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Group 9 Project - Fluorescent Proteins
- 1 Introduction
- 2 The Theory of Fluorescence
- 3 The History of the Green Fluorescent Protein
- 4 Green Fluorescent Protein
- 5 Development of New Fluorescent Proteins
- 6 Current research
- 7 Advantages of Fluorescent Proteins
- 8 Limitations of Fluorescent Proteins
- 9 Links to Current Research
- 10 Glossary
- 11 References
The Green Fluorescent Protein (GFP) was first observed in the Aequorea victoria jellyfish in 1962 and since then this protein has become a vital tool for viewing many biological processes that were once invisible. The GFP can be bound on many of the thousands of different proteins in living organisms, in real time and in vivo or in vitro. Many new colours of fluorescent proteins are now available and allow multiple cell structures or processes to be imaged simultaneously. Fluorescent proteins, in their many colours and properties, have revolutionised modern cell biology as we know it, and continue to play a part in cutting-edge research into normal function and disease.
The Theory of Fluorescence
Fluorescence is the spontaneous emission of radiation (ultraviolet, visible or infrared photons) from an excited atom. The Latin word luminescenz describes all phenomena in which light is released and where a rise in temperature does not occur, whereas incandescence involves heat. The various types of luminescence are classified according to the mode of excitation, fluorescence occurs via the mode of absorption of light to release another wave of light. A prime example is the reaction of luciferase where the oxidation of a substrate by an enzyme releases light. [1] [2]
There are three principles of fluorescence
1. Excitation; energy/ photons are absorbed by the atom of an appropriate wavelength by the fluorophore which becomes excited.
2. Excited state; the electron jumps to a higher energy level.
3. Soon, the electron drops back to the ground state, the atom will emit light as the atom's electron reverts to a lower, more stable, energy level, thus fluorescing.
NB; fluorescence is cyclical therefore a fluorophore usually is excited repeatedly.
Fluorescence microscopes emit a specific wavelength to excite the specimen, the microscope has a filter that only allows the specific emitted wavelength which helps make up the image without other emitted light to get through and scatter desired light. [1] [2]
The History of the Green Fluorescent Protein
Major Milestone Timeline [2] [3]
1565 N. Monardes observed the emission of light by an infusion of wood lignum Nephriticum (first reported observation of fluorescence).
1640 Licetus studied the Bolognese stone and defined it as a non-thermal light emission.
1842 E. Becquerel made the first statement that the emitted light is of longer wavelength than the incident light.
1853 G. G. Stokes introduces the term fluorescence.
1858 E. Becquerel created the first phosphoroscope.
1944 Lewis and Kasha Triplet state
1955 Green fluorescent substance in jellyfish first described.
1962 GFP identified as protein, extracted from Aequorea victoria jellyfish
1979 Osamu Shimomura characterized structure of chromophore.
1985 Prasher clones GFP gene from Aequorea victoria.
1993 The Structure of GFP chromophore confirmed, side amino acid residues corrected from Shimomura's 1979 structure.
2000 onwards The development of GFP variants, as described below in "The Development of New Fluorescent Proteins"
GFP and The Nobel Prize
The 2008 Noble Prize in Chemistry "for the discovery and development of the green fluorescent protein" was awarded jointly to:
Osamu Shimomura- When Shimomura first obtained GFP, he did so by squeezing strips of the jelly fish Aequorea through filtercel and then purified it by ammonium sulfate and column chromatography. [4]
Martin Chalfie- conducted experiments by tagging GFP in six individual cells in the transparent roundworm Caenorhabditis elegans, supporting previous ideas that GFP can be used as a molecular tagger or "light bulb". Chalfie also contributed to the ideas of transfecting cells and with his experiment was able to revolutionize the biosciences.
Roger Y. Tsien- -was able to mutate GFP and create a variety of new colours now termed. Tsien was also able to make the DsRed FP compact thus more versatile and finally created mutants that start fluorescing faster and are brighter. [5]
The Extraction and Purified of GFP from the Aequorea victoria Jelly fish[6] [7] [8] [9]
1. Transfection into cells via vector for amplification (See Current Research section for detailed methodology)
2. Cell are lysed by sonication, then centrifuged
3. Products are put in ammonium sulfate, ethanol and chloroform and mixed it, resulting in GFP at upper aqueous phase and others proteins at bottom. Alternatively, the hydrophobic nature of the GFP molecule can be utilised for purification with a size column
4. To confirm the extraction of GFP, the sample is excitated at 395nm and checking if a 510nm wavelength emission results.
Further links to purification methods:[32] , [33]
Green Fluorescent Protein
Structure and Classification
GFP is compromised of 238 amino acids and weighs 26.9kDa, discovered in 1962 by Osamu Shimomura in the jellyfish Aequorea victoria, it glowed green when under ultraviolet light. Its structure was found in 1972 by Shimomura, it is an 11-stranded beta-barrel threaded by an alpha-helix running up the axis of the cylinder, it is predicated that the beta-can structure protects the chromophore and is presumably responsible for GFP's stability. This protein is now the most commonly used biological marker tool in molecular biology, medicine, and cell biology. [10] [11] [12]
The Protein Data Bank currently lists 22 GFP's, there are many mutants, this is done by changing amino acids in them, they actually all have similar structures to aequorea GFP. The chromophore is in the centre of the beta can and is a 4-(p-hydroxy-benzylidene) imidazolidin-5-one attached to the peptide backbone through the 1- and 2-positions of the ring.
Tsien [5] has classified GFPs into seven major classes based on their spectral characteristics:
(i) Wild-type GFP. The chromophore is in equilibrium between the phenol and phenolate form. It has two excitation peaks at 395 and 475 nm.
(ii) Phenolate anion. Ser65 has been substituted with Thr, Ala, or Gly. Does not have the 395 nm excitation peak.
(iii) Neutral phenol. Mutation of Thr203 to Ile results in a mutant that only has the 399 nm excitation.
(iv) Phenolate anion with stacked ð-electron system. Mutation of Thr203 to His, Trp, Phe, or Tyr results in yellow fluorescent proteins.
(v) Indole in chromophore. Cyan fluorescent proteins have properties intermediate to those of BFP and EGFP.
(vi) Imidazole in chromophore and phenyl in chromophore. Blue fluorescent proteins have an excitation peak at 383 nm.
(vii) Phenyl in chromophore. It has the shortest excitation wavelength and no apparent uses.
|Advantages of GFP [5] [13]
|Disadvantages of GFP [5] [13]
|Applicable to nearly all organisms and in live tissue
|Gene transfection required which is time consuming
|Can be targeted to specific tissues, cells, organelles, or proteins
|GFP is most efficient temperatures well below 37 °C, thus searches for ones functional in mammalian cells were made.
|Unlikely to diffuse well enough to blur spatial gradients
|GFP only fluoresces in presence of oxygen as the chromophore requires an autocatalytic reaction, thus GFP only works in aerobic environments.
|Good optical properties: visible excitation, high photo-stability
|Some of GFP's limitations are the slow post-translational chromophore formation.
|Cheap and relatively easy to replicate and distribute
|Difficulty in distinguishing GFP from background fluorescence when the GFP is not densely localized or highly expressed.
|It's chromophore does not require a cofactor
|It is highly stable as GFP is resistant to heat, alkaline pH, detergents, photo-bleaching, organic/ non-organic salts, and many proteases.
|Shimomura showed GFP was a relatively small protein which allows little hindrance to the protein or its function.
Tagging GFP onto Targets
GFP is so versatile and can be tagged by means of labelling reagents with functional groups on amino groups via covalent binding, Wang and Hazelrigg discovered that GFP can be used as a fluorescent tag for the N- or C-termini of proteins. Since there are so many proteins it can be labelled on nearly all aspects of the cell. [5] [12] [14]
Development of New Fluorescent Proteins
Since the revolutionary development of GFP, extensive research has been conducted with the aim of producing fluorescent proteins (FPs) which cover a broader colour spectrum. These colour mutant FPs are also being genetically engineered to exhibit faster maturation rates, greater photostability, increased brightness, pH insensitivity and reduced oligomerisation and toxicity Cite error: Invalid
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New FP Colours
Enhanced BFP (EBFP) was one of the first spectral variants engineered from Aquorea GFP, but due to its low brightness and poor photostability it is now unappealing for most research Cite error: Invalid
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name cannot be a simple integer. Use a descriptive title [15]. EBFP2 is the most photostable and brightest blue FP Cite error: Invalid
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The cyan FPs (CFPs) began with the production of enhanced CFP (ECFP) from Aequorea GFP[16]. mCerulean followed in 2004 offering a brighter and better general-purpose CFP [17]. In 2006, a monomeric teal-coloured variant mTFP1 was obtained from a Clavularia soft coral protein[18]. mTFP1 is brighter, less pH sensitive and more photostable than the traditional CFPs, making it an excellent alternative to its predecessors Cite error: Invalid
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Most recently, site-directed mutagenesis of ECFP has produced super CFP (SCFP) which is twice as bright as ECFP when expressed in bacteria. SCFP shows promise for use as a fusion tag or as a biosensor for the detection of calcium ion fluctuations, pH changes, metabolites or enzyme phosphorylation Cite error: Invalid
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Following the discovery of the original Aequorea GFP discussed previously, many other proteins which express in the green region of the spectrum have been isolated from other Aequorea species, copepods, amphioxus and reef corals Cite error: Invalid
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Most of these novel GFPs exhibit no discernable advantage over EGFP Cite error: Invalid
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Yellow FPs (YFPs) are among the brightest and most versatile probes developed in any of the spectral classes Cite error: Invalid
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name cannot be a simple integer. Use a descriptive title. EYFP has proved effective in tracking the distribution patterns of single proteins on the membranes of live cells[22]. mCitrine, derived from the addition of a single mutation in EYFP, is less halide sensitive and twice as resistant to photobleaching as its predecessor[23].
mVenus is a popular YFP mutant with a greatly reduced maturation time, however it has low photostability. The fact that mVenus requires only two minutes in vitro or seven minutes in vivo to produce fluorophores makes it ideal for monitoring cellular processes with fast dynamics such as gene expression[24].
Super YFP (SYFP), the product of site-directed mutagenesis of EYFP, may hold similar applications as those discussed previously for SCFP. Yellow fluorescent protein for energy transfer, YPet, is the brightest YFP variant. YPet is also reported to have very good photostability and superior acidic resistance to mVenus and other YFP derivatives Cite error: Invalid
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Compared to the other areas of the spectrum, few probes have been constructed to emit in the orange and red wavelengths. Probes such as DsRed, TagRFP and tdTomato actually have emission profiles in the orange range of the spectrum and not the red range as suggested by their names Cite error: Invalid
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name cannot be a simple integer. Use a descriptive title. mOrange, a member of the 'Fruits' series (see 'Red' section for details), once dominated this spectrum in terms of brightness, but has average photostability and is unstable at low pH Cite error: Invalid
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name cannot be a simple integer. Use a descriptive title. In an attempt to overcome these problems mOrange2 was engineered from mOrange and shows significantly improved photostability but is still pH sensitive and shows an almost doubled maturation time.
Kusabira Orange (KO), derived as a tetramer from the mushroom coral Fungia concinna, was later modified to give monomer KO (mKO)[25]. mKO demonstrates extremely good photostability and brightness similar to that of EGFP, making it a good candidate for long-term and wide-fluorescence illumination experiments Cite error: Invalid
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In 2007, TagRFP was cloned as a dimer from Entacemaea quadricolor sea anemone and appears to be a useful tool for localisation and FRET studies[26] . Random mutagenesis of TagRFP produced the highly photostable, bright and pH resistant TurboRFP.
Many of the problems associated with Discosoma DsRed FP- including slow maturation, an intermediate green state, and tetrameric character- have been the target of many attempted modifications of this protein through both random and site-directed mutagenesis. The production of the monomeric mRFP1 from DsRed was promising, but reduced emission and quick photobleaching still means that it is less useful than monomeric GFPs and YFPsCite error: Invalid
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tdTomato, another of the 'Fruits' proteins, is the brightest of all available FPs, emits at closer to the true red range and is very photostable Cite error: Invalid
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FPs that emit in the far red area of the spectrum are desirable due to this wavelength of light being less phototoxic and more able to probe deeper into biological tissues Cite error: Invalid
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name cannot be a simple integer. Use a descriptive title. According to Shaner and team, mStrawberry and mCherry are the best reds, with brightness levels of 75% and 50% of EGFP Cite error: Invalid
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Developed in 2004, mPlum is one of the first true far-red probes, emitting at 649nm[27]. mPlum has limited brightness (10% of EGFP) but good stability and is recommended for use in multicolour imaging experiments, the imaging of thicker tissues and as a FRET partner for GFPs and YFPs Cite error: Invalid
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Kutushka, a dimeric protein that emits at 635nm, was developed in 2007 and is commercially available from Evrogen as TurboFP635Cite error: Invalid
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name cannot be a simple integer. Use a descriptive title. Despite being less bright than EGFP, Kutushka has the highest brightness level of any of the FPs in the 650-800nm wavelength area.
mKate (Evrogen, TagFP635) has similar spectral characteristics to Kutushka, brightness on par with mCherry and is reported to be very photostable, making it a good candidate for localisation experiments in this area of the spectrumCite error: Invalid
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For more information on the range of currently available colour palette of FPs visit:
1) The Clontech "Interactive Guide" (found as a link on the righthand side of the page)- allows you to explore their range of comercially available FPs by colour, application, spectra and antibody choice [34]
2) The Nikon Microscopy Education page- gives the basics of FP use in live cell imaging [35]
3) The Olympus FluoView Resource Centre- gives information on the colour palette for confocal microscopy [36]
Other Novel FPs
Infra-Red FPs
The development of a protein that emits in the infra-red spectrum is the holy grail of fluorescent protein engineering. Accurate in vivo imaging of animal tissue using FPs requires emission wavelengths of 650 to 900nm to minimise absorbance by water, lipids and haemoglobin and to reduce light scattering. Infra-red fluorescent proteins (IFPs) which can be genetically expressed would be incredibly valuable for whole-body imaging in cancer and gene therapyCite error: Invalid
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A paper published in 2009[28]reports the production of a monomeric IFP from a bacteriophytochrome which incorporates biliverdin (an intermediate of heme catabolism) as the chromophore. The IFP was effectively expressed in both mammalian cells and mice, demonstrated the spontaneous incorporation biliverdin and produced infra-red fluorescence.
Photoactivated FPs
These proteins display negligible fluorescence until excited by irradiation at a specific wavelengthCite error: Invalid
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name cannot be a simple integer. Use a descriptive title. PA-GFP is one such protein that is activated by light of wavelength 488nm and emits in the green wavelength (38). Other FPs which demonstrate "reversible on/off switching" are Dropna, rsFastLime, rsCherry, rsCherryRev and PAmCherryCite error: Invalid
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Photoconvertable FPs
This class of FPs show "light-driven modulation of fluorescence properties"Cite error: Invalid
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name cannot be a simple integer. Use a descriptive title. These proteins, such as Kaede (as shown in the picture to the right), KikGR, EosFP, Dendra2, mKikGR, tdEosFP, mEosFP and mEosFP2, are useful for the tracking of fusion proteins, organelles or the fate of embryonic cells during developmentCite error: Invalid
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Destabilised GFP variants
These FPs allow the characterisation of the expression timing or lifetime of a target protein due to their rapid turnover by proteolysis resulting in only younger protein chimeras fluorescing[29].
"Fluorescent Timer" Protein
This protein, similarly to the destabilised GFP variants, allows the measurement of protein turnover and expression timingCite error: Invalid
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name cannot be a simple integer. Use a descriptive title. It initially fluoresces in the green area of the spectrum before conversion of the fluorophore after several hours leads to emission in the red[30]. Using the ratio of green to red fluorescence allows the age of the tagged protein to be determined.
Future Engineering Endeavours
Criteria for future engineering endeavours as proposed by Shaner et al.Cite error: Invalid
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• Improved brightness
• Monomeric structure
• High contrast
• Uncomplicated photoconversion
• Reversible photoactivation
• Red-to-green photoconversion
• Improved expression in the far-red or near-infrared regions
Current research
Current research in cell biology with fluorescent proteins is a vast field of study. We shall focus our investigation on a specific technique that has significant clinical consequences. This technique is the production of individual transgenic cells and whole transgenic organisms using spliced fluorescent genes. The principle behind this technique is based on the model for protein synthesis and thus an explanation of the protein synthesis model is an essential requisite. Protein synthesis occurs in 3 stages:
The first stage: (Transcription)
1) Inside the nucleus of a cell, the double stranded DNA helix unwinds in the area of the gene containing the information for synthesizing a specific protein. [31] [32]
2) The enzyme RNA polymerase moves along the unwound DNA strand at the location of the gene using it as a template to create a complementary strand known as a messenger RNA strand (mRNA). This complementary strand is made by linking RNA nucleotides that are complementary to the gene nucleotides on the DNA strand. Note: In any RNA strand, the base thymine is replaced with uracil and the start codon (AUG) and a stop codon (UAA or UAG or UGA) control the length of the strand. [31] [33]
3) The mRNA strand then undergoes modifications in the nucleus to contain only the base sequence that will code for the protein.[32] Most genes contain non-coding regions known as introns and coding regions called exons.[31] [32] Thus its complementary mRNA strand will also contain coding and non-coding regions. The non-coding regions are excised by splicing introns from the mRNA and joining the exons together.[31] [32]
The second stage: (Amino acid activation)
This stage occurs in the cell cytoplasm where the aminoacyl-tRNA synthetise enzyme attaches amino acids to transfer RNA molecules (tRNA). Each type of amino acid is attached to its specific tRNA.[33]
The final stage: (Translation)
1) The mRNA strand binds onto a ribosome in the cytoplasm at the end with the start codon (AUG).[31] [33]
2) A tRNA molecule binds to the start codon on the mRNA within the ribosome.[33]
3) A second tRNA binds to the next codon of the mRNA within the ribosome. [33]
4) The first tRNA is released from the ribosome and the ribosome moves along the mRNA strand one codon at a time (42). Two tRNAs are temporarily bound within a ribosome at any one time and their amino acids linked together forming a polypeptide chain. This process ends when the stop codon is reached on the tRNA molecule and the polypeptide is then released. [32] [33]
5) The polypeptide then undergoes further modifications in the cell cytoplasm particularly in the Golgi apparatus before becoming the final protein.[32]
Transgenic Organism
A transgenic organism is one whose genome contains genes from another species.[32] The aim of creating a transgenic organism is to obtain a favorable characteristic in the organism's phenotype. This desired characteristic of phenotype is obtained by altering an organism's normal genotype to include the gene from another species coding for the desired characteristic. [33] Once a desired gene is incorporated into an animal's genotype, the process of protein synthesis allows the transgenic organism to express this new characteristic in its phenotype. [32] [33]
In the case of fluorescent research, the gene responsible for fluorescence phenotypic characteristics in one organism can be spliced and inserted into the genotype of another organism for experimentation. The recipient organism is then able to produce the fluorescent proteins (coded for by the inserted gene) in its phenotype. [34]
The production of a fluorescent transgenic organism or cell involves several steps summarized below:
1) Organisms containing fluorescent proteins are identified. The chromosome and the gene coding for the fluorescence are identified in the organism.[32]
2) A cell sample is taken from the organism and the gene coding for fluorescent protein synthesis is isolated from its DNA strand. This involves "cutting" the gene out of its DNA strand using enzymes called restriction endonucleases. The restriction endonucleases cut DNA at specific site so the desired gene can be removed from the DNA strand. The cut ends are known as "sticky ends".[31] [32]
3) The gene is then incorporated into a recipient organism's DNA. To do this, the recipient organism's cell DNA must first be cut with the same restriction endonucleases at a desired location (usually just before the stop codon at the end of a gene coding for a particular protein).[31] The foreign gene is then inserted with a promoter sequence by microinjection or vector transfer into the fertilized egg cell (zygote) and as the embryo develops, the entire organism will become transgenic.[34] Alternatively, the foreign gene can be injected into a somatic cell or sex cell of a post natal recipient organism, this in effect will result in the fluorescence of a particular type of cell and thus tissues rather than the entire organism. [31] [34]
4) The inserted foreign gene will be attracted to and connect at the recipient's cut DNA in a process called "annealing".[33] The DNA is now known as recombinant DNA as it contains a combination of the recipient's normal genes as well as foreign genes. [32]
5) DNA ligases are added to the annealed DNA fragments to help strengthen the bonds of the new recombinant DNA. [32] [33] DNA ligases are sealing enzymes found in all living organisms that help make and repair DNA. [32]
6) As the cell develops and its genetic information read, the recombinant DNA will not only code for the production of the normal protein, but will also code for the production of the fluorescent protein that will be attached to the normal protein. [32] This will be true of the recipient cell and all its descendants. [32]
The creation of transgenic organisms with the capability to produce fluorescent proteins has significant clinical consequences particularly in the understanding of inexorable diseases such as cancer and malaria.
Cancer is the leading cause of death worldwide claiming approximately 8 million people in 2009. The WHO estimates this figure to rise to 12 million by 2030. [35] In Australia, cancer claims approximately 42,000 lives every year and an estimated 114,000 new cases are expected in 2010. [36] A cancer or malignant neoplasm is a class of disease in which cells display uncontrolled mitotic divisions, invasion of surrounding tissues and metastasis (spread via the circulatory or lymphatic system). [37] Cancer cells are unspecialized and have no other function other than reproduction. These cells invade tissues and organs diminishing their functioning gradually until failure. [34]
Fluorescent proteins provide a means by which cancer can be studied in vivo. The implications of this are significant as it allows researchers to study the characteristics of this disease as it develops in the body over time. [38] A histologically intact primary tumour from a cancer patient is obtained by surgical means and transfected with a specific fluorescent protein. The transfected tumour cells are then transplanted into immunodeficient (nude) transgenic mammals (usually mice) which themselves have been genetically modified to display a different coloured fluorescent protein.[38] [39] This creates a contrast between the healthy cells of the recipient organism and those of the tumour cells as both display different coloured fluorescence. [40] [41] The fluorescent protein fluoresces for the entire life of a cancer cell and after it divides mitotically, fluoresces in the daughter cells. [40] Using this contrast, researchers are able to visualize, in real time, important aspects of cancer in living animals, including tumour cell angiogenesis, mobility, invasion and metastasis. [39] [41]
This technique of studying cancer has so far yielded some interesting results. GFP expressing tumour cells reveal that angiogenesis occurs early in tumour formation when as little as 60 to 80 tumour cells are present inducing vasodialation and changes to blood vessel morphology. [42] [43] Armed with this knowledge, researchers can experiment with various methods to stop the ability of tumour cells to recruit blood vessels that are vital for their growth and spread thereby stopping the cancer in its tracks. [44] Research by Yamauchi and Yamamoto explored the mechanism by which tumour cells migrate down through narrow capillaries. By expressing red fluorescent proteins in the tumour cell cytoplasm and green fluorescent proteins in the cell nucleus they showed that tumour cells are highly motile and capable of changing their morphology to great extents in order to squeeze down narrow vessels. [45] [46] This is critical information on the metastatic ability of cancer cells and explains to some degree the mechanism by which cancer cells are able to spread to body extremities. [45]
Malaria is the world's most serious infectious disease and is responsible for one million deaths annually worldwide.[47] There are currently between 250 to 500 million people affected by malaria most of them in developing countries.[32] [47] In Africa, malaria is an especially serious problem where a child dies from it every 30 seconds.[47] In Australia, malaria was declared eradicated in 1981 but approximately 700 to 800 cases occur each year from travellers infected elsewhere with northern Australia the primary receptive zone for malaria transmission. [48] Malaria is a parasitic infection caused by four species of the genus Plasmodium: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. The infection begins when Plasmodium sporozoites enter dermis from the bite of an infected female Anopheles mosquito.[31] [32] [33] [48] The parasites then invade blood vessel and travels by blood to the liver where they enter liver cells (hepatocytes).[31] Inside a hepatocyte, the Plasmodium sporozoites divide asexually forming Plasmodium merozoites.[49] The merozoites are then released into the circulatory system where their invade red blood cells (erythrocytes).[49] [50] Once inside erythrocytes, the merozoites continues to proliferate causing each infected red blood cell to burst releasing toxins into the blood stream. [50] Some erythrocytes do not burst as they contain merozoites that do not proliferate but instead develop into male and female gametocytes. When a female Anopheles mosquito bites an infected host, these gamatocytes enter the mosquito abdomen where they form a zygote and develop into new active Plasmodium sporozoites that can begin a new infection cycle. [50] Growing Plasmodium resistance to anti-malarial medicines such as chloroquine and vector resistance to mosquito pesticides has spread very rapidly undermining malaria control efforts across the globe. [47]
Fluorescent proteins provide a unique method of imaging the entire malaria parasite lifecycle from development to transmigration inside both insect and vertebrate hosts. This is achieved by transfecting the malarial parasites with genes encoding for green-fluorescent protein (GFP) or red-fluorescent protein (RedStar) creating a transgenic parasite.[51] Fluorescent microscopy can then be used to viasulize the parasites behaviour inside the mosquito vector and also in cells of the vertebrate host. Using this technique, Rogeiro et. al found that Sporozoites are formed inside oocysts at the mosquito midgut wall. They are then released into the hemolymph and eventually bind to the mosquito's salivary glands, which they then invade. When the mosquito bites its host, the sporozoites are released into the hosts' circulatory system.[52] Tracking the movement of the malarial sporozoites inside the mosquito vector helps researchers understand the behaviour of the parasite and devise methods of preventing transmission to humans by stopping the parasite's advance at the vector level. [51] [52]
Another research focusing on control of malaria found that transgenic mosquitoes were less susceptible to plasmodium infection and had fewer sporozoites in their salivary glands than control mosquitoes. They also found transgenic mosquito parasite transmission was reduced by more than twofold. The team investigated this result to be a consequence of the expression of the SM1 peptide in the mosquito midgut that severely reduced vector competence by inhibiting Plasmodium development. [53] This suggests that gene modifications can inhibit malarial sporozoite development in the mosquito and thus holds great potential for gene experimentation methods to control malaria. Other research has focused on breeding experiments where fluorescent proteins are used to differentiate male and female mosquitoes. Since female mosquitoes only mate once in their lifecycle and are the transmitters of the malaria parasite, sterile males (marked by fluorescent gonads) are sorted and released into the environment where they mate with the infected females. Since the female will be unable to reproduce, the malaria parasite life cycle is ended. [54]
Advantages of Fluorescent Proteins
Some advantages in using fluorescent proteins include:
• The imaging of structures, cells, tissues, organs and even living organisms in vivo.
• There is no known adverse disruption to the normal functioning of the cell biochemistry.
• Fluorescent proteins are easily obtained from fluorescent organisms mainly corals, jellyfish and marine invertibrates.
• The isolation and transfection of fluorescent proteins is a higly successful process that produces minimal hazardous wastes unlike other fluorescent techniques like radioactive dyes and fluorescent nano dots.
• FP's are available in a vast array of colours covering different emission spectra for imaging different tissues.
Limitations of Fluorescent Proteins
Some problems associated with using fluorescent proteinsCite error: Invalid
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• Oligomerisation
• Aggregation
• Cytotoxicity due to FP
• Environmental sensitivity
• Light-induced cytotoxicity
• Potential biological activity exerted by FPs
• No strict criteria under which the FPs are tested- results are often biased and therefore not trustworthy for researchers
• Commercial availability/price
Links to Current Research
AntiCancer Inc. [37]
Bio medicine [38]
Brighter Ideas Inc [39]
Central caribbean marine institute [40]
Clontech [41]
Evrogen [42]
GeneBrite [43]
Glen Research [44]
MetaMouse [45]
Invitrogen [46]
Promega Corporation [47]
Angiogenesis: The formation of new blood vessels
Chromophores: Molecules which are able to absorb light
Codon: A specific sequence of three adjacent nucleotides on a strand of DNA or RNA that specifies the genetic code information for synthesizing a particular amino acid
Erythrocytes: Red blood cells
Exons: A segment of a gene that contains a coding region. The exons provide the instructions for making a protein
Filtercel:An industrial filter used to seperate small particles from big ones.
Fluorochromes or fluorophores: Molecules which are able to absorb and emit light Filtercel
FRET: Fluorescence Resonance Energy Transfer. See the Invitrogen website for a short explanation of this technique: [48]
Gametocyte: A eukaryotic germ cell that divides by mitosis into other gametocytes or by meiosis into gametids during gametogenesis
Genotype: The genetic makeup of an individual
Golgi apparatus: An organelle in the cytoplasm that packages proteins and carbohydrates into vesicles for export from the cell
Hemolymph: A circulating fluid in the bodies of some invertebrates that is the equivalent of blood
Introns: An intron is a DNA region within a gene that is not translated into protein
Ligase: Any of a group of enzymes that catalyze the binding of two molecules
Merozoite: Cell that arises from the asexual division of a parent sporozoan during its life cycle
Metastasis: When cancer cells have broken away from the primary tumor and spread to other organs in the body through the blood stream or the lymphatic system
mRNA: The RNA transcript of a protein-encoding gene
Phenotype: Phenotype is any observable characteristic or trait of an organism
Photoactivation:The absorption of energy from a photon in raising a chromophore from the ground state
Photoconversion: The photochemical conversion of a chromophore, resulting in a change in emission spectrum
Photostability: The stability of a chromophore during excitation
Random mutagenesis: Random incorporation of mutations in DNA
Restriction Endonucleases: An enzyme that cuts double-stranded or single stranded DNA at specific recognition
Ribosome: Ribosomes are the components of cells that make proteins from amino acids
Site-directed mutagenesis: Targeted insertion of a mutation in a DNA sequence, usually by use of of a plasmid vector
Sporozoite: Slender, spindle-shaped organism that is the infective stage of malaria
tRNA: A small RNA molecule that "translates" a codon in mRNA by bringing in its corresponding amino acid during protein synthesis
Zygote: A fertilized egg cell, it is diploid
- ↑ 1.0 1.1 http://vohweb.chem.ucla.edu/voh/classes%5CSpring08%5C156ID35%5Cfluoresence.pdf
- ↑ 2.0 2.1 2.2 Bernard Valeur (2002). Molecular Fluorescence; Principles and Applications. WILEY
- ↑ http://www.conncoll.edu/ccacad/zimmer/GFP-ww/timeline.html
- ↑ Osamu Shimomura (2009). Discovery of Green Fluorescent Protein. Angewandte Chemie. Volume 48 Issue 31, Pages 5590 – 5602. http://info.library.unsw.edu.au/cgi-bin/local/access/ej- access.cgi?url=http://www3.interscience.wiley.com/resolve/openurl?sid=ExLibris%3ASFX&volume=48&spage=5590&issn=1433-7851&genre=article&issue=31&id=doi%3A10.1002%2Fanie.200902240&title=Angewandte+Chemie+(International+ed.+in+English)
- ↑ 5.0 5.1 5.2 5.3 5.4 Roger Y. Tsien Howard (1998) THE GREEN FLUORESCENT PROTEIN. Annual Review of Biochemistry. Vol. 67: 509-544. http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.biochem.67.1.509
- ↑ O Shimomura, FH Johnson, Y Saiga (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59 223-29. http://www.ncbi.nlm.nih.gov/pubmed/13911999
- ↑ Yakhnin A.V.; Vinokurov L.M.; Surin A.K.; Alakhov Y.B (1998) Green Fluorescent Protein Purification by Organic Extraction.. Protein Expression and Purification, Volume 14, Number 3, December, pp. 382-386(5). http://www.ingentaconnect.com/content/els/10465928/1998/00000014/00000003/art90981
- ↑ Sulakshana Jain, Sunita Teotia and Munishwar N. Gupta (2004) Purification of green fluorescent protein overexpressed by a mutant recombinant Escherichia coli.. Volume 36, Issue 1, July, Pages 76-81. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WPJ-4CDHGRP-3&_user=1975841&_coverDate=07%2F31%2F2004&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1338723339&_rerunOrigin=google&_acct=C000004218&_version=1&_urlVersion=0&_userid=1975841&md5=d74ef098e6b095af1e38fd5a7b27d77b
- ↑ Shimomura, O., Johnson, F.H. & Saiga, Y (1962). Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan Aequorea.. J. Cell. Comp. Physiol. 59, 223-239. http://www.nature.com/nmeth/focus/fluorescence/classics/proteins.html
- ↑ Prendergast F, Mann K (1978). "Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskålea". Biochemistry 17 (17): 3448–53.http://pubs.acs.org/doi/abs/10.1021/bi00610a004
- ↑ Zimmer M (2002) Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev. Mar; 102(3):759-81. http://www.ncbi.nlm.nih.gov/pubmed/11890756
- ↑ 12.0 12.1 Hans-Hermann Gerdes and Christoph Kaether (1996) Green fluorescent protein: applications in cell biology.. Volume 389, Issue 1, 24 June, Pages 44-47. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T36-3VYTMHG-9&_user=1975841&_coverDate=06%2F24%2F1996&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_rerunOrigin=google&_acct=C000004218&_version=1&_urlVersion=0&_userid=1975841&md5=6b435914a9efeb38653f8099ef1f0e29
- ↑ 13.0 13.1 Jennifer Lippincott-Schwartz,* George H. Patterson (2003). Development and Use of Fluorescent Protein Markers in Living Cells Science 4 April 2003: Vol. 300. no. 5616, pp. 87 – 91. http://www.sciencemag.org/cgi/content/short/300/5616/87
- ↑ Stéphanie Cabantous, Thomas C Terwilliger & Geoffrey S Waldo (2004). Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein.. Nature Biotechnology 23, 102 – 107. http://www.nature.com/nbt/journal/v23/n1/abs/nbt1044.html
- ↑ Kremers, G J, Goedhart, J, van den Heuvel, D J, Gerritsen, H C. & Gadella, T W J. (2007). Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells. Biochemistry, 46. [1]
- ↑ Cubitt, A B., Heim, R, Adams, S R, Boyd, A E, Gross, L A & Tsien, R Y. (1995). Understanding, improving and using green fluorescent proteins. Trends Biochemical Science, 20. [2]
- ↑ Rizzo, M A, Springer, G H, Granada, B & Piston, D W. (2004). An Improved cyan fluorescent variant useful for FRET. Nature Biotechnology, 22. [3]
- ↑ Ai, H, Henderson, J N, Remington, S J & Campbell , R E. (2006). Directed evolution of a monomeric, bright and photostable version of clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochemistry Journal, 400(3). [4]
- ↑ Zacharias, D A, Violin, J D, Newton, A C & Tsien, R Y. (2002). Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science. [5]
- ↑ Tsien, R Y. (1998). The green fluorescent protein. Annual Review Biochemistry, 67.
- ↑ Miyawaki, A, Griesbeck, O, Heim, R & Tsien, R Y. (1999). Dynamic and quantitative calcium measurements using improved cameleons. Proceeding of the National Academy of Science USA, 96, 10051607
- ↑ Ober R J, Ram S and Ward E S. (2004). Localization accuracy in single-molecule microscopy, Biophysics Journal, 86, [6]
- ↑ Griesbeck, O, Baird, G S, Campbell, R E, Zacharias, D A & Tsien, R Y. (2001). Reducing the environmental sensitivity of yellow fluorescent protein: Mechanism and applications. Journal of Biological Chemistry, 276, [7]
- ↑ Nagai, T, Ibata, K, Park, E S, Kubota, M, Mikoshiba, K & Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell biological applications. Nature Biotechnology, 20, [8]
- ↑ Karasawa, S, Araki, T, Nagi, T, Mizuno, H & Miyawaki, A. (2004). Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochemistry Journal, 381, [9]
- ↑ Merzlyak, E M, Goedhart, J, Shcherbo, D, Bulina, M E, Shcheglov, A S, Fradkov, A F, Gaintzeva, A, Lukyanov, K A, Lukyanov, S, Gadella, T W J. (2007). Bright monomeric red fluorescent protein with an extended fluorescent lifetime. Nature Methods, 4, [10]
- ↑ Wang, L, Jackson, W C, Steinbach, P A & Tsien, R Y. (2004). Evolution of new nonantibody proteins via iterative somatic hypermutation. Proceeding of the National Academy of Science USA, 101, [11]
- ↑ Shu X, Royant A, Lin M Z, Aguilera T A, Lev-Ram V, Steinbach P A & Tsien R Y.(2009). Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome, Science, 324, 19423828
- ↑ Li X, Zhao X, Fang Y, Jiang X, Duong T, Fan C , Huang C, & Kain S R. (1998). Generation of Destabilized Green Fluorescent Protein as a Transcription Reporter. The Journal of Biological Chemistry, 273, 9857028
- ↑ Terskikh A, Fradkov A, Ermakova G , Zaraisky A, Tan P , Kajava AV, Zhao X , Lukyanov S, Matz M, Kim S, Weissman I & Siebert P. (2000). "Fluorescent timer": protein that changes color with time. Science, 290, [12]
- ↑ 31.0 31.1 31.2 31.3 31.4 31.5 31.6 31.7 31.8 31.9 Solomon, EP, & Berg, LR. (2002). Biology sixth edition. New York: Thompson Learning.
- ↑ 32.00 32.01 32.02 32.03 32.04 32.05 32.06 32.07 32.08 32.09 32.10 32.11 32.12 32.13 32.14 32.15 32.16 Campbell, NA, & Reece, JB. (2002). Biology 6th edition. San Francisco: Benjamin Cummings.
- ↑ 33.00 33.01 33.02 33.03 33.04 33.05 33.06 33.07 33.08 33.09 33.10 Mudie, K, & Brotherton, J. (2000). Heinemann biology. Melbourne: Heinemann.
- ↑ 34.0 34.1 34.2 34.3 Glanville, AR. (2008). Scientifica. Elanora Heights: Millennium House.
- ↑ World Health Organization, Initials. (2009, February). Cancer. Retrieved from [13]
- ↑ Cancer Council Australia, Initials. (2010, April 23). Facts and figures. Retrieved from [14]
- ↑ Fidler, IJ . (2003). The Pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited.. Nature Review, 6(3), Retrieved from [15]
- ↑ 38.0 38.1 Yang, M, & Baranov, E. (1999). Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proceedings of the National Academy of Sciences , 97(3), Retrieved from [16]
- ↑ 39.0 39.1 Chishima, T, & Miyagi, Y. (1997). Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression . Cancer Research , 57. Retrieved from [17]
- ↑ 40.0 40.1 Farina, K, & Wyckoff, J. (1998). Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein. Cancer Research , (58), Retrieved from [18]
- ↑ 41.0 41.1 Yang, M., Chishima, T., Baranov, E., Shimada, H., Moossa, A.R., and Hoffman, R.M. Green fluorescent protein: A new light to visualize metastasis and angiogenesis in cancer. Proc. of SPIE Conference on Molecular Imaging: Reporters, Dyes, Markers and Instrumentation 3500, 117-124, 1999. [19]
- ↑ Yang, M, & Baranov, E. (2002). Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model . The National Academy of Sciences, 99(6), Retrieved from [20]
- ↑ Huang, M, & Wang, T. (2002). Establishment of fluorescent lung carcinoma metastasis model and its real-time microscopic detection in scid mice. Clinical and Experimental Metastasis, 19(4), Retrieved from [21]
- ↑ Li, C, & Shan, S. (2000). Initial stages of tumor cell-induced angiogenesis: evaluation via skin window chambers in rodent models. Oxford Journals, 92(2), Retrieved from [22]
- ↑ 45.0 45.1 Yamamoto, N, & Jiang, P. (2004). Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression. Cancer Research, 64. Retrieved from [23]
- ↑ Yamauchi, K, & Bouvet, M. (2005). Real-time in vivo dual-color imaging of intracapillary cancer cell and nucleus deformation and migration. Cancer Research, 65. Retrieved from [24]
- ↑ 47.0 47.1 47.2 47.3 World Health organization, Initials. (2010, April). Malaria. Retrieved from [25]
- ↑ 48.0 48.1 Russel, R. (2008, May). Department of medical entomology - malaria. Retrieved from [26]
- ↑ 49.0 49.1 Jones, M, & Good, M. (2006). Malaria parasites up close. Nature, 12(2), Retrieved from [27]
- ↑ 50.0 50.1 50.2 McGraw Hill, (2004). Malaria: lifecycle of plasmodium. Retrieved from [28]
- ↑ 51.0 51.1 chen, Q. (2008). Implications of imaging malaria sporozoites. Trends in parasitology, 24(3), [29]
- ↑ 52.0 52.1 Rogerio, R, Ménard, R, & Frischknecht, F. (2005). In vivo imaging of malaria parasites — recent advances and future directions . Current Opinion in Microbiology, 8(4).
- ↑ Ito, J. (2002). Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite. nature, 417(452-455 ), Retrieved from [30]
- ↑ Stephenson, T. (2005, October 9). New gm mosquito sexing technique is step towards malaria control, report scientists. Retrieved from [31]
2010 Projects
Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer


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French Rivers Spilling Sediments into the Bay of Biscay and English Channel – April 14th, 201047.2N 2.2W
Sediments from various rivers release thick tan sediments off the coast of France into the Bay of Biscay (left) and the English Channel (above).
Along the west coast, these sediments come from the Dordogne and Garonne Rivers, released through the Gironde Estuary near Bordeaux (lower right quadrant), and from the Loire River, whose mouth is located at St Nazaire, near Nantes. With a length of 1,013 kilometres (629 mi), the Loire is the longest river in France.
Moving northward from the mouth of the Loire, the Couesnon River is visible releasing sediments on France's north coast in an estuary at Mont Saint-Michel. Finally, the mouth of the River Seine can be seen by Le Havre, near the upper right corner, spilling sediments into the Bay of the Seine in the English Channel.

subset: commoncrawl


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Growth stocks can be some of the most exciting picks in the market, as these high-flyers can captivate investors' attention, and produce big gains as well. However, they can also lead on the downside when the growth story is over, so it is important to find companies which are still seeing strong growth prospects in their businesses.
One such company that might be well-positioned for future earnings growth is Etsy, Inc. (ETSY - Free Report) . This firm, which is in the Internet - Services industry, saw EPS growth of 24.6% last year, and is looking great for this year too.
In fact, the current growth estimate for this year calls for significant earnings-per-share growth. Furthermore, the long-term growth rate is currently an impressive 26.5%, suggesting pretty good prospects for the long haul.
And if this wasn't enough, the stock has actually seen estimates rise over the past month for the current fiscal year by about 80.1%. Thanks to this rise in earnings estimates, ETSY has a Zacks Rank #1 (Strong Buy) which further underscores the potential for outperformance in this company. You can see the complete list of today's Zacks #1 Rank stocks here.
So if you are looking for a fast growing stock that is still seeing plenty of opportunities on the horizon, make sure to consider ETSY. Not only does it have double-digit earnings growth prospects, but its impressive Zacks Rank suggests that analysts believe better days are ahead for ETSY as well.
More Stock News: This Is Bigger than the iPhone!
It could become the mother of all technological revolutions. Apple sold a mere 1 billion iPhones in 10 years but a new breakthrough is expected to generate more than 27 billion devices in just 3 years, creating a $1.7 trillion market.
Zacks has just released a Special Report that spotlights this fast-emerging phenomenon and 6 tickers for taking advantage of it. If you don't buy now, you may kick yourself in 2021.
Click here for the 6 trades >>


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The WS-C2960C-8PC-L is one of the Catalyst 2960 Series fixed-configuration switches. It provides Power over Ethernet (PoE), 10/100-Mbps Fast Ethernet, and 10/100/1000-Mbps Gigabit Ethernet connectivity with enhanced LAN services for midsize and branch office networks.
Benefits for Organizations
Organizations can use this switch to extend an existing network, bringing wired connectivity to more employees and supporting new wireless deployments.
Major Capabilities
- LAN Base IOS software: The switch's software includes a rich Layer 2+ suite of intelligent services, including:
- Advanced quality of service (QoS)
- Granular rate limiting
- Access control lists (ACLs)
- Multicast services
- Multiple switch management options:
- Embedded GUI device manager for all operating systems
- Windows-based Cisco Network Assistant, available at no cost from the Cisco website
- Enhanced operational usability: Cisco Catalyst Smart Operations simplifies LAN deployment, configuration, and troubleshooting. These features enable automated installation and replacement of switches, faster upgrades, and easier troubleshooting with reduced operational cost.
- Security: Security is delivered through a wide range of authentication methods, data encryption technologies, and Network Admission Control (NAC) based on users, ports, and MAC addresses. Cisco TrustSec prevents unauthorized access and helps ensure that users get only their designated privileges.
Product Specifications
The 8PC-L model provides:
- Eight 10/100-Mbps PoE ports providing up to 15.4 W per port (124 W total) for desktop, wireless access point, IP telephony, or closed-circuit TV camera connectivity
- Two dual-purpose uplink ports, each having one 10/100/1000 Ethernet port and one small-form-factor pluggable (SFP) Gigabit Ethernet port, with one active at a time
More information about this product is available on the Cisco website.
Product Service and Support
Cisco will provide five years of hardware and software service and support ("SMARTnet") for this product.
Obtaining Your Product
When your request has been approved, you will receive a confirmation email from TechSoup Canada. Your product will be delivered within eight weeks. For details, see The Cisco Donation Program.
- AC input voltage/frequency:
- 100 to 240 V
- 1.6 to 0.8 A
- Environmental constraints:
- Operating temperature: 23 to 104°F (-5 to 45°C)
- Storage temperature: -13 to 158°F (-25 to 70°C)
- Operating/storage relative humidity: 5 to 95 percent (noncondensing)
- Operating altitude: Up to 10,000 feet (3,000 m)
- Storage altitude: Up to 15,000 feet (4,000 m)


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Shipping & Returns
Orders received before 10am, Monday - Friday will be dispatched the following business day. Orders received on a Saturday or Sunday or Public Holiday will be dispatched the next business day.
We ship orders using trusted delivery partners Australia Post & DHL.
Standard Delivery - Free Shipping
Parcels sent within Australia will be sent by Parcel Post or DHL with tracking at no charge with delivery in 2 or more business days after dispatch.
Sending within NSW:
up to 2 business days
up to 5 business days
Sending outside of NSW:
up to 3-6 business days, depending on destination points
up to 7-10 business days, depending on destination points
Express Delivery - $24.95
Express Post may be selected for $24.95, and will be sent within Australia by Australia Post for next business day delivery (if order is received by 10am on a business day).
Express Post Covers 80% of Australian business addresses, private addresses and Post Office Boxes.
The Service operates between all capital cities (except Darwin and in Perth CBD only) and some major centres. Outside metropolitan areas guaranteed service is to and from townships only. If your item is for an address outside the Express Post network, Australia Post will use the fastest possible transport links, but it won't be covered by the next business day delivery guarantee.
International Delivery
International shipping (outside of Australia) for orders up to $699 is $80.
International shipping (outside of Australia) for orders over $700 is complimentary.
We are sorry to hear the product doesn't satisfy your needs.
We understand things happen and you might need to return, exchange or obtain a credit note for the product.
If this is the case, please email firstname.lastname@example.com the receipt, specify the reason and ensure the product has not been used - all packaging must be in the condition you received it. Carlotta + Gee will respond within 24 (business day) hours of receiving your request.
Return and Exchange needs to be within 60 days and a Credit Note is available for 12 months. Please note that the shipping and handling costs will be on the customer sending the items to and from. We'll provide the return address once we receive your email. If the product is faulty then Carlotta + Gee will cover this cost of sending the product and replacing with a new one.
Please note C+G will need to receive the item along with a receipt first. They'll then action within 7 days of receiving the item(s).
Remember we are always available via email for any questions or concerns. firstname.lastname@example.com
Thanks - C + G


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With the recent rescue of the 33 Chilean miners, we've got mining on the brain. How do they create those crazy tunnels deep in the earth? With these spinning, churning, crushing machines of mayhem and destruction, that's how.
For some more tunneling, drilling, and mining fun, check out 9 incredibly impressive mining diagrams, ferocious oil drill bits, the extreme hardware that lets us collect oil and gas, 12 of the world's most fascinating tunnel networks, and some spectacular sewer systems. [Oobject]


text:
Found 1 resultsAuthor Title Type [ Year]
Filters: First Letter Of Title is D and Author is Whistler, Toni [Clear All Filters]
"Detection of Pneumococcal DNA in Blood by Polymerase Chain Reaction for Diagnosing Pneumococcal Pneumonia in Young Children From Low- and Middle-Income Countries", Clin Infect Dis, vol. 64, no. suppl_3, pp. S347-S356, 2017.,


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Egypt security forces arrest top Sinai militant
Egyptian security forces said they arrested Saturday a top wanted militant in the Sinai Peninsula, while authorities reported a failed attempt to disrupt traffic on the strategic Suez Canal but gave scant detail.world Updated: Sep 01, 2013 08:21 IST
Egyptian security forces said they arrested Saturday a top wanted militant in the Sinai Peninsula, while authorities reported a failed attempt to disrupt traffic on the strategic Suez Canal but gave scant detail.
Canal authority chairman Mohab Mamish said a "terrorist element" had tried to disrupt navigation in the waterway by targeting a Panama-flagged ship. In comments carried by official news agency MENA, he said the attempt was "completely unsuccessful" and the container carrier unharmed. He did not say how the ship was targeted.
Authorities have taken extra security measures to safeguard the waterway as lawlessness and violence gripped Sinai, where militants and smugglers rove relatively freely and target security forces and posts.
Mamish said military troops dealt "firmly" with the situation, and that navigation was largely uninterrupted. Earlier Saturday, residents of Port Said, a city that lies along the waterway, said they heard a loud bang. It was not immediately possible to reach witnesses later Saturday.
Egyptian authorities have heightened their security arrangements in recent weeks, following the military coup that ousted Islamist President Mohammed Morsi on July 3.
They accuse opponents of the coup of trying to destabilize the country, and have waged a security crackdown against members of the Muslim Brotherhood, from which Morsi hails, and other allies, arresting hundreds.
Pro-military media and state TV have frequently described the crackdown on Morsi supporters as a "fight against terrorism."
Morsi supporters have held near-daily protests since the coup but deny they are violent. After a bloody dispersal of their major sit-ins earlier this month, however, some retaliated by attacking police stations, government buildings and churches.
In Sinai, violence has only worsened since Morsi's overthrow.
The militant arrested there Saturday is suspected of leading an al-Qaida-linked group in an ambush where 25 off-duty policemen were lined up and shot last week, security officials said. The attack was one of the area's worst militant strikes on security forces.
The man, Adel Mohammed, also known as Adel Habara according to one official, has already been sentenced to death in absentia for killing soldiers in the Nile Delta last year.
Habara's arrest could potentially undermine militant activities in the area, where over two dozen security men have been killed alone since July. The security official said two other suspects were arrested along with Habara.
Authorities have been engaged in a long-running battle against militants in the northern half of the strategic region, which borders the Gaza Strip and Israel.
Earlier Saturday, security officials said 31 suspected militants have been arrested since Thursday, including two caught seeking treatment for wounds sustained in clashes with police.
In other violence nearby, a riot police officer was shot in the chest Friday while on patrol in the city of El-Arish, the capital of North Sinai governorate, the Interior Ministry said Saturday.
According to one security official, four militants have also been killed since Thursday. All officials spoke on condition of anonymity because they were not authorized to speak to the media.
Since the ouster of Morsi, his supporters have been organizing regular protests calling for his return. Several thousand took to the streets on Friday in scattered protests in different parts of Egypt, though demonstrations have largely waned in recent days.
In a sign authorities are responding to the easing of the pro-Morsi rallies, the government once again shortened a military-imposed curfew by two hours, making the evening lock-up in nearly a dozen Egyptian provinces only seven hours.
However, the full 7 p.m. to 6 a.m. curfew will remain in place for Fridays, when supporters of Morsi usually organize large rallies. Hundreds of his supporters, including leading Brotherhood members, have been arrested in a crackdown on the group.
On Saturday, authorities arrested another top figure from the Brotherhood in the Mediterranean city of Alexandria. Former lawmaker Sobhi Saleh was taken into custody on charges of inciting violence and disrupting public order, according to security officials.
The son of another Brotherhood figure, Saad Emara, was also detained and ordered held for 15 days, pending investigations for charges of inciting violence.
Also on Saturday, Health Ministry spokesman Khaled el-Khateeb raised the death toll in violence during Friday's pro-Morsi protests in several cities to eight. El-Khateeb said 221 were also injured - most of them in clashes between Morsi supporters and local residents.
Those killed on Friday included one police officer and one civilian shot dead in a drive-by shooting targeting a police station in an upscale neighborhood of Cairo.


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Traffic sucks, so why not start your morning off with some music? You provide the toast and we'll provide the jams.
Bet you wish someone had thrown a Theremin under the tree for you this year. Float away on a cloud of space age electronic goodness as Clara Rockmore exhibits her virtuosity on one of the most obscure musical instruments of all time.


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Last year, Razer announced their entry into the PC space: The Razer Blade, a laptop which the company touts as "the world's first gaming laptop." Priced at the low cost of $2800, it packs a 17.3″ LED backlit display with a full HD resolution, a 2.8GHz i7 2640M processor, an Nvidia GeForce GT555M GPU, and — now, this is unique — an LCD panel near the keyboard area, along with dynamic, programmable keys above it. Cool, right?
The founder of VoodooPC, Rahul Sood definitely thinks so. After taking the Blade for a spin, he professed some pretty strong positive feelings for the device, stating that the company "takes over where Voodoo left off", going on to praise the device's hardware build quality and design. He also noted that Razer will be able to create an "Apple-esque movement" in the PC space, and here's why:
Besides the amazing craftsmanship, the quality of materials, unique ID, beautiful packaging, and the fit and finish, the Razer brand is something else. Razer built their brand on a thriving community, they have evangelists all over the world who live and breathe their products very similar to Apple in their early days. People overlook this important ingredient too often, the small community Apple once fostered became a movement that helped make them who they are today.
So overall is it worth the ~$2800? – *@#! yes it is.Anyone who compares this to your average "thick-brick-super-hot-loud-laptop-with-faster-hardware" doesn't have a clue what they're missing.


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Education in Malaysia
This is because there is no word for "parody" in Malay.
Avoid falling into the trap of heart!
Schools in Malaysia educate the children primarily about the smatijove, and also to become high achievers; eg. to be able to create the HIGHEST pile of pirated VCDs, the LONGEST line of cows, or even the BIGGEST Ramly burger. To achieve the best of the best results, school children are made to carry huge bags containing books that must weigh at least 57% of said child's own body weight. This builds up their back muscles and backbone so that they will not only look great, but have excellent overall posture. If you disagree with this, you are not patriotic! Note that much like in the military, students are required to wake up at 0600 hours every day. Uniforms are specially designed for dual purposes: firstly, to look as unattractive as humanly possible (because to have clothes that actually fit would be a sin, although some die-hard Malaysians claim that the normal uniform, which covers all parts of the body except the face and the hands, is already far too sexy). The uniform also functions as a portable torture device, trapping students in their own body heat in a country that is already close to 38°C on a normal day. Some schools go the extra mile by forcing some to wear blazers on top of their uniform. Efficiency of the uniform is maximized by strict rules against untucked shirts or rolled-up sleeves. Name tags are also utilized so that teachers don't actually have to remember your name.
The teachers in school have total control of you. They can whip, rotan, and smack you like there's no tomorrow when you do something stupid, such as forgetting to bring your name tag. No, you cannot sue a teacher, because your parents will never be on your side. Rest assured, though, that students do fight back. A popular way of coping with disciplinary action is to take drugs in the toilet, go back to the teacher's office and stab the teacher with a pair of scissors (I am not kidding).
The education system will at one point stream the children according to what they're "good at". Children who are weird or nerdy will be streamed towards science, while those who are too stupid to amount to anything useful will be streamed to the arts. Those who are only good at sports... well, who cares. As you can see, the Malaysian education system is kind enough to dictate your future for you.
In accordance with the institutionalized racism practiced in Malaysia (officially known as "Bumiputra Rights", where special privileges are given to the majority of the population), exam scores do not actually matter in examinations. Often you will see non-bumiputra students getting 7A's for their SPM (the Malaysian equivalent of the SAT's) finally working as pirated vcd sellers, while bumiputra students are given a free ride to study overseas, with their single A's. Malaysia is a one-of-a-kind country; it is probably the only place in the world where one has to fill in their race when sitting for a public examination. Upon writing 'bumiputra', you are automatically granted sixty marks. Bizarrely, non-bumiputras generally still manage to get the highest scores in the country.
After School Tuition is a must, as school teachers are generally too incompetent to teach their students anything useful. All Malaysian students must go to tuition classes to get the latest leaks in exams.
The education system of Malaysia is unique, being the only system in the world to penalize high achievers and reward idiots. Brains are exported overseas while the least brightest are nurtured locally, which is the main reason why Malaysians will be unable to differentiate between left and right by 2020. All Malaysians attend 11 years of compulsory brainwashing at government facilities called public schools. Upon graduating, ethnic Malays either enroll in top-quality world class Malaysian universities or creativity classes such as The Art of Mat Rempit and The Basics of Porno DVD Production, while non-Malays generally get the fuck out of the country.
A student sitting for the SPM examination is analogous to a cow, as this examination assesses the ability of students to swallow and regurgitate information. Subjects can be easily passed by memorizing the textbook without actually understanding anything. While other international examinations are increasing in standard every year, the SPM examination has shown a steady decline since the 70's. The reason is simple: if the standard is lowered, more students (Malays) will be able to score A's, and this would appear as if Malaysians are getting smarter every year.
For example, take a look at actual questions for SPM Math:
- 1960s: The equation 3x^2+px+120=0, where p >0 has roots α and β. α-β=3. Evaluate the value of p and (αβ)^2. Hence, calculate the third derivative of y=3x^2+px+120.
- 1980s: The quadratic equation x^2+px+q=0 has roots -2 and 6. Find the value of (p+q), hence, form a quadratic equation with roots p, q.
- 2000s: A quadratic equation has the roots 2 and 3. State the sum of 2 and 3.
- 2020s: If the number 2 is a color, would you like that color? Why?
The SPM has 6 compulsory subjects: Malay, substandard English, 'Modern' Math, Immoral Education for the kaffirs and Plane Hijacking for Muslims, PseudoScience and Fabricated History.
While Bumis learn how to slam planes into buildings, non-Muslim students have to tolerate 2 years of ineffective brainwashing during Moral lessons. The dynamics behind Moral Studies is simple: if you can memorize a pre-determined definition of a pre-determined "moral value", this means that you are a good person. If you cannot memorize the definitions, then you deserve to burn in hell.
Civics was introduced in 2005 by heroic politicians concerned for students' welfare , as well as teaching the students to love Malaysia ("Malaysia" is synonymous with "National Party") . Through the textbooks, Malaysian students are taught that Malaysia is a peaceful and multi-racial country unlike the US, where only whities exist (incidentally, this is also the lie given to tourists who otherwise could not be convinced to visit Malaysia instead of its slightly less backward neighbour, Singapore). Unfortunately, the lessons have backfired, as students largely use the lessons to catch up on sleep.
To protect young minds from the horrors of real life, "Sejarah", which is a parody of History, is also taught in schools. Topics included are Islam, Islam, the history of Islam, the contributions of Islam, Ways In Which The Sultans Of Old Whored Out The Country For A Few Pieces Of Gold, Malays, contributions of Malays, Why We Like Islam, and a short chapter briefly explaining all other historical events on earth since the beginning of man.
Language Dillema[edit]
Following a policy reversal of teaching Math and Science in English, nation-wide SPM test scores for rural areas increased by 0.005%, which government experts claim has huge significance even though most citizens in rural areas never have, and never will, travel more than 6.5 kilometers outside of their home town. Universities outside the country refuse to accept any SPM candidates and the Malaysian university ranking has plummeted another 20 places, sitting behind Zimbabwe, Nigeria and Somalia.
Cellphones in School[edit]
Malaysia is the one and only country where the Education Minister initially suggested that cellphones be allowed in schools, citing several benefits, only to change his mind just a few days later. Some reasons for the ban are that teachers worry about exposing too much cleavage, only to have the students take a shot and report it. Another worry is that students seem to like sending crude messages and disturbing pictures to each other after finishing their test papers too early and have another 2 hours to wait..
URM (Universiti Rasuah Malaysia)[edit]
Due to high demands, a new campus at Kuala Lumpur is now open. Required education for Datuks, Tan Sris, YB's, etc., both current and future. Courses available are:
- Foundation Study of Rasuah - It only takes a year to build up some basic skills (ie: apple-polishing, bargaining with police, playing golf with big people, and many more), this course will be conducted in Rojak (a mixture of six languages, more practical) and we have experts to guide you.
- Datukship - We teach you the methods to becoming a Datuk, Tan Sri, etc. You need a semester for this lah.
- Making a Fortune - We teach you 'government connections'. Three semesters are needed and you will be guided by ex-politicians/ex-businessmen (senior), to help you make a fortune in Malaysia.
- Foundation of Material Inspection - The one and only course which offers a"close one eye" inspection skill on buildings and amenities. Sign of a successful inspection are leakage, collapsing structures, mess up wires, etc...
- Certificate in Smartijove studies - Apply early as this space is quickily filled up every year. Quota: 99% Malays, 1% non-malays and other indegeneous people.
- Diploma in Racism - The only professor is Babi Hamaludin and nazi ass-is. You will be taught how to fuck over the Chinese and Indians
- Diploma in Politicism - We will teach you how to become a successful and influential politician, how to say "1Malaysia!" while simultaneously fucking the Chinks and Indian bastards up the ass, and how to deal with those annoying Al-Jazeera bitches that keep calling Malaysia an un-democratic country. Those who know how to pronounce "election" as "erection" will have a higher chance of being accepted.
- Ph. D in Mahathirism & Melayu-ism - Doing researches about important Malaysian subjects such as Money Politics, Ketuanan Melayu, Pendatang-ism, Melayu-ism and Dictatorshipism.
For more information, please call Kerishamuddin through mail to Kementerian Seks Malaysia.
IPTA (Public University)[edit]
Standard Bumiputra Rights apply.
Kolej (College)[edit]
Malaysia has private
universities colleges such as Monash and Taylor's Kolej (College). The lecturers speak in half broken english and the mispronounciations are hilarious. They claim to have PHDs and Doctorates but, usually bought from diploma such as Irish International University (which Ireland claims to have no knowledge of). Any questions they don't know the answer to will be answered: "cannot" + "lah" at the end.
Shorts and sandals are considered to be the uniform of most commoners. Shoes and proper clothings that you didn't sleep in are frowned upon. Kolej students spend their time in non-sexual relations such as mamak eating group massacres (ie: a group of 20+ malays to go eat at a local mamak) here they discuss their school work and watch wrestling.
Kolej in Malaysia is equivalent to a flea market in the hood; people are most likely to sell you something in between classes. They'll have excuses like "it's for charity" or some shit but we all know the money goes to the Chinese cook thats grinning behind the cafeteria booth. Everything costs extra money; your fees are not enough.
DISCLAIMER: The chinky chong who wrote this article has no PhD himself, neither has he done any research or has any publications in his life, but reserves the right to criticize the rest of the country. What about his broken english you say? It's called "chinklish", which he thinks is proper. To justify himself, Chinky ass here carries a certificate from his chinky sharman that says he has the widespread Bodohland-CHINK slit eye disease called Syok sendiri aka head up own ass syndrome.
DISCLAIMER-DISCLAIMER: The above racist fascist pig has obviously no merit to his argument and thus you may now disregard it and retain your (obviously biased) view on local Unis in Malaysia. He is also probably Malay and therefore does not recognize 'Freedom of speech' for other people, though he obviously exercises his freedom of speech. He probably thinks it's his Allah-given right.
THIRD DISCLAIMER: The shorts and sandals thing is true, though. Also I've fixed up the English a bit.
Art Colleges[edit]
Art students in Malaysia are not allow to study human body drawing, it was banned some 20 years ago because the government see it as GATAL (erotic, dirty and lustful). No no, you are not allow to see nudes (that explains why the country is so desperate about pr0n on the internet). If you go to an art college in Malaysia, human anatomy sessions mean drawing male models wearing football pants. The Malay-Muslim art students are also not allowed to draw women wearing the Malay traditional sarong, because that is also consider GATAL. Today, there is a controversy going on. Malay-Muslim art students came up with a brilliant idea of drawing invisible women dressed only in Victoria Secret panties and bras. Whether or not this will be allowed or banned, we shall know in the near future.
Government Scholarship[edit]
Again, standards Bumiputra Rights apply.


text:
Stephanie J. Ouellette
1954 - 2020
Plant a Memorial Tree
Celebrate the memory of someone special by planting trees in their name. Your gift will serve as a lasting, living memorial, while preserving the health of forests in areas of critical need. We have partnered with American Forests and Canadian Institute of Forestry for the planting of our memorial trees. Your gift will also include a digital certificate that will be emailed to you.
|Plant Memorial Tree
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CVV2 is a security measure we require for all transactions. Since a CVV2 number is listed on your credit card, but is not stored anywhere, the only way to know the correct CVV2 number for your credit card is to physically have possession of the card itself.
Visa, MasterCard & DiscoverVisa, MasterCard & Discover cards show the CVV2 in the signature area on the back of the card. (it is the last 3 digits AFTER the credit card number).
American ExpressAmerican Express cards show the 4 digit CVV2 printed above and to the right of the imprinted card number on the front of the card.
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224 Milton Ave.
Ballston Spa, New York 12020
518 885-7110
Wilton Funeral Home Inc
395 Ballard Road
Wilton, New York. 12831


text:
Jennifer Lopez says she loves herself enough to walk away from a bad marriage.
In her first interview since splitting from hubby Marc Anthony after seven years of marriage, Lopez tells Vanity Fair she's still a "hopeless romantic and passionate person when it comes to love."
"It's not that I didn't love myself before," the thrice-married performer tells Vanity Fair for its September cover story. But she says, "sometimes we don't realize that we are compromising ourselves."
U.S. & World
"To understand that a person is not good for you, or that that person is not treating you in the right way, or that he is not doing the right thing for himself—if I stay, then I am not doing the right thing for me," Lopez adds. "I love myself enough to walk away from that now."
According to E!, which published more transcripts of the interview, Lopez, 42, confirms reports that she and Anthony were constantly fighting.
"Marriage is tough," she told VF. "It's not going to be perfect...It's definitely challenging."
The Bronx-born actress, singer and likely returning "American Idol" judge was previously married in 1997 to restaurateur Ojani Noa and in 2001 to back up dancer Cris Judd.
Besides revealing her thoughts on marriage, Lopez also talks about her past high-profile relationship with P. Diddy and broken engagement to Ben Affleck.
Despite the failed relationships, Lopez calls love her "biggest dream" and says she remains an "optimist."
She also tells VF that Anthony – the father of her 3-year-old twins -- will remain in her life. According to TheWrap.com, Lopez is sticking with the new Latin American talent competition she's producing with her pop star ex.
"I will always respect Marc as a singer and performer," Lopez tells VF. "We actually work great together, and he was always very supportive. Together we could make magic—and we did. He will always be in our lives. He will always hold a special place in my heart as the father of my children."
Check out Lopez's full interview when the new issue of Vanity Fair drops Wednesday.


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Eurodisco star Gina T is performing Friday, November 12th at the Club 740 in Downtown, Los Angeles in a concert sponsored by Keep On Music (KOM), an organization committed to preserving and spotlighting Italo/Eurodisco -- often aka Vietnamese New Wave.
Gina T is a gold and platinum singer as well as songwriter for other artists. She was born Gina Tielman on October 24, in Bussum, Noord-Holland, Netherlands to Indonesian parents. Music runs in her family; a grandfather, an uncle and her father, Ponthon, were members at various times of The Tielman Brothers, an amazing Indorock band.
Her mother, Joyce, was also a famous singer and her brother Nino (aka Mr. Double T) was involved in T'N'T Partyzone, Die Kranken Schwestern and Culture Beat.


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What if, instead of hanging out with Ted Turner and going back in time to fight Hitler, Captain Planet and the Planeteers were the stars of an R-rated anime in which they threw out their sweet vests and became disaffected eco-terrorists hooked on endangered, psychoactive toads?
Illustrator Benjamin Ang has imagined such a situation where perhaps this amphibian addiction is the case. Look how disgruntled Ma-Ti is for having the most mediocre power! I bet he only summons cone shells. Also, Captain Planet eats a hydroelectric dam whenever he's summoned. It's in his contract.
Related: Don Cheadle as Captain Planet.
G/O Media may get a commission
[Via Comics Alliance]


text:
We all know that loyal co-worker who gave his or her all while working. Year after year, this person never failed and is now retiring. Celebrate him or her with an honorable retirement save the date magnet with artsy banner, announcing the great legend and the upcoming party. Invite the whole company with these stylish magnet invites.
Measurements: 3.5" X 5"
Thickness: 25-mil
Finish: Glossy/Digital Print
Includes: 4.5" x 5.75" Envelopes
Select your quantity from the available options in this listing. Magnets are printed in units of 12 and up. The following custom fields are available for this design. Please list your personal party information in the Customization Area on the right.
The information we need:
- Name(s) on Magnet:
- Date of Event:
- Time of Event:
- Years of Service:
Other info to be printed on magnet (optional - list only if needed)
- Location of Event
- Invitation to Follow: Y/N
- RSVP number:
- Other text: (optional - anything we missed)
Extra Stuff: We know you want to see what your magnet will look like! You will receive an e-mail proof of your design, typically within 24-48 hours after your order has been placed. If you do not get an email, check your spam folder.
We will not print until you say you are happy! Remember, you must approve your proof before we will print your order. If we do not hear from you, we will try to reach you by phone. Customer is fully responsible for final proofreading approval prior to print. Customer is fully responsible for final proofreading approval prior to print.
*Be sure to see our FAQs Section and Terms of Service for answers to commonly asked questions, rights and obligations.
A Great Legend Retirement Save The Date Magnets
Magnets and Included Envelopes
Minimum Order is 12
Click Quantity Arrow for More Options
Sold in Units of 12 Up To 120


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By creating an account at a touch of Dutch you will be able to shop faster, be up to date on an orders status, and keep track of the orders you have previously made.
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1 x Aardappel Puree 1 x Chili con Carne (base) 1 x Nasi Speciaal (base) 1 x Custard 1 x Sambal Manis 1 x Butter Almond Christmas Stollen 1 x Kippensoep 1 x Sambal Brandal 1 x Soep Pakket$60.03
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What's New?[more]Onsite CafeCome down and say hiGift WrappingGift Wrapping availableCall UsOn : +61 3 9769 7999Fast ShippingFast package delivery
a touch of Dutch Online was the natural 'next step' for our business as demand for our products grew rapidly over the years. Launched late 2013 we have undergone a facelift in early 2015 to cater for mobile shoppers as well.
Please have a look around and make sure to keep checking back as we're always changing and as always; have fun! :)InformationAccount InfoContact Details
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text:
hi, permission to post
i used/still using in my 7a-ge:
flywheel from 5af, lightened and balanced to 6kg
Clutch from 4af
stock pistons of Honda Sir (High Compression)
4ag bigport head minus tvis (ported and polished)
red top stock fuel injector (green)
red top stock ecu
fabricated phantom grip LSD
timing belt from Nissan (part# 1177BS25 13028-54AO1 however, you need to trim about 5mm from one edge as original is wider)
Jasmag 4-2-1 exhaust
finally stock cams of blue top.
FRANKENMOTOR as they say.
I've read through the whole thread and done a fair bit of searching, I know you use the Porsche 944 timing belt on a 7A-GE 16v with the 7A-FE oil pump however I can't seem to find which timing belt to use with the 16v oil pump? I know they're 116 teeth instead of 117 like the 944, but can't locate a part number.
Any one got some info for me?
Gates T018.


text:
Welcome to the CRG Discussion Forum!Forum registration problems: Make sure you enter your email correctly and you check your spam box first. *Then* email KurtS2@gmail for help.Classified ads are not allowed on the forum.
709660, this vin is about 30 some numbers away from my copo, i am curious as to what this car might be
any information on this car 124379N694229...Eddie
I took the time and added all the loose POP's to the list. About 500 cars on the list.


text:
I have been using PHW V1 and all seems good, but if I were to try and deploy it via a web page so customers could receive support, it all seems to rely on them being in a windows environment. Is there (are any for) a Java based server that the customer can download as a web page so it will work on mac, linux or other environments like a more traditional VNC environment?
I see most of the other systems out there like gotmypc, beamyourscreen etc use a Java install instead of running an exe. Any thing in development for PHW?
The setup I'm using is to use a repeater so any java "server" client need to implement this?


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Mommy Makeover
A "Mommy Makeover," is a group of surgeries designed to change the physical effects of childbirth and bring the body back to how it was before the pregnancy. Post-pregnancy plastic surgery, skin treatment, and cellulite removal are all combined in a custom procedure, tailored to your needs.
Mommy Makeovers are totally personalized plans for your treatment. Consultation with Dr. Peled will let you find the best way to proceed, based on your aesthetic preferences, financial situation, overall health, and how much time you are willing to devote to this project. This is not intended for weight loss, but will help you regain your original, pre-pregnancy body.


text:
Student Classifications
Curricular Student
Individuals are classified as curricular students when they declare a major, that is, when they are admitted to a curriculum of the College. The student must be a high school graduate, have earned a General Educational Development (GED) diploma, have completed an approved developmental program, or have been otherwise determined qualified for admission. The applicant's academic record must contain all of the information required for admission to the College. A curricular student may be either a full time or part-time student working toward completion of a certificate or associate degree at the College.
Noncurricular Student
Students who have not requested admission to a curriculum may still enroll in courses by identifying the reason for enrolling at NOVA. Students are expected to declare a major prior to completing 30 credit hours of coursework. Students may be classified as noncurricular for purposes of the following:
- upgrading employment skills for one's present job
- developing skills for a new job
- exploring a new career
- seeking personal satisfaction or general knowledge
- taking developmental studies courses
Other noncurricular students may include the following:
- a transient (visiting) student. Such students may be enrolled at NOVA while maintaining primary enrollment with another college or university.
- a nondegree transfer student. Such students may be enrolled at NOVA to take only a certain number of courses for transfer to another college or university prior to completing the graduation requirements of a specific curriculum at NOVA.
- a high school junior or senior or the home school equivalent. See Dual Enrollment for more information.
- a student with general or curricular requirements pending. The student may not have met all of the general or specific admission requirements as stated in the College Catalog but may be accepted by the College to take courses for one semester only, with approval from the College.
- a student whose desired program has restricted enrollment. The student may meet admission requirements of a specific curriculum but be temporarily denied entry because of an enrollment limitation. The student may enroll in other courses while waiting for entry into the chosen curriculum, with approval of the College.
Full-Time Student
A full-time student is enrolled in 12 or more credits of coursework in a semester or during the summer.
Part-Time Student
A part-time student is enrolled in fewer than 12 credits of coursework in a semester or during the summer.


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TUFF rolls onto Toronto subway platform screens, Sept. 6 – 16
The Toronto Urban Film Festival, North America's largest commuter film festival is rolling out its seventh annual edition. For 11 days, from September 6 – 16, 2013, Toronto's subway system will become a city-wide underground theatre, screening the world's best one-minute, silent films to over one million daily commuters. For more information on all films and a screening schedule, download a pdf of the 2013 printed program.
TUFF will screen 82 outstanding silent films from 20 countries on the Pattison Onestop network of screens located in subway stations across the city. Films play every 10 minutes on all 290 screens. In addition, commuters can watch TUFF uninterrupted at designated "Film Zone" screens on the north and south platforms of the Bloor subway station, as well as at Dundas and St. Andrew subway stations.
New this year, 'TUFF Above Ground' will animate the Toronto downtown cityscape from September 9 – 15, 2013 with Subdivision by media artist David Clark. Clark's series of six eight-second videos, commissioned by TUFF, will be featured on Pattison Outdoor's largest media beacon, located at the corner of Yonge and Edward Streets, just north of Dundas Square, an area that experiences more than 100,000 pedestrians on a daily basis.
TUFF's Spotlight program on Friday, September 13th highlights the work of experimental filmmaker Christina Battle through a new series of four short videos. The series, titled we're not exactly sure what just happened, offers an alternative visual backdrop to the essentially meaningless transcripts from CNN's reporting on April 19, 2013 while the city of Boston was on lockdown and police searched for the then at large Boston City Bomber. This series was commissioned by TUFF.
'For the Love of Animation' on Saturday, September 14th includes two noteworthy programs: '4 from TAAFI', a co-presentation with the Toronto Animated Arts Festival International; and a specialAnimation Spotlight presenting four works from the award-winning, Burnaby B.C.-based multimedia artist Dimitri Kozma.
This year's festival also includes 'Too TUFF for the TTC', an online-only program of three great films not appropriate for screening in a public transit setting. All TUFF films can be seen online, and viewers are encouraged to cast their vote for the Viewers' Choice Award from September 4th to 12th. The top 10 Viewers' Choice films play on the TTC on Sunday, September 15th.
More informations please check : http://www.torontourbanfilmfestival.com
To mark the end of another successful year, the award-winning films will screen on Monday, September 16th, highlighting the brilliant work of TUFF's talented filmmakers.
Returning to the festival is The Screening Room at The Drake Hotel. Relax, kick-back and enjoy 85 minutes of back-to-back TUFF from Friday, September 13th to Sunday, September 15th from 11:00 am to 5:00 pm in Room 222. Admittance is free.
TUFF is co-produced by Pattison Onestop and Art for Commuters, and is funded by NBCUniversal Canada and the Ontario Arts Council.
Where to find TUFF: http://www.torontourbanfilmfestival.com