text:
Purpose: Said Board to determine the suitability of alternate materials & methods of construction & to provide for reasonable interpretations of Building Code. Hear & determine appeals from the ruling of the Building Official concerning any codes adopted by Ordinance 3862.7
Contact: Development Services Department
Regulations for Appeals Process:
Building Appeals Board in Volume 1, Section 113, California Building Code:
Accessibility Appeals Board in Section 19957.5, Health and Safety Code:
El Dorado County Government Code Section 15.12 for building appeals:
Meetings: Meetings as necessary but not less than once a year. Agenda: 10/16/2013
Agenda: 09/18/2013
Minutes: 09/18/2013
Agenda: 12/12/2012
Agenda: 3/27/2008
© County of El Dorado


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Indiana County's Foremost Leaders in Business
The Indiana County Business Hall of Fame recognizes past and present business leaders for their lifetime achievements in promoting and advancing the economic climate of Indiana County.
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Radio Great Casey Kasem To Be Buried IN NORWAY? Oslo Officials "Not Sure"!
Reports are that officials in Norway are consulting with lawyers
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HERE'S MORE On The "Concerns" Of Norway Officials...
Here's A Video On Jean Kasem's "Plan" For Casey...
Here's A Video Of Casey's Life And Career...


text:
Will Sandra Bullock be the first actress to win an Oscar as best actress and a Razzie as worst actress, in the same year?
Bullock won her Razzie for "All About Steve," a comedy with Bradley Cooper, of "The Hangover." And the pair were also named worst screen couple. Not only that she showed up in person and accepted the award.
See her acceptance speech below. She brought a DVD copy of the film for everyone in the audience.
But she has also been nominated as best actress in the best picture nominee "The Blind Side," one of her rare dramatic roles. A CNN news anchor just called her the Oscar front runner.
"Transformers: Revenge of the Fallen," meanwhile, which received an Oscar nomination for sound editing, also received a Razzie award as worst film of the year.
Take a look at the complete list of Razzies here, and join me here later as I live blog the Oscars, from an undisclosed location.


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Located in the interior mountains of beautiful British Columbia, just a few hours northeast of Vancouver
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PlayStation Vita Still Has Minor Sony SupportThe Vita still has some life in it, but it might not be around for much longer.
'Call of Duty: Black Ops 3' Dev Details Mapping ToolsMapping and modding tools will come to the PC version of "Call of Duty: Black Ops 3" in 2016.
No 'Sea Of Thieves' Apprearance Till 2016The game that will supposedly be Rare's best, won't be showing in shores till next year.
'Phoenix' Jean Grey Joins 'Marvel: Puzzle Quest'The iconic member of the X-Men joins the match-3 RPG as one of its rare five-star characters.
'Need For Speed' Is Reborn As A Street Racing GameThe reborn Need For Speed may not be perfect, but it's most of what fans have been starving for: It's essentially Need For Speed: Underground 3.
Game On For Activision"Candy Crush" joins "World of Warcraft," "Call of Duty" and "Skylanders" in Activision Blizzard's portfolio.
'Shadow Of The Beast' Gets New TrailerThe PS4 exclusive still has no release date, but gets a bloody new trailer.
'WWE 2K16' Possibly Coming To PCOne of the best wrestling games in a long time might be coming to PC, though 2K Games have yet to confirm.
'Super Mario Maker' Dev Talks Amiibo Inspirations, Cursors As Game Stays Strong At Top 40The developer for "Super Mario Maker" shares the creation process involving Amiibo and inspirations for cursors used in the game.
'Real Boxing 2' Is Now 'Creed' Tie-In GamePlayers will step into the shoes of Adonis Johnson, as they train with Rocky and take on other boxers.
'Naruto Shippuden: Ultimate Ninja Storm 4' Has DLC PlansThere will be two "scenario" packs and one new characters pack.
New Xbox One Experience Gets An Update For PreviewMicrosoft has rolled out a new preview for its New Xbox One Experience preview program members.
Check Out These Scary Games For HalloweenIt's recommended that you play these games with the lights off.
Laura Bailey, Video Game Star Who's Heard, Not SeenLaura Bailey, the voice behind Jaina Proudmoore in "World of Warcraft," Chun Li in "Street Fighter" and Spartan Vale in "Halo 5: Guardians" talks about what it's like to be a video game star.
eSports Fans: An Advertiser's DreamCompetitive gaming attracts a demographic that marketers love: young, affluent men. No wonder television deals and other lucrative partnerships are forming.
'Batman: Arkham Knight" PC Port Has 99 ProblemsThe "Batman: Arkham Knight" PC port woes continue.
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PlayStation VR Platform Gets 'Gran Turismo,' 'Tekken 7,' 'Driveclub'The PS4-tailored PlayStation VR is already looking at a healthy lineup of compatible games despite the lack of announced price and release date.
Nintendo Reveals Details Of First Smartphone Game MiitomoNintendo has revealed details of its first smartphone game called Miitomo, which will be free to download but won't be released until March 2016.
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Never heard of Invisible Illness Awareness Week? You're not alone. Jody Smith sheds a little light to make it more visible
Discuss the article on the Forums.
Latest from Prof Hooper and Margaret Williams, DSM V submission
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Page 2 of 2
Page 2 of 2


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Swift Springstrider
105,021pages onAdd New Page
this wiki
this wiki
- Swift Springstrider
- Item Level 20
- Binds when picked up
- Unique
- Mount
- Requires level 20
- Requires Apprentice Riding (75)
- Use: Teaches you how to summon this mount.
Cost Edit
- 500 from Noblegarden Merchant or Noblegarden Vendor.
- Can also be found in Brightly Colored Eggs, but chances are very low.[citation needed]
Patch changes Edit
- Patch 4.3.0 (29-Nov-2011): Added.
External links Edit
|Mount item
|Summoning spell


text: Posted November 23, 2012 I'd just like to say that whole you don't run if you are innocent thing is BS. If your wife gets murdered, you are the prime suspect, cops keep coming to your house and your lawyer tells you they are going to come arrest you, you wouldn't necessarily wait around and hope the justice system works itself out. I'm not saying this in any relation to the OJ case, just in general. Share this post Link to post Share on other sites


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Salem Daily News, The (Newspaper) - November 27, 1890, Salem, Ohio THE SAI.EM DAILY NEWS. II. NO. 281. SALEM. OHIO, THURSDAY, NOVEMBER 27. 1890. TWO CENTS. WILL YET THE EAUT1I. Smith, the Outoati or Harder, Will Probably be svtvod from the Gal- Chronicle of Kecent Events In liuckeyedom. BEEMBLIX INVESTIGATION. Governor Cxinpbell Testifian u'.aoi 1'asscreeX. Tho Indians w-.' surir anl defiant la. manner. One of t.U- n siU he Ual -u tho timo when I to boat our. fpo brains of children and drink woman's blood, anl t'.iit tho f.'in AM-> oo'uirr: when they would do it In tho j last few d'ays t-hillip; h.is hal twoiity cattle killed bv the "Three half-breeds from White river I the boarl. Sidney Dillon, -ilroady a di- i rector, was elected I About twentv reporters i crathored in tho corrido-s ouf-id the Union Pacific olnVe-s in the Equitable building yesterday and sent in a reou-.'st to Mr. Uoalil. Llo au- swcrod of shook hands w'.th al! the in ani hurried bade to the desk lately occupied by President Adams. When asked tbe significance o: the ly as indicating here." APPKUSB'i WADE HAMPTON SHOT. South Ssuiit -r by nis While Out HlllltiilC. I COLUMBIA, S. C., Nov. 27. telegram received here last night states that Son- ator Wade Hampton, while out gunning I on his favorite bunting grounds in Washington County, MisMssiopi, was accidentally shot by bis son, MeDuffle Hampton. The sort iirod towards his father and wound.'! him in the _ f XJilO I ULAC3 OL1UU "3 U. 1IV i. U1' LI 1 "3 GVO 1J.IS a bad state o, j inJHries. bow6VPr. are not believed to be i serious, and it >s not thought likely SUHOS.VL. i that he will lo-.o hn The ac- cident ir, ths sain) looalitv where Senator a few years ago lost one of his stopped at Phi.lips' houss Monday night i ch.lnKes }n the Union Pacific directory, and said they to find the set- I fae 3aid that ho not kno.tV that thpy tloment destroyed when thoy got home. tad any matorial sisal Seance. Ho had Phillips siyi -veryhjly who has boon j boyn tbo Union Pacific in among tho In'ii-.ns length of tim i that there Gould Said to be Keachtos Oat for Control f Caul Kallroadb NEW YOKX, Nov. persons who believe that Mr. Gould's plans for reformation of corporate practices are comprehensive to include tbe Atlantic seaboard, are satisfied that he will direct his attention iu time to the anthracite coal producing railroads. His interest in Delaware, Western is well known and it has been common gossip that he has had a feud with the capitalists who control the Philadelphia Reading railroad, foi reasons not conuecteu with the coal trade. Mr. Gould has denied emphatically that ho is responsible for the reports that have been circulated of his hostility to Postmaster General Wanamakor, who is a prominent member of the syndicate supporting tho Reading property. But tbe action of tho Reading iu relation to coal mining furnishes an opportunity for him to exorcise his persuasive pow- Tho journals report tho coal businobs to be in a state of dullness, with ofi-oal accumulating at tide- water and interior points. Uy some coal Kion tbe ilo.uling is charged with mining operations which will result in a coal oiitiuuk its quota, as in- formal'y t-gri-i'd upon b-jtwoen the dif- ferent companies. Tho production Is said to bo in of the demand, and f c Reading is chargsd with pressing its worn with au blfeec unfavorable to actual Overwhelming Sentiment la Fa- vor of His Retirement From tlie of the Irish Na- tionalists iu Parliament. and hid returned to his without exoop'io.i, says that there is going to be an uprising, and that very quickly. Tha at Sliort Bull's headquarters at 1'asi croek has running for a montii .ml Phillips thinks it is a point of for all tin bands. Ho aro a thousand armod now. The Indians say they will not up Sbort Tlnll an'1 xv'tll of bribery impressed me vary s-riou.s- one Q{ th_ ghot A Government 1 His Arrrsted for St-lllntr Liquor. CisciXN'Aii, Nov. startling developments have come to iiorht in con- nection with tbe arrost of Jo-siah Burd- sal, nephew of Appraiser Bardsul, for illegally selling packages of liquor. It is known beyond a doubt that the ap- praiser himself was directly connected with the illegal transaction, and that the nephew was acting as agent for his uncle in disposing of tho liquor. Young Burdsal made a confession under oath implicating his undo. In addition to this, several business men will testify that Burdsal offered to sell them sample jugs containing whisky. It seems from recent developments the appraiser planned the whole thing and used his nephew as a tooL Burlsal arrested yesterday and is now in tho custody of the United States marshal Trotter Sold fnr S2U.OOO. YOITXGSTOWS. O., Nov. 27. Allie the well-known trotting stal- lion, wwned by Stanhope Bros., of Kins- man, O., has been sold to C. McCann, of LeTcington, Ky., He was sired by Red Wiiices. datn Al lie West, and began his trotting career the past season, securing aTOcord on the track at Cleveland, which was considered four seconds slow, of The Stanhopo Bros, purchased him two years ago for intending to use him for stud purposes only, and not believing then that he would show any great trotting qualities. ____________ Groivenor May Hot a ATHENS. U.. >7ov. Gros- venor, being asked if he had knowledge of the probability of bis anpoiatment as a member of tbe Inter-State Commerce Commission, replied that, unsolicited by himself, Speaker Reed, Major McKinley and 165 numbers of Congress, to- gether with Senator Sherman, had joined in asking the President for his appointment to such position upon tbe occurrence of vacancy, and that his friends had reason for that the President -would comply snob request. ________________ 27. i-'t-of. LI. Will Hare to Dttforff CLETKLAXD, Jfov. 27. Solders' jury on Wednesday rendered verdict for tbe plaintiff? in the suit of Doming A Co. against James Robertsoa for tbe fall amount sued for. namely. The pULctifis are floor merchants of Oberlin. and bad in their employ as Cleveland a young man named Stinjpson. The latter gambled away the firm's money the amount above stated in tbe gambling rooms, and sjit was brouzbt to enforce restii-jiioa of njoney lost. Corn Tairf nith Shot. C. U- NOV. Taylor, lifd of bavinjr bis com han't on ai aizbt with a 3 rr.-- a ns :a a wacon. tad whiie one '-a.- ia of Sliing a --ora. vnc hire-.i man Sred. IS" r awa-. and i; is be- much as to sav "an T'idiin with bad heart and ara you go icg to do ab ;ul Indians wuo were formerly and Irie.ndly and wouia are now surly-looking, dumb as oysters and carry their be.ts full of cartridges and an excellent Winchester over their All or good Indiana, they olaiTn ha-ve gone into the agencies, which is considered a sure pointer tbat trouble is at hand, and know it The seat of war is Short Bull's camp, wbicb is located sooth of "White river in the Pine Eidge Agency, few miles froKi the of creek. Here they are lodges, containing tA least thai many men, bo- sides women and children. Short following ars of ugly, wilder claas, who have tbe firmest sort of faith in the new Messiah craze. Tbe seriOES foatnre of this situation tbat an order has pone on t to tbe army officers to capture tJie leaders of Messiah craze. Tbe army is now mas- sing in the southern portion of tbe wrath reservation, and tbe of Short Boll swear tbat if any attempt is made to capture bin there wili be bloodshed forthwith, and Pbiliips and VTaJdron are they mean wbtt tbey fay. Tbe res-Jit is tbat thr; in Nowlin, Jaciscn and Z: batch conn ties, to tee aorta. wSil be left unprotected and long before troops caa be got iato tbat section the Indians will scatter in sroaii sqaads and burn and siltage ani massacre settlers drive off tbeir is if tbey follow op former customs. Tbis U tbe new obase of tbe Indian as it has taken its babyhood old love. Asked if it was his purpose to consol- idate all the roads wast of the Missis- sippi river, he said, "Yes, so far as traffic operations go." Ho believed in concentrating the mi.iagoment and saving of a large exponso. The roads were now losing money by very low rates. Without advancing ratos ma- terially they should at luast be stiffened. "Have you boon gunning for Wana- "I nover owned a gun in iny life. I have been all over this country with Mr. Course In to Matter for Him thv of All In LOSDOX, Nov. 27. Is is impossible to exaggerate the political excitement now prevailing in consequence of the Par- nell Such a crisis has never been witnessed in English politics be- foro, except o-i the of tho over- throw of a party by a vote hi tho tlouse of Commons or at the wills. In fact, the fate of both the p.irty and of home rule is recousiized as depending upon tbo ouci-osw- of the present imbroglio The clubs and the House lobbies are flilod with groups of all It t-> but t; 'u-.i.a i.f tM J -vl, .r1 iv .V >v- h vs tjis to nothing more than a jack-knife in my pockot" __ KYIiNES ON THE TKAIL. NBW Fnruous I-Zn Uunt the Churlwy R-iiw. NEW YORK, Nov. 27. Inspector Byrnes yesterday about tbe alleged finding of Charley Ross that three or four months ago his attention was called to the case and he endeavored to eet some information rolatiye to it, but those interested kopt all sources of in- formation closed, tu'csumably to obtain tho reward offorod by the boy's father. snid the inspector, "a per- son brought to rrj9 who told sub- stantially tho samystory." The inspec- tor then began a tiio-ough investigation of the case and adtulued that at the present time one of men is in Bos- ton trying to unravel tho mystery there, it being alleged that the boy is now in prison there. A AM MOTH STKUCTUKE. for tin.- liuildloK Groat KrtflKH to Cuuiivul York ami NKW YORK, Nov. 27.-Tho coinmis- sionors of toe New York audNow.lersoy Bridge Company held an nnthusiastic moetinR Tuebday, and the presence of Colonel F. K. liain, manager of tho Elevated railroad system, was thought to indicate a business relationship be- tween the bridge and the Manhattan company. Colonol Ham spoke in glow- ing terms of tbe big bridge, and added that, it would bo connected with every railroad in New York and a continuous lino from Boston and Montreal 10 west- ern points ho did not think improbable. It is estimated that the bridge can bo built in thirty months, and depots, ap- proaches and all witi.iu thrco years. Tho work will boim in March next Tho cost of this structure will be S50.000.000. There will bo eight tracks on the bridge. It will, ol course, be a boxes and tbe floor. Awarded Nor. Oscar Friedman was a verdict for damages tbe Wabash Westers railroad la Jadire McConaell's court yesterdaj. Friedman was peramneatly inicred wreck on tbe line tliie. Mo., two JWM Attcmpt to Wreck Train. MILWAUKKK, Wis., Nov. 27. at- tempt to wreck a passenger train on the Wisconsin Central road was made early Wednesday morning noar Stevens Point. Passenger train No. 3. northbound, ran off an open switch, ditching tho engine and baggage car and injuring tho engi- neer and fireman. Examination proved that the switch had boon tampered with. The real object ia not known, but was probably robbery, in the event of a gen- wal smash up. Tlirnmhefl tho Editor. SALEM, Mass., Nov. W. Gil- bert, a former scout with ''Buffalo Bill." and a well-known citizen, ycsterdav thrashed C. Cochran, editor of the Call, on tbe street for publi.-ning an article refl'-eting on an enterprise with which Gilbert is connected. Toe latter surrendered- himself to tiio police and was released on his own r'-cognizance. Startling: Hf-vebulmn 1'rotnlKfd. Nov. 27. reported robbery Tuesday night of William Clarke, of Shoboygan, of Sl'J.ODO. takes on an entire new phase and rumors are rife to the effect that no highwaymen held him cp. but tbat the money has mysteriously disappeared as a part of a singular plan. Tbe -police department promises startling revelations soon. Work of a Canuck CralMr. OTTAWA, Out., The flshariei protection cotter Cruiser has working this season in Georgian Reports were received by tbe Marine Department tbat a (Treat deal of illegal flsbing was feeing done by American nth- ing boats tbe cruiser was dispatched to suppress rt Sbe succeeded in seizing suspension bridge, and its elevation in the center will bo fout abovo tide- water. All lines of roiiroad terminating in Jersey City uud ciuuo will run thoir ciiis" directly uvor tho bridge and into the heart of the city. Kcvitll QUEUEC, Nov. entire Roman Catholic diocese, of liimouski is in opon revolt against its episcopal head. Bishop Langovin. Tho bishop, who is an ex- tremely bitter political partisan, has been engaged in wreaking vongoance upon tho priests' whoso people voted for the Promier, whoje politica ibe bishop heartily detests. The laity, as well as tbe clergy, are in revolt against the bishop, who will doubtless be called upon to resign, as those with whom he is now at war are all-powerful with Car- dinal Taschereau, as well as the Vatican. debaters .-.bsorbing MI' j 'U iiuiy be U-'.u" raised him trymon's itioi. "grand old :nan" eur in so st a letter, m.iv wr- for over nir'in roacait'r I'remier- ship, but wbicb places hiw in front" rrink of t'no-' vvl o r.it b right than to be Times and one or two other papers whie.h havn alwavs shown malig- nity toward Mr. aro only ones which vmiUirn to tho meed of praise for hi, coiidnT. on tills sion. and th'nr carpiuT v are drowned by tho ixco'.aiti ition-) which come up from all o-vrt-i o. the country, and in which tnon :if all parties join iiliko- As Mr. itlujsiov enterod the llouse yesterday ho w is by ft sudden whirlwind of applause that seemed to startle hini w'th its unusual vigor. It oamu from pirty benches, tbo Conservativns join'.i r t Liberals In paying tribute to th.i -nnn who hadso signally demonstrated thit no consider- ations of personal o- p iriy oxigenoy could shako him fruta '..o of his dutv. The eonlvnit botwe -n position oc- c.npied by Mr. thab in which Mr I'.irnoll no iisds is a very iminfill one. If is just to that Mr. Parnall :H not hiinsolf wholly responsible f'ir !i last mishap to bin nccoptanuo of a re-election Lo Iho Ionler-oinof thn Irish party at a moment .ti'ii had already l.is ucsiro for his rctivemcnt; thus rna-sn.' P.t noil's a.c- tion sooni to place, hiii D'.rHon.illty ttbovo Oho interost of his cou'Hry. Justin Mo Carthy baa not su.x-oi-a-id to the sa'is- factiou of his u-.iU.Mgii-s in explaining why ho did not show -'arnnll the M'H'k'.v Iflttei- Imforn thn oprifi'-.i1? of Tuesday's Nationalist meeting. Itsoems evident, that bo did not make tho Irish load-r fully aware of tho nature of Mr. Gladstone's uo-n-iirniuation. Thp.t Parnell has met with a blow Tuesday is apparent to The postponement of H nai action In tae past few days. M FT. RECOVERY. 0., Nor. traap tailor named Tom Caranangb, while at- tempting to a ride on a passenger traia on tbe Lake Erie A Western rail- road at Cold water, a -rilUge east of this place, Ml from tto rear ooack and hUsiCBlL It has bia raviojr MMiac maail ratwuiMd kit novnet. i Iiiv LONDON, Nov. Advices from St Petersburg fatate tbat the Czar has or- dered an investigation of tho stories re- garding the kiimeraH, or apartments where men and women aro temporarily imprisoned at Tomsk, Siberia. It is said that the condition of affairs in tho kameras is horrible: that the women prisoners many of thorn of respectable training, am at tho mercy of tho guards and also of other prisoners who bribe tbo guards for admission to tbe female kameraa _ gard to the Gladstone until M. in- day, by yesterday's mrscfng of itos, reveals tha existence of dissension in thoir ranks, but rill tho Irish mnm- bors who are willing to talk declaro 1'arnell roust and will rotiro. Tho de- lay is attributed by noinc tf) a dusire to hear from thn envoys in America before taking final acUoa. but this is hardly more than a nrUter of courtesy to the absent lenders. World's CHICAGO, Nov. meeting of tbe National Comroiwon oo that most of th" had lo ieavo i'l their night clothes, but the? pro- vided wilh clothing and sholtor by neighbor.s. Tho loss is about wbich is widely dislrtbiile-1. Tbo flro started in a cigar store kopt by a Mrs. and was caused by tbo upsetting of a lamp during a carousal in horapart-- meats The woman has to WASHINOTOV. Nov. Cblof Post- office Inspector R.ithbono yesterday di- rected the postm-Htor at Austin, Tes., to dismiss immediately H. H. Kinncy. the aosistant at that place. Kinney destroyed certain evidence in bis possession after it bad been de- manded of him by a postoffice inspector a-bo wanted to use it in prosecuting jase arising under tho Anti-Lottery law. ST. -il. U. Morso. salesman and coi for tho Jesse French Orr.'in charged bjr bis th fmbesizlin? S12.0JW. 5s now a Morse sold pianos and "ircann fer cash and then turnel in iao made on tbe liioe-paynaent plan, lie made lo tbe bouse in and bet oa aorso races th' of "ho cash. So have operation of anNnz trom a dog to a leg of Oeiaios, wbich was bv Dr. A. 5L Pselps attheCharf-y Biack- dog are ors-i together asd <-vcr Vbeir boacs have ja a to caiic. lUan bed conference yesterday witk tbe tab-committee of iloa-; Con- nitteeom Appropriatioirs. -n cnarge appropriation? for feosi iUatn toii Ike committee that a t weald ajaonat ,31 ;
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Candidates from BE/BTech/MTech in CSE/EE from any recognized university
Candidates having Excellent academic background from premium engineering institutes
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PlayMaker 44 Note Keyboard290150 9335679019499
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Teach your child how to play the piano with ease thanks to the PlayMaker 44 Note Keyboard.
Key Features
- The PlayMaker keyboard offers a lesson function that encourages your child to play and keeps them engaged.
- The PlayMaker Keyboard features 22 demo songs, 12 rhythms and tones, a record and playback function, tempo control, chord accompaniment, four animal sounds, and a headphones output.
|Barcode (GTIN)
|Portable Keyboards
If you feel your product has failed a Consumer Guarantee (as defined by Australian Consumer Law) then don't worry you can easily return it to your local Harvey Norman store. We'll even pay for the postage if you live in an area where there is no store. Our returns policy covers any purchased item (conditions apply).
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I ask myself the same questions everyday. My husband told me how it makes him so sad to see me so sad. So I am trying to enjoy our time together and make each day a gift. I hope that is helpful. You are not alone.
Por favor publique sus preguntas en Español y un intérprete le va responder.


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Every action
Has a consequence
The cause's effect
I walk to the shops - I cross the road - he waits at the light I turned on
He's late - his phone rings uselessly on his desk - she answers it
Which means that
She tells them to try later - the caller gives up - finds someone else.
The someone else, he stays with her - he isn't there to stop the fire - and a whole bright life is gone.
Ripples - they spread.
But then, I should never go out - do nothing - then I can't be the cause
Me not being there has just as much of an effect
Think about it.
You can touch a hundred people's lives
Simply by existing


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ROBERTO MANCINI says Arsene Wenger should stop moaning about Manchester City's
Arsenal's boss had another dig about City's £400million spon-sorship deal with
But ahead of today's clash with the Gunners, Mancini says there are no plans
to stop spending.
He said: "There were a lot of teams in the past spending money for big
players, including Arsenal.
"All the managers talk about the money we have all the time. But we have 11
players like all the other teams.
"I don't know why we should only talk about the money."
Wenger hopes City will be under the spotlight as UEFA implement Financial Fair
Play regulations.
But Mancini added: "If we want to stay at the top, we need to improve and buy
"This summer we needed to buy good players to improve. We had the chances to
get them and we were successful."
Video: Mancini backs Arsenal title bid


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GoPro's CEO Just Dropped $229 Million to Fulfill a Promise to His College Roommate
J Thoendell stashed this in Business
Source: http://www.bloomberg.com/news/articles/2...
Billionaire Nick Woodman's fortune dropped $229 million this week after he fulfilled a promise made to his college roommate more than a decade ago.
The GoPro Inc. founder returned 4.7 million shares to the San Mateo-based camera maker on May 11, according to a regulatory filing. He agreed in 2011 to repay the company for stock options it granted to Neil Dana, who attended the University of California at San Diego with Woodman and was GoPro employee No. 1.
To save this post, select a stash from drop-down menu or type in a new one:


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On April 29, the US co-chair of the OSCE Minsk Group James Warlick met with Foreign Minister Edward Nalbandian, SIA informs that the US co-chair has wrote on his Twitter page.
"Productive and positive talks today with FM Nalbandian in Paris. Armenia is open to opportunities for Nagorno-Karabakh peace." tweeted Warlick.
Note that Azerbaijani Foreign Minister Elmar Mammadyarov also will today meet with the co-chairs of the OSCE Minsk Group in Paris to discuss steps toward Nagorno Karabakh peace.


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Pyrogenium Plex contains a specific combination of synergistic homeopathic remedies known for their antiseptic and anti-inflammatory properties to help relieve symptoms associated with congestion in the upper respiratory tract' specifically the nasal cavity and sinuses. Studies show that inflammation in these areas can be caused by viral' bacterial or fungal infections as well as allergic reactions' resulting in sinus tenderness and swelling' toothaches and headaches. Nasal congestion can interfere with ears' hearing' speech development and sleep. Pyrogenium Plex is indicated for nasal congestion' sinus inflammation and pain' nasal discharge and burning' infectious rhinitis and associated headaches.
These statements have not been evaluated by the Food and Drug Administration (FDA). These products are not meant to diagnose' treat or cure any disease or medical condition. Please consult your doctor before starting any exercise or nutritional supplement program or before using these or any product during pregnancy or if you have a serious medical condition.
Pyrogenium (Rotten Peat Moss) 6 CH
Hepar sulfur (Crude Calcium Sulfide) 5 CH
Hydrastis canadensis (Goldenseal) 5 CH
Kalium bichromicum (Potassium Bichromate) 5 CH
Kalium iodatum (Potassium Iodine) 5 CH
Arsenicum album (Metal Oxide) 5 CH
Adults: Ten drops three to four times daily.Children under 12: Five drops three to four times daily.
Consult your healthcare professional.
Ratings & ReviewsReview This Item
Overall Rating for Pyrogenium Plex - 1 fl. oz (30 ml)
Displaying reviews 1-1 of 1
By Lori
from Cleveland, Ohio
It works!
I get sinus headaches and stuffy headed migraines. Pyrogenium Plex by Seroyal takes away the stuffy head and pounding soon after I took it. My son has also used Pyrogenium Plex when hes felt a sinus infection coming on and it stopped it. It works!
√ I would recommend this item to a friend.
Displaying reviews 1-1 of 1
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Additionally, PureFormulas is unable to directly recommend products in relation to specific health conditions. Please contact your trusted healthcare practitioner for direct product recommendations.
If you have any general questions regarding orders or our policies and programs, please contact our Customer Happiness Team @ 1.800.383.6008
If you are pregnant, nursing, taking any medications or have any medical condition, consult your doctor before use. Your healthcare professional is the best source for guidance before beginning an exercise or nutritional supplement program.


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Welcome to Alpha Manufacturing - we're a UK based precision Sheet Metal Fabricator. As a leading precision sub-contract sheet metal and fabrication company, we've been serving many industries since 1989 offering sheet metal fabrications, metal design and engineering services. Additionally, we provide a quick turnaround of metal prototypes using our full range of expert sheet metal fabrication techniques.
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UPDATE 3-Gambling firm GVC raises offer to $1.6 bln for rival Bwin.party
* GVC makes 122.5p/share offer for Bwin
* Says Cerberus Capital to provide 400 mln-euro loan
* Cost savings to exceed 135 mln euros per annum (Adds shareholder quotes)
By Aastha Agnihotri and Neil Maidment
LONDON, July 27 (Reuters) - Online gambling firm GVC Holdings returned on Monday with a new 1 billion-pound ($1.55 billion) bid for rival Bwin.party Digital Entertainment , looking to trump a recently accepted offer from 888 .
Bwin, put up for sale last year, accepted a cash and stock deal worth almost 900 million pounds from online casino and poker firm 888 this month, shunning an earlier 908-million-pound offer from GVC and Canada's Amaya Inc, which it deemed too complex and with less attractive growth prospects.
The tussle for Bwin is the latest in a flurry of merger activity in the industry, a trend likely to continue as firms seek to expand to offset increasing taxes and tighter regulation in Britain. Last week Ladbrokes and Gala Coral agreed a $3.4 billion all-share merger.
Bwin, which has struggled with the decline of regulated poker markets in Europe and to make cost savings since its creation via a merger of sports betting group Bwin and online poker group PartyGaming in 2011, confirmed on Monday that it had received a new offer from GVC and would respond in due course.
888 declined to comment on whether it planned to raise its offer, though analysts said they widely expected it to do so. Continued...


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An American academic is creating a searchable database of 12 million historic copyright-free images.
Kalev Leetaru has already uploaded 2.6 million pictures to Flickr, which are searchable thanks to tags that have been automatically added.
The photos and drawings are sourced from more than 600 million library book pages scanned in by the Internet Archive organisation.
The images have been difficult to access until now.
Mr Leetaru said digitisation projects had so far focused on words and ignored pictures.
"For all these years all the libraries have been digitising their books, but they have been putting them up as PDFs or text searchable works," he told the BBC.
They have been focusing on the books as a collection of words. This inverts that. Stretching half a millennia, it's amazing to see the total range of images and how the portrayals of things have changed over time.


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Sell your mobile phone for cash. Recycle and sell old, used mobile phones or tablets to TopDollarMobile. Payments are made in just five days and postage is free with their freepost envelopes.
|Sale - Value Over £10
Give as you Live is only available when the basket value is £10 or greater. Donations may only apply to the sale price and not include VAT, delivery and other such costs. Please do not use voucher codes from other websites as this may affect your donation.
Unfortunately there are currently no offers available for this retailer.


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Tote Bag
The Eye of the Peacockby Bel Menpes
Our quality crafted Tote Bags are hand sewn in America using durable, yet lightweight, poly poplin fabric. All seams and stress points are double stitched for durability. Available in 13" x 13", 16" x 16" and 18" x 18" variations, the tote bags are washable, feature original artwork on both sides and a sturdy 1" wide cotton webbing strap for comfortably carrying over your shoulder.
Jewel bright iridescent turquoise, sapphire blue, deep and pale golds make up this abstract design created from my own photographs of the tail of a beautiful Peacock.
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Experienced (non manager) Customer service Technology jobs in Petersfield
Found 0 jobs
We do have the following jobs available for Technology, Customer service, Experienced (non manager)
Showing 1 - 3 of 7
Assistant Buyer - Mobile Phones (Carphone Warehouse)
This new role offers the opportunity to help the development of existing & new elements across the mobile phone portfolio within the Phone Buying Team
Technical Support Tier II
Provide support to 1st line via standard comms channels (Hipchat) for live issues.
Test Centre Administrator - Isle of Wight
The appointed individual will be responsible for the efficient delivery of tests to candidates.
Related courses
Telephone Skills Diploma – Online
This course is ideal for those seeking to deliver professional telephone standards on a consistent basis.
Customer Service Diploma - CPD & IVCAS Accredited
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Information Technology Infrastructure Library (ITIL Foundations V3)
Become skilled in ITIL to enable your company to use leading-edge IT capabilities to provide world class services and maximise value


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Think of what you need to be fit. You need to work on your endurance. You need to build strength. You need to eat the right amount of food and make good choices.
Say you looked just at strength. You would need to choose the right exercises, how many sets, the number of reps, and so forth. These would define your strength program.
Search engine optimization is broad like fitness. You want to address all of its parts, and you can delve further into each one. You can look at site architecture, but then drill down by looking at how fast the pages load and if the search engines can crawl them all. You could work on off-page SEO by building backlinks and engaging in social media.
Keywords are one of the broad areas for SEO. You need to address the right keywords for your business. Keyword research allows you to best express the intent of your pages. The best option, Keyword Planner, which is also free, can confirm that you aim for the right keywords.
You then need to support this with the right content and HTML. At Wakefly, we have a process called keyword maintenance. We use this to get suggestions for meta data and other factors. We grab the first round of ideas through tools called Searchlight Conductor and the Screaming Frog SEO spider tool. They give suggestions on titles, meta descriptions, H1 tags, and alt tags for images.
Each one of these will sway SEO success in different ways:
- Titles are the main element beyond the content. They show the subject of the page in the search engine listing.
- Meta descriptions, though they do not change ranking, can allow a higher clickthrough rate by giving a summary used only by the search engines.
- H1s also affect the rank, acting as a sort of page title seen by the visitor.
- We look if keywords are bolded or italicized, which gives a slight edge.
- We also look at elements like anchor text, which helps to signal relevance and clout for a set of keywords.
It helps to be aware of the best practices for these elements. For instance, the character limits for the titles and meta descriptions are 55 and 115. This has decreased over time as smartphones and tablets become more popular.
Using Keyword Maintenance
Creating and updating the on-page SEO for keywords is vital. This can feel overwhelming though. Using keyword maintenance allows us to keep up with it.
Though tools will help, you need to use a good human mind in the end. A tool may suggest too many keywords per meta tag or tell you to use a phrase that looks awkward in the meta description. An expert will use these tools to quickly identify problems but will have to use his or her judgment hereafter. Adding these elements also demand someone familiarity with the content management system that runs the website to make the changes.
An online marketing firm such as Wakefly can help you with these issues. We will use keyword maintenance, along with our other tools and methods, to grow your business. Learn more by getting in touch with us today.


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Deco Black Retro Office Chair
Product Description
- Retro 'Retro' style chair
- Supple faux leather in black
- Bright chrome arms and base
- Matching arm covers
- Reclining function with tilt tension
- Gas lift seat height adjustment
- Also available in black
- Requires easy self assembly
- Seat Width: 48 cm
- Seat Depth : 45 cm
- Seat Height : 47-54 cm
- Back Height : 63 cm
- Back Width : 48 cm
Deco Retro Style Black Office Chair
Retro 'Retro' style chair upholstered with supple faux leather and soft sit cushions. Features a gas seat height adjustment and reclining function with adjustable tension control. Finished with bright chrome armrests and matching arm cover.
- Recommended Daily Use: 8 Hours
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10338 Malcolm Circle
Cockeysville, Maryland, 21030
Commute to Downtown Cockeysville
Some errands can be accomplished on foot.
About your score
10338 Malcolm Circle has a Walk Score of 54 out of 100. This location is Somewhat Walkable so some errands can be accomplished on foot.
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Sparco Side mount Steel - Black | (004902)PART# SPAR 004902
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When mounting your Sparco Competition Seat, the use of Side Mounts is necessary for proper installation. Side Mount Aluminum Sparco side mounts are used to mount competition seats. Side mounts have mutliple side holes that provide a customizable mounting position. All Aluminum construction available in anodized black only.
- Black anodized for superior protection
- Used to mount competition seats. /li>
- Side mounts have multiple side holes that provide a customizable mounting position.
- Weight - 5 lbs per set
- Universal
What's in the box?
- 1 Side mount Steel
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At least five times . . . with the Arian and the Albigensian, with the Humanist skeptic, after Voltaire and after Darwin, the Faith has to all appearance gone to the dogs. In each of these five cases it was the dog that died.
-- G.K. Chesterton, The Everlasting Man


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Hi. My sons do not want their friends to see "their mothers", (me) profile picture.?
Use an arbitrary Icon instead....something that won't really mean anything like a cartoon character or a flower. I have children and they told me the same thing.
Why do mothers love their sons more?
Because the trachea is very tiny, so that type of food for children is very harmful.A way you can resolve a choking is by:Pom ping somebody on the abdominal decreases the volume of the chest cavity...
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How can I encourage my sons to grow up gay I hear gay men are nice to their mothers.?
dress em like ladies, they'll get the hintcan i borrow one of yours? you only need one to take care of you right?? I need my son to stay straight so i can have blood related grandbabbies
Mothers who get their sons circumcised, have you considered your daughters?
I think some people here need to educate themselves on infant male circumcision. Please read these links:http://consciouswoman.org/2009/06/23/int…http://www.circinfosite.com/http://www.nocirc.org...
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Don't mothers teach their daughters and sons to cook anymore?
As I'm a "wrinkly" [an old person!!] I've seen many men whose wives have died and the widowers have not even known how to put the kettle on, let alone, boil an egg. So as soon as my offspring were...
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0 10th, Chetek WI 54728 $12,500
Features of 0 10th Chetek WI 54728
- Lots for Sale
- Status: Active
- Estimated Age: Not Available
- Est. Acreage: 1
- Est. Acreage: >= 1, >= 1/2
- School District: Chetek-Weyerhaeuser
- County: Barron
- Property Taxes: $176
- Property Tax Year: 2013
- Listing Company: Woods & Water Realty Inc, Blue Diamond
- Listing Agent: Chris Butterfield
Property Description for 0 10th Chetek WI 54728
0 10th Chetek WI 54728 - Nice Open Parcel Of Land In The Country With Easy Access To Town. Property Can Be Sold As Three Lots Together Consisting Of 3.3 Acres Or Each Lot Separate At 1.110 Acres. Perfect Location With Many Opportunities To Build Or Farm. Lot 4 - 0 10th Ave.
Interior Features
- Water Waste: No Waste Disposal System
Building and Construction
- Construction Type: E
Land Features
- Topography: Level, Open, Some Tilled
- Zoning: Residential
- Waterfront/Access: N
|MLS Number
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|Previous List Price
|Sold Price
|Mar 22 2016 11:30AM
|Jan 16 2015 4:02PM
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Buyer understands that the information which is contained on Data pages is obtained from a number of different sources and which has not been independently verified or confirmed by the various real estate brokers and agents who have been and are involved in this transaction. If any particular measurement or data element is important or material to buyer, Buyer assumes all responsibility and liability to research, verify and confirm said data element and measurement. REALTOR *MLS* Equal Housing Opportunity


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Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins
Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative.
CHO cell clones, expressing 300 μg/ml IGF-E5 in batch culture, were isolated more easily and quickly compared to the classic limiting dilution method. The intensity of the detected fluorescent signal was found to be proportional to the amount of IGF-E5 secreted, thus allowing the highest producers in the population to be identified and picked. CHO clones producing up to 9.5 μg/ml of Tissue-Plasminogen Activator (tPA, 67 kDa) were also generated using FLSSM. In addition, IGF-E5 high-producers were isolated from 293SF transfectants, showing that cell selection in semi-solid medium is not limited to CHO and lymphoid cells. The best positive clones were collected with a micromanipulator as well as with an automated colony picker, thus demonstrating the method's high throughput potential.
FLSSM allows rapid visualization of the high secretors from transfected pools prior to picking, thus eliminating the tedious task of screening a high number of cell isolates. Because of its rapidity and its simplicity, FLSSM is a versatile method for the screening of high producers for research and industry.
Chinese Hamster Ovary (CHO) cells continue to be a choice mammalian system for producing recombinant biopharmaceuticals despite relatively low yields and high costs. This is largely due to their ability to properly fold and post-translationally modify complex recombinant proteins such as antibodies for human administration
"Blind" selection of high producers using limiting cell dilution in 96 well plates after transfection, is a common method. Following the formation of drug resistant colonies, blind cloning requires amplifying and individually testing hundreds (sometimes thousands) of these drug resistant colonies or cell populations, most of which will eventually be discarded as non/low producers. Although the technique is well established and reliable, it is a labor-intensive, time-consuming process. The advent of natural, low toxicity fluorescent markers such as the Green Fluorescent Protein (GFP) family opened a new era for reporter genes and detection of recombinant protein expression. Fluorescent markers are used in fusion proteins, as co-expression indicators, or in dicistronic configuration to help visualize cells expressing the proteins of interest within a mixed population
A different approach is to attempt at labeling the secreted product itself, the main difficulty being to retain the product around the cells in order to identify high producers. This strategy can be traced back to the selection process for myeloma mutants which involved plating the cells in semi-solid medium containing agarose
In the present study, we demonstrate how FLSSM is used to isolate CHO and 293SF cells secreting high-levels of recombinant proteins other than antibodies. FLSSM was compared to the traditional cell dilution in cluster wells to isolate high producers of a small protein (25 kDa) derived from the insulin like growth factor-1 (IGF-1). High producers were isolated with FLSSM more rapidly and with less effort. FLSSM was also used to isolate stable CHO cells expressing Tissue Plasminogen Activator (tPA, 67 kDa). In its simplest form, this method only requires a fluorescent antibody against the secreted product, but it can also be used with automated scanning and picking systems. FLSSM is a versatile method for the screening of high producers for research and industry.
Isolation of clones secreting IGF-E5 using the conventional procedure
The structure of the recombinant IGF-1 protein (IGF-E5), employed to compare the conventional procedure of clone isolation (dilution in 96-well plate) with FLSSM, appears in Fig.
Figure 1
Expression and purification of IGF-E5
Expression and purification of IGF-E5. A: Structure of IGF-E5. This recombinant protein was made by fusing the complete amino acid sequence of the murine IGF-1 to a His Tag (6H) and to an artificial peptide referred as E5-Coil. The latter possesses a strong affinity for a complementary peptide, the K-Coil
Selection of clones expressing IGF-E5 by FLSSM
To test if we could isolate high producer clones more efficiently by direct labeling with a fluorescent antibody specific for the recombinant product, a pool of CHO cells expressing IGF-E5 was generated as described above. Fourteen days after electroporation and hygromycin selection, the resistant cell pool was plated in semi-solid medium containing a fluorescent anti-His Tag antibody. By microscopic observation, about 5% of cells were associated with a fluorescent signal (Fig.
Figure 2
Detection of secreted IGF-E5 from stable CHO transfectants by FLSSM
Detection of secreted IGF-E5 from stable CHO transfectants by FLSSM. A, C and E: transmitted light images. B, D and F: green fluorescence at 510 nm from anti-His Tag/FITC antibody present in semi-solid medium; fluorescent dots formed by reaction of anti-His Tag/FITC with secreted IGF-E5 bearing His Tag moiety. A and B: longer exposure from single cell showing a cloud of fluorescent dots. C and D: positive CHO-IGF-E5 colony: signal is weak, but sufficient for detection of high producing cells. E and F: negative control; CHO cells secreting an unrelated product do not react with anti-His Tag antibody.
Figure 3
Screening and quantification of high-producing CHO-IGF-E5 clones by spot blot
Screening and quantification of high-producing CHO-IGF-E5 clones by spot blot. A: initial screening of all colonies selected by FLSSM and picked by Quixell micromanipulator (A trough EE). std: 20 ng spot of purified IGF-E5 for reference. B: Quantitative spot blot. Top row: purified IGF-E5 standards, numbers indicate amounts spotted in nanograms. Bottom row: 2 best clones isolated by FLSSM (L and Y) and best cell population isolated by classic dilution in wells (4-B4). Duplicates not shown. C: plot of the fluorescence signal obtained by scanning the standard spots using a Typhoon™ scanner, R2 = 0.99.
The five clones showing the strongest signal were seeded at 5 × 105cells/ml and supernatants sampled at day 3 were analyzed by quantitative spot blot. Production at 30°C from the two best clones were then compared in a final spot blot, with the starting pool as well as with the best cell population (4-B4) obtained from the conventional cloning method described above (Fig.
Correlation of fluorescence intensities and secretion level
To confirm that the secreted recombinant protein levels by the CHO-IGF-E5 clones correlated well with their fluorescence intensities in semi-solid medium, we compared the IGF-E5 secretion level and the fluorescence of seven different clones under identical conditions. The clones were plated in semi-solid medium for fluorescence scanning and, in parallel, were cultured in liquid medium for a 3-day batch assay at 30°C, followed by quantitative spot blot analysis. Duplicate semi-solid medium wells for each clone were scanned with a CellCelector™ (Aviso) and the combined fluorescence for all colonies above threshold were summated. Results (Table
Table 1
Correlation between cell colony fluorescence and recombinant protein secretion from CHO-IGF-E5 clones selected by FLSSM.
aNames of stable CHO clones secreting various levels of IGF-E5 (Fig. 3A).
bProduction from a 3-day batch culture at 30°C was measured by quantitative spot blot against purified IGF-E5. The production was normalized to the highest producer (clone L).
cRank for IGF-E5 production (column 2) in decreasing order. Clones J and E were both assigned rank 6, because their production level was not significantly different.
dFluorescence was measured by scanning the wells at 510 nm using the CellCelector™. The fluorescence signal of all the colonies above background was summed and normalized to the highest value (clone L).
eRank for the total fluorescence (column 5) in decreasing order and independently from column 4.
CHO clones expressing tPA and 293SF expressing IGF-E5
To verify if clones producing a different recombinant protein could be isolated by FLSSM, a stable pool of CHO cells expressing tPA (67 kDa), controlled by the CMV promoter, was generated. The cells were then plated at low density in semi-solid medium containing an antibody against tPA labeled with Alexa488. The fluorescent signal from positive CHO-tPA colonies was overall much stronger than that from CHO-IGF-E5 positive cells. Indeed, a precipitate around colonies was visible even in phase contrast microscopy (Fig.
Figure 4
FLSSM applied to different products and cell lines
FLSSM applied to different products and cell lines. A, A' and C: transmitted light images. A and B: colonies from CHO-tPA transfectant pool. B: detection of secreted tPA using an anti-tPA antibody conjugated to Alexa488 in semi-solid medium. Note the presence of positive and negative colonies in the same field. Arrow indicates positive, high-producing clone; A': insert blown up from A showing that a precipitate around the positive colony is visible in transmitted light microscopy. C and D: colony from 293-IGF-E5 transfectant pool. D: detection of secreted IGF-E5 (small fluorescent dots at the colony periphery using an anti-His Tag antibody conjugated to FITC in semi-solid medium).
To determine if our method could be extended to other cell lines besides CHO, the human embryonic kidney 293SF cell line
In this work we have demonstrated that FLSSM works equally well for detecting small and intermediate-sized proteins, compared to large molecules such as antibodies. We have shown that the method also worked for 293 cells. A previous study
Other methods that can isolate cell lines expressing soluble and non-fluorescent recombinant proteins have been described
Additional File 1
Identification of high-secreting CHO clones: comparison between surface detection by flow cytometry and FLSSM. Additional data showing labeling of CHO-IGF-E5 cells with the anti His Tag-FITC antibody followed by flow cytometry.
Click here for file
The recombinant IGF-E5 is a good test for this method because of its relatively small size (25 kDa). We observed that the fluorescent signal with IGF-E5 was significantly weaker compared to that from tPA and an IgG (data not shown). Many factors contribute to the intensity of the detected signal: secreted product concentration, molecular size, shape, diffusion speed in methylcellulose, the detection antibody's affinity for the secreted product and the size of the colonies. The CHO-IGF-E5 positive cells were picked early after plating as single cells or very small colonies, which likely affected the survival rate of the clones. Despite this weaker signal and the very small size of the CHO-IGF-E5 colonies picked, several high CHO-IGF-E5 producers were obtained by merely analyzing a few dozen clones from FLSSM. In contrast, more than 400 cell populations had to be screened by the classic method to find a similar producer. In addition, the relative fluorescence intensity of the CHO-IGF-E5 colonies in semi-solid medium was proportional to the level of secretion of the corresponding clones in batch culture, strongly supporting the notion that high-secretors are the brighter ones. Our best CHO-tPA clone isolated by FLSSM produces 9.5 μg/ml of tPA in batch culture, which is significantly lower than our best CHO-IGF-E5. This value however is comparable to the best clones we characterized in our laboratory using the standard dilution method (data not shown). Although it is difficult to compare data obtained from different laboratories, our tPA production level is in the range of those (10 to 30 μg/ml) reported in literature for CHO cells in batch culture, without medium or growth culture optimization
It should be pointed out that the intensity of the fluorescent signal in FLSSM should only be used as a relative index to compare different clones expressing the same product in a particular host. Case in point, the fluorescent signal surrounding CHO-tPA clones was much stronger than the signal from CHO-IGF-E5 clones, even though the best IGF-E5 clones were much higher producers. However, for a given cell host, secreted product and detection antibody, the fluorescent signal should be proportional to the level of expression, allowing isolation of the highest producers from a mixed population.
We observed some toxicity (reduced growth and lack of colony formation) when the cells were labeled with certain antibodies. Ideally, azide and thimerosal-free antibodies should be used to minimize toxicity. These and other potential contaminants present in commercial antibodies can easily be removed by microdialysis. Even though we have found that FLSSM allows detection of high producers at the single cell level, it is more advantageous and reliable to pick positive colonies of a minimal size. Accordingly, conditions favoring good colony growth should be optimized, such as medium composition, plating density and time of fluorescent antibody addition. For 293SF-IGF-E5 selection, we saw that plating cells in semi-solid medium without the detection antibody permitted initial colony growth. By adding the fluorescent label in a liquid medium overlay 3–7 days later, chances to successfully isolate high-producers improved.
Regardless of the cell selection method used, loss of expression during clone amplification is always possible if high-level recombinant protein expression is toxic and negatively affects cell growth. To that effect it would be advisable, if possible, to use an inducible, tightly-regulated promoter (such as the Tetracycline-based system
Fluorescence labeling in semi-solid medium (FLSSM) allows rapid visualization of the high secretors from transfected cell pools prior to picking, thus eliminating the tedious task of screening a high number of cell isolates. It can be used to select for growth characteristics at the same time as high protein production. FLSSM should be applicable for the isolation of most secreted protein producers, provided that the product is secreted at a sufficient level and that an antibody against it is available. It is far less labor intensive than traditional methods and represents large savings in time, supplies and incubator space. In addition, the method has been tested successfully on micromanipulator and robotic systems.
Plasmid construction
Restriction and DNA modifying enzymes were purchased from New England Biolabs, Ltd (Pickering, Ont, Canada). Plasmids were constructed using standard molecular biology procedures and they were purified with the Qiagen plasmid Maxi Kit (Qiagen, Valencia, CA). To construct pMPG-IGF-E5, the coding sequence of IGF-1 was isolated by PCR from the murine cDNA of that gene (GenBank ID:
Cell culture
CHO cells adapted to serum-free suspension culture (CHO-SF, Invitrogen, Grand Island, NY), were grown in protein free chemically defined medium for CHO cells (CD-CHO medium, Invitrogen) supplemented with 1× HT supplement (Invitrogen), 4 mM glutamine and 50 μg/ml dextran sulfate (MW: 500 000, Amersham Pharmacia Biotech, Uppsala, Sweden). 293SF
Transfection and stable clone production
CHO-SF and 293SF were transfected by electroporation in the presence of linearized plasmid using a BTX T820 electrosquare porator (Genetronics, San Diego, CA) according to the manufacturer's recommendations. For the clone isolation using the "standard method", after electroporation, the CHO-SF were diluted into growth medium and incubated overnight in 6-well plates. The next day, they were separated into 96-well plates (1000 and 3000 cells/wells) in growth medium supplemented with 600 μg/ml hygromycin B (Invitrogen). Resistant colonies, isolated from plates in which less than 30 wells contained resistant colonies to maximize the odds of isolating a true clone, were then transferred to 24-well plates in the presence of hygromycin B. Supernatant from confluent 24-well plates were analyzed by dot blot for the presence of IGF-E5. To generate clones using the FLSSM method (see below), after electroporation, a pool of stable transfectants was generated by growing the cells in the presence of 600 μg/ml (CHO-SF) or 25 μg/ml (293SF) hygromycin B.
Cell plating in semi-solid medium
Immediately before plating in semi-solid medium, cells were dispersed in a 35 mm Petri dish through a fine microtip (Costar, Bethesda, MD) and observed under the microscope. The process was repeated until only single cells were observed. CHO-IGF-E5 and CHO-tPA stable cell pools were mixed in medium containing Clonematrix methylcellulose, XL Reagent (Genetix, Boston, MA) and CDCHO-AGT 2× (Invitrogen), at a 1× final concentration and supplemented with 8 mM L-glutamine (Invitrogen) and fluorescent antibody (see below). 293SF-IGF-E5 stable cell pools were mixed in medium containing Clonematrix, 2× LC-SFM medium (1× final concentration), 1%FBS (HyClone) and 4 mM L-glutamine. The cells (CHO and 29SF) were agitated and vortexed at medium speed to ensure uniform distribution of all components before plating. They were then seeded in 24-well dishes (Sarstedt, Newton, NC) or 60 mm Petri dishes (Greiner, Monroe, NC), made of untreated plastic, at densities of 500, 1000 and 2000 cells/m. After plating, the cells were observed under the microscope to verify they were single and well dispersed inside the dish.
Cell labeling and colony picking
Anti-His Tag-FITC antibody (Abcam, Cambridge, MA) was used at 4 μg/ml to detect IGF-E5 secreting cells in semi-solid medium. The anti-tPA (Cedarlane, Hornby, Ont) antibody was first dialyzed on a microcon YM-50 spin filter (BioRad, Missisauga, ON) against PBS (HyClone), and conjugated to Alexa488 fluorochrome with a Microscale Protein Labeling Kit (Molecular Probes, Eugene OR) according to the manufacturer's recommendations. The labeled antibody was used at a concentration of 1 μg/ml to detect tPA-secreting cells. Positive cells and colonies were picked either with a Quixell micromanipulator (Stoelting, Wood Dale, IL) using 50 μm glass capillaries, or with a CellCelector™ colony picker (Aviso, Jena, Germany) equipped with the methylcellulose tool. For Quixell picking, the most fluorescent cells were determined visually. For CellCelector picking, the plates were scanned at 10× magnification and the brightest objects were determined using the included analySIS software (now called Cell D). Single cells and colonies were deposited into 96-well dishes (Corning, Lowell, MA). Images of fluorescently labeled cells were taken on a Leica DMIL inverted microscope using a color 3CCD cooled camera (Optronics, Goleta, CA) or a monochrome Retiga Exi (QImaging, Surrey, BC) and Openlab software (Improvision, Coventry, England).
Analysis of protein production by dot blots and spot blots
For dot blot analysis, the culture supernatant was diluted 5 times with distilled water. The samples were denatured by adding an equal volume of 2× lysis buffer (0.125 M Tris-HCl pH 6.8, 4% SDS, 20% glycerol and 0.1% bromophenol blue) and heated at 95°C for 5 min. The samples were spotted on a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences Buckinghamshire, UK) under vacuum on a dot-blot apparatus (Bio-Rad Laboratories Canada Ltd., Mississauga, ON). The membrane was incubated with an anti-His Tag antibody (Serotec, Oxford, UK) followed by a horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham Biosciences). The signal was revealed by chemiluminescence using the ECL Western Blotting detection reagents (Amersham Biosciences). For spot blot analysis
Analysis of protein production by Western blot
Stable CHO transfectants (5.0 × 105) were resuspended into 1 ml of growth medium in 24-well plates. The next day, the plates were transferred to an incubator at 30°C and incubated at this temperature for up to 14 days. At selected time points, an aliquot of growth medium was mixed with an equal volume of lysis buffer 2× supplemented with 5% β-mercaptoethanol. The samples were heated for 5 min at 100°C and separated on a SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and the presence of the IGF-E5 was demonstrated using anti-His Tag antibody by chemiluminescence as described above.
Purification of IGF-E5
Actively growing CHO cells from clone 4-B4 (1.0 × 106/ml) in a T-Flask were incubated at 30°C for 7 days. After separation of supernatant and cells by centrifugation, Complete protease inhibitor (Roche Diagnostics, Mannheim, Germany) and a final concentration of 10 mM imidazole were added to the supernatant. The supernatant was loaded onto a 5-ml HisTrap column (Amersham Biosciences) using a peristaltic pump and the bound IGF-E5 was eluted with increasing concentrations of imidazole (10 to 200 mM). The purified IGF-E5 was dialyzed against PBS and concentrated on a centrifugal concentrator (Centricon YM-10, Millipore Corporation, Bedford, MA). After determining the protein concentration with the DC protein assay (Bio-Rad laboratories, Hercules, CA), the purified IGF-E5 was stored at -80°C in 5% glycerol. The purity of the protein was evaluated by SDS-PAGE followed by staining with Coomassie Fluor™ Orange protein gel stain (Molecular Probes, Eugene OR) and analyzed on a Typhoon trio+ scanner (Amersham Biosciences). The nature of the purified IGF-E5 was confirmed by Western blot (as described above) using as primary antibody an anti-His Tag antibody or a goat anti-IGF-1 antibody (USBiological, Swampscott, MA).
Correlation of production and fluorescence in FLSSM
Seven CHO-IGF-E5 clones of various production levels (as per initial spot blot screening) were seeded at 1.0 × 104 cells/ml in semi-solid medium containing anti His Tag-FITC antibody in duplicate wells of a PetriWell24 plate (Genetix) and, in parallel batches at 1.0 × 106 cells/ml in liquid growth medium. Duplicate wells of a 24-well dish (Sarstedt, Montréal, QC) were incubated at 30°C for 3 days. Samples were clarified by centrifugation and analyzed by quantitative spot blot as described above. Semi-solid medium plates were scanned on a CellCelector™ (Aviso). The product of area and mean fluorescence for all the colonies above background threshold was determined with the Cell D software (Aviso/Olympus) and summed for each separate clone using an Excel spreadsheet. The results were normalized to the brighest clone (L). Quantitative spot blot results for batch production were normalized to the highest producer (L).
Authors' contributions
AC developed the FLSSM method, did the microscopic analysis, generated clones, quantified their expression and drafted the manuscript. CN generated the IGF-1 expressing clones and quantified their expression, BG produced the tPA expressing plasmid, developed the spot blot quantification and quantified the CHO-tPA clones. IB constructed the IGF-1 ligand, MP produced the IGF-1 ligand and developed a purification method for it. AG supervised the work of BG and participated in the design of tPA production and quantification, BM provided the original idea for the study and interpreted the data, RG conceived of the study, participated in its design and co-drafted the manuscript. All authors read and approved the final manuscript.
We would like to thank Lucie Bourget for technical assistance and Roseanne Tom for critically reviewing the manuscript. Supported in part by a grant from the National Cancer Institute of Canada. This is a NRC publication, number 47836.
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