text:
Parallel Programming
Advanced Computer Concepts For The (Not So) Common Chef: Terminology Pt 1
Before we start, I will use the next two blogs to clear up some terminology. If you are familiar with these concepts, I give you permission to jump to the next section. I suggest any software readers still check out the other blog about threads. There is a lot of confusion, even among us software professionals.
Advanced Computer Concepts For The (Not So) Common Chef: Introduction
While talking to a very intelligent but non-engineer colleague, I found myself needing to explain the threading and other components of the Intel® Xeon Phi™ ⅹ100 and ⅹ200 architectures. The first topic that came up was hyper-threading, and more specifically, the coprocessor's version of hyper-threading. Wracking my brain, I finally hit upon an analogy that seemed to suit: the common kitchen.
Power Management Policy
By Taylor Kidd, Intel Corporation
This article is essentially a collection of blogs I wrote on the same subject. The differences are simply a degree of formalism.
Preface: Power Management: Policy
Section: Power Management: What a Power Management Policy is
Section: Power Management Policy: You Mean there's More than One?
Intel® Xeon Phi™ Coprocessor – Applications and Solutions Catalog
The PDF document attached to this article contains a growing list of available, downloadable or work-in-progress code that can be run, or actively being optimized to run on Intel® Xeon Phi™ Coprocessors.
Состояния управления электропитанием: P-состояния, C-состояния и пакетные C-состояния
(Вы можете скачать PDF-версию этой статьи во вложении.)
Предисловие. Что, почему и откуда? 1
Глава 2: P-состояния, Снижение энергопотребления без ущерба для производительности. 3
Глава 3: С-состояния ядер. Подробности. 5
Hybrid MPI and OpenMP* Model
In the High Performance Computing (HPC) area, parallel computing techniques such as MPI, OpenMP*, one-sided communications, shmem, and Fortran coarray are widely utilized. This blog is part of a series that will introduce the use of these techniques, especially how to use them on the Intel® Xeon Phi™ coprocessor. This first blog discusses the main usage of the hybrid MPI/OpenMP model.
Performance BKMs: There's more than one hammer
I don't know if any of you have noticed but Intel® has a tendency to emphasize its own homegrown tools. This isn't bad as Intel has some of the best. Still, if someone has a favorite hammer, there's a tendency to use that hammer for just about everything.
Submissions open: High Performance Parallelism Gems
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text: In case this wasn't posted elsewhere.... There will be a WISH meet-up and photo op at Pop Century Food Court starting at 4 pm Friday, Jan. 11, followed by a group photo outside the Food Court on the steps at 4:30 pm. Everyone is welcome to come by, say hello, meet fellow WISHers and capture the moment for posterity! Family and friends are welcome. Name tags will be available (seriously, who can pronounce anyone's screen name?!) Please come say hello and put a face to a screen name! Maura

subset: commoncrawl


text:
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Ubuntu 8.04 "Hardy Heron" Alpha 3
Published on 10 January 2008 05:51 PM EST
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Michael Larabel
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The third Alpha release in the road to Ubuntu 8.04 "Hardy Heron" is now available for download.
Ubuntu 8.04
Alpha 3 has improved PolicyKit integration and Firefox 3 Beta 2. Download links and more information is available from the
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text: Jul 17, 2013 16:46 Party Central: Shots of Bourbon Party Central: Shots of Bourbon Advocate story July 17, 2013 Comments The music was in the Superdome, but the action is always on Bourbon Street. For locals and visitors alike, the French Quarter strip was a hotspot during the 19th Annual Essence Festival. The 2013 Essence Festival had the largest attendance in the festival's history, drawing more than 540,000. Get The Advocate in your inbox Subscribe


text:
~|| I Wanna Fly...
Credits & Info
- Date
- 08/13/2011
- File Info
- Song
- 3 min 34 sec
- Score
- 4.24 / 5.00
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Licensing Terms
- Attribution:
- You must give credit to the artist.
- Noncommercial:
- You may not use this work for commercial purposes unless you make specific arrangements with the artist.
- Share Alike:
- If you alter, transform, or build upon this image, you may distribute the resulting creation only under a license identical to this one.
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- Rated 4.24 / 5 stars
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- 8,112 Plays | 735 Downloads
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Author Comments
Rated 5 / 5 stars
Man the piano work on this is amazing, melody is great, well done bro!
Keep doing your thing, you will get recognised soon I bet!
Good job!
Rated 5 / 5 stars
Das Piano.........!!!!!!! Unfassbar genial Thomas,ich liebe dein Gefühl für Melodien und die dazu stets passende Zusammensetzung der Instrumente als auch verschiedener Melodien!
Perfekter Track!
Rated 5 / 5 stars
Are you famous yet :P
Why can't ppl find you! I mean!
this aint ordinary!
i think your work is good enough to be in movies and games and stuff!
Keep it up! Hope the best for ya :)
Rated 5 / 5 stars
really like it, always been a huge fan of your work.
10/10, 5/5
Rated 4.5 / 5 stars
as always great but
The clapping is a bit much but nothing that makes me want to say that this is a bad song in the least. So you still get a 5/5 and a 9/10 from me


text:
Tickets & Events
KST Presents
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Welcome the spring season with kid-friendly music spun by our guest DJ, dancing, hands-on art activities, and more.
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KST Presents
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newMoves Contemporary Dance Festival 6
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KST Presents
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Full Bloom Summer Dance Party is KST's annual benefit bash and celebration of summer in the city! Enjoy live performances, food and drinks from the East End's best bars and restaurants, and the city's hottest dance floor. Proceeds support low-cost summer programming at KST.


text:
Join us at an upcoming event:
The core goal of the Woodstock District Chamber of Commerce is connecting you. The most effective way to get connected in the Woodstock business community is to come to an event, introduce yourself to a few people and find out how your business may be able to help theirs. Meet suppliers, wholesalers, retailers and potential clients by joining us at an upcoming event.
Upcoming Events
5:30 pm Business After Five
Business After Five
Apr 14 @ 5:30 pm – 7:30 pm
Join WDDS at the Toyota Motor Manufacturing plant for networking, refreshments and light snacks! Door prizes include an Apple iPad Mini, Fitbit Flex Wireless Activity and Sleep Wristband. Everyone will go home with gift bags[...]
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Group Plans that Work
Apr 23 @ 8:00 am – 9:30 am
Are you finding and retaining quality employees? Having the right group plan in place might help. If you have just one other employee or thousands you can benefit from having a group and/or retirement benefit[...]
5:00 pm Business After Five
Business After Five
May 12 @ 5:00 pm – 7:00 pm
Networking ~ Refreshments ~ Door Prizes Located in Woodstock, Ontario, Steele Health and Wellness Centre helps you feel great and live life to the fullest! From the everyday warrior, to the performance athlete, your goals[...]
Emerging Stronger 2015Your Chamber of Commerce has partnered with the Ontario Chamber of Commerce to release Emerging Stronger 2015, the fourth edition our economic agenda for Ontario Emerging Stronger 2015 identifies the immediate steps that government and the private sector must take to...
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Whether you're ready to join today, or looking for more information on Chamber benefits, we'd be happy to speak with you.


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text:
The present invention provides ABC transporter gene of P. fluorescens encoding a protein which enhances the secretion of foreign proteins extracellularly, a recombinant expression vector containing the transporter gene, a microorganism transformed therewith and a process for preparing foreign proteins which comprises the steps of incubating the transformant, inducing expression of the proteins and leading the proteins to be secreted extracellularly. In accordance with the present invention, foreign proteins can be secreted extracellularly in a simple and efficient manner by employing a secretion system associated with the ABC transporter gene.
1. An isolated ATP binding cassette transporter (ABC transporter) from Psedomonas fluorescens SIK W1 (KCTC 2689P) which consists of DNA sequence SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 linked in sequential order.
2. An expression vector which comprises the ABC transporter of claim 1.
3. An expression vector of claim 2, where the expression vector is pACYC184.
4. A recombinant microorganism transformed with the expression vector of claim 3.
5. Escherichia coli XL1-Blue which is transformed with the expression vector of claim 3.
6. An expression vector which produces foreign protein, being transformed into a recombinant microorganism together with an expression vector comprising the ABC transporter of claim 1, to secrete foreign protein extracellularly.
7. An expression vector pHOPE which produces lipase, being transformed into a recombinant microorganism together with the expression vector comprising the ABC transporter of claim 1, to secrete lipase extracelluarly.
8. An expression vector pPROT705 which produces protease, being transformed into a recombinant microorganism together with the expression vector comprising the ABC transporter of claim 1, to secrete protease extracelluarly.
9. An expression vector comprising a foreign protein gene and the ABC transporter of claim 1, being transformed into a recombinant microorganism, to secrete foreign protein extracelluarly.
10. A microorganism transformed with the expression vector of claim 9.
11. A process for preparing a foreign protein comprising a step of culturing a microorganism transformed with two expression vectors, where the first expression vector comprises foreign protein and the second vector comprises ABC transporter to secrete the foreign protein extracellularly.
12. The process of claim 11 wherein the expression vector for ABC transporter is pACYC184.
13. The process of claim 11 wherein the microorganism is Escherichia coli, Pseudomonas sp. , Xanthomonas sp. or Erwinia sp.
14. The process of claim 11 wherein the transformant is cultured at 15-27° C.
15. A process for preparing a foreign protein comprising a step of culturing a microorganism transformed with an expression vector which expresses both foreign protein and ABC transporter, to secrete the foreign protein extracellularly.
16. The process of claim 15 wherein the microorganism is Escherichia coli, Pseudomonas sp., Xanthomonas sp. or Erwinia sp.
17. The process of claim 15 wherein the transformant is cultured at 15-27° C.
18. The Escherichia coli XL1-Blue of claim 3 which has the Korean Collection for Type Cultures accession number of KCTC 0586BP.
The present invention relates to ABC transporter gene cluster, tliDEF, in Pseudomonas fluorescens("P. fluorescens"), more specifically, to ABC transporter gene of P. fluorescens encoding proteins which enhance the secretion of foreign proteins extracellularly, a recombinant expression vector containing the transporter gene, a microorganism transformed therewith and a process for preparing foreign proteins which comprises the steps of incubating the transformant, inducing expression of the proteins and leading the proteins to be secreted extracellularly.
For the last scores of years, various genes from microorganism to human have been isolated and sequenced. In line with the development of biotechnology, foreign proteins have been produced by expressing these genes in host organism such as animal, plant, microorganism, etc. and used it various fields of medicaments, enzymes for biochemical synthesis, food additives and agriculture.
Escherichia coli("E. coli"), a representative host organism used for introducing gene, has been employed as a research tool for a long time, since it has been throughly investigated to use conveniently. Further many E. coli strains for various purposes have been developed so far, which accelerate the use of E. coli for the production of useful proteins necessary for industrial purposes and research purposes as well. E coli has an advantage of excessive production of desired protein, since it accumulates foreign gene-derived proteins in a level of 20-50% of total proteins. However, there are many cases that proteins are produced not as an active form but as an inactive inclusion body. Production of inclusion body occurred when proteins can not be secreted extracellularly and then excess amount of proteins accumulated in the cell. These proteins are accumulated as an inclusion body before they were refolded to have a biological activity(see: Mitraki et al., Bio/Technology, 7:693(1989)). Many attempts have been made to solve these problems, for examples, change of fermentation condition(see: Schein, Bio/Technology, 7.1141(1989)) or insertion of chaperon preventing inclusion body from being produced(see: Hockney, Trends Biotechnol., 12:456(1994)).
As an alternative approach, a method for secreting intracellular protein to extracellular medium has been investigated. If tale expressed proteins are secreted extracellularly before the accumulation in the cell, inclusion body is not formed any more. Expressed proteins can be ideally manufactured from microorganism by secreting the proteins in the cell, removing the microorganism from culture media, and obtaining the desired proteins from the media. If the expressed proteins are not secreted to extracellular media, inactive inclusion body is formed, or if the inclusion body is not formed, mechanical, chemical and biochemical methods are required to lyse cells and desired proteins should be purified from many other proteins contained in the cell. This accompanies great loss of expense and loss of proteins. Accordingly, it is of great importance to improve is method for secreting proteins expressed in microorganism to extracellular medium.
In an association with protein secretion, general export pathway(GEP) has been extensively studied, by which N-terminal sequence of protein is recognized by sec protein in the cell to secrete the protein extracellularly(see: Pugsley, Microbiol. Rev., 57:50(1993)) The industrial use of GEP has been considered, since GEP can lead intracellular proteins to attach to cell surface or to be secreted extracellularly(see: U.S. Pat. Nos. 5,156,959; 5,541,087; 5,658,755; and, 5,665,590). By way of GEP, protein is secreted extracellularly in case of Gram-positive bacteria having only one cell membrane, however, it should pass inner membrane, stay at periplasm and bypass outer membrane in case of Gram-negative bacteria like E. coli having two cell membranes. Many proteins(≧14) are involved in the export through only outer membrane, which in turn, reveals a serious problem that secretion efficiency is relatively low(see: Akrim et al., Mol. Microbiol., 10:431(1993); Pugsley and Possot, Mol. Microbiol., 10:665(1993)).
Another pathway, mediated by ABC transporter(ATP binding cassette transporter), has been also known in the art, where ATP is hydrolyzed to secrete protein(see: Fath and Kolter, Microbiol. Rev., 57:995(1993)). Protein thus secreted has a C-terminal targeting signal, which is recognized by ABC transporter, to secret the protein extracellularly. Protein secretion is carried out by the aid of ABC transporter protein located at cell membrane and proteins of both Gram-positive and Gram-negative. bacteria can pass cell membrane at once. That is, in case of Gram-negative bacteria, secretory protein makes inner membrane fuse partly with outer membrane, which allows the protein to pass 2 membranes at the same time. Accordingly, the secretion pathway based on ABC transporter is more efficient in Gram-negative bacteria such as E. coli.
Until now, many ABC transporters have been successively known in the art, those associated with cyclolysin(see: Glaser et al., EMBO J., 7:3997(1988)), leucotoxin(see: Highlander et al., DNA, 8:15(1989)), protease and lipase, since a secretion system associated with α-hemolysin(see: Felmlee et al., J. Bacteriol., 163:94(1985)) was discovered in E. coli. Especially, ABC transporter secreting α-hemolysin has been extensively studied and it is proposed as a universal secretory system, based on the results that other proteins can be secreted by C-terminal targeting signal(see: Blight and Holland, Trends Biotechnol., 12:450(1994) In this connection, ABC transporters cloned from Pseudomonas sp., Erwinia sp. and Serratia sp., are practically applied for the secretion of protease extracellularly(see: Duong et al., Gene, 121:47(1992); Letoffe et al., EMBO J., 9:1375(1990); Letoffe et al., T. Bacterol., 173:2160(1991)). However, little attraction has been, paid to ABC transporter for secreting lipase.
So far, two kinds of ABC transporters secreting, lipase extracellularly have been reported in the art: First, a gene secreting lipase was cloned from Serratia marcescens(see: Akatsuka et al., J. Bacteriol., 177:6381(1995)); second, ABC transporter secreting protease from P. aeruginosa was revealed to secrete lipase of P. fluorescens(see: Duong et al., Mol. Microbiol., 11; 1117(1994)). Naturally, ABC transporter has not been practically applied in industry since only the ABC transporters from Serratia marcescens and protease from P. fluorescens are available to secrete lipase. In addition, ABC transporter for lipase secretion has not been found in P. fluorescens, though the lipase from the microorganism was cloned in many research groups(see: Lee et al, Biochim. Biophys. Acta., 1169:156(1993); Tan et al., Appl. Environ. Microbiol., 58:1402(1992); John et al., Appl. Environ Microbiol., 58:1776(1992)).
Accordinqly, there are strong reasons for exploring ABC transporter gene from P. fluorescens, and to develop a novel process for preparing foreign proteins by ABC transporter in a simple and efficient manner.
The present inventors first cloned a gene encoding ABC transporter from P. fluorescens, and constructed an expression vector containing the said gene and lipase or protease coding gene which can be expressed in Gram-negative bacteria such as E. coli and Pseudomonas sp., to secrete the lipase or protease proteins. As a result, it was found that these proteins can be produced and accumulated in extracellular medium with a high efficiency. Since the ABC transporter enhancing the secretion of foreign proteins functions at a low temperature of below 25° C., it can prevent inclusion body formation, which allows universal use of the ABC transporter for industrial purposes.
The first object of the invention is, therefore, to provide ABC transporter gene from P. fluoresens which enhances the secretion of foreign proteins to extracellular medium.
The second object of the invention is to provide an expression vector containing the ABC transporter gene which is designed to secrete foreign proteins extracellularly.
The third object of the invention is to provide recombinant expression vectors which are constructed to express and secrete foreign proteins extracellularly by ABC transporter.
The fourth object of the invention is to provide a transformant harboring the ABC transporter expression vector.
The fifth object of the invention is to provide a process for preparing foreign proteins by secreting the proteins expressed in the transformant extracellularly by ABC transporter.
The above and the other objects and features of the present invention will become apparent from the following description given in conjunction with the accompanying drawings, in which:
FIG. 1 shows a gene arrangement of ABC; transporter on Pseudomonas; fluorescens DNA and restriction maps of various plasmids containing these genes.
FIG. 2a is a graph showing the changes of OD600 value against incubation time of E. coli transformants.
FIG. 2b is a graph showing the changes of lipase activity against incubation time in culture medium.
In describing the present invention, the term ABC transporter gene′ is employed to mean a gene encoding ABC transporter which promotes secretion of foreign proteins expressed in a microorganism extracellularly. ABC transporter gene from P. fluorescens is further illustrated in detail.
ABC transporter gene of the present invention was cloned from total DNA of P. fluorescens. The ABC transporter gene can be cloned in P. fluorescens SIK W1(KCTC 2689P), P. fluorescens B52, P. fluorescens LS107d2, P. fluorescens(Serratia marcescens Sr41) or P. fluorescens FT(JP 1997224679-A 1 Sep. 02, 1997) which produce lipase or protease.
Plasmid vectors which are replicable in transformants and have resistance to antibiotics such as (ampiciIin, chloramphenicol, kanamycin or tetracycline, and have promoter such as lac, tac, trp or PCm, can be used to clone the ABC transporter gene. Among these vectors, pUC18, pUC19(see; Yanisch-Perron et al., Gene, 33:103(1985)) or pBluescript(Stratagene, USA) may be used as high-copy-number plasmids, and pBR322(see: Rodriguez et al., Gene, 2:95(1977)), or pACYC184(see: Chang and Cohen, J. Bacteriol., 134:1141(1978)) may be used as low-copy number plasmids. Since the origin of replication of pUC18, pUC19 or pBluescript is different from pACYC184, replication and maintenance of high-copy-number plasmids are possible when these plasmids are introduced into microorganism with pACYC184.
E. coli can be usually used as a host for recombination of the plasmid vectors and analysis of DNA sequence as well, inter alia, E. coli XL1-Blue, JM109 and DH5 which are widely distributed and commercially available, are preferred. These E. coli can be used to express interested genes, and Pseudomonas sp. Such as P. fluorescens, P. putida, P. aeruginosa and P. fragi can be also used for the same purpose. According to the present invention, a gene encoding lipase was first screened to clone ABC transporter gene, since the ABC transporter gene is frequently located adjacent to the genes encoding secretory proteins: Total DNA of P. fluorescens was digested with restriction enzymes, and DNAs of proper size was collected and inserted into vector to construct genomic library for cloning of lipase gene. If the genomic library is spread on a medium confirming lipase activity, like LAT media containing LB, tributyrin and ampicilin, active halos appear in E. coli with lipase gene. Consequently, lipase gene can be selected. After cloning of lipase gene, upstream and downstream DNA sequence of lipase gene was analyzed to confirm whether ABC transporter gene was contained or not.
If the ABC transporter gene had existed, it was investigated whether all components of ABC transporter gene are contained or not. If ABC transporter gene cluster had not been contained in cloned vector, other components were selected by Southern hybridization: Total DNA was digested with EcoRI, HindIII, BamHI, KpnI, SalI, SacI, etc., electrophoresed, and hybridized band was eluted with probe homologous to a part of ABC transporter gene. The extract was inserted into a vector, introduced into E. coli and colony hybridization was performed to screen E. coil harboring recombinant vector containing desired gene. Recombinant vector containing the other region of ABC transporter gene was obtained by isolating vector from E. coli selected by colony hybridization. ABC transporter gene(tliDEF), protease, protease inhibitor and lipase gene(tliA) were cloned from P. fluorescens. ABC transporter gene was a double helix DNA of 4540 bp long composed of 3 open reading frame(ORF), and protease, protease inhibitor and lipase gene of P. fluorescens was located upstream and downstream of ABC transporter gene, respectively.
After cloning of ABC transporter gene as described aboves, a transformant which secretes foreign protein to extracellular medium was constructed by introducing both foreign protein encoding gene and tliDEF into Gram-negative bacteria such as E. coli and Pseudomonas sp. Two kinds of transformants may be constructed as follows: The first one is constructed by transforming with one expression vector containing genes encoding foreign protein and ABC transporter gene, and the other one is constructed by transforming with two different recombinant expression vectors containing the said genes in a separate manner.
In the former case, it has an advantage of using an antibiotic to maintain expression vector. However, the expression of foreign protein gene and ABC transporter gene can not be controlled independently, since the said two genes are regulated by one promoter. In the latter case, though the expression of each recombinant vector can be regulated independently, two antibiotics are essentially required. Since ABC transporter should be located in cell membrane to secrete foreign proteins to extracellular medium, expression and secretion can be maximized effectively if the expression of ABC transporter gene is carried out constitutively and the expression of foreign protein gene is induced at the initial stage of log phase. In accordance with the present invention, a recombinant vector pABC-ACYC containing ABC transporter gene of P. fluorescens, pHOPE containing lipase gene, and pPROT705 containing protease gene were constructed, respectively, and found that lipase or protease was secreted extracellularly by transforming a microorganism with two recombinant expression vectors. A transformant was constructed by transforming E. coli XL1-Blue with ABC transporter-expressing vector of pABC-ACYC. The transformed E. coli was named Escherichia coli XL1-Blue/pABC-ACYC′, and deposited with the Korean Collection for Type Cultures(KCTC, #52, Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea) an international depository authority, on Mar. 9, 1999 as accession numbers of KCTC 0586BP.
Incubation is carried out at below 27° C., preferably 15-27° C., more preferably 18-25° C. Expression vector containing foreign protein gene was introduced into E. Coli or Pseudomonas and incubated at low temperature, since ABC transporter gene of P. fluorescens functions at low temperature and secrete foreign proteins optimally. Moreover, incubation at low temperature prevents the formation of inclusion body and induces the production and secretion of foreign proteins. This system has an advantage of producing foreign protein with high efficiency, by introducing expression vector into low temperature-resistant microorganism such as P. fluorescens. Further, high expression of foreign protein or ABC transporter can be realized by adding an inducer of IPTG when the recombinant microorganism containing induction vector reached log phase.
Proteins may be isolated from medium by saltinq out employing highly concentrated salt solution or detergents, use of ion chromatography, protein precipitator or combination thereof. Lipase isolated from nutrient broth may be used immediately after centrifugation, or concentrated through ultrafiltration, lyophilized and solidified. Lipase of P. fluorescens can be easily isolated by inactivating other proteins at a high temperature since it is resistant to heat treatment.
The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention. In particular, the ABC transporter gene of the invention covers a DNA sequence from P. fluorescens disclosed herein and its functional equivalents as well which are prepared by insertion mutation, chemical mutation, random mutation, or PCR technique aided mutation.
Cloning of ABC Transporter Gene
Isolation of Total DNA Containing Lipase and ABC Transporter Genes
In order to screen ABC transporter gene, total DNA was isolated from P. fluorescens as follows: 100 ml of nutrient broth was inoculated with P. fluorescens SIK W1 (KCTC 2689P) and incubated at 25° C. for about 24 hours. The culture was centrifuged, and suspended with 15 ml of saline-EDTA buffer(containing 0.15M NaCl and 0.1M EDTA). After incubating at 37° C. for 30 minutes with 3 mg lysozyme, cells were shaked vigorously and lysed by adding 0.2 ml of 25% SDS(sodium dodecylsulfate). Foreign proteins and impurities were extracted 5 times with 10 mM Tris-buffered phenol and extracted 2 times with phenol-chloroform(1:1, v/v) solution. DNA was collected by swirling glass rod after adding 1/10(v/v) of 3M sodium acetate and 2 volumes of ethanol to the resultant solution. Total DNA was washed with 70% (v/v) ethanol, air-dried and resuspended in TE buffer(10 mM Tris, 1 mM EDTA, pH 8.0).
Cloning of Lipase Gene
Since the ABC transporter gene is frequently located adjacent to the genes encoding secreted proteins in DNA of microorganism, a gene encoding lipase, to clone ABC transporter gene, was screened as follows: Total DNA collected in Example 1-1 was partially digested with restriction enzyme Sau3AI, and the fragments of about 4 to 20 kb were isolated by sucrose gradient centrifugation. This DNA fragments are digested with BamHI and ligated to pUC19 vector dephosphorylated with calf intestinal phosphatase. Recombinant plasmids containing various DNA fragments were used to transform E. Coli and genomic library was constructed. Genomic library thus constructed was screened by plating on LAT medium which is used to measure lipase activity, and 12 colonies showing lipase activity were selected. Since clear halo may appear on the LAT plate by the activity of esterase, 12 colonies were placed on ROM plate(nutrient broth, 0.001% Rhodamin-B, 1.0% olive oil and 1.5% agar) to confirm lipase activity, and only one out of the 12 colonies was revealed to possess lipase activity. Recombinant vector isolated from this colony confirmed that 10.2 kb DNA was inserted into the vector and a gene encoding lipase was located in the middle of inserted DNA. This recombinant vector was named 'pJH9' (sec: FIG. 1).
Cloning of ABC Transporter Gene for Lipase Secretion
Upstream and downstream of lipase gene in pJH9 was sequenced and it was determined that: though the ABC transporter gene secreting lipase was located upstream region, only some components of the ABC transporter gene were contained in pJH9. Therefore, Southern hybridization was carried out to find other component of the ABC transporter gene: Total DNA of P. fluorescens obtained in Example 1—1 was digested with HindIII, BamHI, and KpnI, respectively, and electrophoresed on 1% agarose gel and then transferred onto a nitrocellulose membrane by electroblot. Probe homologous to 1.3 kb fragment produced by HindIII-BamHI digestion of pJH9 was synthesized, and hybridized with nitrocellulose membrane. As a result, hybridized bands were detected in HindIII- and BamHI-digested chromosomal DNA among DNA of P. fluorescens(≧13 kb for HindIII, 4.5 kb for BamHI). The 4.5 kb BamHI DNA fragments were eluted and ligated into BamHI-digested pUC19, and the cloned plasmid were transformed into E. coli, and plated on LB plate, then colony hybridization was performed to select transformant containing ABC transporter gene, and 2 colonies containing the same plasmids were finally selected. Cleavage map of insert revealed that 3′ region of the insert was overlapped with the 5′ region of the insert in pJH9. This recombinant vector was named 'pSCRAP' (see: FIG. 1).
Analysis of Cloned Gene
To analyze DNA sequence of cloned recombinant vectors(i.e., pJH9 and pSCRAP), the vectors were digested with various restriction enzymes on the basis of cleavage map of 2 vectors, and the fragments were subcloned into M13mp18 and M13mp19. DNA sequence was analyzed with ABI PRISM BigDye primer cycle-sequencing kit(Perkin-Elmer, USA) by using AmpliTaq DNA polymerase, where synthetic nucleotides were also employed for sequencing each side of 2 strands of DNA. Analysis of 8.5 kb DNA revealed that this DNA contained 6 open rending frames and 3 among them were associated with lipase ABC transporter gene. Genes encoding protease and protease inhibitor, and gene encoding lipase were located upstream and downstream of ABC transporter gene, respectively(see, SEQ ID NO:1). DNA sequence of 8580 bp containing these 6 genes was shown in SEQ ID NO:1, where 292-1725 bp is a region of gene encoding protease PrtA (see: SEQ ID NO:2), 1775-2143 bp, protease inhibitor Inh (see: SEQ ID NO:3), 2215-3951 bp, ABC transporter TliD (see: SEQ ID NO:4), 3992-5293 bp, ABC transporter TliE (see: SEQ ID NO:5), 5296-6741 bp, ABC transporter TliF (see: SEQ ID NO:6) and 6788-8218 bp, thermostable lipase TliA(see: SEQ ID NO:7). From the analysis of DNA sequence, it was found that promoter is located upstream of the ABC transporter gene and transcription terminator is located downstream of lipase gene, which indicates that lipase and ABC transporter genes constitute an operon to produce proteins translated from the same RNA.
Construction of Transformant Secreting Lipase
A 2.9 kb HindIII-EcoRI-digested fragment containing lipase gene from pJH9 was inserted into pUC19 and named pHOPE′ (see: FIG. 1). When this cloned plasmid was expressed in E. coli transformed therewith as a control, most of lipase existed in the cell and very small amount of lipase was produced extracellularly(≦1 U/ml). Accordingly, a recombinant vector containing ABC transporter gene was constructed to secrete lipase extracellularly: First of all, 2.3 kb BsrBI-BamHI-digested fragment was isolated from pSCRAP, and ligated into EcoRV/BamHl site of pACYC184 vector to constitute a recombinant vector pBsB-ACYC containing a gene encoding ABC transporter TliD(see: FIG. 1). After the recombinant vector pBsB-ACYC was digested with BamHI and SalI, BamHI-XhoI-digested fragment of pJH9 was inserted to construct a recombinant vector pABC-ACYC containing genes encoding ABC transporter TliD, ABC transporter TliE and ABC transporter TliF(see: FIG. 1). In addition, a transformant was constructed by transforming E. coli XL1-Blue with ABC transporter-expressing vector pABC-ACYC. The transformed E. coli was named Escherichia coli XL1-Blue/pABC-ACYC′, and deposited with the Korean Collection for Type Cultures(KCTC, #52, Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea) an international depository authority, on Mar. 9, 1999 as an accession number of KCTC 0586BP. Then, transformants were prepared by simultaneously transforming two vectors of pABC-ACYC expressing ABC transporter and pHOPE expressing lipase into E. coli XL1-Blue, and inoculated in a test tube containing 5 ml LB, and incubated at 25° C. for 2 days. As a result, it was determined that 19 U/ml of lipase was secreted extracellularly. On the contrary, original strain P. fluorescens secretes only 1.7 U/ml of lipase when it is incubated for 7 days under the same condition.
Secretion of Protease by ABC Transporter Gene
Though protease gene located upstream of ABC transporter gene does not constitute an operon with ABC transporter genre, protease secreted extracellularly by the ABC transporter gene was examined as follows: To prepare a recombinant vector containing protease gene located upstream of ABC transporter gene, polymerase chain reaction was carried out by employing a forward primer of 5′-AAGAATTCACTTCTGAAAGTTGCTGGCGT-3′ (see;. SEQ ID NO:8) and a reverse primer of 5′- TTAAGCTTGTGGTTTATCTTCCTTGAACC-3′ (see: SEQ ID NO:9), respectively, where the former possesses EcoRI recognition site and the latter possesses HindIII recognition site. Amplified PCR product contained protease and protease inhibitor genes. Amplified gene was digested with EcoRI/HindIII and inserted into EcoRI-HindIII-digested pUC18 to give a recombinant vector 'pPROT705' (see: FIG. 1). The recombinant vector 'pPROT705' and pABC-ACYC containing ABC transporter gene were introduced into E. coli to construct transformants, and the transformants were incubated at 25° C. to secrete protease to extracellular medium. On the other hand, protease was not detected at all in culture media when only the recombinant vector pPROT705 expressing protease and protease inhibitor was introduced into E. coli and incubated.
Secretion of Lipase by Culturing of Transformants
To confirm whether lipase is secreted extracellularly or not, E. coli transformants harboring ABC transporter-expressing vector pABC-ACYC constructed in Example 3 and lipase-expressing vector pHOPE were cultured. As a control, E. coli carrying pSTV29 free of ABC transporter gene and lipase-expressing vector pHOPE was incubated under the same condition. And then, activity of lipase secreted extracellularly was examined: 2 transformants were inoculated on LB media, incubated at 25° C. sufficiently, and inoculated again in 100 ml of LB media to reach a concentration of 1% (v/v). When OD600 value reached 0.3-0.6, IPTG was added to induce lipase expression and incubated again at 25° C. for about 2 days. Cells were harvested by centrifuging the culture media, and lipase existed in supernatant was assayed by pH-STAT method that titrates fatty acids hydrolyzed from lipid(see: FIGS. 2a and 2 b) . In FIGS. 2a and 2 b,(o) represents E. coli carrying ABC transporter-expressing vector pABC-ACYC. constructed in Example 3 and lipase-expressing vector pHOPE; (), E. coli carrying Pstv29 and lipase-expressing vector pHOPE. Changes in OD600 value depending on culture time of these transformants and activity of lipase in media were shown in FIGS. 2a and 2 b, respectively. Consequently, it was found that a transformant() carrying pSTV29 and pHOPE could not secrete lipase extracellularly, while a transformant(o) carrying pABC-ACYC and pHOPE secreted lipase to the extracellular medium.
Homologous Expression of TliDEFA in P. fluorescens
To increase the extracellular production of lipase, the lipase and ABC transporter gene were introduced into P. fluorescens. Broad-host-range vector, pDSK519(see: Keen, N. T. et al., Gene, 70(1):191-7(1988)) was used for the construction of pAJH5 containing tliA and pAJH10 containing tliDEFA(see: FIG. 1). The inserts of two plasmids excluded a native promoter upstream of tliD and were under the control of lac promoter of the vectors. P. fluorescens harboring pAJH5 or pAJH10 was grown at different temperatures and harvested at the stationary phase when they reached an OD600 value of around 2.7. P. fluorescens harboring tlia or tliDEFA secreted the lipase only at below 30° C. as was in E. coli harboring tliDEFA. These results demonstrated that the ABC transporter was functional at lower temperature in its original host and definitely accountable for higher production of the lipase at lower temperature in P. fluorescens. P. fluorescens harboring pAJH10 secreted much more lipase than E. coli harboring pAJH10 at 25° C. P. fluorescens harboring pAJH5 also secreted the small amount of lipase at 25° C. by chromosomal ABC transporter gene, although no lipase was secreted by E. coli harboring pAJH5.
Much more lipase was secreted by P. fluorescens complemented by ABC transporter and lipase gene than P. fluorescens complemented only by the lipase gene. P. fluorescons secreting lipase with one copy of tliDEF in the chromosome seemed to secrete more lipase by increasing the copy number of tliDEF. In order to investigate the effect of gene dosage, lipase(tliA) and ABC transporter/lipase(tliDEFA) genes were inserted into another broad-host-range vector, pRK415(see: Keen, N. T. et al., Gene, 70(1):191-7(1988)), resulting in pAJH3 and PAJH11, respectively. P. fluorescens carrying the various plasmids and sets of dual plasmids were grown at 25° C. The secreted lipase was detected by the activity and Western blotting of each culture supernatant. P. fluorescens containing dual vector sets(pAJH3 and pAJH10; pAJH11 and pAJH10) secreted more lipase than P. fluorescens containing only one vector indicating that increased gene of ABC transporter and lipase enabled P. fluorescens to secrete more lipase.
Expression of tliDEFA in Various Pseudomonas sp.
To investigate whether tliA and tliDEF could be expressed in other Pseudomonas, pAJH5 and pAJH10 were introduced by conjugation into various Pseudomonas species; P. aeruginosa, P. fluorescens, P. fragi, P. putida, and P. syringae from Pseudomonas rRNA subgroup I; P. acidovorans(Comamonas acidovorans) from subgroup III. They were all grown in LB at 25° C. for the expression of tliDEF, because tliDEF was functional at below 30° C. P. aeruginosa and E. coli were also grown at 37° C. because they grew too slowly at 25° C. The lipase activity of the culture supernatant of different Pseudomonas species was measured (see: Table 1). When P. fluorescens harboring pAJH10 were grown in LB, they reached around OD600=2.7 ±0.2(DCW-1.9 q/L) and secreted the lipase up to 999±70 U/ml which corresponded to 130 μg/ml of lipase based on the protein concentration of culture supernatant and the percentage(74%) of lipase in the supernatant on SDS-PAGE. Recombinant P. fluorescens harboring tliDEFA secreted the lipase 70 times more than P. fluorescens harboring only tliA(14±4 U/ml), while original P. fluorescens secreted no lipase(<1 U/ml) in LB and 2-3 U/ml of lipase in nutrient broth after prolonged incubation at low temperature.
Other Pseudomonas species carrying no plasmid did not secrete the lipase in LB(<1 U/ml). Recombinant P. syringae and P. putida harboring pAJH5 secreted the lipase, where only tliA was expressed in these strains. It meant that these hosts had inherent ABC transporter for the lipase although there has been no report on the lipase secreted by ABC transporter or ABC transporter itself in these species. Recombinant P. fluorescens, P. fragi, P. syringae, and P. putida secreted much more lipase by the supplemented pAJH10 containing tliDEFA. The ABC transporter was functional in these Pseudomonas species but not functional in P. aeruginosa and P. acidovorans.
It will be apparent to those skilled in the art that certain changes and modifications can be made to this invention without departing from the spirit or scope of the invention as it is set forth herein.
As clearly illustrated and demonstrated above, the present invention provides ABC transporter gene of P. fluorescens encoding a protein which enhances the secretion of foreign proteins extracellularly, a recombinant expression vector containing the transporter gene, a microorganism transformed therewith and a process for preparing foreign proteins which comprises the stops of incubating the transformant, inducing expression of the proteins and leading the proteins to be secreted extracellularly. In accordance with the present invention, foreign proteins can be secreted extracellularly in a simple and efficient manner by employing a secretion system associated with the ABC transporter gene.
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Craftsy Member since September 2013
I'm enrolled in these Craftsy classes:
Finishing School: Edges & Bindings
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2014 Block of the Month: Craftsy Color Theory
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Latest Gillette Stories
FOXBOROUGH, Mass., Sept. 28 /PRNewswire/ -- Foodies and football fans are invited to watch as three New Englanders vie for the winning dish in Ocean Spray's 2nd Annual Tailgating Recipe Contest Oct. 2, at Gillette Stadium.
NEW YORK, Sept. 23 /PRNewswire/ -- Procter & Gamble (P&G) has long been a committed leader in advertising that reaches the Hispanic TV viewing audience and their history of delivering brand messaging has often been adjusted to meet the market's changes.
BOSTON, Sept. 22 /PRNewswire/ -- Gillette (NYSE: PG) and The Kraft Group today announced an extension to their strategic marketing and business partnership that includes exclusive naming and sponsorship rights of Gillette Stadium through the 2031 football season.
CINCINNATI, Aug. 12, 2010 /PRNewswire/ -- The Procter & Gamble Company (NYSE: PG) today announced plans to use renewable, sustainable, sugarcane-derived plastic on selected packaging on its Pantene Pro-V®, COVERGIRL® and Max Factor® brands.
GILLETTE, Wyo., July 14 /PRNewswire-FirstCall/ -- Black Hills Corp. (NYSE: BKH) today announced that its electric utility subsidiary, Black Hills Power, sold a 23 percent ownership interest in the Wygen III power generation facility to the City of Gillette for $62 million.
BOSTON, May 19/PRNewswire-FirstCall/ -- If lifeguarding at the community pool or working indoors at the local mall this summer doesn't appeal to you -- but earning $50,000 and becoming an overnight celebrity road tripping with your best friend does -- then grab your camera and prepare a one-minute video application for the Gillette® Fusion® ProGlide(TM) "Ultimate Summer Job" contest.
NEW YORK, May 4 /PRNewswire/ -- For the second year in a row, Spike TV is teaming up with EA SPORTS(TM), a label of Electronic Arts Inc., (Nasdaq: ERTS) and Gillette to show the world what happens when sports superstars meet their contemporaries in the video game world, as it presents the Gillette®-EA SPORTS(TM) Champions of Gaming on Thursday, May 6 at 11:35 PM, ET/PT.
BOSTON, April 28 /PRNewswire/ -- New Hampshire homebrewer Rob North triumphed over nearly 200 other entrants to win the third annual Samuel Adams Patriot Homebrew contest.
BOSTON, April 23 /PRNewswire-FirstCall/ -- (NYSE: PG) - Gillette® [PG], the world's leading grooming brand, today announced that it will sponsor the NASCAR Sprint Cup Series race at Pocono Raceway on June 6, 2010. The event will be titled the Gillette Fusion ProGlide 500 Presented by Target.
NEW YORK, April 15 /PRNewswire-FirstCall/ -- Gillette [PG], the world's leading grooming brand, and EA SPORTS, a label of Electronic Arts Inc., (Nasdaq: ERTS) crowned four new champions last night at the Gillette - EA SPORTS Champions of Gaming Tournament on Xbox 360® in New York City.
Latest Gillette Reference Libraries
A safety razor, designed to protect the user from serious injury, is a razor where the facial skin is protected from all but the very edge of the blade. Prior to using a safety razor, most men used a straight razor. These razors, although still available today, are not used very often due to the skill and attention required for use. Jean-Jacques Perret invented the safety razor. A rare safety razor design called "Comfort", although not truly a safety razor, was a landmark design. In...
- The governor of a province or people.


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Jim FloraIllustration // Thursday, February 02, 2012
Jim Flora (b. 1914 - d. 1998) was a prolific commercial illustrator, most recognized for his distinctive album cover art for RCA Victor and Columbia Records during the 40's and 50's, as well as his self-authored and illustrated children's books. In addition to his commercial work, he was a fine artist with a diabolical bent who created hundreds of paintings, drawings, etchings, and sketches during his lifetime.
Aaron Crawford's Art and MonstersIllustration // Thursday, February 02, 2012
Aaron Crawford, a full-time artist and illustrator living in Atlanta, GA, creates nightmarish characters inspired by horror movies, depicting stomach churning scenes replete with skulls, demons, fangs, and the terrifyingly deformed. Crawford's brightly colored and dynamic monsters force the viewer into an alternate reality where the grotesque and evil aren't ashamed to flourish.
Olaf HajekIllustration // Thursday, February 02, 2012
Olaf Hajek is an award-winning illustrator currently living and working in Berlin. His fantastical and highly textural works combine elements of South American folklore, mythology, religion, history, and geography to create images that strike a balance between sober reality and mystical hallucination.
The Work of Pavel SabelnikovIllustration // Thursday, February 02, 2012
Pavel Sabelnikov or PAL is an ordinary observer, whose interests cover painting, vector and street graphics, illustration, wall decoration, sketching and aggressive inline skating. Its big in Latvia.
The Work of Egene KooJuxtapoz // Thursday, February 02, 2012
Egene Koo is an illustrator based in Korea. Her beautifully detailed oil paintings are a unique fusion of nature, fashion, and portraiture, depicting richly colored wildlife in the same space as over-sized high heels and well-dressed models with wide-eyed cat and rabbit faces, an unusual combination of elements that is both dreamy and unsettling.
Chapter XXXIII: The Kervin Brisseaux MethodIllustration // Wednesday, February 01, 2012
Automatons, Betaveltronics, The System is Incomplete. Kervin Brisseaux's architectural illustrations are like hyper-galactic explosions of animatronic dreams-capes from the not so distant future of illustration and technology.
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What do you get when you cross Kreayshawn with the wrath of 10,000 tiny eyeballs, lightning bolts and the feature cover of your favorite magazine?? Hattie Stewart is an illustrator out of London with a series of your favorite international celebrities getting terrorized by her playful shapes and charachters. Hattie draws.
Adolfo CorreaIllustration // Wednesday, February 01, 2012
The work of Adolfo Correa is the result of his inner search to experiment styles, shapes, colours and techniques in order to learn and, as a result, develop his own potent and functional style for the world of graphics, advertising and the visual expression of contemporary design.
The Work of Alice X. ZhangIllustration // Wednesday, February 01, 2012
Alice X. Zhang is a New York-based designer and illustrator. She graduated from the Rhode Island School of Design with a major in Graphic Design, but heartily wishes she had chosen illustration instead. Always drawing, learning, and experimenting, her wildly colorful and dynamic digital paintings take inspiration from literature, cinema, and television.
Rob BridgesIllustration // Tuesday, January 31, 2012
Rob Bridges is an illustrator based in Lexington, Kentucky. He earned his BFA from the Cornish College of the Arts in Seattle, WA, and has worked as a senior creative in a design firm. Since 2005 his interest has grown to include book illustration and he has been highly influenced by artists such as Arthur Rackham and Maurice Sendak.


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Pretty fun game
Gravity Rush caught me by surprise. It's currently free on PSN for Plus members and the title is not exactly inspiring. I downloaded it for the sake of it without really intending to play it. The first attempt at playing the game didn't exactly grab me, but being slightly inebriated at the time, I resolved to try again under more sober circumstances.
The second time around was much more interesting. The story is told through a mixture of a slightly interactive comic and cut-scenes and although it starts off slowly it eventually builds up momentum. In fact, the pacing of the story is quite good and through the 21 chapters of the game, you never feel as though the story is treading water.
The main character, Kat is like-able enough, her dialogue and internal monologue portray her as a strong, if somewhat naive, heroine. Her mysterious appearance on this floating world is never really explained in too much detail as the game's story tends to focus more on the events that are happening at the moment. Her ability to manipulate gravity is the game's main mechanic and while it can seem almost like flying, the difference is that you are always being pulled in a definite direction rather than flying through the air at will.
While I enjoyed messing around with her gravity abilities, I never really got into the combat part of the game. I just tried to get it over with as soon as possible so I could be free to fling Kat through the city and collect gems or talk to the city people.
The story is quite light-hearted for the most part and it does have a distinctive Japanese flavour to the writing which is funny without being too goofy.


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Gary Goldhammer is an award-winning, thought provoking and business-driving digital communications strategist with more than 21 years of experience in online marketing, advertising, public relations, public affairs and journalism. He has built and led regional/global teams, creating innovative best-in-class programs for clients including Adobe, eBay, HP, Nike and The Walt Disney Company. He currently serves as Executive Vice President, West Coast Client Service, for Velocidi, a digital agency with offices in New York and San Francisco.
November 05, 2012
Giving clients a social media strategy used to make agencies and clients look smart, now it just makes us look pathetic. Social media is nothing more than a buzzword, a security blanket, an emperor with no clothes.
July 02, 2012
A LONG, LONG TIME AGO... I can still remember how the L.A. Times used to make me cry. Hardly a week would go by it seemed without them scooping me on some local story. I scooped them once in a while, too, but our battles were more like the Celtics and Lakers in the '60s and '70s – always in the...
February 03, 2012
Google's mission is simple, bold, and in the annals of silicon culture, tantamount to sacred gospel: "Organize the world's information and make it universally accessible and useful." Today, however, there is a New Testament being written: "Organize Google's information about the world and make it selectively accessible and mostly useful."
April 04, 2011
I'm uncomfortable because every time – I mean every damn time – the questions devolve into a turf battle over whom should "own" social media (as if any one discipline can or should "own" a public conversation.) If it's the Public Relations Society of America (PRSA), it's PR people beating the drum. If it's a group of marketers, well take a guess what they think.
March 27, 2011
Every few months when I'm back in the Midwest, I make it a priority to visit a Wal-Mart. I go up to people and ask if they are on Twitter or Facebook. I ask how they get their news and if they've ever "liked" a brand or checked-in on Foursquare. The answers are eye opening and refreshing.


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University of Miami Long-sleeve Tee & Boxer Gift Set
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Christina Hendricks is belle of the ball at 'Mad Men' celebration
Christina Hendricks turned heads at the "Mad Men" premiere party Wednesday donning a stunning ball gown.
Christina Hendricks turned heads at the "Mad Men" premiere party Wednesday donning a stunning ball gown.
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"Cinderella" star Lily James' red carpet styles are as glamorous as the outfits that her characters wear in the Disney film and in "Downton Abbey," where she plays Lady Rose.
Queen Maxima looked equally as beautiful in her royal blue custom-made gown that she wore in 2013 while attending a state banquet.
Model turned actress Suki Waterhouse reveals in a new interview that she names her pimples and takes seven-hour-long baths.
Always the fashion icon, Kate Middleton has amazing pregnancy style sticking to several of her favorite designers to dress up her bump.
Actress Olivia Wilde has found a way to combine her two passions, sustainability and fashion.
Shailene Woodley along with Suki Waterhouse, Ashley Judd and Zoe Kravitz opted for sexy outfits at the New York premiere of "Insurgent."
Pregnant Kate Middleton and Princess Victoria are just some of the many members of the wide range of monarchies who stepped out this week creating the latest royal fashionable moments.
Victoria Beckham, Ricky Martin and more support Elton John in his boycott of Dolce & Gabanna after saying children born out of IVF were "synthetic."
All the news as Duchess Kate's due date draws close...
With just weeks left until Kate Middleton gives birth, the new favorite name for the second royal baby is Alice.
New mom Doutzen Kroes shows off her enviable bikini body eight months after giving birth while on vacation with her husband in Miami.
While carrying out royal engagements on Monday, Kate Middleton shared an update on her second baby.
Those lucky enough to attend aren't just watching the models walk down the runway. They are also able to see who's who in the coveted front row.


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Barefoot by the Sea
Tessa Galloway, the gardener at Barefoot Bay's new resort, begins an complicated romance with John Brown, who is in the witness protection program and needs a job and a wife in order to be reunited with his twin toddlers.
Publisher: New York, NY : Forever, 2013
Edition: 1st ed
Characteristics: xii, 434 p. ;,18 cm
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Are these hydrangeas?
aligailJune 13, 2010
I have a large blue hydrangea in my front yard. There are two smaller plants that sort of look like hydrangeas nearby - and I am wondering whether they could be baby hydrangeas from the big plant? If so, I think I need to move them somewhere else because I don't think they are getting enough sun.


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It always sucks when your crush doesn't like you back, but
you shouldn't waste anytime dwelling on that fact. The most important thing is
to distract your mind and make yourself so busy that you literally don't have
time to think about him. Here's how to move on without looking back.
Spend more time with your friends and
family. Closing yourself off only makes it worse. Get out there and laugh
with the ones who know you best.
Get back into an old hobby. No, we
don't mean stalking him on Facebook. Step away from social media for a sec and
do activities that put a smile on your face.
Take your frustrations out through
exercising. Pick yourself up and hit the gym or run around the block. A
daily dose of endorphin-raising activities will kick your brokenhearted blues
Look for someone new to crush on who is
nicer and cuter. He's out there somewhere. Even if you don't find him right
away, keeping your eyes open helps you lose focus of the old flame.
Focus on your schoolwork. Crushes only
last so long. That gleaming sense of accomplishment from boosting your GPA
outlasts any heartache.
Learn to cook or bake something new. We know you've got oodles of recipes on your pinboards,
and we've got tons here and here.
Try 'em out, sweetness!
Clear out the mess under your bed and in your
closet. A clean start should start with a clean room. Any cutesy momentos
that remind you of him? Toss 'em fast and relish in an uncluttered mind.
Start reading a new book. Preferably
one that doesn't draw parallels between the dreamy hero (ahem, Twilight) and your former crush. Pick
one that puts your mind as far away from him as possible.
Crank up the tunes and dance. Because
life's too short to be a wallflower at your own party – even if it's just a
personal dance party in your room.
» 14 hotties to drool over
» Spring date ideas he'll love
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Brinkmann Outdoors Steel Vertical Chicken Roaster
of beer or another liquid to flavor the meat and keep it moist Package Contents: Brinkmann Vertical Steel Chicken Roaster 812-9009-S ... Roasting a tender and juicy chicken is simple with the Brinkmann Vertical Steel Chicken Roaster ... 812-9009-S. This Roaster from Brinkmann has a large base that accommodates an opened can of beer ... Roasting Rack is dishwasher safe for quick and easy cleanup. Brinkmann Outdoors 812-9009-S: Steel ... Vertical Chicken Roaster, Silver Specifications for Chrome Plated Steel Chicken Roaster - ... Vertical: Assembled Depth (in.): 6.97 in Assembled Height (in.): 4.02 in Assembled Width (in.): 6.97 ... No Features of Brinkmann Vertical Steel Barbecue Chicken Roaster: For holding a chicken upright...
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Mona V.
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Feb. 12, 1929
Jun. 9, 2007
Mona was born in Lamesa, Texas to John and Edna Rogers, and passed away in Clovis, California.
Mona's father was a cotton and alfalfa rancher in Lamesa till 1945, when at that time he retired and moved his family to Kerman when Mona was 14 years old.
She attended Kerman High School until she left to marry her sweetheart from Texas, Dan Marley. They had met when he was home on leave and she was working at a candy store in Lamesa. They had grown up in the same town but did not meet until then.
After she moved to Kerman, Dan was shipping out of San Francisco and came to see her. They were married when he got out of the service in 1947.
They had two sons Randy in 1948, and Mike four years later. They both attended Central High School.
Dan opened Dan's Dairy Supply and then bought the store building. He sold big drums of soap and dairy supplies to local dairymen.
Mona started Buffy's Junque Shop in the same store building. They phased out the dairy supply in 1971 - 72 after a robbery when their son Randy was killed and Dan was shot in the shoulder.
Dan and Mona were also in real estate since 1959, until they retired.
Mona was preceded in death by her son Randy Marley. She was the youngest of eight children, four girls and four boys who have also preceded her in death.
She is survived by her husband, Daniel B. Marley of Clovis, California; her son, Michael Brent Marley; and two grandchildren, Brent Marley and Megan Soto; and one great grandchild, Mason Albitre.
Visitation will be held at Boice Funeral Home on Wednesday, June 13, 2007, from 12 noon to 7:00 p.m.
A Funeral Service will be held at Boice Funeral Home, Clovis, California, on Thursday, June 14, 2007, at 10:00 a.m.
Remembrances may be made to Woodward Park Church of Christ, Building Fund, 7886 N. Millbrook, Fresno, Ca 93720.
Published in the Fresno Bee on 6/12/2007.
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I was wondering if I could send some webservice calls while my application is in the background. How does skype do it? Even if I press the home button my call stays connected.
Building on what rckoenes stated, applications are allowed to register background tasks to be completed after the user hits the home button. There is a time limit of 10 or 15 minutes for these tasks to complete. Again, you can register a task to complete immediately after the user hits home, this does NOT allow you to execute code say an hour after they exit the app.
UPDATE: if your app supports versions of iOS previous to iOs 4, you should also check to ensure that multitasking is supported before registering a background task. Use something along the lines of:
Try This... Excellent code for running app in background with no time limit. (I tested it for downloading more than 600 mb data from web-service.)
Update ::
you can found more information regarding multitaksing in this apple doc Background Execution and Multitasking.
Please test on device.


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Huey D. Johnson: Green Plans: Blueprint for a Sustainable Earth
- Devparna Roy
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A quarter of a century ago, the United Nations' World Commission on Environment and Development delivered a frank message about the dangerous state of the global environment and called on all citizens of the world to unite and make an effort to avert environmental disaster. In 1987, the Commission published the famous Brundtland Report, which contained the concept of "sustainable development" and defined "sustainability" as "development that meets the needs of the present without compromising the ability of future generations to meet their own needs." However, despite occasionally succeeding as a rallying cry and even as a negotiating tool, the term "sustainable development" is vague and has become a buzzword. This term is often put forth as a solution to major problems facing humankind, but rarely is it accompanied by any explanation or definition of how the goal of "sustainable development" is to be met. The book under review offers a practical guide of how certain countries are usin ...
- Title
- Huey D. Johnson: Green Plans: Blueprint for a Sustainable Earth
- Journal
Journal of Agricultural and Environmental Ethics
Volume 26, Issue 2 , pp 513-516
- Cover Date
- 2013-04-01
- 10.1007/s10806-012-9388-9
- Print ISSN
- 1187-7863
- Online ISSN
- Publisher
- Springer Netherlands
- Additional Links
- Topics
- Authors
- Devparna Roy (1)
- Author Affiliations
- 1. Polson Institute for Global Development, Department of Development Sociology, Cornell University, Ithaca, NY, 14853, USA


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Hi, i'm new here although i've been a big fan of Lush for years and the many talented boys and girls who contribute. I'm fun, open and honest and love a good time with friends old and new.
The Shard building does remind me of a giant cock though x
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Secure Data Processing in a Hybrid Cloud
Free registration required
Executive Summary
Cloud computing has made it possible for a user to be able to select a computing service precisely when needed. However, certain factors such as security of data and regulatory issues will impact a user's choice of using such a service. A solution to these problems is the use of a hybrid cloud that combines a user's local computing capabilities (for mission- or organization-critical tasks) with a public cloud (for less influential tasks). The emergence of cloud computing has created a paradigm shift by allowing parallel processing of massive amounts of data. Cloud computing has further segmented traditionally provided software services into SaaS, PaaS and IaaS. This segmentation allows users to choose the appropriate kind of computing service precisely when needed.
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Weather Alert
Severe Thunderstorm Watch issued March 31 at 4:18PM CDT expiring March 31 at 10:00PM CDT in effect for: Choctaw, Haskell, Latimer, LeFlore, McIntosh, Muskogee, Okfuskee, Pittsburg, Pushmataha, Sequoyah
NEW YORK (AP) -- Katie Couric's next move appears to be online.
An ABC News executive who requested anonymity because talks aren't complete said Friday that the veteran news anchor has an opportunity at Yahoo! that is too good to pass up.
Couric has been working at ABC News, but published reports have said that Yahoo! is seeking her to host an interview show to be aired on the company's website.
A spokesman for Couric declined comment on Friday.
Couric is in her second season on an ABC daytime talk show, which is not expected to be renewed.


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Top three trends in the automobile industry
Kippreport gets insights from Mike Belk, CEO and president of Daimler Middle East and LevantMarch 26, 2015 12:02
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Abu Dhabi Airports CEO leaving to take up U.K. defence ministry role
Kuwait February inflation edges up to 2.9 pct
MIDEAST STOCKS-Gulf markets sink after Yemen intervention; Dubai worst hit
HSBC's Islamic banking unit plans sukuk sale in Malaysia
Oil rises more than 1 pct, boosted by weaker dollar
UAE's Shah gas project seen achieving full capacity in Q2
National Bank of Fujairah issues 500 mln dirham tier 1 capital
Dubai builder Arabtec wins $283 mln contract from Saudi Aramco
Gold holds 4-day rally on softer dollar, U.S. rate hopes
Oil rises more than 1 pct, boosted by weaker dollar
BP Plc, shut out of pre-qualifcation bidding to develop Abu Dhabi's largest onshore oil fields, hopes to hear soon whether it can participate in the process, Chief Executive Bob Dudley said on Sunday.
November 12, 2012 6:31 by Reuters
"I'm positive about it … I'm optimistic that cooperation will go on for a long time," Dudley said on the sidelines of an oil and gas event in the UAE capital.
Dudley said he hopes to hear about the concessions up for renewal "in the next week or so," adding that British Prime Minister David Cameron had played a constructive role in promoting British business in the Gulf Arab state.
Cameron visited the UAE last week in a trade and diplomacy tour to the Gulf.
Thousands of Kuwaitis protest against voting changes
November 12 2012
Egypt getting ready for IMF deal this week
November 12 2012


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You know those insecure feelings you get when you throw a party… that anxiety that no-one will turn up… You've got more than enough supplies for everyone you've invited as well as for some uninvited hangers-on. The food is arranged … Continue reading
Posted in audience, conference, conference presentation Tagged audience, conference paper, Pat Thomson 3 Comments
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I like lippiness. When I was a teacher I liked the lippy kids the most. They had a bit of spark and energy. They were often smart and funny. And of course I've been more than a bit lippy myself … Continue reading
conference blog- let's flip the format!
It's the end of the standard conference day. You've seen every session. Keynote. Panel. Three papers. Keynote. Three papers. Panel. Each session Chair has tried to allow time for discussion and most of them have succeeded – sort of. A … Continue reading
conference blog – questions of etiquette
There are conventions about conference behaviour just as there are in every other area of academic life. Here's three conference basics. 1. If I'm late for a session, should I go in or wait outside? It's just about acceptable to … Continue reading
conference blog: beware the conference app
This is a guest post written by Dr Julie Rowlands from Deakin University, Australia. Greetings from the ECER conference at Porto. As a recently appointed academic after a mid-life career change, this is my first international conference. It is very … Continue reading
why doctoral researchers should get support for conferences
I've recently been told by a number of doctoral researchers that their institutions are pretty mean about funding them to go to conferences to give papers. I'm pretty scandalized by this as it seems to me that it ought to … Continue reading
rules for conference presentations
Having just returned from a conference where the presentations were a little mixed – to say the least – I was reminded of the reality that conference presentations are not the same as the conference paper. The paper is the … Continue reading
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Frederick Sieber
member for 6 years, 3 months, 23 days
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Picture is I think gravestone of my 3ggfather Marcus Gillett in Wellwood Cemetery, Mexico, New York.
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E. Charles Ash
RE: William Kenyon Alden Cemetery
Unfortunately, I don't have any additional information about William F. Kenyon. An obituary of Mr. Kenyon would be the most likely source of information about his ancestry - and it is possible that the Freeborn County Genealogy Society or the Freeborn County Museum's library research service might be able to assist you.
Added by
E. Charles Ash
on Feb 02, 2015 8:33 AM
Phyllis Meyer
RE: Lila Evans
Added by
Phyllis Meyer
on Dec 04, 2014 11:37 AM
Phyllis Meyer
RE: Lila Evans Clark
I got the death date from a small death notice that was in a Buffalo newspaper
Added by
Phyllis Meyer
on Nov 19, 2014 7:59 AM
Sumner Ely
I just moved, and my personal computer is still in storage. But I've add this info to my "Findagrave To DO" list. I will check to see if I have other images from other sides of the monument for Hannah.. I might have overlooked Sumner. I'll let you know.
Added by
on Nov 19, 2014 5:25 AM
Frank H Gillett
I added the info and obit.
Yes a gravestone would be able to tell us Gillett or Gillette, but unfortunately as I mentioned in his memorial there is no stone for him or his wife. I photographed the entire Fly Creek Valley Cemetery, after creating all the memorials with the photos I went and compared it with the Fly Creek burial list, some have lot numbers some don't, if you walk trough the cemetery you will see quite a few empty lots or with just a stone in it, more so than in other cemeteries in our area.
And yes I am way behind with my photos, working on Edmeston Union at the moment.
Greetings from Richfield Ingrid
Added by
on Nov 02, 2014 4:37 PM
Suzanne Andrews
Bachmann request
I found Fred Bachman's gravesite. He is buried with his in-laws, Ludwig & Rosalia Milbrandt as well as with Augustina Milbrandt Bachman Jones and 3 children. Augustina apparently remarried to Thomas L. Jones and he is also buried in the plot.
The spelling of the names varies. I found some articles on genealogybank.com about how Fred Bachman died. If you would like me to send them to you, I will be happy to do so.
I have made memorials for the above people and children. If you would like these transferred to you please let me know.
Note: The child born in 1901 does not have a marker, however, he/she is listed in the cemetery records as being buried with the other two children and Fred Bachman.
One other note: When I looked for Augustina, I found her father listed as Wilbur and also as Joe. According to the death notice for Ludwig Milbrandt, he died in his son-in-law's (Fred Bachman) home as did Rosalia Milbrandt. I made the assumption that these are Augustina's parents. If I am wrong, please let me know. I did not link them to her memorial just in case, but would like to do so if this is correct. (The articles on Fred's death indicate Auagustina's mother was living with them at the time of his death.
Let me know if you have any questions.
Added by
Suzanne Andrews
on Oct 17, 2014 9:38 PM
Patricia Foster
Orley Welcome Bullock
Thank you for transferring the memorial.
Added by
Patricia Foster
on Jul 29, 2014 11:28 AM
Patricia Foster
Orley Welcome Bullock
Morning Mr. Sieber,
I wonder if you would be willing to transfer the memorial for Orley Welcome Bullock to me. He is a distant relative, my great grandmother's sister's son.
My # is 47276586
Thanks so much.
Patricia Foster
Added by
Patricia Foster
on Jul 29, 2014 4:11 AM
Patricia Foster
Orley Welcome Bullock
Morning Mr. Sieber,
I wonder if you would be willing to transfer the memorial for Orley Welcome Bullock to me. He is a distant relative, my great grandmother's sister's son.
My # is 47276586
Thanks so much.
Patricia Foster
Added by
Patricia Foster
on Jul 29, 2014 4:11 AM
Ron Kinney
RE: Eliza and Frederick Gillett, Cherry Creek Central
You're welcome, glad to help out.
Added by
Ron Kinney
on Jun 27, 2014 4:35 PM
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Acquia is seeking an Accounting Intern for the upcoming Summer Semester (up to 40 hours per week). In this role, you will report to the Accounting Manager and work closely with both general accounting & billings/revenue team. The ideal candidate will be disciplined and2/17/2015 Burlington, MA Save This Job
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Click On Book For Reading
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These two book are the complete guide of Qurbani. The order of qurbani in the Quran and Ahadith . When do the qurbani .The types of animals use in qurbani.
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The age of Qurbani animal.How we buy the Qurbani Animal. Distribution of meat of Qurbani in Islamic point of view.Qurbani (or Udhiyya as the Shariah refers to it) has become synonymous with Eid-ul-Adha, the festival of sacrifice. It is a time when all Muslims all over the world commemorate the ultimate sacrifice of Nabi Ebrahim (Alehissalam) and Nabi Ismail (Alehissalam). However it is disheartening to learn that Muslims fail to respect the basic rights of animals and are ignorant of Islamic injunctions pertaining to sacrifice. Many a times Muslims through their actions unconsciously transgress the Shari'ah. This guide hopes to be a reminder to Muslims of these basic rules and the responsibilities of those performing Qurbani. Almighty Allah says in the Holy Quran (Chapter 22 Verse37): 'It is nor their flesh, nor their blood that reaches Allah, but to Him is acceptable observance (piety) of duty on your part.The wisdom of the Islamic rules of slaughtering is to take the animal's life in the quickest and the least painful way; the requirements of using a sharp knife and of cutting the throat relate to this end. The main intent is to be kind to the animal and spare it unnecessary suffering insofar as this is possible.The Prophet (Salay ala Muhammad)saw a man who was sharpening his knife after laying down a sheep to be slaughtered, the Prophet (Salay ala Muhammad) rebuked him saying: 'Do you intend to make it die two deaths? Why did you not sharpen your knife before laying it down?Qurbani can be made from the 10th of Zil Haj after the Eid Khutba till sunset on the 12th of Zil Haj (3 days). It is preferable to perform Qurbani on the 10th day of Zil Haj. Animals bought with the intention of Qurbani but were not slaughtered by the 12th of Zil Haj should be donated to charity while alive. Qurbani is Waajib on every Muslim, male or female that has reached puberty, is sound of mind and has more than the NISAAB (+/- R3000,00) at that time. Qurbani is a Waajib obligation every year on the occasion of Eid-ul-Adha and not a once in a lifetime duty. Qurbani can be undertaken on behalf of another person provided that prior consent is received from the person. It is permissible to make Qurbani in the name of Rasoolallah (Salay ala Muhammad) or any other deceased Muslim as naffil Qurbani on condition that the person making the Qurbani has discharged his own (Waajib) Qurbani. The animal should be chosen from healthy stock without any visible defect.
Sheep and goats must be at least one year old and count as one share.
Cattle must be at least two years old and count as 7 shares per animal.
Camels must be at least five years old and count as 7 shares per animal.
Animals should not be blinded, emaciated (sick), toothless or with broken limbs, preferably the ears and tail should be whole, but not a necessity.
Sharpen your knife prior to putting the animal down for slaughter.
Ensure that the correct size knife is used to slaughter the animal swiftly.
Feed the animal, give the animal water and sooth the animal, show kindness and mercy to the animal.Ensure the area of slaughter is clean and clear of live animals.
Make provision for Blood drainage, fly control and general cleanliness.
Lead the animal to its slaughter humanely. Once Umar (RA)saw a man dragging a sheep by its leg to be slaughtered, he said: 'Woe to you! Lead it to its death in a decent manner.' Do not abuse or torture the animals.
Do not perform the slaughter process in front of other live animals.
Maintain strict hygiene standards.According to the Shari'ah, the legal purification of the flesh of animals requires that the following conditions be met.Make your intention.
It is preferable to make the throat of the animal face the Qibla; (Mustahab)
Using a sharp knife swiftly cut the windpipe, the gullet and the two jugular veins, do not sever the entire head.Recite the name of Allah (S. W. T.) Tasmiyah, while slaughtering the animal (Preferably aloud).It is preferable for one to perform the Qurbani if it belongs to him provided he/she is able to slaughter properly otherwise he/she should entrust it to someone to slaughter on his behalf. (Women are also permitted to perform the slaughter).
Skinning of the animal should only commence once the animal stops kicking.
Keep meat and offal separate, clean and refrigerate as soon as possible.It is preferred that the meat be divided into three parts. One part for own use, one part for relatives and one part for the poor and needy (preferably Muslims).The meat and skin cannot be given as payment for services rendered BUT may be given as a gift.The skin may be kept for personal use, or given to someone else or donated to an organisation.Give such meat as you would yourself like to receive and eat.Ensure that meat is given to reputable organisations for distribution to the needy.Do not waste. Answers of the different Question about qurbani. The Best way of qurbani. This Book is very important for All Muslim.You read this book and share with friends on facebook and twitter.
A book about the waist of power of men or women . This is very bad habit in some unmarried men and women that they waist their power with different unnatural ways which has many side effects for their health and life of after merriage. The waist of power by unnatural ways is a big sin in Islam so every muslim must avoid this bad habit. This thing is directly effects their sexual parts of body. This book cover the all these topics. the problems and their treatment. You try to read this book and share with friends on Facebook and twitter. you can download this complete book also.
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A book about the topic lucky husband and lucky wife. The main topic in this book are the rites of husband and the rites of wife. About the lovely life of husband and wife.Husband and wife of the two pillar of life building if even one of that pillar is unstable the whole life is unstable. The interest of Films and Dramas is very bad for Both husband and wife they implement that rules which they see in that Films and dramas and unstable their life.The story of injured snake, Good news in dream. The hate is change in Love.The punishment of the music in the day of judgment.You must read this book completely and share with friends on Facebook and twitter and download this book in computer free.
Click On Book For ReadingDownload This Book
A beautiful Book about the power of a man or women in young part of life.This book contain many topics like good and different ways of sex with wife,rites of wife and rites of husband, what is Nikkah, what are the benefits of Nikkah , and how much the side effects of relation of girl and boy without Nikkah, So this Book is the treasure of knowledge. Read complete this Book and share with friends on Facebook and twitter .
Click On Book For ReadingDownload this Book
Click On Book For ReadingIt may be helpful to understand a little about divorce and the typical effects it has on men, women and children. The divorce rate in the United States is the highest in the world. Fifty percent of marriages end in divorce. Sixty-seven percent of all second marriages end in divorce. As high as these figures are, what is also true is that the divorce rate appears to be dropping. The reasons for this change are not clear. Many people cannot afford to divorce, many people cannot afford to marry. Another reason is that "baby boomers," who account for a large proportion of our population are no longer in their 20s and 30s, the ages when divorce is most prevalent. The societal expectation is that divorced life is less satisfying than married life. Divorce is associated with an increase in depression--people experience loss of partner, hopes and dreams, and lifestyle. The financial reality of divorce is often hard to comprehend: the same resources must now support almost twice the expenses.Fifty percent of all children are children of divorce. Twenty-eight percent of all children are born of never married parents. Divorce is expensive. Aid for Dependent Children (AFDC) resources are drained by the needs of divorced and single parent families; including the cost of collecting child support.
So must read this whole book and share with friends on Facebook and twitter. You can also download this Book free of any cast and very easily.
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These Books are complete guide of wozu. Wozu is necessary for prayers.These books cover many topics like the best and sunnat way of wozu, water wastage is not allowed in wozu. Their are many benefits for wozu in 24 hours like stable the eye sight. The second Book is research of scientists that prove that wozu is best part of our life and good for our health. The benefits of Islamic Tooth paste( miswak).The benefit of washing hands , washing face, washing nose and washing feet. So read these two books completely and share with friends on Facebook and twitter.And download free.
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Two Beautiful book about the method of Namaz e Eid and method of Namaz e Janaza. Namaz e Janaza is very important and pray after the death of Muslim male or female. In This Book complete guide for namaze janaza. Second book about the Eid prayer which pray at Eid day which will come at the end of Ramdhan month and 10th zilhaja.You must read these two book completely and share with friends on Facebook and twitter. and download these book free of cast.
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These Books (Guide line to Make Kafan and bathing of Dead body) contain very Important topics which must to be known by every muslim. These Book are complete guide of Kafan , the size of kafan , length, width, and colors, Your Must read These Two Books and share with friends On Facebook and Twitter.