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sporting brands
Posted by Mark J. Miller on September 15, 2011 11:18 AM
It's generally best for a passionate male sports fan to keep his obsession out of the bedroom when it comes to relations to the opposite sex, but a company in England is helping to change that predicament.
Premier Lingerie has partnered with two Premier League clubs to produce lingerie with the team's clubs and colors. And this isn't just so the fellas can live out some sort of Harlequin-style soccer fantasy — it's for the female footie fan.
"Women now make up about 20 percent of football supporters but only three to five percent of club merchandise is aimed at them," said Paul Robinson, director of Premier Lingerie, to the Daily Mail. "A lot of clubs simply stock pink T-shirts with the club's logo on them. That's not good enough. Not enough clubs are doing anything sassy for their female fans, so that's where we're helping."
Premier Lingerie recently partnered with Premier League team Newcastle United on the heels of signing a deal with the Wolverhampton Wanderers before the holidays last year. It is in conversations with other teams about producing similar lingerie lines, the brand's website notes.
We may all have Katy Perry to thank for this.
Back at the 2009 MTV Music Awards in Berlin, Perry showed up in lingerie that was the color of the uniform of West Ham United, the favorite team of her British comedian husband, Russell Brand. The also prominently featured the logo of the team as well. The Hammers picked up on the brand extension and briefly sold Katy Perry-inspired lingerie at its own club shops.
The Newcastle United collection, by the way, includes briefs, stockings, suspender belts, bras, and camisoles (bring your own whistle). It also brings a whole new meaning to the British expression, "take off your kit!"
More about: Premier Lingerie, Premier League, UK, Sports, Lingerie, Fashion, Wolverhampton Wanderers, Newcastle United, West Ham United, Russell Brand, Katy Perry
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... to get to therapeutic levels in my body? And has anyone out there who has had a seizure (partial) found a drug, it worked, and is living a 'normal' life with no side effects??
I am transitioning off dilantin(generic) and on to trileptal . How long does it take for trileptal?
- Posted:
- 7 Jul 2012 by nana9848
- Topics:
- dilantin, therapeutic, trileptal, seizures, side effect, generic
Answers (1)
16 Jul 2012
Hello nana,
Theoretically, after 5 doses you should have reached a steady blood level. In answer to your other question about living a normal life, I believe after you are seizure free for two years, you may begin to drive again. Not sure, depends on where you are. I have been on anti-seizure meds for my bipolar disease and I live a normal or as normal it can get for a crazy person. So, I'm not sure what normal is. Normal apparently means different things to different people. Philosophically, what is normal? Anyway, I hope I didn't confuse you as much as myself.
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Stewartia pseudocamellia
I'm in northern coastal Washington. I need a four (or at least 3) season specimen tree. It will be in a fairly exposed location. We get pretty strong windstorms several times each year. I was thinking of Stewartia pseudocamellia, but I don't know much about it's wind behavior. Is it prone to breakage, etc.?
Any other suggestions for a multi-season tree for such a location?
Stewartia might take about 30 years to grow 30' tall, if you like that one try it. Tallest ones measured in Seattle 41'-42'+, after many years of growth.
Although De Wilde's reportedly had problems with winter injury in (I guess) their fields out off Meridian, tree is native to and grown in much colder places than Bellingham. Usual critical factor for it in PNW is lack of drenching rains in summer. Must be placed in soil that remains moist.
Downtown Everett has some nice ones growing out of small openings in pavement over what appears to be the most seemingly inhospitable Alderwood glacial till.
With a discarded cigarette butt mulch.
So, a soil remaining moist (as this location may, due to shading and density of the soil) could be the most important attribute - trumping other concerns including organic content.
Of course, the city may sometimes come along and water the openings - although I have seen no sign of this myself, the soil being dry on top when viewed later in summer.
Look for stock differentiated as Korean stewartia as that tends to be ornamentally superior, with more effective flowers and fall color, narrower growth lending itself better to planting sites with less abundant space - as with dogwoods and many other decorative trees you really cannot lop side branches back to control spread without spoiling the tree's appearance.
Or you could try something else entirely.
Here is a link that might be useful: 25 Trees that thrive in the maritime Northwest's Dry Summers.
I've grown Stewartia pseudocamellia var. koreana in Bellingham for 15 years with no hardiness or breakage problems. Wonderful fall color, peeling bark, flowers, and structure-- an all-year tree. It does grow slowly, a little over a foot a year, and needs a position where the soil doesn't bake in summer. I watered it a lot the first few years but now only after 2 or 3 weeks of drought. Mulching helps, as does allowing moss to take over on the ground beneath the tree. Really the only trouble I go to for this plant is rubbing lichen from its branches each year.
Even better for bark effects and fall color is Stewartia monadelpha, but the flowers aren't as showy. It seems to be a bit more tolerant of dryness.
The USDA puts Bellingham in Zone 8. Mulching makes a huge difference, when a soil is bare the sun bakes moisture out of it - you can actually see it rising up, as mist. When a root zone becomes shaded by branches or groundcover plants, this also produces a much better situation for roots of plants not liking hot and dry conditions. Sensitive types like rhododendrons may get root rot and die off even if a warm soil is kept damp.


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What are you waiting for?!
I thought it was 50% off.
75% off for today only. I'm glad it's not Victoria II because I took the plunge and bought that yesterday.
This is a great deal though because I pre-ordered and paid full price. I may even get the music DLC at that price.
Bought dynasty shields, mongol faces and ruler designer. Great offer! (already had CK2)
I bought 2 of the music DLC's yesterday. I feel like steam owes me 78p now :/
buying ruler designer and the 3 song packs
Bought it just now! I shall have tons of fun!
Oohh wait...I got exams in about 2 weeks
Oh well, got the shields, had a spare 38p sitting around.
That is a pretty amazing deal for the game imo.
I'm such a victim and got both CK1 complete and CK2 with DLCs for 15 euro. I want to ask though is there a way to get the serial for CK1 so I can enter it here in the forum?
Check my Crusader Kings gfx mod: New muslim and pagan CoA frames!
* Click here if you value your derp time for herp.
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Not a fan of relatively new games getting discounted by so much so fast, but luckily CK2 is one of those pre-orders I never regretted, and still don't. Also picked up the music DLC today, so all is well!
Sine anime nihil sumus
I'm thinking they may be doing it to convince peoples that haven't taken the plunge to get in now so they still have a bit left to buy the SoI expansion/dlc when it comes out.
May have to look into buying EUIII again since its still 50% off :P
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The invention pertains to the genetically down regulating a lignin pathway p-coumarate Co-enzyme A ligase (CCL) in trees.
1. An isolated promoter comprising SEQ ID NO:6.
2. A polynucleotide construct comprising the promoter of
3. The construct of
4. A method of expressing a polynucleotide coding sequence in the epidermis of a plant, comprising:
(a) introducing the DNA construct of
(b) regenerating a transgenic plant from the plant cell, wherein the polynucleotide coding sequence is expressed in the epidermis of the plant.
5. The method of
6. A transgenic plant produced by the method of
This application is a divisional of U.S. patent application Ser. No. 08/969,046, filed Nov. 12, 1997, now U.S. Pat. No. 6,455,762, issued Sep. 24, 2002.
The present invention was made at least in part with the support of the United States Government awarded by a grant from the U.S. Department of Agriculture (Grant No. 95-37103-2061). Therefore, the Government may have certain rights in the invention.
The invention relates to genetically modifying trees through manipulation of the lignin biosynthesis pathway, and more particularly, to genetically modifying trees through the down regulation of p-coumarate Co-enzyme A ligase (CCL) to achieve faster growth, and/or altered lignin content, and/or altered lignin structure, and/or altered cellulose content and/or disease resistance of the trees and to the use of promoters of the CCL genes to drive gene expression specifically in xylem tissue or specifically in epidermal tissues.
Genetic engineering of forest tree species to conform to desired traits has shifted the emphasis in forest tree improvement away from the traditional breeding programs during the past decade. Although research on genetic engineering of forest trees has been vigorous, the progress has been slow due.
The ability to make trees grow faster and be disease resistant to produce the highest volume of wood in the shortest period of time has been and continues to be the top objective of many forest products company worldwide. The ability to genetically increase the optimal growth of trees would be a commercially significant improvement. Faster growing trees could be used by all sectors of the forest and wood products industry worldwide.
Lignin, a complex phenolic polymer, is a major component in cell walls of secondary xylem. In general, lignin constitutes 25% of the dry weight of the wood, making it the second most abundant organic compound on earth after cellulose. Although lignin plays an important role in plants, it usually represents an obstacle to utilizing biomass in several applications. For example, in woodpulp production, lignin has to be removed through expensive and polluting processes in order to recover cellulose.
Thus, it is desirable to genetically engineer plants with reduced lignin content and/or altered lignin composition that can be utilized more efficiently. Trees that could be genetically engineered with a reduced amount of lignin would be commercially valuable. These genetically engineered trees would be less expensive to pulp because, in essence, part of the pulping has already been performed due to the reduced amount of lignin.
Trees with increased cellulose content would also be commercially valuable to the pulp and paper industry.
Disease resistance in plants is also a most desirable plant trait. The impact of disease resistance in trees on the economy of forest products industry worldwide is significant.
Promoters that target specific plant tissue could be useful in manipulating gene expression to enable the engineering of traits of interest in specific tissue of plants, such as, xylem and epidermal tissues.
Although studies have revealed several general properties of plant p-coumarate Co-enzyme A ligase (CCL), the role of CCL in regulating the synthesis of monolignols in response to different states of development and environmental stress in tree species remains largely unknown. Furthermore, multiple CCL isoforms are normally present in plants, channeling phenolic compounds to the biosynthesis of not only lignin but also other phenylpropanoids, such as flavonoids. Since CCL isoforms have not been previously cloned from tree species for the identification of their biochemical functions, the presence of CCL isoforms remains so far as a challenge to a specific control of metabolic flux to the lignin biosynthesis in tree species.
The invention provides a method to genetically alter trees through the down regulation of p-coumarate Co-enzyme A ligase (CCL). Such down regulation of CCL results in faster growth, and/or reduced lignin content, and/or altered lignin structure, and/or altered cellulose content and/or disease resistance. The invention also provides for genetically engineered trees which have been altered to down regulate p-coumarate Co-enzyme A ligase (CCL) to achieve faster growth, and/or reduced lignin content, and/or altered lignin structure, and/or increased cellulose content and/or increased disease resistance. The invention also provides tissue specific promoters of the CCL genes that can be used to manipulate gene expression in target tissue such as xylem and epidermal tissues.
It is one object of the present invention to down regulate p-coumarate Co-enzyme A ligase (CCL) in trees.
It is another object of the present invention to provide a method to genetically alter trees to grow faster.
It is another object of the present invention to provide a method to genetically alter the growth of trees through manipulation the lignin pathway p-coumarate Co-enzyme A ligase.
It is another object of the present invention to provide genetically altered trees with an accelerated growth characteristic.
It is another object of the present invention to provide transgenic trees with an accelerated growth characteristic which have been genetically altered by down regulating lignin pathway p-coumarate Co-enzyme A ligase.
It is another object of the present invention to provide a method to genetically alter trees to reduce their lignin content.
It is another object of the present invention to provide a method to genetically alter the lignin content of trees through manipulation of a lignin pathway enzyme.
It is another object of the present invention to genetically engineer trees which have reduced lignin content through manipulation of lignin pathway p-coumarate Co-enzyme A ligase.
It is another object of the present invention to provide genetically altered trees with a reduced lignin content.
It is another object of the present invention to provide transgenic trees with reduced lignin content which have been genetically altered by down regulating the p-coumarate Co-enzyme A ligase (CCL).
It is another object of the present invention to provide a method to genetically alter trees to change their lignin structure through manipulation of lignin pathway p-coumarate Co-enzyme A ligase.
It is another object of the present invention to provide trees with altered lignin structure.
It is another object of the present invention to provide a method to increase the cellulose content in trees.
It is another object of the present invention to provide a method to increase the cellulose content of trees through the manipulation of a lignin pathway enzyme.
It is another object of the present invention to provide trees with increased cellulose content.
It is another object of the present invention to provide transgenic trees having increased cellulose content from the down regulation of CCL.
It is another object of the present invention to provide a method to genetically alter trees to increase their disease resistance.
It is another object of the present invention to provide a method to genetically alter trees to be more disease resistant through manipulation of the lignin pathway p-coumarate Co-enzyme A ligase.
It is another object of the present invention to genetically alter trees to increase their disease resistance to fungal pathogens.
It is another object of the present invention to provide trees with increased disease resistance.
It is another object of the present invention to provide transgenic trees with increased disease resistance through down regulation of the lignin pathway p-coumarate Co-enzyme A ligase.
It is another object of the present invention to provide a method using a promoter of a CCL gene to target gene expression in specific plant tissue.
It is another object of the present invention to provide a method using a promoter of a CCL gene to target gene expression specifically in plant xylem.
It is another object of the present invention to provide a method using a promoter of the CCL gene to target gene expression specifically in the epidermal tissues of plants.
It is another object of the present invention to provide a CCL gene promoter that targets gene expression specifically in the xylem of plants.
It is another object of the present invention to provide a CCL gene promoter that targets gene expression specifically in the epidermal tissues of plants.
Other features and advantages of the invention will become apparent to those of ordinary skill in the art upon review of the following drawing, detailed description and claims.
Before one embodiment of the invention is explained in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description of the preferred embodiment. The invention is capable of other embodiments and of being practiced or being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
The invention pertains to genetically down regulating a lignin pathway p-coumarate Co-enzyme A ligase (CCL). Trees which have been genetically transformed to down regulate CCL will hereafter be called transgenic trees. Such down regulation can result in faster growing trees. Such down regulation can result in a reduction in the lignin content of the trees and/or altered lignin structure. Such down regulation can result in increased cellulose content. Such down regulation can result in increased tree disease resistance. Further, by using a specific promoter of CCL, targeted tissue gene expression can be achieved in either the xylem or the epidermal tissues of the plant.
Lignin is synthesized by the oxidative coupling of three monolignols (coumaryl, coniferyl and sinapyl alcohols) formed via the phenylpropanoid pathway as shown in FIG. 1. Reactions in the phenylpropanoid pathway include the deamination of phenylalanine to cinnamic acid followed by hydroxylations, methylations and activation of substituted cinnamic acids to coenzyme A (CoA) esters. The CoA esters are then reduced to form monolignols which are secreted from cells to form lignin.
The products of the phenylpropanoid pathway are not only required for the synthesis of lignin but also required for the synthesis of a wide range of aromatic compounds including flavonoids, phytoalexins, stilbenes and suberin.
In angiosperms (hardwoods), lignin is composed of both coniferyl and sinapyl alcohol and is classified as guaiacyl-syringyl lignin. Grasses synthesize a third precursor (p-coumaryl alcohol) which is polymerized along with coniferyl and sinapyl alcohol. In gymnosperms (softwoods), lignin is composed of mainly coniferyl alcohol and is classified as guaiacyl lignin.
In the phenylpropanoid pathway, CCL activates a number of cinnamic acid derivatives, including p-courmaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid and sinapic acid. The resulting products, CoA esters, serve as substrates for entry into various branch pathways, such as lignin, flavonoids, phytoalexins, stilbenes and suberin. The esterification reactions catalyzed by CCL require high energy and the reactions are not likely to occur without CCL. CCL is important in making a continuous flow of the lignin biosynthesis pathway. CCL is also important because it is located at the branching points of the phenylpropanoid metabolism. CCL is suggested to play a pivotal role in regulating carbon flow into specific branch pathways of the phenylpropanoid metabolism in response to stages of development and environmental stress.
The basic properties of CCL are quite uniform. CCL depends on ATP as a cosubstrate and requires Mg2+ as a cofactor. The optimal pH for CCL ranges from pH 7.0 to 8.5 and the molecular weight of CCL isoforms from various plant species ranges from 40 kd to 75 kd. Most CCLs have high affinity with substituted cinnamic acids. CCL has the highest activity with p-coumaric acid.
CCL cDNA sequences have been reported for parsley, potato, soybean, loblolly pine, Arabidopsis, Lithosperum and tobacco. CCL genes have been isolated and sequenced for parsley, rice, potato and loblolly pine. The analysis of CCL cDNAs and genes indicates that CCL is encoded by multiple/divergent genes in rice, soybean, and Lithosperum, very similar genes in parsley, potato, tobacco and loblolly pine, and a single gene in Arabidopsis. CCL promoters have been isolated and sequenced for parsley, rice and potato.
Alignment of deduced amino acid sequences of cloned plant CCL sequences reveals two highly conserved regions. The first conserved region (SSGTTGLPKGV),(SEQ ID NO:7) proposed to designate a putative AMP-binding region, is very rich in Gly, Ser and Thr and is followed by a conserved Lys. The second conserved region (GEICIRG), (SEQ ID NO:8) contains one common Cys residue. The amino acid sequences of CCL from plants contain a total of five conserved Cys residues.
The CCL genes of parsley, potato and rice contain five exons and four introns. The CCL genes also share the same exon/intron splice junction sites but have different lengths of introns. The genomic sequences of loblolly pine CCL are composed of four exons and three introns. It has been found that two similar CCL genes of the same species may differ slightly in length of intron as shown in two parsley genes (PC4CL1 and PC4CL2) and in two loblolly pine genes (LP4CL1 and LP4CL2).
By Northern blot analysis, it has been shown that CCL is expressed in leaf, shoot tip, stem, root, flower and cell culture. Two similar CCL cDNAs in parsley, potato and tobacco have been shown to be expressed at similar level in response to the environmental stress and during different developmental stages. Two distinct CCL cDNAs in soybean and Lithosperum have shown different expression levels when pathogens or chemicals were applied to the cell cultures. It appears that the expression of the CCL genes is developmentally regulated and inducible by many environmental stresses at the transcription level.
Genetic transformation with a CCL sequence can result in several significant affects. The description of the invention hereafter refers to aspen, and in particular quaking aspen (Populus tremuloides Michx) when necessary for the sake of example. However, it should be noted that the invention is not limited to genetic transformation of aspen. The method of the present invention is capable of being practiced for other trees, including for example, other angiosperms, other gymnosperm forest tree species, etc.
Preferably, the CCL down regulation is accomplished through transformation with a homologous CCL sequence in an antisense orientation. However, it should be noted that a heterologous antisense CCL sequence could be utilized and incorporated into a tree species to down regulate CCL if the heterologous CCL gene sequence has a high nucleotide sequence homology, approximately higher than 70%, to the endogenous CCL gene sequence of that tree species.
In addition, trees transformed with a sense CCL sequence could also cause a sequence homology-based cosuppression of the expression of the transgene and endogenous CCL gene, thereby achieving down regulation of CCL in these trees.
B. Isolation of CCL cDNAs
The present invention utilizes a homologous CCL sequence to genetically alter trees. The preferred embodiment of the invention as further described below utilizes a cDNA clone of the quaking aspen CCL gene.
Two aspen (Populus tremuloides Michx) cDNAs encoding two distinct CCL isoforms, PtCCL1 and PtCCL2 have been cloned. PtCCL1 cDNA is lignin pathway-specific and is different from PtCCL2 cDNA, which is involved in flavonoid synthesis. The cloning of PtCCL1 and PtCCL2 cDNAs and the identification of their biochemical functions will be discussed in more length below. PtCCL1 and PtCCL2 genomic clones including their 5′-end regulatory promoter sequences were also isolated. The promoter of PtCCL1 (PtCCL1p) directs xylem tissue-specific gene expression in a plant, whereas the promoter of PtCCL2 (PtCCL2p) drives the expression of genes specifically in epidermal tissues of stem and leaf of a plant. These tissue specific promoters will be discussed in more length in Section I below.
Two CCL cDNAs, PtCCL1 and PtCCL2, have been isolated from quaking aspen using either a conventional cDNA library screening method or a PCR-based cDNA cloning method. It should be noted that the methods described below are set forth as an example and should not be considered limiting. These CCL cDNA clones are available from Michigan Technological University, Institute of Wood Research, Houghton, Mich.
Young leaves and shoot tips are collected from greenhouse-grown quaking aspen (Populus tremuloides Michx). Differentiating xylem and sclerenchyma are collected from three to four year old quaking aspen. The bark is peeled from the tree exposing the developing secondary xylem on the woody stem and the sclerenchyma on the inner side of the bark. Developing secondary xylem and sclerenchyma are scraped from the stem and bark with a razor blade and immediately frozen in liquid nitrogen until use.
Total RNA is isolated from the young leaves, shoot tips, xylem and sclerenchyma following the method of Bugos RC et al. (1995), RNA Isolation from Plant Tissue Recalcitrant to Extraction in Guanidine, Biotechniques 19(5):734-737. Poly(A)+RNA is purified from total RNA using Poly(A)+mRNA Isolation Kit from Tel-test B, Inc. A unidirectional Lambda gt22 expression cDNA library was constructed from the xylem mRNA using Superscript S System from Life Technologies, Inc. and Gigapack Packaging Extracts from Stratagene. The PtCCL1 cDNA was obtained by screening the cDNA library with a 32P-labeled parsley 4CL cDNA probe. The parsley 4CL cDNA (pc4CL2) has Genbank accession number X13325, (SEQ ID NO:15).
The PtCCL2 cDNA was obtained by RT-PCR. The reverse transcription of total RNA isolated from shoot tips was carried our using the Superscript II reverse transcriptase from Life Technologies. Two sense primers (R1S, 5′-TTGGATCCGGIACIACIGGIYTICCIAARGG-3′ (SEQ ID NO:9) and HlS, 5′-TTGGATCCGTIGCICARCARGTIGAYGG-3′ (SEQ ID NO:10)) are designed around the first consensus AMP-binding region of CCL that was previously discussed. One antisense primer (R2A, 5′-ATGTCGACCICGDATRCADATYTCICC-3′) (SEQ ID NO:11) is designed based on the sequence of the putative catalytic motif GEICIRG (SEQ ID NO:8). One fifth of the reverse transcription reaction (4 Tl) is used as the template in a 50 Tl PCR reaction containing 1� reaction buffer, 200 TM each deoxyribonucleotide triphosphate, 2 TM each R1S and oligo-dT (20 mer) primers, and 2.5 units of Taq DNA polymerase. The PCR reaction mixture was denatured at 940 C for 5 minutes followed by 30 cycles of 940 C/45 seconds, 500 C/1 minute, 720 C/90 seconds and is ended with a 5 minute extension at 720 C. 2 T1 of the PCR amplification products are used for a second run PCR re-amplification using primers H1S and R2A. A 0.6 kb PCR fragment is cloned using the TA Cloning Kit from Invitrogen and used as a probe to screen an aspen genomic library to obtain the PtCCL2 genomic clone. Two primers (2A, 5′-TCTGTCTAGATGATGTCGTGGCCACGG-3′ (SEQ ID NO:12) and 2B, 5′-TTAGATCTCTAGGACATGGTGGTGGC-3′ (SEQ ID NO:13)) are designed based on the genomic sequence of PtCCL2 at around the deduced transcription start site and stop codon for the cloning of PtCCL2 cDNA by RT-PCR as described above using total RNA isolated from shoot tips.
The DNA sequences of PtCCL1 and PtCCL2 cDNA were determined using�Taq Cycle Sequencing Kit from Amersham.
The PtCCL1 cDNA has an open reading frame of 1620 bp which encodes a polypeptide of 540 amino acid residues with a predicted molecular weight of 59 kd and pI of 5.8. The nucleotide sequence of the aspen CCL cDNA clone PtCCL1 is set forth as SEQ ID NO:1. The deduced amino acid sequence for the aspen CCL1 protein is set forth as SEQ ID NO:2.
The PtCCL2 cDNA has an open reading frame of 1713 bp which encodes a polypeptide of 571 amino acid residues with a predicted molecular weight of 61.8 kd and pI of 5.1. The nucleotide sequence of the aspen CCL cDNA clone PtCCL2 is set forth as SEQ ID NO:3. The deduced amino acid sequence for the aspen CCL2 protein is set forth as SEQ ID NO:4.
The aspen PtCCL1 cDNA shares a 59-74% identity at the nucleotide level and 59-81% identity at the amino acid level with other prior reported CCL cDNAs and genes, whereas the PtCCL2 cDNA shares a 60-73% identity at the nucleotide level and 57-74% at the amino acid level with other CCL cDNAs and genes as set forth in the following table.
The results of sequence analysis, phylogenetic tree and genomic Southern blot analysis indicate that PtCCL1 and PtCCL2 cDNAs encode two distinct CCLs that belong to two divergent gene families in aspen. The deduced amino acid sequence for the PtCCL2 protein contains a longer N-terminal sequence than the PtCCL1 protein but shows profound similarity in the central and C-terminal portions of protein to the PtCCL1 protein.
PtCCL1 and PtCCL2 cDNAs display distinct tissue-specific expression patterns. The PtCCL1 sequence is expressed highly in the secondary developing xylem and in the 6th to 10th internodes whereas the PtCCL2 sequence is expressed in the shoot tip and leaves. These tissue-specific expression patterns were investigated by fusing promoters of PtCCL1 and PtCCL2 genes to a GUS reporter gene. The tissue specific promoters for PtCCL1 and PtCCL2 will be discussed in more length in Section I below.
The substrate specificity of PtCCL1 and PtCCL2 is also different from each other as determined using recombinant proteins produced in E. Coli. PtCCL1 utilized p-coumaric acid, caffeic acid, ferulic acid and 5-hydroxyferulic acid as substrates. PtCCL2 showed activity to p-coumaric acid, caffeic acid and ferulic acid but not to 5-hydroxyferulic acid.
Specifically, PtCCl1 and PtCCL2 were used to construct expression vectors for E.coli expression. The substrate specificity of PtCCL1 and PtCCL2 were tested using fusion proteins produced in E.coli. Two plasmids, pQE/CCL1 and pQE/CCL2, were constructed in which the coding regions of PtCCL1 and PtCCL2, respectively were fused to N-terminal His tags in expression plasmids pQE-31 and pQE-32 (QIAGEN, Chatsworth, Calif.). The recombinant proteins of PtCCL1 and PtCCL2 produced by E.coli are approximately 59 kd and 63 kd, respectively.
The two recombinant proteins were tested for their activity in utilizing cinnamic acid derivatives. PtCCL1 recombinant protein showed 100, 58, 71, 18 and 0% relative activity to p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid and sinapic acid, respectively. PtCCL2 recombinant protein exhibited 100, 14, 27, 0 and 0% relative activity to p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid and sinapic acid, respectively. Neither recombinant protein showed detectable activity to sinapic acid.
The results of the tissue-specific expression pattern and substrate specificity suggests that in addition to the general function of CCL, PtCCL1 apparently is more related to lignin synthesis in the xylem tissue and PtCCL2 apparently is more likely involved in flavonoid synthesis and UV protection.
It should be noted that the isolation and characterization of the PtCCL1 and PtCCL2 cDNA clones is described in Kawaoka A, Chiang VL (1995), The Molecular Cloning and Expression of Syringyl- and Guaiacyl-Specific Hydroxycinnamate: CoA Ligases from Aspen (Populus tremuloides ), Proceedings of the 6th International Conference on Biotechnology in the Pulp and Paper Industry, Vienna, Austria; and in Hu, Wen-Jing, Isolation and Characterization of p-coumarate Co-enzyme A ligase cDNAs and Genes from Quaking Aspen (Populous temuloides Michx), Ph.D Dissertation, Michigan Technological University, Houghton, Mich. (1997); which are both herein incorporated by reference.
C. Transformation and Regeneration
Several methods for gene transformation of plant species with the CCL sequence are available such as the use of a transformation vector, agroinfection, electroinjection, particle bombardment with a gene gun or microinjection.
Preferably, a CCL cDNA clone is positioned in a binary expression vector in an antisense orientation under the control of double cauliflower mosiac virus 35S promoter. The vector is then preferably mobilized into a strain of Agrobacterium species such as tumefaciens strain C58/pMP90 and is used as the DNA delivery system due to its efficiency and low cost.
For example, with reference to
The gene construct pACCL1 is available from Michigan Technological University, Institute of Wood Research, Houghton, Mich.
The binary vector construct is mobilized in Agrobacterium tumefaciens using the freeze-thaw method of Holsters et al., Mol. Gen. Genet. 163:181-187 (1978). For the freeze-thaw method, 1.5 ml of overnight cultures Agrobacterium tumefaciens strain C58/pMP90 is pelleted at 5000�g for 3 minutes at 40 C and suspended in 1 ml of ice cold 20 mM CaCl2. To the suspension is added 10 1l binary vector DNA (from an alkaline lysis minipreparation) and mixed by pipetting. The microcentrifuge tube is then frozen in liquid nitrogen for 5 minutes and thawed at 370 C for 5 minutes. After being cooled on ice, 1 ml of LB is added and the mixture is incubated at 280 C for 2 hours with gentle shaking. 200 1l of the cells is spread onto LB plates containing gentamycin and kanamycin and incubated at 280 C for 2 days. Colonies grown on the selection plates are randomly picked or miniprep and restriction enzyme digestion analysis is used to verify the integration.
The resulting binary vector containing Agrobacterium strain is used to transform quaking aspen according to Tsai et al., Agrobacterium-Mediated Transformation of Quaking Aspen (Populous temuloides) and Regeneration of Transgenic Plants, Plant Cell Rep. 14:94-97 as set forth below.
Explants of young leaves from cuttings of aspen are obtained by cutting leaf disks of approximately 7 mm square from the young leaves along the midrib of the leaves. The explants are surface sterilized in 20% commercial bleach for 10 minutes followed by rinsing 3 times with sterile double-distilled water.
All of the culture media used includes the basal medium of woody plant medium (WPM) as described in Lloyd et al., Proc. Int. Plant Prop. Soc. 30:421-437 (1980) and supplemented with 2% sucrose. 650 mg/L calcium gluconate and 500 mg/L MES are added as pH buffers as described in Tsai, Plant Cell Reports, 1994. All culture media is adjusted to pH 5.5 prior to the addition of 0.075% Difco Bacto Agar and then autoclaved at 1210 C and 15 psi for 20 minutes. Filter sterilized antibiotics are added to all culture media after autoclaving. All culture media are maintained at 23�10 C in a growth chamber with 16 hour photoperiods (1601E�m−2�S−1) except for callus induction (as will be described later) which is maintained in the dark.
The sterilized explants are then inoculated with the mobilized vector with an overnight-grown agrobacterial suspension containing 20 μM acetosyringone. After cocultivation for 2 days, the explants are washed in 1 mg/ml claforan and ticarcillin for 2 hours with shaking to kill Agrobacterium. The explants are blotted dry with sterile Whatman No. 1 filter paper and transferred onto callus induction medium containing 50 mg/L kanamycin and 300 mg/L claforan to induce and select transformed callus. The callus induction medium is the basal medium with the addition of 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 0.5 mg/L and 1 mg/L, respectively, to induce callus.
The kanamycin-resistant explants are then subcultured on fresh callus induction media every two weeks. Callus formation occurs after approximately four weeks. Formed callus are separated from the explant and subcultured periodically for further proliferation.
When the callus clumps reach approximately 3 mm in diameter, the callus clumps are transferred to shoot regeneration medium. The shoot regeneration medium is the basal medium containing 50 mg/L kanamycin, 0.5 mg/L thidiazuron (TDZ) as a plant growth regulator and cefotaxime at 300 mg/L to kill Agrobacterium. Shoots were regenerated about 4 weeks after callus is transferred to regeneration medium. As soon as the shoots are regenerated, they are immediately transferred to hormone-free elongation medium containing 50 mg/L kanamycin and, whenever necessary, cefotaxime (300 mg/L), to promote elongation. Green and healthy shoots elongated to 2-3 cm in length are excised and planted separately in a hormone-free rooting medium containing 50 mg/L kanamycin. The efficient uptake of kanamycin by shoots during their rooting stage provides the most effective selection for positive transformants. Transgenic plants are then transplanted into soil medium of vermiculite:peatmoss:perlite at 1:1:1 and grown in the greenhouse.
The above described transformation and regeneration protocol is readily adaptable to other tree species. Other published transformation and regeneration protocols for tree species include Danekar et al., Bio/Technology 5:587-590 (1987); McGranahan et al., Bio/Technology 6:800-804 (1988); McGranahan et al., Plant Cell Reports 8:512-616 (1990); Chen, phD Thesis, North Carolina State University, Raleigh, N.C. (1991); Sullivan et al., Plant Cell Reports 12:303-306 (1993); Huang et al., In Vitro Cell Dev. Bio. 4:201-207 (1991); Wilde et al., Plant Physiol. 98:114-120 (1992); Minocha et al., 1986 Proc. TAPPI Research and Development Conference, TAPPI Press, Atlanta, pp. 89-91 (1986); Parsons et al., Bio/Technology 4:533-536 (1986); Fillatti et al., Mol. Gen. Genet 206:192-199 (1987); Pythoud et al., Bio/Technology 5:1323-1327 (1987); De Block, Plant Physiol. 93:1110-1116 (1990); Brasileiro et al., Plant Mol. Bio 17:441-452 (1991); Brasileiro et al., Transgenic Res. 1:133-141 (1992); Howe et al., Woody Plant Biotech., Plenum Press, New York, pp. 283-294 (1991); Klopfenstein et al., Can. J. For. Res. 21:1321-1328 (1991); Leple et al., Plant Cell Reports 11:137-141 (1992); and Nilsson et al. Transgenic Res. 1:209-220 (1992).
D. Phenotype Changes
The results of the transformation can be confirmed with conventional PCR and Southern analysis. For example, transferring CCL cDNA in an antisense orientation down regulates CCL in the tree. Expression of the CCL has been found to be blocked up to 96 percent in some transgenic trees.
After acclimation, the transgenic aspen display an unusual phenotype, including big curly leaves, thick diameters, longer internodes, more young leaves in the shoot tip and a red pigmentation in the petioles extending into midvein leaves. Red coloration of the developing secondary xylem tissues is observed after peeling of the bark in the transgenic plants.
E. Accelerated Growth
Down regulation of CCL alters growth of the transgenic trees. For example, transformation with an antisense CCL sequence accelerates the growth of the tree. Enhanced growth is markedly noticeable at all ages. In particular, the transgenic trees show enhanced growth in the form of thicker stems and enlarged leaves as compared to control trees. These characteristics are retained in the vegetative propagules of these transgenic trees. Table 2 sets forth exemplary data with respect to several lines of transgenic quaking aspen grown in the greenhouse after eight months. Volume represents the overall quantitative growth of the tree.
The averages for height, diameter, volume and average length between internodes for the control plants are as follows:
With respect to height alone, for those transgenic plants (11-1, 11-4, 11-8, 11-9, 12-6, 16-2, 16-3) having a statistically larger height than the control plants, the average height was 286.83 cm as compared to the control plant average height of 248.95 cm.
With respect to diameter alone, for those transgenic plants (11-1, 11-7, 11-8, 11-9) having a statistically larger diameter than the control plants, the average diameter was 1.30 cm as compared to the control plant average diameter of 1.045 cm.
With respect to volume alone, for those transgenic plants (11-1, 11-8, 11-9, 12-1, 16-3) having a statistically larger volume than the control plants, the average volume was 120.2 cm3 as compared to the control plant average volume of 71.2 cm3.
With respect to average length of internodes alone, for those transgenic plants (11-1, 11-2, 11-3, 11-4, 11-5, 11-7, 11-8, 11-9, 11-10, 12-6, 16-2, 16-3) having a statistically larger average length of internodes than the control plants, the average average length of internodes was 3.39 cm as compared to the control plant average average length of internodes of 2.70 cm.
As demonstrated in Table 2, while there are variations in growth among the transgenic trees, the average length of the internodes for the transgenic trees is consistently and significantly higher than that of the control plants. Variations in the growth of the transgenic trees is normal and to be expected. Preferably, a transgenic tree with a particular growth rate is selected and this tree is vegetatively propagated to produce an unlimited number of clones that all exhibit the identical growth rate.
F. Lignin
Down regulation of lignin pathway CCL results in production of trees with reduced lignin content.
The following table shows the reduction of lignin content and CCL enzyme activity in several transgenic aspen which have been transformed with an homologous antisense CCL sequence.
Lignin content was determined according to Chiang and Funaoka (1990) Holzforschung 44:147-155. CCL enzyme activity was determined according to Ranjeva et al. (1976), Biochimie 58:1255-1262.
The data in Table 3 demonstrates a correlation between down regulation of CCL and reduction in lignin content.
Transgenic trees with reduced lignin content have an altered phenotype in that the stem is more elastic to the touch and the leaves are typically curlier.
It should also be noted that for those transgenic trees (12-1, 12-2, 12-3 and 16-1) with the approximately 40% reduction in lignin content and the corresponding approximately 95% reduction in CCL enzyme levels, all of those transgenic trees had a consistent deep red coloration in the wood of the plant. Accordingly, the deep red color can be used as an identifier of reduced lignin content.
Down regulation of lignin pathway CCL also results in production of trees with an altered lignin structure. Based upon thioacidolysis (Rolando et al. (1992) Thioacidolysis, Methods in Lignin Chemistry, Springer-Verlag, Berlin, pp 334-349) of plants 12-3 and 16-1, coniferyl alcohol and sinapyl alcohol lignin units are significantly reduced in these two trees as compared to the control tree, as shown in the following table.
The alteration of the frequency of the structural units in lignin of these transgenic trees is evidence that the overall structure of lignin in these plants has been genetically altered.
G. Cellulose Content
Down regulation of lignin pathway CCL results in increased cellulose content of the transgenic plants. Analysis of control and transgenic aspen for carbohydrate content demonstrate a higher cellulose content in the transgenic trees than the control trees. Particularly, the transgenic trees that have over 40% lignin reduction have about 10-15% higher cellulose content than the control. Data is set forth in the following tables for trees that were transformed with homologous CCL in an antisense orientation:
The procedure for carbohydrate analysis utilized is as follows. About 100 mg of powdery woody tissue with sizes that pass a 80-mesh screen was hydrolyzed with 1 mL of 72% (W/W) H2SO4 for 1 hr at 300 C. Samples were then diluted to 4% (W/W) H2SO4 with distilled water, fucose was added as an internal standard, and a secondary hydrolysis was performed for 1 hr at 1210 C. After secondary hydrolysis, the sugar contents of the hydrolysates are determined by anion exchange high performance liquid chromatography using pulsed amperometric detection. Sugar contents are expressed as % of the weight of the woody tissue used. The above procedures are similar to those in a publication by R C Pettersen and V H Schwandt, 1991, J. Wood Chem & Technol. 11:495-501.
H. Increased Disease Resistance
Down regulation of lignin pathway CCL results in production of trees with increased disease resistance, and in particular, with increased fungal pathogen resistance.
In particular, greenhouse transgenic aspen plants showed a disease resistance to fungi such as those which induce leaf-blight disease.
I. Promoters
Two distinct genes encoding CCL and their promoters were cloned. The promoter of PtCCL1 can drive gene expression specifically in xylem tissue and the promotor for PtCCL2 confers gene expression exclusively in the epidermal tissues. These promoters can be used to manipulate gene expression to engineer traits of interest in specific tissues of target plants. The significance of the promoters is the application of the xylem-specific promoter to direct the expression of any relevant genes specifically in the xylem for engineering lignin content, lignin structure, enhanced tree growth, cellulose content and other value-added wood qualities, etc. The importance of the epidermis-specific promoter is its ability to drive the expression of any relevant genes specifically in epidermal tissues for engineering disease-, UV light-, cold-, heat-, drought-, and other stress resistance traits in trees.
Specifically, the promoters of the PtCCL1 and PtCCL2 were conventionally isolated as follows. An aspen genomic library was screened with PtCCL1 cDNA and PtCCL2 partial cDNA fragment to isolate genomic clones of PtCCL1 and PtCCL2. Eleven and seven positive genomic clones were identified for PtCCL1 and PtCCL2 gene, respectively. Among 11 positive clones for PtCCL1, PtCCL1g-4 contained a full length coding sequence and at least 2 kb 5′ flanking regions. The restriction map of PtCCL1g-4 is set forth at FIG. 3.
With respect to PtCCL2, restriction map analysis was performed onλDNA of positive genomic clone PtCCL2g-11. The restriction map of PtCCL2g-11 is set forth at FIG. 4.
Approximately a 2.3 kb 5′ flanking region of PtCCL1 was digested from PtCCL1g-4 using Xba I and Sac I sites and cloned into pGEM7Z Xba I and Sac I sites. The subcloned PtCCL1 promoter was named p7Z-4XS and the restriction map of P7Z4XS is set forth at FIG. 5. The 5′ unilateral deletion of p7Z-4XS was generated for DNA sequencing by exonuclease III/S1 nuclease digestion using Erase-a-Base System (Promega, Madison, Wis.). The deletion series was sequenced using a primer on pGEM7Z vector.
A 1.6 kb Hind III and EcoR I fragment containing a 1.2 kb 5′ flanking region of PtCCL2 and 0.4 kb coding region of PtCCL2g-11 were subcloned in pBluescript II SK+ Hind III and EcoR I sites. The restriction map of the resulting clone, pSK-11HE, was determined by digesting the plasmid with several restriction enzymes, as in set forth at FIG. 6. In order to determine the sequence of the PtCCL2 promoter, pSK-11HE was further digested into small fragments according to the restriction map and subcloned into vectors with suitable cloning sites. The DNA sequence was determined using M13 universal primer and reverse primer on the vector.
The DNA sequences of the two promoters was determined and analyzed using qTaq cycle sequencing Kit (USB, Cleveland, Ohio), and GENETYX-MAC 7.3 sequence analysis software from Software Development Co., Ltd. The nucleotide sequence of promoter region of PtCCL1 is set forth as SEQ ID NO:5 and the nucleotide sequence of the promoter region of PtCCL2 is set forth as SEQ ID NO:6. The promoter gene constructs PtCCL1p and PtCCL2p are available from Michigan Technological University, Institute of Wood Research, Houghton, Mich.
Tissue-specific expression can be achieved by conventionally fusing the promoters of PtCCL1 or PtCCL2 to a gene of interest and transferred to a plant species via Agrobacterium. For the sake of example, the promoters of PtCCL1 and PtCCL2 were fused to a GUS reporter gene as detailed below. However, it should be noted that genes other than the GUS reporter gene can be fused to these promoters for tissue specific expression.
In order to construct PtCCL1 promoter-GUS binary vector, a 1 Kb fragment covering 5′-flanking region and 117 bp coding region of PtCCL1 was subcloned into pGEM7Z Sph I and EcoR I sites for constructing promoter-GUS binary vector. In this 1 kb DNA fragment, it is found that one Xho I site locates at 486 bases proximal to the translation start site and the EcoR I site at 117 bases downstream the translation site. This 0.6 Kb fragment was subcloned into pGEM7Z Xho I and EcoR I sites and used as a template in PCR amplification.
In order to construct a promoter-GUS transcriptional fusion, a BamH I site was introduced in front of the translation start site of PtCCL1 by PCR. PCR amplification was performed using p7Z-4XE as the template, M13 universal primer on pGEM7Z vector as 5′ end primer and PtCCL1p-1 primer containing a BamH I site at the end is complementary to a sequence upstream of the translation start site. The reaction was carried out in 100 1l reaction mix containing 1�pfu reaction buffer, 200 1l leach dNTPs, 100 1M each primer and 5 units of pfu. The PCR reaction mixture was denatured at 940 C for 5 minutes followed by 30 cycles of 940 C (1 minute), 550 C (1 minute), 720 C (1 minute, 30 seconds) and was ended with a 5 minute extension at 720C.
The amplified 0.6 Kb fragment was cloned and sequenced to confirm the sequence. The engineered 0.6 Kb fragment was ligated to p7Z-4SE which was digested with Xho I and BamH I. In order to incorporate a Hind III site in the 5′ end of PtCCL1 promoter, the 1 kb Sph I-BamH I PtCCL1 promoter region was the cloned into pNoTA (5 prime→3 prime Inc., Boulder, Colo.) Sph I and BamH I site. The 1 Kb PtCCL1 promoter was then released from pNoTA vector with Hind III and BamH digestion and subsequently transcriptionally fused to pBI101 Hind III and BamH I sites in front of GUS. The resulting binary vector was named PtCCL1p-GUS and is set forth at FIG. 7.
In order to construct PtCCL2 promoter-GUS binary vector, pSK-11HE was digested with Sph I and EcoR I to release 0.2 Kb Sph I and EcoR I fragment. The 0.2 Kb fragment was cloned into pGEM7Z Sph I and EcoR I sites. A primer, PtCCL2p-3′ (5′-CATCGGATCCTGAGATGGAAGGGAGTKTCT-3′ (SEQ ID NO:14)) was designed to be complementary to a sequence upstream of the translation start site of PtCCL2 and to incorporate BamH I site at the end. Amplification was performed using p7Z1 SE as a template, M13 universal primer as the 5′ end primer and PtCCL2p-3 as the 3′ end primer. A PCR reaction was carried out and the amplified PCR product was cloned and sequenced to check the fidelity of the PCR amplification. The 0.2 Kb Sph I-BamH I DNA fragment with correct sequence was fused to pSK-11HE linearized with Sph I and BamH I. The resulting plasmid was named pSK-11HB. The promoter of PtCCL2 was then excised from pSK-11HB with Hind III and BamH I and ligated to PBI101 Hind III and BamH I site to make PtCCL2p-GUS transcriptional fusion binary vector as shown in FIG. 8.
The PtCCL1p-GUS and PtCCL2p-GUS constructs are then mobilized into Agrobacterium tumefaciens strain C58/pMP90 by freeze and thaw method as explained previously.
Leaf disk transformation of tobacco with these two Agrobacterium constructs is conducted according to the method of Horsch R. B. (1988) Leaf Disk Transformation, Plant Molecular Biology Manual, A5:1-9. Histochemical GUS staining of promoter-GUS transgenic tobacco plants demonstrated that the PtCCL1 promoter restricted GUS expression in xylem tissue whereas PtCCL2 promoter regulated GUS expression in epidermal cells.
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Commentary: George's own library
MOUNT VERNON, VIRGINIA -- It was fitting that the buzz around George Washington's homestead recently was about the first president's overdue library books, just as the estate's guardians were plotting a new presidential library in the founding father's name.
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I'm looking to purchase a Bugg (Boston Terrier/Pug) Mix puppy. Please contact with any information. Thank you
Beautiful accident lol my boxer got hooked with my buddies black puggle (pug/ beagle mix) pups are 8 weeks old have first shots and deworm. Mom is 45lbs
Boxer and dad is a 17lb puggle. call 5082218634
Mar 26 -
Hartford :
Enfield - Dogs & puppies
Ruff Wear Bark'n Boots Skyliner for Dogs
Charcoal color
Size XS
4 boots in box
Just purchased for $50, worn once by Cairn terrier/Pug mix
The Ruffwear Bark'n Boots Skyliner boots will provide everyday traction and paw protection from a wide range
of temperatures, ...
Mar 14 -
Allentown :
Bethlehem - Accessories
Beautiful mix pugsund puppies
They look just like a dachshund and they are mixed with pug
Male and female
First set of shots and de worming done
Adoption fee is $450 firm
Great with kids and other dogs please call or text (631) 488-3561
This ad was posted with the ...
Mar 14 -
New York City :
Centereach - Dogs & puppies
2 little girls, very sweet, well socialized, groomed
Will be vet checked and include shots and dewormings
Comes with blanket, collar and toy and food
Born Feb 1, 2015 and will be ready March 27th
Please text for more info
Mar 12 -
Buffalo :
Richburg - Dogs & puppies
Patti & Pets
[email removed]
365 Days a Year Open for Pet Boarding & Much More
Small furry family pets, cats, dogs, rabbits, & more ( sorry no reptiles)
Dogs must have up-to-date vaccines, rabies tag, NY dog license tag
Dogs can include English & French bulldogs, ...
Mar 10 -
Central New York :
Ithaca - Animal services
I have 5 pug mix puppies...mother is fawn, half pug half pom..father is purebred black pug. I have 1 black male, the rest are fawn 2 male 2 female. They will come vet checked,wormed, and first set of shots. They are beautiful,loving and family raised. They are like my babies ...
Feb 24 -
Buffalo :
Nunda - Dogs & puppies
I have 5 pug mix puppies..2 females and 3 males. The mother is half pug and half pom and father is purebred black pug. So the pups are 3/4 pug. I have 1 black one which is a male..the rest are fawn..2 males 2 females.. Very beautiful and loving..family raised pups. They will ...
Feb 23 -
Buffalo :
Nunda - Dogs & puppies
there r 4little boys one girl daddy is a full blooded toy poodle mom is toy poodle and pug mix pups r small and look all poodle one baby is a party poodle looks like a baby cow he is rare and is 350.00 all others r. 250.00 u would be responsible for all vet care Inc first ...
Feb 13 -
Rochester (NY) :
Rochester - Dogs & puppies
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zach in Utah
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UK Team Claims Breakthrough In Universal Cancer Test 63
from the coming-to-a-patent-office-near-you dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
This could be a giant step forward in cancer diagnostics, but media reports are - of course - sensationalizing beyond evidence.
In the study, the types of tumors tested share some similarities that might mean findings true of them would not be true of "all cancers". Specifically, none of the lesions tested were tumors of mesenchymal origin. No sarcomas, no fibromas, no leukemias. That's a broad range to not examine, and it means that generalizing this as a test for "all cancers" is premature. Additionally, none of the tumors tested were types that tend to show up in places that lymphocytes have trouble getting to (like the brain, eye, and portions of the reproductive tract).
It is good that they tested against COPD (a chronic inflammatory condition), but it does not appear as if they could distinguish between less-aggressive tumors and inflammatory conditions (I can't tell for sure because of the paywall). It may be that this is a test that is a good indicator of chronic inflammation (seen in many cancers as well as other conditions) rather than a cancer-specific test.
Regardless of the limitations of the preliminary sample set, the findings are very exciting and a potentially amazing discovery in cancer medicine. Kudos to the hardworking scientists involved!
The University of Bradford has filed patents for the technology and a spin-out company, Oncascan, has been established to commercialise the research.
And they already have a website [oncascan.com] running.
I'd say under 5 years before we start seeing this applied in the wild.
Computers can figure out all kinds of problems, except the things in the world that just don't add up.


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text: Starting July 1 It Will Cost More for a Driver's License in South Dakota – How to Avoid the Hike By Marc Elliott 3 days ago South Dakota motorists will pay more for their driver's license starting July 1. But some people may be able to avoid it. Read More|Comment Category: Homepage, News Tags: Driver's license increase, South Dakota driver's license Send to a friend! Share on Facebook Share on Twitter


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Many varieties of fruit, meat and vegetable are disappearing from our plates, says Rachel Nuwer. Why is this happening, and can we stop the rot?
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Many varieties of fruit, meat and vegetable are disappearing from our plates, says Rachel Nuwer. Why is this happening, and can we stop the rot?
ComplexInsight's insight:
Its probably not often we stop to think of the impacts of agriculture on the evolution of foodstuffs and what varieties of plants are being lost, along with their distinct tastes, benefits and properties. Compared to pre 1900 agriculture an estimated 75% of global farmed plant diversity is now gone and lost. Diversity is key to sustainable agriculture over the long term, so the points made in the article are particularly worrying. Thought provoking article from the BBC / worth reading.
Trophic cascade effects occur when a food web is disrupted by loss or significant reduction of one or more of its members. In East African Rift Valley lakes, the Lesser Flamingo is on top of a short food chain. At irregular intervals, the dominance of their most important food source, the cyanobacterium Arthrospira fusiformis, is interrupted. Bacteriophages are known as potentially controlling photoautotrophic bacterioplankton. In Lake Nakuru (Kenya), we found the highest abundance of suspended viruses ever recorded in a natural aquatic system. We document that cyanophage infection and the related breakdown of A. fusiformis biomass led to a dramatic reduction in flamingo abundance. This documents that virus infection at the very base of a food chain can affect, in a bottom-up cascade, the distribution of end consumers. We anticipate this as an important example for virus-mediated cascading effects, potentially occurring also in various other aquatic food webs.
Via Chris Upton + helpers, Ed Rybicki
Scientists say there was a widespread extinction of bees 66 million years ago, at the same time as the event that killed off the dinosaurs.
The demise of the dinosaurs was almost certainly the result of an asteroid or comet hitting Earth. But the extinction event was selective, affecting some groups more than others. Writing in Plos One journal, the team used fossils and DNA analysis to show that one bee group suffered a serious decline at the time of this collision.
ComplexInsight's insight:
Any study explaining why a species went extinct 65 million years ago will at first glance seem disconnected from current events. However bees are critical to agriculture and ensuring biodiversity. Understanding extinction events that impacted different species of Bees in the past help us better understand what could happen in the future as Bee's are currently being severely impacted by diesel pollution, modern farming practises (especially insecticides), changing ecosystems and new pests.
Diesel exhaust is pretty nasty stuff. Pass an overloaded 18-wheeler clouding up the highway, and that acrid plume of hydrocarbons will overpower even your best little tree air freshener.
ComplexInsight's insight:
New findings show that diesel exhaust impedes bees. Article on Gizmodo.
Farmers will not be allowed to spray widely used insecticide blamed for declining bee population A widely used insect nerve agent will be banned from use on corn and sunflowers in Europe from the end of 2013 as part of an effort to protect bees,...
ComplexInsight's insight:
If you have been following any agricultural news in recent years - you may have seen titles such as Honey Bees's Disapearing or Bee Population Collapse. After several researchers linked the declining Bee population to fipronil insecticide useage- the EU has finally implemented a ban. Hopefully similar actions will follow elsewhere worldwide and Bee populations can begin to recover. Bees are an essential natural component to most agricultural systems and fipronil usuage and its impact and endangering of ecosystem it was supposed to help preserve needs to become a lesson we take to heart.
EAST LANSING, Mich. — An international team of scientists has developed crop models to better forecast food production to feed a growing population – projected to reach 9 billion by mid-century – in the face of climate change.
ComplexInsight's insight:
A recurrent and central theme for us at Complex Insight is how important simulations are to tackling major real world challenges. In a paper appearing in Nature Climate Change, Bruno Basso, Michigan State University ecosystem scientist and other members of the Agricultural Model Intercomparison and Improvement Project unveiled an all-encompassing modeling system that integrates multiple crop simulations with improved climate change models.The new simulation model better predicts global wheat yields while reducing political and socio-economic influences that can skew data and planning efforts..In engineering simulation there has long been a trend to model based design - hopefully other areas of public policy can leverage work such as this to follow in a similar direction.
Farmers in the U.S.and China should use less fertilizer, freeing it up for application where such nutrients can do the most good...
Via LilyGiraud
An international collaboration... has figured out how to make a longer cotton fiber... could potentially have a multi-billion-dollar impact on the global cotton industry and help cotton farmers fend off increasing competition from synthetic fibers...
"This technology allows improvement of fiber quality in upland cotton, which is widely grown everywhere," said Alan Pepper, an associate professor in the Texas A&M Department of Biology... "This will increase the competitiveness of natural cotton fibers versus synthetic fibers, which have been snagging an increasing amount of the market share every year."
The overwhelming majority of cotton harvested in the U.S. and worldwide is upland cotton, or Gossypium hirsutum, with more than 6.5 million acres planted in 2012 in Texas alone... A higher-end cotton called Gossypium barbadense is more desirable because of greater fiber length and strength but is late-maturing, low-yielding and more difficult to grow because it requires dry climates with significant irrigation and is less resistant to pathogens and pests.
"For a long time cotton breeders have been trying to develop upland cotton with the fiber qualities ofbarbadense cotton... Globally, everybody's trying to do it. Economically, it's a huge deal, because every millimeter you add to fiber length adds that much to the price of cotton when the farmer sells it." The researchers' method increased the length of the fiber by at least 5 millimeters, or 17 percent, compared to the control plants in their experiment...
The cotton plants developed in the project technically are genetically modified organisms (GMOs)... "What we're doing is a little different," Pepper said. "We're not actually adding in a gene from another species. Rather, we're knocking down the effect of one of the genes that's already in the plant... This was pure basic science, seeking to understand the biological function of a gene... And sure enough, the phytochrome 'knock-down' plants had all these phenotypic changes associated with it [phytochrome], and one of them was longer fiber."
The discovery was especially important to Ibrokhim Abdurakhmonov, the lead author of the study who received his master's degree in plant breeding from Texas A&M in 2001 and is now a professor in his native Uzbekistan. The landlocked agricultural nation that borders Afghanistan historically has relied heavily on cotton... Uzbekistan currently accounts for around 10 percent of world cotton fiber exports.
"Sustainability and biosecurity of cotton production is pivotal for the Uzbekistan economy because agriculture accounts for 24-to-28 percent of the country's gross domestic product... The increased value of longer and stronger lint, at 10 cents per pound, would be at least $100 per acre more income from the lint for each new cultivar using this technology. New markets for longer, finer, stronger and more uniform cotton lint fiber, as well as early maturity and increased yield potential could further increase estimated economic value. Our anticipation of possible improvement of resistance to abiotic stresses via phytochrome RNA interference further adds to its commercial potential." ...
Press release: http://www.science.tamu.edu/articles/1157/
Original article: http://dx.doi.org/10.1038/ncomms4062
Via Alexander J. Stein
The extensive exploitation of marine resources by modern fisheries (...) has wide-ranging effects on marine ecosystems. Across the world's oceans, size-selective harvesting by commercial fisheries has been a key driving force behind changes in phenotypic traits such as body size and age at maturation (1–3). These changes have altered the trophic structure of the affected ecosystems, disturbed predatorprey relationships, and modified trophic cascade dynamics (3, 4). Phenotypic changes can involve both ecological and evolutionary reactions to the effect of fishing, and there has been much debate about the relative roles of these reactions. This is important because genetic changes could result in long-term reductions in catches. Recent work has provided evidence for fisheries-induced evolutionary changes, with important implications for the sustainability of fisheries.
How Fisheries Affect Evolution
Science 6 December 2013:
Via Complexity Digest
Biotech, farmer associations key for climate adaptation - panel Reuters AlertNet (blog) LONDON (Thomson Reuters Foundation) - An increasingly extreme climate is presenting new challenges to farmers across the world, and biotechnology and greater...
ComplexInsight's insight:
The potential for genetically modified crops and relation to climate change - which recently helped drive Monsanto to acquire The Climate Corporation is once again in the headlines. At the recent Iowa discussion, five farmers from Malawi, India, Portugal, Argentina and Kenya said they were strong believers in using biotech crops to survive and thrive in the face of a changing climate, and said that farmers needed to share ideas and help each other improve farming techniques. Trust.org does a great job in summarizing the ideas discussed at the event. Worth reading.
Nearly 80% of bumblebees imported from Europe carry pathogens that pose a threat to UK native honeybees and bumblebees, say scientists.
ComplexInsight's insight:
As the EU move to ban fipronil additional findings indicate more challenges for UK bumblebee. As local populations of bumblebees fell - more bees were imported from Europe. The European imported bees have now been found to carry parasites which pose a threat to the remaining UK native honeybees. With a million colonies of bees imported globally the findings indicate potential problems for many native bee species in other countries. The research highlights problems not only with current import controls and how obvious economic remedies can often further complicate the situation further.
Impact of increased demand for animal protein products in Asian countries: Implications on global food security - Cao & Li (2013) - Animal Frontier
In Asia, the consumption of animal products has been steadily increasing, thus creating a greater demand for feed crops. As the quality of life progresses, demand for animal protein also increases.
In the next 5 to 10 years, even greater challenges will be encountered in meeting an ever increasing demand for animal protein products in developing countries. Therefore, there is an urgent need for new technological innovations in livestock production to ensure global food security and the stability of global economy...
Via Alexander J. Stein
ComplexInsight's insight:
Good insight into the future complexity of food demand and geospatial constraints and driving factors that in increasing demand for animal protien products in developing countries and its relationship to global food security.
With projections of 9.5 billion people by 2050, humankind faces the challenge of feeding modern diets to additional mouths while using the same amounts of water, fertilizer and arable land as today. Cornell researchers have taken a leap toward meeting those needs by discovering a gene that could lead to new varieties of staple crops with 50 percent higher yields.
The gene, called Scarecrow, is the first discovered to control a special leaf structure, known as Kranz anatomy, which leads to more efficient photosynthesis. Plants photosynthesize using one of two methods: C3, a less efficient, ancient method found in most plants, including wheat and rice; and C4, a more efficient adaptation employed by grasses, maize, sorghum and sugarcane that is better suited to drought, intense sunlight, heat and low nitrogen... If C4 photosynthesis is successfully transferred to C3 plants through genetic engineering, farmers could grow wheat and rice in hotter, dryer environments with less fertilizer, while possibly increasing yields by half...
Underlying study: http://dx.doi.org/10.1093/pcp/pcs147 ;
Via Alexander J. Stein
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Since Java holds characters internally in UTF-16, what if you need to output in a certain encoding that includes characters that are not in unicode at all?
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Date for Your Diary - Romsey Show 2015 - Saturday September 12th.
This year's show is now over for another year. Thank you to everyone who came along and made this a memorable year in every sense.
NOTICE TO ALL ROMSEY AGRICULTURAL & HORSE SHOW SOCIETY MEMBERSAnnual General MeetingRomsey Town Hall, Thursday 26th February 2015, 7.00pmAll Members invited Agendas have been sent this weekRomsey Show Annual ReportNow available to all Members – included with your AGM noticeMembers' QuestionnaireIncluded in the AGM mailing is the attached QuestionnaireWe are looking for your opinions, ideas, suggestions, etc to help our Social Working Group provide more of what you wantWe would love it if you could complete and return the form to us in the Show OfficeWe look forward to hearing your viewsClick here to download pdf of Questionnaire and background informationClick here to download word document to email back to usQUIZ NIGHT
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Arab Spring
On The Media
Arab Governments No Longer Ignoring Regional Atrocities
Friday, November 18, 2011
The regime of Syrian President Bashar Al Assad is becoming increasingly isolated. Eight months into the Syrian uprising, with estimates of more than 3,500 people killed, the Arab League voted to suspend Syria's membership in the organization. Bob speaks with Foreign Policy blogger Marc Lynch, who says the idea that Assad would lose legitimacy among fellow Arab leaders for killing his own people may seem obvious, but it is actually a revolutionary shift in the regional mentality.
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Bees in a tree stump
red013(6)October 1, 2011
I have bees living in a rotting tree stump next to my children's playset.
I was blowing leaves today and I was swarmed by the little highly agitated and aggressive bees. They are not real big and are yellow and black in color. I figured them for a type of ground yellowjacket.
I was hoping to find out how to rid myself of them. I can't let the kids play out there while they're around.
Any info would be great.
Konrad___far_north(3..just outside of Edmonton)
Yes...sounds like yellow jacket, get some wasp killer and spray a few second into the hole at night, then stuff quickly the hole with some paper, you might have to repeat the next day if some still fly. If nothing gets done, most likely by next spring all are dead.


text:
Chronic Fatigue Syndrome Health Center
Medical Reference Related to Chronic Fatigue Syndrome
Chronic Fatigue Syndrome
Chronic fatigue syndrome (CFS) is a complex disorder characterized by profound fatigue and other related symptoms. Standards for the diagnosis of CFS have been defined by researchers at the Centers for Disease Control and Prevention (CDC). These include excessive fatigue that does not respond to bed rest,combined with four or more of the following symptoms: substantial impairment in short-term ...
Chronic Fatigue Syndrome - When To Call a Doctor
It's important to talk to your doctor about any symptoms you may have of chronic fatigue syndrome. Early detection of chronic fatigue syndrome (CFS) and early treatment of its symptoms can result in a quicker recovery.
Chronic Fatigue Syndrome - Treatment Overview
Treatment for chronic fatigue syndrome (CFS) focuses on making you feel better so that you can resume a normal life. Simple measures you can take at home-such as improving your sleep habits and getting gentle exercise - are important parts of treatment.
Chronic Fatigue: Changing Your Schedule - Topic Overview
You may find it helpful to change your schedule to take advantage of times when you have more energy and feel less tired. Keep a diary for a week or so,and record the times of day when you have energy and when you are tired. If there is a pattern to how your energy levels change during the day,try to plan your work,school,or other activities around that pattern. If there are certain times ...
Chronic Fatigue: Getting Support - Topic Overview
People who have chronic health problems,such as chronic fatigue syndrome (CFS),often benefit from support groups. These groups can be a good source of information and tips for managing your illness and a chance to share your frustrations and problems with others who are in a similar situation. Ask your doctor or contact a local hospital for the location of a support group near you. Think ...
Chronic Fatigue: Antidepressants - Topic Overview
Antidepressants do not cure CFS. They may relieve symptoms such as fatigue,sleep problems,difficulty concentrating,and pain. But they will not affect the course of the illness or the speed of recovery. Antidepressants that are commonly used to treat chronic fatigue syndrome (CFS) include: Selective serotonin reuptake inhibitors (SSRIs),such as fluoxetine (for example,Prozac),paroxetine ...
Chronic Fatigue Syndrome - What Happens
In some cases, chronic fatigue syndrome (CFS) develops after an illness such as mononucleosis (mono) or flu, or after a period of unusual stress. But it may also develop without warning, even if you have not been sick.
Chronic Fatigue Syndrome: Using Cognitive-Behavioral Therapy
Cognitive - behavioral therapy is good news for people who have chronic fatigue syndrome (CFS). Studies show that this type of therapy can help you feel better and improve your quality of life.1, 2 What is cognitive - behavioral therapy? Why is this therapy helpful? How does the therapy work? Where to go from hereReturn to topic:Chronic Fatigue Syndrome. ...
Chronic Fatigue Syndrome - Symptoms
The main symptom of chronic fatigue syndrome (CFS) is a devastating tiredness or exhaustion that has lasted at least 6 months and does not improve much with rest. This fatigue also is so severe that it interferes with your daily activities.
Chronic Fatigue Syndrome - Topic Overview
What is chronic fatigue syndrome (CFS)? Chronic fatigue syndrome causes such devastating tiredness or fatigue that you can't perform all of the normal, daily activities you used to.


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