The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
23 Related JoVE Articles!
Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation
Institutions: Agency for Science, Technology and Research, Singapore, A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, National University of Singapore, Singapore.
Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12
. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13
. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6
. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.
Genetics, Issue 62, ChIP, ChIA-PET, Chromatin Interactions, Genomics, Next-Generation Sequencing
Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells
Institutions: Erasmus MC - University Medical Center.
Fluorescent in situ
hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist
RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.
Biochemistry, Issue 88, Fluorescent in situ hybridization (FISH), combined DNA-RNA FISH, ES cell, cytogenetics, single cell analysis, X chromosome inactivation (XCI), Xist, Bacterial artificial chromosome (BAC), DNA-probe, Rnf12
Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
Institutions: University of Akron.
Recombinant protein engineering has utilized Escherichia coli (E. coli)
expression systems for nearly 4 decades, and today E. coli
is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing a T7 lac
inducible vector and E. coli
expression hosts, starting from transformation to scale-up and purification.
Bioengineering, Issue 83, protein engineering, recombinant protein production, AviTag, BirA, biotinylation, pET vector system, E. coli, inclusion bodies, Ni-NTA, size exclusion chromatography
Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides
Institutions: Georgia Institute of Technology.
Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo
, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae
/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.
Cellular Biology, Issue 45, RNA-containing oligonucleotides, ribonucleotides, RNA/DNA hybrids, yeast, transformation, gene targeting, genome instability, DNA repair
Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA
Institutions: University of Toledo Health Science Campus.
Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova & Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al.
Here we demonstrate a freely available Internet resource -- the Genomic MRI
program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al.
2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.
Genetics, Issue 51, bioinformatics, computational biology, genomics, non-randomness, signals, gene regulation, DNA conformation
Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun
Institutions: Harvard University, Harvard Medical School, Delft University of Technology.
In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5’ mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11
, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana
epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.
Environmental Sciences, Issue 86, Plant Leaves, Synthetic Biology, Plants, Genetically Modified, DNA, Plant, RNA, Gene Targeting, Plant Physiological Processes, Genes, Gene gun, Gibson assembly, Nicotiana benthamiana, Alternative splicing, confocal microscopy, chloroplast, peroxisome
Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology
Institutions: California Institute of Technology, California Institute of Technology, Massachusetts Institute of Technology, University of Minnesota.
Ideal cell-free expression systems can theoretically emulate an in vivo
cellular environment in a controlled in vitro
This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3
To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo
cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli
based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5
The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli
, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6
Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7
The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8
Accompanying mathematical models are available.9,10
The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."
Cellular Biology, Issue 79, Bioengineering, Synthetic Biology, Chemistry Techniques, Synthetic, Molecular Biology, control theory, TX-TL, cell-free expression, in vitro, transcription-translation, cell-free protein synthesis, synthetic biology, systems biology, Escherichia coli cell extract, biological circuits, biomolecular breadboard
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Magnetic Tweezers for the Measurement of Twist and Torque
Institutions: Delft University of Technology.
Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.
Bioengineering, Issue 87, magnetic tweezers, magnetic torque tweezers, freely-orbiting magnetic tweezers, twist, torque, DNA, single-molecule techniques
Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
Institutions: University of California, Los Angeles .
In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek's microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus
, consequently the name Taq
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to:
● Set up reactions and thermal cycling conditions for a conventional PCR experiment
● Understand the function of various reaction components and their overall effect on a PCR experiment
● Design and optimize a PCR experiment for any DNA template
● Troubleshoot failed PCR experiments
Basic Protocols, Issue 63, PCR, optimization, primer design, melting temperature, Tm, troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics
Designing a Bio-responsive Robot from DNA Origami
Institutions: Bar-Ilan University.
Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1
, they can be utilized in parallel computing2,3
, recognize and bind molecular or cellular targets4,5
, catalyze chemical reactions6,7
, and generate calculated responses in a biological system8,9
. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12
, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13
, and several variations on this theme have since been realized using various techniques11,12,14,15
. However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16
. In this technique, the folding of a long (>7,000 bases) single-stranded DNA 'scaffold'
is directed to a desired shape by hundreds of short complementary strands termed 'staples'
. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18
The current paper will focus on the caDNAno 2.0 software19
developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20
. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21
to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.
Bioengineering, Issue 77, Genetics, Biomedical Engineering, Molecular Biology, Medicine, Genomics, Nanotechnology, Nanomedicine, DNA origami, nanorobot, caDNAno, DNA, DNA Origami, nucleic acids, DNA structures, CAD, sequencing
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
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
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii
has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii
by deleting the gene encoding the KU80 protein1,2
. The Δku80
strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro
and in vivo
and exhibit essentially a 100% frequency of homologous recombination. The Δku80
strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4
Here, we report methods for using type I and type II Δku80Δhxgprt
strains to advance gene targeting approaches in T. gondii
. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT
) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80
strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii
and related significant human pathogens that cause malaria (Plasmodium
sp.) and cryptosporidiosis (Cryptosporidium
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
Studying DNA Looping by Single-Molecule FRET
Institutions: Georgia Institute of Technology.
Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.
Molecular Biology, Issue 88, DNA looping, J factor, Single molecule, FRET, Gel mobility shift, DNA curvature, Worm-like chain
Laser Capture Microdissection of Mammalian Tissue
Institutions: University of California, Irvine (UCI).
Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.
Issue 8, Basic Protocols, Laser Capture Microdissection, Microdissection Techniques, Leica
Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay
Institutions: Thermo Scientific Solaris qPCR Products.
The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.
Cellular Biology, Issue 40, qPCR, probe, real-time PCR, molecular biology, Solaris, primer, gene expression assays
Electroporation of Mycobacteria
Institutions: Barts and the London School of Medicine and Dentistry, Barts and the London School of Medicine and Dentistry.
High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.
Microbiology, Issue 15, Springer Protocols, Mycobacteria, Electroporation, Bacterial Transformation, Transformation Efficiency, Bacteria, Tuberculosis, M. Smegmatis, Springer Protocols
Isolation of Genomic DNA from Mouse Tails
Institutions: University of California, Irvine (UCI).
Basic Protocols, Issue 6, genomic, DNA, genotyping, mouse
Transformation of Plasmid DNA into E. coli Using the Heat Shock Method
Institutions: University of California, Irvine (UCI).
Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42°C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37°C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.
Issue 6, Basic Protocols, DNA, transformation, plasmid, cloning
Molecular Evolution of the Tre Recombinase
Institutions: Max Plank Institute for Molecular Cell Biology and Genetics, Dresden.
Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells.
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.
Cell Biology, Issue 15, HIV-1, Tre recombinase, Site-specific recombination, molecular evolution
Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit
Institutions: University of California, Irvine (UCI).
Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.
Issue 6, Basic Protocols, plasmid, DNA, purification, Qiagen
Principles of Site-Specific Recombinase (SSR) Technology
Institutions: Max Plank Institute for Molecular Cell Biology and Genetics, Dresden.
Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.
Cellular Biology, Issue 15, Molecular Biology, Site-Specific Recombinase, Cre recombinase, Cre/lox system, transgenic animals, transgenic technology


The Minister of Education, Mallam Adamu Adamu disclosed in Abuja yesterday that President Muhammadu Buhari will establish six universities of science and technology across the six geo-political zones of the country.
Adamu said at a forum that the future of the world depended on science and technology.
“This government has already decided to even add more; it is going to establish six new universities of science and technology,” he said.
“And, at the lower level, there will be a technical school in each state of the Federation and then vocational centres at the wards and local government levels.
“You know, we have a problem of access; if you look at it last year, one million people sat for the universities but, only 150,000 were admitted.
“So, we still need to build more universities to create more space for those who want to get in.
“And, then, the fact that the six new universities are being created for science and technology underscores the decision by the government to put more emphasis on science.
“Whether we agree or not, oil has limited time before it finishes; the whole economies of the world are going towards the knowledge economy.
“Actually, it is the cultivation of science, technology and engineering studies that will hold the economy in the future.”
He said over the years, government had laid emphasis on science and technology by giving more access to students in such fields.


Logo Design Package
from R3 499
Specifications: One full colour vector Logo design
Concepts: 3 initial concepts presented, one to be finalised
Final Artwork: Supplied as low resolution (web) and high resolution (print) formats
BASIC BRAND STYLE GUIDE
A basic Brand Style Guide detailing the various logo formats included and their applications.


I have a 1997 freightliner fld 120 with a detroit 60 ...
I have a 1997 freightliner fld 120 with a detroit 60 series and I have a problem with the AC. I have no power outside at the compressor . I also need to know which wires go to the ac switch on the dash. Is there a fuse or circuit breaker on a firewall?Freightliner Fld 120 Fuse Panel Diagram Ebook
Freightliner Fld 120 Fuse Panel Diagram Ebook Summary Books : Freightliner Fld 120 Fuse Panel Diagram Ebook [EPUB] Freightliner Fld 120 Fuse Panel Diagram Ebook contains important information and a detailed[PDF Download] Freightliner Fld 120 Fuse Panel Diagram ...
Freightliner Fld 120 Fuse Panel Diagram Free Download Chapter 1 : Free Download Freightliner Fld 120 Fuse Panel Diagram Freightliner Fld 120 Fuse PanelFreightliner Fld 120 Fuse Panel Diagram Simple Wiring For ...
Freightliner Fld 120 Fuse Panel Diagram Simple Wiring For Fld120 Diagrams is one from many image from this website. you can find the latest images of Freightliner Fld 120 Fuse Panel Diagram Simple Wiring For Fld120 Diagrams on this website and other images. we hope this picture can be useful for you.Freightliner Fld 120 Fuse Panel Diagram. Wiring. Wiring ...
Freightliner fld 120 fuse panel diagram moreover 99 freightliner fuse panel diagram 2009 freightliner fuse panel diagram 2002 freightliner fuse panel 2005 freightliner fuse panel location freightliner fl70 fuse box diagram freightliner relay diagram freightliner columbia fuse box diagram freightliner fl70 fuse panel diagram freightliner ...1999 Freightliner FLD 120 JustAnswer
On this 1999 freightliner fld 120, 60 series. I have a light problem. When the head lights are on the marker and tails are not only on tractor. The trailer is fine. Already changed the light switch. IFreightliner Fld120 Wiring Diagrams WordPress
92 Freightliner Fld120 Fuse Panel Diagram.doc. MSWord Document We diagrams,1999 freightliner wiring diagram,free freightliner wiring. N 14C, Manual 10.Freightliner Fld 120 Dash
1994 freightliner fld 120 dash. ... Up For Sale Freightliner FLD 120 1998 DETROIT S60 Great Shape Walk Around Review ELD EXEMPT TRUCK SE Duration: 3:24.Fuse box location | TruckersReport Trucking Forum | #1 ...
I finally learned from a Freightliner mechanic that the 98 Frieghtliner is one of few models with a buss bar circuit panel. I was looking for a fuse box to troubleshoot my non working cruis control. Now I get to dismantle the dash and pull my cruise switches and replace them instead of replacing a small fuse.How to Check Fuses Freightliner
Do you know how to check the fuses in your Freightliner Cascadia? Check out this video to see how to check and replace fuses in your truck! Tyler Patrick fro...
freightliner fld 120 fuse panel diagram Gallery
2001 freightliner classic wiring diagram
kenworth t800 fuse box location gmc c5500 fuse box wiring
1997 freightliner fl80 fuse box diagram
1995 freightliner fld fuse panel location freightliner
freightliner mt45 wiring schematics freightliner columbia
1999 freightliner fld120 fuse box 1999 freightliner fld112
1996 freightliner fld120 wiring diagram 39 wiring
freightliner fld120 wiring diagrams freightliner m2 wiring
kenworth t800 ke wiring diagram kenworth fuse panel
2007 freightliner m2 fuse box location 2007 get free
1996 freightliner fl60 fuse box 1996 freightliner classic
i bought a 1999 freightliner century off ebay vin
1999 freightliner fl60 wiring diagram
freightliner m2 blower motor wiring diagram 43 wiring
2000 freightliner fld120 wiring diagram 2000 freightliner


Physician Key Opinion Leaders (KOLs) Embrace Virtual Meetings with Field Medical Professionals During COVID-19 Pandemic
Alucio’s Beacon product enhances virtual scientific exchange with a fully-integrated video conferencing module
San Francisco, CA (August 12, 2020) - As expected, the growth in usage of video conferencing technology during the COVID-19 pandemic has been dramatic. For example, a popular video conferencing service recently reported a 30x increase in usage of its platform in April 2020 as compared to the prior month.¹
Physician Key Opinion Leaders (KOLs) are also embracing the technology as a way to stay engaged in scientific exchange with life science companies. In fact, a recent study published by The Medical Science Liaisons Society and H1 found that 77% of KOLs were somewhat to very receptive to engaging with Medical Science Liaisons (MSLs) virtually.² Moreover, the same study also found that 66% were interested in continuing to meet virtually with MSLs beyond the COVID-19 pandemic.³
Alucio™, a fast-growing provider of cloud-based software for the life sciences industry, is working to help MSLs stay connected with KOLs and other healthcare professionals during these challenging times. Alucio’s flagship product, Beacon, features a fully-integrated and secure video conferencing module that enables users to take control of their virtual meetings and make them much more effective.
The product streamlines interactions by enabling presenters to quickly find and organize the content they need both before and during a virtual engagement to respond to questions without leaving the application. Additionally, the solution is very user friendly for participants since it does not require them to download any software.
“Beacon provides everything needed to prepare for and conduct KOL virtual meetings effectively and efficiently, with a clean, logical, and well-organized layout,” said Nika Sajed, PharmD, a principal medical scientist and Alucio advisory board member.
In addition to its virtual meeting capability, Beacon includes a host of other well thought out features for MSLs and other field medical professionals. These include the rapid publishing and synching of medical and scientific content via the cloud to various devices, and flexible and smart file management, with powerful search and filtering capability. Built-in version control also ensures that users access the correct documents and remain compliant from a regulatory standpoint.
Jessica Wong, Alucio’s VP - Product Management & Strategy, commented “MSLs clearly tell us that they want to be able to conduct better virtual meetings with their KOLs. Beacon enables them to make their virtual engagements much more seamless and effective. We’re pleased to be able to support these users now and into the future.”
To learn more about Beacon and to schedule a product demonstration, please contact the company at firstname.lastname@example.org.
Alucio is dedicated to the development of innovative software applications specifically for the life sciences industry. Alucio’s flagship product, Beacon, is a cloud-based content management and presentation platform that enhances scientific exchange between life science companies and healthcare professionals. For more information, please visit the company’s website http://www.alucio.io or contact: Lisa Amin at email@example.com (925) 788-3533.
- “Zoom Sees Sales Boom Amid Pandemic”, Natalie Sherman, BBC News, June 2, 2020
2,3. “How KOLs Prefer to Engage with MSLs During the COVID-19 Pandemic”, Medical Science Liaison Society and H1, June 30, 2020


Polyphonic Packets is the future of remote production, streamlining collaboration, and enhancing efficiency with cutting-edge technology intergraded into seamless connectivity.
Streamlining workflows and enhancing remote collaboration with cutting-edge technology, ensuring top-notch performance across various industries.
Delivering robust, reliable, and innovative tools that meet the highest standards of performance.
Creating cutting-edge solutions that empower remote production and streamline collaboration with precision and efficiency.
Private cloud solutions harness the internet’s power to seamlessly interconnect live events, creating a cohesive crew that delivers top-notch quality for both live and streaming audiences, ensuring a memorable show.


30 HR METAL MINIATURE|
is an iron clock with a 30 hour time only balance wheel movement. The theme of
this clock is Columbus.
Close up view of the top of the clock.
Middle of the clock with Columbus and the dial.
Bottom of the clock, lots of detail and 1492.
Back of the clock.Cost is:$175.00


Parker Hannifin Corp. is sponsoring a contest that encourages college engineering students to develop a hydraulically powered bicycle. The fluid power giant is working with five-student teams at Cleveland State University, the University of Akron, University of Cincinnati, Purdue University, California Polytechnic University, the University of California-Irvine, Western Michigan University and Murray State University. Winning entries may earn $2,000 grants for each team member. To learn more, go to www.parker.com or e-mail firstname.lastname@example.org.
The company says it anticipates high-definition video for home security and other uses will be the next mature technology integrated into the IoT domain, hence the introduction of its MatrixCam devkit.
Siemens and Georgia Institute of Technology are partnering to address limitations in the current additive manufacturing design-to-production chain in an applied research project as part of the federally backed America Makes program.
Most of the new 3D printers and 3D printing technologies in this crop are breaking some boundaries, whether it's build volume-per-dollar ratios, multimaterials printing techniques, or new materials types.
Focus on Fundamentals consists of 45-minute on-line classes that cover a host of technologies. You learn without leaving the comfort of your desk. All classes are taught by subject-matter experts and all are archived. So if you can't attend live, attend at your convenience.


nc description:Here you can download nc with version 1.1.
This software was developed by Florian Delizy.
Distribute by license GPL and price FREE.
Download time for this software with internet channel 512Kb/sec would be 0 seconds.
You can download this software from www.vim.org domain.
Related software for 1.1 ncVocabulary Highlighter
HPS WinTail Build 782
Alternate Text Browser
Portable Presentation Pointer
EControl Syntax Editor


Post-traumatic optic chiasma and optic track oedema
c/o blurring of vision after trauma
Loading Stack -
0 images remaining
There is altered signals in the optic chiasma and left optic radiation. There is also altered signal in the posterior high parietal subcortical region.
There was definite history of injury followed by sudden development of diplopia and blurring of vision.
No obvious history of fever.
Other differentials would be demylination.


Dubna – PhD programs in Computer Science
We found 1 university offering 1 PhD program in Computer Science in Dubna.
Study the PhD programs in Computer Science in Dubna
Years of study
Where can the PhD в Computer Engineering find a position?
PhD in Computer Engineering in the private or public sector hold positions such as computer application engineer, computer build engineer, computer vision engineer, hardware engineer, as well holding occupations in military services or defense establishments and space establishments.
Why should you obtain a PhD degree in Computer Engineering?
Experts of Free-Apply.com Education Company recommend holding a Doctor's of Philosophy in Computer Engineering, as it gives a wide scope for choosing a career. The average computer engineering salary is one of the highest and gives financial stability to employment.
Russia, Dubna – PhD programs in Computer Science statistics
Free-Apply.com provides information about 1 PhD program in Computer Science at 1 university in Dubna, Russia. Furthermore, you can choose one of 1 Bachelor program in Computer Science at 1 university, 1 Master program in Computer Science at 1 university, and 1 PhD program in Computer Science at 1 university.
Reasons to study in Russia
No 22 in the world education ranking
No 54 in the world ease of doing business ranking
No 63 in the world economy ranking
No 105 in the world safety ranking
Higher education statistics of the largest cities in Russia
The largest cities offering PhD programs in Computer Science in Russia.
|Nizhniy Novgorod||2||~ $1,512||Search|
|Saint Petersburg||2||~ $1,620||Search|
Russia – Average monthly personal finance statistics
100% discount for the 1st year
Apply now and get a 100% tuition fee discount for the first year of studies


- 姓 名：
- 职 称：
Haifeng Zhang is a visiting scientist at Center on Frontiers of Computing Studies (CFCS), Peking University. His research areas include reinforcement learning, game AI, game theory and computational advertising. Before visiting CFCS, he was a research fellow at University College London working with Prof. Jun Wang. He received his PhD from Peking University in 2018, supervised by Prof. Wenxin Li, and his bachelor's degrees in computer science and economics from Peking University in 2012.


A distinct entorhinal cortex to hippocampal CA1 direct circuit for ... A distinct entorhinal cortex to hippocampal CA1 direct circuit for olfactory associative learning | Nature NeuroscienceAxxess Gmos 100 Wiring Schematic - axxess gmos 04 wiring diagram axxess gmos-04 wiring diagram axxess gmos-04 wire diagram metra gmos-04 wiring diagram gmos-04 installation Axxess Interface Wiring Diagram, Axxess GMOS-04 16Pin Diagram, Axxess GMOs 100 Wiring Schematic, GM Bose Wiring-Diagram, Chevy Factory Radio Wiring Diagram, 2005 Silverado Speaker Wiring Diagram,. 2) Connect the Green wire to the parking brake wire of the aftermarket radio. 3) Connect the Blue/Pink wire to the VSS or speed sense wire of the aftermarket radio. 4) Connect the Green/Purple wire to the reverse wire of the aftermarket radio. When completed, plug the 14-pin harness into the GMOS. Applications GMOS-100 Caution: Metra recommends disconnecting the negative battery terminal before beginning any installation. All accessories, switches, and especially air bag indicator lights must be plugged in before reconnecting the battery or cycling the ignition..
gmos 04 wiring diagram web in axxess gallery sample best of b2network co on gmos04 integrate and deltagenerali me with subwoofer luxury 1 can bus interface harness collection collections new prerequisite for kiosystems amazing images stuning gmrc 01 to arcnx premium sound lan install chevy hhr network 2014 17 161642 amp at canopi roc grp org unusual health shop (gmos04) onstar. Jvc car stereo wiring diagram color on images unfastened down load all through aftermarket with radio metra diagrams 1, particular scosche wiring harness diagram ninety six in lamp socket with metra diagrams four, axxess gmos 04 wiring diagram webtor me inside coachedby sizeable metra interface diagrams five, metra bose integration and skip. Axxess GMOS-100i Installation Instructions Manual Usb updatable,is a replacement interface designed to retain onstar, retain the warning chimes that are normally lost when the oem radio is removed, retain factory installed satellite radio (xia-lcd is required and sold separately), and factory steering w..
Gmos 04 Installation Diagram Of Html. welcome to my blog here I will show you a little more what you are looking for Gmos 04 Installation Diagram Of Html below there are some photos and a little about Gmos 04 Installation Diagram Of Html that you can see and read, hopefully in accordance with what you are looking for.. Axxess GMOS-LAN-034. GMOS-LAN-034. Not Rated Yet 2006-Up GM LAN11 Data Interface Harness $100.67. Free Shipping. Compare. $100.67. Add to Cart Most vehicles are built with their own wiring harness specifically connecting their OEM hardware such as an In-Dash receiver. Replacing vehicle OEM receiver usually requires an additional wiring. Product Features: Wiring Harness for Replacing Select GM OEM Radios Compatible with GM CLASSII bit vehicles Replacement harness for the GMOS-100 or GMOS-100i Home Shop by Brand ».
Wiring schematics for 2004 chevy colorado also 59622 firstgen wiring diagrams in addition 2007 nissan altima fuse box 2010 nissan altima fuse box diagram pertaining to 1998 nissan altima fuse box diagram further 348917 trailer wiring question also c4 corvette brake diagram also dodge dakota 1997 dodge dakota code p0740 also p 0996b43f80cb1d98 as well as p 0900c15280251da6 along with chevrolet.
Why We Will Need Genetically Modified Foods - MIT Technology Review David Lobell, a professor of environmental earth system science at Stanford University, has a calm demeanor that belies his bleak message about how global ...
Car Stereo Wire Harnesses - Radio Wires for all Car Audio - Wiring ... Click for more info about Car Stereo Radio ISO Extension Wiring and Antenna Harness Adaptor for
Amazon.com: Axxess GMOS-LAN-01 LAN OnStar Interface for Non ... Amazon.com: Axxess GMOS-LAN-01 LAN OnStar Interface for Non-Amplified Systems: Car Electronics


19 Results in Interaction Design
M.S.O.E.’s UXCD program will help you develop the skills to design the next digital innovation and solve user interface issues.
A Bachelor of Media Design specialising in Interactive Design prepares you for the technical demands of the web design and mobile development industries.
The Graphic Design and Interactive Media degree provides the practical knowledge and insight you need to develop effective, stunning graphic design and web interfaces. You wil ... +
Our multimedia design program provides an intensive background in design and offers specialized classes in digital media, motion graphics, animation and film. We also offer a ... +


NOLEJ, the generative AI edtech start-up, has announced a strategic partnership with OpenAI, the AI research and deployment business behind GPT-4, GPT-3.5, ChatGPT, DALLE, Codex, and other projects.
In mid-April, at the ASU GSV Cup, where they have been chosen as a finalist in the GSV cup, the largest edtech competition, NOLEJ will introduce the commercial edition of NOLEJ AI, a generative AI for educators, powered by GPT-3.5, following a successful Beta test with over 2500 educators.
Teachers now have 50X more time to create interactive microlearning materials thanks to NOLEJ AI. Teachers can upload static information (video, audio, or text), and NOLEJ AI will then create interactive videos, flashcards, and quizzes ready to use and accommodate different learning preferences. According to hundreds of independent studies, such interactive micro-learnings beat conventional static formats, increasing completion rates by 85% and retention rates by 75%.
By utilizing their expertise in generative learning built on OpenAI's technologies and cutting-edge AI systems, NOLEJ intends to revolutionize AI-powered education further. The alliance plans to produce new technologies to improve learning and give educators new opportunities.
Bodo Hoenen, the co-founder of NOLEJ, said: "We are thrilled to be collaborating with OpenAI to continue our mission of powering the future of learning."
By working with OpenAI strategically, NOLEJ joins organizations like Shutterstock, Duolingo, and Kahn Academy.


Accounting software development
30 profesionistë freelancer dërguan një ofertë mesatare prej $470 për këtë punë
I am experienced Accounting solutions developer. I have developed number of different Account and Trading solutions according to the client requirements.
Hey, Good Day! I am a proficient developer with 5+ yrs of experience working on desktop applications using .net framework. Let`s discuss more on requirements. Looking forward to work with you. Thanks


VUDU Internet application platform
December 17, 2008
VUDU has launched its VUDU RIA (Rich Internet Application) platform, a standards-based platform that brings Web-hosted rich applications and services to consumer appliances such as the popular VUDU Internet movie player. RIA combines the openness and ease of development of Web applications, lightweight hardware requirements compatible with today’s consumer Internet appliances, and a lean-back user experience optimised for television.
VUDU has created an initial set of applications and services in a new area of the VUDU home page, called VUDU Labs. Available to all VUDU users, the applications include casual games, implementations of Flickr, Picasa and the entire YouTube library, as well as a new 'On Demand TV' area with more than 120 channels.


1 results containing "Aspin, Jehoshaphat. [from old catalog]"
Larger image available anywhere
Larger image available only at the Library of Congress
Not Digitized (1)
A familiar treatise on astronomy, explaining the general phenomena of ...
All ways to connect
DonateInspector General |
External Link Disclaimer |


Bespoke Assembly Machines
Bespoke automated assembly lines to safeguard your production
Assembly Automation System
Automated Manufacturing Solutions | Integrated Automation and Robotics
APL has a wealth of experience in designing and manufacturing bespoke automated assembly lines/bespoke assembly machines to safeguard your production.
APL solutions vary from fully automated assembly line machines to manual assembly benches with poke yoke devices for quality assurance, whichever meets your specific needs and requirements.
We build all machines in-house. With nearly 30 years experience in assembly automation, our automated bespoke assembly solution is designed to provide the best method for reducing labour costs and increasing productivity.
For more information on any of our products please get in touch using the form below. One of our sales team will respond to your enquiry as soon as possible.


Wynn Donates $25 Million to Blindness Research
The Las Vegas casino tycoon Steve Wynn has announced that he donated $25 million to support blindness research at the University of Iowa having been convinced that the university’s scientists are leading the way in a search for a cure.Wynn has said that the researchers are “knocking on the door” of a discovery that could cure blindness for millions.
He believes that scientists will be able to use stem cells to restore vision by growing new cells and transplanting them into patients’ eyes.
Wynn said, “This is an exhilarating, quite exciting place. To a scientist, this is like going to a rock concert. I mean there is stuff going on in these rooms here that, to put it in the common vernacular is really far out.”
The money will be used to help the university build a new laboratory to grow stem cells, hire scientists and accelerate studies already underway. Wynn also pointed out that federal money for research has been tight in the last few years and private donors are now needed more than ever before.


Television Station Information
City: Austin, TX
Owner: Tegna Media
Web Site: http://www.kvue.com/
Station Info: Digital Full-Power - 1000 kW
DTV/HDTV Channel: 33
KVUE is a television station in Austin, TX that serves the Austin television market. The station runs programming from the ABC network. KVUE is a digital full-power television station that operates with 1000 kilowatts of power and is owned by Tegna Media.
Detailed Engineering Information / KVUE FCC Applications


Shareaza 2.0 review by PC Magazine PC Magazine is out with a review today on Shareaza 2.0, which claims to provide the search and download speeds of eDonkey but with a far better user interface. It also allows previewing a file as it downloads. Shareaza is an open-source app that is free of charge and reportedly free of spyware.
“In the end, search and download speeds are much like what you’ll find using the eDonkey client, and the array of available files is similar. The key difference is that Shareaza is much easier to use. It gives you much more information about the files and machines on the network, it is better at tracking the status of your many downloads, its general layout is more intuitive, and it is far more stable.”


Logical Methods in Computer Science – LogiCS doctoral program
The Logical Methods in Computer Science – LogiCS doctoral program is a PhD degree program, funded by the Austrian Science Fund FWF and run jointly by the three Austrian universities TU Wien (Vienna University of Technology), Graz University of Technology and Johannes Kepler...
PhD Positions in Cloud, Fog and Edge Computing
vor 2 Tagen
| Bewerbungsschluss am Dez. 31
The Institute of Computer Science at the University of Innsbruck, Austria seeks for excellent PhD students to carry out research in Cloud, Fog and Edge computing.
You are enthusiastic about developing and investigating innovative research ideas and systems...
PhD position - 3D Objects discovery in 3D scene
vor einem Monat
| Bewerbungsschluss am Okt. 31
Computer science and software
Object detection and localization in images is a problem studied since many years. The latest technological developments now allow the real-time acquisition of depth data coupled to color...


Researchers show for the first time that one of the most abundant methane-producing microorganisms on Earth makes direct electrical connections with another species to produce the gas in a completely unexpected way.
Securing affordable energy supplies, changing the energy mix towards a more sustainable and renewable portfolio, as well as providing energy to those who currently have no access ranks high on the global agenda. In order to discuss these issues as part of Swiss Re's 150-year anniversary event series 'connecting generations', Swiss Re organised a small conference entitled 'Meeting the energy challenge'.
The EU-funded IURBAN project, which kicked off in October 2013, aims to develop an intelligent tool that gives consumers greater control of their energy use and energy suppliers greater flexibility in providing the energy needed.
Engineering researchers have developed new technology and techniques for transmitting power wirelessly from a stationary source to a mobile receiver - moving engineers closer to their goal of creating highway stations that can recharge electric vehicles wirelessly as the vehicles drive by.
A new research project will contribute to the everyday practicality of lithium-ion batteries, thus improving consumer acceptance of electric vehicles. In the course of the project, the researchers will consider the entire development process, from materials to suitable operating strategies.
Researchers studying more effective ways to convert woody plant matter into biofuels have identified fundamental forces that change plant structures during pretreatment processes used in the production of bioenergy.
Nature designed lignin, the tough woody polymer in the walls of plant cells, to bind and protect the cellulose sugars that plants use for energy. For this reason, lignin is a major challenge for those who would extract those same plant sugars and use them to make advanced biofuels. As part of their search for economic ways to overcome the lignin challenge, researchers have characterized the enzymatic activity of a rain forest microbe that breaks down lignin essentially by breathing it.
Thermal radiation from the sun is largely lost on most silicon solar cells. Up-converters transform the infrared radiation into usable light, however. Researchers have now for the first time successfully adapted this effect for use in generating power.
The DTOcean (Optimal Design Tools for Ocean Energy Arrays) initiative will allow a suite of design tools to be developed for the deployment of ocean energy arrays. These tools will be contributing towards open-sea demonstration in ocean energy arrays of the technologies for capturing ocean current energies and wave energies.
The Centre for Carbon Measurement at the National Physical Laboratory (NPL) is partnering with Astrium Services to deliver a new emissions measurement service to enable countries and cities to better quantify their greenhouse gas (GHG) emissions.
The WINDTRUST project kicked off in September 2013 with the objective of improving the competitiveness of the wind energy sector by developing and testing innovative components to further improve turbine reliability.


Type of Document Dissertation Author Hinkelmann, Franziska Babette Author's Email Address email@example.com URN etd-08032011-113739 Title Algebraic theory for discrete models in systems biology Degree PhD Department Mathematics Advisory Committee
Advisor Name Title Laubenbacher, Reinhard C. Committee Chair Herdman, Terry L. Committee Member Jarrah, Abdul Salam Committee Member Tyler, Brett M. Committee Member Keywords
- systems biology
- discrete models
- Mathematical biology
- finite fields
- polynomial dynamical systems
Date of Defense 2011-08-01 Availability unrestricted AbstractThis dissertation develops algebraic theory for discrete models in systems biology. Many discrete model types can be translated into the framework of polynomial dynamical systems (PDS), that is, time- and state-discrete dynamical systems over a finite field where the transition function for each variable is given as a polynomial. This allows for using a range of theoretical and computational tools from computer algebra, which results in a powerful computational engine for model construction, parameter estimation, and analysis methods. Formal definitions and theorems for PDS and the concept of PDS as models of biological systems are introduced in section 1.3.
Constructing a model for given time-course data is a challenging problem. Several methods for reverse-engineering, the process of inferring a model solely based on experimental data, are described briefly in section 1.3. If the underlying dependencies of the model components are known in addition to experimental data, inferring a "good" model amounts to parameter estimation.
Chapter 2 describes a parameter estimation algorithm that infers a special class of polynomials, so called nested canalyzing functions. Models consisting of nested canalyzing functions have been shown to exhibit desirable biological properties, namely robustness and stability. The algorithm is based on the parametrization of nested canalyzing functions. To demonstrate the feasibility of the method, it is applied to the cell-cycle network of budding yeast.
Several discrete model types, such as Boolean networks, logical models, and bounded Petri nets, can be translated into the framework of PDS. Section 3 describes how to translate agent-based models into polynomial dynamical systems.
Chapter 4, 5, and 6 are concerned with analysis of complex models. Section 4 proposes a new method to identify steady states and limit cycles. The method relies on the fact that attractors correspond to the solutions of a system of polynomials over a finite field, a long-studied problem in algebraic geometry which can be efficiently solved by computing Gröbner bases. Section 5 introduces a bit-wise implementation of a Gröbner basis algorithm for Boolean polynomials. This implementation has been incorporated into the core engine of Macaulay2. Chapter 6 discusses bistability for Boolean models formulated as polynomial dynamical systems.
Filename Size Approximate Download Time (Hours:Minutes:Seconds)
28.8 Modem 56K Modem ISDN (64 Kb) ISDN (128 Kb) Higher-speed Access Hinkelmann_FB_D_2011.pdf 2.18 Mb 00:10:06 00:05:12 00:04:33 00:02:16 00:00:11
If you have questions or technical problems, please Contact DLA.


Support your decisions with data.
Sometimes you need to back up your decisions with data. Collecting data is only one part of the job, the other is reading it correctly.
Empowering businesses with advanced data processing solutions to enhance efficiency and drive growth.
We strive to continuously improve our methods and technology to ensure the highest level of quality. Our goal is to be a trusted partner for companies looking for reliable and efficient data processing solutions.


Home | Submit a file | New | Categories | RSS Feeds | Blog
Free Software Advertising
"AthTek WebAPP Kit" snapshot
Fast create rich internet applications (RIA) by PC software projects.
Asoftech Photo Recovery is an easy to use data recovery software available to restore your photos, music and videos from local PC and removable devices. No matter multimedia files have been deleted accidently, or memory card has been formatted.
AutoMe is a reliable and user-friendly windows automation software and macro recorder with task scheduler, color checker. AutoMe is very easy to use, you can record robot, and run it as many time as needed. No programing needed.
9% of users liked this software
Windows 7/Vista/XP/2000, Other
January 31, 2012
Description from the Publisher
AthTek WebAPP Kit is a great programming tool for web software development. It works
together with popular programming languages and enables software developers to create rich
internet applications (RIA) directly by the ordinary software projects. If you are a PC
software developer who wants to add internet versions of your software, AthTek WebAPP Kit
is the spider-man you should never miss.
Users reviews & testimonials
This software is not reviewed yet.
Product tagstiger software builder express, applications, wysiwyg, fast cable internet, software development project, programming, software development, webapp kit, live internet tv app, kit, web page builder, web development tool, athtek netwalk enterprise edition, product development, software development tools, software development kit, internet tv software, web development, builder, bob the builder, athtek code to flowchart, tiger software builder professional, development, internet applications, spider, kit power point, tiger software builder, medical software development, phpdug internet services web hosting, multimedia builder, spider man, rich, spider.com, athtek netwalk edition, tiger software builder express review, web cam software, c++ builder, projects, athtek skype recorder, internet software, software development services
Other downloads from this publisher
AthTek Voice Recorder helps you record and repeat audio from line-in or playlist
AthTek File Master is both file management and file encryption software.
Automatically generate elegant flowchart or NS chart, to let code visual.
AthTek File Master is both an encryption software and file management software.
Video Surveillance Monitor streams video from your camera directly to your local surveillance system or networked computers. Using Video Surveillance ...
[ More downloads from this publisher ]


Nowadays, some business applications are deployed on-premises and some are hosted in the cloud. Such separation makes building automation workflows that combine both on-premises and cloud applications difficult, because usually cloud applications can't access on-premises systems directly.
In this webinar, we demonstrate how cloud applications can securely send data to on-premises systems and databases by triggerring EasyMorph workflows via EasyMorph Gateway. The demonstration will show how to use webhooks and/or Zapier to trigger EasyMorph workflows right from a cloud application. Such integration simplifies marketing automation, IoT data collection, and near real-time operational analytics.
The online webinar will take place on June 23rd at 3pm (GMT+0) via Webex. Registration is required.


Performance-enhancing carbohydrate mouthwash
It has long been established that carbohydrates around training or competition improve performance but researchers from the University of Birmingham, UK wanted to see if the underlying mechanism for this is metabolic or central nervous system mediated. In order to investigate this, the researchers employed simple carbohydrate mouth rinsing, where participants gargled a carbohydrate drink [...]
|All times are GMT -5. The time now is 09:07 PM.|
Powered by vBulletin® Version 3.8.5
Copyright ©2000 - 2017, vBulletin Solutions, Inc.


Creating smarter, healthier transportation systems
Eleni Bardaka guides her students in developing transportation systems that support a community's physical, mental and sustainable well-being.
Plant patch enables continuous monitoring for crop diseases
Researchers have developed a patch plants can “wear” to monitor continuously for plant diseases or other stresses.
Turning yeast cells into labs for studying drivers of gene regulation
The cells are a more efficient platform for studying proteins that play a key role in regulating gene expression.
Biochemical sensor researcher makes MIT Technology Review’s list of top young innovators
Amay Bandodkar, an assistant professor in the Department of Electrical and Computer Engineering, is being recognized for his work on wearable health sensors.
‘Nanodecoy’ therapy binds and neutralizes SARS-CoV-2 virus
Nanodecoys can bind to SARS-CoV-2, helping clear the virus faster.
Combining energy storage and solar offers unexpected power reliability boost
This is encouraging news for renewable energy.
Computer scientist wins UNC System award
Veronica Catete is the first NC State staff member to win the award that recognizes exemplary service to both campus and community.
Three from CSC win NSF CAREER Awards
Alexander Kapravelos, Christopher Parnin and Ruozhou Yu, all assistant professors in the Department of Computer Science, have each received an NSF CAREER Award.
New Board of Governors member tours Fitts-Woolard Hall
NC State faculty and staff members showed off some of the building's state-of-the-art labs to Sonja Nichols, a newly appointed member of the UNC Board of Governors.
New twist on DNA data storage lets users preview stored files
Researchers have turned a longstanding challenge in DNA data storage into a useful tool.


Advanced Technology Development
AeroEdge Research and Development Policy
To keep providing value to the society over several decades, and to achieve discontinuous growth, we are working on the development of elemental technologies as seeds for next-generation businesses.
R&D plan is linked to corporate strategy, technology trends, and a global paradigm shift. The best alliance with other leading edge companies, institutions, academia, governments will be established.
Development of Addtive Manufacturing
In Europe, the research and development of Additive Manufacturing (AM) technology has been accelerated and has already entered the industrialization phase. The aviation industry is the one of leading industry in AM, and AM parts are already being used in commercial aircraft engine parts. In the automotive industry, AM products have already intalled in motor sports and high-end cars, and it is expected that many cars will be equipped with AM products in the near future. AM products are also being used in medical devices such as artificial joints and teeth.
AeroEdge is developing prototypes and consulting services using existing AM technology, and is also working on academic research as our own project. We define aircraft parts as one of the goals, keep understanding the effect of build parameters on mechanical characteristics, acquiring data for development a simulation technology, new design method with AM (DfAM; Design for AM), and utilizing AI in those technology areas.
X-ray image analysis software development
The shortage of skilled workers in non-destructive inspection area of the Japanese aerospace industry is one of the major issue that preventing the its growth. It takes a long time to train highly specialized personnel to carry out the inspection work. In addition, the inspection process forces a long period of concentration and the overuse of the eyes in a dark room environment.
Aiming to solve these problems, we are developing automatic inspection system with digital technology such as AI, which has made remarkable progress in recent years. In parallel, we are taking actions to get certification for the inspection process, which is especially essential in the aircraft industry.
Collaborating with the National Institute for Materials Science (NIMS), AeroEdge has been conducting research and development on new titanium-aluminum alloy and manufacturing technologies for jet engine components.
Titanium aluminum, as a lightweight and heat-resistant alloy, has been applied to low-pressure turbine blades of some commercial jet engines in recent dacades, and further expansion of market is expected to realize a carbon-neutral society. We are working on research to reduce the manufacturing cost of titanium-aluminum and to improve the mechanical properties.


What did Joseph Martin want, receive, and learn?
Joseph Plumb Martin served as a private soldier in the Continental Army for eight years (1776-83), with one hiatus during the winter of 1776-77. Born in 1760, he was raised by his maternal grandparents on their Connecticut farm from age six. He was fourteen at the time of Lexington and Concord. Inspired by the rage militaire directly following the first successful clash of arms with the British invader, Martin decided to join the militia. However, he could not obtain his grandfather's permission until July 1776, when he enrolled as a six-month volunteer in a Connecticut regiment of the Continental Army. He mustered out of the service in December and returned home. In April 1777, Martin again "put my name to enlisting indentures" (41). This final enlistment was for three years, or the duration of the war.


The middle school curriculum is taught by specially trained, uniformed law enforcement professionals called G.R.E.A.T. instructors. It is designed to be taught at the entry level of junior high or middle school. However, it may be taught in the sixth, seventh, or eighth grade. The lessons must be presented consecutively, with no less than one day and no more than two weeks between lessons.
Implementation of the 13-lesson middle school curriculum produces the best results when students first complete the G.R.E.A.T. Elementary School Curriculum. However, if this is not feasible, the middle school curriculum can be taught as a stand-alone program.
One unique feature of this curriculum is the project called “Making My School a G.R.E.A.T. Place.” Its main purpose is to provide students with the opportunity to apply the skills they have learned. Research has shown that school bonding—that is, how connected a child feels to his or her school—is an important protective factor against joining a gang and participating in criminal activities. The goal is to increase the students’ bond to the school and to have a positive impact on how students view their school.
The time frame for the lessons ranges from 30 to 45 minutes, with the following performance objectives:
Welcome to G.R.E.A.T.: A Gang and Violence Prevention Program
Students will identify the relationship among crime, violence, drug abuse, and gangs.
What's the Real Deal?: The Real Deal on Gangs and Violence
Students will analyze information sources and identify realistic, normative beliefs about gangs and violence.
It’s About Us: Being Part of the Community
Students will define their roles and responsibilities in the family, school, and community.
Where Do We Go From Here?: How to Set Goals
Students will write realistic and achievable goals.
Decisions, Decisions, Decisions: Making the Right Choice
Students will practice decision-making skills.
Do You Hear What I Am Saying?: How to Communicate Effectively
Students will practice effective communication skills.
Walk in Someone Else’s Shoes: Thinking of Others
Students will identify active-listening skills, how to recognize the emotional state of others, and how to demonstrate empathy toward victims of crime and violence.
Say It Like You Mean It: Some Ways of Refusing
Students will practice effective refusal skills.
Getting Along Without Going Along: Dealing With Peers
Students will continue to practice effective refusal skills.
Keeping Your Cool: Managing Your Anger
Students will practice anger-management skills.
Keeping It Together: How to Calm Others
Students will identify how anger-management skills help prevent violence and conflicts.
Working It Out: How to Solve Conflicts
Students will practice conflict-resolution techniques.
G.R.E.A.T. Days Ahead: Applying Your G.R.E.A.T. Skills
Students will explain how their G.R.E.A.T. project helped them develop a feeling of commitment and ownership of their school and their community.


Work Medicis vase
Department of Decorative Arts: 19th century
© 2011 Musée du Louvre / Philippe Fuzeau
At the Manufacture de Sèvres exhibition in 1824, Louis XVIII gave his brother the Comte d'Artois (the future Charles X) a monumental Medicis vase with a painted cameo-style decoration by the painter Louis-Bertin Parant. A second example was made in 1826. The two vases decorated the Etruscan Vases Room at the Louvre.
A gift chosen at a porcelain exhibition
Louis XVIII revived a tradition of the Ancien Régime-that of an annual exhibition of porcelain from the Manufacture Royale de Sèvres, enabling the king to choose New Year presents for his family. Louis XVIII thus selected two vases for his brother the Comte d'Artois at the exhibition in January 1818. At the 1824 exhibition, the last present that Louis XVIII presented to his brother was a monumental Medicis vase with a subject drawn from antiquity: Augustus closing the gates of the Temple of Janus. Another vase was made in 1826, this time illustrating a contemporary subject, the entry of Charles X after his coronation.
The fashion for cameo-style painting
The band of decoration on the body of the vase is the work of Louis-Bertin Parant, a specialist in painting imitation antique cameos. The band is painted as a continuous scene, with the figures assembled around the emperor, seen in profile, sitting in a Roman chariot drawn by two horses; other figures surround two standing women (possibly Religion and Faith) crowning a potted lily on an altar. Cameo-style painting was very popular during the Empire, as well as during the Restoration. Parant made several works using the technique, such as Le Déjeuner à l'Apothéose d'Henri IV, kept at the Château in Pau.
In fashion since the Empire, the so-called "Medicis" style of vase was inspired by classical models. The choice of cameo-style painting contributed to this theme from antiquity and made it possible to imitate valuable works in a cheaper material. This work influenced by antiquity is completed by bronze ornaments: the lip is highlighted by a palmette frieze, the rim of the foot is marked by a frieze of laurel leaves and palmettes, and the handles have a fluted decoration. This type of Medicis vase continued to be manufactured at Sèvres; Charles X gave similar models to the composer François Adrien Boieldieu in 1825 and another was delivered to the Galerie de Diane at the Château de Saint-Cloud in 1838.
Manufacture de Sèvres
H. 1.28 m; W. 0.89 m.
Peinture de Louis-Bertin Parant : Entrée de Charles X après son sacre
MR XIV 46
The Louvre is open every day (except Tuesday) from 9 a.m. to 6 p.m.


The company concerned, Canadian Natural Resources, which is one of the largest independent crude oil and natural gas producers in the world, undertakes what is known as cyclical steam stimulation (CSS) to extract the tar sands. This is one of the fastest growing methods of extracting the highly polluting and controversial tar sands. But now it seems it has a fundamental flaw.
The process involves injecting thousands of gallons of boiling hot, high-pressure steam into deep underground reservoirs. This in turn liquefies the bitumen and creates cracks through which the bitumen flows. It is then pumped to the surface.
But now it seems the steam can also crack the so-called “cap rock” above the bitumen deposits, leaving a path open for the bitumen to reach the surface in an uncontrolled fashion.
A year ago, last July, the Canadian press reported how Canadian Natural Resource’s CSS operations had resulted in four separate oil spills which had been leaking for six weeks near Cold Lake in Alberta. One scientist said at the time that everybody in the company and government “is freaking out about this. We don’t understand what happened. Nobody really understands how to stop it from leaking, or if they do they haven’t put the measures into place.”
A year on, according to press reports, the spills still show no sign of stopping.
Ever since the spills started, Canadian Natural Resources stuck with its story that if anything was to blame, then it was long-disused faulty boreholes from previous mining operations, which were bringing the bitumen to the surface.
But finally it has admitted that the spills are being caused, or at least exacerbated, by its own high-pressure CSS operations.
In a detailed report which has just been released, the company has conceded that its high-pressure steaming practices could have fractured the cap rock and be the cause of the spills.
Worrying though, having admitted it is partly to blame, the company says it has no plans to change its operating practices.
The Pembina Institute points out that the admission that the shale “cap rock” could have been breached by CSS is really important, as the project only received regulatory approval on the basis it would not breach the cap rock.
This has massive implications for other CSS operations. Because if the cap rock cannot withstand pressures caused by the steaming, “then the entire CSS project concept needs to be re-evaluated”, argues Chris Severson-Baker, the Managing Director of the Institute. He adds that the discovery also calls into question the legitimacy of other projects for CSS which were given permits by the Canadian regulator.
Moreover, Greenpeace is now calling on all operations using CSS stimulation to be stopped until the ongoing investigations into the causes of the spills are complete. “We need answers before Alberta faces yet another unexplained disaster,” says Greenpeace Climate and Energy campaigner, Mike Hudema.


The natural Amur-Heilong Ecoregion ecosystems are biologically rich, and valued by the human population and countries for their natural qualities and ecosystem services. The crowns of evolution, the Amur tiger and Far Eastern leopard thrive in extensive tracts of intact forests, protected by effectively managed nature reserves and connecting corridors.
The free flowing Amur River, uninterrupted by dams, ensures climate adaptation for regular monsoon floods and, supports freshwater relic communities and enormous shoals of migrating salmons and sturgeons. The Amur River valley, conserved in an internationally recognized Green Corridor, supports last breeding grounds of Oriental stork,eastern cranes and millions of migratory birds along the Northeast Asian Flyway.
Sustainable management of the viable ecosystems is linked to long-term regional strategiesof social-economic development. The Amur-Heilong Ecoregional program serves as a powerful symbol of international cooperation between peoples and countries of the basin.
Protected Areas (PA) of Amur-Heilong Ecoregional Complex
Open full map in the new window
Protected areas cover 12% of the Amur Ecoregion territory: 7,3 million hectares of federal PAs and 8 million hectares of provincial PAs including 17 strictly protected areas, 7 national parks, 8 federal and 79 provincial wildlife refuges, 6 nature parks, 7 ecological corridors, and 2 wetlands. Sikhote-Alin and Dauria Nature Reserves is the UNESCO World Heritage site. Five more nature reserves are UNESCO biosphere reserves: Sokhondinsky NR, Far Eastern Marine NR, Dauria NR, Kedrovaya Pad NR and Khankaisky Nature Reserve. Six territories are included in the list of the Wetlands of International Importance (Ramsar sites): Khanka Lake, Torey Lakes, Khingan-Arkharinskaya Lowland, Zeya-Bureya Plain, Bolon Lake and Udyl Lake.
The Amur-Heilong Ecoregion Complex covers a total area of 1.85 million km2. It includes the basin of the Amur-Heilong River in Russia, China and Mongolia. The area also includes the internal river basin of the Ulz River that drains its water to Torey lakes, the Uda River Basin, part of the Olekma River Basin, the eastern slope of the Sikhote Alin mountains and basins of the Razdolnaya-Suifun River and Tumannaya-Tumenjiang River. Technically the northern part of the Democratic People’s Republic of Korea (DPRK) is part of the Amur-Heilong ecoregion but for political reasons does not fall within the scope of this PIA.
In Russia the AHEC encompasses 5 provinces: the totality of Amurskaya Oblast, Evreiskaya Autonomous Oblast and Primorskiy Krai, part of Khabarovskiy Krai (up to the North of the Uda River), and the Shilka and Argun Rivers Basin of Zabaikalskiy Krai (former Chitinskaya Oblast and Aginskii Buryatskii Autonomic Prefecture). In China the AHEC covers three provinces: the totality of Heilongjiang and Jilin provinces, and part of Inner Mongolia Autonomous Region in Haelar-Dalaihu Basins. In Mongolia the AHEC covers parts of the following four provinces: Dornod, Sukhbaatar, Khentii and Tuv province in Onon and Kerulen Basins.
The majority of WWF’s work is focused on the southern portion of the ecoregion (Sikhote-Alin in Russia and Changbaishan in China), where both the levels of biodiversity and the threats are greatest. Integrated River Basin Management work is concentrated in the headwaters of the Amur River (Zabaykalskiy Krai in Russia and Dornod and Khentii provinces in Mongolia).