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Undercard Overhaul 3: Yet 5 More Angles to Improve WWE TVJanuary 21, 2012
Undercard Overhaul 3: Yet 5 More Angles to Improve WWE TV
In the first and second installments of Undercard Overhaul, we discussed a basic rule of professional wrestling: the very definition of career suicide is the ability to be forgotten.
This industry thrives on spectacle and people have millions (if not billions) of entertainment alternatives if WWE and its performers fail to deliver a compelling product.
With that in mind, let's take a look at five more angles and ideas that can turn WWE's struggling undercard from awful to enduring.
5. S.O.B. Inc.
Tyler Reks and Mason Ryan are two intimidating beasts who can't seem to find a niche. Too big to job convincingly yet too late and too green to fill an "unstoppable monster" role in a company with too many unstoppable monsters already.
It brings up the question: How do they get on TV without resorting to comedy (a la Brodus Clay)?
Mark Henry, Kane, Big Show, Ezekiel Jackson, Sheamus and John Cena are all singles Superstars. That market's closed. There is, however, a distinct lack of big men in tag team competition and WWE still needs a new wave of challengers to fortify a (continually) struggling division.
I think Reks/Ryan (not to be confused with a certain NFL head coach) could drop into that spot and draw.
The answer to my question, "What can they do?" is simple: go into business as S.O.B. Inc., a heel-tinged Acolytes Protection Agency who sell their services as black sunglasses-wearing bad asses, bodyguards and unscrupulous hitmen.
Ten years is long enough to rest the gimmick; let's dust it off and present it to another generation of fans.
Hook them up with Kevin Nash, who has plenty of experience in the bodyguard business and managing groups of up-and-coming wrestlers, preferably in a non-wrestling role, and the tandem's flexibility as hired help allows them to go over without so much as an ongoing feud.
WWE could use S.O.B. Inc. as supporting elements on TV, letting them mix it up most on Superstars and house shows, and give Reks and Ryan a chance to grow into the gimmick before a big push.
(To the IWC: I'm prepared to take some heat over Nash's inclusion in this package. As polarizing as he is, I still feel like he's got something to give the world of professional wrestling. Keep the promos short and focused, use him to highlight his clients rather than himself, get rid of the shoots and let the characters shine. I stand by it.)
4. Jack Swagger, American Ambassador
Jack Swagger using the Ankle Lock has got to be a rib.
At some point, someone in a position of power had to look at this kid and say, "Well, if we can't have Kurt Angle..." (which is blessing and a curse, being compared to one of the all-time greats and Fake Razor and Diesel in the same sentence). He even uses the same style of tights!
Swagger's already there, the comparisons to Angle are inevitable and WWE shouldn't shy away from stories where Jack takes even more pages out of Kurt's playbook.
I'm thinking back to Angle's run as a milk-drinking, chastity-pushing, overbearingly self-righteous American hero as further inspiration for Swagger now that he has the United States Championship.
There has never been a better time for this guy to embrace his All-American American gimmick and be that Kurt Angle.
The belt should be a catalyst for all kinds of patriot-based shenanigans.
I want to see Jack Swagger wearing an Uncle Sam hat, giving us tours of American monuments, visiting national parks and landmarks, giving speeches to school children about the importance of their American heritage (in El Paso!) and cutting promos with Congressmen.
I want him draped in Old Glory on his way to the ring, coming out to "Real American" and kissing babies. (I especially want him to do this in Canada.)
Swagger needs to be a goodwill ambassador who promotes nothing of the sort.
I'm not sure I want him hunting illegals with Bradshaw but there's a lot of room for the All-American American to feud with foreign wrestlers who besmirch (I'm looking at you, Regal) the good name of the United States of America.* (Anything to get supremely talented workers like Tyson Kidd and Yoshi Tatsu on TV.)
What I want to see Swagger do the most, however, is an All-American Challenge. He needs to invite a different foreign wrestler to the ring every week and dare them to beat him in 10 minutes.
If they can't, it would somehow re-affirm Swagger's belief that America is the greatest country in the world and he'd prance around, saluting like an idiot (but an idiot who would be such an interesting and unpredictable self-parody that people would pay to see him).
There's also potential for Swagger to run a World Cup-like wrestling tournament, returning loyalty angles to an industry that was practically built on nationalism and the fear of other cultures.
* I swear I'm not trying to break any records for "most uses of the word 'America' in a single slide."
3. Resurrect the Hardcore Championsihp
The Hardcore Championship was a casualty of the WCW buyout.
Too many titles under one roof made consolidation an absolute necessity so WWE unceremoniously unified it with the Intercontinental Championship barely a month after that same strap also absorbed the European title.
In addition, when every segment devolved into a brawl, the Hardcore Championship became redundant. The 24/7 angle was milked to death and turned into a joke. The belt needed a rest.
It's been almost 11 years, however, and WWE's working environment has changed.
For one, there are no tertiary belts for the curtain-jerkers to fight over and having the Hardcore Championship exclusive to Raw while the Cruiserweight title was exclusive to Smackdown gave people incentive to watch both shows. Brand split or not, each program needs its own identity.
The Hardcore Championship is marketable again, as well. Extreme rules and gimmick matches are few and far between thanks to WWE's commitment to PG programming and a lack of novelty acts on a vanilla card has created a specialized niche where the Hardcore Championship could find success.
WWE is afraid to revisit "hardcore" because it's so closely associated with words like "bloodbath" and "massacre" but it's also an atmosphere where unique offense and innovation flourished.
Unorthodox maneuvers, a variety and clever use of weapons, inventive brawling outside the ring, stunt finishes and the imaginative use of Falls Count Anywhere: these are the things a new era of "hardcore" would have to rely on, removed from the blood and gore that WWE's sponsor/partners are so afraid of.
I'm calling for the return of Bull Rope and Four Corners matches, too. Creative match gimmicks could be used as sleight-of-hand, making fans forget they're not seeing blood.
Modern hardcore wrestlers would have to craft a new identity for the division, one where people watched because they saw things they've never seen before rather than "Guess Who's Wearing a Crimson Mask This Week."
The key is maintaining a low profile. They can't flood the market. Keep "hardcore" special, an attraction, and its many rewards will far outweigh its risks.
2. Diversify the Divas
Staying with championships for a moment, I think the Divas Championship butterfly belt is beautiful and unique. Sadly, there aren't many nice things I can say about the women's division otherwise.
When fans stay through the commercials and take their bathroom breaks during women's matches (some of which don't last long enough to wash our hands), the championship and Divas who pursue it suffer accordingly. Ask Gail Kim.
The quality of competition has bottomed out in recent years thanks to a number of glaring issues, chief among them is this revolving door, "we can get anyone to replace you with a new Diva Search or the worst NXT in history" mentality. WWE now emphasizes glam girls and badly trained models, style over substance.
There are a couple of notable exceptions within the division (the so-called Divas of Doom, for instance, when they're not booked like inept pushovers) but the cardboard cut-out valley girl routine we get from Kelly Kelly, Eve, Alicia Fox and the rest of the Divas is forgettable at best and an insult to every female performer who came before them at worst.
If you've see one makeup-wearing cheerleader, you've seen them all, and no amount of cheap pops or apple-polishing the crowd is going to make me root for a Diva who can't work. (Might've gotten someone over in the '50s, but it doesn't happen today.)
In order to elevate the Divas division, WWE needs to do two things: make its divas memorable and hire wrestlers rather than models.
To address the first point, Divas need identifiable personas beyond the superficial rich girl glitz.
Give us a gangster wannabe; a death-obsessed Goth girl; a man-hating, flannel-wearing "hardcore" diva; a super-heroine like the Hurricane; an angry evangelist on a Right to Censor-like quest to clean up the smut; whatever.
I don't care what gimmick you give them, just give them a character. Quirks. Make these girls memorable for more than just their skimpy outfits because (lemme tell ya, brother) I have the Internet and skimpy outfits aren't so memorable anymore.
As for hiring models, wrestling's a hard business that demands a hard lifestyle and women who get hired with an eye toward Playboy or future careers in other professions aren't likely to have WWE's best interests at heart (and I'm sure they're a pain to train).
There are plenty of talented women out there in the Independents, Japan or SHIMMER, and many still in TNA, that can actually work.
Vince McMahon isn't fooling anybody. Just because Kelly Kelly's booked to win matches doesn't mean she's a good wrestler with solid fundamentals and the ability to tell a story. It just means she got booked to win a match, and that's why long-term fans wanted Beth Phoenix to turn her inside out.
Awesome Kong (Kharma) can't get back fast enough.
1. "I'm Tyler Black and I'm Here to Wrestle"
Seth Rollins is close to making the jump from FCW to the big leagues. He's been touring with WWE's main roster and wrestling dark matches against Tyson Kidd, and anybody who saw him perform as Tyler Black knows the kid can go.
Seth's laundry list of accomplishments won't mean anything if he's brought in as Val Venis 2.0 or the son of "Sparky" Plugg, however, and it's important for WWE to capture the energy, intensity and pure athleticism that is Rollins' trademark (especially in this so-called Reality Era).
The first time we see Rollins on TV, FCW theme intact, he should be in the middle of the ring and declare himself on no uncertain terms: "My name is Tyler Black and I'm here to wrestle."
Invoking the memory of Dean Malenko and Chris Benoit, guys who didn't need a gimmick to get over, Seth (returning to his roots as Tyler) would come in as everything Bryan Danielson should've been without NXT.
Whatever "the American Dragon" is now, however, Tyler Black is the one who needs to make his career as a "pure athlete," someone who's a student of the sport and isn't interested in Santino's sock puppets or the trappings of sports entertainment.
He can capitalize on CM Punk's "Shoot Heard 'Round the World" by saying, "They wanted to call me 'Seth Rollins' and make me the next Nick Dinsmore. I told them 'no.' I'm not here to sing or dance or argue with Michael Cole, and I'd walk out the door if they made me wear face paint and tassels. I came to WWE for one reason and one reason only: This is where the competition is and you can't be the best until you beat the best."
He wouldn't cheat or use dirty tricks. He wouldn't cater to the fans, though, either. He'd cut short, old school promos and pay supreme respect to the craft.
WWE would be gambling on Black to bring out the "Indy" in his opponents, in stark contrast to the company style, as a way of highlighting and showcasing him. (To riff on Paul Heyman, if there's a 99 hot blondes in a room and one ugly redhead, which one stands out?)
Black needs to be that one guy, above all others, who lets his actions speak louder than words. They ought to give him the Texas Cloverleaf, as well, to help fans make a more overt connection. (Malenko built a career out of being "the Ice Man," if Black's using his move, Black must be an "Ice Man," too.)
We could also hope that Black calls out Daniel Bryan, leading to an intense "best of" series across several pay-per-views. He'd say, "You used to stand for something, Daniel. Then you got to WWE and fell for everything."


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Halle, Germany (PressExposure) April 23, 2009 -- Neuber Software has released Visual TimeAnalyzer 1.3 v2009, a Windows application that automatically tracks working time, software and Internet use. The software presents detailed, richly illustrated reports.
Easily log individual users or particular projects, and record detailed accounts of time spent within specific programs. The logger monitors all activities on familyâs PC or companyâs network. Parents have control over their children's PC use. You learn which programs were used for how long, when, and by whom.
Visual TimeAnalyzer analyses software usage at individual workstations or across the network. You can choose detailed reports and analysis graphs for time tracking, time management, project tracking, project management, project cost or attendance. Select user and time periods and see: Weekly statistics, hourly computer use, most used programs, online time, active working time, pause times, opened documents, projects, daily summary (diary), history and intensity of program usage (ideal for project overview), extent of the use of available programs, visited web pages (time, URL, title), and TopTen of the most popular web pages. Keep user data (like passwords and personal documents) safe: Visual TimeAnalyzer does not record keyboard inputs or run background screencaptures.
Visual TimeAnalyzer v. 1.3 runs under Windows Vista,XP,2000,2003,2008, and costs $39(US) for a single license. Volume discounts are available. A free, fully-functional trial version is available for download at http://www.neuber.com/timeanalyzer/.

subset: commoncrawl


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a man of many wonderful things, he only exists in the 27th dimension, and loves to play 'video games' he is usually a giant and sometimes dead.
you like neesen?
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Texting Program will Reduce the ER Wait Times
MetroWest Medical Center has come up with its first texting program that will allow patients to check for the ER waiting time before coming to a hospital.
According to a research published in Medical Care Research and Review, patients who are admitted in those hospitals which have the most advanced type of EMRs spend 22.4% less time in the emergency room (ER), as compared to those hospitals which have basic EMR facilities.
The Boston-based hospital has started the service from Aug. 9. The hospital is also hopeful that this new program will boost up its market share and improve the ER services.
Since the start of this service, as many as 450 patients have inquired about the ER waiting time through their mobiles at Framingham Union Hospital and Leonard Morse in Natick.
In the previous week, the normal wait times were 24 minutes and 6 minutes, respectively, for one day.
The wait time is calculated as the time the patients come in the hospital to the time when they consult a doctor.
To know the ER wait time, the patients need to text 63311 to 437-411 and the wait time will be sent back right away.
Chief Executive, Andrei Soran says, "We're trying to promote better customer service and show people they can be better served by staying [at a hospital] in their own community".


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New Delhi/Mumbai, Sep 25 (UNI) Customers across the country were put through great hardship as bank operations were crippled for the second day today with employees and officers participating in a two-day nationwide strike to press for their demands.
The two-day strike had been called by the United Forum of Bank Unions (UFBU) to protest the proposed privatisation of PSU banks, government's plan to merge banks, reject Raghuram Rajan and Anwarul Hoda committee recommendations and stoppage of unrestricted entry of FDI in banks.
The strike, which had hit the clearing operations, was almost total in Maharashtra though some foreign banks were functioning normally.
Dharnas and rallies were organised in Patna, Muzaffarpur, Gaya, Munger, Purnea, Bhagalpur, Motihari and several other places in Bihar by the bank employees. Clearing house operations in different parts of the state also remained paralysed resulting in the suspension of clearing of cheques worth more than Rs 5,000 crore.
ATM services of several banks were also affected.
This is the second countrywide bank strike this year to press for the employees' demand, causing severe inconveniences to millions of customers across the nation.
In Kerala, agitating employees held demonstrations in front of banks. Even the private sector banks closed their offices in some places. Many banks could not open due to shortage of staff. New generation banks such as ICICI, HDFC, AXIS and Centurion also closed their branches in Kozhikode following intervention by those supporting the strike. Later the strikers, numbering about 300, held a demonstration before the State Bank of India office at Mananchira in Kozikode.
The strike affected an estimated business of Rs 20-25,000 crore in Chhattisgarh.
About 3,000 employees of 65 bank branches in Puducherry struck work and assembled in front of the UCO Bank today where they staged a dharna. ATM services were also totally affected causing hardships to traders and general public.
In Visakhapatnam, several bank employees and union leaders resorted to agitation before State Bank of India main branch in support of their demands. Operations was also affected in Vizianagaram, Srikakulam and Visakhapatnam Districts.


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GraphicRiver Business and Finance Icons 9284807
Category: Stock Vector | Comment: ( 0 )
GraphicRiver Business and Finance Icons 9284807
Stock Vector \ Conceptual \ Business
Internet \ Lock \ Illustration \ Icons \ Gold \ Graph \ Market \ Money \ Symbol
Stock Vector - Business and Finance Icons 9284807 by timurock @ GraphicRiver
Stock Vector \ Conceptual \ Business
Graphics Files Included: Vector EPS; Layered: No; Minimum Adobe CS Version: CS.
Set of 16 blue business and finance icons. This file is fully editable vector illustration. It can be scaled to any size. All its parts are grouped for easy customization.
Web Icons; Web Icons; Landmarks; E-mail Icons; Fitness Icons; Environmental Icons; Christmas Icons.
Keywords: bag, bank, blue, business, buttons, buy, card, cash, chart, coin, credit, currency, dollar, euro, exchange, finances, gold, graph, icons, illustration, internet, lock, market, money, pound, retail, safe, scales, symbol, vector.


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MOVIE SYNOPSIS [ General Audiences ] With producers of USA, this movie of 2014 has been brilliantly directed by Rydrea Walker. You can watch Battle of Evil: Part 2 online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2014.
Rating: 5 by 10 users./
Runtime: 58 minutes
Cody Maddox, Debbie Wheatley, Ashley Laster, Brittney Fisher, Joshua Edwards, Uriah Stallworth, Cordell Woosley, Marshe' Brownlee, Marc. Wallace Jr., Xavius Turner, Whittney Fisher, Isaac Stevenson, Rashard James, Payton Sutls and Anthony Scott.
Drama, Terror.
58-minute film (full movie) directed by Rydrea Walker.
The original audio of Battle of Evil: Part 2 has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2014.
Screenplay written by Rydrea Walker.
Quintrell Little.
Kelly Ciganek.
This film was produced and / or financed by Silent Production and Walker Films - ASL.
Walker Films - ASL.
Without commercial breaks, the full movie Battle of Evil: Part 2 has a duration of 58 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, Battle of Evil: Part 2 in FULLTV Movies.


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Dent's disease, an inherited disorder characterized by hypercalciuria, nephrolithiasis, nephrocalcinosis, rickets, low-molecular-weight proteinuria, Fanconi's syndrome, and renal failure, is caused by mutations in the renal chloride channel, CLC5. The normal role of CLC5 is unknown. We have investigated the intrarenal and subcellular localization of CLC5 in rat kidney by in situ hybridization and immunohistochemistry. By in situ hybridization, CLC5 mRNA was detected predominantly in cortical medullary ray and outer medullary tubule epithelial cells. Polyclonal antiserum was generated against a CLC5 fusion protein, affinity purified, and immunoadsorbed against CLC3 and CLC4 to yield a CLC5 isoform-specific antiserum. By immunohistochemistry, CLC5 protein was localized to the intracellular domain of tubular epithelial cells in the S3 segment of the proximal tubule and the medullary thick ascending limb. By subcellular membrane fractionation and flow cytometry, CLC5 expression was found in outer medullary endosomes. These findings are consistent with a model in which CLC5 encodes an endosomal chloride channel that facilitates acidification and trafficking of renal epithelial endosomes.
- chloride channel
- calcium
- nephron
- endosome
- Dent's disease
dent's disease, a disorder characterized by hypercalciuria, nephrolithiasis, nephrocalcinosis, rickets, low-molecular-weight proteinuria, Fanconi's syndrome, and renal failure (33), has recently been mapped to the renal chloride channel gene, CLC5 (15, 16, 19, 33). Chloride currents expressed by mutant CLC5 in Xenopus oocytes are significantly reduced or absent compared with those of wild-type CLC5 (15), suggesting a causal role for loss of CLC5 function in this disease. The mode of presentation of Dent's disease and its clinical severity is quite variable, with affected members in some families developing end-stage renal disease or rickets and others having only persistent hypercalciuria or low-molecular-weight proteinuria.
Exactly how loss of CLC5 function leads to the broad clinical spectrum encompassing Fanconi's syndrome, hypercalciuria, and low-molecular-weight proteinuria has not yet been explained. We (8) and others have postulated that CLC5 is expressed on the renal tubular endosomal membrane where it would provide the chloride conductance required for endosomal acidification, a prerequisite for efficient membrane protein recycling. Interference with apical membrane transport protein recycling and endocytosis would lead to defective reabsorption of glucose, amino acids, phosphate, uric acid, and low-molecular-weight proteins in the proximal tubule and of calcium in distal nephron segments. Dent's disease is rare, but the problems of "idiopathic" hypercalciuria and nephrolithiasis are significant in the broader population, in a subset of whom CLC5 dysfunction may be a contributing factor. The nephron segment localization of CLC5 may provide important clues to its physiological function.
The intrarenal distribution of CLC5 is controversial. By RT-PCR of microdissected tubules (30), CLC5 was found to be expressed predominantly in the cortical collecting tubule, S3 segment of proximal tubule, and medullary thick ascending limb of the loop of Henle (MTAL). RT-PCR, however, is not entirely specific, because cross-contamination of tubule samples cannot unequivocally be excluded. Also, the technique is not quantitative and so cannot distinguish between physiological levels of expression and minute amounts of contamination. By contrast, a recent in situ hybridization study localized CLC5 mRNA only to type A intercalated cells of the collecting duct (17). No studies of CLC5 protein expression and subcellular localization have been reported. In the present study, we have determined the nephron segment and subcellular localization of CLC5 mRNA and protein by in situ hybridization and immunohistochemistry.
Preparation of riboprobes and Northern blot analysis. A CLC5 cDNA containing the full coding region (corresponding to nucleotides 11–2550, GenBank accession no.D50497; Ref. 24) was cloned by RT-PCR from rat kidney cortex. A 770-bp CLC4 cDNA sequence was cloned in the same manner (corresponding to nucleotides 1034–1798, GenBank accession no. Z49916; Ref. 23). Digoxigenin (Dig)-labeled sense and antisense riboprobes were prepared by in vitro transcription with T3 or T7 RNA polymerase. For in situ hybridization, riboprobes were treated by alkaline hydrolysis to yield fragments of ∼200 bp. Northern blots were performed at high stringency using the Dig Genius System (Boehringer-Mannheim, Indianapolis, IN) as described previously (35).
Preparation of tissue cryosections. Adult male Sprague-Dawley rats were anesthetized with pentobarbital and perfused via the infrarenal aorta with 4% paraformaldehyde followed by 750 mosmol/kg of sucrose-PBS. Perfused kidneys were then removed, equilibrated in 30% sucrose overnight at 4°C, embedded in Tissue-Tek (Sakura, Torrance, CA), and frozen in isopentane. For immunohistochemistry, 5-μm thick sections were cut on a cryostat, mounted on Superfrost-Plus glass slides (Fisher, Pittsburgh, PA), and air dried for 20 min. For in situ hybridization, unperfused kidneys were used, which were prepared similarly except that cryosections were 10 μm thick.
In situ hybridization. Our protocol was modified from previously published methods (25). Cryosections were fixed for 15 min in 4% paraformaldehyde at room temperature, washed three times in PBS, acetylated for 10 min, washed twice in PBS and once in 2× SSC. Sections were then prehybridized at room temperature for 6–8 h. Hybridization was performed in a humidified chamber overnight at 70°C with 400 ng/ml hydrolyzed riboprobes in the presence of 50% formamide. High-stringency washes were performed at 70°C with 2× SSC for 1 and 5 min, followed by 0.2× SSC twice for 30 min. Immunologic detection of Dig-labeled probes was performed using the Dig Genius System. Sections were rinsed in washing buffer and incubated for 1 h in blocking solution, followed by 1 h in blocking solution containing alkaline phosphatase-conjugated anti-digoxigenin antibody at 1:500 concentration. Following two 30-min washes in washing buffer, sections were transferred to detection buffer for 5 min. For the color reaction, sections were incubated upside-down in the dark with detection buffer containing nitro blue tetrazolium chloride, 30 mg/ml, and 5-bromo-4-chloro-3-indolylphosphate, 15 mg/ml, for up to 8 h, washed, air dried, mounted with Vectashield (Vector Laboratories, Burlingame, CA), and examined under light microscopy.
Generation and affinity-purification of the C1 antiserum. A cDNA encoding a 108-amino acid polypeptide from the region of the carboxy terminus of rat CLC5 (residues 570–677, GenBank accession no. Z56277; Ref. 30) that shares the least identity to the homologous region of either of the closely related isoforms CLC3 (62% identity, GenBank accession no. D17521; Ref. 14) and CLC4 (65% identity, GenBank accession no. Z36944; Ref.11) was cloned into the bacterial expression vector, pMAL-c2 (New England Biolabs, Beverly, MA), downstream of the maltose-binding protein (MBP) coding sequence and a recognition site for the specific protease, factor Xa. Synthesis of the MBP-CLC5 fusion protein under the control of a lacZα promoter was induced by isopropyl-β-d-thiogalactopyranoside. The protein was purified by binding to an amylose column and used to immunize two rabbits by footpad injection, followed by four booster injections, and tested for reactivity to the fusion protein by ELISA.
Serum from the rabbit with the highest titer of specific antibody was collected and affinity purified in a two-step, positive-negative selection procedure. Antibodies with specificity for MBP were adsorbed away by incubation of serum with an MBP-galactose fusion protein (MBP-gal) immobilized on polyvinylidene difluoride (PVDF) membrane. Antibodies with specificity for CLC5 were then selected by incubation of the negatively selected serum with MBP-CLC5 fusion protein bound to PVDF and elution with 0.2 M glycine-HCl, pH 2.6. The eluate was neutralized to pH 7.4 and dialyzed against PBS, to yield C1 antiserum.
Preparation of the isoform-specific C2 antibody fraction. To select a CLC5 isoform-specific antibody fraction, C1 serum was subjected to a further round of selection in which cross-reacting antibodies to the CLC3 and CLC4 isoforms were removed by immunoabsorption. MBP fusion proteins containing the homologous carboxy-terminal regions of CLC3 (residues 584–691) and CLC4 (residues 571–678) were immobilized on PVDF and incubated with C1 overnight at 4°C. The unbound serum fraction was designated as C2.
Western blotting. Protein samples were denatured by boiling in 2% SDS, electrophoresed in discontinuous polyacrylamide gels, electrophoretically transferred to PVDF membrane, immunoblotted with the C1 antibody at 1:100 dilution or C2 at 1:25 dilution, and detected using the ECL enhanced chemiluminescence system (Amersham, Arlington Heights, IL). The following items were used as negative controls. 1) C1 serum was preincubated overnight at 4°C with equimolar MBP-CLC5 fusion protein (peptide-blocked C1). 2) Preimmune serum was diluted 1:1,000 to the equivalent immunoglobulin concentration to C1 (preimmune serum).3) Antibodies with specificity for MBP (anti-MBP) were isolated from the eluate of the first adsorption step in affinity purification described above, and used at 1:5,000.
The specificity of the C1 and C2 antibodies was tested by immunoblotting against MBP-CLC3, -CLC4, and -CLC5 digested with factor Xa, which proteolytically cleaves these fusion proteins into separate MBP and CLC polypeptides. Immunoblots were also performed on crude kidney membranes prepared from rat and mouse kidneys by a standard differential centrifugation technique (6). The 47,000g pellet and supernatant were considered to be the crude membranes and cytosolic fraction, respectively.
Immunohistochemistry. C1 antibody staining was performed using fluorescein isothiocyanate (FITC) or cyanine-3 (CY3) with the tyramide signal amplification system (NEN Life Science Products, Boston, MA). Cryosections, 5 μm thick, were thawed and rehydrated in PBS. Sections were permeabilized for 5 min in 1% SDS, washed three times, then incubated for 1 h in 5% goat serum/1% BSA in PBS at 37°C to reduce nonspecific background staining. Sections were then blocked sequentially with a drop of avidin and biotin (Vector Laboratories), each for 5 min at 37°C. After one rinse in PBS, sections were incubated overnight at 4°C with a 1:50 dilution of the primary antibody (C1). Sections were sequentially washed in high-salt PBS then regular PBS and then incubated at 37°C with 1:800 dilution of the secondary antibody, biotinylated goat anti-rabbit IgG, for 1 h. Sections were then washed and incubated with 0.3% H2O2-90% methanol for 30 min at room temperature to block endogenous peroxidase activity. After four washes in PBS, tyramide amplification was carried out according to manufacturer's recommendations, except that FITC-conjugated tyramide was used at 1:100 and the CY3-conjugated tyramide was used at a 1:250 concentration. Sections were mounted in Vectashield (Vector Laboratories) and viewed by epifluorescence microscopy.
C2 antibody staining was performed without amplification at a 1:2 dilution and detected with FITC- or Texas Red-conjugated secondary antibody at 50 ng/μl concentration. To evaluate the specificity of staining of C1 and C2 antibodies on tissue sections, C1 and C2 were peptide-blocked by overnight preincubation at 4°C with up to fourfold molar excess of MBP-conjugated CLC5 peptide.
In double-labeling experiments, the polyclonal antibody to calbindin-D28k (CBP) (2) (generous gift from Dr. René Bindels) was used at a concentration of 1:250, polyclonal anti-bumetanide-sensitive Na-K-2Cl cotransporter (BSC1) (13) at 1:500, and polyclonal anti-thiazide-sensitive NaCl cotransporter (TSC) (18) at 1:500; and the E11 monoclonal antibody against the 31-kDa subunit of the vacuolar H+-ATPase (9) (generous gift from Dr. Stephen Gluck) was applied undiluted. Artifacts due either to cross-reactivity of the second secondary antibody to the first primary antibody (when the two primary antibodies were derived from the same species) or to spectral overlap between the two fluorophores were excluded by one or more of the following control experiments. 1) The pattern of staining of sections with each primary antibody when applied individually was demonstrated to be identical to the staining by the same antibody in double-labeled sections.2) The staining pattern was unchanged when the order of application of the two primary antibody was reversed. 3) The first primary antibody was applied at a very low concentration (undetectable without amplification) and amplified with tyramide, whereas the second primary antibody was detected with unamplified fluorophore-conjugated secondary antibody.
Flow cytometry analysis of endosomes. True endosomes were isolated from rat kidneys and labeled with C1 or C2 antisera by the indirect sandwich technique, as previously published (7). In brief, the kidneys of adult male Sprague-Dawley rats were harvested without pretreatment or 10 min after injection of intravenous FITC-dextran (average molecular mass, 10 kDa) as an entrapment marker for endosomes or with nonfluorescent dextran as a negative control. The cortex and outer medulla were isolated by macroscopic dissection and homogenized, and membranes of low buoyant density were enriched by successive rounds of differential centrifugation followed by separation on an 18% Percoll gradient at 48,000g. Light endosomes, which have previously been shown to represent a true endosomal population (7), were removed from the top of the gradient.
Blocking of nonspecific binding sites was performed on aliquots of endosomes by incubation with 50% clarified normal goat serum for 2 h at 4°C. Endosomes were then incubated overnight at 4°C in C1 or C2 antisera at dilutions ranging from 1:33 to 1:10,000, washed, incubated for 4 h at room temperature with the phycoerythrin-conjugated goat anti-rabbit secondary antibody and washed again. Flow cytometry was performed on a Becton-Dickinson FACStar flow cytometer using 100-mW excitation of blue 488-nm light from a 5-W argon laser, as described previously (12). Data were collected on 2,000 individual endosomes in a list mode file. The binding of antibodies to different endosomal populations was compared using Kolgomorov-Smirnoff summation statistics (34).
Intrarenal localization of CLC5 by in situ hybridization. The intrarenal distribution of CLC5 mRNA was determined by high-stringency in situ hybridization. A CLC5 riboprobe that encompasses the full coding region was prepared and shown to be isoform specific by high-stringency Northern blot. As shown in Fig.1 A, the CLC5 probe hybridizes to a single band of ∼9.5 kb, the expected size for CLC5 (30), with no cross-hybridization to the predominant 4.4-kb transcript for CLC4 (11). Figure 2 shows rat kidney cryosections hybridized to antisense and sense CLC5 probes. Prominent staining with the antisense probe was seen within tubule epithelial cells in the medullary rays and outer stripe of outer medulla, consistent with the distribution of the S3 segments of proximal tubules. In the inner stripe of the outer medulla, strongly positive staining was seen in a distinct subset of tubules that appeared to be the thick ascending limbs of the loop of Henle. Staining in the inner medulla and papilla was sparse. Minimal background staining was observed in control sections hybridized with the sense probe.
Generation of a polyclonal antisera to CLC5. An affinity-purified polyclonal antibody (C1) was prepared against a CLC5 fusion protein, so that the cellular and subcellular localization of CLC5 could be further refined. As shown by Western blotting in Fig. 1 B, the C1 antibody binds to a single peptide of ∼80 kDa (the predicted molecular mass for rat CLC5 is 83 kDa) in both mouse and rat kidney. As expected for a membrane transport protein, immunoreactivity is confined to the crude membrane fraction and is absent from the cytosolic fraction. C1 also recognizes the 55-kDa intact MBP-CLC5 fusion protein against which the antisera was originally raised. Preimmune serum, as well as C1 serum blocked with peptide, showed no immunoreactivity in negative control blots. This indicates that C1 contains antibodies specific for CLC5. C1 binds neither to the negative control construct, MBP-gal, nor to a 42-kDa degradation fragment found in the MBP-CLC5 peptide sample. MBP antiserum was used to demonstrate that the 42-kDa fragment is in fact the MBP peptide, spontaneously cleaved off from its CLC5 portion. This confirms that our affinity purification method effectively removed all traces of MBP-reactive antibody from the C1 serum.
Although we specifically selected the region of the CLC5 protein against which our antiserum was raised on the basis of least similarity to homologous isoforms, the C1 antiserum does in fact cross-react with CLC3 and CLC4 (Fig. 1 C). We therefore further prepared a CLC5 isoform-specific antibody fraction by immunoadsorption of C1 against CLC3 and CLC4 fusion proteins immobilized on solid supports. The resultant C2 serum fraction was found to be highly specific for CLC5 by Western blot (Fig.1 C).
Immunohistochemical localization of CLC5. Immunofluorescence was performed on perfusion-fixed kidney sections with C1 and C2 antisera. To confirm the identity of specific nephron segments, double labeling was performed with either anti-CBP antibody (marker for distal convoluted tubule and connecting segment), anti-BSC1 (marker for apical membrane of thick ascending limb), anti-TSC (marker for apical membrane of distal convoluted tubule), or anti-vacuolar-type H+-ATPase (marker for apical membrane of type A and basolateral membrane of type B intercalated cells of collecting duct). As shown in Fig.3, C1 staining is present in tubule epithelial cells of the renal cortex and outer medulla but absent from glomeruli, blood vessels, or the interstitium. C1 staining is seen strongly on the apical membrane and subapical region of proximal convoluted and straight tubules with a faint intracellular signal. The specificity of C1 binding was confirmed by demonstrating that preincubation with CLC5 peptide blocks binding in both the cortex and outer medulla (Fig. 3, b andd). In double-labeled sections (Fig.4,a–d), C1 staining is clearly absent from calbindin-D28k-positive cells in the renal cortex but is present on the apical membrane of calbindin-D28k-negative cells, which are presumed to be intercalated cells of the connecting segment (Fig. 4 a). C1 stains intracellularly in distal convoluted tubule cells, identifiable by apical TSC staining (Fig. 4 b). C1 staining is absent from cortical BSC1-positive tubules (data not shown), but in the outer medulla, intracellular C1 staining is observed in MTAL cells that are also positive for apical BSC1 (Fig.4 d). In the cortical collecting duct, C1 staining colocalizes with the vacuolar-type H+-ATPase to the apical membrane of type A intercalated cells and the apical and basolateral membranes of type B intercalated cells (Fig.4 c). In summary, C1 staining is present on the apical membrane domain of the proximal tubule and type A intercalated cells, on the apical and basolateral membranes of type B intercalated cells, and in the intracellular compartments of the proximal tubule, MTAL, and distal convoluted tubule.
Because C1 cross-reacts with CLC3 and CLC4, we repeated our immunohistochemistry studies with the highly CLC5-specific C2 antiserum. C2 staining is seen intracellularly in tubules of the medullary ray and outer medulla and is absent from the inner medulla (Fig. 3). In double-labeled cortical sections, C2 staining is seen to localize to S3 segments of proximal tubule but, in contrast to C1, is absent from distal convoluted tubule, connecting segment, cortical thick ascending limb, and cortical collecting duct (Fig. 4,e–i). In outer medulla, C2 stains MTAL and colocalizes to tubules with apical BSC1 staining (Fig. 4 j).
In summary, our results indicate that CLC5 is expressed in the intracellular compartments of the S3 segment of the proximal tubule and the MTAL (Fig. 5).
CLC5 is expressed in outer medullary endosomes. To determine whether intracellular CLC5 is located in endosomes, rats were preloaded with intravenous FITC-dextran as a freely filtered luminal entrapment marker (or nonfluorescent dextran as a negative control) and apical membrane-derived endosomal vesicles isolated from the outer medulla, where the majority of CLC5 immunoreactivity was identified. CLC5 epitopes exposed on the cytoplasmic side of the endosomal membrane were labeled with either C1 or C2 primary antibody and indirectly conjugated with a phycoerythrin-labeled secondary antibody, and the vesicles were subjected to two-color fluorescence flow cytometry. The results are displayed as three dimensional histograms in Fig.6 A. Vesicles that were isolated from rats loaded with nonfluorescent dextran and not labeled with any primary antibody exhibit minimal background fluorescence, as shown by the low values on both horizontal axes in Fig. 6. Vesicles derived from rats loaded with FITC-dextran demonstrate significant fluorescence in the bandwidth for FITC emission (left horizontal axis in Fig. 6), demonstrating entrapment of the fluorophore by endocytosis, as shown previously (7). When FITC-dextran-loaded vesicles were labeled with C1 antiserum, substantial antibody binding was observed, as evidenced by the high phycoerythrin fluorescence values (right horizontal axis). Furthermore, there was a close correlation between phycoerythrin and FITC fluorescence, indicating colocalization of C1 immunoreactivity and entrapped FITC-dextran to the same vesicle population. The dilution curve of the amount of antibody bound to endosomal vesicles plotted against the dilution of C1 used was nonlinear (data not shown) and highly suggestive of specific binding.
To confirm that CLC5 is expressed in endosomes, these experiments were subsequently repeated using the CLC5 isoform-specific antibody fraction, C2 (Fig. 6 B). Outer medullary light endosomal vesicles demonstrated a bimodal phycoerythrin fluorescence distribution (righthorizontal axis), with approximately one-third of vesicle binding in a highly specific manner to C2. Furthermore, the size distribution (as estimated by side scatter, lefthorizontal axis) of the subpopulation of C2-positive endosomal vesicles was similar to that of the whole population, excluding vesicle aggregation as an artifactual cause of increased fluorescence. These data confirm that CLC5 protein is expressed on endosomes.
Using in situ hybridization and immunohistochemistry, we have localized CLC5 within the rat kidney. In initial immunohistochemical studies using C1 antiserum, staining was seen in several different nephron segments on both the plasma membrane and in intracellular compartments. The staining observed with C1 could not, however, be assumed to represent only CLC5 expression, because although attempts were made to maximize its specificity, C1 was found to cross-react with CLC5's closest homologs, CLC3 and CLC4 (Fig.1 C). Accordingly, it was necessary to purify our antiserum by immunoadsorption with CLC3 and CLC4 peptides to remove cross-reacting antibodies.
Using the purified antiserum, C2, CLC5 was found to be strongly expressed in the intracellular compartments of both S3 segments of proximal tubules and the MTAL. These findings are likely to represent true CLC5 staining, because 1) C2 was shown to be CLC5 isoform-specific by Western blot analysis (Fig.1 C);2) the results are in full agreement with our in situ hybridization, which was performed at high stringency, under which conditions we have clearly demonstrated that our CLC5 riboprobe does not cross-react with CLC4 mRNA (Fig.1 A); and3) our results are in general agreement with previous work in the rat kidney in which CLC5 mRNA was localized by RT-PCR (30). Because our immunoadsorption technique would be expected to remove antibodies reactive to epitopes common to CLC5 and either CLC3 or CLC4, we cannot formally exclude the possibility that CLC5 may exist in several antigenically distinct forms and that reactivity to one or more of these forms may have been lost with immunoadsorption. Some CLC5 staining may therefore have been lost with C2 antiserum.
Our results by both in situ hybridization and immunohistochemistry do not agree with those of a recent in situ hybridization study in which CLC5 was reported to be expressed only in type A intercalated cells (17). There are two main differences in in situ hybridization methodology between that study and ours, which might perhaps explain this discrepancy. First, Obermüller et al. (17) perfusion-fixed their kidneys with paraformaldehyde, which, although this does indeed improve tissue morphology, in our experience appears to hinder access of riboprobe to cellular RNA and reduces signal-to-noise ratio. Second, our hybridization conditions were more stringent than those used by Obermüller et al. (42°C in 50% formamide; Ref. 17).
By flow cytometry analysis of subcellular membrane fractions, we found that CLC5 was expressed in the endosomal compartment of cells in the outer medulla (as expected for the S3 segment of proximal tubule and MTAL). Very few electrophysiological studies of rat kidney endosomal chloride channels have been performed (21, 27), and these report widely differing properties. It is difficult to speculate whether any of the reported channels correspond to CLC5, since the electrophysiological properties of CLC5 have only been characterized briefly, only at the whole cell level, and only in the unphysiological setting of heterologous cell expression systems.
The proximal tubule is the site of sodium-coupled reabsorption of solutes such as glucose, amino acids, and phosphate as well as the site of endocytosis of low-molecular-weight proteins. It is therefore not surprising that CLC5 is expressed in this tubule segment, given that a partial Fanconi's syndrome and low-molecular-weight proteinuria are prominent features of Dent's disease. The localization of CLC5 to the endosomal compartment in this tubule segment is consistent with a role in providing counter-ion conductance for the electrogenic vacuolar H+-ATPase pump, thereby permitting adequate endosomal acidification. Indeed, optimal endosomal acidification has been shown to require the presence of cytoplasmic chloride ions and to be inhibited by nonspecific chloride channel blockers (1). However, if CLC5 is a strong outward rectifier (as observed when expressed on the plasma membrane ofXenopus oocytes; Ref. 30), then it would not be predicted to provide a significant conductance for chloride entry into the endosome. This apparent paradox may be explained if the in vivo physiological properties of CLC5 differ from those observed in Xenopus oocytes, either simply because of artifacts arising from the use of an amphibian expression system or because CLC5 normally associates with other peptides to form a multi-subunit channel with different functional properties. Since endosomal acidification is necessary for the normal recycling of plasma membrane proteins (5, 20), the proximal reabsorption defects in Dent's disease might therefore be due to defective endocytosis of luminal low-molecular-weight proteins, and defective endosomal recycling of plasma membrane transporters responsible for sodium-coupled reabsorption of solutes such as glucose, amino acids, and phosphate.
Our results also raise some interesting hypotheses for the pathogenesis of hypercalciuria in Dent's disease. Transepithelial calcium transport and its regulation by calciotropic hormones is conventionally believed to be primarily mediated by the distal convoluted tubule and the connecting segment (3, 10, 28, 29), yet C2 staining was absent from these cells. As discussed previously, it is possible, although unlikely, that C2 may have missed CLC5 expression that was detected, for instance in the distal convoluted tubule, with C1. C2 staining, and therefore CLC5 expression, is, however, clearly present in the MTAL. This staining is unlikely to represent cross-reactivity with CLC-K1 or -K2 (also expressed in the MTAL), as these channels share less than 20% homology with CLC5 in the carboxy-terminal region. The thick ascending limb is not only an important site for paracellular calcium reabsorption, but it may also mediate active, transepithelial calcium transport in certain species, when stimulated by various peptide hormones (4, 31, 32). The possible mechanisms whereby loss of MTAL CLC5 function could lead to hypercalciuria are therefore:1) defective endosomal recycling of an MTAL plasma membrane calcium transporter, directly preventing transepithelial calcium reabsorption;2) defective recycling of MTAL plasma membrane transport proteins in the sodium chloride reabsorption/potassium recycling pathway, leading to loss of the transepithelial voltage gradient, a Bartter-like syndrome, and reduced paracellular calcium reabsorption. The absence of metabolic alkalosis in these patients, however, argues against the latter possibility. Finally, hypercalciuria could be a secondary phenomenon in Dent's disease, for example, as a consequence of proximal tubule phosphate leak, hypophosphatemia, and suppression of parathyroid hormone (PTH) secretion, or could alternatively be due to a primary alteration in vitamin D metabolism, such as enhanced 1-hydroxylation of 25-hydroxyvitamin D in the proximal tubule (22). Such a hypothesis would be consistent with the finding in Dent's disease of an absorptive component to the hypercalciuria in all patients, normal or low intact PTH levels, and normal or high 1,25-dihydroxyvitamin D levels (26).
In conclusion, CLC5 is predominantly expressed intracellularly in tubule epithelial cells of the renal outer medulla and is found on endosomal membranes derived from this region. The finding that CLC5 is expressed in endosomes of the MTAL and S3 segment of the proximal tubule lends significant weight to the hypothesis that endosomal dysfunction may be a unifying cause of the clinical features of Dent's disease.1
We thank Dennis Brown, Urs Berger, Daniella Riccardi, Jason Xu, and Joe Mindell for generously sharing time and expertise, Olivier Devuyst for sharing unpublished data and helpful comments, and Barry M. Brenner for support and advice.
Address for reprint requests: A. S. L. Yu, Renal Division, Brigham and Women's Hospital, 77 Ave. Louis Pasteur, Boston, MA 02115.
This work was supported by a Joseph E. Murray Research Award from the National Kidney Foundation of Massachusetts and Rhode Island (to A. S. L. Yu).
↵1 Shortly after this manuscript was accepted for publication, a study by W. Günther, A. Lüchow, F. Cluzeaud, A. Vandewalle, and T. J. Jentsch (Proc. Natl. Acad. Sci. USA 95: 8075–8080, 1998) reporting the immunolocalization of CLC5 in rat kidney was published. In this study, polyclonal antibodies were raised against two CLC5 peptides that share identity over at least 12 of 15 amino acid residues with either CLC3 or CLC4. The authors attempted to remove cross-reactivity by immunoadsorbing their serum against CLC4 peptide and testing the specificity of the resultant antibodies against Western blots of membranes isolated fromXenopus oocytes injected with CLC3, CLC4, and CLC5 cRNA. However, these studies were inconclusive, because no evidence was presented that CLC3 or CLC4 protein was expressed in the oocytes, and indeed this group have previously reported absence of functional expression of CLC3 and CLC4 in this preparation (J. Physiol. 482: 19S–25S, 1995). Of note, their immunohistochemistry findings with these antibodies are strikingly similar to our initial findings with our C1 antibody (which cross-reacts with CLC3 and CLC4), namely, staining of the subapical domain of proximal tubule with predominance in the S1 segment, and staining of the apical membrane of α-intercalated cells, as well as apical and basolateral membranes of β-intercalated cells of the collecting duct. In the light of our quite different findings with the CLC5-specific C2 antibody, it is possible that their antibodies may have stained primarily CLC3 or CLC4.
- Copyright © 1998 the American Physiological Society


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Light, bright, and beautiful, our 2016 Sauvignon Blanc is your perfect summer date. Notes of green apple, wildflowers, and honeydew melon are balanced elegantly with rich minerality. Bright acid on the palate creates a lovely tangy apple flavor. Enjoy chilled with fun folks and good food.


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The End Product
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Hi there and welcome to the Stencil Girl and Colourcraft Blog Hop!
I am sharing two cards today with quick instructions for the backgrounds.
As this is a blog hop we have a Giveaway for you, of course!!!
One lucky winner will receive both a $25 Gift Certificate to StencilGirl Products
AND a $25 Gift Certificate to Colourcraft!
AND a $25 Gift Certificate to Colourcraft!
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Susanne Rose <-- you are here
This is how you can make the backgrounds for these cards:
I made two gelli prints with black acrylic paint and the lovely Flower stencils. The first print is the one in the middle, the second is the ghost print on the right. The left one is where I've cleaned the stencils. I didn't use this for my cards, but I save it for later.
Now I added water with a spray bottle and Brusho powders. I used purple, gamboge, olive green, sea green and orange. I cut the backgrounds for my cards with a stitched frame die and build the cards together.
I embossed the kraft card base with WOW! Black Glint powder and a stamp from STAMPlorations. I adhered my Brusho background at an angle with foam dots. Finally I added the sentiment.
I cut the sentiment with the Tim Holtz Word Block dies from Sizzix from some Brusho - scrap paper.
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Black and Mild Cigars are a popular Pennsylvania made plastic tip cigars brand. They are created with Middleton's pipe tobacco for a smooth, mild smoke. Black and Mild Cigars are available in several flavors too like apple and cream. The experience is unique, worthy of anyone who already enjoys these cigars
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Why Vis?. David H Rogers, Scalable Data Analysis and Visualization Sandia National Laboratories [email protected] July 19, 2012 SAND Number: 2012-5928C. What's the Goal of Visualization?. "It's stories, all the way down." David Rogers "Simple design. Intense content." Edward Tufte.
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David H Rogers,
Scalable Data Analysis and Visualization
Sandia National Laboratories
July 19, 2012
SAND Number: 2012-5928C
"It's stories, all the way down."
David Rogers
"Simple design. Intense content."
Edward Tufte
Charles Minard's story of Napoleon's March to Moscow, War of 1812. Band shows size of the army along the advance and retreat routes, superimposed over a map, with temperature and time scales below. Tufte, The Visual Display of Quantitative Information
After a few minutes of rendering, the new plot appeared, and I was a bit taken aback by what I saw. The blob had turned into a surprisingly detailed map of the world. Not only were continents visible, certain international borders were apparent as well. What really struck me, though, was knowing that the lines didn't represent coasts or rivers or political borders, but real human relationships. Each line might represent a friendship made while travelling, a family member abroad, or an old college friend pulled away by the various forces of life.
Paul Butler, in a blogpost: http://www.facebook.com/notes/facebook-engineering/visualizing-friendships/469716398919
In-Situ Visualization for Large-Scale Combustion Simulations, Yu, Wang, Grout, Chen, Ma IEEE Computer Graphics
An Exploratory Technique for Coherent Visualization of Time-Varying Volume Data, Tikhonova, Correa, Ma, EuroVis2010Combu
Powerful set of capabilities, plus design
Here's an example …NodeBox= Power + Beauty
Sandia National Laboratories is a multi- program laboratory operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin company, for the U.S. Department of Energy's National Nuclear Security Administration un- der contract DE-AC04-94AL85000.
SAND Number: 2012-5928C


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Search Tags: men's snowboard, guys snowboard, all mountain boards, classic boards camber boards, mens Burton Process Snowboard, guys Burton Process Snowboard, employee-picks1617, feb15-17-sale, 1MAR17sale,
Explores all obstacles across the mountain, enjoying anything coming across them on the hill.
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Burton Process
Part Number: 10692103
From Burton: Re-evolved to shed ounces and amplify pop, Mark McMorris picks the Burton Process for its twin freestyle playfulness and all-terrain prowess. The FSC™ Certified Super Fly II™ core creates a lighter, lift-off-ready deck, while Squeezebox core profiling transfers energy towards the tip and tail for snappier ollies and effortless stability. Available in two versions, choose the relaxed and floaty Flying V™ or precise control of camber. Softer than the Custom, the Process is suited to the freestyle rider, but still capable of all-mountain action as evidenced by the pros who push it.
+ BEND: Camber
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Buckman's in Whitehall, PA
Would you recommend this product to a friend?
There's a reason Pros like Mark McMorris rides the Process. This board can handle any type of terrain. It's the type of board one would want to ride if they enjoy spending time in the park as well as cruising down groomers. The process is a soft and playful board but its camber makes it a little more responsive.
solid deck
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Buckman's in Whitehall
Huge fan of this guy. I rode the process last year and loved it. Stiff enough to rip groomers but soft enough to play around in the park. By far one of my favorite boards in the 2017 Burton line.
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Beyond the Psalms, Into the Messianic Age of Redemption, The Psalms of Hallelujah, by Dr. Robert Lehton
The word for praise is Hallelujah found primarily in the book of Psalms. The Jewish worshipper uses hallelujah in Judaism as part of the Hallel prayers, and likewise in Christian's prayers of praise. Yeshua (Jesus) conducted his final Seder in the "upper room" and the scripture says, "then they sang a song," and this undoubtedly would have been the Hallel.
22 "And as they were eating, he took bread, and after blessing it broke it and gave it to them, and said, "Take; this is my body." 23 And he took a cup and when he had given thanks he gave it to them, and they all drank of it 24 And he said to them, "This is my blood of the covenant, which is poured out for many. 25 Truly, I say to you, I will not drink again of the fruit of the vine until that day when I drink it new in the kingdom of G-d. 26 "And when they had sung a hymn (Hallel), they went out to the Mount of Olives." (ESV)
This is volume six of six studies on the Book of Psalms and will be a launching pad of God's grace, love and redemption to mankind. "Beyond the Psalms, Into the Messianic Age of Redemption" is an exegesis study of man's deepest yearning for redemption and restoration of his body, soul, and spirit.
Dr. Lehton holds a two-year Master's Degree (MRE) and also post doctorate Educational Specialist degree (Ed.S.) from New Orleans Baptist Theological Seminary. He likewise holds a doctorial degree in Clinical Psychology (Psy.D.) from Florida Institute of Technology.
Paperback-506 pages


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The base 2.0-litre manual-only Tribute Limited at $30,990 drive-away saves $3078. Drive-away savings on the more popular quartet of auto-only 3.0-litre V6s vary from $3298 to $3549.
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Without a dual-range transfer box, the Tribute was re-classified for 2003 as a compact rather than medium off-roader. For added on-road grip, the driver can activate an electro-magnetic coupling to lock the system in 4WD via a dash-mounted switch.


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Demand Generation Campaigns
Demand generation is a mix of outbound and inbound marketing activities that create brand awareness and drive demand for your service or product. The primary demand gen goal is to increase brand awareness and increase your position on SEO and PPC channels to increase visibility - and ultimately drive leads into the pipeline. Demand generation can empower your company to tap new markets, promote new products and build customer relationships throughout the buyer journey.
Adopting new technologies is a key component of developing a scalable demand gen program that can adapt to the changing search engine landscape. Captora was purpose-built to help companies target and launch high-performing campaigns at scale and intelligently optimize cross-channel campaign portfolios. Learn more about which marketing technologies will help improve demand gen program in this eBook.
Download the Modern Marketer's eBook


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8:00 pm - 10:00 pm
Marymoor Park
6046 West Lake Sammamish Pkwy NE
Redmond, WA 98052
Discover LUZIA, where a waking dream transports you to an imaginary Mexico. Experience a wondrous world that inspires you to explore your senses, enveloped in light and nurtured by rain.
Inspired by the cultural richness of Mexico, Cirque du Soleil's production LUZIA features original music written by Canadian composer Simon Carpentier. The LUZIA album takes Carpentier's original music written for a live acoustic setting and passes it through the filter of the electro-pop of Nortec Collective's Bostich + Fussible, infused with brassy Mexican and Latin American sounds.


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- A male gamer
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My name is mary, i got you from www.vgchartz.com and i want to have a
good relationship with you, please i need your cooperation,
am yours mary
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Nice avatar. Just saw your "Hero of Time" movie thread. I was surprised at first, but after reading further I side with Nintendo.
Yes another Pikmin fan is on this site.
wow you must be really happy. That username has yet t be taken. Welcome to the site!
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Nintendo sues The Hero of Time movie producersin Nintendo Discussion on 13 January 2010
For those guessing that the question about the Zelda fan-film, The Hero of Time, taken down last December, a nasty surprise: at the beginning of January, Nintendo officially filled a lawsuit at the Federal Court of Canada against BMB Finishes, the group formed by the producers of the fanfilm, for copyright infringement. Nintendo claims that the group, although "peacefully warned earlier,...
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Residential Accomodation for Newport Coming Soon!
12th Dec 2016
We are delighted to announce that The Boys' Brigade NI District has received funding from the Alpha Programme to commence work on the residential phase of our project at Newport. This is very welcome news and a great boost to our fundraising efforts.
The Alpha Programme was established in 2008 by Alpha Resource Management, a dedicated waste management and green energy company based at Mullaghglass, a Lagan Group Company. Tax on landfill waste, introduced in 1996 enables operators of landfill sites to contribute money to community projects that meet environmental objects. The grant programme is managed by Groundwork NI.
Commenting on the announcement, Drew Buchanan, MBE Chairperson of the Trustee Body of The Northern Ireland District of The Boys' Brigade said:
'We are delighted to receive this funding from the Alpha Programme. It will make a real difference to us. We can now deliver phase 3 of our Headquarters, Conference, Training and Residential Centre and we are thrilled to be able to commence the conversion of three of the classrooms into dormitory style residential accommodation and also a further classroom into a modern training facility. This funding brings us a step closer to bringing our residential training to our new home at Newport. We look forward to commencing residential training at Newport; to continue the training and developing of Boys and Young Men for leadership roles in Boys' Brigade and to creating lasting memories and a legacy of leadership training at Newport.'
The Boys' Brigade was one of three projects in the Lisburn area selected for funding in the October round of applications and will receive £49,764 towards the renovation of three existing classrooms into dorm style accommodation.


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|Digium TE235B 2-Port Digital PCIe Card w-Echo Cancellation T1-E1-J1-PRI 1TE235BF
|Digium TE235B 2-Port Digital PCIe Card w/Echo Cancellation T1/E1/J1/PRI (1TE235BF) The Digium TE235 is a dual port modular PCI Express card with hardware echo cancellation that supports T1, E1, J1, and PRI digital circuits. As part of Digium's next generation digital telephony cards, it offers higher performance and reliability, includes field upgradeable firmware, low profile form factor, the ability to handle high jitter environments and decreased interrupt times, improving computer processor performance. New: Quantity Digium TE235B Features Provides 2 RJ45 port for connecting T1/E1/J1 circuits Supports industry standard telephony and data protocols including PRI (North American and Standard Euro) protocol families for voice, PPP, Cisco, HDLC, and Frame Relay data modes Supports both E1, T1, J1 environments and is selectable on a per-card or per-port basis Improves I/O speed by up to 10 times resulting in reduced CPU usage and increased card density per server Includes hardware echo cancellation module Fully compatible with existing software applications and is fully integrated with the Asterisk Open Source PBX/IVR platform Half-length, low-profile PCIe Card Note: For use only with PCI Express (PCIe) 1.0 compliant slot - x1, x4, x8, or x16 (slots 4, 5, 6, and 7 below) Applications Dual span digital cards are perfect for building Asterisk solutions with direct connections to the phone network or legacy equipment. Common applications include: Direct PSTN connections for office phone systems Small call center ACDs with T1/E1 trunks Toll-bypass adjuncts for legacy TDM PBXs SIP trunk interface for legacy TDM systems Compatibility Asterisk and DAHDI supported (DAHDI 2.8 or later required for TE235) Switchvox supported (Switchvox 5.8.1 or later required for the TE235)
CATEGORY-> Electronics -> Audio
BRAND-> Digium
SKU/UPC-> digium-te235b
STATUS-> instock
Price: 1100.75


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Hospice Family Care
About Hospice Family Care4514Englewood Avenue
Lubbock, TX 79414
Phone: (806) 765-6111
Fax: (806) 762-0828
Service Area for Hospice Family Care
We don't have the exact service area for Hospice Family Care. Based on the average area most agencies cover, the agency is very likely to provide services to the area in green and less likely to provide services further outside this area. Be sure to contact the agency to make sure they provide services in your area.
Other Nearby Home Health Care Agencies
|Apex Homecare - Lubbock, TX
|< 1 mile
|Children's Home Healthcare - Lubbock, TX
|1 mile
|Direct Health Care - Lubbock, TX
|1 mile
|Crown Point At Home - Lubbock, TX
|2 miles
|Freedom Concepts - Lubbock, TX
|2 miles
|CareMinders - Lubbock, TX
|2 miles


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@Rocker wrote:
Your post implies a hatred for Germany, replicated throughout UKIP and the like. Historically, it's understandable, but the world has moved on from the events of a hundred years ago.
They lynched the last black man in Georgia in 1956, but the most powerful country in the world now has a black president.
Perhaps it's time for the rest of us to move on too.
I'm a fan of the Euro and I want the UK and Poland join it. Poland has a very large part of its infrastructure embedded from Germany so I would be keen on fiscal union between Poland and Germany. I cannot stand the UKIP.
But I stand by my comments about Germany, they are insane in that they have no connection with the reality that the Eurozone is on the point of collapse.
Even the Germans believe that a large number of countries will be leaving the Eurozone, so any talk about new members is ridiculous.
The Poles and the Serbians are on side, and the Finns and Greeks, it's time to step out of the trenches, son, and face the new world.
That new world has spectacularly failed. Six countries bailed out by the IMF; really impressive.
The Poles will only join the Euro when the Eurozone qualifies as being suitable for Poland. As it stands the Euro will not be ready for Polands entry for five years at least, according to the Polish FM.
The Finns and Greeks will probably leave the Euro and Serbia isn't even going to be eligible for EU entry.


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I love how luxe a faux fur collar can look. It takes a military inspired jacket and makes it easy to dress it up. I paired my Need Supply jacket with this chic, sheer-top dress from Cameo and a beanie, for a look that can take you from day to night. We are back in LA, but I'm holding on to our east coast holiday adventure; it was a special one for sure. Excited to end the week in a new year and looking forward to a spectacular 2015. Got your resolutions ready?
Photography by Lydia Hudgens


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Wow, this is powerful and true! Thank God for putting this huge burden on the hearts of these sisters to take their message around the country. And Im glad they're not keeping quiet about it!!! May our God Bless them all!
© 2017
Tucson Weekly |
7225 Mona Lisa Rd. Ste. 125, Tucson AZ 85741 |
(520) 797-4384 |
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These are species that grow as woody shrubs and keep their foliage year round.
Pruning is both an art and a science. It takes practice, experience and learning from your mistakes to become a proficient pruner. The pruning information about this plant should be considered as a guideline for getting started. Your particular climate, soils, watering and fertility schedules, sun exposure, space requirements and weather are all factors that influence how and when you choose to prune. We're providing a starting place for you, and over time you will learn the particularities of this plant in your garden. Don't be afraid to get started – Salvias, in general, are quick to rebound if inappropriately pruned.
Deadheading – the removal of spent flowers, is a practice that will always benefit the plant's health and appearance. This can be done at any time. Pruning involves removal of entire stems of spent growth. Becoming "spent" means that flowering stems stop blooming and begin going to seed.
(Sacred White Sage) Bees, hummingbirds and spiritual blessings are all connected to this elegant shrubby sage, which is an important herb to indigenous Californians and deserves a place in every salvia garden. Stiff and almost fleshy, its leaves are tight rosettes of brilliant, silvery white. The flower spikes soar above the foliage, with hundreds of small white-to-lavender flowers that are one of the most important sources of pollen and nectar for pollinators. This Salvia is also the source of leaves for Native American smudge sticks used in purification rituals.
Slow growing but not difficult, this California native requires good drainage and full sun. In its dry-summer/wet-winter range, it often grows on rocky, south slopes. Very little water is needed once the plant becomes established.
Our strain is well adapted to the moist environment of coastal Northern California, and performs well in a wide variety of climates. We select only the whitest and most compact plants for vegetative propagation, insuring a tidy shrub that will not overgrow its space.
Historically, Sacred White Sage has been used in medicinal teas and ground into flour for cooking. We burn the leaves in our home to sweeten and purify the air. This is a beautiful and powerful plant.
(Cleveland Sage or California Blue Sage) A California native plant garden is not complete without a Cleveland Sage. This particular cultivar has deeper blue flowers with a purple overlay as well as deep purple calyxes. Due to its height and drought resistance, it is ideal for back of border in a dry garden.
At 5 feet tall and wide, this plant is also a good xeric screen for fences, boundary lines and separations in your yard. Its tidy dome of fragrant leaves and flowers is rarely without honeybees, butterflies or hummingbirds.
There is much confusion in the naming and identification of Salvias native to California, especially Cleveland Sage. However, we have done our due diligence and believe that the plant we offer under this name is the one first grown by the Saratoga Horticultural Research Foundation in 1990.
(Greek Sage) Most of the dried culinary sage sold in the United States is Greek Sage. Frescoes on the island of Crete, dating to 1400 BC, depict this plant used by the Phoenicians and Greeks for cooking and medicine. It is an ancient and beloved friend of mankind.
In the garden, Greek Sage provides a pleasant lavender fragrance, especially on warm days, and has spikes of pink-to-lavender flowers. Similar to most culinary sages, it loves full sun and well-drained soil. However, it tolerates moist ground. This compact plant, which grows 24 inches tall and 18 inches wide, is a good choice for fragrant borders and patio containers as well as kitchen gardens.
Grow this drought-resistant, heat-tolerant plant in well-drained soil that is on the dry side. Butterflies, honeybees and hummingbirds will thank you with frequent visits.
Although some cooks find Salvia oficinalis culinary sages tastier, Salvia fruticosa is easier to grow. It comprises 50 to 95% of the commercial market. We think it offers an interesting change of taste.
(Black Sage or Honey Sage) One of the most common and fragrant native shrubs in Central California's Coast Ranges, Black Sage is ideal for dry gardens. Admirably adaptable, it tolerates soils ranging from the most marginal to ones that are loamy and provide excellent drainage. It is a survivor.
The elegant long wrinkled leaves are powerfully aromatic. Its small white-to-lavender whorls of flowers, which bloom from summer into fall, are vital sources of nectar and pollen for honeybees and hummingbirds.
Use this garden workhorse for a large scale groundcover, as a background planting for other more dramatic Salvias or as a vital plant in a wildlife garden. It likes full sun and is heat tolerant.
Our strain is originally from seed collected at the far northern edge of its range, and is hardy to at least 20 degrees F.
(Apricot Hummingbird Sage or Cerro Alto Pitcher Sage) Large clusters of warm, apricot-colored blossoms top the tall, thick flower spikes of this sage. It is named after a peak in the mountains behind the crashing waters of Big Sur on California's Central Coast.
The flowers darken as they age atop mid-green bracts. Cerro Alto's basal foliage mounds and spreads by underground runners. In favorable conditions, it can spread 3 feet across. The leaves are less lobed than those of the species, but are still sticky and richly scented.
This drought-tolerant, heat-resistant sage is adaptable to light conditions ranging from full sun to partial shade and grows particularly well in morning sun and afternoon shade. It blooms from winter into spring. As with other types of Salvia spathacea it likes the temperatures of USDA Zones 8 to 11.
This is the strongest growing, most vigorous clone of Hummingbird Sage we have seen. It makes a fine groundcover in woodland, native and dry gardens where it also works well in perennial borders and containers. Plant it in rich, well-drained soil and provide average watering based on local conditions.
(Bee's Bliss Sage) If you are looking for a California native sage to use as a groundcover, Bee's Bliss is a fine choice. Low-growing, widespreading and colorful, it is ideal for choking weeds.
Long-blooming spikes of lavender-colored flowers rise a foot above the mat of fine, fragrant, gray-green foliage that is perennial in warm-winter areas.
Honeybees and hummingbirds love this hybrid, which was selected in 1989 at the University of California Botanic Garden by California native plant specialist Roger Raiche. Berkeley artist and gardener Marcia Donahue named it.
Bee's Bliss is likely a cross of California Purple Sage (Salvia leucophylla), which is also called California Gray Sage, with either Creeping Sage (Salvia sonomensis ) or Cleveland Sage (Salvia clevelandii).
This is a superior, drought-resistant groundcover requiring full sun, good drainage and little-to-no water other than what it receives from nature. It's ideal for slopes and native-plant gardens. Claims of cold hardiness vary, but 18 degrees F is a safe bet even though lower temperatures have been reported.
(Celestial Blue Sage) Fast growing and adaptable, this sage is a chance hybrid between Cleveland Sage (Salvia clevelandii) -- also called California Blue Sage -- and California Rose Sage (Salvia pachyphylla). It may also be related to California Purple Sage (Salvia leucophylla).
Celestial Blue has lovely royal blue flowers and purple bracts. Sun-loving, heat tolerant and drought resistant, it was discovered at Las Pilitas Native Plant Nursery in Southern California.
This fragrant sage blooms and blooms throughout the heat of summer. Tolerant of everything but wet feet during summer, it withstands winter temperatures as low as 10 degrees F for a short time as well as lows in the 20-degree range for days.
Use this pretty plant in tough soils, on banks and in areas where watering is difficult or undesirable. Butterflies, honeybees and hummingbirds love it, but deer leave it alone. This cultivar is one of the best Salvias for cut-flower arrangements.
(Starlight Sage) This is a white-flowering hybrid of White Sage (Salvia apiana) and Black Sage (Salvia mellifera), two California natives often seen growing together in the wild. Similar to Black Sage, it blooms from spring into summer, attracting honeybees. In contrast, White Sage is a winter-to-spring bloomer.
The foliage of Starlight Sage closely resembles the silvery whitish green of White Sage, but it doesn't have that plant's typical pink or lavender flowers. But similar to its parents, it is powerfully fragrant, drought tolerant and heat resistant.
This compact, tough sage comes from the world famous Rancho Santa Ana Botanic Garden in Southern California, which is renowned for its collection of California native plants.
Highly recommended. Limited availability.
Hummingbirds love Salvia (sage) nectar and are attracted to it by the bright colors of tubular sage blossoms. In particular, these little whirlybirds can easily spot flowers in the red spectrum, which is prevalent among sages. Here are some hummingbird gardening tips.
If you live in suburbs or rural areas where deer plunder gardens, Salvias (sages) can be part of your plan for discouraging these hungry visitors. Here are some tips.
Xeric plants are excellent for water conservation. They grow well in dry gardens with little to no supplemental watering once established. In fact, overwatering can harm these plants, which are native to dry environments such as deserts and chaparral.
At Flowers by the Sea, we identify all xeric plants with a blue plant marker that warns against overwatering. Here are some tips for growing and understanding our xeric, or blue tag, plants:
1) In a humid region, you may find it difficult to grow plants native to semi-arid and arid environments. Yet xeric plants may succeed if you have a persistently dry area, such as under a roof overhang or in the shelter of a tree.
2) Xeric plants are excellent for locations far from garden hoses, such as along sidewalks -- areas often referred to as "hellstrips."
3) Shipping is hard on xeric plants, which suffer from confinement in small containers as well as boxes. You may see some mold, spots on leaves or withered foliage when they arrive. But xeric plants perk up with proper care while hardening off in partial shade before planting.
4) When amending soil before planting, remember that xeric plants not only need excellent drainage but also flower better in low fertility soil. Fertilize sparingly and use a mix with more phosphorous than nitrogen to encourage flowering and discourage lax overgrowth of foliage.
5) Organic matter, such as compost, is an excellent soil amendment for xeric plants, because it keeps their roots healthy by improving aeration and drainage.
6) When your xeric plants are established, water infrequently to encourage deep root growth and to avoid fungal problems. However, it's a good idea to gently spray dust off foliage about once a week.


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Emerging Vectorborne Diseases in the Southeastern United States: An Evaluation for National and Global Security
Printer-friendly versionSend by email
Vectorborne diseases are historically common in the southeastern US. The region is now
Award date:
Dec 19, 2013
Award amount:
Organization name:
Duke University Medical Center
Principal Investigator:
Woods, Christopher W
Grant program category:
Event Support
Grant program name:
Biotechnology Meeting Grant
Research Triangle
Vectorborne diseases are historically common in the southeastern US. The region is now
threatened by both the introduction of new vectorborne diseases and the re-emergence of
endemic diseases. The Southeast Research Center of Excellence on Emerging Infections
and Biofense (SERCEB) is holding a meeting of local and international experts to discuss
this threat to our national and global health security.
Fiscal year:


text: Hot For GOLD or higher VIP users Eziriz .NET Reactor 18.104.22.168 What is .NET Reactor?.NET Reactor is a powerful code protection and software licensing system for software written for the .NET Framework, and supports all languages that generate .NET assemblies. Its' main features are : Intellectual property protection NecroBit Protection Native Code generation Obfuscation Control Flow Obfuscation String Encryption Anti Tampering Resource Encryption and Compression Dependency Merging Protects full applications and libraries (dlls) Powerful licensing system Create trial versions of your software Set an expiration date Limit use to a fixed number of days after installation Limit by number of uses Limit to use for no more than a set number of minutes each invocation Limit trial version functionality Easily turn your trial version into a fully licensed version License your software Permanently (non expiring license) By time period (i.e rent your software) By number of uses (i.e software as a service) Software development kit Integrate license checking into you assembly and extend licensing fuctionality Extend licensing functions with bespoke extensions. - [+] Added option to merge all obfuscated types inside a single namespace (random generated namespace or a user defined one) - [+] Significantly improved obfuscation of virtual generic methods and generic method parameters- [+] Improved event obfuscation. Now events are removed without affecting the runtime behavior - [+] Improved NecroBit Protection- [+] Improved Control Flow Obfuscation- [+] Improved runtime performance of assemblies protected with String Encryption- [+] Improved protection for Universal Apps and Universal/Portable Class Libraries - [+] Now the menu item 'Tools'->'Generate Command-line Switches' generates encrypted passwords for strong name and digital certificate- [+] Further minor obfuscation improvements- [!] Fixed Windows Phone 8.1 protection issue- [!] Fixed Windows Phone 8.1 deployment issue- [!] Fixed protection problem on assemblies with ARM platform target- [!] Fixed generic methods merging issue- [!] Fixed problem affecting the combination Control Flow Obfuscation level 9 and cross assembly obfuscation- [!] Fixed SQL assembly protection issue- [!] Fixed network drive path issue- [!] Fixed rare licensing problem affecting the combination 'Number Of Uses' + 'Run Without License File'->'True' + 'Individual Licensing Behaviour'->'True'- [!] Fixed protection issue on classes containing properties decorated with JSON attributes- [!] Fixed minor bugs Download Link(s)File(s) size: 15.9 MB


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The Marketing Art Director is responsible for leading, concepting and executing innovative and compelling marketing materials to promote and elevate the image of the agency. These responsibilities include case studies, website assets, thought leadership pieces, presentations, and paid media materials. In addition, this role will also have a hand in creating the new agency identity, helping to establish and maintain the brand's creative standards across all channels. Reporting to the SVP of Marketing, the Art Director will work with the Integrated Marketing team as well other groups in the organization, including Creatlve leadership, Business Development, and other executive leadership. Therefore, the ideal candidate will have a strong balance of creative, interpersonal and strategic thinking skills.
Education/ Training
To learn more about us, visit our website at www.ampagency.com Qualified applicants should submit their resume, salary history and requirements to Nicole Peterson at firstname.lastname@example.com