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Shares of Boston, Massachusetts based Wayfair Inc. (W) fell sharply on Tuesday amid a strong advance in stocks across the board. Wayfair's stock fell 15.80%, sinking $4.44 per share, to close at $23.66 on volume of 2,331,513 shares. The company reported widening losses against substantially higher revenues for the third quarter, compared to the same period in 2013.
Founded on August 8, 2002, Wayfair, Inc. operates an online home furnishing store. It offers a selection of home furnishings and decor across all styles and price points. The company operates an e-commerce business model, which offers visually inspiring browsing, compelling merchandising and easy product discovery for various products across five distinct brands, including Wayfair.com, Joss & Main, AllModern, DwellStudio and Birch Lane. For 2013, Wayfair had total revenues of $915.84 million, and a net loss of -$40.91 million, although sales growth was 52.4% over the previous year. The company's stock trades on the NYSE.
The company announced it's third quarter results earlier in the week, that showed a mixed picture. Despite the sharp increase in total revenues, as well as a strong increase in its customer base, the company's net loss expanded to -$24.1 for the most recent quarter, verses a loss of just -$3 million for the same quarter a year earlier.
The company launched its IPO on October 2, opening with a price of $36 per share. As Tuesday however, the stock has fallen by more than a third in just a few weeks. Despite the strong increase in total revenues, investors were focusing primarily in the expanding net loss.
On Monday, Wayfair announced it's third quarter 2014 earnings results. For the quarter ended September 30, the company reported total revenues of $336.2 million, up 41.7% over the $237.3 it reported for the third quarter of 2013. Year-to-date total revenues increased to $910.3 million, compared to $620.5 for the same period in 2013.
However the net loss from operations expanded to -$24.1 million in the third quarter, compared a loss of just -$3 million for the third quarter of 2013. For the nine months ended September 30, the year-to-date loss from operations totaled -$75.5 million, compared to just -$11.4 million for the same period in 2013. Investors reacted to the news by dumping the stock.
Still the company is accentuating the positive. Direct Retail Revenue grew to $285.5 million, a 57.1% increase over the $181.7 million for the third quarter of 2013. In addition, the company reported that the number of active customers in the Direct Retail business reached 2.9 million as of September 30, 2014, up 61.0% year over year.
"Moving forward, we remain highly focused on driving growth by acquiring new customers and increasing repeat purchases from existing customers," said Niraj Shah, Wayfair CEO, Co-Chairman and Co-Founder. "As in the past, we intend to do this by investing in our brands and the customer experience through improved site experiences, product offering and logistics. Overall, we are excited about our performance in the quarter and the strong continued growth at Wayfair."
Other News About Wayfair
Wayfair Can Compete With Amazon In Home Products
Wayfair can carve a niche in the home products market.
Wayfair Announces Closing of Initial Public Offering and Full Exercise of Underwriters' Option to Purchase Additional Shares
Wayfair announced the closing of its previously announced IPO of 12,650,000 shares.
Other Stocks in the News
JPMorgan Chase (JPM) Stock Is Declining Today on Analyst Downgrade
Bank was downgraded to "market perform" from "outperform" by analysts at Keefe Bruyette.
Why Sprint Is in Trouble
The nation's third-largest carrier is losing customers left and right.


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From Early Man to Adam Smith: Economics Explains World History!
Submitted by TimDaly on Thu, 08/02/2012 - 1:02pm
Session describes active-learning lessons that show how middle school students can use economics to better understand societies throughout world history. Many lessons are adaptable to high school use.
Sat, 11/17/2012 - 10:30am - 11:30am
John LeFeber, Council for Economic Education, New York, NY
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subset: commoncrawl


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This home was amazing! It was just the right size for our family, being able to make our own meals, using the pool when we wanted and just really feeling comfortable as if we were in our own home. We really enjoyed it and will be coming back next year in March! You will truly have fun!
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Thoroughly enjoyed my stay in this lovely villa. Very spacious, clean and comfortable. We loved the pool and spa area especially. The pool is perfect for families of all sizes. Also in a very convenient area regarding nearby shops/restaurants etc. Walmart virtually at the end of the estate which really helped. Overall, a fantastic holiday and stay in this villa! Would highly recommend!!


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Happy Friday!! This week I am sharing what it's like to raise a boy. This is all just for fun no serious-ness and a little glimpse into my life in "boyland". Let me know what you think.
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Movie night is our son, Caleb's, favorite night of the week. It's it also the hubs favorite night as well, but you didn't hear that from me, LOL. So this week I wanted to share with you what it looks like for us here at 523. I even put together an outfit to correspond with our 101 Dalmatians movie. Let me know what you think.
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Happy Friday!! This week I am linking up a recipe for pork tenderloin cooked in the crockpot. 4 ingredients is all it takes for this easy dinner.
Stopping in to say hello and a warm welcome from The Girlz Korner.


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The invention relates to the field of glycoprotein processing in transgenic plants used as cost efficient and contamination safe factories for the production of recombinant biopharmaceutical proteins or pharmaceutical compositions comprising these. The invention provides a plant comprising a functional mammalian enzyme providing N-glycan biosynthesis that is normally not present in plants, said plant additionally comprising at least a second mammalian protein or functional fragment thereof that is normally not present in plants.
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28. A whole plant comprising a gene sequence which encodes and stably expresses a mammalian β1,4 galactosyltransferase and a gene sequence which encodes and stably expresses an antibody or a functional fragment thereof, wherein the plant produces an antibody or a functional fragment thereof comprising a galactosylated N-linked glycan.
29. The whole plant of
30. The whole plant of
31. The whole plant of
32. The whole plant of
33. The whole plant of
34. The whole plant of
35. The whole plant of
36. The whole plant of
37. The whole plant of
38. The whole plant of
39. The whole plant of
40. A transgenic plant cell which comprises a gene sequence which encodes and stably expresses a mammalian β1,4 galactosyltransferase, wherein the plant cell is capable of regenerating into a whole plant.
41. The plant cell of
42. The plant cell of
43. The plant cell of
44. The plant cell of
45. The plant cell of
46. The plant cell of
47. The plant cell of
48. A method for producing a whole plant which produces an antibody chain or a functional fragment thereof comprising a galactosylated N-linked glycan, the method comprising the steps of:
regenerating a whole plant from a transgenic plant cell, wherein the cell comprises a stably expressed gene sequence which encodes a mammalian β1,4 galactosyltransferase: and crossing the regenerated plant with a further transgenic plant which expresses a recombinant antibody or a functional fragment thereof.
49. The method of
50. A method for obtaining an antibody chain or functional fragment thereof comprising a galactosylated N-linked glycan, the method comprising the steps of: fractionating the harvested material of
51. The method of
The invention relates to the field of glycoprotein processing in transgenic plants used as cost efficient and contamination safe factories for the production of useful proteinaceous substances such as recombinant biopharmaceutical proteins or (pharmaceutical) compositions comprising these.
The creation of recombinant proteins as e.g. medicaments or pharmaceutical compositions by pharmaco-molecular agriculture constitutes one of the principal attractions of transgenic plants; it is also the domain where their utilization is accepted best by the public opinion. In addition to the yield and the favourable cost which may be expected from the field production of recombinant proteins, transgenic plants present certain advantages over other production systems, such as bacteria, yeasts, and animal cells. Indeed, they are devoid of virus which might be dangerous to humans, and can accumulate the proteins of interest in their �organs of storage�, such as seeds or tubers. This facilitates their handling, their transportation and their storage at ambient temperature, while affording the possibility of subsequent extraction according to needs. Moreover, the transgenic plant, or some of its parts, can be utilised as vector of medicaments or of vaccines. In 1996, the team of Charles Arntzen (Boyce Thompson Institute for Plant Research, Cornell University, New York) has demonstrated the production of a recombinant vaccine against the thermolabile enterotoxin of Escherichia coli by the potato. Its efficacy has been demonstrated in mice and through clinical trials carried out on volunteers having consumed 50 to 100 grams of raw transgenic potatoes over a period of six months. Another team, at Loma Linda, in California, has successfully tested in mice a vaccine against cholera formed in the potato. Traditional vaccination against germs responsible for enteropathies is regarded as �too costly� to be generally implemented in developing countries. However, the production of oral vaccines for example no longer in the potato but in the banana, would, at a very low cost, enable general implementation of vaccination against diarrheas of bacterial origin, which cause the death of three million children every year. In the developed countries, one can imagine that children would certainly prefer a banana or strawberry vaccine to the doctor's needle. More generally, molecular pharming could enable developing countries to produce, at low cost, substantial quantities of therapeutic proteins utilizing the capacities of their agriculture, without it being necessary to invest in pharmaceutical factories.
Although the advantages of plants as factories of proteinaceous substances are explained mostly in the light of biopharmaceuticals, plants are also useful for production of other proteins, e.g. industrial enzymes and the like, because of their capability of glycosylation leading e.g. to higher stability. Today, the utilisation of plants for the production of proteins or glycoproteins for therapeutic use has gone widely beyond the domain of science fiction since soy, tobacco, the potato, rice or rapeseed is the object of investigations for the production of vaccines, proteins or peptides of mammals such as: monoclonal antibodies, vaccine antigens, enzymes such as canine gastric lipase, cytokines such as epidermal growth factor, interleukins 2 and 4, erythropoietin, encephalins, interferon and serum albumin, for the greater part of human origin. Some of these proteins have already proven their efficacy in human volunteers, however, their potential immunogenicity and their possible allergenic character still restrict their development. Several heterologous proteins have successfully been produced in plants. Among these proteins are monoclonal antibodies, hormones, vaccine antigens, enzymes and blood proteins (Dieryck et al., 1997; Florack et al., 1995; Ma et al., 1995) Matsumoto et al., 1163; Saito et al., 1991; Thanavala et al. 1995) A major limitation of plants, shared with other heterologous expression systems like bacteria, yeast and insect cells, is their different glycosylation profile compared to mammals. In contrast to bacteria, having no N-linked glycans, and yeast, having only high mannose glycans, plants are able to produce proteins with complex N-linked glycans. Plant glycoproteins have complex N-linked glycans containing a α1,3 linked core fucose and β1,2 linked xylose residues not found in mammals (Lerouge et al., 1998) (
In short, analyses of glycoproteins from plants have indicated that several steps in the glycosylation pathways of plants and mammals are very similar if not identical. There are however also clear differences, particularly in the synthesis of complex glycans. The complex glycans of plants are generally much smaller and contain beta-1,2 xylose or alpha-1,3 fucose residues attached to the Man3 (GlcNAc)2 core. Such residues on glycoprotein are known to be highly immunogenic. This will cause problems for certain applications of recombinant proteins carrying these sugars. In addition, although common and often essential on mammalian glycoproteins, sialic acid has never been found in plant glycans. This is particularly relevant since experiments have shown, that the absence of terminal sialic acid on glycosidic side chains can dramatically decrease biological activity in vivo. Most likely, asialo-glycoprotein-receptors in the liver can bind to asialo-glycoprotein, and thereby cause a clearance of the glycoprotein from the circulation, which is reflected in a reduced metabolic half life and low bioactivity in vivo.
The invention provides a plant comprising a functional mammalian enzyme providing N-glycan biosynthesis that is normally not present in plants. It is especially the �plant� character of the glycans that makes glycoproteins produced in plants less suited for pharmaceutical use. This �plant� character imparts undesired antigenic and immunogenic characteristics to the glycoprotein in question, which would require a strategy intended to prevent immunogenicity of glycoproteins produced by transgenic plants. The aim of the strategy is to modify the genome of vegetable cells in such a manner that they ripen their proteins like human cells would. Numerous genes of glycosyl transferases of mammals have already been cloned, which is not the case in plants. In view of the ease of transformation of vegetable systems, the temptation is strong to �complement� the Golgi apparatus of plants by glycosyl transferases from mammals in order to �humanize� or �mammalize� the glycans of the glycoproteins they produce. The success of such a strategy is nonetheless not evident. In particular, the galactosylation and subsequent sialylation of recombinant glycoproteins in a vegetable cell depends not only on the transfer and the expression of the gene of the galactosyl and the sialyl transferase: these foreign enzymes must also be active in the vegetable cell, without detrimental effects to the plant cell, and last but not least, without detrimental effects to the transgenic plant as a whole.
To mammalise the glycosylation of plant for the production of tailor made glycoproteins in plants a xylosyltransferase and fucosyltransferase can be knocked out and at least one of several mammalian glycosyltransferases have to be expressed. Providing the xylosyltransferase and fucosyltransferase knock-outs and thereby reducing the unwanted glycosylation potential of plants is a feasible option because for example an Arabidopsis thaliana mutant mutated in the gene encoding N-acetylglucosaminyltransferase I was completely viable (Von Schaewen et al., 1993). As N-acetylglucosaminyltransferase I is the enzyme initiating the formation of complex glycans (Schachter, 1991), this plant completely lacks the xylose and fucose containing complex glycans.
In a preferred embodiment, the invention provides a plant comprising a functional (mammalian) protein, e.g. a transporter or an enzyme providing N-glycan biosynthesis that is normally not present in plants additionally comprising at least a second mammalian protein or functional fragment thereof that is normally not present in plants. It is provided by the invention to produce in plants a desired glycoprotein having a mammalian-type of glycosylation pattern, at least in that said glycoprotein is galactosylated. Again, desired glycoproteins may be any useful glycoprotein for which mammalian-like glycosylation is relevant.
In a preferred embodiment, the invention provides a plant according to the invention wherein said functional mammalian enzyme providing N-glycan biosynthesis that is normally not present in plants comprises (human) β1,4-galactosyltransferase. An important mammalian enzyme that is missing in plants is this β1,4-galactosyltransferase. cDNA's encoding this enzyme has been cloned from several mammalian species (Masri et al., 1988; Schaper et al., 1986). The enzyme transfers galactose from the activated sugar donor UDP-Gal in β1,4 linkage towards GlcNAc residues in N-linked and other glycans (
Also modification of the N glycosylation machinery of immortalized tobacco BY2 cells has been reported. Introduction of GalT into this cell line results in the production of galactosylated N-linked glycans of endogenous proteins Palacpac et al., 1999). However, cells from this BY2 cell line can not be regenerated into viable tobacco plants. In addition and as described elsewhere in this patent application, the largest population was an abnormal hybrid type glycan (GlcNAc2Man5GlcNAcGal) suggesting premature action of the introduced galactosyltransferase and an abnormal Golgi morphology and localisation of the calactosyltransferase in the BY2 cell line. This provides further evidence that this cell lines is significantly different from normal tobacco plant cells. No reports have been made until now wherein a fully grown. non-mammalian organism such as an insect or plant, is disclosed having the capacity to extend an N-linked glycan, at least not by the addition of a galactose. Surprisingly, the invention now provides such a non-mammalian organism, a plant having been provided a a (functional) mammalian enzyme providing N-glycan biosynthesis that is normally not present in plants thereby for example providing the capacity to extend an N-linked glycan by the addition of a galactose. In a preferred embodiment, the invention provides such a plant wherein said enzyme shows stable expression. It is even provided that beyond said second mammalian protein a third mammalian protein is expressed by a plant as provided by the invention. The experimental part provides such a plant that comprises a nucleic acid encoding both an antibody light and heavy chain or (functional) fragment thereof. Of course, it is not necessary that a full protein is expressed, the invention also provides a plant according to the invention expression only a fragment, preferably a functional fragment of said second mammalian glycoprotein, said fragment having at least one activity of the whole protein and further being characterised by for example a truncated polypeptide chain, or a not fully extended glycan, for example only extended with galactose. In a preferred embodiment, the invention provides a plant according to the invention wherein said second mammalian protein or functional fragment thereof comprises an extended N-linked glycan that is devoid of xylose and/or of fucose. As can be seen from
The N-terminal cytoplasmic, transmembrane and stem region of glycosyltransferases determine the localisation of the enzyme in the ER or Golgy membrane. To provide natural or desirable glycosylation, glycosyltransferases can be expressed in plants as they occur in mammals, but can also be expressed as a fusion protein between two, or part of two, different glycosyltransferases. In this case the localisation is determined by one enzyme and the catalytic activity by a second enzyme. As example, a fusion between the cytoplasmic, transmembrane and stem region of plant xylosyltransferase and the catalitic domain of mammalian galactosyltransferase, providing an enzyme with galactosyltransferase activity and localisation of the xylosyltransferase.
If one would desire to further separate glycoproteins comprising extended N-linked glycan that is devoid of xylose and/or of fucose, or to produce these in a more purified way, several possibilities are open. For one, several types of separation techniques exist, such as (immuno)affinity purification or size-exclusion chromatography or elecrtrophoresis, to mediate the required purification. Furthermore, another option is to use as starting material plants wherein the genes responsible for xylose and/or fucose addition are knocked-out.
In another embodiment, the invention provides a plant according to the invention wherein said N-linked glycan comprising galactose is further comprising sialic acid added thereto. In particular, the transfer of genes coding for sialyl transferases, enzymes which catalyze the addition of sialic acid on the glycan, into vegetable systems leads to even more stable glycoproteins during in vivo usage and hence better adapted to a possible therapy. The invention herewith provides the transfer of a sialic acid biosynthesis pathway to plants. In this invention when referring to plants the whole spectrum of plants ranging from algae to trees is intended unless otherwise specified. Plants in general lack sialic acid, a sugar residue needed for the enhanced function of certain glycoproteins like antibodies and hormones, in their N-linked glycans and also the substrates for sialylation have never been found. The invention provides plants that have the capacity to produce NeuAc containing N-linked glycans on their proteins. To establish this, up to 5 different heterologous genes are expressed in plants (see Table 1). To provide plants with the biosynthetic capacity to produce sialic acid, genes encoding up to five enzymes acting in the sialic acid biosynthesis pathway are transformed to plants. These enzymes from bacterial and mammalian origin are known: GlcNAc-2 epimerase, NeuAc synthase, CMP-NeuAc synthetase, CMP-NeuAc transporter and NeuAc transferase. All genes encoding the enzymes are if desired supplied with a (FLAG) tag to follow expression, and are transformed to e.g. tobacco and corn.
In another preferred embodiment, the invention provides a plant according to the invention wherein said N-linker glycan comprising galactose is further comprising or extended with glucoronic acid, glucoronyl, sulfate, sulfon, fucose, or other compound capable of extending galactose with linked to said galactose. This is particularly relevant since experiments have shown, that the absence of terminal sialic acid on glycosidic side chains can in general dramatically decrease biological activity in vivo. Most likely, asialo-glycoprotein-receptors in the liver can bind to asialo-glycoprotein, and thereby cause a clearance of the glycoprotein from the circulation, which is reflected in a reduced metabolic half life and low bioactivity in vivo. The presence of for example GlcA or another extending group but sialic acid has the same effect as the presence of sialic acid, it hinders the binding of a thus modified protein to the asialo-glycoprotein receptor of for example liver cells, thereby effectively increasing half-life, and thus clearance time, of such proteins, when used as therapeutic substance, i.e. as pharmaceutical composition. The invention thus provides an organism derived, herein in particular a plant-derived glycoprotein or functional fragment thereof comprising an extended N-linked glycan at least comprising galactose, said galactose further extended with a compound capable of extending galactose with, such as Glca to function in a similar way as silaic acid. For example, the invention provides plants that have the capacity to produce GlcA containing N-linked glycans on their proteins. To establish this, a gene encoding for example glucuronyltransferase (Terayama et al., PNAS 94:6093-6098, 1997) is expressed in plants according to the invention using methods known in the art or herein disclosed.
In this aspect, the invention is not limited to plants but also provides other organisms like animals, fungi or yeast, or cell lines like mammalian cell lines or insect cell lines with the capacity to produce a glycoprotein (essentially non-sialiated) according to the invention wherein said N-linked glycan comprising galactose is further comprising or extended with for example glucuronic acid linked to galactose; which in essence has the same effect as the presence of sialic acid. The invention is not limited to extending the galactose by glucuronic acid which has the essentially the same effect as the presence of sialic acid in that it increase biological half-life and clearance time. Also sulfate, fucose or any other compound can be linked to galactose, thereby extending the carbohydrate group, by expressing a sulfotransferase, fucosyltransferase or other enzyme that transfers sulfate, fucose or other compound to galactose residues can be used to increase half-life. The invention thus provides a method to increase half-life or improve clearance time of a pharmaceutical composition comprising as active component a glycoprptein, comprising providing said glycoprotein with a compound, attached to galactose, that replaces or provides sialic acid function and thus provides at least reduced reactivity with a asialo-glycoprotein-receptor, preferably wherein said receptor is at least present on a liver-cell. Also more than one compound can be transferred to galactose, for example glucuronic acid that is extended by sulfate by expressing a sulfotransferase that transfers sulfate to glucuronic acid. The invention is not limited to those cases in which extension of galactose by other compounds than sialic acid has the same effect as extension with sialic acid. Extension of galactose by other compounds than sialic acid can have a function by its own for example in interaction with other compounds, cells or organisms. Furthermore, it has the advantage that components, otherwise extended by sialic acid, but now for example with glucoronic acid, or sulfate of fucose groups, for that matter, can easily be recognised and thus distinguished from like endogenous compounds extended with sialic acid. For example, a pharmaceutical composition comprising a glycosylated protein, such as a glycoprotein hormone, or erytrhopoetin (EPO), normally provided with sialic acid, but now with for example a sulfon, or with glucoronic acid, can easily be recognised, facilitating detection of the foreign compounds. As an example,
Extending galactose with other compounds than silalic acid can also have advantages for the production of recombinant proteins in plants. It can make the glycoprotein or glycan of the glycoprotein more stable by preventing galactosydases and/or other glycosydases from degrading the N-glycan. It can, by doing that, increase the galactosylation. It can also be of use in a purification procedure, for example by facilitating affinity purification by specific antibodies, lectins or other compounds if desired, the compound by which galactose is extended or further comprised can, after purification of the recombinant glycoprotein, be removed, by for example a specific glycosydase, sulfatase, phosphatase, or other suitable enzyme.
In another preferred embodiment, the invention provides a plant according to the invention wherein said N-linked glycan comprising galactose is further comprising other sugar residues not directly linked to galactose, for example core alphal,6 linked fucose or betal,4- or betal,6 linked N-acetylglucosamine (GlcNAc). To establish this, a gene or genes encoding for example core alphal,6 fucosyltransferase or/and GlcNAc-transferase III, GlcNAc-transferase IV, GlcNAc-transferase V and/or GlcNAc-transferase VI are expressed in plants according to the invention using methods known in the art or herein disclosed.
In general, herein is provided a method to tailor N-linked glycosylation for the production of heterologous glycoproteins in plant species with typical plant like glycosylation patterns-similar to those as shown in
In the detailed description the invention provides a plant as defined herein before according to the invention which comprises a tobacco plant, or at least a plant related to the genus Nicotiana, however, use for the invention of other relatively easy transformable plants, such as Arabidopsis thaliana, or Zea mays, or plants related thereto, is also particularly provided. For the production of recombinant glycoproteins, use of duckweed offers specific advantages.
The plants are in general small and reproduce asexually through vegetative budding. Nevertheless, most duckweed species have all the tissues and organs of much larger plants including roots, stems, flowers, seeds and fronds. Duckweed can be grown cheaply and very fast as a free floating plant on the surface of simple liquid solutions from which they can easily be harvested. They can also be grown on nutrient-rich waste water, producing valuable products while simultaneously cleaning wastewater for reuse. Particularly relevant for pharmaceutical applications, duckweed can be grown indoors under contained and controlled conditions. Stably transformed Duckweed can for example be regenerated from tissues or cells after (co)-inoculating with Agrobacterium strains containing each a (binary) vector which comprises one or more nucleotide sequences of interest encoding N-glycosylation modifying enzymes and/or genes encoding commercially interesting heterologous glycoproteins. The duckweed plant may for example comprise the genus Spirodella, genus Wolffia, genus Wolffiella, or the genus Lemna, Lemna minor, Lemna miniscula and Lemna gibba.
Expression in tomato fruits also offers specific advantages. Tomatoes can be easily grown in greenhouses under contained and controlled conditions and tomato fruit biomass can be harvested continuously throughout the year in enormous quantities. The watery fraction containing the glycoproteins of interest can be readily separated from the rest of the tomato fruit which allows easier purification of the glycoprotein. Expression in storage organs of other crops including but not limited to the kernels of corn, the tubers of potato and the seeds of rape seed or sunflower are also attractive alternatives which provide huge biomass in organs for which harvesting and processing technology is in place.
Herewith, the invention provides a method for providing a transgenic plant, such as transgenic Nicotiana, Arabidopsis thaliana, or corn, potato, tomato, or duckweed, which are capable of expressing a recombinant protein; with the additional desired capacity to extend an N-linked glycan with galactose comprising crossing said transgenic plant with a plant according to the invention comprising at least one functional mammalian protein, e.g. a transporter or an enzyme providing N-glycan biosynthesis that is normally not present in plants, harvesting progeny from said crossing and selecting a desired progeny plant expressing said recombinant protein and expressing a functional (mammalian) enzyme involved in mammalian-like N-glycan biosynthesis that is normally not present in plants. In a preferred embodiment, the invention provides a method according to the invention further comprising selecting a desired progeny plant expressing said recombinant protein comprising an extended N-linked glycan et least comprising galactose. In the detailed description a further description of a method according to the invention is given using tobacco plants and crossings thereof as an example.
With said method as provided by the invention, the invention also provides a plant expressing said recombinant protein and expressing a functional (mammalian) enzyme involved in mammalian-like N-glycan biosynthesis that is normally not present in plants. Now that such a plant is provided, the invention also provides use of a transgenic plant to produce a desired glycoprotein or functional fragment thereof, in particular wherein said glycoprotein or functional fragment thereof comprises an extended N-linked glycan et least comprising galactose.
The invention additionally provides a method for obtaining a desired glycoprotein or functional fragment thereof comprising for example an extended N-linked glycan at least comprising galactose comprising cultivating a plant according to the invention until said plant has reached a harvestable stage, for example when sufficient biomass has grown to allow profitable harvesting, followed by harvesting said plant with established techniques known in the art and fractionating said plant with established techniques known in the art to obtain fractionated plant material and at least partly isolating said glycoprotein from said fractionated plant material. In the detailed description (see for example
The invention thus provides a plant-derived glycoprotein or functional fragment thereof comprising an extended N-linked glycan at least comprising galactose, for example obtained by a method as explained above. Such a plant-derived glycoprotein with an extended glycan at least comprising galactose essentially can be any desired glycoprotein that can be expressed in a plant. For example, antibodies, FSH, TSH and other hormone glycoproteins, other hormones like EPO, enzymes like antitrypsine or lipase, cellular adhesion molecules like NCAM or collagen can be produced in plants and be provided with essentially mammalian glycosylation patterns. Expression of such proteins can be performed by using a method known in the art. For example, by stable expression via Agrobacterium mediated transformation, electroporation or particle bombardment, but also by transient expression using a virus vector like PVX or other method, glycosyltransferases or an other protein extending glycan biosynthesis, and/or said glycoprotein could be expressed under control of a specific promoter to facilitate expression in certain tissues or organs.
Herewith, the invention also provides use of such a plant-derived glycoprotein or functional fragment thereof according to the invention for the production of a pharmaceutical composition, for example for the treatment of a patient with an antibody, a hormone, a vaccine antigen, an enzyme, or the like. Such a pharmaceutical composition comprising a glycoprotein or functional fragment thereof is now also provided. The invention is further explained in the detailed description without limiting it thereto.
One important enzyme involved in mammalian N-glycan biosynthesis that is not present in plants is β1,4-galactosyltransferase. Here, for one, the stable expression of β1,4-galactosyltransferase in tobacco plants is described. The physiology of these plants is not obviously changed by introducing β1,4-galactosyltransferase and the feature is inheritable. Crossings of a tobacco plant expressing β1,4-galactosyltransferase with a plant expressing the heavy and light chain of a mouse antibody produced antibody having terminal galactose in similar amounts as hybridoma produced antibodies. Herein it is thus shown that the foreign enzyme can be successfully introduced in plants. A clear increase in galactose containing glycoproteins is observed. Moreover, this feature is inheritable and there is no visible phenotypical difference between the galactosyltransferase plants and wild type. A mouse monoclonal antibody produced in these plants has a degree of terminal galactoses comparable to hybridoma. produced antibody. This shows that not only endogenous proteins become galactosylated but also a recombinantly expressed mammalian protein.
Materials and Methods
Plasmids and Plant Transformation
A plant transformation vector containing human β1,4-galactosyltransferase was constructed as follows: a 1.4 kb BamHI/XbaI fragment of pcDNAI-GalT (Aoki et al., 1992; Yamaguchi and Fukuda, 1995) was ligated in the corresponding sites of pUC19. Subsequently, this fragment was re-isolated using surrounding KpnI and HincII sites and cloned into the KpnI and SmaI site of pRAP33 (named pRAP33-HgalT). Using AscI and PacI sites the CaMV35S promotor-cDNA-Nos terminator cassette of pRAP33-HgalT was cloned in the binary vector pBINPLUS (van Engelen et al., 1995). Modifications to the published protocol are: After incubation with A. tum., leaf discs were incubated for three days in medium containing 1 mg/ml of NAA and 0.2 mg/ml BAP and the use of 0.25 mg/ml cefotaxime and vancomycine to inhibit bacterial growth in the callus and shoot inducing medium. 25 rooted shoots were transformed from in vitro medium to soil and, after several weeks, leaf material of these plants was analysed.
Northern Blotting
The β1,4-galactosyltransferase RNA level in the transgenic plants was analyzed by northern blotting (Sambrook et al., 1989) RNA was isolated from leafs of transgenic and control plants as described (D� Vries et al., 1991). Ten μg of total RNA was used per sample. The blot was probed with a [32p]DATP labeled SstI/XhoI fragment, containing the whole GalT cDNA, isolated from pBINPLUS-HgalT.
Glycoprotein Analysis
Total protein extracts of tobacco were prepared by grinding leafs in liquid nitrogen. Ground material was diluted 10 times in SDS page loading buffer (20 mM of This-HCl pH 6.8, 6% glycerol, 0.4% SDS, 20 mM DTT, 2.5 ig/ml Bromophenol Blue). After incubation at 100� C. for 5 min insoluble material was pelleted. Supernatants (12.5 μl/sample) were run on 10% SDS-PAGE and blotted to nitrocellulose. Blots were blocked overnight in 0.5% Tween-20 in TBS and incubated for 2 hours with peroxidase conjugated RCA120 (Ricinus Communis Agglutinin, Sigma) (1 μg/ml) in TBS-0.1% tween-20. Blots were washed 4 times 10 minutes in TBS-0.1% tween-20 and incubated with Lumi-Light western blotting substrate (Roche) and analysed in a lumianalyst (Roche). A rabbit polyclonal antibody directed against Horseradish peroxidase (HRP, Rockland Immunochemicals) was split in reactivity against the xylose and fucose of complex plant glycans by affinity chromatography with bee venom phospholipase according to (Faye et al., 1993). A rabbit anti LewisA antibody was prepared as described (Fitchette Laine et al., 1997). Blots were blocked with 2% milkpowder in TBS and incubated in the same buffer with anti-HRP, anti-xylose, anti-fucose or anti-Lewis-A. As secondary antibody alkaline HRP-conjugated sheep-anti-mouse was used and detection was as described above.
Plant Crossings
Mgr48 (Smant et al., 1997) is a mouse monoclonal IgG that has been expressed in Tobacco plants. The construct used for transformation was identical to monoclonal antibody 21C5 expressed in tobacco (van Engelen et al., 1994). Flowers of selected tobacco plants with high expression of β1,4-galactosyltransferase were pollinated with plants expressing Mgr48 antibody. The F1 generation was seeded and plants were screened for leaf expression of antibody by western blots probed HRP-conjugated sheep-anti-mouse and for galactosyltransferase expression by RCA as described above.
Purification of IgGl from Tobacco
Freshly harvested tobacco leaves were ground in liquid nitrogen. To 50 g of powdered plant material, 250 ml of PBS, containing 10 mM Na2S2O5, 0.5 mM EDTA, 0.5 mM PMSF and 5 g polyvinylpolypyrrolid, was added. After soaking for 1 hour (rotating at 4� C.), insoluble material was removed by centrifugation (15 min, 15,000 g, 4� C.). The supernatant was incubated overnight (rotating at 4� C.) with 1 ml of proteinG-agarose beads. The beads were collected in a column and washed with 10 volumes of PBS. Bound protein was eluted with 0.1 M glycine pH 2.7 and immediately brought to neutral pH by mixing with 1 M Tris pH 9.0 (50 μl per ml of eluate). Purified antibody was quantified by comparison of the binding of HRP-conjugated sheep-anti-mouse to the heavy chain on a western blot with Mgr48 of known concentration purified from hybridoma medium (Smant et al., 1997).
Hybridoma Mgr48 and plant produced Mgr48 was run on 10% SDS-PAGE and blotted as described above. Detection with RCA was as described above. For antibody detection, blots were probed with HRP-conjugated sheep-anti-mouse and detected with Lumi-Light western blotting substrate as described above.
Human β1,4-galactosyltransferase galactosylates endogenous proteins in Nicotiana tobacum.
Human β1,4-galactosyltransferase (Masri et al., 1988) was introduced in tobacco plants by Agrobacterium mediated leaf disk transformation of plasmid pBINPLUS-HgalT containing a cDNA that includes a complete coding sequence. Twenty-five plants selected for kanamicin resistance were analysed for mRNA levels by northern hybridization (
As it is known that galactosylated N-glycans are poor acceptors for plant xylosyl- and fucosyltransferase (Johnson and Chrispeels, 1987), the influence of expression of β1,4-galactosyltransferase on the occurrence of the xylose and fucose epitope was investigated by specific antibodies. A polyclonal rabbit anti-HRP antibody that reacts with both the xylose and fucose epitope shows a clear difference in binding to isolated protein from both control and transgenic plants (
Recombinantly Produced Antibody is Efficiently Galactosylated.
The effect of expression of β1,4-galactosyltransferase on a recombinantly expressed protein was investigated. Three tobacco plants expressing β1,4-galactosyltransferase (no. GalT6, GalT8 and GalT15 from
Using proteinG affinity, antibody was isolated from tobacco plants expressing mgr48 and from the two selected plants expressing both mgr48 and β1,4-galactosyltransferase. Equal amounts of isolated antibody was run on a protein gel and blotted. The binding of sheep-anti-mouse-IgG and RCA to mgr48 from hybridoma cells, tobacco and crossings GalT8xmgr48-11 and 12 was compared (
Construction of Plant Expression Vectors With cDNA's Encoding α2,6 Sialytransferase, β 1,3-Glucuronyltransferase and β1,4-Galactosyltransferase.
The available β1,4-galactosyltransferase vector was not in a suitable format to easily combine with α2,6-sialyltransferase and β1,3-glucuronyltransferase clones. Therefore, by using PCR, the coding region of β1,4-galactosyl-transferase cDNA, α2,6-sialyltransferase cDNA and β1,3-glucuronyl-transferase cDNA have been cloned in plant expression vectors. Constructs are made in which galactosyltransferase is combined with either sialyltransferase or glucuronyltransferase in one vector, in order to enable simultaneous expression of the enzymes in transgenic plants after only one transformation. The galactosyltransferase expression is controlled by the 35S promoter, whereas expression of sialyltransferase and glucuronyltransferase is controlled by the 2′promoter.
There is a need for an accessible and standardised source of FSH for therapeutic and diagnostic purposes, which is guaranteed to be free of LH activity.
FSH preparations normally are derived from ovine or porcine pituitaries, which always implies the presence of (traces of) LH, and the risk of contamination with prion-like proteins. Substitution of brain derived FSH for plant produced recombinant FSH may be a good method of eliminating these problems. However, production of bioactive animal glycoproteins in plants, especially for therapeutic purposes, requires modification of plant-specific sugar sidechains into a mammalian type of glycans. The invention provides recombinant bFSH by infecting stably transformed tobaccoplants capable of forming mammalian type of glycans, with recombinant Tobacco Mosaic Virus TMV containing the genes for bFSH or bFSHR.
Construction of Single Chain (sc) BFSH into pKS (+) Bluescript Vector, Construction of sc-bFSH-TMV and sc-bFSH-HIS-TMV
In order to circumvent the need of simultaneous expression of the two separate genes of bFSH-alpha and bFSH-beta subunits in plants, we decided to construct a bFSH fusion gene. By overlap PCR we fused the carboxyl end of the beta subunit to the amino end of the alpha subunit (without a linker). In addition, we constructed a second sc-bFSH version carrying a 6� HIS tag at the C-terminus of the alpha subunit, which will allow us to purify the recombinant protein from the plant. Both, sc-bFSH and sc-bFSH-HIS constructs were subcloned into the cloning vector pKS(+) bluescript. The correctness of the clones was confirmed by sequence analysis.
Sc-bFSH was subcloned into the TMV vector. Two positive clones were chosen to make in vitro transcripts and Inoculate N. Bentahamiana plants. After a few days, plants showed typical viral infection symptoms, which suggested the infective capacity of the recombinant TMV clones. In order to test whether the sc-bFSH RNA is stably expressed in systemically infected leaves, 8 days post inoculation RNA was isolated from infected N. benthamiana leaves and a reverse transcriptase polymerase chain reactions using bFSH specific primers was performed. In all cases we obtained a PCR fragment of the expected size, indicating the stability of our Sc-bFSH-TMV construct. Extracts of infected plants are used for Western blot analyses and ELISA to determine whether Sc-bFSH is expressed and folded properly.
Abbreviations Used:
GlcNAc, N-Acetylglucosamine; Fuc, fucose; Gal, galactose; GalT, �1,4-galactosyltransferase; RCA, Ricinus Cummunis Agglutinin; Tables
Major differences between mammalian and plant complex N-linked glycans. Drawn are typical N-linked glycans. Numerous variations, both extended or truncated, occur in mammals and plants.
Comparison of RNA levels and product of β1,4-galactosyltransferase. Upper panel: Northern blot of total RNA isolated from 25 transgenic plants, including a not transformed control plant (0), detected with a human β1,4-galactosyltransferase probe. Lower panel: Western blot of the same plant probed with RCA to detect terminal galactose residues on glycoproteins. M. indicates the molecular weight marker.
Western blot showing the binding of lectin and antibody to protein isolated from wild-type and a β1,4-galactosyltransferase plant (no.8 from
Western blot showing RCA and sheep-anti-mouse-IgG binding to purified antibody produced in hybridoma culture (Hyb), tobacco plants (plant) and tobacco plants co-expressing β1,4-galactosyltransferase (GalTil and GalT12). H.C.: heavy chain, L.C. light cain.
Tobacco cell cultures expressing galactosyltransferase produce unnatural hybrid N-glycans while tobacco plants expressing galactosyltransferase have natural, mammalian like galactosylation. To get natural galactosylation, galactosyltransferase should act after mannosidase II and GlcNAcTransferase II.
Western blot showing the expression of GlcAβ1,3Gal structure in transgenic tobacco by binding of an antibody (412) directed against the glucuronic acid-galactose (GlcAβ1,3Gal) stucture to protein isolated from 8 plants expressing human β1,4 galactosyltransferase and rat β1,3 glucuronyltransferase and a wildtype controll plant (−).
Aoki, D., Lee, N., Yamaguchi, N., Dubois, C., and Fukuda, M. N. (1992). Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain. Proceedings Of The National Academy Of Sciences Of The United States Of America 89, 4319-4323.
Asano, M., Furukawa, K., Kido, M., Matsumoto, S., Umesaki, Y., Kochibe, N., and Iwakura, Y. (1997). Growth retardation and early death of beta-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells. Embo j 16, 1850-7.
Boyd, P. N., Lines A. C., and Patel A. K. (1995). The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-lH. Mol immunol 32, 1311-8.
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De Vries, S., Hoge, H., and Bisseling, T. (1991). Isolation of total and polysomal RNA from plant tissues. In Plant Molecular Biology Manual, B. Gelvin, R. A. Schilperoort and D. P. S. Verma, eds. (Dordrecht: Kluwer Academic Publishers), pp. B6/1-13.
Dieryck, W., Pagnier J., Poyart, C., Marden, M. C., Gruber, V., Bournat, P., Baudino, S., and Merot, B. (1997). Human haemoglobin from transgenic tobacco [letter] Nature 386, 29-30.
Faye, L., Gomord, V., Fitchette Laine, A. C. and Chrispeels, M. J. (1993). Affinity purification of antibodies specific for Asn-linked glycans containing alpha 1→3 fucose or beta 1→2 xylose. Anal Biochem 209, 104-8.
Fichette Laine, A. C., Gomord, V., Cabanes, M., Michalski, J. C., Saint Macary, M., Foucher, B., Cavelier, B., Hawes, C., Lerouge, P., and Faye, L. (1997). N-glycans harboring the Lewis a epitope are expressed at the surface of plant cells. Plant J 12, 1411-7.
Florack, D., Allefs, S., Bollen, R., Bosch, D., Visser, B., and Stiekema, W. (1995). Expression of giant silkmoth cecropin B genes in tobacco. Transgenic Research 4, 132-141.
Herman, T., and Horvitz, H. R. (1999). Three proteins involved in Caenorhabditis elegans vulval invagination are similar to components of a glycosylation pathway. Proc Natl Acid Sci U S A 96, 979-9.
Hollister, J. R., Shaper, J. H. and Jarvis, D. L. (1998). Stable expression of mammalian beta, 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells. Glycobiology 8, 473-80.
Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G., and Fraley, R.T. (1985). A simple and general method for transferring genes into plants. Science, USA 227, 1229-1231.
Ioffe, E., and Stanley, P. (1994). Mice lacking N-acetylglucosaminyltransferase I activity die at mid-gestation, revealing and essential role for complex or hybrid N-linked carbohydrates. Proc Natl Acid Sci U S A 91, 728-32.
Jarvis, D. L., and Finn, E. E. (1996). Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors. Nat Biotechnol 14, 1288-92.
Jenkins, N. Parekh, R. B., and James D. C. (1996). Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol 14, 975-81.
Johnson, K. D., and Chrispeels, M. J. (1987). Substrate specificities of N-acetylglucosaminyl-, fucosyl-, and xylosyltransferases that modify glycoproteins in the Golgi apparatus of bean cotyledons. Plant Physiology 84, 1301-1308.
Lerouge, P., Cabanes Macheteau, M., Rayon, C, Fischette Laine, A. C., Gomord, V, and Faye, L. (1998). N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol 38, 31-48.
Ma, J. K., Hiatt, A., Hein, M., Vine, N. D., Wang, F., Stabila, P., van Dolleweerd, C., Mostov, K. and Lehner, T. (1995). Generation and assembly of secretory antibodies in plants [see comments.] Science 268, 716-9.
Masri, K. A., Appert, H. E., and Fukuda, M. N. (1988). Identification of the full-length coding sequence for human galactosyltransferase (beta-N-acetylglucosaminide: beta 1,4-galactosyltransferase). Biochem Biophys Res Commun 157, 657-63.
Matsumoto, S., Ikura, K., Ueda, M., and Sasaki, R. (1163). Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Molecular Biology 27, 1163-1172.
Melo, N. S., Nimtz, M., Conradt, H. S., Fevereiro, P. S., and Costa, J. (1997). Identification of the human Lewis(a) carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium myrtillus L.). FEBS Lett 415, 186-91.
Palacpac, N. Q., Kimura, Y., Fuijyama, K., Yoshida, T., and Seki, T. (1999). Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells. Biosci Biotechnol Biochem 63, 35-9.
Palacpac, N. Q., Yoshida, S., Sakai, H., Kimura, Y., Fuijyama, K., Yoshida, T., and Seki, T. (1999). Stable expression of human beta 1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc Natl Acad Sci U S A 96, 4692-7.
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Saito, K., Noji, M. Ohmori, S., Imai, Y., and Murakoshi, I. (1991). Integration and expression of a rabbit liver cytochrome P-450 gene in transgenetic Nicotiana tabacum. Proceedings Of The National Academy Of Sciences Of The United States Of America 88, 7041-7045.
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Schaper, N. L., Shaper, J. H., Meuth, J. L., Fox, J. L., Chang, H., Kirsch, I. R. and Hollis, G.F. (1986). Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A 83, 1573-7.
Smant, G., Goverse, A., Stokkermans, J. P. W. G., De Boer, J. M., Pomp, H., Zilverentant, J. F. Overmars, H. A. Helder, J., Schots, A. and Baaker, J. (1997) Potato root diffusate-induced secretion of soluble, basic proteins originating from the subventral esophageal glands of potato cyst nematodes. Phytopathology 87, 839-845.
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van Engelen, F. A., Schouten, A., Molthoff, J. W., Roosein, J. Salinas, J., Dirkse, W. G., Schots, A., Bakker, J., Gommers, F. J., Jongsma, M. A., and et al. (1994). Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenetic tobacco. Plant Mol Biol. 26, 1701-10.
von Schaewen, A., Sturm, A., O'Neill, J., and Chrispeels, M. J. (1993) Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans. Plant Physiol 102, 1109-18.
Yamaguchi, N., and Fukuda, M. N. (1995). Golgi retention mechanism of beta-1,4-galactosyltransferase. Membrane-spanning domain-dependent homodimerization and association with alpha- and beta-tubulins. J Biol Chem 270, 12170-6.
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Oasis of the Seas Cruise Review by LARRYKCMO: AWESOME OASIS
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Another great cruise on Royal Caribbean Oasis of the Seas from Port Everglades to the Eastern Caribbean sailing October 5, 2013. This was my second cruise on the Oasis and my 49th cruise since 1992. The Oasis and the Allure have become my favorite ships.
Some of the great features of the Oasis class ships are: Aqua theatre shows, Ice shows, great production shows, very comfortable beds, very clean and well maintained public areas, public restrooms well located with push button doors, central park, boardwalk, royal promenade, diamond and concierge lounges for Crown & Anchor members, thousands of deck chairs on upper decks. Viking Crown lounge.
Some of the not so nice features are: very limited closet space, small bathroom, very loud music.
On day 3 as we sailed from Nassau to St. Thomas, as we were about two hours out to sea, the captain announced a medical emergency for a passenger who needed a hospital. To the credit of the captain, we reversed course, More and returned to Nassau so the stricken passenger could be transported to a hospital. The captain announced later that the passenger was in the hospital and doing well. We arrived in St.Thomas about four hours behind schedule, but we are grateful to the captain for his wise decision to help this passenger.
The Oasis class ships are the biggest, but it is very easy to move around the ship, and electronic interactive displays are located near the elevator banks, to help everyone find their way around. It is amazing how Royal Caribbean has found a way to move 5 to 6 thousand passengers so easily.
These big ships are not for everyone, but many will greatly enjoy the tremendous features of these fantastic Oasis class ships. Less
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Characters that could not fit in above: Marjorie, Bridget, Ivy, Jimmy, Heather, (guests: car-girl, sandwich-girl, etc)
I just like their personalities the best out of the cast.
Dave beats out Brian by just a little bit....in the first season and beginning of the second I really didn't like Adam...but he's grown on me
To me the greatest scenes in the shows are when Dave and Brian are together...Watson and Gomez have a good chemistry and are both really funny together
Dave, Deena, and Adam are all my favorite right now. I cant choose one!
I like Dave and Nicole the best because I think that all of the other characters can somehow interlock with all of them. They are the threads that hold the group's friendship together.
Do you really need that answered?
She talked Dave into an open marriage he didn't want, went out and did her thing despite Dave's feelings, and when Dave finally found someone to do the same with she flips out on him.
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in Self-help, Mental Health/Wellness, Happiness and Spirituality. Find out more.
Working with The Magic Challenge
Thank you for this challenge Carmen. It is one I am going to continue to do privately, over again! :)
A.T. Project Blog2Heal Participant
About the book "I Will NOT Forgive"
The moment I started reading I couldn't stop. I was captivated by the style and the inspiring stories to which we can easily relate and learn so much from. A must read!
Gabriela Embon, Life Coach "Rewire Your Brain for SUCCESS"


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Meetings on intangible cultural heritage (co-)organized by UNESCO
32 element(s)
9th session of the Intergovernmental Committee24/28-11-2014, Paris (France)
Training Workshop on the the implementation of the 2003 Convention10/14-11-2014, Niamey (Niger)
Workshop in Mozambique: towards a national strategy for the safeguarding of cultural intangible heritage27-10-2014/01-11-2014, Chinhambudzi, Manica and Maputo (Mozambique)
Workshop on community-based inventorying in Myanmar27-10-2014/03-11-2014, Nyaung Shwe (Myanmar)
Expert meeting on safeguarding intangible cultural heritage and sustainable development at the national level29-09-2014/01-10-2014, Istanbul (Turkey)
Review meeting of the global capacity-building strategy for safeguarding intangible cultural heritage in Europe and Central Asia23/26-09-2014, Sofia (Bulgaria)
Capacity-building workshop on ICH community-based inventorying05/15-09-2014, Bamako (Mali)
How to provide policy advice effectively?
25-06-2014, UNESCO Headquarters (France)
Curaçao national consultation meeting 24/25-06-2014, Willemstad (Curaçao)
Tunisia: INV workshop21/28-06-2014, Kerkennah (Tunisia)
National Consultation for Strengthening Capacities to Safeguard Intangible Cultural Heritage for Sustainable Development in Ethiopia10/11-06-2014, Adama (Ethiopia)
Review meeting: safeguarding intangible cultural heritage of Belize, Jamaica and Trinidad and Tobago 10/11-06-2014, Kingston (Jamaica)
Meeting of category 2 centres in the field of intangible cultural heritage 06-06-2014, Paris (France)
5th session of the General Assembly02/05-06-2014, Paris (France)
Needs assessment and national consultation Workshop for strengthening capacities to safeguard Intangible Cultural Heritage for sustainable development in Ethiopia26-05-2014/12-06-2014, Ethiopia (Ethiopia)
Morocco: inventory workshop. Marrakech20/27-05-2014, Marrakech (Morocco)
Myanmar pursues its march towards safeguarding intangible heritage19/24-05-2014, Mandalay (Myanmar)
Regional workshop: conceptual and legal frameworks for the safeguarding of the intangible cultural heritage in the Arab region 05/09-05-2014, Kuwait (Kuwait)
Community-Based Inventorying of Intangible Cultural Heritage in Tajikistan16/23-04-2014, Dushanbe (Tajikistan)
Iraq: Workshop on the Implementation of the 2003 Convention13/17-04-2014, Erbil (Iraq)
Workshop on community-based inventorying in Dominican Republic31-03-2014, Baní (Dominican Republic)
From 31 March 31 to 6 April, the Dominican Republic hosts a workshop on intangible cultural heritage community-based inventories within the framework of a regional project for strengthening national capacities for the effective safeguarding of intangible cultural heritage in Cuba, Dominican Republic and Haiti, with the support of the Intangible Cultural Heritage Fund, thanks to the generous contribution of Norway.
Conducted by two members of the UNESCO network of facilitators, María Ismenia Toledo (Venezuela) and Enrique Pérez López (Mexico), the workshop brings together 30 specialists, students and bearers of intangible cultural heritage for a training on the approach promulgated by the 2003 Convention for the Safeguarding of the Intangible Cultural Heritage to inventories and the role played by communities. The theoretical training is complemented with a technical part on data collection and a field exercise with the Chuineros of Cañafístol in Baní, in the province of Peravia, whose improvised songs in couplets are at risk of disappearing.
This activity has been made possible thanks to the support of the Vice Ministry of Cultural Heritage and the Cultural Centre of Perelló, located in Baní, which was instrumental in obtaining the Chuineros' free, prior and informed consent.
Joint meeting of the Bodies of the Convention27/28-03-2014, Paris (France)
Morocco: workshop on the implementation of the 2003 Convention26/30-03-2014, Agadir (Morocco)
Using Natural and Cultural Heritage in Sustainable Development – Synergy for Development24/26-03-2014, Bergen (Norway)
Strengthening sub-regional Cooperation and National Capacities in Seven Southern African Countries for implementing the 2003 Convention for the Safeguarding of Intangible Cultural Heritage18/20-03-2014, Harare (Zimbabwe)
Elaboration of nomination files to the ICH Lists in Bhutan04/08-03-2014, Paro (Bhutan)
Inventory workshop in Mauritania04/11-03-2014, Aleg (Mauritania)
Workshop on community-based inventorying in Guatemala25-02-2014/04-03-2014, Guatemala (Guatemala)
Elaboration of Nomination Files to the ICH List in Sri Lanka11/15-02-2014, Galle (Sri Lanka)
Training on the implementation of the 2003 Convention for the Safeguarding of Intangible Cultural Heritage at the national level in Eastern Cuba11/14-02-2014, Santiago de Cuba (Cuba)
Tunisia: workshop on the implementation of the 2003 Convention at the national level 26-01-2014/01-02-2014, Matmata (Tunisia)
Training on the use of the mechanisms of the 2003 Convention for the Safeguarding of Intangible Cultural Heritage in Lao PDR20/24-01-2014, Vientiane (Lao People's Democratic Republic)


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State Line Drive-in will charge no admission to its showings this weekend of "Cloudy with a Chance of Meatballs II." It will be the first movie shown since the drive-in converted from 35 mm film to digital this winter.Comments
Police say an autopsy has found no evidence of foul play.Comments
An injured survivor told his relatives that the path up the mountain was unstable just before the avalanche. As soon as the avalanche hit, rescuers, guides and climbers rushed to help.Comments
Signs spotted around the area that will make you laugh - or at least scratch your head.Comments
"I think the fear of God needs to be put back into them."Comments
Former coaches at Science Hill High School.Comments
Miss something? The Buzz is here to fill you in on 10 essential news and sports stories that might have slipped past you.Comments
Tony Benton's role as interim CEO of Washington County facilities will include the CEO role at Johnson City Medical Center, along with oversight for the operations of Franklin Woods Community Hospital and Woodridge Hospital.Comments
A bankruptcy filing and an expected quick liquidation will spell the end for Johnson City's Coldwater Creek women's clothing store.Comments
With so many apparently seeking to change the County Commission and expressing opinions on the number of commissioners, health insurance and compensation, it is time for these citizens to get a first-hand understanding.Comments


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Canada's Insurance and Risk Magazine
Insurance professionals need to bear in mind catastrophe models are imperfect, and should account for factors such as a concentration of exposures, location of critical suppliers and the time since the last catastrophe occurred in an area, brokerage firm Lockton stated in a recent report.
"Recognize models will always be wrong and there will always be nonmodeled factors," Lockton advises in the report, titled Catastrophe Models: Learning from Superstorm Sandy.
Hurricane Sandy was downgraded to post-tropical storm status when it made landfall in New Jersey Oct. 29, 2012.
"Each catastrophe has unique circumstances that no model will ever capture. For instance, governors of the states impacted by Sandy ordered property insurers to enforce all-other-peril (AOP) deductibles, rather than percentage hurricane deductibles post-Sandy."
The report notes that a $500,000 home with a 5% deductible for hurricane would have a $25,000 deductible, but the all-other-peril deductible for the same home might be as low as $500.
Kansas City, Mo.-based Lockton noted that longer time intervals between events "increase the uncertainty in loss estimates."
It used as an example a series of earthquakes in 1811 and 1812 centred near New Madrid, Mo., on the Mississippi River about 200 km south of St. Louis. One of the quakes was later estimated to be about 7.7 on the Richter scale. Quoting from the U.S. Federal Emergency Management Agency, Lockton said a similar earthquake today "would cause US$300 billion of direct economic damage, while (FEMA) also (suggests) indirect losses could reach $600 billion.
"Insurers would bear 60-80% of the economic damages from this event, due to the relatively high take-up rate of earthquake insurance in the New Madrid region."
According to the U.S. Geological Survey, the earthquakes 200 years ago "rank as some of the largest in the United States since its settlement by Europeans" and "were by far the largest east of the Rocky Mountains in the U.S. and Canada."
Although there were no seismographs in North America at that time, USGS said the shaking associated with those shocks was 10 times as large at the San Francisco earthquake in 1906, adding chimneys were toppled in St. Louis and Cincinnati.
Lockton suggested in its report that a similar earthquake today could result in damage to more than 3,500 bridges and could render 15 major bridges unusable. It could also cause 425,000 breaks or leaks in gas pipelines.
"Pinpoint the location of critical suppliers and infrastructure dependencies," the report advises. "Is your client dependant on a utility company located in a CAT-prone area?"
It also advises insurance carriers to ask about sources of indirect losses, such as power failures and transportation disruption. Another question to ask is whether covered locations are in areas prone to flooding or near a levee in a high-risk area.
"Does your client have a concentration of exposures very near each other, which could easily consume the limits of insurance?"
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Interview: Ian Fetterman - Pumpkin Carver Extraordinaire
November 24th, 2014
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November 20th, 2014
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Book Review: Axes of Evil - Edited by Alex S. Johnson
November 22nd, 2014
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Book Review: Prisoner 489 - Author Joe Lansdale
November 17th, 2014
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Book Review: Equilibrium Overturned - Authors Anthony Rivera and Sharon Lawson
November 13th, 2014
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Film Review: The Return Of Chandu (1934)
SYNOPSIS: "The Black Magic cult of Ubasti, based on the isle of Lemuria, believes that Nadji, a princess of Egypt, is a reincarnation of their long-dead goddess, Ossana, and intend to sacrifice Nadji so that Ossana may be resurrected. Nadji has taken refuge at the California home of Frank Chandler, an American raised in the east and possessed of White Magical powers, who calls himself Chandu. Vindhyan, high priest of the cult's California outpost, learns … Continue reading


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What did you learn from the Curly Girl Challenge?
Where do I start? I was doing everything wrong because I wasn't aware that curly hair needed different treatment from regular hair. And trust me, no stylist was coaching me any differently. Most were pouring alcohol-filled products all over my head, and trying to form a non-frizzy helmet that would look OK until I walked out the door and into the wind and weather (where all bets were off). I learned that the right products make the most notable difference. I also learned little tricks like sleeping on a satin pillowcase and absorbing excess water with non-terry cotton fabric, scrunching, duck-billed pins.....the gift of curl knowledge has changed my hair forever.
How has it changed your hair?
My hair has changed in many ways. I have a sleeker look (I still get some breakage, thus some fly-aways), and more depth of color. Stylists used to cut my hair in an unflattering layered cut. Technically, the cut should have worked, but I had one clump of hair, growing from the back of my crown, that was so poorly conditioned it stuck straight out, almost like a perpendicular curtain of hair. Deva One Conditioner sorted that out after one use. The moisture balance in my hair has radically improved. My silhouette is extremely flattering, and only frizzy when I'm in gale-force winds. Even then, I still have more curl definition than I used to have by a long shot. Many more strangers stop me and compliment my hair. Knowing what to do and what to use gives me confidence that I look great when I walk out my door every day.
Some may call it "edgy," we just call it natural: Solange's wedding hairstyle. Read more »
Whether neatly patted down with every strand in place or finger-combed and slightly "undone", the Afro will forever be a natural hair fave around these parts! Read more »
It's the best method I've found that keeps your natural curl pattern in tact and hydrated, with very minimal frizz. Read more »
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Special Events
Wicomico County Recreation, Parks & Tourism hosts a variety of special events throughout the year, from signature festivals drawing thousands like Pork in the Park to traditional events continued annually such as the Fishing Derby – Wicomico Recreation, Parks & Tourism Department works together to make these events a success. We hope you'll save the date to join us for one or many of our special events this year!
Gear up for your Thanksgiving Day feast at the 6th Annual Turkey Trek 5K at Pemberton Historical Park on Thursday, November 27. The race will wind through Pemberton's beautiful forests, meadows and wetlands. Enjoy pie after the race - it is Thanksgiving after all! Read More
Each year, community members dedicate over 5,000 hours of time to Wicomico Recreation, Parks & Tourism programs and events. These volunteers are honored through the Volunteer Appreciation Banquet each winter. This year's banquet will be held on Tuesday, December 9. Read More
Clean out your house and fill up your wallet! Wicomico Recreation will hold its annual Indoor Community Yard Sale at the Wicomico Youth & Civic Center on Saturday, January 3. Table and booth rentals are available now. Read More
Search our parks to find the perfect setting for your next outing in Wicomico County.Search Parks
Whenever possible we offer an online registration option for our recreation programs.Register Today
Sign-up for our email announcements to receive information about the latest recreation programs and sporting events.


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Lauren J. Walter
Best Bistros In Charlotte
Pommes frites, moules, croque monsieur – all of those French specialities and more can be found in the bistros of Charlotte.
Walking Tour Of Charlotte's NoDa District
Charlotte's northernmost neighborhood is NoDa. Take a walk through one-of-a-kind shops and delicious restaurants. There are even gallery crawls twice a month where you can enjoy some great artwork.
Best Places For A Fall Marriage Proposal In Charlotte
Planning an engagement proposal can be challenging. Luckily, Charlotte has an abundance of romantic spots that can make asking the question that much easier. There are charming gardens, beautiful beaches and trendy restaurants.
Best Upcoming Musicals In Charlotte
Musical theater is alive and well in Charlotte. From national Broadway touring companies to local productions, you can get caught up on all of the hit songs and dance routines in your own backyard.
Best Places To Go With Your Family For The Weekend In Charlotte
Weekends are a great time to spend with your children exploring Charlotte. Whatever you enjoy, Charlotte has something for you to experience as a family.
Best Piano Bars In Charlotte
Piano bars are few and far between in Charlotte. It takes some effort to track down the right venue, but once you do, you can have a fabulous time singing along with some amazing performers.
Best Fall Salads In Charlotte
With the weather turning cooler, the restaurants in Charlotte are changing their menus. If you're into salads, there are some delectable selections that will have you craving greens.
Best Places To Buy A Hat In Charlotte
Shopping for hats should be fun, and Charlotte has a collection of stores that will allow you to try on and purchase the latest and greatest options.
Best Pizza In Charlotte
If you are looking for the perfect slice of pizza, Charlotte has plenty of options available. New York style or crispy thin crust, you can find it in the Queen City.
Best Camping Sites Near Charlotte
The Charlotte region offers a plethora of fine camping sites for those who prefer sleeping closer to nature. From spots over by the NASCAR track, to camping on a mountainside, Charlotte has something that will suit everyone's taste.


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Jared Kaplan, Contributing Editor
WATCH: 'AMC' Star Denyse Tontz in New Music Video
'ATWT' Actor Dylan Bruce Set for 'Flowers in the Attic' Sequel
TV Network Executives Join Forces to Improve Diversity in Media
WATCH: Maurice Benard in 'The Ghost and The Whale' Trailer
Former 'Guiding Light' and 'As The World Turns' Actor Passes
'The 100′, Starring Soap Alum Paige Turco, Scores Big for the CW
Judith Light Joins ABC's 'The Winklers'Read More »
Sean Kanan Leaving 'GH' Role for Old 'B&B' Role!
ABC Countersues Prospect Park Networks!
BREAKING: Prospect Park Networks Files for Bankruptcy


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The BMW X6 is very dependable and I love the car.
By L (FL)
overall rating 10 of 10
Simply Amazing!
By csydware1 (TX)
overall rating 10 of 10
great ride
By Richie (NY)
overall rating 10 of 10
Consumer Ratings Countdown: LuxuryPosted 6/6/2014 11:18 AM
The BMW X6 is very dependable and I love the car.
Find a Top 10 List - New Car and Used Car
Looking for a new sedan that gets great MPG? Or a used SUV with high crash test ratings? We've got a list for that.
Most Fuel EfficientBased on fuel-economy estimates from the EPASee the list
Top Consumer RatedFrom current owners and other reviewersSee the list
Best Safety RatedBased on government crash test dataSee the list
Highest HorsepowerCars and trucks that pack a punchSee the list
Most PopularThese vehicles get the most reviews on KBB.comSee the list
Most ExpensiveBased on KBB.com's Fair Purchase PriceSee the list


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About Us:
Frontier Nursing University (FNU) has been educating nurses and midwives for over 70 years. Its innovative community-based distance education programs enable registered nurses to advance in their chosen specialty while earning their master's or doctoral degrees online.
Established in May 2010, The George Washington University School of Nursing (SON) develops nursing leaders who are actively engaged in health promotion, patient advocacy, and health care innovation.
Count Your Blessings at Thanksgiving
5 Ways to Handle the Holidays When Your Life's in Chaos
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Соруright 2014Minority Nurse is Registered in the U.S. Patent and Trademark OfficeSpringer Publishing Company, LLC


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Some services survive an audit more easily than others, but there are things that you can do now to ensure safer travels through audit country. Join coding guru Becky Zellmer, CPC, CBCS, MBS, for this 1-hour audio conference where she will teach you how to shore up your documentation for E/M and eye codes, prove medical necessity, survive an audit, and more.
Here's what you will learn:
Who should attend? Compliance officers, managers, coders, billers, physicians, and others providing eye care services.
- Lynn Anderanin, CPC, CPC-I, COSC
- Barbara J. Cobuzzi, MBA, CPC-OTO, CPC-H, CPC-P, CPC-I, CHCC
- Jugna Shah,
- Michael A. Ferragamo, Jr., MD, FACS
- Gail Madison-Brown,
- Thomas Force,
Becky Rodrian (formerly Becky Zellmer), CPC, CBCS, MBS, has 16 years experience in coding, billing, auditing, reimbursement and collections. She has a diverse background, having performed duties in medical offices from patient care to collections. She has had the pleasure of auditing and educating physicians nationally. She is currently employed at SVA Healthcare Services as a Medical Billing and Coding Supervisor.
Becky has educated through teleconferences and national seminars... More Info
More Events By The Speaker


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Southside Gallery features artists Jere Allen, Ron Dale
OXFORD TOWN – Southside Gallery is now exhibiting paintings by local artist and former University of Mississippi painting professor Jere Allen and new sculptures (pictured here) by local artist and former University of Mississippi ceramics professor Ron Dale. An artists' reception is scheduled for Thursday, Nov. 17, from 6:30 to 8:30 p.m. For more information on the new gallery exhibit, Page 7 in this week's issue of Oxford Town has it or call Southside at 662-234-9090. (November 17, 2011, Page 7)
Oxford Town #950
IN THIS WEEK'S ISSUE: RAIN, the ultimate Beatles tribute band (on the OT cover this week) play the Ford Center tonight (Oct. 27); Thacker Mountain Radio is set for an outdoor version of its broadcast on the courthouse lawn this evening at 6; Sister Sparrow plays Rooster's Blues House on Saturday, Oct. 29; the popular annual event "One Night Stand" returns to the Ole Miss Motel on Oct. 29; Taylor Farmers' Market wraps up its season this Saturday morning with its annual Harvest Festival; the Fourth Annual Square Toast event takes place on the Oxford Square on Nov. 3; and OT columnist Jim Dees pens a moving tribute to the late Betty Harrington. CLICK HERE to download the complete issue in PDF format. (October 27, 2011)
'One Night Stand' returns to Ole Miss Motel
OXFORD TOWN – The concept of the fifth annual "One Night Stand" art exhibit is when 15 artists take over 12 rooms at the Ole Miss Motel, swapping out the standard motor-in landscapes for their own paintings and photographs and essentially becoming the curator of their own small gallery. Details of the popular event to be held on Oct. 29 can be found on Page 16 in this week's issue of Oxford Town by Lifestyles Columnist George Atkins. (October 27, 2011, Page 16)
Oxford Town #949
IN THIS WEEK'S ISSUE: The New Orleans-based band, e.company (on the OT cover), who have been compared to the likes of Maroon 5, are headed to Proud Larry's tonight (Oct. 20) and OT music writer Tom Speed has them covered; African Art is being featured at the University Museum and a reception is planned for Oct. 25; The Ole Miss choral Department welcomes Joan Szymko on Oct. 20 at Nutt Auditorium; and OT outdoor columnist Michelle McAnally turns us on to the wonderful world of Clear Creek and everything it has to offer. CLICK HERE to download the complete issue in PDF format. (October 20, 2011)
African art exhibited at University Museum
OXFORD TOWN – The University of Mississippi Museum continues its presentation of works from its permanent collection with a selection of African art from the Richard K. Meyer Collection. An opening reception is set for Oct. 25 during the Oxford Art Crawl and the public is invited to attend the free event. Page 5 in Oxford Town issue #949 has more information about the exhibit and reception. (October 20, 2011, Page 5)
Oxford Town #946
IN THIS WEEK'S ISSUE: Artist Stan O'Dell's artwork (featured on the OT cover this week) will be shown through October at Southside Gallery beginning Tuesday, Oct. 4; Musician Jeff Callaway is featured by OT Lifestyle Columnist George Atkins; Thacker Mountain Radio returns to the Lyric Theater this week featuring Silas Reed 'N' Da Books as musical guests (among others); Genghis Barbie is set for a free concert at Nutt Auditorium tonight (Sept. 29) at 8; and the Oxford Film freak features two former Ole Miss students who are making waves in Hollywood these days. CLICK HERE to download the complete issue in PDF format. (September 29, 2011)
"LES FEMMES ENCHANTEUR" – Southside Gallery
OXFORD TOWN – Oxford figurative artist Stan O'Dell's latest exhibit, "Les Femmes Enchanteur," opens Oct. 4 at Southside Gallery. The exhibit celebrates the beauty, grace and mystery of women. Pages 8 and 9 in this week's issue of Oxford Town has more information on the upcoming exhibit and amazing photo samples — just a taste – of O'Dell's paintings. (September 29, 2011, Page 8, 9)
Oxford Town #945
IN THIS WEEK'S ISSUE: 20 pages packed with features and events set to take place all around the L-O-U community including the Nashville touring group Ten Out of Tenn will play Proud Larry's on Monday, Sept. 26; Local musician Sky Shelton is set for a CD release party at the Lyric Theater tonight (Sept. 22); Print artist Katherine Rhodes Fields will launch her new exhibit "Scratch and Sniff" on Tuesday, Sept. 27, at the Powerhouse; Jason Isbell and The 400 Unit return to Oxford and Proud Larry's on Saturday, Sept. 24; and OT outdoor columnist Michelle McAnally turns us on to disc golf opportunities all around our area. CLICK HERE to download the complete issue in PDF format. (September 22, 2011)
Scratch This
OXFORD TOWN – Local artist and University of Mississippi printmaking instructor Katherine Rhodes Fields invites people to scratch and sniff her latest artwork at a reception to be held for her on Tuesday, Sept. 27, from 6 to 9 p.m. at the Powerhouse Community Arts Center located at 413 South 14th St. Turn to Page 7 in this week's issue of Oxford Town to read about Fields' new exhibit. (September 22, 2011, Page 7)
Oxford Town #942
IN THIS WEEK'S ISSUE: Thacker Mountain Radio cranks up its 2011-2012 season at Off Square Books tonight featuring author Clyde Edgerton; Blue Mountain returns to Proud Larry's this Saturday night after the Ole Miss game; Cirque du Soleil is set for Tupelo's BankcorpSouth Arena from Sept. 6-8; New Orleans rocker Lynn Drury returns to Proud Larry's on Wednesday, Sept. 7; and Oxford Town Lifestyle columnist George Atkins explores one of BYU's favorite foods, Mormon Funeral Potatoes. Click her to download the complete issue in PDF format. (September 1, 2011)


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Fox Victory Backpack
SALE $47.60 was $59.50 Item #3715305519
- "Honor Amongst Riders" graphic canvas backpack
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Latest News
Brain circuits involved in stress-induced fevers identified
When we feel mentally stressed, we often also feel physiological changes, including an increase in body temperature. This increase in body temperature is known as psychological stress-induced hyperthermia. Stress for people in today's society can last a long time and cause a chronic increase in body temperature, a condition called psychogenic fever. Researchers now have identified a key neural circuit connection in the brain that's responsible for the development of psychological stress-induced hyperthermia.
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Experience with family verbal conflict as a child can help in stressful situations as an adult
Federal legislation ignores PTSD toll on civilians, analysts say
Mental disorders due to permanent stress?
Education empowers Canadians, but raises risks of overwork, work-family stress
Brain protein influences how the brain manages stress; suggests new model of depression
Mentally stressed young women with heart disease more likely to have reduced blood flow to heart
Office stress? Workers may wait before acting out, study finds
Pregnant women with PTSD more likely to give birth prematurely, study finds
Brain's response to threat silenced when we are reminded of being loved and cared for
Brain dissociates emotional response from explicit memory in fearful situations


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This blog gives an overview of how to run Chocolatey commands on a remote Azure VM using Custom Script Extension.
Recent Posts
Red Teaming: Using Cutting-Edge Threat Simulation to Harden the Microsoft Enterprise Cloud
To help combat emerging threats, Microsoft employs an innovative Assume Breach strategy and leverages highly specialized groups of security experts, known as the Red Team, to strengthen threat detection, response and defense for its enterprise cloud services.
Azure SQL Database introduces breakthrough business continuity and Disaster Recovery administration
Last week we introduced breakthrough visualization experiences for the business continuity capabilities in Azure SQL Database. Setting up and administering your geo-replication is easier and more intuitive than ever before.
How to Sync SQL Server Data with Azure Search
In this blog post, I want to explain how to use a capability in SQL Server called Integrated Change Tracking to efficiently sync data changes from SQL Server to Azure Search.
Azure DocumentDB – edit and query documents in the Azure portal
We've released a number of great enhancements to further improve the DocumentDB management experience. Our October 23rd portal update lights up several new capabilities!
Azure Search Updates: Multilanguage, Azure Portal Index Management & More!
We have made available a new prototype. The purpose of preview versions moving forward is to allow you to have early access to potential new features to test and provide feedback on.
Announcing Azure Event Hubs General Availability
Event Hubs is a new capability now commercially available in Microsoft Azure Service Bus that provides a platform-as-a-service solution.
Preview for greater SQL Server compatibility coming soon to Azure SQL Database
Today at the annual PASS Summit conference, we announced a new preview of Azure SQL Database coming later this year that represents another major milestone for our database-as-a-service.
Introducing PowerShell support for Azure Site Recovery
Last week we announced PowerShell support for recovery between two Hyper-V sites managed by Azure Site Recovery. The ASR cmdlets can be used to enable protection for virtual machines and performing recovery operations on virtual machines or recovery plan containing virtual machines.
Getting Started with Live Streaming using the Media Services SDK
Azure Media Services just announced the public preview of its Live streaming service. This tutorial will show you how to set up live streaming with Azure Media Services .NET SDK.
Innovation in Cloud Security Enables Customers to Move to Microsoft Azure with Confidence
Just as Azure continues to release new cloud services at a rapid pace, it is also driving innovation in data protection, threat defense, network security, and identity and access management.


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Mortal Kombat PS Vita: 1st Launch Trailer
Mortal Kombat comes to the PS Vita with hard-hitting violent and tons of new features that will appeal to any MK fan.
Mortal Kombat moves at a brisk 60 frames per second, but with the speed comes a tradeoff. The textures aren't quite as detailed as those of the console versions, but many of the potential flaws are masked by the fact that the game is running on the PS Vita's 5-inch display which makes them harder to see. – PCMag
Mortal Kombat PS VITA Launch Trailer
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Driving the Greatest Caribbean gaming scene, Caribbean Game-Zone (CGZ.tv), is a passion that I hope changes lives across the Caribbean for the better. Like, Follow, & Subscribe to CGZ!
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Flat to let, Innellan Dunoon
One bed flat for rent. Situated in Innellan near Dunoon. Five min walk to Innellan school and a five min bus ride to Dunoon. Very quiet area with stunning views across the clyde. One hour from Glasgow with western ferries just a short drive away. Bea
- Date available: 30 Nov 2014
- 1 Beds
- Flat


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Media Morsels
A joint venture between the Metro, the American Heart Association and the Ketchum-Downtown YMCA, Metro Fit offers Angelenos options for becoming more physically fit. According to a press release, the campaign suggests "walking to and from transit, utilizing Metro subway steps, discovering local attractions on Metro and on foot and combining bicycling with public transit."
For more information on this program—and on how many steps you'll walk from a transit stop to area "neighborhood adventures"—visit www.ExperienceLA.com.
A man in Santa Rosa, California, has sued an all-women fitness center, saying it discriminated against him by refusing to sell him a membership. According to news reports, the state's Department of Fair Employment and Housing sued in Sonoma County court on Phillip Kottle's behalf, alleging that Elan Health and Fitness Center, which has facilities in San Anselmo and Petaluma, California, failed to provide "full and equal accommodations" for men.
You can't get your fitness business venture off the ground without capital. Did you know there are numerous ways to garner financial assistance? An online resource called BusinessFinance.com categorizes the funding criteria of more than 4,000 business capital sources. It also features a special section for start-ups that helps you figure out exactly how much money you need to get your brilliant idea off the ground. For more information, visit www.businessfinance.com.
For the latest research, statistics, sample classes, and more, "Like" IDEA on Facebook here.
Joy Keller
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|Extreme Interval Training
In this course you'll learn goal-focused intervals and over 50 dynamic exercises and drills to create extensive and intensive training formats.
|Cut to the Core
This is a raw, unedited video filmed live at the 2009 IDEA World Fitness Convention™. Cut to the Core is packed full of core-focused exercises that aim to improve the way you look, feel and live.
|September 2011 IDEA Fitness Journal Quiz 4: Plyometric Training
This continuing education quiz is an in-depth look at plyometric training. Plyometric exercises—jumping, bounding, hopping, arm pushing, and catching and throwing weighted objects such as machine balls—are movements that involve rapid eccentric and concentric muscle actions.