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My bio experience so far
So far I have put about 500 miles on my truck running my home made bio. The first 350 ish miles I ran about a 30% blend. The next 150 miles I have ran a 50% blend. So far my truck runs great. Actually it runs much better than running #2 and 1oz/gal 2 stroke like I did for about 5k miles. Over the past few days I have noticed sounds that before I did not hear. Like now I can hear a drone at 65 mph that use to be silenced by the rattling of the injectors. Now with the cruise control set to 65 on flat land and the radio off, I have to really listen for the injectors. They sure are quiet now. Idling and taking off from a light is about the same amount of noise.
I have not yet changed my filters. I think I will change them after this tank is gone. When I dropped my tank to do the hutch mod, I looked in there and did not see any sludge on the walls or in the bottom. So maybe my tank is pretty clean? or maybe my filters are about to plug and I just don't know it... I was getting b5 from my local truck stop, so I wonder if that helped to keep my tank clean.
Those of you that want to make your own Bio need to read read and then read again. Then after you start making a batch or 3, read it again and again. Stuff will make more sense after you have seen it happen a time or 3. Also take your time. If you need lots of bio, add wash tanks to the mix. Dont rush anything. My buddy got going in bio right before I did and he kept trying to cut corners and rush things and ended up having to spend lots of time fixing his bio (emulsions, lots of soap, or reprocessing) Figure that from the time you start a batch it will take about 1 week to finish, whether it is 5 gallons or 50 gallons. I know the SVO guys will chime in here and say, sounds complicated SVO is easier and better. Yes I agree, but 1k to buy in is much cheaper then 1k for a filter setup and 3k to 5k for a conversion. With my processor, I can easily convert it to a svo setup with a few turns of a wrench. In my world I can not justify converting my truck. Plus if and when the Honda dies, a TDI will be in the driveway
I'm not really chiming in, Just want to say that I run a one gallon of diesel to 4 gallons of WVO mix (don't know what cat. I fall under) but the truck run way quiter, lots cooler, and I get more mpg than running diesel. I spent about $75 on my filtration system and if I run out of WVO I can still just drive home on diesel. I guess you got a pretty good handle on things but if you get that mix wrong it will cost big $$$$. My friend has a cummins in his shop with over $4000 of damage cause bio diesel. I heard of complaints of bio eatting and clogging fuel filters, has anyone else? I have a $3 see through desposeable filter at the beginning of my system. I change it once a month (some times sooner). Has been running great for 9 months. I'm not knocking bio by any means, I know it works just be careful of the mix, this cummins is not the first to fall to a bad mix of bio.
How long have you been running the mix?
Have you had your oil checked out to see how its doing?
when the engine is cold veggie oil can get past the rings into the crankcase and make a mess of your oil. I have seen pictures of the sludge that forms in there after long oil change intervals. lots of people mix vo with diesel and yea it works, but I am too chicken to try it.
Dirty Bio will kill your injectors for sure. I wash my bio extra because I am paranoid about it. If bio is done right you can run it no prob. people tend to rush it and not clean it correctly and that is when the trouble starts!
Hey Tubular, sounds like youre dead nuts on on the week thing. I just put my first 18 gallons in my truck this morning, and yep, she took a week to do right.
Now as far as trucks being damaged by bio, I have discussed this with Scuffy, and some others, and have come to the conclusion that bad bio is just that, bad bio. Not finished, full of soap, fatty acids, etc. Looked good but wasnt tested, things of that nature. Well, if you dont test, you dont know, and if you dont know, you hurt the truck.
I should clarify, I run WVO in a 84 250 with a 6.9. I bought for 1k just for the purpose of being a test subject for alt. fuels and additives a year ago. I have two tanks on this truck snd start and runtill warm on reagular diesel then switch over, then before shut off I switch back to clean out the injectors. I haven't done it to my 99 cause it only 1 tank, but the I deas the same 84 to 99, injesctor squirts, piston compresses, ignition, and so on. The only difference is the turbo whitch shouldn't make any, and the computer controlling the motor as opposed to mechanical operation. The first oil change and the dipstick seem fine but I'm due for an oil change so I'll have to see what happens but I'd feel confident putting in in a later 7.3. I think the worst that could happen is messing the injectors up sause I think the injectors are made differently from the 6.9. Will not that cheap of a mess up, if you were determined try, not really the end of the world. You seem to have a handle on the bio so I wouldn't change, was just agreeing with the quiter injectors. Besides running cooler did I mention I get more mpg on WVO? How about the bio?
i don't run bio, i run 100% vegi in a two tank heated system. Is your vegi tank steel or plastic?
I have stock dual tanks, and I just use the stock rear tank and fliter it before it goes through the relay switch. I would like to build a preheat tank useing the radiator heat. How is yours heated?
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The CIA actions amounts to a blatant violation of the constitutional doctrine of separation of powers, the Fourth Amendment, and a swath of congressionally-enacted statutes.
"I was briefed Tuesday by CIA Inspector General David Buckley on the results of an IG investigation," Senate Intelligence Committee Chair Dianne Feinstein (D-Calif.) wrote in a statement published on her website. "The investigation confirmed what I said on the Senate floor in March — CIA personnel inappropriately searched Senate Intelligence Committee computers in violation of an agreement we had reached, and I believe in violation of the constitutional separation of powers."
Feinstein — who had been the biggest apologist in the U.S. Senate for U.S. intelligence agencies before a March 11 speech condemning the CIA for its surveillance of Senate Intelligence Committee staffers — termed the CIA admission "positive first steps." On March 11, Senator Feinstein accused the CIA of violating the principles of separation of powers, the Fourth Amendment, the Computer Fraud and Abuse Act, and Presidential Executive Order 12333.
The CIA admission follows categorical denials that it had either hacked into Senate computers or deleted information. "As far as the allegations of CIA hacking into Senate computers," CIA Director John Brennan told a Council on Foreign Relations confab on March 11, "nothing could be further from the truth. I mean, we wouldn't do that. I mean, that's — that's just beyond the scope of reason in terms of what we would do."
Click here to read the entire article.
Photo of CIA Director John Brennan: AP Images

subset: commoncrawl


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Ditto for Jimmy Bunn.
"The entire team is probably going to take their SI frustration out on them," the longtime fan said, "and it won't be pretty."
Bunn graduated from OSU in 1987, and he has worn something orange every day since. Watch. Class ring. Tie. Shirt.
Even when he went to Arizona State to attend law school, he says he drove back to OSU for almost every home game from 1987-90.
The past eight years, he and his three daughters have had season tickets in the west end zone. While he doesn't believe Saturday's game will mean more in light of what happened this week — "It's the same old opportunity to spend time with my orange family, to continue brainwashing my daughters to love OSU as much as I do and to watch the Cowboys' next step to two conference championships in three seasons," he said — he knows that the SI articles will be a hot topic of conversation.
"I keep in touch with much of my orange family via social media year around, so ... we'll discuss who had the best SI-related tweets this week," Bunn said.
So, who did?
"That's me," Bunn said.
Cowboy fans are admittedly in better spirits than they thought they might be when news of this SI investigation broke last weekend.
"Sunday, we were all worried," said Trebilcock, an attorney who was the OSU student body president in 1993-94 and is now a state representative for District 98. "Now, we're all laughing."
Like other Cowboy fans, he wants any problems that are legitimized to be fixed. Even so, he won't be thinking much about that Saturday.
It's a day to simply soak in the orange.
That's the reason Trebilcock will drive to Stillwater from Broken Arrow, a trip he wasn't planning to make until the SI report put his alma mater under attack, a trip he hasn't made in nearly two years.
"It's kind of a coming together," he said, "and I think there will be a lot of people there who haven't been there in quite awhile."
Jenni Carlson: Jenni can be reached at (405) 475-4125. Like her at facebook.com/JenniCarlsonOK, follow her at twitter.com/jennicarlson_ok or view her personality page at newsok.com/jennicarlson.


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The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment that was obtained with a 1- or 3-step PCR protocol and that carries an antibiotic cassette flanked by a region homologous to the target locus into a strain that expresses the lambda Red recombination system.
This system has been modified for use in Pseudomonas aeruginosa. Chromosomal DNA deletions of single genes were generated using 3-step PCR products containing flanking regions 400–600 nucleotides (nt) in length that are homologous to the target sequence. A 1-step PCR product with a homologous extension flanking region of only 100 nt was in some cases sufficient to obtain the desired mutant. We further showed that the P. aeruginosa strain PA14 non-redundant transposon library can be used in conjunction with the lambda Red technique to rapidly generate large chromosomal deletions or transfer mutated genes into various PA14 isogenic mutants to create multi-locus knockout mutants.
The lambda Red-based technique can be used efficiently to generate mutants in P. aeruginosa. The main advantage of this procedure is its rapidity as mutants can be easily obtained in less than a week if the 3-step PCR procedure is used, or in less than three days if the mutation needs to be transferred from one strain to another.
The availability of an increasing number of sequenced genomes has generated the need for the development of efficient methods that will enable functional analysis of newly identified genes. The construction of knockout mutants by gene replacement has traditionally been a time consuming process because it requires several subcloning steps. A more time efficient mutagenesis method that does not require cloning was developed recently and has been used in various bacteria and fungi [1-3]. The methodology was first described in E. coli [2] and Aspergillus nidulans [1]; and subsequently applied successfully to Yersinia [4,5], Salmonella [6,7], Shigella [7] and Serratia [8].
The procedure involves the deletion of chromosomal genes via homologous recombination between the chromosomal region of interest and a polymerase chain reaction (PCR)-product that contains an antibiotic cassette flanked by a region of homology with the target DNA. In E. coli, a linear DNA is obtained in a 1-step PCR using primers that contain a region that is homologous with a 36-nucleotide (nt) region of the target gene. An efficient recombination between the PCR product and the chromosome is achieved by induction of the lambda phage Red operon. The Red operon encodes the nuclease inhibitor Redγ(gam) and the site specific recombinases Redα(exo) and Redβ(bet), which mediate homologous recombination [9]. Although molecular biology techniques used in E. coli are often applicable to other bacteria, adaptations are frequently required. For example in Yersinia pseudotuberculosis, 55-nt homology extensions were generally not sufficient to allow recombination, while Y. pseudotuberculosis PCR products with extensions of approximately 500-nt, generated using a 3-step PCR procedure, could trigger reproducibly gene disruption [4].
Here we describe an adaptation of the lambda Red-based methodology for use with P. aeruginosa, in which chromosomal single gene deletions were generated using a 3-step PCR product containing 600- to 400-nt flanking regions that are homologous to the target sequence. We further examined the feasibility of using 1-step PCR product with only 100-nt homology extension for obtaining mutants. Finally we tested the ability of this method to delete large chromosomal regions.
Gene disruption using a 3-step PCR product
The mutagenesis was carried out by electroporating a polymerase chain reaction (PCR)-product that contains an antibiotic cassette flanked by sequences homologous to the targeted DNA into a strain expressing the lambda Red operon. The presence of the specific PCR product promotes the deletion of the chromosomally located targeted region via homologous recombination between the genomic region and the flanking sequences of the PCR product. In order to use this system in P. aeruginosa, we first cloned the lambda Red operon into the pUCP18 vector. We also cloned the sacB gene into this vector to enable a rapid cure of the plasmid following mutagenesis. The generated vector is called pUCP18-RedS (Genbank EU073163) (Additional file 1). Initially, the pqsC gene was selected for knockout because the mutant phenotype can be differentiated from the wild-type strain by its inability to produce the Pseudomonas aeruginosa blue pigmented phenazine, pyocyanin, lack of which results in a yellowish culture (unpublished data). We used a 3-step PCR procedure to generate a fragment containing the kanamycin resistance cassette flanked by regions surrounding the pqsC gene (Fig 1A). In the first step, P. aeruginosa genomic DNA was used to amplify the upstream and downstream regions of the target sequence with primers FuppqsC/RuppqsC-kan and FdownpqsC-kan/RdownpqsC (all primer sequences are shown in Additional file 2). In each pair, one primer contains at its 5' end a 20 bp-region homologous to the kanamycin cassette. Simultaneously, the pUC4K plasmid was used to amplify the kanamycin resistance gene. In the second step, the three fragments and primers FuppqsC/RdownpqsC were mixed and a product containing the upstream, kan and downstream regions was obtained. The third step is performed with primers FuppqsC/RdownpqsC to obtain a large quantity of PCR product.
Additional File 1. Schematic map of pUCP18-RedS. The schematic representation of pUC18-RedS (Genbank EU073163) includes the Red operon (gam, bet, exo) – araC fragment, sacB encoding the levansucrase, and bla encoding the resistance to ampicillin.
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Additional File 2. Primers used in this study. The table lists all primers used in the various mutagenesis steps described in this paper.
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Figure 1. Principles of 3-step and 1-step PCR. A) Single gene deletion using a 3-step PCR. The first step involves amplifying independently the upstream and downstream regions of the target gene and the kanamycin resistance cassette. Then the three fragments obtained are mixed together and a third amplification performed to augment the yield of the product. B) Single gene deletion using a 1-step PCR. The first step is performed using pUC4K (Genbank X06404) as template and a mixture of long and short primers. The long primers (F100-kan/R100-kan) contain at their 5' extremity 100-nt homology to the flanking regions of the target gene and at their 3' extremity 22–24 nt homology to the kanamycin resistance cassette. The short primers Fup/Rdown are homologous to the 5' extremities of the long primers. C) Deletion of large chromosomal region using a 3-step PCR. The first step requires the amplification of MAR2xT7 transposon with the upstream and downstream region of genes of interest. Two transposon mutants are used as template to generate the up and down regions. Then the two fragments are mixed together to generate the desired PCR product and a third step performed to augment the yield of the product.
We first assessed whether flanking regions of ~600-nt were sufficient to enable homologous recombination between the target gene and the PCR product, as this is generally sufficient when a typical suicide vector is used. Primers FuppqsC/RuppqsC-kan and FdownpqsC-kan/RdownpqsC were designed to amplify regions 651-nt upstream and 610-nt downstream of pqsC, and to leave intact the 5' and 3' ends of the gene in the resulting recombinant mutant (Fig. 1A). The 2.2 kb PCR fragment obtained was electroporated into PA14/pUCP18-RedS previously induced by arabinose for the expression of the lambda Red proteins. Aliquots of 2 or 4 μg of electroporated DNA allowed the selection of 10–20 KmR recombinants; that colony yield was approximately doubled when 8 μg of DNA was used. All KmR colonies obtained were analyzed by assessing their ability to produce pyocyanin. Approximately 80% of the colonies potentially carried the insertion, as revealed by their inability to produce pyocyanin (Fig. 2). The correct integration of the kanamycin cassette was then confirmed by PCR in five independent colonies. Figure 3A shows results obtained for one representative ΔpqsC recombinant. A PCR product of 868 bp was obtained using primers Fupout-pqsC/Rinkan (Additional file 2) and the mutant genomic DNA as template, while no PCR product was obtained when the wild type genomic DNA was used. In addition when the wild type and ΔpqsC genomic DNA were used as template, primers Fupout-pqsC/Rdownout-pqsC (Additional file 2) amplified a product of 2757 bp and 2550 bp (720 bp of pqsC was replaced by 927 bp of the kanamycin cassette) respectively (Fig 3A). The correct and unique insertion of the kanamycin resistance cassette was verified by Southern. HindIII-digested genomic DNA of three independent colonies was hybridized with the 5' end of the kanamycin cassette, and only one band of 8016 bp was obtained confirming that the cassette was inserted into pqsC (Fig 3B). Finally, to ensure inclusion of proper borders in the inserted kanamycin cassette, 700-bp regions upstream and downstream of the cassette were sequenced. The sequences indicated proper insertion of the cassette and that no mutations were incorporated during the 3-step PCR procedure as the flanking sequences were identical to the original.
Figure 2. ΔpqsC, ΔlasR and trpE-phnAB-ΔkynBU mutants do not produce pyocyanin. The production of pyocyanin, the P. aeruginosa blue phenazine pigment, is assessed in the mutants generated in this study after overnight growth in LB at 37°C.
Figure 3. Verification of the ΔpqsC mutant by PCR and Southern. A) Verification by PCR. To confirm that the pqsC gene is mutated, two primer pairs Fupout-pqsC/Rin-kan and Fupout-pqsC/Rdownout-pqsC were used, with Fupout-pqsC and Rdownout-pqsC hybridizing outside the DNA region used for the recombination event. Primers Fupout/Rin-kan allow DNA amplification only if the kanamycin cassette is inserted, while the primer pair Fupout/Rdownout amplify a fragment of different size when mutant or wild type genomic DNA is used. B) Verification by Southern. The HindIII-digested total genomic DNA of three ΔpqsC generated mutants was hybridized using the kanamycin cassette as a probe. One band of 8016 bp is obtained when the cassette is inserted into pqsC. The HindIII-digested pUC4K and PA14 genomic DNA was used as positive and negative controls, respectively.
Using 3-step PCR, we also successfully obtained two supplementary mutants: ΔlasR and ΔkynBU. The ΔlasR mutant was generated using 490-nt upstream and 445-nt downstream flanking homologous regions resulting in the replacement of 93 bp of lasR by the kanamycin cassette. As expected the lasR mutant generated was defected in pyocyanin production (Fig 2). Five independent colonies were analyzed and the correct insertion was confirmed in all five colonies using primers Fupout-lasR/Rinkan and Fupout-lasR/Rdownout-lasR (Additional file 2 and Fig. 4A). The ΔkynBU mutant was generated in both wild type and trpE-phnAB- backgrounds using 471-nt upstream and 419-nt downstream flanking homologous regions. In these mutants 1041 bp region of kynBU was replaced by the 927 bp in length kanamycin cassette. The trpE-phnAB-ΔkynBU mutants were functionally validated also using pyocyanin as readout. In fact, P. aeruginosa is unable to produce pyocyanin if the bacterium is impaired in its capacity to synthesize 4-hydroxyl-quinolines (HAQs). Recently it was reported that two distinct pathways supply anthranilate as the precursor of HAQs [10]. Anthranilate can be either derived from the conversion of chorismate to anthranilate by an anthranilate synthase: TrpEG and PhnAB; or produced by the degradation of tryptophan through the kynurenine pathway: KynBU. While kynBU and trpE-phnAB- mutants were still able to produce pyocyanin in rich medium (Fig 2) due to the fact that the anthranilate or kynurenine pathways supplied anthranilate respectively, no trace of pyocyanin was present in any trpEphnAB-ΔkynBU recombinants tested (Fig 2). Also, DNA amplification was performed in five independent ΔkynBU and trpEphnAB-ΔkynBU colonies using primer pairs Fupout-kynBU/Rinkan and Fupout-kynBU/Rdownout-kynBU (Additional file 2) and confirmed the insertion of the kanamycin cassette in all five ΔkynBU and trpEphnAB-ΔkynBU recombinants (Fig 4A). Note that PCR products of similar size were obtained with primers Fupout-kynBU/Rdownout-kynBU when wild type or mutant genomic DNA was used as templates, as 1024 bp of kynBU were replaced by 927 bp of kanamycin cassette. Altogether these results demonstrate that the lambda Red technique is an efficient system for generating mutants in P. aeruginosa.
Figure 4. Verification of the ΔlasR, ΔkynBU, trpE-phnAB-ΔkynBU and ΔHSI-II mutants by PCR. A. Verification of single gene deletion. To confirm that the target gene is mutated, two primer pairs Fupout/Rin-kan and Fupout/Rdownout were used. The ΔlasR mutant was verified with primer pairs Fupout-lasR/Rin-kan and Fupout-lasR/Rdownout-lasR, ΔkynBU and trpE-phnAB-ΔkynBU with Fupout-kynBU/Rin-kan and Fupout-kynBU/Rdownout-kynBU. B) Verification of deletion of large chromosomal region. To confirm that the MAR2xT7 transposon replaced the 24 kb HSI-II locus, primer pairs Fdown-MAR2xT7/Rdown-PA14_42880 and Fup-PA14_43100/Rdown PA14_42880 were used. No amplification is achieved when wild type genomic DNA is used.
Gene disruption using a 1-step PCR product
The mutagenesis process could be simplified by using a PCR fragment with a shorter region of homology to the target gene. This would enable the product to be obtained in a 1-step PCR using long primers that are homologous to the kanamycin resistance cassette at their 3' ends and homologous to the target gene at their 5' ends. Thus in order to reduce the length of homologous extension necessary to select for P. aeruginosa recombinants, the following four sets of primers were designed to amplify the pqsC::kan locus: F-pqsC600/R-pqsC600, F-pqsC300/R-pqsC300, F-pqsC150/R-pqsC150 and F-pqsC100/R-pqsC100 (Additional file 2). The PCR products generated contained the antibiotic cassette flanked with 600, 300, 150 or 100-nt homologous regions to the pqsC-surrounding region; when 8 μg of each product was electroporated, 35, 50, 60 and 40 KmR colonies were obtained, respectively, and out of those 30, 46, 50 and 34 positive recombinants were identified based on their inability to produce pyocyanin, which corresponds to a 83–92% positive rate. Five recombinants from each transformation event were analyzed by PCR and the expected products were obtained systematically using primers Fupout-pqsC/Rinkan and Fupout-pqsC/Rdownout-pqsC (Additional file 2). These results show that in the case of pqsC mutagenesis the number of positive recombinants was not dependent on the length of the homologous region used and that a 100-nt homologous sequence was sufficient to allow recombination.
To further test the feasibility of 1-step PCR for this application, we designed and purchased primers (Sigma Aldrich) that contain at their 5' extremity 100-nt homology to the flanking regions of kynBU (F-kynBU100-kan/R-kynBU100-kan) or lasR (F-lasR100-kan/R-lasR100-kan) and at their 3' extremity 22–24 nt homology to the kanamycin resistance cassette (Additional file 2). The kanamycin resistance cassette was amplified using pUC4K as the template and a mixture of two sets of primers: F100-kan/R100-kan and Fup/Rdown (Fig 1B). The resultant PCR product was used for electroporation. ΔkynBU recombinants, but not ΔlasR mutants, were obtained using 8 μg of PCR product and confirmed by PCR as described above. Our inability to obtain the ΔlasR mutant with this protocol indicates that the 1-step PCR method cannot be considered highly reliable and that 100 nt is likely close to the minimal length of homology needed.
Transfer of deletion to other mutant backgrounds
The lambda Red methodology was combined with 1-step PCR to transfer a knockout mutation from the original strain in which it was generated to other strains. We amplified the lasR::kan locus using the DNA of the previously obtained mutant as the template and the primer pair F-lasR/R-lasR. The amplification generated a PCR product containing 490-nt and 445-nt upstream and downstream flanking target regions. Eight-microgram aliquots of PCR product were electroporated into three PA14 isogenic mutants (ΔmvfR, ΔpqsE and ΔpqsA), and all three strains successfully acquired the mutated locus, which was demonstrated by DNA amplification using primer pairs Fupout-lasR/Rinkan and Fupout-lasR/Rdownout-lasR.
A comprehensive non-redundant PA14 transposition mutant library was recently constructed by random insertion of the MAR2xT7 transposon which confers gentamycin-resistance [11]. We tested whether the lambda Red methodology would allow the transfer of loci where the MAR2xT7 transposon has been randomly inserted. The gentamycin cassette that had been inserted into pqsC was amplified using primers F-pqsC600/R-pqsC600 to generate a product with 379-nt upstream and 471-nt downstream pqsC-surrounding regions. Similar to the deletion experiments above, 2, 4 or 8 μg samples of DNA were electroporated into PA14/pUCP18-RedS and yielded 14, 14 and 50 GmR recombinants, respectively. Five recombinants were tested for the inability to produce pyocyanin in cultures and the presence of the MAR2xT7 transposon was checked by PCR amplification using primers F-pqsC600/R-pqsC600, respectively (data not shown). These results demonstrate that the lambda Red technique can be used in conjunction with a mutant library to achieve rapid transfer of mutated genes into various PA14 isogenic mutants and thereby enable the creation of multi-locus knockout mutants.
Deletion of a large DNA region using the lambda Red system
Lambda Red-based methodology has the potential of enabling large regions of chromosomal DNA, including large gene clusters and operons and pathogenicity islands, to be deleted. We examined whether the lambda Red system could be also used to delete the entire P. aeruginosa HSI-II locus [12], an approximately 24 kb genomic region encoding a putative type VI secretion system located between PA14_43110 and PA14_42880 genes. The PCR procedure followed to obtain the PCR product necessary to delete the HSI-II locus is presented in Fig 1C. First, genomic DNA of mutants of the PA14 non-redundant transposon library: PA14_43100::MAR2xT7 and PA14_42880::MAR2xT7 were used as template to generate the PCR fragment containing the gentamycin cassette flanked by the HSI-II locus-borders. Primers Fup-PA14_43100/Rup-MAR2xT7 were designed to amplify MAR2xT7 and the 890 nt upstream sequence when inserted into PA14_43100; while Fdown-MAR2xT7/Rdown-PA14_42880 were used to amplify MAR2xT7 and the 955 nt downstream sequence when inserted into PA14_42880 (Additional file 2). In a second PCR-step, the resulting upstream and downstream fragments were mixed with the primers Fup-PA14_43100 and Rdown-PA14_42880 and a PCR product containing MAR2xT7 flanked by the borders of the HSI-II locus obtained. PCR product aliquots of 2, 4 or 8 μg were electroporated into PA14/pUCP18-RedS, and 256, 280 and 300 recombinants were selected on gentamycin, respectively. All mutants tested were producing pyocyanin (Fig 2). DNA amplification using primers Fup-PA14_43100/Rup-MAR2xT7 and Fup-PA14_43100/Rdown-PA14_42880 confirmed deletion of the target region in 5 independent colonies; Fig 4B shows the PCR products obtained for one representative recombinant. While no DNA amplification with the primers Fup-PA14_43100/Rdown-PA14_42880 flanking the HSI-II locus was obtained when wild type genomic DNA was used as a template, a fragment of 2844 bp was amplified when mutant genomic DNA was used (Fig 4B).
The principal advantage of lambda Red-based mutagenesis is its rapidity. The technique can be performed in less than a week if the 3-step PCR procedure is used, and in less than three days if just the transfer of an existing mutated locus is needed. The protocol has been also further simplified by the use of Choi et al's method for preparing electrocompetent cells [13].
Our results demonstrated that 100-nt homology between the PCR product and the target gene is sufficient in some cases to produce recombination. Nevertheless to maximize the chances of obtaining mutants, the 3-step PCR technique should be used to generate a product with 600- to 400-nt homology extension to the target gene. Also, note that fragments of the same length may have different efficiency of recombination rates due to differing sequence contexts.
The technique has yet never failed; however further experiments should be conducted to additionally test the ability of this methodology to transfer genes marked with an antibiotic cassette from PA14 to various non-isogenic P. aeruginosa strains.
To simplify the process, transposon mutants in the PA14 transposon mutant library can serve as templates for amplification of genes of interest already interrupted by an inserted MAR2xT7 transposon carrying the gentamycin cassette. We routinely use this method to produce multi-locus knockout mutants. Moreover, PA14 transposon mutant library can also serve for the rapid deletion of large chromosomal regions. The PAO1 tetR transposon mutant library is an additional useful resource [14]. Thus both libraries together with any mutant that carries a selective marker can be used to generate easily and rapidly multiple-mutation strains. To allow the construction of multiple mutations in the same chromosome, this method can be used in conjunction with previously described methods for marker excision (Flp/Frt) [15], which would allow recycling of the same selection marker in the same strain.
The lambda Red-based technique can be used efficiently and rapidly to generate mutants in P. aeruginosa. Chromosomal single gene deletions were generated using PCR product containing flanking regions that are homologous to the target sequence. To maximize the efficiency of mutants' generation, PCR products with 600- to 400-nt homology extension to the target gene should be used. This technique, in conjunction with previously generated P. aeruginosa transposon libraries, can also serve to delete large chromosomal regions.
Strains and growth conditions
Pseudomonas and E. coli strains were grown in Luria Bertani medium (LB) at 37°C with aeration and when necessary pseudomonas cultures were supplemented with kanamycin (400 μg/ml), gentamycin (15 μg/ml) or carbenicillin (300 μg/ml). Pseudomonas strains used in this study include ΔpqsA [16], ΔpqsE [17], ΔmvfR [18], PA14_42880::MAR2xT7 and PA14_43100::MAR2xT7 [11]. The trpE-phnAB- double mutant was constructed in this study: 1293 bp of phnA and 300 bp of phnB were deleted by allelic exchange in the auxotroph trpE::Mar2xT7 mutant background. The vector pKOBEG-sacB [4] contains the Red operon expressed under the control of the arabinose inducible pBAD promoter and the sacB gene that is necessary to cure the plasmid [2,4]. Since pKOBEG-sacB was not capable of replication in P. aeruginosa, the Red operon-araC fragment obtained after digestion with KpnI and HindIII was cloned into the multi clonal site of the E. coli – P. aeruginosa shuttle vector pUCP18 (Genbank U07164) [19] to create the plasmid pUCP18-Red. In addition, the NdeI sacB fragment from pKOBEG-sacB was cloned into pUCP18-Red, previously digested with NdeI, to create pUCP18-RedS (Genbank EU073163) (Additional file 1). P. aeruginosa recombinants were easily cured from pUCP18-RedS by streaking the mutant strains on NaCl-free LB agar plates supplemented with 10% sucrose.
Southern Blot
Approximately 5 μg of DNA from P. aeruginosa and 1 μg of pUC4K DNA was digested by HindIII. The 5' end of the kanamycin cassette was amplified using primers 5'-gccacgttgtgtctcaaaat-3' and 5'-gcatttctttccagacttgttc-3', and the PCR product was labeled using the ECL kit (Amersham) and used for hybridization following manufacturer's instructions.
All PCRs were performed using High Fidelity Taq polymerase (Roche), 1 μM of each primer, 100 μM of dNTPs. DMSO 8% was added to the reaction when P. aeruginosa genomic DNA was used as template. Most of the primers were generated by the Massachusetts General Hospital DNA oligo core facility. The long primers were obtained from Sigma Aldrich.
A kanamycin resistance cassette with long flanking regions homologous to the target gene was generated by a previously described 3-step PCR protocol [4,20] with minor modifications. P. aeruginosa genomic DNA was first used as a template to amplify the regions flanking the target gene with the primers Fup/Rup-kan and Fdown-kan/Rdown (Additional file 2 and Fig. 1A). Rup-kan and Fdown-kan contain, respectively, at their 5' extremity 19 and 22-nt regions that are homologous to the 5' and the 3' terminals of the kanamycin resistance gene. The plasmid pUC4K [21] was used as a template to amplify the kanamycin cassette with the primers F-kan/R-kan (Additional file 2). In a 2nd PCR, 10 ng, 25 ng or 50 ng of each of the three fragments were mixed with the primers Fup/Rdown. A third PCR was finally performed using again the Fup/Rdown primers and the product of the second PCR as the template in order to increase the yield of the sought band (Fig. 1A). Five hundred microliters (10 reactions) of the 3rd PCR were ethanol precipitated, dissolved in 10 μl of water and dialyzed for 1 h to eliminate salt that can interfere with electroporation.
The 1-step kynBU or lasR PCR products were generated using pUC4K (Genbank X06404) as template with a mixture of long and short primers (Fig 1B). The long primers contained at their 5' extremity 100-nt homology to the flanking regions of kynBU (F-kynBU100-kan/R-kunBU100-kan) or lasR (F-lasR100-kan/R-lasR100-kan) and at their 3' extremity 22–24 nt homology to the kanamycin resistance cassette. The short primers F-kynBU/R-kunBU and F-lasR/R-lasR were homologous to the 5' extremities of the kynBU or the lasR long primers, respectively.
The DNA concentration was measured by spectrophotometer at 260 nm and the DNA molecular weight was visually confirmed on agarose gel.
Gene disruption
P. aeruginosa PA14/pUCP18-RedS was grown in LB with carbenicillin to an optical density at 600 nm (OD600) of 0.4 at 37°C. Then cells were induced for 1.5 h by treatment with 0.2% L-arabinose; the bacteria were rendered electrocompetent by four washings with 10% sucrose at room temperature and were condensed to a concentration of about 1011 /ml. Electroporation was carried out using 100 μl of bacteria solution and no more than 10 μl of DNA. The electroporated cells were diluted in 2 ml LB and incubated for 2 h at 37°C. Transformants were selected by plating the electroporated cells on antibiotic-imbued plates and further verified by PCR for the correct insertion using two set of primers Fupout/Rinkan and Fupout/Rdownout, with the latter pair hybridizing outside the DNA region used for the recombination event.
Polymerase Chain Reaction (PCR), nucleotide (nt), base pair (bp), kan (kanamycin), Kanamycin resistant (KmR).
Authors' contributions
BL performed all experiments. BL and LGR designed experiments and wrote the paper. BL and LGR have read and approved the final version of the manuscript.
This work was supported by the National Institute of Health R01AI063433.
Chaveroche MK, Ghigo JM, d'Enfert C: A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans.
Nucleic Acids Res 2000, 28(22):E97. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.
Proc Natl Acad Sci U S A 2000, 97(12):6640-6645. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Murphy KC: Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli.
J Bacteriol 1998, 180(8):2063-2071. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Derbise A, Lesic B, Dacheux D, Ghigo JM, Carniel E: A rapid and simple method for inactivating chromosomal genes in Yersinia.
FEMS Immunol Med Microbiol 2003, 38(2):113-116. PubMed Abstract | Publisher Full Text
Lesic B, Bach S, Ghigo JM, Dobrindt U, Hacker J, Carniel E: Excision of the high-pathogenicity island of Yersinia pseudotuberculosis requires the combined actions of its cognate integrase and Hef, a new recombination directionality factor.
Mol Microbiol 2004, 52(5):1337-1348. PubMed Abstract | Publisher Full Text
Husseiny MI, Hensel M: Rapid method for the construction of Salmonella enterica Serovar Typhimurium vaccine carrier strains.
Infect Immun 2005, 73(3):1598-1605. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Beloin C, Deighan P, Doyle M, Dorman CJ: Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein.
Mol Genet Genomics 2003, 270(1):66-77. PubMed Abstract | Publisher Full Text
Rossi MS, Paquelin A, Ghigo JM, Wandersman C: Haemophore-mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules.
Mol Microbiol 2003, 48(6):1467-1480. PubMed Abstract | Publisher Full Text
Shulman MJ, Hallick LM, Echols H, Signer ER: Properties of recombination-deficient mutants of bacteriophage lambda.
J Mol Biol 1970, 52(3):501-520. PubMed Abstract | Publisher Full Text
Farrow JM 3rd, Pesci EC: Two distinct pathways supply anthranilate as a precursor of the Pseudomonas quinolone signal.
J Bacteriol 2007, 189(9):3425-3433. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Liberati NT, Urbach JM, Miyata S, Lee DG, Drenkard E, Wu G, Villanueva J, Wei T, Ausubel FM: An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants.
Proc Natl Acad Sci U S A 2006, 103(8):2833-2838. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M, Gifford CA, Goodman AL, Joachimiak G, Ordonez CL, Lory S, Walz T, Joachimiak A, Mekalanos JJ: A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus.
Science 2006, 312(5779):1526-1530. PubMed Abstract | Publisher Full Text
Choi KH, Kumar A, Schweizer HP: A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation.
J Microbiol Methods 2006, 64(3):391-397. PubMed Abstract | Publisher Full Text
Jacobs MA, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, Ernst S, Will O, Kaul R, Raymond C, Levy R, Chun-Rong L, Guenthner D, Bovee D, Olson MV, Manoil C: Comprehensive transposon mutant library of Pseudomonas aeruginosa.
Proc Natl Acad Sci U S A 2003, 100(24):14339-14344. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP: A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants.
Gene 1998, 212(1):77-86. PubMed Abstract | Publisher Full Text
Déziel E, Lépine F, Milot S, He J, Mindrinos MN, Tompkins RG, Rahme LG: Analysis of Pseudomonas aeruginosa 4-hydroxy-2- alkylquinolines (HAQs) reveals a role for 4-hydroxy-2- heptylquinoline in cell-to-cell communication.
Proc Natl Acad Sci U S A 2004, 101(45):1339-44.
Epub 2004 Jan 22.PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Déziel E, Gopalan S, Tampakaki AP, Lepine F, Padfield KE, Saucier M, Xiao G, Rahme LG: The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing-regulated genes are modulated without affecting lasRI, rhlRI or the production of N-acyl-L-homoserine lactones.
Mol Microbiol 2005, 55(4):998-1014. PubMed Abstract | Publisher Full Text
Cao H, Krishnan G, Goumnerov B, Tsongalis J, Tompkins R, Rahme LG: A quorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism.
Proc Natl Acad Sci U S A 2001, 98(25):14613-14618. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Schweizer HP: Escherichia-Pseudomonas shuttle vectors derived from pUC18/19.
Gene 1991, 97(1):109-121. PubMed Abstract | Publisher Full Text
Wach A: PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae.
Yeast 1996, 12(3):259-265. PubMed Abstract | Publisher Full Text
Taylor LA, Rose RE: A correction in the nucleotide sequence of the Tn903 kanamycin resistance determinant in pUC4K.
Nucleic Acids Res 1988, 16(1):358. PubMed Abstract | Publisher Full Text | PubMed Central Full Text


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When was Raquel Welch Born?Raquel Welch Birthday:
September , 5th 1940
What sign is Raquel Welch ?Virgo
Where was Raquel Welch born ?Chicago City
Raquel Welch was born on September 5, 1940 and is an American actress and author. Welch, whose real surname was Tejada, was considered as one of the top sex symbols in cinema during the 1960s and 1970s. The picture of her wearing an animal skin bikini in the 1966 film One Million Years B.C. has become iconic, becoming one of the most popular pin-ups and posters during that time. During her lengthy career, Welch has won one Golden Globe award for Best Actress in a Musical or Comedy, as well as a Golden Globe nomination for her appearance in the TV show Right to Die. Aside from acting, Welch had also written a book and had launched a business selling signature wigs.


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Article ID: 196996 - View products that this article applies to.
NOTE: These procedures only apply if you have installed Outlook with the Corporate Workgroup/Other option. This option allows you to use Messaging Application Programming Interface (MAPI) services. To determine your installation type, on the Help Menu click About Microsoft Outlook. In About Microsoft Outlook you should see "Corporate" if you have the Corporate Workgroup installation.
Some users have multiple e-mail accounts on the same Microsoft Exchange Server. Although you can only have one service in your Microsoft Exchange Server profile, it is possible to access all of your accounts with Microsoft Outlook 2000 without having to log on separately to each account.
This article describes how to set up Outlook to access two Microsoft Exchange mailboxes from one profile.
More information
Given two accounts on a Microsoft Exchange Server (A and B), follow these steps to be able to log on to account A, and have access to the mailbox for account B:
User A can send messages and meeting requests on behalf of user B by entering the name of user B in the From Field. These messages contain the names of both user A and user B.
Article ID: 196996 - Last Review: June 22, 2014 - Revision: 5.0
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A 15 degree down bag sized for 6ft, purple (they all are), full down draft tube over full length zipper, no snags, tears, holes of any kind. Very good condition. This bag is a few years old, meticulously cared for, never stored stuffed, has a full 6" loft just like brand new. If you aren't sure what it is, it's not what you're looking for.Asking $195.00Thanks.Will post pics a little later.
Edited by tachus1 on 10/22/2013 19:20:56 MDT.
Interested. You have no PM setup so please advise how to contact. Trying to figure out which version this is -- do you know fill type or overall weight or what year it is from ? Thanks
Mark,Sorry about that. I didn't know I had to set up the PM. I'll be glad to take email and answer any questions you have about this bag. The DL version stands for "DryLoft". It is essentially a Gore product which was used in making this bag almost water proof yet breathes remarkably well. I have slept out in a pretty heavy snow without a bivy or tarp and had no issues except putting my jacket over my face. I think the fill is 800, but it may be 750. I measured the loft and I can detect no loss at all. This bag has an athletic cut and is very efficient. With overstuffed draft tube and collar and the DL membrane, a lot of people push the limits of this bag into winter. The weight I think is 2lb 6oz. I bought it new in 1997 in Montana. It looks new today. Down stays in the bag, never stored stuffed. Probably has about 20-30 nights use. I'm just lightening the load around the house here. I've collected a lot of stuff over the years. My email is:email@example.com,Rogerps: I'll check into setting up that PM stuff :)
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Boston Celtics 96, New York Knicks 93
A buddy of mine asked me last night, "If you're so convinced that David Stern has an agenda and the NBA is fixed then why are the Knicks down 2-0?" While that's a great question, I won't answer it until they've played Game 3 because I think the tables will turn when these teams head to Madison Square Garden. Call it a hunch, but I can't imagine the Knicks losing another close game on the league's biggest stage. We might even see some questionable officiating, but that's a stretch. *fart noise*
So let's ask ourselves, what was the highlight of this game?
A) Rajon Rondo dropping 30 points and proving again that he is without a doubt the best point guard in the league.*
B) Mike D'Antoni basically calling this game a moral victory and making me want to fire him into the sun on a rocket.
C) Carmelo Anthony couldn't play defense against a girl's middle school team.
That's an obvious answer, of course, but while LaLa's husband is being praised for scoring 42 of his team's 96 points, he's really not getting enough credit for being one of the most lethargic players on the court at any time. Keep in mind, I only caught glimpses of this game in between ordering Three Olives bombs and cursing Gilbert Arenas' name, but I can recall at least 5 times that I asked someone: "Does this guy even give a sh*t?"
And the quick and easy answer is Yes, and he's going to win in Game 3 and we will praise his efforts because he is rumored to be a superstar. So they say.
(P.S. – Quote of the game: "The Celtics didn't do anything special. They won two games on their home court. Now it's our turn to go to our home court and try to do the same thing." – Carmelo Anthony. Someone want to explain to Melo that he's two non-special performances away from a summer in the Hamptons?)
*Arguable, I know. But for as much as I dislike the Celtics, I think it speaks volumes about my praise for little RuPaul.


text: [ Colo. Man Accused Of Supplying Rawlins Drug Dealer By Associated Press March 14, 2012 RAWLINS, Wyo. (AP) — A Colorado man recently sentenced to prison on drug charges is accused of supplying a Rawlins cocaine dealer. Fifty-year-old Manuel Ersmo Trujillo of Johnstown was sentenced to 18 years behind bars in Colorado on Jan... Read More|Comment Category: Associated Press, Wyoming News Tags: colorado, drug dealer, Manuel Trujillo, Rawlins Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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Heritage in our hands
In early 2006 the Countryside Agency, on behalf of the National Heritage Memorial Fund, provided a grant for a Local Heritage Initiative (LHI) project in the Essex town of Coggeshall.
This was to fund a project aimed at raising awareness among the local community of the importance of Coggeshall Grange Barn, an adjacent building of some significance. Linking this with other aspects of Coggeshall's history was also part of the brief.
The importance of Bryan Saunders and his tools collection was recognised and some funds were set aside to enable the collection to be fully identified, photographed and catalogued.
After this important procedure, the aim was to exhibit the collection in an appropriate setting at Grange Barn, thus making it available to the public in the best possible context.
A team of four volunteers was assembled for the task and included skilled carpenters and joiners, well able to identify the tools, plus someone to organise the entry of all of the details on to the National Trust's database.
Work started in mid-2007 and the catalogue was completed a year later in 2008.
The collection was displayed in part inside the barn in an area set aside for it, and was opened by Janet Saunders who so generously donated the collection, in May 2009.


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5 top gardening books this season
All ready to get out into the garden? It's prime time for planting, for sure, but you can still curl up with a good book. Here are five of the season's best offerings, and each will both inform and entertain. You might not even have to leave the house to start reading; most are available electronically. -- JESSICA DAMIANO, email@example.com
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20 crazy-cute pet costumes 11 Halloween events for dogs Pumpkin specialties to fall for Haunted attractions near Long Island and NYC Peek inside Long Island's most lavish homes Brian Wilson concert, more NYC weekend picks 15 new theme park rides, attractions in 2015 29 chains Long Island wants now 20 best places to see fall foliage in the Northeast 12 secrets of North Shore Animal League 23 vegetarian recipes 23 vegetarian recipes


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Lily Allen to perform at Glastonbury fest 2014
Singer Lily Allen has confirmed an appearance at the Glastonbury Festival 2014.
The singer, who made her comeback with new single "Hard out here", confirmed the news on Nick Grimshaw's Radio 1 Breakfast Show Tuesday, reports contactmusic.com.
"It got confirmed yesterday," she said Tuesday.
"I'll be doing 16 weeks of festivals over the summer and then tour," she said, adding that Glastonbury would be her first "big" gig of 2014.
"It's the best festival, I think - that, and Fuji Rock in Japan."
Glastonbury Festival, to be held June 25-29, 2014, will see a congregation of various arts like contemporary music, dance, comedy and theatre.
(Posted on 20-11-2013)


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> TV Series DVD Boxset
> Ken Burns' America 7 Dvd Box Set
Ken Burns' America 7 Dvd Box Set
Language: English Boxed Set: Yes Discs: 7 pcs Release date: Condition: New Weight: 0.70kg Format: Support both NTSC & PAL No. of Season: Region: Region Free Genre: Documentary
Cast and Crew
Summary of Ken Burns' America 7 Dvd Box Set
For 25 years, filmmaker Ken Burns has been producing films that are unafraid of controversy and tragedy. This collection of documentary films will show you America as you have never seen it before.
The set includes the following 7 DVDs:
1.Brooklyn Bridge
2.The Statue of Liberty
3.Empire of the Air
4.The Congress
5.Thomas Hart Benton
6.Huey Long
7.The Shakers


text:
- Infowars - http://www.infowars.com -
Illinois attorney general seeks gun ban review
Posted By kurtnimmoadmin On January 8, 2013 @ 4:53 pm In constitution,Old Infowars Posts Style,Tile | Comments Disabled
January 8, 2012
The Illinois attorney general has filed a petition to have a judicial ruling striking down a ban on concealed handguns reviewed.
Lisa Madigan's petition asks the full U.S. Court of Appeals for the Seventh Circuit to review the December appeals panel ruling that the state's longtime ban on carrying concealed weapons in public violates the Second Amendment, the Chicago Tribune said Tuesday.
If the court accepts the petition the case will be re-heard by all 10 appellate judges; if the petition is rejected the attorney general could ask the U.S. Supreme Court for review.
Read more
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Color Treated Hair Care
Published on by Aiden
Color treated hair can be difficult to maintain. Some shampoos may strip your color, fade the intensity and leave your hair dry and frizzy. Some conditioners may be too heavy and weigh down your hair while not giving it the essential protein it needs.
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For very dry and damaged hair, colored hair, or hair in need of lots of TLC, I recommend Alterna TEN Hair Masque as a once a week indulgence! This masque is the perfect fusion of ten key elements from around the globe to transform any hair from ordinary to extraordinary. Another FIVE STAR customer rated product!
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text: Albert Wilder | IBDB: The official source for Broadway Information IBDB: The official source for Broadway Information Official Broadway League sites: - sites - broadway.org broadwayleague.com broadwayfanclub.com kidsnightonbroadway.com stagespecs.org tonyawards.com Quick Search For: In This Category: All Show People/Org. Theatre Season Albert Wilder Performer Male Productions ProductionsDate of ProductionsThe Girl and the Kaiser[Musical, Operetta, Original] Performer: Albert Wilder [Hans Lange]Nov 22, 1910 - Jan 14, 1911Lady Teazle[Musical, Original] Performer: Albert Wilder [Careless]Dec 24, 1904 - Feb 11, 1905Maid Marian[Musical, Opera, Romantic Comedy, Original] Performer: Albert Wilder [Sir H. Ven deVere]Jan 27, 1902 - Mar 22, 1902 Find out where Albert Wilder and are credited together © 2001-2015, The Broadway League, All Rights Reserved. (Copyright and Disclaimer)


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Evangelistic Rally — Day 4
Alastair McAlees from Inverclyde Christian Initiative, opened the service and asked those gathered at the Greenock Town Hall to sing the traditional Scottish hymn, "The Lord is my Shepherd." The hymn was beautiful and so fit the moment. The Times Square Church choir followed with an exhuberant "Hosanna."
A Times Square Church member started her testimony by honoring her parents. She came from a dysfunctional family, where her dad was an alcoholic and her mom who was suicidal. She has known God since she was six. At the age of 16, she looked at her father and told him, that she loved him for the very first time in her life. As her mother took her and her siblings away, her dad got on his knees and pleaded for them not to go. She felt compassion for him and realized he needed God too. She learned not to look at the situation but to look to God. To look to the hope that can only be found through Christ.
Another member who used to be a drug addict came to New York as a photographer at the age of 23, to seek fame and fortune and he got both very fast. He was living the dream life that he wanted. His life changed overnight when he went to a party at a loft where people were cooking up cocaine making free-base and crack. As his life plummeted, he did things that he wasn't proud of. He was so strung out that he was seeing satanic symbols on the wall of in his living room and was hearing demonic voices.
One day, he hired a make up artist who attended Times Square Church. This person was warned by her agency not to speak about God. But when she saw him. She had to share about the hope there is in God. She invited him to church. Each time they made an appointment to meet at church, he would be a no show because he would be strung out on drugs. She didn't give up. She kept calling him. One night when he was supposed to meet her, he was all alone and passed out till the next morning. He began counting the money that he had to see how much drugs he could buy. He thought he had lost her phone number. He looked at the wall and started to see satanic symbols and he cried out with all his heart to God, "God please tell me, you are real. I always believe you are real. If you are real right now prove it to me, show me where her phone number is." In a quick second, he felt a peace upon him and God led him to go to a box where her phone number was. He stood there and the Holy Spirit came down on him and it was the warmest, most wonderful feeling. God told him, "Open your eyes and always believe in me." When he opened his eyes, the room was bright with lights and it lasted for about 30 seconds.
He looked across his studio space, and saw a charity photo campaign that he did. God said to him, "I used you to execute my will on anti-racism." He looked at the billboard and remembered he had framed pictures from the photo shoot. He wanted to grab those pictures to bring in to the room where he saw satanic symbol. He got on his feet and grab his tool chest, he fell on his knees and said, "God I am a drug addict, I can't stop doing drugs…I need you God." He pulled out a hammer and two nails, and the nails fell perfectly pointed to the inside of his palm. And he thought about Jesus on the cross." The Holy Spirit came over him and said, "you don't have to do drugs anymore." He confronted a lot of people that he had wronged. The following Tuesday, he gave his life to Christ at Times Square Church.
Why should I turn from sin?
This message is for the church and those that don't know Christ as Lord and Savior, based on 1 Corinthians 6. Sin is a willful decision to embrace or practice a way of living, which God says it's wrong. Sin was infused into our race when our first parents Adam and Eve consciously decided to defy what God had spoken but instead did what they thought would bring them happiness. We begin to justify wrong and contaminate what is right. Sin is when we make our own decision to become our own God. We are living in that type of hour where evil is being called good, and good is being called evil. Pastor Carter said, "I pray in our country and in Scotland that we don't make that tragic mistake of thinking that we can charter another course and somehow ever prosper in that course." "For the wages of sin is death; but the gift of God is eternal life through Jesus Christ our Lord," (Romans 6:23)
God has a marvelous plan for everyone. We are called to be extraordinary by the power of God. "And the eyes of them both were opened, and they knew that they were naked; and they sewed fig leaves together, and made themselves apron," (Genesis 3:7). The moment you sin, the sense of well-being is lost. "He replied, I heard you walking in the garden, so I hid. I was afraid because I was naked," (Genesis 3:10). Sin brings shame. We become afraid of life, circumstances, and ultimately afraid of God. Sin brings sorrow, a deep feeling of unsatisfaction no matter how much we try to work. But it still will not bring fourth what God gives us. You can try anything or achieve anything, but it will still produce thorns and thistle; it will never satisfy.
As exemplified by the story of Cain and his brother Abel which is also in Genesis, sin brings envy and murder, sin brings lies and indifference and turns life into death. "How do we say that we are worshipping God, and we are children of God with the heart of and don't care if the people around us go to hell? That's not possible." We can't live and not care about the people around us who don't know Christ. In Christ, we're intended to be what Christ wants us to be. Everything changes when we have the love of God. The old nature is put away but God gives us a new nature.
So why should you turn from sin? Firstly, because God loves you. You have an opportunity to do what is right, and the Bible says in Philippians 4:13 we can do all things through Christ who loved us. Secondly, because this is a supernatural life. You don't have to figure it out, you just have to believe, and it is all to the glory of God and for the souls of men.
Lastly, when you turn from sin, death is turned into life. In Christ you become the person God wants you to be. The scripture says, "Death has turned into life." A new song comes; it's a song of our life that comes from within. There are so many young people that need to be taken out from the dark pit. God makes all things new. The song doesn't happen all at one time but it happens line upon line. Eventually the song is finished and it begins to be sung. "Death is swallowed up in victory. Scotland, rise to the occasion. Church of Jesus Christ, rise to the occasion. The hour is dark but Christ is still the light of the world. The power of evil still roams our streets but the power of God is greater."
God gives a new hope and a new mind. We don't have to prove the existence of God to anyone. David says, "I will dwell in the house of the Lord forever." This is why we should turn from sin. God has promised to give us a new life and a new song.
Pastor Carter made an altar call for those who do not know Christ and for Christians who wants to renew their commitment to Christ. As people made their way to the altar and we began to sing "Draw me close to you," Pastor Carter said, "I know there is somebody else here in the sanctuary. This is really your moment. You are very nervous and your heart is pounding that's because the Holy Spirit calling you. This is a defining moment of your life. You will not regret coming down here." He said, "For those who are wavering even if it's just one more person, we will take that time and wait for you. You are that important to God that we will wait for you." There is no cost when coming to Christ because we are getting the treasure of heaven. We began singing an old hymn, "And Can't Be." As we sang, the young man walked up to the altar. He told the young man, "Young man, the Lord is going to put His hands on you. He is going to use you for His glory because He waited for you tonight. He knew something about you." Pastor Carter then led them to prayer. God be glorified!


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Ask the Building Maintenance Experts
Solutions for Your Toughest Problems
ICAN Q & A Home
Wednesday, October 07, 2015
[Friday, June 07, 2013]
How much toilet paper and paper towels should I use for 25 bathrooms with 3 stalls in each one of them.?
Equipment, Chemicals & Supplies
- Latanya Jones
My math says 75 rolls of tissue for single roll dispensers (150 for double) and 25 stacks or rolls of towels, to start.
However, I suspect that you are really concerned with the potential usage and that is something only time and experience will reveal.
Stock the rest rooms, leave spare product where it is not easily stolen or wasted by vandalism, and track the usage.
If this is a contract job, do not lock yourself into a cost projection until you have a sound idea of the usage, and this may take a few months to develop. Offer to provide product on a cost plus basis until true usage is determined.
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text: Patrick Barnum | IBDB: The official source for Broadway Information IBDB: The official source for Broadway Information Official Broadway League sites: - sites - broadway.org broadwayleague.com broadwayfanclub.com kidsnightonbroadway.com stagespecs.org tonyawards.com Quick Search For: In This Category: All Show People/Org. Theatre Season Patrick Barnum Performer Male Productions ProductionsDate of ProductionsThe Hand of the Potter[Play, Tragedy, Original] Performer: Patrick Barnum [McGranahan (Detective)]Dec 05, 1921 - Dec 1921 Find out where Patrick Barnum and are credited together © 2001-2015, The Broadway League, All Rights Reserved. (Copyright and Disclaimer)


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Beth Trask
Project Director
Areas of expertise: Corporate partnerships, Innovation Exchange
Beth Trask works to incorporate Environmental Defense Fund's corporate partnership innovations into new companies and industries.
With a focus on California's business community, Beth collaborates with companies to leverage new and proven best practices that provide distinct business benefits and produce significant environmental results.
In particular, she specializes in marketing sustainability initiatives to employees, customers, and other key constituents.
Masters in Public Administration, Harvard University, 2005.
Beth began her career working for local government and business associations in northern California and later went on to develop national and international conservation initiatives.
Blog Posts
Updates on Beth's Work
Beth Trask


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Out magazine's fashion department is to be commended for the majority of its work. I am still reeling from the Stephen Sprouse-inspired spread in the March 2009 issue. Their choices are normally thoughtful, and when they're not spot-on trend, they still serve up pretty amazing editorials.
But that does not give them the excuse to be insensitive and stupid. And really, that's the only thing we can think of to call the latest installment of their "Live The Life" series, which aims to teach viewers how to emulate the style of Malcolm X.
Yes, Malcolm X, The Muslim activist whose incendiary public speaking rivaled Martin Luther King, Jr.'s brand of passively resistant protest in the Civil Rights Movement. The man who was shot 16 times by members of his own faith because they didn't like what he was saying. Yeah. That guy.
First let us say that while Malcolm X was a well-dressed man, his wardrobe should be secondary (tertiary, even) to his commitment to the Civil Rights Movement and to his political clout. But Out's junior fashion editor Brent Coover says quite plainly, "Enough politics. Let's get down to playing dress up." Which, ew.
Secondly, Malcolm X was not the originator of the tie clip, or any other items of 60s men's style. If the magazine wanted to cite the man as a sartorial paragon of that era that would have been fine, because he kinda was. But we have a sneaking suspicion that the only reason Out picked Malcolm X as an example is because they need a few more thousand pageviews this month to keep whoever is holding the purse strings at Here Media happy. And if anything else, this video is going to shock and anger more than a few people, which, sadly, is one of the best ways to drive traffic on the Internet. And as much as we hate to ask you to watch this video, there's really no other way that you'll fully understand just how ridiculous it is.
[Via The Awl]


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We were married at around 3pm (I think) 10 years ago today. I married a guy, who, I had found out, worshipped the ground I walked on, came from a loving family, would do anything for me, at any cost and loved me more than life itself. (Gosh how things change! Just kidding!)
We had planned to have a reasonably lavish (that's 'lavish' not 'lavosh', the unleavened yeast free bread) wedding at an inner city hotel because a lot of guests were travelling from interstate and this hotel would provide the ceremony, reception and accommodation. About 6 months before the wedding, my husband lost his job and although he only took a couple months to find another one, we knew we couldn't afford it.
Instead, we had a beautiful ceremony and reception here at our home for about 40 guests with the wedding catered by a wonderful guy who went above and beyond with the quality and quantity of food. We didn't skimp on the photos or the honeymoon and had a wonderful week soaking up the sun in Bali. The photographer was fantastic as well, photographing my husband getting ready and myself getting ready at the apartment we shared before we bought our house (my sister had conveniently taken over the lease), at the ceremony, about 6 different locations around where we live and later on (around midnight) at our city motel.
It was a lovely wedding and a lovely reception and it's been a wonderful 10 years. We've had our ups and downs but we've got 2 beautiful children, 2 dogs and a pretty wonderful life. I couldn't really imagine it any other way. Love you honey and Happy Anniversary!!!
This was our wedding song:


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Nokia Announces the Asha 210 QWERTY
The Nokia Asha 210 brings the QWERTY back to life! It's been awhile since we have seen the likes of Nokia's Asha series handsets rocking a keyboard! The latest iteration of the feature phone came in the form of the touch screen Asha 310. Way back in 2011, right around the same time Nokia was introducing their Lumia 800 and 710 devices they also took to emerging markets and announced three different Asha Series 40 handsets, two of which sport full QWERTY keyboards. The QWERTY Asha is back and looking better than ever. World, meet the Nokia Asha 210!
The Nokia Asha series devices are mid to low range feature phones that offer up awesome connectivity for end users in emerging markets. The latest Asha however is a looker, and has a few social tricks up its sleeve. Spec's wise, the Asha 210 is not something you'd hang your hat on, however what it lacks in high end specs is made up for is pure streamlined connectivity. The Asha 210 starts with a standard bar shape design that is built around an 11.8 mm thin body that houses a 2.4 inch 320×240 LCD with 169 pixels per inch. Internally the Asha 210 packs in a 1200 mAh battery, 32 MB of RAM and expandable microSD memory options up to 32 GB. The processing power has not been ousted at this point however Engadget reports it clocks in just under 1 GHz. Around back of the Asha 210 you'll find an embedded 2 mega pixel camera with 176×144 video recording at 10 frames per second. Other connectivity will allow for FM radio, Bluetooth 2.1, Wi-Fi, micro USB and SyncML. The Asha 210 also adds in a dedicated WhatsApp button which will end up being a Facebook button in certain markets as well as a dedicated camera button on the front side of the phone for easy one touch access to your 2 mega pixel shooter. The best part about the Asha 210 is it will only cost potential customers a mere $72 bucks! Of course this is before taxes and subsidies. The device will also ship in dual sim variations and will begin leaving its nest sometime this quarter. Lastly, as you can see the Asha 210 will be offered up in yellow, blue, pink, white and black!
Anyone interested in an Asha 210? It looks beautiful! Sound off in the comments below and let us know what you think of the new Nokia Asha 210 and remember that we carry a wide variation of all the best in Nokia accessories known to man!facebook, keyboards, Wirelessground.com, QWERTY keyboard, feature phones


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Tim received his undergraduate degree at Xavier University in Cincinnati, Ohio where he graduated summa cum laude in 1975. He completed his M.D. at The College of Medicine at The Ohio State University (Columbus, Ohio) in 1980. He then moved to New York City where he completed Residency and Fellowship training in Brooklyn, New York 1986. He has since been practicing Emergency Medicine first in NYC from 1986-1999 and then in Hawaii from 1999-2011.
Tim started on a conscious path of self discovery in the mid 1980's which included practicing various techniques, readings, taking courses and seminars, etc. In 2005 he met Guilherme Alves and began studying the DeRose Method which has been his exclusive type of practice since then. Tim began training as an Instructor in 2009 and completed examinations and requirements for becoming an Instructor in the DeRose Method in October 2010.
We have different specialties that adjust to every person or particular situation:
• DeRose Method Company
• DeRose Method Academy
• DeRose Method School
• DeRose Method Personal
• DeRose Method Proficiency
This modalities fits any need, you can practice DeRose Method in the comfort of your home or even in your company, with personal or small group classes and with and adaptable duration, from 15 min to an hour. Make an appointment for a trial class.


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With built-in wireless networking and robotic intelligence the Smarter Kettle 2.0 tracks traffic and alarms to boil water precisely when you want it.
Pricing not available
Aiming to save power and water, the Miito induction kettle is now seeking crowdfunding to bring it to market.
At last, a nice cup of tea and the iPhone can co-exist in harmony. The KettleCharge works at home or while camping.
To cut down on electricity wastage from boiling too much water, the Miito uses induction technology to heat water right in the cup.
Alcatel, Huawei and ZTE are hoping that low prices for quality products will lure US consumers online.
Columbia University researchers discover a new source of alternative energy by harnessing the power of evaporation and soil bacteria spores.
No need to move to a violent, post-apocalyptic desert wasteland to rock out like a warrior. Learn how to make a flamethrower ukulele that would impress even the likes of Doof.
The Charcoal Companion Half Circle Pro Griddle opens up the grill to new possibilities. The simple accessory makes breakfast easy to find.
Put the kettle on without ever leaving the warmth of your bed. The iKettle is a Wi-Fi enabled, smartphone-controlled, water-boiling gadget.
The popular housewares brand just added a new set of small kitchen appliances to its product family. We go hands-on.


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Social media: a force for good?
Stephen Fry, Biz Stone and Reid Hoffman discussed the phenomenon of social media and its future impact.
Event venue
Nesta, London
Event date
19 November 2009 - 12:00pm
Stephen Fry, actor, journalist and celebrity 'Tweeter' and self-confessed technophile; Biz Stone, Founder and Chief Executive of Twitter; and Reid Hoffman, Founder and Chief Executive of LinkedIn gathered at Nesta for a lively discussion on the possibilities, pitfalls and impact of social media.


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Archive for the 'Doyusha' tag
Die-cast news roundup
Here are some follow-ups to recent items posted in this space…
Fresh Cherries and 1:64-scale Malibu International cars have been on sale
at Wal-Mart for 50 cents apiece over the last few weeks. Most seem to have
evaporated, although availability may vary from store to store. Even if
you're not into them: A.) they make great traders, particularly with
international trade partners who don't see these castings at all, and; B.) At
50 cents apiece, could it really hurt to stockpile a few?
Norev of France continues to release its line of small-scale promotional
models for the European car market; latest is the family-size Citroën C5,
available in sedan and wagon form in a variety of colors. We've got ours coming shortly, but pictures indicate that if you've liked previous Norev
models, there will be no surprises here. As always, we recommend the Dutch
site www.tinytoycars.com for all of your Norev needs.
Doyusha is following up their first two waves of kei-cars (sub-660cc) with
the Suzuki Wagon R and the Wit, available now, as well as the upcoming
Mitsibishi eK and i, as well as the Honda Diva and Vamos Lowdown. Of these
six, only two have been done in small-scale before (the i and the Wagon R,
both by Tomica). Either www.toyeast.com or else HobbyLink Japan should have these available.
TACCAR has launched a second wave of historic Nissan Skylines with opening hoods: a '65 GT-B (the street car that really started the whole hype about Skylines), as well as a 1995 R33 and a 1999 R34 are each available. ToyEast
and Hobbylink Japan should have these available.
Tomica's superb Limited Vintage series continues: Coming later this year will be a 1966 Subaru 1000, a 1964 Toyopet Corona, a 1970 Toyota Carina and a 1969 Honda 1300-77. Few of these will be recognizable to Western eyes, but
the quality of the models is so remarkable that it almost doesn't matter.
Toy East and HLJ, listed above, should have these as they come out.
The second wave of M2 Autothentics are out; these are slightly hot-rodded
versions of the first six castings, adding custom wheels, lake pipes and paint. Our favorite of the lot: the black '51 Ford Crestliner with red
steelies, which replaces the odd-colored two-tone example in the home collection. Coming up soon: three new castings, all Mopars! The '54 Dodge
Royal and a pair of De Soto Fireflites (1955 and '57) will be in the third wave, in stock form, sometime later this year.
(This post originally appeared in the June 26, 2008, issue of the Hemmings eWeekly Newsletter.)
Venerable model company launches small-scale models of small-scale cars
Doyusha is offering a pair of die-casts replicating a couple of odd Daihatsu machines — the Tanto and the Move.
Yet another model company has jumped into Japan's ever-increasing collector-oriented die-cast fray. Kyosho, Konami, Tomica (with their Vintage Limited line), CMs, Aoshima, Yow Modellini…the list goes on and on, but now there's one more to add to the list. Doyusha, long a maker of model kits of old-school Japanese cars, has launched their small-scale die-cast offensive with a pair of Daihatsu machines — the Tanto, and the Move. Four colors of each model will be available at the outset. As seems to be the trend (now a tradition?) in collectors' Japanese die-cast, they come in a glossy box that gives no indication of what's inside.
Don't confuse kei-cars with K-cars. Far from the Plymouth Reliants and early minivans that we think of in this country, Japanese kei-cars have long been a way to lessen the tax burden and proof of parking-spot ownership in that country. The real cars are limited to no more than 3.4 meters long, two meters high, and use a 660cc engine putting out no more than 63hp. Tomica models of kei-class cars (like the Subaru R1 and Mitsubishi i, for example) tend to clock in at around 1:55 or larger and are nearly three inches long, so Doyusha's Daihatsus, if they are true 1:64 scale, will be itty-bitty — barely two inches long.
If the Daihatsu name is vaguely familiar, it should be. They were on American soil for about half a decade, and had a rough time in the U.S. market in the late '80s and early '90s with the Charade hatchback and Rocky off-roader. Daihatsu has been owned by Toyota since the late 1960s.
We haven't seen these in person yet, but the photos certainly look promising. Check out the pictures yourself at
http://www.doyusha-model.com/list/diecast/keicar1daihatsu.html, but you'd better brush up on your Japanese language skills if you want any hope of reading the Web site. We would scare up our old Japanese-model haunts, www.hlj.com or www.toyeast.com, if you wanted to get any of these.
(This post originally appeared in the February 7, 2008, issue of the Hemmings eWeekly Newsletter.)


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Capital: Bangkok
Languages: Thai, English
Useful links: Kids Culture Corner: Thailand | CIA World Factbook
The following information is generalized and compiled from questions posed to the agents and interviewers in Thailand. Although au pairs from this country may or may not have had these experiences/beliefs, Au Pair in America wishes to share this general information with our families.
I really feel like I have a family in America after being an au pair. My host family is going to visit me and my family in Sweden this winter and I cannot wait to see them!Sandra, au pair


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Narrow your search
In a just-held briefing, NASA says that Pluto's "heart" will be named for the dwarf planet's original discoverer, a dark spot on moon Charon is called "Mordor," and Pluto is a relative newborn.
Pluto, the focus of NASA's New Horizons spacecraft, has a softer side in the form of a distinctive heart-shaped feature on its surface.
The PBS and BBC One series "Big Blue Live" offers a close look at a blue whale's massive ticker. Just be thankful YouTube doesn't have a virtual smell feature...for now.
The Cassini spacecraft says its goodbyes to icy Dione in stunning style with some of the best images of the moon ever taken.
The company's latest activity tracker aims to outdo the original by monitoring your heart rate and stress level.
Women are more likely to pick up on romantic cues when they feel sated, a new study says. It probably doesn't hurt if you also pick up the check.
Tesla issues an over-the-air update after security researchers hack the car via its entertainment system.
Yub nub! Let Internet star Munchkin brighten your day as she dresses in an Ewok costume and waddles through the grass with her walking stick to a "Star Wars" soundtrack.
Watch Stephen Colbert try to coax some love for Pluto out of Neil deGrasse Tyson while they eat Klondike bars and discuss drafting in Nascar.
A lot of people heart iHeart, it seems -- enough to sign up free with the digital radio provider owned by a terrestrial radio giant.


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Kenley Collins
What have you been up to since your time on "Project Runway"?
I've been writing music and singing in a band called Jetblack Bullseye while managing a restaurant.
What garment from your season of "Runway" do fans compliment you on the most to this day?
My finale feathered wedding dress.
What do you think you are best remembered for from your season?
My headpieces.
Favorite client?
What celebrities would you love to dress?
Gwen Stefani.
What are your strengths as a designer at this point in your career? Any weaknesses you.re still working on?
My strength is staying true to what I like. Sometimes that can also be my weakness.
How has your design aesthetic changed since your time on the show, if at all?
I'm still very me, but I think I've matured.
What are your favorite pieces and staple items in your closet?
My 1960s Christian Dior sheer gold sparkling stockings given to me by Elisa on Project Runway All Stars.
Which fashion rule do you think is begging to be broken?
I don't like any fashion rules. I think if it looks fabulous, wear it.
How do you choose what to pack for "All Stars"?
There was no choosing. I brought it all.
After being on the show, what "Project Runway" challenges do you think are the best and worst of all time?
We had to make clothes out of car parts. I remember there was a lot of blood in the workroom that day. I think they're all stressful with the time given.
Who is your favorite fellow "Project Runway" designer from any season, including yours, and why?
Jay, because he set the bar high for the rest of us.
What goes through your head when you see yourself on television?
I crack up.
What is the one thing fans would be surprised to find out about you?
Most are surprised to see that I'm singing more than designing, but you need a break from it, especially after designing bridal for a while.
What makes you All-Star material?
My strong point of view.


text: The electronic forms can be filled out simply by tabbing through the cells and entering the information. Each form that you complete will need to be opened and saved separately under a different file name. For directions on how to fill out the forms, please refer to the instructions booklet link below.


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Article ID: 2536089 - View products that this article applies to.
On This Page
This hotfix rollup package resolves compatibility issues of Asset Intelligence in Microsoft System Center Configuration Manager 2007 Service Pack 2 (SP2) with other Microsoft volume license products. This hotfix rollup resolves the following issues and is dated May 2011:
To resolve these issues, install the following hotfix rollup package on the affected System Center Configuration Manager 2007 SP2 primary site server. The installation wizard for the package creates another package for clients. To deploy the package to all clients after you run the package on the primary site server, you have to create an advertisement.
Note The hotfix package can be installed on a System Center Configuration Manager 2007 SP2 site server that is running an x86-based or x64-based version of an operating system.
For more information about installation guide, click the following article number to view the article in the Microsoft Knowledge Base:
(https://support.microsoft.com/kb/2477182/ )System Center Configuration Manager 2007 Hotfix Installation Guidance
Hotfix informationA supported hotfix is available from Microsoft. However, this hotfix is intended to correct only the problem that is described in this article. Apply this hotfix only to systems that are experiencing this specific problem.
If the hotfix is available for download, there is a "Hotfix download available" section at the top of this Knowledge Base article. If this section does not appear, submit a request to Microsoft Customer Service and Support to obtain the hotfix.
Note If additional issues occur or if any troubleshooting is required, you might have to create a separate service request. The usual support costs will apply to additional support questions and issues that do not qualify for this specific hotfix. For a complete list of Microsoft Customer Service and Support telephone numbers or to create a separate service request, visit the following Microsoft Web site:
http://support.microsoft.com/contactus/?ws=supportNote The "Hotfix download available" form displays the languages for which the hotfix is available. If you do not see your language, it is because a hotfix is not available for that language.
PrerequisitesTo apply this hotfix, you must have System Center Configuration Manager 2007 Service Pack 2 (SP2) installed.
Registry informationTo use the hotfix in this package, you do not have to make any changes to the registry.
Restart requirementYou do not have to restart the computer after you apply this hotfix.
Note You have to close Configuration Manager administration console before you install this hotfix package.
Hotfix replacement informationThis hotfix does not replace a previously released hotfix.
File informationThe English (United States) version of this hotfix installs files that have the attributes that are listed in the following tables. The dates and the times for these files are listed in Coordinated Universal Time (UTC). The dates and the times for these files on your local computer are displayed in your local time together with your current daylight saving time (DST) bias. Additionally, the dates and the times may change when you perform certain operations on the files.
System Center Configuration Manager 2007 file information notes
Note It is expected that the article ID in SCCM2007AC-SP2-KB2528650-x86-ENU.msp does not match the ID of this article. This is an artifact of the hotfix build process.
Collapse this tableExpand this table
Microsoft has confirmed that this is a problem in the Microsoft products that are listed in the "Applies to" section.
For more information about software update terminology, click the following article number to view the article in the Microsoft Knowledge Base:
824684For more information, click the following article number to view the article in the Microsoft Knowledge Base:
(https://support.microsoft.com/kb/824684/ )Description of the standard terminology that is used to describe Microsoft software updates
(https://support.microsoft.com/kb/2444668/ )Hotfix rollup for Asset Intelligence compatibility issues in System Center Configuration Manager 2007 SP2 with other Microsoft volume license products: November 2010
Article ID: 2536089 - Last Review: May 27, 2011 - Revision: 2.0
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|Mark Osteen
Vocals; Tenor, Alto and
Soprano Saxophones
Anthony Villa
Gary Kerner
Greg Mack
|For over a decade Cold Spring Jazz Quartet has appeared regularly in nightclubs, restaurants and concert venues in the Baltimore-Washington area, winning a broad and loyal following. Favorite venues have included The Tremont Hotel, Ixia Restaurant, and the Minoan Room at Meli. Recent private engagements have included Stella Maris's annual wine-tasting event, concerts at Cylburn Arboretum and at the Greenmount Street Festival.For smaller rooms, the group performs as a drumless trio. But at any size, CSJQ can create a variety of different sounds and moods—from a swing-and-standards combo to a bebop group or blues band—depending upon the audience and venue.CSJQ's debut CD, Same Place. Different Time, includes passionate performances of classic bop and instrumentals such as Bud Powell's "Webb City" and Dexter Gordon's "Cheesecake." It also features exciting original compositions by Brian Smith and Mark Osteen. Osteen's vocals, which distinguish CSJQ from most straight-ahead jazz groups, lend songs such as "Doxy" and "The Old Country" a distinctive flavor.The group's second CD, Urban Pastoral, reflects the group's versatility and range of influences, showcasing CSJQ's contemporary polish while paying tribute to the band's deep roots in bebop and blues.The "City" side opens with Peggy Lee's "I Love Being Here with You," and features Osteen's insouciant vocal. The next five tracks offer a variety of urban sounds, from Latin jazz (Gary Kerner's smooth samba, "Highpockets," as well as a smoking rendition of Jobim's "Favela" ) and bebop (Monk's "Hackensack," Horace Silver's "Tippin'") to the risque city blues of "Doodlin,'" which spotlights Osteen's sassy singing.The "Country" side presents a set of sharp original compositions—Brian Smith's "Perception," Osteen's haunting ballad "Fallow," and multi-rhythmic "Black Olive," along with Smith's driving "The Big 'C'"—punctuated by two standards: a snappy take on "Is You Is" and a concluding, lyrical duo version of "You Don't Know What Love Is."
Urban Pastoral: it's current. It's classic. Above all, it's cool.
Contact Mark Osteen (410-339-7721) or e-mail email@example.com for further information or to book an engagement.
Photo: Leo Howard Lubow


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June 25, 2013
Kim Tran (left) talks to Carol Hatfield as she sells her some tomato and parsley plants Saturday during the opening of the Jonesboro farmers market hosted by the University of Georgia's Clayton County Extension Office.
Stories this photo appears in:
Jonesboro farmer's market makes best of sluggish start
Opening day plagued by few vegetables, customers
Umm Taufeeq saw the sign for a farmers market in downtown Jonesboro and thought it would be a good opportunity to buy some fresh vegetables, but luck was not on her side.
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