutions: The Ohio State University, Tohoku University.
The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5
. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9
. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10
, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.
Cell Biology, Issue 48, BRCA1, homologous recombination, breast cancer, RNA interference, DNA repair
A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Institutions: University of Maryland .
Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1
. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2
. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e.
cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro
and in vivo
for their therapeutic potential3-5
. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6
. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo
. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 °C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 °C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus
endolysin LysK and Streptococcus pneumoniae
endolysins Cpl-1 and Pal lose activity spontaneously at 42 °C, 43.5 °C and 50.2 °C, respectively8-10
. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11
. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1
). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.
Immunology, Issue 69, Molecular Biology, Genetics, Microbiology, directed evolution, thermal behavior, thermostability, endolysin, enzybiotic, bacteriolytic, antimicrobial, therapeutic, PlyC
Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
Institutions: Alberta Health Services / Calgary Laboratory Services / University of Calgary, University of Calgary, University of Calgary, University of Calgary, University of Calgary.
Staphylococcal Cassette Chromosome mec
typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus
(MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec
by targeting and identifying the individual mec
gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec
types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec
types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec
assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.
Infection, Issue 79, Microbiology, Genetics, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Bacteria, Bacterial Infections and Mycoses, Life Sciences (General), Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcal cassette chromosome mec (SCCmec), SCCmec typing, Multiplex PCR, PCR, sequencing
Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
Institutions: Auburn University , Keesler Air Force Base.
A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus
strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus
. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3
. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus
strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.
Bioengineering, Issue 75, Microbiology, Infectious Diseases, Infection, Medicine, Immunology, Cellular Biology, Molecular Biology, Genetics, Anatomy, Physiology, Bacteria, Pharmacology, Staphylococcus, Bacteriophages, phage, Binding, Competitive, Biophysics, surface properties (nonmetallic materials), surface wave acoustic devices (electronic design), sensors, Lytic phage spheroids, QCM-D, Langmuir-Blodgett (LB) monolayers, MRSA, Staphylococcus aureus, assay
Stress-induced Antibiotic Susceptibility Testing on a Chip
Institutions: Fraunhofer USA Center for Manufacturing Innovation, Harvard Medical School, Boston University, Boston University.
We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step - bacterial growth - is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane (PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.
Bioengineering, Issue 83, antibiotic, susceptibility, resistance, microfluidics, microscopy, rapid, testing, stress, bacteria, fluorescence
Invasion of Human Cells by a Bacterial Pathogen
Institutions: University of Bath.
Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus
. The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus
would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2
at 37°C. For S. aureus
this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 μl tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.
Infection, Issue 49, Bacterial pathogen, host cell invasion, Staphylococcus aureus, invasin
Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice
Institutions: David Geffen School of Medicine at University of California, Los Angeles (UCLA), PerkinElmer, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine.
Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo
optical and μCT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus
strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo
bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, μCT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical μCT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo
bioluminescent/fluorescent imaging with μCT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.
Infection, Issue 92, imaging, optical, CT, bioluminescence, fluorescence, staphylococcus, infection, inflammation, bone, orthopaedic, implant, biofilm
Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo
Institutions: University of Tübingen.
Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5'-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo
fluorescent primer extension method to reliably and rapidly detect the 5' ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g.,
by toxin-antitoxin system components) in S. aureus, E. coli
and other bacteria.
Molecular Biology, Issue 92, Primer extension, RNA mapping, 5' end, fluorescent primer, transcriptional starting point, TSP, RNase, toxin-antitoxin, cleavage site, gel electrophoresis, DNA isolation, RNA processing
Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy
Institutions: University Medical Center Utrecht.
We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus
on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+
. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.
To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSMα operon to GFP. When these reporter strains of S. aureus
are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.
Infection, Issue 77, Immunology, Cellular Biology, Infectious Diseases, Microbiology, Genetics, Medicine, Biomedical Engineering, Bioengineering, Neutrophils, Staphylococcus aureus, Bacterial Toxins, Microscopy, Fluorescence, Time-Lapse Imaging, Phagocytosis, phenol soluble modulins, PSMs, Polymorphonuclear Neutrophils, PMNs, intracellular expression, time-lapse microscopy, flow cytometry, cell, isolation, cell culture
One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures
Institutions: Maastricht University Medical Center, Erasmus Medical Center.
Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1
. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4
. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5
. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa
and Escherichia coli
as well as Gram-positive bacteria including Staphylococcus
spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp.,
and Streptococcus pneumoniae
could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus
spp. and (facultative) aerobe Gram-negative rods6
. This assay was based on a study in which PCR was used to measure the growth of bacteria7
. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1
). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.
Immunology, Issue 65, Infection, Medicine, Microbiology, Bacteria, real-time PCR, probes, pathogen detection, blood culture, 16S rDNA gene, antibiotic resistance, antibiotic susceptibility testing
Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity
Institutions: Michigan State University.
This protocol describes a fluorescence microscope-based screening of Arabidopsis
seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis
is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2
), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.
Genetics, Issue 62, EMS mutagenesis, secretory pathway, mapping, confocal screening
Forward Genetic Approaches in Chlamydia trachomatis
Institutions: Duke University Medical Center.
, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis
despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype–phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.
Immunology, Issue 80, genetics, chemical mutagenesis, whole genome sequencing
Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)
Institutions: University of Technology, Sydney.
Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.
Molecular Biology, Issue 91, super-resolution microscopy, fluorescence microscopy, OMX, 3D-SIM, Blaze, cell division, bacteria, Bacillus subtilis, Staphylococcus aureus, FtsZ, Z ring constriction
Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Institutions: Rensselaer Polytechnic Institute.
has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae
has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Institutions: University of Toronto, University of Toronto, University of Regina.
Phenotypes are determined by a complex series of physical (e.g.
protein-protein) and functional (e.g.
gene-gene or genetic) interactions (GI)1
. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2
. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7
, but GI information remains sparse for prokaryotes8
, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10
Here, we present the key steps required to perform quantitative E. coli
Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9
, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format.
Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g.
the 'Keio' collection11
) and essential gene hypomorphic mutations (i.e.
alleles conferring reduced protein expression, stability, or activity9, 12, 13
) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14
. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9
. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2
. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e.
slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2
as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9
Genetics, Issue 69, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis
Institutions: Youngstown State University.
Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5
-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ
replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter
sp. YSU, and randomly incorporates itself into this host’s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli
) strain. The R6Kγ
replication origin allows the plasmid to replicate in pir+ E. coli
strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ
replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter
sp. YSU or of a wider variety of bacterial strains.
Microbiology, Issue 92, Auxotroph, transposome, transposon, mutagenesis, replica plating, glucose minimal medium, complex medium, Enterobacter
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains
Institutions: Georg-August University.
Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis
. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.
Cellular Biology, Issue 83, Bacillus subtilis, evolution, adaptation, selective pressure, beneficial mutation, intraspecies competition, fluorophore-labelling, Fluorescence Microscopy
In Vivo Modeling of the Morbid Human Genome using Danio rerio
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo
complementation in zebrafish. Zebrafish (Danio rerio
) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo,
and can be genetically manipulated.1
These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells
Institutions: UT Health Science Center at San Antonio, UT Health Science Center at San Antonio, UT Health Science Center at San Antonio, UT Health Science Center at San Antonio, UT Health Science Center at San Antonio.
In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
transgenic mice carry a recoverable λ phage vector encoding the LacI
reporter system, in which the LacI
gene serves as the mutation reporter. The result of a mutated LacI
gene is the production of β-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI
reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli
. After incubating infected E. coli
on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI
gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI
gene will show the location of the mutations in the gene and the type of mutation.
transgenic mouse model is well-established as an in vivo
mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-
(LSK) cells and other subpopulations of the hematopoietic system.
Infection, Issue 84, In vivo mutagenesis, hematopoietic stem/progenitor cells, LacI mouse model, DNA mutations, E. coli
Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes
Institutions: University of Massachusetts, Amherst, University of Massachusetts, Amherst, University of Massachusetts, Amherst.
RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans
. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans
genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans
Developmental Biology, Issue 79, Caenorhabditis elegans (C. elegans), Gene Knockdown Techniques, C. elegans, dsRNA interference, gene knockdown, large scale feeding screen
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing
Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis
Institutions: University of British Columbia - UBC, University of British Columbia - UBC.
The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of pathogenic microorganisms. The cuticle is made up of a tough crosslinked polymer called "cutin" and a protective wax layer that seals the plant surface. The waxy layer of the cuticle is obvious on many plants, appearing as a shiny film on the ivy leaf or as a dusty outer covering on the surface of a grape or a cabbage leaf thanks to light scattering crystals present in the wax. Because the cuticle is an essential adaptation of plants to a terrestrial environment, understanding the genes involved in plant cuticle formation has applications in both agriculture and forestry. Today, we'll show the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.
Plant Biology, Issue 16, Annual Review, Cuticle, Arabidopsis, Eceriferum Mutants, Cryso-SEM, Gas Chromatography
An Introduction to Worm Lab: from Culturing Worms to Mutagenesis
Institutions: University of Texas at Arlington.
This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus
, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.
Basic Protocols, Issue 47, Mutagenesis, Caenorhabditis elegans, Pristionchus pacificus, ethyl methane sulfonate (EMS), 4, 5', 8-trimethylpsoralen (TMP).
Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model
Institutions: University of Southern California, Los Angeles.
Studies using the Saccharomyces cerevisiae
aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2
. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.
Genetics, Issue 55, saccharomyces cerevisiae, life span, aging, mutation frequency, genomic instability

domain: biology


text:
Halsey is honoring her late friend Juice Wrld the best way she knows how.
During her Pandora Live concert in Brooklyn on Tuesday night, the "Graveyard" singer honored the late rapper by performing their "Without Me" remix, released in January. Before performing the song, the 25-year-old star gave an emotional message to the audience.
In a video shared by an audience member, Halsey is heard dedicating the song to her friend, who died on Sunday at the age of 21. "The remix of this song is the better version of this song," she told the crowd. "And I think everybody knows that deep down inside. But I really want to play it tonight for him."
As the musician continued, "So, while we have every single one of us together in one place, I just wanted to take a moment to remind you guys that life is short. We should appreciate every single moment that we have together." Before she began to sing, Halsey made a special request. As she said, "Promise me to sing along loud?"
The song served as her show's finale. Another video shared by the same audience member shows confetti falling from the ceiling as Halsey slows down the pace of the last few lines, her hands stretched up above her head.
The audience sang along as the song closes, and then the singer thanked them all and makes her way off stage.
It was a fitting tribute for the late rapper, and clearly an emotional moment for Halsey.
Juice Wrld died on Sunday, Dec. 8, after suffering a seizure following a flight from California to Chicago. The cause of death is still under investigation.
The "Nightmare" singer reacted to her friend's death on Instagram on Sunday.
Sharing a photo of her and the late star, Halsey wrote, "Sweet boy. The fastest brain in the game. Your creativity, endless. Immortal. Rest in power. I can't even explain the sadness."

domain: entertainment


Houston — Ridley Terminals in Prince Rupert, British Columbia, exported 967,772 mt of thermal and metallurgical coal in October, up 6% from September, data from the Prince Rupert Port Authority showed Nov. 30.
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The month-on-month increase was the largest in six months, largely driven by a jump in thermal exports.
October were also at a five-month high, and from the year-ago month exports were up 11%.
Through October, Ridley has exported about 9.3 million mt, up 23% from the same point last year.
Total thermal exports were 413,869 mt in October, up 75.1% month on month and up 14.3% from the year-ago month.
Through 10 months, the terminal has shipped over 4.3 million mt of thermal coal, up 61.4% from the corresponding 2019 point.
Met exports totaled 553,903 mt in October, down 18.1% from September and up 8.7% from October 2019.
Through October, met exports totaled about 5 million mt, up 1.8% year on year.
Petcoke exports were 231,721 mt in October, the most in 39 months. From September they rose, 227% and year on year, they were up 29.9%.
Over 10 months, petcoke exports totaled over 1.2 million mt, down 6% from the same point last year.


text:
"MELBOURN is a parish, in the hundred of Repton and Gresley. The village, which is a considerable one, and of some antiquity, is situate seven miles and a half S.S.E. from Derby, and about six N.N.E. from Ashby-de-la-Zouch. Camden notices this place as having a castle, wherein John Duke of Bourbon, of the blood royal of France, was incarcerated nine years, having been taken prisoner at the battle of Agincourt."
[Description from Pigot and Co's Commercial Directory for Derbyshire, 1835]
Melbourne is also a small town and a township in the parish of the same name. The parish lies 126 miles north of London. King's Norton is a hamlet in the parish, one mile north of Melbourne.
You can see pictures of Melbourne which are provided by:
You can see the
administrative areas in which Melbourne has been placed at times in the past. Select one to see a link to a map of that particular area.
You can see maps centred on OS grid reference SK386252 (Lat/Lon: 52.822957, -1.428625), Melbourne which are provided by:

domain: history


text:
Austria's Nicole Schmidhofer wins Lake Louise World Cup season-opener
Roni Remmi of Collingwood, Ont., is top Canadian finisher
Nicole Schmidhofer claimed the first World Cup victory of her career Friday by winning the season-opening downhill.
The 29-year-old Austrian was the fastest woman in the final training run and carried her speed over into the race in Lake Louise, Alta.
"I never thought my first World Cup victory will be a downhill and for sure not in Lake Louise," Schmidhofer said.
She was third in super-G in 2017. In a decade of racing downhills at the Alberta ski resort, however, Schmidhofer had never finished higher than eighth.
"In recent years, the jumps were bigger and higher and a little bit of a problem for me," the five-foot-two racer said. "This year, I saw the slope and maybe the chances will be better.
"I thought 'make it like training from the top to the bottom."'
Schmidhofer posted a top time of one minute 48.13 seconds in flat light because of cloudy skies.
Watch as Schmidhofer opens season with victory at Lake Louise
She won super-G gold at the 2017 world championship, but her best World Cup result prior to Friday was a super-G silver in 2013.
Switzerland's Michelle Gisin, the first woman to kick out of the start hut Friday, was second in 1:48.26.
"I really wanted to take bib one because when we start really late, it's good to get down early," Gisin said. "The visibility wasn't really good for anyone, so I'm super-glad I did a great run."
Weidle takes 2nd
Germany's Kira Weidle was third, half a second back of Schmidhofer, for the 22-year-old's first podium.
"It means a lot to me," Weidle said. "Last year I was eighth here in Lake Louise, which was already good, but I'm really happy to do one step forward and be on the podium. That's crazy."
Another downhill is scheduled for Saturday followed by Sunday's super-G.
So Schmidhofer's celebration of her first win in 128 World Cup starts was going to be mellow.
"Not beer because tomorrow is also a race," she said. "Good coffee and cake."
Slovenia's Ilka Stuhec, who swept both downhills in 2016, was sixth in her first start in over a year. The reigning women's world downhill champion did not race in 2017-18 because of a knee injury.
Ontario's Remme, top Canadian
Defending overall World Cup champion Mikaela Shiffrin of the U.S. was ninth.
Roni Remme of Collingwood, Ont., was the top Canadian in 35th.
"I brought some intensity to my ride," said the 22-year-old Remme. "It was tough with the lighting because of the clouded skies. Every time I see the track, I feel that I learn something new."
The women's speed season opened without stars Sofia Goggia of Italy, the reigning Olympic downhill champ, and American Lindsey Vonn.
Goggia fractured her ankle in training in October. Vonn injured her knee in a training fall in November.
Vonn has won a record 18 World Cup races at Lake Louise, including 14 downhills.
Although the 34-year-old has said this season will be her last, Vonn announced Friday she intends to return to Lake Louise in 2019 for one more series of speed races.
"I would regret it for the rest of my life if I didn't take that one last chance to push out of the starting gate in Lake Louise," Vonn said.
Watch as Lindsey Vonn extends her farewell tour
With 82 career World Cup wins, Vonn is five away from the record held by Sweden's Ingemar Stenmark.
In Beaver Creek, Colo., Ben Thomsen of Invermere, B.C., placed 38th in a men's downhill.
Watch as Switzerland's Feuz overcomes difficult conditions to seal win
Watch women's alpine skiing from Lake Louise

domain: news


text:
98109, 98108, 98104, 98107, 98106, 98101, 98103, 98102, 98105, 98154, 98122, 98121, 98126, 98125, 98178, 98174, 98177, 98144, 98146, 98195, 98199, 98118, 98119, 98116, 98117, 98115, 98112, 98164, 98134, 98136, 98133, 98111, 98113, 98114, 98124, 98127, 98129, 98139, 98141, 98145, 98161, 98165, 98170, 98175, 98181, 98185, 98191, 98194
Pay after Deletion means just that, once you are enrolled you only pay when and if items are ACTUALLY deleted from your credit report.
Get answers to all your credit repair questions by joining our credit repair tidbits facebook group
” You need to weigh the cost advantage of paying somebody else to do those things for you, versus doing those things by yourself and what your time is worth.” Here’s how credit repair companies work and, if you’re thinking about using one, why you should think about credit therapy initially.
The companies typically guarantee to “manage all the heavy lifting” of dealing with the credit reporting firms, states McClary. Credit repair organizations are different from credit therapy companies, which are generally a free resource from nonprofit monetary education organizations that evaluate your finances, debt and credit reports with the objective of mentor you to improve and manage your financial circumstance.
To help prevent scammers, it is essential to research any credit repair work organization before accepting deal with it. There are “all sort of methods” to veterinarian credit repair companies, McClary says. Here’s someplace to begin. Guide clear of business that seem too great to be real. If the business says it can do any of the following, it’s a warning and likely an indication of a rip-off: Get rid of precise unfavorable details from your reports Legally create a new credit identity for you The company demands you pay before it supplies services Guarantee improved credit Once again, we suggest looking for out a credit therapist first.
Lots of credit repair work business begin by requesting a copy of your credit report from each of the 3 significant consumer credit bureaus Equifax, Experian and TransUnion. The company will evaluate your credit reports for negative marks, like Then, it will set a strategy for challenging mistakes and negotiating with creditors to remove those items.
Beware here. If you’ve had difficulty managing credit in the past, a brand-new account may not be the best option. Plus it’s not a terrific idea to take on more credit if you do not require it. The quantity you’ll pay and how it’s determined will differ depending on the company, but there’s a rule they need to follow.
Depending upon the business, you might pay a one-time flat cost, or spend for each negative mark the company removes from each of your reports. This might start around $35 per deletion and could range to $750 or more. The business may also charge by the month, ranging from $50 to $130 or more.
Consider how much work your reports require. If there are simply a couple of unfavorable products, you’ll likely be better off applying any costs toward paying for debt and contesting any errors in your credit reports yourself. The credit bureau generally has 30 days after receiving your disagreement to examine and confirm info.
The credit bureau is needed to send you the outcomes of the examination within five business days of the completion of the examination. But if the credit bureau figures out the dispute is “frivolous” it can select not to examine as long as it communicates that to you within 5 days.
However many consumers do not understand where to begin, says Gerri Detweiler, education director at Nav, which aims to help entrepreneur understand and handle their service credit. Here are some methods you can deal with enhancing your credit on your own. Consumers must “work out extreme caution” when thinking about dealing with a credit repair work business, McClary states.
Even if you find a business you’re comfortable with, the services might not work for your circumstance. Plus you ‘d likely conserve cash if you disputed any incorrect details in your credit reports yourself. The Credit Repair Work Organizations Act, or CROA, makes it unlawful for credit repair business to lie about their services and outcomes, and sets some extra rules.
Credit reporting companies have a number of requirements under CROA, some of that include Giving you a composed contract that explains your legal rights and the services they’ll offer Permitting you to cancel within 3 days without charge Fulfilling the guaranteed service prior to charging you or receiving fees If a credit repair business refuses to address your questions, demands payment in advance or fails to put promises in writing, “that’s part of the ‘smell test’ that tells you there’s something incorrect there,” McClary says.
We recommend considering credit counseling first. Ultimately, you might have the ability to rebuild your credit yourself. Dealing with your credit?.
Choosing the finest credit repair business to assist you enhance your credit history is a big choice. A common credit repair service will cost you a few hundred dollars and will take up to a year to complete their service. Picking the wrong credit repair business can wind up wasting cash and perhaps more significantly, your time.
Improving your credit rating can be the difference between whether you enter a new home, car, or perhaps a job. A higher credit history might likewise help you refinance your existing payments so you wind up paying less every month. A relied on credit repair company can lend their proficiency and help you on your way to achieving these outcomes.
The procedure of fixing your credit can involve a lot more than simply contesting your credit with the credit bureaus. You must ensure to utilize a credit repair work company that can fulfill all of your credit needs. When the majority of people believe of credit repair work they think about a process that has actually ended up being called credit report repair.
This component of credit repair work has actually assisted lots of individuals with incorrect, incomplete, or unverifiable info on their credit reports enhance their credit history. While credit report repair is an excellent start, lots of people find that it is inadequate. Credit report repair when the credit bureaus are unable to confirm that the doubtful items in your credit reports need to be there.

domain: finance


text:
St. John Neumann beat Steel High 51- 49
Saturday, March 08, 2014 11:08 PM
Inappropriate photo? Alert us.
St. John Neumann beat Steel High 51- 49 during the first round of the PIAA State Tournament on March, 8, 2014 at Northern High School in Dillsburg. l' Janique Simmons shoots for two against St. John Neuman in the first period.

domain: sports


text:
Sarah Holstein, MD, PhD, discusses new approaches to induction therapy for patients with multiple myeloma.
Sarah Holstein, MD, PhD
Sarah Holstein, MD, PhD
The benefit of adding monoclonal antibodies to upfront induction therapy in patients with multiple myeloma is currently being looked at in ongoing trials.
The current standard therapy of lenalidomide (Revlimid) plus bortezomib (Velcade) and dexamethasone (RVd) is quite beneficial for newly diagnosed patients, said Sarah Holstein, MD, PhD, with triplets quickly replacing doublets in this setting. However, quadruplet therapy is currently showing promise in clinical trials.
In May 2018, the FDA approved daratumumab (Darzalex) in combination with bortezomib, melphalan, and prednisone (VMP) for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant. This approval was based on findings of the ALCYONE study, which showed that the addition of daratumumab to VMP demonstrated a 50% reduction in the risk of progression or death compared with VMP alone (HR, 0.50; 95% CI, 0.38-0.65;P<.001).1
An interim safety analysis of daratumumab plus RVd was presented at the 2017 ASH Annual Meeting.2These safety results concluded that there were no added safety signals with the addition of daratumumab to the triplet. Investigators noted that the overall safety profile was consistent, and enrollment has begun on the main phase of the randomized study.
Additionally, daratumumab is currently being investigated in combination with carfilzomib (Kyprolis), lenalidomide, and dexamethasone (KRd). The addition of daratumumab to this triplet has shown to be well tolerated in patients with newly diagnosed myeloma, according to updated results of a phase Ib study presented at the 2017 ASH Annual Meeting.3
In an interview withTargeted Oncologyduring the 2018 Pan Pacific Lymphoma Conference, Holstein, an associate professor at the University of Nebraska Medical Center, discussed these new approaches to induction therapy for patients with myeloma.
TARGETED ONCOLOGY:Can you provide an overview of your presentation?
Holstein: I provided a historical overview of where we have come with respect to induction regimens, dating back to some of the old regimens such as vincristine, doxorubicin, and dexamethasone (VAD) and melphalan. I spoke about how we have moved from doublet to triplet therapies, with the current standard of care being combinations of immunomodulatory drugs and proteasome inhibitors.
I then spoke about the newer studies, which are investigating the effects of adding a monoclonal antibody therapy to the triplet. We are now moving into the quadruplet therapy era. I also touched on the role of transplant.
TARGETED ONCOLOGY:What need is aiming to be filled with the introduction of new induction therapies?
Right now, the current standard of care is RVd, which is lenalidomide, bortezomib, and dexamethasone. There are ongoing studies asking the question of whether we can improve upon that by adding the second-generation proteasome inhibitor carfilzomib instead of bortezomib. However, we know that especially for patients with high-risk disease, even though they initially do well, they have a much higher rate of relapse and live a shorter time progression free. The question is going to be, “Can we do something that will significantly change the outcomes for high-risk patients?”
TARGETED ONCOLOGY:What are some triplets or quadruplets that are looking most promising?
There is a lot of excitement about KRd, which is a triplet of carfilzomib, lenalidomide, and dexamethasone. There is also some interest with lenalidomide, ixazomib (Ninlaro), and dexamethasone in the older patient population as it is an all-oral regimen, which might be well tolerated. We are eagerly awaiting the results of a phase III study that has finished accruing, which compared lenalidomide plus dexamethasone to the triplet of lenalidomide, ixazomib, and dexamethasone to see whether that improved survival outcomes.
With respect to the quadruplets, the most interest so far is with adding antibodies such as daratumumab to the backbone of a proteasome inhibitor plus an immunomodulatory agent. This includes daratumumab plus RVd and daratumumab plus KRd. Initial studies are showing very high response rates, but we are going to need the larger studies to see if we are making significant changes in overall survival.
TARGETED ONCOLOGY:In what instance would you use a triplet of quadruplet over a doublet?
At this point, I am not using quadruplets outside the context of a clinical trials, because I don't think we are quite there yet. For the majority of my patients, I really try to use a triplet. It is really the patients who are quite elderly or frail who I will sometimes start on a doublet and see how well they can tolerate it. A lot of times, in that case, if I can get them past the first couple of cycles successfully, I will then try to add in that third drug.
TARGETED ONCOLOGY:What would be your takeaway message about induction therapy for patients with myeloma?
The current standard of care for all patients at this point in time should be RVd, that there is still a role for upfront transplant for the majority of transplant-eligible patients, and that there is going to be some exciting changes in what the standard of care will be over the next couple of years. The future likely will be quadruplet therapy.
TARGETED ONCOLOGY:Is there any else you would like to highlight?
I didn't have time to work it into the talk, but it is still really important to use maintenance therapy posttransplant for patients. There, the current standard of care is lenalidomide, but we are waiting on some exciting data about the use of ixazomib as maintenance therapy. There are also ongoing studies looking at regimens such as lenalidomide plus daratumumab. Maintenance therapy is also going to be changing over the next couple of years.

domain: medical


New Delhi: Hundreds of disabled people Friday continued their protest at Jantar Mantar here as the Parliament session ended without passing the Disability Rights Bill.
This was despite assurances by both the government and the opposition to pass the measure in this session.
The group has been protesting over a month while demanding passage of the bill. The Bill would now lapse as the last parliamentary session of the United Progressive Alliance (UPA)-II came to an end.
Currently, there are more than 70 million disabled in the country.
"This is an extremely sad day for us (disabled in the country) and it is very unfortunate that the political leaders despite making promises and assurances did not make efforts to pass the bill in parliament", Javed Abidi, convenor, Disability Rights Group, told IANS.
He said despite meeting leaders from the government and the opposition, and getting assurances, it was politicised.
"We saw the extremely political Telangana Bill get passed. Then why did the parliamentarians not do the same with the Disability Rights bill, which is non-political and non-contentious" Abidi said.
"A lot of hard work was involved in it," he added.
"I do not understand why politicians could not gather the political will to pass the bill. They should understand that because of them, the most marginalised section will have to pay the price."


In a lopsided 161-5 vote, the UN General Assembly on Tuesday approved an anti-Israel resolution backed by Arab nations. The resolution calls on Israel to not only subject its nuclear facilities to international oversight, but also to renounce possession of nuclear weapons.
According to Associated Press, the resolution pointed out that Israel is the only country in its region to have not signed the Treaty on the Non-Proliferation of Nuclear Weapons. The resolution demands that Israel “accede to that treaty without further delay, not to develop, produce, test or otherwise acquire nuclear weapons, to renounce possession of nuclear weapons” and put its nuclear facilities under the inspection regime of the U.N.’s International Atomic Energy Agency.
The resolution was introduced by Egypt. The only countries voting against the measure were Israel, the United States, Canada, Palau, and Micronesia. Eighteen countries abstained.
As the resolution comes from the General Assembly, it is not legally binding. However, it does carry weight due to the General Assembly representing all 193 UN countries.
Israel is widely considered to possess nuclear weapons but has never confirmed whether or not that is the case. Israel instead maintains strategic ambiguity, saying only than that it will not be the first country to introduce nuclear weapons to the region. Israel also argues that Iranian efforts at developing nuclear weapons are the real threat to the region. Iran maintains that its nuclear program is for purely peaceful purposes.
The resolution, titled “The risk of nuclear proliferation in the Middle East,” promoted a Middle East nuclear-free zone.
U.S. representative Robert Wood criticized the resolution, saying that the measure “fails to meet the fundamental tests of fairness and balance. It confines itself to expressions of concern about the activities of a single country.” He said that such measures only serve to undermine progress on eliminating nuclear weapons from the region.


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Clonque beach is a stoney/gravelly beach ideal for rock pooling.
Small car park around coastal track behind Fort Tourgis.
Dog Friendly Beach?
We have no current information on dog restrictions for Clonque Beach.
Visit Clonque Beach
Clonque Beach Photos
7 Day Weather Forecast
Expect a temp in the range 6.6℃ to 7.5℃ with wind speed 7.7 m/sec (NE)
Expect a temp in the range 6.4℃ to 7.3℃ with wind speed 7.5 m/sec (NE)
Expect a temp in the range 7.8℃ to 8.2℃ with wind speed 2.9 m/sec (NNE)
Expect a temp in the range 10.4℃ to 10.7℃ with wind speed 9.4 m/sec (W)
Expect a temp in the range 9.2℃ to 10.3℃ with wind speed 6.8 m/sec (W)
Expect a temp in the range 9.6℃ to 10℃ with wind speed 9.2 m/sec (WNW)
Expect a temp in the range 8.7℃ to 9.4℃ with wind speed 10.1 m/sec (NW)
Saint Anne weather forecast from Yr, delivered by the Norwegian Meteorological Institute and NRK
Where to stay ...
You may also like ...
In this 'you may also like' section we attempt to answer what else can I do? Here you have a list by order of being the closest some more beaches, things to see and do, places to eat and upcoming events.

domain: travel


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LONDON, May 29 (Reuters) – Markets will see if re-elected Turkish President Tayyip Erdogan turns to policy-economic legitimacy or step up policies widely seen as unsustainable In the midst of this, the top person he has appointed as policy maker is attracting attention.
Erdogan has used Sunday’s election victory to launch a major cabinet reshuffle, widely said to have the potential to transform his financial and economic portfolios and central bank leadership as he enters his 30th year in power. expected.
Foreign investors have turned away from Turkish bonds and stocks in recent years after a boom-bust cycle, rampant inflation and a currency crisis that saw the lira drop more than 90% over the past decade. On Monday, the lira fell to new record lows.
Erdogan’s stepped-up intervention to stabilize the lira and his insistence on ultra-low interest rates are increasing pressure on Turkey’s fiscal budget and financial sector. As reserves dwindle, few doubt that change is inevitable. But how deep and meaningful it will be remains unclear.
“In an environment of highly centralized decision-making, markets will only open if a change in the economic team marks a decisive shift in policy-making,” said Emre Akchamak of East Capital, an asset manager that invests in Turkey. will look to change the economic team,” he said. KK.
Days before Sunday’s vote, ruling party lawmakers met to discuss how to adopt the new policy of gradual interest rate hikes and a targeted lending program. surfaced.
“The final decision relating to Finmin’s (finance minister) position is likely to send some strong signals about economic policy in his new term,” Barclays’ Erkan Ergzell said, citing former finance minister Mehmed Simsek as his predecessor. He pointed out that efforts were made to get the minister back. Highly rated by the market.
“If the market sees the appointment of Simsek, or a similarly orthodox figure, near, it will strengthen hopes of an early return to orthodoxy.”
Some fear that the revolving door’s scars on key economic and monetary policy institutions will remain. These personnel changes come as President Erdogan sidelines the technocrats and hollows out the institutional capacity of the central bank and the Ministry of Finance.
Over the past four years, four governors have single-handedly held power at the central bank.
Fund manager and Ninety-One analyst Roger Mark said: “While surrounded by supporters who appear to have been chosen on the basis of loyalty, the danger is that the voices of common sense that still remain within the AKP will become too distant. , the inability to influence Erdogan’s decision-making,” he said.
Despite acknowledging some of the problems facing the country of more than 80 million people, Erdogan offered little indication of the shape of economic policy in the coming weeks and months.
In his victory speech in Ankara, Erdogan said, “The most urgent task in the coming days is to alleviate the problem of inflation-induced price rises and compensate for the loss of prosperity,” adding that inflation will be around 44%. expected. % in April will fall in the same way interest rates have fallen.
Tunkay Tursk, founder of Tunkay Tursk Research and Consulting, said President Erdogan’s speech, which emphasized “internationally respected fiscal management”, raised hopes for a policy change.
“But for this rise to be permanent, we need to appoint a cabinet, clarify the names of those involved in economic management and announce a new economic roadmap, if any,” Tursk said. said Mr.
Pre-election optimism about a reorientation of economic policy has all but vanished as Erdogan’s strong first-round performance roiled polls and set him up for 30 years in office.
Thomas Gillette, Sovereign Ratings Director at Scope Ratings, said partial adjustments to the policy mix were still possible, but would require consistent planning and execution to be effective.
“But Erdogan has shown little sign of such a U-turn,” Zillet said.
Reported by Karin Strohekker and Libby George in London; additional reported by Kanan Sevgiri in Gdansk; edited by William McLean
Our standards: Thomson Reuters Trust Principles.


A bomb sniffing dog found two AGM-114 hellfire missiles on a passenger flight bound to Serbia from Lebanon.
The Lebanese army said that the missiles were being sent to the American company that produced them. The missiles were meant for training purpose and did not contain any explosives. The return was in accordance “with administrative and legal measures after the training ended,” AFP quoted the army as saying Tuesday.
Serbian N1 TV reported that the said package with two guided missiles was discovered on Saturday by a sniffer dog after the flight landed at Belgrade airport.
Serbian media said documents listed the final destination for the AGM-114 Hellfire missiles as Portland, Oregon.
The US State Department has approved a $800 million Foreign Military Sale of Hellfire missiles to Iraq. The Defense Security Cooperation Agency notified Congress of this sale on January 6, 2016, the Pentagon said Thursday
Lockheed Martin has won a support contract worth $318 million to provide Hellfire II missile hardware and component production requirements for the US Army and the Air Force. The contract awarded by the US Department of Defense will also see Lockheed Martin offering support to the foreign customers of Hellfire II missile – Korea, Egypt, Pakistan, Iraq, India, Saudi Arabia, Tunisia, and Indonesia, the DoD statement said
The US Defense Security Cooperation Agency (DSCA) has approved sale of up to 200 Hellfire air-to-ground missiles to France for use in Mali and North African operations. The $30 million sale also includes related military gear such as conversion kits, blast fragmentation sleeves, and installation kits and containers, were also approved
Lockheed Martin Missiles and Fire Control has been awarded a $80,250,000 contract modification for the Hellfire production ramp-up effort. This includes production line set-ups, acquisition of additional tooling/test equipment, and establishment of additional suppliers
The US State Department has approved $34 million sale of additional 70 AGM-114R Hellfire missiles to Netherlands. The Defense Security Cooperation Agency delivered the required certification notifying Congress of this possible sale on 11, July, 2017
The US State Department approved $150 million for the sale of 1,000 AGM-114-R1/R2 Hellfire Semi-Active Laser (SAL) missiles with logistics support services and other related program support to the United Kingdom. The Defense Security Cooperation Agency delivered the required certification notifying Congress of this possible sale today
The US State Department has approved sale of 4000 AGM-114 R/K Hellfire Category III Missiles to the United Arab Emirates for $476 million. The Defense Security Cooperation Agency delivered the required certification notifying Congress of this possible sale on May 11, 2016
France tests nEURON stealth combat drone with Rafale Jet, AWACS Aircraft
Indian Diplomacy Triumphs over US's Anti-Russia Sanctions Threat
GA-ASI tests Air-To-Space Lasercom System for Military Drones
First Radar Antenna for US Army’s LTAMDS to defeat Hypersonic Threats Built...
Mobile Phone Led Israelis to Destory Pantsir-S Air Defence Battery in Syria
China’s Z-10 Attack Helicopter Upgraded with Powerful Engine
Russia Developing New Attack Helicopter ‘Airborne Combat Vehicle’
Leonardo Delivers First 2 C-27J Transport to Kenya Air Force
Several joint production and direct procurement programs could be halted if the US and Europe carry through with their threat...
Sanctions-hit Iran has found ingenious ways to develop military hardware
Artificial Intelligence (AI) is rapidly permeating the defence industry to aid and improve human decision-making
Upgrade of Russias Sukhoi Su-30SM fighters to equip them with armaments, radar, sensors and engines from the more powerful Su-35...
US companies sanctioned by China for supplying weapons to Taiwan may be denied rare earth elements (REEs), which have critical...
While the US F-35 stealth aircraft has become one the fastest selling fighter jets in the world aircraft market, thanks...


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Practices’ viability is threatened by Capita’s failings, warns BMABMJ 2017; 359 doi: https://doi.org/10.1136/bmj.j5123 (Published 03 November 2017) Cite this as: BMJ 2017;359:j5123
- Abi Rimmer
- The BMJ
The viability of general practices is threatened by failings by the private company Capita, which provides primary care support services for NHS England, the BMA has warned.
In a letter sent on 30 October to Simon Stevens, chief executive of NHS England, the BMA highlighted what it called Primary Care Services England’s “ongoing issues due to poor delivery” by Capita. However, Capita said that the BMA’s letter did not accurately reflect its role and involvement in providing the services.
Richard Vautrey, chair of the BMA’s general practitioners committee, wrote that significant issues were affecting the practice support system, which had been “causing much distress to practices and putting patients at risk” for over two years.
In the letter, Vautrey said that the BMA was very concerned about preparations for Capita to run new systems for cervical screening and for …


Now showing items 1-4 of 4
Enabling distributed radar data retrieval and processing in distributed collaborative adaptive sensing environments
This thesis describes the integration of radar network and grid computing technologies to provide a set of grid services to manipulate and store data from different radars, to execute algorithms on different platforms in ...
A grid portal based tool for the deployment and management of grid infrastructures
The deployment of grid infrastructures is a time consuming and arduous task, therefore, the goal of this work was to establish an easy, “out of the box”, grid por- tal environment that allows administrators to go through ...
A grid portal for environmental monitoring applications
Grid portals hide cyber-infrastructure complexity via easy-to-use interfaces, creating gateways to computing resources and data. This project report describes the development of a grid portal for a image processing ...
Utilizing high-performance computing to improve performance and investigate sensitivity of an inversion model for hyperspectral remote sensing of shallow coral ecosystems
This research presents a sensitivity analysis of a semi-analytical inversion model for hyperspectral remote sensing of shallow coral ecosystems. Using this inversion model, five parameters describing water column bioptical ...


Citation: Patent number: 5,130,563 Issue date: July 14, 1992
Abstract: A neural network for processing sensory information. The network comprise one or more layers including interconnecting cells having individual states. Each cell is connected to one or more neighboring cells. Sensory signals and signals from interconnected neighboring cells control a current or a conductance within a cell to influence the cell s state. In some embodiments, the current or conductance of a cell can be controlled by a signal arising externally of the layer. Each cell can comprise an electrical circuit which receives an input signal and causes a current corresponding to the signal to pass through a variable conductance. The conductance is a function of the states of the one or more interconnecting neighboring cells. Proper interconnection of the cells on a layer can produce a neural network which is sensitive to predetermined patterns or the passage of such patterns across a sensor array whose signals are input into the network. The layers in the network can be made sensitive to distinct sensory parameters, so that networks which are sensitive to different wavelengths or polarizations of light energy can be produced.


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This event is LIVE at Barbes
, from 9pm not midnight. No advance tickets necessary, first come first served, get there early.
Streaming free online. Click "Get Tickets" for all options.
sings and writes like a classic, and the timeless quality of her music is maybe what makes her sound as relevant and "modern" today as she did 20 years ago. She is always authentic, knows how to convey emotion of the deepest order, and we were lucky to host her in our backroom somewhat regularly when we first opened, 19 years ago. We are totally thrilled to have her back..There are no advance tickets for this show, and no streaming. Admission is first come first served.

domain: artistic