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Chat with Kevin Seifert
Welcome to SportsNation! On Tuesday, ESPN.com NFC North blogger Kevin Seifert will drop in to chat about the NFL.
Seifert joined ESPN.com in 2008 after covering the Minnesota Vikings for the Minneapolis Star Tribune for eight years. He lives in the Twin Cities with his wife and children.
Send your questions now and join Seifert Tuesday at 2 p.m. ET!
More Kevin Seifert: Blog | Chat | Search | NFL index
Kevin Seifert (1:59 PM)
Hi everyone. Let's start this here chat thing.
The Delivery Guy ((At the front door with your Moo-shu Pork))
Any late round picks or undrafted free agents jumping out at the minicamps around the NFCN?
Kevin Seifert (2:03 PM)
Lots of talk about the WR the Bears drafted from Washington State, Marquess Wilson. He could be a find if he can exhibit maturity.
Craig (Braintree, MA)
I know Joe Webb has played wide receiver some. How is his physicality? How do you see his ability to block?
Kevin Seifert (2:03 PM)
He's a big dude. 6-4 and 220 pounds at least. He has the physicality. Technique and experience are what will hurt him.
Jordan P (Milwaukee, WI)
The Packers defense has been down for a few years now, are there any rumblings that Dom Capers may be on the hot seat if the Packers D has another poor season?
Kevin Seifert (2:04 PM)
Mike McCarthy was very loyal to him after last season. You would think at some point Capers' defense will have to get back to where it was in 2010, but I can't say for sure that he will be on the hot seat if it doesn't.
Greg M (Chicago)
Kevin, any chance a rookie LB could start for the Bears in Week 1? Or have the FA's they signed been guaranteed to start?
Kevin Seifert (2:06 PM)
No, they aren't even guaranteed to make the team let alone start. There is a real chance for Bostic or Greene, or both, to start. The Bears are in good shape with a veteran presence if they need it, but most teams want young players on the field as soon as they show a sign they're ready.
Feek Freak (Detroit)
How much does Charles Woodson want to come to the Lions? We already have 2 safeties where would he fit in?
Kevin Seifert (2:08 PM)
That's why the second part of the question hasn't been answered: How much do the Lions really want Woodson? Don't they want Glover and Delmas to start? I know Delmas has had a lot of injury problems, but signing Woodson would basically be telling Delmas that you don't think he is going to make it through the season.
Seth (Fairfield)
Aaron Rodgers + a dominant Running game = unstoppable?
Kevin Seifert (2:09 PM)
Let's pull back the reigns on "dominant running game" just because the Packers drafted two tailbacks. But yes, if you combine an All-Pro quarterback with a running game that dominates opponents, it would be hard to stop.
Jose, illinois [via mobile]
What's the status on Henry Melton's contract? Any progress?
Kevin Seifert (2:09 PM)
Not that I'm aware of, but there is plenty of time for the sides to agree to a deal. Melton won't have a problem playing for $8.45 million this season if that's what it comes to.
Mike (Utah)
Whose going win the starting RB spot? Seems to me that it isn't a foregone conclusion that Lacy wins...
Kevin Seifert (2:10 PM)
Not that I'm aware of, but there is plenty of time for the sides to agree to a deal. Melton won't have a problem playing for $8.45 million this season if that's what it comes to.
Kevin Seifert (2:10 PM)
Not that I'm aware of, but there is plenty of time for the sides to agree to a deal. Melton won't have a problem playing for $8.45 million this season if that's what it comes to.
Kevin Seifert (2:11 PM)
Sorry, we got a little out of order there. On the Packers' RB situation, I think Lacy might be the favorite to start eventually but you're right, it's not a done deal.
Chris (Des Moines, IA)
Is Carimi making a mistake by not showing up to Bears camp?
Kevin Seifert (2:13 PM)
Depends on what he wants his outcome to be. If he wants to get cut so he can move to another team, then he's doing the right thing. Maybe he reads the writing on the wall -- playing out of position under a new coaching staff with a general manager that didn't draft him -- and is preparing for his next job.
Mike Huckleberry (firstname.lastname@example.com)
Kevin, is it true that you drive a Prius?
Kevin Seifert (2:14 PM)
It is not. But I could be talked into it...
Jason (Orlando)
Kevin Seifert (2:14 PM)
John (Pa)
Do you think the Vikings are gonna suffer on offense with the loss of Percy Harvin?
Kevin Seifert (2:15 PM)
They're going to use Cordarrelle Patterson in a lot of the same way they used Harvin last year, I think. But in the end, no one can be the kind of player Harvin is. So Christian Ponder is going to have to make more difficult throws than he did when Harvin was there to clean up the mess.
Bryan (Richmond, VA)
Hey Kevin, thanks for the weekly chats.Over a guy like let's say, Stephen Burton, how do you think Joe Webb compares? Does he have a shot to make the team as the 4th or 5th receiver, or did the Vikings move him to this position so they can cut him and blame it on his ability (or lack thereof) to play WR....
Kevin Seifert (2:16 PM)
He probably has more athleticism, but frankly Stephen Burton has been playing WR a lot longer than Joe Webb and should have an advantage there. It can take some time to make the adjustment. That's why it's too bad the Vikings waited this long.
Paul (Denver)
Do you think Marques Wilson plays well enough to grab the 4th WR spot? He's certainly got the skill.
Kevin Seifert (2:17 PM)
Especially if Devin Hester remains as a returner only. Again, we don't want to get too carried away with ourselves, but Wilson would have been a mid-round guy if not for his issues last year with Mike Leach at Washington State.
Chris (St Paul, MN)
What does Carl Gerbshmidt think of the new Vikings stadium?
Kevin Seifert (2:18 PM)
I'm told he has vowed never to set foot there. Which is fine.
Ben (Minnesota)
The Vikings and Bears both finished 10-6 last year. Who in your opinion had the better offseason, and who will have a better record this year?
Kevin Seifert (2:19 PM)
Fair question. The Bears are much more of an unknown, having parted ways with Brian Urlacher and hired a mostly-new coaching staff. But I'll take my cue from the Vikings themselves, who didn't extend Leslie Frazier's contract. If they aren't confident in where things are going, than I won't be, either. Bears.
Drew Fox (riverside, ca) [via mobile]
Hey Kevin do you think the Vikings need to give a different back a chance to back up A.P.Gerhart doesn't seem to be dynamic enough
Kevin Seifert (2:20 PM)
Whoever has that job isn't going to play much or if at all as long as Peterson is healthy on the roster. Kind of amoot point.
Corey (D.C.)
Is Bostic strickly a MLB, or could he possibly play outside? What is the Bears' hope for him?
Kevin Seifert (2:21 PM)
He could play outside but his natural position is inside. I'm sure that's where the Bears hope he will ultimately end up.
Jared (Nort' Dakota)
Will John Carlson have an impact for the Vikes this year? Do they move toward a Belicheck 2 TE type system?
Kevin Seifert (2:21 PM)
Any impact would be better than last year. I don't see them moving that way, though.
JB (RvA)
Have the Packers done enough in the offseason to overcome to label of being a finesse team that will fold if smacked in the mouth by a physical defense and running oriented team? (ex. Giants & 49ers past two playoffs)
Kevin Seifert (2:23 PM)
Well, Datone Jones was a pretty good run-stopper at UCLA. But other than that and releasing Charles Woodson, they haven't really flipped much of their personnel. So if you think that label is accurate, I'm not sure that you can say it's been addressed adequately.
Sam (Wi)
Remember when the Bears passed up Tyler Eifert and picked Kyle Long instead? Good times.
Kevin Seifert (2:23 PM)
That'll be one to track. But it could take a few years. If Long turns out to be a good 10-year starter for a team that has struggled forever to put together a solid offensive line, well, there's something to be said for that.
Martin (NYC)
anything positive coming from allen park that doesn't have a history of injury or "upside" in the phrase?
Kevin Seifert (2:24 PM)
Ha. Glover Quin is a real leader and an exceptional person, from what I've been told. How's that?
Barry (Detroit)
Do you think AD fate is sealed to be one of the the greatest backs in history not to get a ring?
Kevin Seifert (2:25 PM)
Little early for that, but his window of dominance will close sooner rather than later.
Bob Lifetime Fitness Champlin(not working out) [via mobile]
So you're saying the Bears coach from Canada is better than Les Frazier?
Kevin Seifert (2:25 PM)
Ha. Marc Trestman is also from Minnesota! I'm just saying the Vikings didn't express any confidence in Frazier, and presumably where they think he will take them in 2013, so why should I?
Kyle Long (The Kitchen)
How do hatters (Packer, Lion, and Viking fans) take their crow? Fried, sauteed, grilled, or poached?
Kevin Seifert (2:26 PM)
How about straw? Baseball?
Paul (Denver)
With Washington's natural rushing ability, do you think he lights a fire under McClellin?
Kevin Seifert (2:28 PM)
I'm not sure about that, but I know there are plenty of people who want to see if McClellin is going to emerge as an impact player or if the first player taken in the Phil Emery regime will prove a flop.
Jason (Orlando)
Yes, how do we "Hatters" take our crow?
Kevin Seifert (2:28 PM)
A question for the ages...
Jon (Chicago)
Am I off base in thinking Emery drafts the same type of player regardless of the round? He doesn't worry about off-the-field stuff and just drafts the guy with the most upside. Good strategy for late rounds, but seems awfully risky for round 1.
Kevin Seifert (2:29 PM)
He's definitely into that Athletic Index score. There are plenty of ways to evaluate college players, and that's one of them.
Eric (Chicago) [via mobile]
Kevin in reference to who has had a better offseason I'd say the Bears by far since they didn't trade one of their best playmakers, injured or not. Harvin is a type of player that doesn't come around very often. But there is the issue with attitude. Urlacher is up there in age. What's Harvin, 27?
Kevin Seifert (2:30 PM)
Harvin is 25. Amazing. That's why I've always said there has to be more to the story. Why would a team trade a special player like that, at such a young age, because of a few dustups? Got to be more to it than that.
Ben (Sandy, UT)
I love Lambeau and go to a game every other year, however, I won't even go to games after October let alone February. I just can't see a Super Bowl in Green Bay and even if you were to build the necessary infrastructure, it would be a waste as it would remain empty for 8 months out of the year.
Kevin Seifert (2:31 PM)
It's not going to happen. Moot point. But I agree that most of the fans who typically get tickets to the Super Bowl wouldn't want any part of this. That doesn't mean the game wouldn't sell out, but the tickets wouldn't go to the people the NFL would most want to sell them to.
Bob (Toyota Dealer)
What's it going to take to get you into this brand new Prius?
Kevin Seifert (2:32 PM)
I don't need that rust inhibitor, I'll tell ya that right now.
Ben (Minnesota)
If the Vikes lose in a close one to Pitt in England, can the owners be blamed for taking away home field advantage?
Kevin Seifert (2:33 PM)
I'm not sure if you blame the owners or the NFL or whoever, but there is no doubt the Vikings have a better chance to beat the Steelers in the Metrodome than they do at Wembley.
Bob Lifetime Fitness Champlin(not working out) [via mobile]
Speaking of faith in Les Frazier, why should there be any faith in Jay Cutler. A starting QB entering the final year of his deal, talk about rare.
Kevin Seifert (2:34 PM)
There is that, although the money involved with a QB contract is a lot bigger than with coaches.
Sam (Wi)
Perry, Ansah or McLellin for more sacks in 2013?
Kevin Seifert (2:35 PM)
I'm saying Ansah, playing next to Suh and Fairley.
Dirty Frank [via mobile]
You have said in the past that Emery traded Olsen. Is it not true that 3Rd round pick was used to get Marshall. I say he did a swell job with that move.
Kevin Seifert (2:36 PM)
FOR AT LEAST THE ELEVENTY MILLIONTH TIME, I didn't write that. It was written by someone with a different name whose post appeared on the NFCN blog when I was gone. I know that was GM Jerry Angelo and assistant GM Mike Martz.
Andrew (MN)
Being of Scandinavian decent, I am offended by the Vikings mascot. Do you think they will every change it to stop being so negative toward Norwegians?
Kevin Seifert (2:36 PM)
Dan Snyder says the Vikings will never change it. He said we can put that IN CAPS.
Eric (Chicago) [via mobile]
I may be alone in saying this but I hope the Bears don't sign Cutler to an extension.
Kevin Seifert (2:37 PM)
The question is always if the grass is greener on the other side. Look at how long the Bears had to wait and how much they had to pay just to get a quarterback of Cutler's skills. That always has to be part of the evaluation.
Dustin (Nashville)
If Derek Sherrod is a bust, who should the Packers have drafted in 2011 instead?
Kevin Seifert (2:38 PM)
If he is a bust, it's because of a terrible injury. So we don't hold that against the Packers and second guess the decision. At least those of us who can't peer into the future.
Mike (MN)
When do you continue to focus on the lack of contract extension for Frazier? The owners got burned with Childress, had an option on Frazier and made a prudent business decision. I don't think the owners are throwing in the towel on the 2013 season.
Kevin Seifert (2:39 PM)
If they were 100 percent convinced that Frazier was the right guy for the job, they would have extended him. That doesn't mean they've thrown in the towel. It means they're waiting and seeing and hoping for the best.
Jason (Orlando)
How about a compromise in terms of a Wisconsin Super Bowl: Miller Park?
Kevin Seifert (2:39 PM)
For that matter, why not Soldier Field? It's less than 4 hours from Lambeau.
Cody (Behind You)
Could you please clarify why your original article on CJ's "broken" fingers yesterday had so many grammatical errors? Did your editor fall asleep?
Kevin Seifert (2:41 PM)
Broken fingers of my own?
Brian (Philly)
How would you fix the Pro Bowl? I say small group events throughout the Saturday before... catching drills, obstacle courses, relays, definitely a tug of war competition...
Kevin Seifert (2:42 PM)
I've always thought it would be fun to see players out of their element. Maybe a Fear Factor-like competition? But I'm guessing the further it gets away from football, the fewer people that would have interest.
Cody (Behind You)
Now that was one of the suggestions in the comments, but I feel you are not being truthful.
Kevin Seifert (2:43 PM)
I wasn't aware of the typos. Looks good now.
Andrew (WI)
Should we be concerned that just under a third of our playroll is Clay and Aaron?
Kevin Seifert (2:43 PM)
That's part of what goes into having two of the best players at their respective positions.
Sando (Not Working)
Why don't you go chat with your blog followers in the Comments section of the posts like me? You have some pretty interesting characters over there
Kevin Seifert (2:44 PM)
I do occasionally but frankly it's hard to keep up. How do you guys get any work done when you're in there? Wait. I don't want to know.
Jay C. (Under Clay Matthews)
Any suggestions on how to avoid pressure?
Kevin Seifert (2:45 PM)
Get a better offensive line. (The Bears are trying.) Get rid of the ball quicker. (Marc Trestman is mandating.) Voila!
Jacob (Poland)
Speaking of the current Top 20 Greatest Coaches Countdown, who do you rank higher, Lombardi or Halas?
Kevin Seifert (2:46 PM)
I wasn't asked to participate and I don't know who wins out. But it would be hard to pick against Lombardi if I had to make a choice.
Cam Elks (crystal lake,IL)
Why do you take the time to answer stupid comments when people actually ask you real football questions?
Kevin Seifert (2:47 PM)
Because frankly 90 percent of the questions I get are the same. It's OK to occasionally toss in a fun one.
Kevin Seifert (2:48 PM)
Ok, a few more here.
cory (seattle)
What are the chances that Woodson will end up signing with the broncos? Do you see the broncos signing any of the last available "big name" FA?
Kevin Seifert (2:49 PM)
I think they see him as someone who would give them more experience, which they clearly lacked in their playoff loss last year. The guess is he is hoping to increase their offer by taking the Raiders visit and putting out that the Giants were interested.
Jason (Orlando)
Are Detroit fans going to throw a fit reading about Grant, Lombardi, and Halas, or complain that Monte Clark isn't listed?
Kevin Seifert (2:49 PM)
Ha. George Wilson deserves some mention.
Dirty Frank [via mobile]
Could the Bears have 3 players with a 1,000 yards receiving, Marshall, Jefferyand Forte?
Kevin Seifert (2:50 PM)
That might be tough, especially if they run the ball, but I could see two of them.
Ryan (Milwaukee)
Which would you consider more annoying as a fan, the Packers putting up with Finley for so long, or the Vikings bailing on Harvin (possibly too early)?
Kevin Seifert (2:52 PM)
Finley has been productive enough, and had enough impact on the overall offense, to make "putting up" with him worth it. Harvin is an elite player who departed when he was 25. I'd be more annoyed that the Vikings couldn't make it work.
Paul (Denver)
Will Stafford be under the most pressure in his short career in '13? Their tackle situation seems very dicey.
Kevin Seifert (2:52 PM)
Fortunately he usually gets rid of the ball fairly quickly. But there will be a premium on that, for sure.
rob (minneapolis)
which fanbase makes you shake your head the most?
Kevin Seifert (2:53 PM)
If rotates on a fairly regular basis, in all honesty.
Kevin Seifert (2:53 PM)
OK everyone, thanks so much. Chat with you soon.
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Samsung Galaxy Nexus Won't Turn On
I've been having problems with my Galaxy Nexus. I'e had it for about 2 yrs now and about a year it started having troubles turning on. I took it in and got it foixed and everything was great for awhile. Lately it's been taking numerous tries to turn it on, it runs super slow, and periodically restarts itself.
The other night I decided to uninstall a number of non-esstintial programs (ones I'd downloaded from Google Play) and update the ones I had. WHile it was doing that I went to bed with it plugged into the wall charger liek I do every night. When I woke up the white light was flashing to say I had a message etc. Despite repeated tries I could not get the phone to turn the screen on. So like usual when this happens I took the battery out and restarted teh phone. The first two times it would start up and get to the Google icon that first comes up but then would get stuck for 10-15 min. Since then it hasn't turned on at all (been 24hrs now). When I plug it in don't even get the white battery charging symbol come up. I've looked around online and tried much of the ideas I've come across. I've taken the battery out, left it for 10-15 min, plugged it in to the wall (and comp), put the battery back in, and tried turning it back on. I've also done that same thign while holding down button combinations etc.
Any help would be much appreciated.
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Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmpR Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10–107 cells (10–107 copies for tetQ gene), while the quantitative range for total bacteria was 102–107 cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.
For microbiological examinations, techniques such as immunoassays, biochemical tests and hybridization using nucleotide probes have been developed and used [1]. However, since these methods required bacterial culture, they are mostly laborious and time-consuming. Recently, advances of novel molecular biology techniques enabled culture-independent methods. PCR is a typical example and is widely spread for the microbiological diagnosis of periodontal diseases. The gene encoding the small subunit of 16S ribosomal RNA (16SrDNA) has been frequently used as a target of the PCR examination because of its structural characteristics [2,3]. Nucleotide sequences of some portions of the 16SrDNA are highly conserved through evolution, while other regions have species-specific variable sequences. The conserved sequences can provide PCR primers for amplification of 16SrDNA from all of the bacterial species and the species-specific regions can be used for identifications of unique bacterial species. The 16SrDNA-based PCR is a rapid, sensitive and specific molecular technique, which can be used for various purposes. However, the method still has a problem in quantification.
Antibiotic treatment is indispensable for periodontal therapy. For evaluation of the treatment, accurate quantification of periodontal bacteria is needed. In addition, we have to be alert to the increase of antibiotic resistance. It has recently been reported that a tetracycline-resistant gene, tetQ, has spread intensely through conjugal gene transfer [4,5]. Tetracycline-resistant Prevotella and Bacteroides strains isolated from periodontal pockets have been reported to possess either or both of the tetQ and tetM genes frequently [6–8]. The nucleotide sequence of the tetQ gene was almost identical among the clinical isolates, supporting the horizontal transfer of the gene [9]. Although PCR methods have already been used for the detection of tetQ gene in clinical plaque samples [7,10], quantitative monitoring of gene presence is required for analysis of its dynamics in periodontal pockets.
Recently, real-time PCR has been developed for the purpose of DNA quantification. Real-time PCR using the GeneAmpR Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA) or LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) is accomplished by the continuous measurement of the PCR products. The method allows easy, rapid and quantitative detection of microorganisms in clinical samples. Some attempts have already been made for periodontal bacteria [11,12]. In the current study, we used the GeneAmp Sequence Detection System with TaqMan and SYBR Green chemistry for the quantification and identification of periodontopathic bacteria. We compared the TaqMan and SYBR Green chemistry in their specificity and sensitivity for further improvement of the real-time PCR assays in microbiological diagnosis of periodontal diseases. In addition, quantitative monitoring of tetQ gene was included in the examination.
Materials and methods
Bacterial strains and growth conditions
The bacterial strains used in this study were Actinobacillus actinomycetemcomitans ATCC 43718 (Y4), Actinomyces viscosus ATCC 15987T, Escherichia coli K-12, Porphyromonas gingivalis ATCC 33277T, Treponema denticola ATCC 35405T, Fusobacterium nucleatum ATCC 25586T, Prevotella intermedia ATCC 25611T, Prevotella nigrescens ATCC 33563T. They were obtained from the American type culture collection (ATCC, Rockville, MD, USA). Each P. gingivalis, P. endodontalis, P. intermedia and P. nigrescens strain was cultured anaerobically in GAM broth (Nissui Seiyaku, Tokyo, Japan) supplemented with 0.0005% hemin and 0.0001% vitamin K3. A. actinomycetemcomitans, A. viscosus and T. denticola were grown as previously described [13,14]. E. coli was cultured aerobically in Luria–Bertani medium. In addition to the type strains, a tetracycline-resistant P. nigrescens was selected from laboratory stock of clinical strains by using the medium containing 4 µg ml−1 tetracycline-hydrochloride.
DNA extraction
Total bacterial DNAs were extracted from cultivated strains and clinical plaque samples by using InstaGene Matrix (Bio-Rad Lab., CA, USA) according to the instructions. Plaque samples and cultivated bacteria suspended in 1 ml PBS (–) (Gibco BRL, MD, USA) were pelleted and resuspended in 200 µl of InstaGene Matrix. The suspension was incubated at 56°C for 30 min and then 100°C for 8 min. After the incubation, the suspension was centrifuged and 5 µl of the resulting supernatant was used for real-time PCR. To determine the quantitative range of real-time PCR, DNAs were prepared from 10–107 cells of cultured bacteria.
tetQ gene
A cloned version of the tetQ gene was kindly provided from Dr. Progulske-Fox A. and Dr. Burks J. (Department of Oral Biology and Periodontal Disease Research Center, University of Florida). The optical density (260 and 280 nm absorbance) of the purified recombinant plasmid was measured to estimate the amount and copy number of the plasmid.
Plaque samples
Eight patients with adult type periodontitis, during the supportive periodontal treatment in Okayama University Dental Hospital, were selected for sample collection. All patients had not received any periodontal therapy in the previous 3 months and not received any systemic or local antibiotics within the preceding 6 months. Plaque sample was obtained from one periodontal pocket of 4–5 mm in each patient by using the paper points as described previously [15]. After the initial sampling, 2% minocycline–HCl paste (Periocline, Sunstar Inc., Osaka, Japan) was injected into the pocket without any other periodontal therapy. Plaque samples were also obtained from the minocycline-treated periodontal pockets after 1 week.
Primers and probes
The sequences of primers and probes used in this study are listed in Table 1. For identification of unique bacterial species, primers and probes were designed from the species-specific region on the 16SrDNA, while the conserved sequences were used for amplification of total bacteria. For the design, Primer Express (version 1.0) and OLIGO (version 4.0) software were used. TaqMan probes were labeled with the reporter dye 6-carboxyfluorescein (FAM) at the 5′ end and with the quencher dye 6-carboxytetramethylrodamine (TAMRA) at the 3′ end.
Real-time PCR
The real-time PCR was performed using GeneAmpR 5700 Sequence Detection System (PE Applied Biosystems). The reaction mixture (50 µl) for TaqMan assay contained 2× TaqMan Universal PCR Master Mix (PE Applied Biosystems), 20 pmol of forward and reverse primers, 10 pmol of TaqMan probe and 5 µl of extracted DNA. While, the reaction mixture for SYBR Green assay contained 2× SYBR Green PCR Master Mix (PE Applied Biosystems), 20 pmol of forward and reverse primer and 5 µl of extracted DNA. Thermocycling program was 40 cycles of 95°C for 15 s and 60°C for 1 min with an initial cycle of 95°C for 10 min. All amplifications and detections were carried out in a MicroAmp optical 96-well reaction plate with optical caps (PE Applied Biosystems). At each cycle, accumulation of PCR products was detected by monitoring the increase in fluorescence of the reporter dye from the TaqMan probes or dsDNA-binding SYBR Green. After the PCR, a dissociation curve (melting curve) was constructed in the range of 60°C to 95°C. All data were analyzed using the GeneAmp 5700 SDS software.
Results and discussion
Specificity of primers and probes
For specificity testing, extracted DNAs from 107 cells of periodontal bacteria listed in Section 2 were examined as templates with the primers and probes prepared for A. actinomycetemcomitans, P. gingivalis and P. intermedia, respectively. PCR amplifications were observed correctly when the target strains were used as templates. The primers and probes did not increase the fluorescence with the mismatched strains (data not shown). Dissociation curves of all PCR products showed a sharp peak at the expected Tm of the products (data not shown). These results indicate that each real-time PCR specifically amplified the target DNA. Since additional hybridization of the probe is required for generation of the fluorescence, TaqMan assay is thought to be more specific than SYBR Green assay in general. However, SYBR Green chemistry demonstrated sufficient specificity for the three periodontal bacteria.
Primers and probe for tetQ gene did not increase the fluorescence with any type strains used in this study. PCR amplification was observed with the tetracycline-resistant P. nigrescens and the recombinant plasmid carrying the tetQ gene (data not shown). For total bacteria, only the SYBR Green method was employed because enhanced specificity was not required.
Quantification of cultured bacteria and tetQ gene
DNA extracts from 10–107 cells of three periodontal bacteria, A. actinomycetemcomitans, P. gingivalis and P. intermedia, were subjected to both TaqMan and SYBR Green assays. In both TaqMan and SYBR Green assay, detection and quantification for each strain was linear over the examined range. A typical result of A. actinomycetemcomitans is shown in Fig. 1. Lyons et al. reported the quantitative range of P. gingivalis as 102–108 cells using nested PCR with a TaqMan probe [11], and Sakamoto et al. demonstrated quantitative detection of five periodontal bacteria in the range of 103–108 cells using the LightCycler™ system [12]. Our study demonstrated a wider quantitative range of real-time PCR. However, since the previous studies did not examine the smaller amount, their actual limits of the quantitative detection may be smaller than the reported data. A copy number of the 16S ribosomal operon in each strain could possibly contribute to the differential sensitivity for the detections. The number of 16S ribosomal operons in P. gingivalis revealed to be four through the TIGR (The Institute for Genomic Research) database. Since genome projects for A. actinomycetemcomitans and P. intermedia are still under progress, the 16S ribosomal numbers were analyzed through genomic blast search and were estimated to be six and five, respectively. Remarkable differences of the copy number were not seen, and therefore, we estimate that the detection limit is similar among these three species.
In order to examine if the quantification was influenced with contamination of another bacterial species, 105 cells of seven periodontal bacteria, other than the target, were added to each serial dilution (102–107) of the target strain. Fig. 2A shows the influence of other bacterial contamination to the quantification of A. actinomycetemcomitans, which was examined by the SYBR Green assay. The standard curve of the contaminated samples was almost the same with the normal standard, demonstrating limited influence of the contamination, if any at all. In the same way, no influence was observed to the quantification of P. gingivalis and P. intermedia in both TaqMan and SYBR Green assay (data not shown).
Quantification of total bacteria was examined using the DNAs from eight periodontal bacteria prepared in this study. The different strains showed similar results of linear detection in the range of 102–107. A typical result for A. actinomycetemcomitans is shown in Fig. 2B. The similar results reflect the similar copy numbers of the 16S ribosomal RNA operon in the strain tested as has been discussed above. An average of the copy number in both the Pasteurellaceae family (including genus Actinobacillus) and the Bacteroidaceae family is similar (5.8), and E. coli possesses seven copies [16]. However, since the copy number of the 16S ribosomal operon can vary from one to as many as 15 copies [16], total bacterial counts evaluated by the number of 16SrDNA may be slightly fluctuated according to the differences of microflora especially in case of polymicrobial communities such as dental plaque. The limit of quantitative detection for the total bacteria was 100 cells, while the quantitative detection of unique bacteria was less than 10. This was caused by a high level of background (Fig. 2B). Since the primers can hybridize to any bacterial species, even small contamination of bacteria in the experimental manipulation or the contamination of E. coli DNA in the recombinant Taq polymerase might be a cause of the conspicuous background.
The quantitative range for tetQ gene was also examined by both TaqMan and SYBR Green assay. Serial dilutions of the recombinant plasmid, which contained the tetQ gene, were prepared and used as templates. The results were almost the same between the two methods and the detection of tetQ was linear in the range of 10–107 copies of the gene (data not shown).
Examination of the plaque sample
The real-time PCR using the SYBR Green chemistry was applied to the clinical plaque samples as has been described above. The original 5 µl of extracted DNA samples and 5 µl of diluted samples (10−1) were run in duplicate. Total bacterial numbers in the periodontal pockets did not consistently decrease after the local application of minocycline (Fig. 3A). The number increased in three patients and did not change in one patient, while the number decreased in four patients. On the contrary, the number of A. actinomycetemcomitans, P. gingivalis and P. intermedia decreased remarkably. Fig. 3B shows the percentage of A. actinomycetemcomitans, P. gingivalis and P. intermedia among total bacterial counts before and after the antibiotic therapy. Local delivery of tetracycline has been effectively used for periodontal therapy [17,18]. The qualitative change of the microbial community may account for the effect.
The tetQ gene in the plaque samples was evaluated by the SYBR Green assay using the standard of the recombinant plasmid. The number of tetQ genes increased after the minocycline therapy in four patients, and the gene could not be detected in the remaining four patients at the both sampling times (Fig. 4). Preus et al. [19] and Wade et al. [20] reported the similar results that locally delivered tetracycline selected for the resistant bacteria. Although several PCR methods had already been reported for the monitoring of tetQ gene [7,10], real-time PCR seems to be useful as well.
The current study demonstrated the potential use of real-time PCR for the analysis of clinical plaque samples. However, besides the method itself, sampling procedures would be another issue. Since the real-time PCR is very sensitive, the sampling procedure could greatly affect the results. Although bacterial sampling using paper point employed in this study is a well-established method [15,21], technical difficulties have also been reported [21–24]. Sample collection methods appropriate for the real-time PCR need to be investigated.
Both TaqMan and SYBR Green assays showed sufficient sensitivity, specificity and quantitativity. Since the TaqMan assay required additional manipulation and cost for the probe, the SYBR Green assay may be suitable for routine clinical examinations. In addition, quantitative monitoring of the antibiotic resistance genes should be included in the microbiological diagnosis.
We thank Dr. Progulske-Fox A. and Dr. Burks J. (Department of Oral Biology and Periodontal Disease Research Center, University of Florida) for providing the tetQ gene. This study was supported by Grants-in-Aid for Scientific Research (B12557192, C13672186 and C13671901) from the Japan Society for the Promotion of Science.


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I stand at the window watching John talking to the lemon tree in our dried up garden.
"Six years you have done this to me, you bastardly tree, was it not me that planted you. I took your little tender seed and placed it into the most fertile soil known to man and fed you water so pure that the clouds became jealous, and all while you were growing and now you have no lemons! No fucking lemons!"
He pauses during hia remonstration towards the tree to move in a little closer to get a better comprehension of it. I wonder what sorts of drugs he must have just taken.
"You have measly little flowers, only the poorest of insects and weakest of birds would bother to pollinate you and likely because of their diminishing eyesight. You cretin of a tree, look at you; you grow now 3 metres tall and 3 metres wide yet your lamentable little leaves can't harbour any shade. Not even enough shade for a rabbit… nooooo, the rabbit has to suffer through the ghastly heat as it ponders about how such a big tree can provide no shade and during all this it sweats out so much bodily fluids that its skin stretches tight against its bony structure resembling a cadaverous body and then its brains begins to boil and it collapses of exhaustion and dies. You bastardly little bunny killing tree, I will get my revenge for all those little helpless rabbits you killed.
"Hah, but worst of all… worst of all is that you do not even fruit, except for those pitiful little green lemons that worms dare not resort to, not even common bacteria. They fall to the floor and remain there resolving not to take part in putrefaction, just reminding me of how superfluous you are. Your sole purpose in life is to fruit beautiful fleshy sour lemons with yellow skin that catches the eye, but you can't do that can you, nooooo!
"You remind me of a eunuch, a fat smelling eunuch with boils on his face and on his baldhead that sits there on a lone bench in a park repulsing passers by who often have to put a piece of cloth to their mouth and nose to prevent themselves from gagging.
"I mean to cut you down tree! I will cut you down with a blunt axe so that you agonise in your slow death. Then once your trunk collapses with a satisfying thud to the ground I will unearth your roots and will place them into my oven. I shall put my oven onto such a great temperature that your roots turn to a fine ash that can even be dropped through a perfect set of teeth. I will then toss them into seas that haven't been sailed in for many centuries so that you shall all but be remembered.
"Oh and lets not forget those undecayed fruits of yours that will still be lying helplessly on the soil. I shall torture them to the extent that they will be envious of even those poor souls who were subject to the brazen bull."
He comes back into the house with his held down and muttering under his breath about the "bunny-massacring tree". I watch with an amused grin as he comes back past with a heavy axe in his hand.
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Get all the latest in MMA at http://MMAWeekly.com -- Check out the UFC 135 Jones vs. Rampage weigh-ins from Denver. Things got a little heated when several of the fighters posed for staredown photos! watch video >>
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All Topics » Homeland Security » Program management
Program management (31 - 40 of 255 items)
Alternatives to Detention: Improved Data Collection and Analyses Needed to Better Assess Program Effectiveness
GAO-15-26: Published: Nov 13, 2014. Publicly Released: Nov 13, 2014.
From fiscal year 2011 through fiscal year 2013, the number of aliens who participated in the U.S. Immigration and Customs Enforcement's (ICE) Alternatives to Detention (ATD) program increased from 32,065 to 40,864, in part because of increases in either enrollments or the average length of time aliens spent in one of the program's components. For example, during this time period, the number of ali...
Combating Terrorism: U.S. Efforts in Northwest Africa Would Be Strengthened by Enhanced Program Management
GAO-14-518: Published: Jun 24, 2014. Publicly Released: Jun 24, 2014.
Nearly half of the funds allocated for the Trans-Sahara Counterterrorism Partnership (TSCTP) since fiscal year 2009 have been disbursed, but TSCTP program managers are unable to readily provide data on the status of these funds. As of late 2013, U.S. agencies reported disbursing nearly $140 million of the approximately $288 million allocated for TSCTP between fiscal years 2009 and 2013. Mali, Maur...
Explosives Detection Canines: TSA Has Taken Steps to Analyze Canine Team Data and Assess the Effectiveness of Passenger Screening Canines
GAO-14-695T: Published: Jun 24, 2014. Publicly Released: Jun 24, 2014.
In January 2013, GAO reported that the Transportation Security Administration (TSA) collected and used key canine program data in support of its National Explosives Detection Canine Team Program (NEDCTP), but could better analyze these data to identify program trends. For example, GAO found that in reviewing short notice assessments (covert tests), TSA did not analyze the results beyond the pass a...
Civilian Intelligence Community: Additional Actions Needed to Improve Reporting on and Planning for the Use of Contract Personnel
GAO-14-692T: Published: Jun 18, 2014. Publicly Released: Jun 18, 2014.
Limitations in the intelligence community's (IC) inventory of contract personnel hinder the ability to determine the extent to which the eight civilian IC elements—the Central Intelligence Agency (CIA), Office of the Director of National Intelligence (ODNI), and six components within the Departments of Energy, Homeland Security, Justice, State, and the Treasury—use these personnel. The IC Chie...
Combating Terrorism: State Department Can Improve Management of East Africa Program
GAO-14-502: Published: Jun 17, 2014. Publicly Released: Jun 17, 2014.
State Department's (State) Partnership for Regional East Africa Counterterrorism (PREACT) supports U.S. counterterrorism efforts in East Africa. PREACT's five goals focus on improving partner nations' military capacity, rule of law, border security, ability to counter violent extremism, and ability to counter terrorist financing. PREACT has funded activities such as providing training for terroris...
Maritime Security: Progress and Challenges with Selected Port Security Programs
GAO-14-636T: Published: Jun 4, 2014. Publicly Released: Jun 4, 2014.
GAO's prior work has shown that the Department of Homeland Security (DHS) and its component agencies—particularly the Coast Guard and Customs and Border Protection (CBP)—have made substantial progress in three key areas of port security since the September 11, 2001 terrorist attacks (9/11), but some challenges remain.Maritime domain awareness and information sharing. DHS agencies along with o...
Trusted Travelers: Programs Provide Benefits, but Enrollment Processes Could Be Strengthened
GAO-14-483: Published: May 30, 2014. Publicly Released: May 30, 2014.
As of January 2014, there were about 2.5 million people enrolled in U.S. Customs and Border Protection's (CBP) four trusted traveler programs—which provide expedited travel for preapproved, low-risk travelers and cargo—and enrollments more than quadrupled over the past 5 fiscal years. About 43 percent of trusted travelers were enrolled in Global Entry, operating at select air ports of entry (P...
Federal Protective Service: Protecting Federal Facilities Remains A Challenge
GAO-14-623T: Published: May 21, 2014. Publicly Released: May 21, 2014.
The Federal Protective Service continues to face challenges ensuring that contract guards have been properly trained and certified before being deployed to federal facilities around the country. In September 2013, for example, GAO reported that providing training for active shooter scenarios and screening access to federal facilities poses a challenge for FPS. According to officials at five guard...
Homeland Security Acquisitions: DHS Could Better Manage Its Portfolio to Address Funding Gaps and Improve Communications with Congress
GAO-14-332: Published: Apr 17, 2014. Publicly Released: Apr 29, 2014.
GAO found that the funding plans for all 35 of the Department of Homeland Security (DHS) acquisition programs it reviewed changed to some degree from fiscal years 2012 to 2014, though the instability affected some programs more than others. Program officials reported that funding instability negatively affected 17 of the 35 programs, contributing to schedule slips, cost growth, and capability redu...
Civilian Intelligence Community: Additional Actions Needed to Improve Reporting on and Planning for the Use of Contract Personnel
GAO-14-257T: Published: Feb 13, 2014. Publicly Released: Feb 13, 2014.
Limitations in the intelligence community's (IC) inventory of contract personnel hinder the ability to determine the extent to which the eight civilian IC elements—the Central Intelligence Agency (CIA), Office of the Director of National Intelligence (ODNI), and six components within the Departments of Energy, Homeland Security, Justice, State, and the Treasury—use these personnel. The IC Chie...


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Yesterday HRM Online reported on a suit filed by Doriana Silva, who claimed creating thousands of fake profiles for Ashley Madison, the dating site for married people, caused her serious and permanent wrist damage.
Today the company has responded describing the suit as "frivolous" and claiming Silva is exaggerating her injuries.
"Ms. Silva's allegations are without merit, and we consider this to be a frivolous claim brought by an opportunistic Plaintiff," Ashley Madison's parent company Avid Media wrote.
The statement said Silva worked for 91 days, complaining about the wrist pain one day after her probationary period ended.
"An independent insurance auditor failed to find her credible and as such refused her claim," the statement said, adding that two medical professionals diagnosed Silva with a strain and prescribed rest. "Not until Ms. Silva travelled back to Brazil, did her medical diagnosis change."
"Several good faith attempts to accommodate Ms. Silva's alleged restrictions were rejected by her; Avid was then sued by Ms. Silva, with her extortionary demands escalating over time from $120,000 (USD) to $20 million," the company said.
The company said photos from Silva's social media showing her jet skiing and vacationing contradict her claim that the injury to her wrist is affecting how she can live her life.
Silva's lawyer Paul Dollak said his client is "alarmed" that Ashley Madison accessed her Facebook account without her permission and after she stopped working for the company, adding that the photos "have nothing at all to do with her ability to keyboard or ongoing injuries."
Ashley Madison refused to compensate Silva for her injuries, and she wasn't eligible for workers' compensation, so she had no other option but to sue, he claimed.
While compensation for personal injuries is usually calculated based on how much a person has suffered, Silva is seeking a share of the money earned through the profiles she created, Dollak has said.
When it comes to onboarding, HR should be able to manage, un ...
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A long-service employee quit last month amid claims of sexua ...
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Global uncertainty has workers worried yet employers continu ...
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My biggest concern with fraudulent claims such as the one in this article is that some bleeding-heart arbitrator, labour lawyer or judge will find a way to make an award for this claim. I believe the courts should be awarding costs to employers when people are found to make fraudulent claims for sick compensation, disability or workers compensation.
Wow..I don't know what's worse, a site that helps married people cheat or one of their employees suing after creating fake profiles for them. She probably won't succeed in her suit but claiming that the company does fake profiles is bad for the brand. From a business stand point, if she's lying they should sue her.
HRM Online forum is the place for positive industry interaction and welcomes your
professional and informed opinion.
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email the editor.
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Ray W wrote:
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> Mentor, Ohio
> P.S. anyone interested in a swingset and large sandbox?
> ---------------------------------------------------------------------
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Resource Scheduling
- Status Completed
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Project Description
Hi All,
Looking for a Staff Scheduling App. Something very Basic as i will be building on this. I basically need a GNATT type form. Multiple Staff down the side and the ability to create tasks (Allocatiing a amount of days to the task, and grouping tasks by Project Name) and then drag and drop on the GNATT Chart. It must also have the ability to show Day, Week, Month, and 3 Month Calander views
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Retailers posted their biggest rise in underlying sales for 10 months in January, suggesting that gloomy forecasts of a dire first quarter may be wide of the mark.
But high street chains warned that they received a boost from pent-up demand after the heavy snowfall in December, a surge in spending ahead of the VAT increase and continued heavy discounting. They also benefited from comparisons with weak sales in January 2010, when sales also suffered from heavy snowfalls.
Nevertheless, the British Retail Consortium and KPMG Retail Sales Monitor survey showed a healthy 2.3 per cent rise in like-for-like sales, which represents the best performance since the 4.4 per cent growth in March 2010.
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He added: "Growth this January was driven by a relatively short but strong burst of non-food buying early in the month. Clearance discounts and a last chance to beat the VAT rise got people buying things like furniture and electricals in the first few days."
Total retail sales jumped by 4.2 per cent in January, said the BRC. Many sub-sectors, including homewares, furniture and DIY, performed strongly, although sales growth at grocery and footwear chains slowed.
Helen Dickinson, the head of retail at KPMG, said: "The divergence between food and non-food narrowed in January, partly due to the VAT rise, and interestingly it was the sectors that had been struggling over the previous quarter, including furniture, DIY and home-related areas that picked up more strongly than clothing."
The BRC-KPMG survey is the latest indicator that consumer spending is holding up better than expected, after the 0.5 per cent fall in UK economic output in the final quarter of 2010. Yesterday, the accountancy firm BDO's High Street Sales Tracker found that like-for-like non-food sales jumped by 9.1 per cent at mid-market retailers in January, although growth slowed significantly in the second half of the month. In a separate survey, MasterCard said total sales rose 3.8 per cent, driven by a bullish performances at department stores, clothing and furniture chains.
However, the BRC warned that non-food sales growth waned in the second half of January, as pressures on household budgets, from record petrol prices to the rise in VAT, hit home.
Mr Robertson said: "Later in the month sales of non-food goods slowed, particularly for bigger items, as the reality of worries about jobs and personal finances returned to customers' minds."
Ms Dickinson also had her cautious hat on. She said: "Falling disposable incomes will continue to exert pressure on sales in the first quarter of the year, traditionally the weakest period of trading as consumers cut back on spending post-Christmas."
- More about:
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Category:Barford St Martin
From Wikimedia Commons, the free media repository
English: Barford St Martin, a Wiltshire village with 467 adult residents, is situated about two miles from Wilton, on the junction of the A30 and the B3089. Barford is known as one of the Nadder Valley Villages, named for the River Nadder, which flows through the village.
Nederlands: Barford St. Martin is een dorp (village) en civil parish in de unitary authority Wiltshire, in het Engelse graafschap Wiltshire. De civil parish telt
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Good to hear, for you at least. I haven't turned on the TV or radio yet to
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they all went home at 1200 yesterday. Hope they will be OK. They were
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Beaufort, SC
> [Original Message]
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> To: Garden Chat <firstname.lastname@example.com>
> Date: 7/9/2005 5:52:37 AM
> Subject: [CHAT] Dennis update
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> ---------------------------------------------------------------------
> To sign-off this list, send email to firstname.lastname@example.com with the
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Are you looking for Metal Detector in Queensland? Hotfrog showcases more than 10 popular businesses related to Metal Detector in Queensland! Find a specific region or suburb using the left navigation.
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Polycom Puts Video Conferencing at the Center of the Room
The vendor's new Centro and Trio products are designed to give people a more natural way to collaborate visually.Polycom officials want to move the focus of video conferences from the end of the conference room to the center. The company is rolling out an array of new telepresence devices that officials said are designed to fit how people sit and communicate when they gather together. "When people collaborate, they tend to work around the table," Michael Frendo, executive vice president of engineering at Polycom, told eWEEK. "They don't sit and look at screens at the end of the room." That was the driving force behind the company's new RealPresence Centro video conferencing product, and also played a role in the thinking behind its new RealPresence Trio smart hub for group collaboration. Both were among the products unveiled at an event in New York City Oct. 7 marking Polycom's 25th anniversary.
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All cities in Germany


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Science & Medicine, Natural Sciences, Society & Culture, TechnologyFollow
|Natural Sciences
|Science & Medicine
Whether the topic is popcorn or particle physics, you can count on BrainStuff to explore -- and explain -- the everyday science in the world around us.
October 30th, 2009
Episode 428 of 1093 episodes
Desktop computers and laptops only have so many USB ports, so what do you do when you want to plug in more USB devices and they're all full? You buy a USB hub. Find out how these hubs work, and which kind you should get, in this episode of BrainStuff.
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Welcome to the daily audio workout for your head.


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Research in the journal Obesity found that obese people on a low-calorie diet who ate more carbohydrates at dinner lost more weight and fat and felt fuller than those on a regular low-calorie regimen. Researchers said the group that ate more carbs at dinner had smaller decreases in leptin, a hormone that regulates satiety and hunger, than the control group.
Full Story:Los Angeles Times (tiered subscription model)


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At Lambert, Edwards & Associates, we are always on the lookout for the "best athletes" in the industry or region to join our team. If you think you're a star and would like to join LE&A's team, feel free to send us information about yourself and previous experience. We look forward to hearing from you!
Opportunity: Investor Relations Professional
Opportunity: Public Relations Director – Detroit
Our internship program runs during the following three seasons:
January – May (Resume deadline: November 15)
May – August (Resume deadline: February 15)
September – December (Resume deadline: July 15)
Interested Junior and Senior students should send a resume, cover letter, season desired and preferred office location to:
Grand Rapids Office: Amanda Passage
Detroit Office: Ashleigh Chatel