text:
I got pretty nervous when they hit a couple of 3s at the start and got up on us by 10 points. But I had confidence in my teammates that would be able to do what they needed to do to bring us back. —South Sevier junior forward Patrick Baker
RICHFIELD — The Sevier Valley Center was full of cheers and tears on Saturday night.
Thunderous cheers for one whale of a state title game, and tears of joy — and defeat — when the final buzzer sounded.
South Sevier shook off a shaky start to turn back arch-rival Richfield 52-47 in an intense showdown for the 2A boys basketball championship, played before a packed house of 5,000-plus raucous and rowdy fans who pleaded for a victory and protested seemingly every time the officials blew their whistle.
The Rams (24-1) finished off a dream season by claiming their second straight state title, rattling off 24 consecutive victories — but this last one was anything but easy.
"We were ranked No. 1 to start the season, and we got beat that very first game of the year against that Wasatch Academy bunch," South Sevier coach Scott Hunt said. "When you're the defending state champions and ranked No. 1, you are everybody's biggest game and our kids have got to get up for that every single game, night in and night out. And that's a real testimony to these kids' will and strength and courage and character that they bring it every night.
"They won 24 in a row and finished it off with the state championship, having that target on their back all season long."
The hometown Wildcats of Richfield (17-7), playing in the school's first boys state championship game since 1993, jumped out to a quick 6-0 lead on a pair of 3-point bombs by Zak Jensen, and the crowd went bonkers.
"That was my fault," Hunt said. "We felt like their strength was Tyler Anderson and Jordan Kemp. So we started in kind of a gimmick defense, which I knew the Jensen kid could potentially hurt us in that and he did. He knocked those two 3s and got them going, and that was a pretty bad mistake on my part that defensive assignment I gave our kids."
Richfield extended its lead to as many as 10 points at 13-3 before South Sevier began battling back. The Rams, led by senior guard Kaden King, tournament MVP Race Parsons and junior forward Patrick Baker, steadily chipped away at the Wildcats' lead, then finished the first half with an 11-0 run to take a 27-22 lead at intermission.
"They battled back," Hunt said proudly of his team. "They've been winners all year; they're warriors, and there's no quit in these kids. They inched their way back, inched their way back.
"That second quarter was a big turning point in that ballgame when we went back into our old, trusty half-court trap, and they didn't really like that trap very well. We got a few steals and that got us going a little bit, got some momentum going and loosened us up a little bit and we went into halftime with the lead, and of course the kids felt a lot better at that point in time."
And when South Sevier extended its lead to 10 points, 41-31, by the end of the third period, it looked like the Rams might be home free.
Not true.
"And yet the second half was still just a dogfight," Hunt said. "We knew with Richfield it was going to be. We got stuck on 41 and couldn't score forever.
"The thing of it is is that they play such good defense that finding points is like pulling teeth. It really is. And they know us so well; they're in our region and we've played 'em twice."
Indeed, Richfield refused to fold, as Anderson fueled a fierce comeback that brought the Wildcats back within three points twice down the stretch, the last time at 46-43.
But Parsons fed King for a layup, King and Colton Jones each hit free throws, and Parsons finally put it away with two foul shots with 11.6 seconds left to seal the deal.
Parsons finished with 16 points with 11 rebounds, five assists and four steals; Baker banged in 15 points and grabbed 10 boards, and King contributed 10 points in his last prep game.
Anderson led Richfield with 12 points, Kemp added 10 and Jensen finished with eight for the Wildcats.
"I got pretty nervous when they hit a couple of 3s at the start and got up on us by 10 points," Baker said. "But I had confidence in my teammates that would be able to do what they needed to do to bring us back.
"Richfield-South Sevier game, plus playing for the championship, you couldn't ask for a better crowd."
Though held to 16 points, more than 8 below his season average, Parsons said the only numbers that counted were the ones on the scoreboard.
"That was a very tough game," he said. "Richfield's a tough team and we knew it was going to be a hard-fought battle, a knock-down, drag-out game, and my hat goes off to Richfield. They're a very good team and they could very easily be right where we are.
"They hit some shots, but it wasn't enough to beat us and that's all that matters. ... You look up at that scoreboard and it says 52-47 and we won. I don't care how many I score, we won the game. ... We have two very good seniors and I'll miss 'em with all my heart."
One of those seniors is King, who was mighty glad to finish his prep career in style — with a second straight state title.14 comments on this story
"It feels great," he said. "When I look back, I really don't remember it but it's just all coming back, that great feeling. I love it. It feels better to me, being my senior year and ending on a good note.
"Richfield came out way hot and they were hitting about every shot they shot. But we just kept playing our game. I had no doubt at all the whole game. That's one thing our coach said, 'No doubt.' "
email: email@example.com
MVP — Race Parsons, South Sevier
Kaden King, South Sevier
Tyler Anderson, Richfield
Jordan Kemp, Richfield
Cody John, Wasatch Academy
Devin Cluff, Manti


text:
Social Question
Can these mini bananas be eaten raw?
My grand daughter LOVES bananas. Rick was at the store and found some mini-bananas. I was trying to remember what they're called…Plantian or something. Starting looking around and found where a mom said her baby did fine with regular bananas, but when she gave her mini bananas her baby wound up with a rash on her chin. Then there was this comment about it on Yahoo answers:
"Those mini bananas are called plantains and they cannot be eaten raw. You have to cook them. They are usually used to make plantain or banana fritters and plantain chips. THis might be why your lo broke out."
They're SO CUTE and my granddaughter would just love them…but I really don't want to kill her. Mom and Dad wouldn't talk to me for a WEEK if I did that. :}~

subset: commoncrawl


text:
D.C. Jewish Film Festival announces 2014 lineup
WASHINGTON (AP) - The Washington Jewish Film Festival is announcing its lineup of 64 movies that will be featured this year at theaters across the Washington area beginning next month.
The festival opens Feb. 27 with a screening of Avi Nesher's film "The Wonders" about a bartender who doubles as a graffiti artist. Nesher, an Israeli filmmaker, will receive the festival's annual Visionary Award for his work. Several of his films will be screened throughout the festival.
Another highlight will include a sneak preview of John Turturro's new film "Fading Gigolo," which recently premiered at the Toronto Film Festival. Turturro directs and stars in the film, which also features Woody Allen. "Fading Gigolo" will be the festival's Centerpiece Evening film on March 8.
The festival also will feature a focus on Polish cinema.


text:
Top CEOs: Companies with 25-100 employees
Don Murphy
Barfield Murphy Shank & Smith PC
'A CPA is often referred to as the most trusted adviser.'
A CPA is often referred to as the most "trusted adviser," and we treasure that title. That's who we want to be.
So says managing shareholder Don Murphy, and it's a philosophy that has served his firm well in recent years.
At Barfield Murphy Shank & Smith, the firm's core business, about 20 percent of the net revenue, comes from the commercial construction sector. With commercial construction languishing, the firm has asserted its role as adviser to continue serving its clients.
"The first thing I have done is to help them figure out how to right size their companies," he said. "If they were doing $30 million before and now they are doing $10 million, we need to set them up to be that $10 million company. Do they need five people here or do they need two? Should they renew the lease on their office space? Sometimes these people just need somebody to talk to."
This approach has helped win trust, which in turn has helped the film woo a couple of hundred new clients during the downturn, enough to keep revenues flat – a big win at a time when a nosedive could easily have been in the cards.
At the same time, the firm has been pushing ahead in another direction: It's been going green.
In fact, Barfield Murphy took up the environmental banner before others in the field, adopting a paperless office eight years ago. Internal files are digital. Clients share documents via secure websites. It saves time and money and it shows off the kind of creative thinking that is helping this firm succeed.
- Adam Stone
Lee Lichlyter
Geographic Information Services
'We have a philosophy that we call ''customer karma."'
Lee Lichlyter loves what he does because every day is different.
"You get new challenges, different things to do, different decisions to make and it keeps you constantly on your toes," said Lichlyter, who heads Geographic Information Services, a 20-year-old, geo-spatial business. "Along the way, I realized that while I love technology, what I loved doing the most was building a high performance organization."
Though he has only been in Alabama five years, he has seen significant growth.
"We have gone from about 30 to 100 employees and our revenue growth, over the last three years, has approximately tripled," Lichlyter said.
Growth has derived strictly from selling work to new clients and having to hire more people. Repeat business from customers in federal and local government, the U.S. Department of Defense and private sector like retail and energy companies has also contributed to the success.
"We have a philosophy that we call 'customer karma', and the idea is that if we take care of our customers even when it may not be the best thing for us, it will pay off in the long-run," Lichlyter said.
In great part, he attributes his vision of having the premier consulting company in the industry to his team.
"We like people who are intellectually relentless, which means they are constantly thinking, learning and love to solve problems," he said. With employees in 22 states, it has required innovation communication and work culture, including web conferences and bringing everyone to Birmingham a few days each year to reconnect and reenergize.
And while Lichlyter thrives on every day being different, he especially appreciated the one where they celebrated the company's 20th anniversary. One of the most recent accomplishments was creating an employee stock ownership plan last year.
"Employee owners take more personal responsibility for the success of the company," he said. "Know what you do really well and focus on getting better at that every day."
- Jamie Lober
Joan Ragsdale
MedManagement LLC
'My role has focused on expansion.'
As a health care lawyer, Joan Ragsdale applied her knowledge of compliance as she branched out to work with physicians and hospitals through her Alabama-based company, MedManagement LLC.
Ragsdale is CEO of the majority woman-owned business, which provides a service called physician adviser services.
"My role has focused on expansion and developing relationships from a regional and national perspective," she said.
MedManagement has clients in about 10 states, which is handled by a highly efficient team of less than 100 workers. This includes the same core group of owners since the company was founded.
Ragsdale travels to meet with clients and helps facilities comply with Medicare, Medicaid and federal regulations, as well as routing the appropriate documents to the right person to avoid issues.
"Ideally, we work with them concurrently with the care being rendered so we can help them make certain that documentation is in place to support them when they are audited," Ragsdale said.
"From the beginning of 2010 to the end of 2010, we quadrupled the revenue attributed to those physician adviser services," she said. The physician billing, practice management and information technology services have also done well. "We had modest increases with the 2009 and 2010 economy; we were still pleased."
Ragsale said her first priority is to do the right thing and she tries to be a good steward of her resources.
"Growth is driven by the idea of expanding services and meeting an unmet need in the marketplace," she said.
Ragsdale subtly tries to get the word out about her business.
"We have focused on updating our website and where we are on search engine capability and are beginning to work on getting our name out nationally," she said. These mediums have been effective as well as her reputation for being responsive and punctual. "If you get a phone call or question, make sure that you return it and get back to people quickly."
Ragsdale wants to be nationwide by late 2012 and anticipates doubling their client base in 2011.
"Growth trajectory is dependent upon us communicating what we do, how we do it and the value."
Being mission-driven and offering quality initiatives as she renders care in a way that is compliant with reimbursement policies and guidelines has surely helped.
"I have been fortunate to learn from people that had their heart in the right place."
- Jamie Lober
Bruce Denson
Cobbs Allen & Hall Inc.
'I enjoy the people and I love the challenges.'
Since Cobbs Allen & Hall is primarily in sales, Bruce Denson is focused on selling and ensuring he and his employees work "smartly."
"I manage areas that need managing," said Denson of CEO Cobbs Allen & Hall Inc. "We want to be a different type of insurance broker than the norm and want to offer services to our clients that will help them improve their balance sheet."
Denson attributes his success to holding himself and his people accountable. He wants to continue growing the firm through new business sales and offering value-added services. That might entail Denson recommending a wellness coordinator or human resources person to help clients have a safer place to work.
As an insurance broker, he represents insurance companies and sells to both the public and business. They sell coverage ranging from property to employee benefits. Most recently, he wrote insurance for individuals and businesses affected by the April 27 tornadoes that tore through Alabama.
Though managing his business is a priority, Denson makes time for himself.
"My personal interest is that I love to stay in shape and play golf, so I work out a minimum of five days a week," Denson said.
He enjoys going to the YMCA or spending time with a personal trainer. He also likes going on trips with his wife. "We have a good time; in fact, we just got back from the Bahamas after spending a week there with some friends," he said.
The company's growth has been organic, although it also reflects the flux of a challenging economy, according to Denson.
"When we re-engineered the firm in 1992, roughly a year before I got here, we only had about $1 million in revenue, now, we have $26 million," he said. "It is all based on new business sales so we do not have 100 percent retention. We are going to lose a little business either through loss of accounts to another broker or in tough economic times. People's sales and payroll are less than they were the year before."
Though Denson has been in the business since he was 22-years-old, he started at Cobbs Allen & Hall at the top in 1993.
"I enjoy the people and I love the challenges," he said. "It is about more than just selling an insurance policy."
- Jamie Lober
Bill Ballard
'My philosophy is win at everything I do.'
Bill Ballard built WIAT – CBS 42's success on the strength of his 100 employees' character and strong work ethic.
"I look for intelligence, character, strong work ethic and a successful track record because it makes my life easier," said Ballard, the station's president and general manager.
As a result of hiring good people, working hard, listening to the community and making the right decisions, he has reaped the rewards of turning the station around from losing money and overcoming the challenge of getting past the stigma of formerly being WBMG-TV with outdated infrastructure.
"We had share growth 22 out of the last 26 quarters, and our gross revenue and cash flow have been growing consecutively almost every quarter since I have been here," Ballard said. "Now we are amongst the top 25 in the country out of roughly 186 CBS stations and my goal is to be the most watched television station in our market."
The station has grown by about 20 people since 2003, a growth trend that he hopes will continue.
Asked why Ballard joined the station, he said there was nowhere to go but up and that's become a reality.
"I was offered a job selling cardboard boxes and said, if I am not going to make any money I might as well have fun, so I got a job for radio with less money," he said.
From there, he shifted to television, which lead to a series of promotions as he was willing to move. Today, he manages CBS 42 and has ownership and an equity position in the station. He's responsible for all aspects of the station from dealing with the federal government and the FCC to hiring talent and engineering staff.
In the future, Ballard foresees the news department doing more investigative reporting.
"My philosophy is to win at everything I do," he said. "I am competitive, enjoy the city of Birmingham and central Alabama and am the longest-serving general manager in town."
- Jamie Lober


text:
At A Time for Heroes, Ambassadors Play Starring Role
At this year's A Time For Heroes carnival in Los Angeles, California, the stars of Hollywood were out in full force. From Interscope Recording Artist Jessica Sanchez to actors and actresses from Disney's "Teen Beach Movie," celebrities took the time to pay tribute to EGPAF's important work and enjoy a day of fun in the sun. But our favorite stars were the EGPAF Ambassadors who gathered at A Time For Heroes to share their stories and the mission of the Foundation.
On Fox 11 in Los Angeles, EGPAF Ambassadors Martha Sichone-Cameron and Jamie Gentille shared their stories, explained why more focus on pediatric HIV is critical, and told the host why they were so excited to be a part of A Time For Heroes.
Los Angeles Local News, Weather, and Traffic
On the American Urban Radio Networks, EGPAF Ambassador Hydeia Broadbent told her story and explained why A Time For Heroes is so important for EGPAF and its work to eliminate pediatric AIDS around the world and help mothers and children stay healthy and happy. Listen to the interview here.
We'd like to thank our Ambassadors for their passion, grace, and candor. We are so proud to work with them every single day in our fight against pediatric HIV.
Jane Coaston is Media Relations Coordinator for the Foundation, based in Washington, D.C.


text:
» Category: General
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text:
IBM Storwize V7000 Eguide: All-in-one Data Storage
Your storage system is important to your business. That is why a storage virtualization solution based on IBM Storwize V7000 will deliver immediate benefits to your IT operations. This guide will walk you through the features and benefits of Storwize V7000 and illuminate the differentiators that make Storwize V7000 an easier, faster, all-in-one storage solution to manage all your data with a simple interface and in real time.
The guide explains storage virtualization as the key to managing data effectively and explores the ways IT pros can benefit from implementation.
Multiple links to resources are provided throughout the guide, and it wraps up with a summary of benefits in deploying Storwize V7000.


text: Hi all, I mentioned in a previous thread that I had some wood stacked up in a shady spot and it started to grow some shrooms on it. I went ahead and split it today and there was some water damage in it but seemed kind of solid. Maybe slightly "spongy", if that's the right description? Do you think it's ok to burn in an insert? -MP


text:
Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed.
Methods and Findings
We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo.
Citation: Chen C, Wang S, Wang H, Mao X, Zhang T, Ji G, et al. (2012) Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain. PLoS ONE7(8): e43845. https://doi.org/10.1371/journal.pone.0043845
Editor: Michel R. Popoff, Institute Pasteur, France
Received: April 9, 2012; Accepted: July 26, 2012; Published: August 29, 2012
Copyright: © Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from National Nature Science Foundation for China, Ministry of Science & Technology of China (973 and 863 Program Projects), Shanghai Commission of Science & Technology (Key Laboratory and projects) as well as special program projects for infectious diseases and new drug development from Ministry of Science & Technology of China (2010ZX09401-407). Dr. Jianxin Dai is recipient of Pujiang Scholar Award from Shanghai Commission of Education. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Botulinum neurotoxins (BoNTs) comprise a group of highly lethal toxins consisting of 7 serotypes (BoNT/A-G) produced by the anerobic bacteria, Clostridium botulinum [1], [2]. Four of the BoNT serotypes (A, B, E, and F) cause human botulism, a neuroparalytic disease which results from ingestion of pre-formed toxin present in contaminated food and from toxin produced in vivo from infected wounds [3]. Owing to their extreme potency and lethality, BoNTs are included in the list of category A select agents and toxins [4]. Each BoNT isoform is synthesized as a single polypeptide chain with a molecular mass of ∼150 kDa. The inactive precursor protein is cleaved either by clostridial or tissue proteases into a 50-kDa light chain (LC) and a 100 kDa heavy chain (HC) linked by an essential interchain disulfide bridge and by the belt, a loop from the HC that wraps around the LC [5]. The LCs act as zinc metallopeptidases, which solely hydrolyze one of three SNARE proteins depending on the serotype: BoNT A and E cleave synaptosomal-associated protein of 25 kDa (snap-25) and BoNT/B and F cleave the vesicle associated membrane protein (VAMP) [6], [7], [8], [9]. resulting in a blockade of neurotransmission and flaccid paralysis [10]. The heavy chain is divided into two functionally distinct regions: a C terminal binding domain (Hc) and a N terminal translocation domain (HN) [11]. The binding domain initially interacts with low affinity to a group of gangliosides on the presynaptic plasma membrane [12], after which it binds to a protein acceptor. Interestingly, the BoNTs' serotypes that exhibit highest sequence similarity share the same protein receptor, i.e., BoNT types A, E, and F bind SV2 [13], [14], [15], whereas BoNT types B bind SytI and II [16]. The existence of two classes of binding sites distinguished by different affinities and the discovery of protease-sensitive binding to neurons resulted in a double-receptor concept. In a first step complex polysialogangliosides accumulate BoNTs on the plasma membrane surface; and in a second step, protein receptors mediate their endocytosis.
Monoclonal antibodies (mAbs) have been intensely explored as inhibitors of the recognition step between BoNTs and their cellular receptors. [17] However, the two-receptor model makes it difficult to development of a mAb-based antitoxin for botulism. Single mAb recognizing only one epitope can hardly block the binding between BoNT and cell completely. It might be the reason why single mAb can only neutralize at most 10 to 100 times the 50% lethal dose (LD50) of toxin in mice [17]. Combination of two or three mAbs recognizing nonoverlapping epitopes can neutralize BoNT A very potently [18]. Although the binding areas of antibodies were mapped, however, the epitopes which these antibodies bind to are not finely defined. However, not all randomly paired mAbs have the potent synergistical neutralizing function. It is more difficult to select mAb pairs with desirable synergistical function from a panel of neutralizing mAbs whose epitopes are not clear. Because both the protein receptor and ganglioside receptor are essential for the entrance of BoNTs into neurons [19], we predicted that two monoclonal antibodies, which recognized protein and ganglioside receptor binding domain respectively, can neutralize synergistically against BoNTs.
In this study, we selected three neutralizing mAbs, which recognize different non-overlapping epitopes of BoNT/B, and compare the neutralizing effects among different antibody combinations. We found that 8E10, response to ganglioside receptor binding site, could collaborate with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination between two mAbs, 5G10 and 2F4, blocking protein receptor binding sites, did not achieve synergistical effects. We also found that the binding epitope of 8E10 was conserved among BoNT A, B, E and F. It could cross-protect the challenge of different serotypes of BoNTs in vivo. The results provide a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.
1. A panel of neutralizing antibodies specific for BoNT/B-Hc
To generate mAbs capable of neutralizing BoNT/B, gene encoding protective antigen Hc fragment of botulism neurotoxin serotype B (BoNT/B-Hc) was optimized and synthesized. The reconstructed Hc proteins were expressed in E.coli BL21 (DE3) in soluble form and purified for preparation of a toxoid immunogen. Sixteen hybridomas were obtained and screened for the production of anti-BoNT/B Hc mAbs, using ELISA with purified BoNT/B Hc (Fig. 1A). Cell culture medium corresponding to the positive wells was subjected to western blot assay for testing the binding specificity of these mAbs (Fig. 1B). The results showed that all of the positive hybridomas were able to bind to native or denatured BoNT/B Hc specifically. The supernatants of these clones were then tested for their ability to protect mice against 4 LD50 of BoNT/B for 4 days. Among them, 7 mAbs (8D1, 8E10, 1F4, 2F4, 5G10, 2H12 and 2B2) could neutralize BoNT/B in vivo (Fig. 1C).
The binding activity of mAb was determined by ELISA (A) and Western blot analysis (B). Neutralizing mAbs were selected by in vivo neutralization assay(C). For the initial screening of the positive hybridoma that produced the neutralizing antibody, 200 µl of culture supernatant from hybridomas grown in a 24-well tissue culture flask for 3–5 days, was pre-incubated with 4 LD50 of BoNT/B for 1 hour at 37°C, and the reaction mixtures intraperitoneally injected to each of six mice. More than 4 survivals in four days were considered as positive. Serum of the immunized mice in the dilution of 1∶100 was set as control. Left: PBS, 1D12, 8D1, 8E10, 4E5, Serum. Middle: PBS, 2F4, 2H12, 2G11, 3F11, 2H7, Serum. Right: PBS, 5G10, 1F4, 2B2, 3D8, 3C1, 8H5, Serum. The error bars represented standard deviations.
2. Blocking activities of the neutralizing mAbs
The first step in BoNT attachment to nerve membranes starts with C-terminus of Hc binding to gangliosides of neuron membranes. Ganglioside binding could either bring the toxin and the protein receptor into close proximity or cause a conformational change at the protein receptor binding site to enhance interactions [2]. To identify binding domain which was blocked by the seven neutralizing mAbs, we used ELISA-based analysis of GT1b or SytII binding with BoNT/B Hc pre-incubated with or without mAbs. The results indicated that mAb 8E10, 1F4 and 2B2 blocked the interaction between BoNT/B Hc and GT1b (Fig. 2A), whereas 8D1, 2F4, 5G10 and 2H12 interrupted the binding between BoNT/B Hc and Syt II (Fig. 2B).
Plates were coated with GT1b (A) or recombinant syt II-LD (B) respectively. HRP-conjugated BoNT/B Hc was pre-incubated with neutralizing mAbs at molar ratio of 1∶100. Bound BoNT/B Hc was detected by adding TMB as a substrate.
To further investigate if the epitopes recognized by the antibodies were overlapped, we tested mAbs using competitive binding ELISAs. The optimal dilution of each HRP-conjugated mAb was determined by testing serial two-fold dilution in BoNT B Hc-coated ELISA plates and selecting this as the working dilution to generate an optical density of around 1.5. The selected working dilution was 1/8000 for mAbs 2B2 and 2F4, 1/16000 for mAbs 8D1, 1F4, 8E10, 5G10 and 1/4000 for 1F4, 2H12. The results of competitive ELISAs were reported in Table 1.
HRP-conjugated 8E10 inhibited the binding of 1F4 or 2B2 to the antigen and vice versa, showing that 8E10, 1F4 and 2B2 recognized overlapping epitopes. HRP-conjugated 5G10 inhibited the binding of 2H12 to the antigen and vice versa, indicating that 5G10 and 2H12 recognized overlapping epitopes. The same competition was also observed between 2F4 and 8D1. MAbs were thus categorized into 3 groups. MAbs binding to the same epitopes or overlapping epitopes were put into the same group (Table 2). Group 1 consisted of mAbs 8E10, 1F4 and 2B2, group 2 included mAbs 5G10 and 2H12 and group 3 included mAbs 2F4 and 8D1. Because the antigen binding affinity of the mAbs 8E10, 5G10 and 2F4 was higher than that of other 4 neutralizing mAbs (Table 1), they were selected for fine epitope mapping based on phage-display.
3. Epitope mapping of mAb 8E10, 5G10 and 2F4
After three successive rounds of panning on 8E10, 5G10 and 2F4 respectively, we randomly selected and purified 100 positive phages. DNA sequence analysis of the phage-encoded peptides revealed 4–6 amino-acid consensus sequences, which was aligned with the sequence of BoNT/B displayed in Table 3. All phages selected by 8E10 have a common motif of SXWY. Alignment of this sequence on the sequence of BoNT/B provides a best fit with 1259SKWY1262. 5G10 and 2F4 bind to SDXFY and KXSP, which correspond exactly to residues 1201SDEFY1205 and 1114KDSP1117 of BoNT/B respectively. The peptides containing 1259SKWY1262, 1201SDEFY1205 and 1114KDSP1117 motif in the BoNT/B were synthesized and denoted as P1, P2 and P3 respectively. The ELISA assay results showed that mAb 8E10, 5G10 and 2F4 bound to KLH-conjugated P1, P2 and P3 peptides (P1, P2, P3-KLH) specifically since these mAbs did not cross-react with the other peptides recognized by the other two mAbs and control peptide-KLH (CP-KLH)(Fig. 3A). The capability of the synthesized peptides to block the binding of mAb 8E10, 5G10 and 2F4 to BoNT/B Hc was also determined. The results revealed that peptide P1, P2, P3 effectively inhibited the binding of mAb 8E10, 5G10, 2F4 to BoNT/B Hc respectively. In contrast, control peptide had not inhibitory effect on their interaction (Fig. 3B). These results further demonstrated that the motifs listed above were the epitopes of 8E10, 5G10 and 2F4 respectively.
Binding of synthetic peptide with mAb. Peptides P1, P2, P3, containing the motifs recognized by 8E10, 5G10 and 2F4 respectively, were synthesized and conjugated with KLH, which were indicated as P1-, P2-, P3-KLH. The binding abilities of mAbs were detected by ELISA. A non-associated peptide was set as control peptide (CP), and conjugated with KLH as CP-KLH. (B) Competitive inhibition of BoNT/B Hc binding with mAbs by synthetic peptides. CP-KLH: synthetic peptide THPHLPRALMRS was coupled to KLH (bearer protein) to generate CP-KLH; P1-KLH: synthetic peptide YFCISKWYLKEV was coupled to KLH (bearer protein) to generate P1-KLH. P2-KLH: synthetic peptide PISDSDEFYNTI was coupled to KLH (bearer protein) to generate P2-KLH; P3-KLH: synthetic peptide IKLKKDSPVGEI was coupled to KLH(bearer protein) to generate P3-KLH.
BoNT/B Hc has been crystalized in complex with trisaccharide sialyllactose (a mimic of GT1b) (PDB 1F31) and with SytII peptide (PDB 2NM1). The crystal structure reveals two distinguished domains which bind to GT1b and SytII respectively. Therefore, it is possible to compare the binding site of protein and ganglioside receptor to BoNT/B Hc with the localization of the mAb 8E10, 5G10 and 2F4 epitope identified in this study. The illustration of structure of BoNT/B Hc is showed in Fig. 4. The epitopes of mAbs were colored and overlaid on the three-dimensional structure of BoNT/B. The epitope of 8E10 (colored in red) is just located inside the GT1b-binding domain (colored in yellow) (Fig. 4 A). The residues "1114KDSP1117" recognized by 2F4 (Fig. 4B, blue) cover part of the Syt II binding pocket formed by residues K1114, S1116, P1117, V1118, Y1183, E1191, K1192, F1194 and F1204 (Fig. 4B, green). The 5G10 epitope comprises 5 amino acids "1201SDEFY1205" (Fig. 4B, cyan), which overlaps another side of Syt II binding site formed by residues P1117, W1178, Y1181, F1194, A1196, P1197 and F1204 (Fig. 4B, green). However, the epitopes of 5G10 and 2F4 are not overlapping with each other. These results provide the structure basis of neutralizing functions of these mAbs. The mAb 8E10 neutralizes BoNT/B through blocking the interaction between BoNT/B Hc and its ganglioside receptor on neurons. 5G10 and 2F4 inhibit the binding of BoNT/B Hc with protein receptor SytII.
The model was established using the software Discovery Studio 2.0 (Accelrys, San Diego, CA) based on the crystal structure of BoNT/B Hc (PDB 1F31) from the Protein Data Bank. BoNT/B Hc is shown in a surface representation. (A) The residues reported as GT1b-binding site are colored yellow (Nat. Struct. Biol. 1998), and the residues recognized by 8E10 are colored red. (B) The SytII-binding site residues are colored green, and the residues of 5G10 and 2F4 are indicated in cyan and blue.
4. Binding inhibition assay in vitro
In order to determine the toxin neutralization by IgG in vitro, we used SytII transfected and ganglioside treated PC12 cells, a neuroendocrine cell line of which wild type is lack of functional toxin receptors [20], to serve as a target cell model. Flow cytometry results showed that FITC-labeled recombinant BoNT/B Hc could bind to SytII transfected and ganglioside treated PC12. 8E10 could completely block the interaction between BoNT/B Hc and ganglioside treated PC12 (Fig. 5A). 5G10 and 2F4 could inhibit BoNT/B Hc to bind to the SytII transfected PC12 effectively (Fig. 5B). However, as to the SytII transfected and ganglioside treated PC12, none of the mAbs above could completely block its binding to BoNT/B Hc. In contrast with the single mAb, the combination of 8E10 with 5G10 or 2F4 could completely inhibit the binding of BoNT/B Hc to the double treated PC12, while the combination of 5G10 with 2F4 could not block the binding of PC12 to BoNT/B Hc (Fig. 5C). The results of confocal microscope showed that the combination of 5G10 and 2F4 could only decrease the fluorescence signal on the membrane of double treated PC12 similar to the effects of single antibody treatment. In contrast, the mixture of 8E10 and 5G10 or 2F4 abrogated the fluorescence signal from the cell membrane completely (Figure S1). These results demonstrated that mAbs, which recognized ganglioside or SytII binding domain respectively, had the potent to neutralize BoNT/B synergistically.
The single or pairs of mAbs were mixed with FITC-BoNT/B Hc at a molar ratio of 1∶100 before staining PC12 cells treated as indicated. Inhibition of mAbs for neuron binding activity of BoNT/B was indicated by the flow cytometer. (A) ganglioside-treated cells and BoNT/B Hc with or without single mAb (8E10, 5G10 or 2F4) (B) the binding of SytII+ cells and BoNT/B Hc with or without single mAb (8E10, 5G10 or 2F4), (C) the binding of ganglioside-treated syt II+PC12 cells and BoNT/B Hc with or without single mAb (8E10, 5G10 or 2F4) or pairs of mAbs (8E10+5G10, 8E10+2F4, 5G10+2F4). For pairs of mAbs, the molar ratio between BoNT/B Hc and mAbs is 1∶50∶50.
5. Protection of mice from BoNT/B toxicity using neutralizing mAbs
In vivo toxin neutralization was studied using a standard mouse protection assay. We incubated 100 µg mAbs with BoNT/B for 1 hour prior to intraperitoneal injection into BALB/c mice and the groups of mice with 6 animals for each group were used to test dose level. Mice were observed for morbidity and mortality over 30 days. Complete protection was observed with doses up to 20 LD50s for 8E10, and 40 LD50s for 5G10 and 2F4. Partial protection, as indicated by increased survival compared with antibody-free control mice, was afforded with higher doses (Table 4). At 80 LD50s, mice receiving 5G10 and 2F4 survived 72 hours, compared to 6 hours for the control mice. As combination of mAbs have demonstrated synergy in BoNT/A neutralization [18], the different pairs of mAb were studied at increasing doses of toxin to explore potent combination. We mixed 50 µg each of the mAbs (total of 100 µg) with different LD50s BoNT/B and tested the combination by intravenous injection. At 640 LD50s, only 1 of 6 mice receiving the pair of 5G10+2F4 survived whereas none of mice receiving two of the pairs of mAbs (8E10+5G10 or 8E10+2F4) died. 3 of 6 mice that received the pair of 8E10+5G10, and 2 of 6 mice that received the pair of 8E10+2F4 survived for 96 hours in the group challenged with 1,280 LD50s. In contrast, the control mice died in less than 3 hours. The survival rate of the group received 8E10+5G10+2F4 is the same as that of the group received the pair of 8E10+5G10. No mice survived in the group challenged with 2,560 LD50s (Data not shown).
6. Neutralizing potency of mAb 8E10 against different serotypes of BoNTs
Given the binding epitope of 8E10 is located at the conservative motif of BoNT A, B, E, F (Fig. 6A), we wondered whether it could bind and neutralize across these serotypes. To evaluate this, we first expressed the Hc domain of BoNT A, E and F, and determined the binding kinetics of the 8E10 antibody to the BoNT/A, B, E, and F Hc by surface plasmon resonance (SPR) and showed the results in Table 5 and Figure S2. The results show that the binding affinity between 8E10 and BoNT/A or F is a little bit lower than the binding affinity between 8E10 and BoNT/E, whereas the affinity between 8E10 and BoNT/B is the highest. The fact that all four serotypes of BoNTs bind 8E10 with nanomolar efficiency demonstrates that all serotypes tested are of high affinity. The results of ELISA (Fig. 6B) and western blotting (Fig. 6C) showed that 8E10 could bind with native and denatured Hc of BoNT A, B, E, F, indicating that 8E10 recognized the conserved linear epitope among these BoNTs. In addition, we compared the potency of BoNT/A, B, E and F neutralization in vivo by mAbs 8E10. All of 6 mice challenged with 20 LD50s of BoNT/B were protected by 100 µg of 8E10. Same dosage of 8E10 protected 5/6 of mice challenged with 20 LD50s of BoNT/A or F, respectively and 4 of 6 mice also survived under the challenge of 20 LD50s of BoNT/E (Fig. 6D). In contrast, none of the mice survived longer than 24 hours after challenged with BoNTs without the injections of mAbs.
(A) Amino acid sequence alignment of the carboxyl-terminal region of BoNT (A, B, E, F). The residues, which are conservative among BoNTs/A, B, E and F are presented as bold letters. (B) ELISA of 8E10 to BoNT Hcs of different serotypes A, B, E and F. (C) BoNT Hcs (A, B, E, F) were separated by SDS-PAGE under reducing conditions and visualized by western blotting with 8E10. (D) Cross-neutralizing effects in vivo. 100 µg of 8E10 was pre-incubated with 20 LD50s of BoNT/A, B, E, F for 1 hour respectively. The mixtures were injected into mice, and the final death tally was determined 4 days after injection.
For the last two decades, the relationship between the structure and function of BoNT has been studied intensively. With the complex structure of BoNT/B and its receptors, protein receptor Syt-II [21] and its ganglioside receptor GT1b [22], we can combine these data to provide the structure basis of the "double receptor" interaction proposed by Montecucco et al. [23]. Bio-functional assays also provided the evidence that both protein receptor SytII and ganglioside co-receptor were necessary for the infective process of BoNT/B [24]. This led us to the hypothesis that a combination comprised of a pair of neutralizing antibodies that bound to different receptors binding domains of the toxin would be more effective neutralizers than either member of the pair alone. We generated three mAbs, 8E10, 5G10 and 2F4 which could neutralize the challenge of 20 LD50 of BoNT/B in vivo. Finely epitope mapping revealed that 8E10 recognized ganglioside binding domain in BoNT/B, 5G10 and 2F4 bound with 2 non-overlapping epitopes surround the SytII binding domain respectively. In addition, we found that the combination between 8E10 (recognizing ganglioside binding domain) and 5G10 or 2F4 (recognizing protein receptor binding domain) could result in a more than 30–60 folds increase in potency compared with that of any the single mAb. However, for the combination of 5G10 and 2F4, which both blocked protein receptor binding sites by non-overlapping epitopes, no synergistic effect was observed. This indicates that double blocking the ganglioside and protein receptor binding domain simultaneously with two mAbs is helpful for the synergistic effects of the two antibodies' combination against BoNT/B.
The combinations of antibodies, which recognize nonoverlapping epitopes, synergistically cooperate in neutralization potency have been reported previously [18]. In that study, random combinations between 2 of the 3 neutralizing mAbs significantly prolonged the time to neuroparalysis compared with single mAbs. However, in our study, 2 mAbs, which bind to the same function domain of BoNT/B, had much less synergistic effects than those whose epitopes located in different function domains of BoNT/B. In addition, a BoNT/B-specific triplex antibody combination exhibited cooperative neutralizing effects to the toxin in vivo that were no better than those of the pairs of antibodies (Table 4). These findings are different from the conclusion of the previous report. Fine epitope mapping showed that the two mAbs (3D12 and S25) which bind the BoNT/A HCC (C terminal of Hc) overlap the putative sialoganglioside binding site and cover a large portion of HCC. The other mAb C25 bound a conformational epitope that consisted of the sequence from the N- and C-terminal subdomains of BoNT/A Hc, which overlapped with a putative inositol phosphate binding site that may be important for attachment to the lipid membrane. This function domain is essential for the binding to anionic lipid in the environment of lipid raft, which is important for the translocation processing of the toxin. [25], [26]. Interestingly, the neutralizing potency of the pair of mAbs (C25+3D12), which bind to translocation domain and sialoganglioside binding domain respectively, is 10 times higher than that of S25+3D12, whose epitopes are located at the same function domain. This finding provides evidence that the synergetic effects of mAbs, which recognize different function domains of BoNT/A, are better than the effects of those recognizing the same function domain. Although we didn't observe the cooperative effects between the mAbs binding to SytII binding domain of BoNT/B, we did prove that the combination of two mAbs recognizing different receptors' binding domain in BoNT/B has a synergistic effect. Accordingly, the data indicate that the combination between 2 mAbs recognizing different function domains in BoNTs could be the general principle for the potent synergistic effect.
Neutralizing mAbs binding multiple serotypes of botulinum neurotoxin are rare have been reported previously [27]. The cross-reactive mAbs bound to a relatively conserved epitope at the tip of the BoNT HN. This is a functionally important epitope for intoxication, as mAb binding leads to potent BoNT neutralization. In this study, a neutralizing mAb 8E10 binding the conservative domain on Hc was reported. The structure of Hc from BoNT/B (PDB 2NM1) [21], BoNT/E (PDB 3FFZ) [28] and BoNT/F (PDB 3FUQ) [15] supports the view that the Hc fold is highly conserved. GT1b binds on a cleft formed by W1266 and Y1267 on one face and E1203, H1253, and F1252 on the other for BoNT/A [29]. The structure of Hc from BoNT/B in complex with the trisaccharide sialyllactose, a mimic of GT1b (PDB 1F31), displays a similar binding cavity with corresponding residues W1261, Y1262, and H1240 [22]. In addition, the crystal structure of Hc from BoNT/F displays a ganglioside-binding pocket with corresponding residues W1250, Y1251, and H1241 [15]. Amino acid sequence alignment shows that S1264, W1266 and Y1267, conserved among all BoNT serotypes [30], constitute key residues of a lactose-binding motif (H. . .SXWY. . .G) that contribute to the crevice binding with GT1b. The implication is that the GT1b-binding pocket for all these BoNTs is similar. We found in this study that the motif 1259SKWY1262 is the recognizing epitope of 8E10, which could also cross react with the Hc of BoNT A/B/E/F (Fig. 6). This conserved epitope may partially explain how 8E10 could cross-protect the challenge of different serotype of BoNTs in vivo. Although its synergistic effect with the mAbs blocking protein receptor binding domain in BoNT A, E or F has not been detected, we can predict that 8E10 could act synergistically with the mAbs recognizing protein receptor binding domain against BoNT A, E or F. This study provides a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.
Materials and Methods
1. Ethics Statements
Mice were purchased from Animal Center of Chinese Academy of Sciences and maintained under pathogen-free conditions. All animal experimental procedures were carried out in strict accordance with the guidelines of the Animal Experiment Committee of the International Joint Cancer Institute, and were approved by the Animal Experiment Committee of the International Joint Cancer Institute.
2. Holotoxin, Antigens, protein and peptides
BoNT/A, BoNT/B, BoNT/E and BoNT/F were provided by Lanzhou Institute of Biological Products (Lanzhou, China). DNA encoding BoNT/A Hc (residues 868 through 1296), BoNT/B Hc (residues 853 through 1291), BoNT/E Hc (817 through 1255), BoNT/F Hc (847 through 1280), and Syt II-LD fragment (residue 37 through 86) were synthesized and subcloned into a pET expression vector, expressed in E. coli BL21(DE3). The recombinant histidine tagged BoNT/A, B, E, F Hc and Syt II-LD fragment were isolated and purified by nickel affinity gel column chromatography and their molecular weight and purity were verified by gel electrophoresis [31], [32].
Peptides used in this study, P1 (YFCISKWYLKEV), P2 (PISDSDEFYNTI), P3 (IKLKKDSPVGEI), P4 (THPHLPRALMRS), were synthesized by Yeli Bio-Scientific Inc. (Shanghai, China).
3. Animals and cell lines
6- to 8-week-old female BALB/c mice were purchased from Animal Center of Chinese Academy of Sciences and maintained under pathogen-free conditions. PC12 (rat adrenal pheochromocytoma cells) was purchased from Cell Bank of Chinese Academy of Science (Shanghai, China). To generate cells that express syt II (syt II), full-length mouse syt II (Genechem. Shanghai, China) was subcloned into pCDNA3.1 (CLONTECH, Mountain View, CA) and transfected into PC12 cells via electroporation. Transfected cells were selected with 1 mg/ml G418, and several independent monoclonal cell lines were established. For experiment in which cells were preloaded with gangliosides (Merk Chemicals, Darmstadt, Germany), cells were grown to 80% confluence followed by incubation in serum-free media plus 250 µg/ml gangliosides. 24 h later, the serum-free/ganglioside media was replaced with complete media, and the cells were incubated with FITC labeled BoNT/B Hc.
4. mAb preparation
6-week-old female BALB/c mice were subcutaneously immunized twice at 3-week intervals with 10 ìg of BoNT/B Hc emulsified in Freund's complete or incomplete adjuvant (Sigma-Aldrich, Shanghai, China). Three days after a final immunization with BoNT/B Hc antigen alone, spleen cells from the mice and mouse myeloma NS1 cells (Cell Bank, Chinese Academy of Science, Shanghai, China) were fused and maintained according to the standard procedure [33]. The hybridomas producing anti-BoNT/B antibodies were screened by an indirect enzyme-linked immunosorbent assay (ELISA), using the purified recombinant BoNT/B Hc as a coated antigen. For ascites production, 5×106 hybridoma cells were injected intraperitoneally into BALB/c mice, which had been primed with 0.5 ml pristane (Sigma-Aldrich, Shanghai, China). Ascites fluid was collected, and the mAbs purified, using HiTrap Protein G coupled to Sepharose 4B (Amersham Bioscience, Uppsala, Sweden). mAb isotypes were determined by the Mouse Typer Isotyping Panel Kit (Bio-Rad, Hercules, CA). The binding specificities of the mAbs were determined by western blotting. The binding strength of neutralizing mAbs to the BoNT/B Hc was analyzed by surface plasmon resonance technology using a Biacore T100 instrument (Amersham Bioscience; Uppsala, Sweden) [34].
5. Binding specificity and cross-reactivity assay by western blot and ELISA
0.1 µg of purified BoNT/B Hc were mixed with 2× SDS sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 1.8% ß-mercaptoethanol), boiled and loaded onto 10% polyacrylamide gels. After electrophoresis, the samples were transferred on nitrocellulose sheets, probed with all of these 16 positive binding mAbs respectively (1∶1000), and stained with a goat anti-mouse IgG coupled to horseradish peroxidase (HRP) (BD Bioscience, San Jose, CA) and developed with ECL Plus.
To determine the cross-reactivity of 8E10, Easy Wash 96-well plates (Corning, Corning, NY) were coated at 4°C overnight with 100 µl/well purified BoNT A/B/E/F Hc (at 5 µg/ml) respectively (BSA was set as negative control). After blocked with blocking buffer (5% non fat dry milk in PBS) for 2 hours at room temperature, 1 ng of 8E10 in 100 ul PBS was added and incubated for 2 hour at 37°C. The wells were washed and incubated at 37°C for 1 h with HRP-conjugated goat anti-mouse IgG at a 1∶3000 dilution. After extensive washing, 3, 3, 5, 5-tetramethylbenzidine (TMB) was added (50 ul/well) and incubated for 10 min at room temperature in the dark and the reaction was stopped by the addition of 2 M H2SO4 (50 ul/well). Absorbance of the samples in the plates was read in an automated ELISA microplate reader at 450 nm. For western blot, purified BoNT A/B/E/F Hc were subjected to SDS PAGE, transfer to nitrocellulose membrane and probed with 8E10 as described above.
6. Kinetic analysis
BIAcore measurements were performed with the Biacore T200 instrument (GE Healthcare) at 25°C in running buffer (10 mM Tris pH 8.0, 100 mM NaCl, 0.005% surfactant P20, 50 µM NiCl). CM5 chips were coated with BoNT/B Hc. 7 neutralizing mAbs were passed over the chip surface at concentrations ranging from 4.4 nM to 22.2 nM for 240 seconds at a flow rate of 30 ul/min and dissociation was recorded during 60 minutes. The chip was regenerated with 20 µl of 35 mM EDTA at 50 µl/min. Binding kinetics were evaluated using the BiaEvaluation software package (GE Healthcare) using a Langmuir model 1∶1.
To determine the binding abilities of mAb 8E10 and BoNT/A/B/E or F Hc, Protein A was cross-linked to the dextran surface of a CM5 sensor chip. Protein A was immobilized using amine coupling with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride and N-hydrosuccinimide to a density of 1000–2000 response units (RU). mAb 8E10 was captured to approximately 100 RU. The analyte BoNT/A/B/E or F Hc was passed over the chip surface at concentrations ranging from 4.4 nM to 22.2 nM as indicated for 240 seconds at a flow rate of 30 µl/min and dissociation was recorded during 60 minutes. Binding kinetics were evaluated as described above.
7. Competition ELISAs
To identify the binding domain which was blocked by the neutralizing mAbs, BoNT/B Hc was conjugated with horseradish peroxidase (HRP) using HRP Plus Activated Conjugation Kit (Thermo, Rockford, IL) according to the instruction of the kit. Easy Wash 96-well plates (Corning, Corning, NY) were coated at 4°C overnight with 100 µl/well recombinant syt II-LD (at 5 µg/ml) or GT1b (at 10 µg/ml) in PBS, washed with PBS/0.05% Tween-20 (Sigma-Aldrich, Shanghai, China) and then blocked for 1 hour at 37°C with PBS/0.05% Tween-20/5% bovine calf serum/3% goat serum (Sigma-Aldrich, Shanghai, China). HRP-conjugated BoNT/B Hc was pre-incubated with neutralizing mAbs at molar ratio of 1∶100 for 1 hour at 37°C before added into coated plate. 3, 3, 5, 5-tetramethylbenzidine (TMB) was used as a substrate (Sigma-Aldrich, Shanghai, China) to detect bound BoNT/B Hc.
To determine the binding epitope of the mAbs were overlapping or not, competition was tested by the competition-ELISA [35] with some modifications. Mixtures of constant quantities of the HRP-mAbs (8D1, 8E10, 1F4, 5G10, 2H12, 2B2 and 2F4) conjugate in the optimal working dilutions with dilutions of the competing non-labelled mAb were incubated on BoNT/B Hc coated plates for 1 hour at 37°C. Follow a final wash cycle, 50 ul of substrate and stopping solution were added and absorbance values were determined as previously described. A control without competing antibody (PBS) and a control containing the same mAb (self-competition) were included in the test. Results were expressed as percent binding of HRP-conjugated mAb. The amount of binding obtained in the absence of unlabeled antibody was set at 100% for each HRP-conjugated mAb.
8. Epitope mapping by biopanning
Anti-BoNT/B antibodies (8E10, 5G10 and 2F4) were separately immobilized on 96-well plate. Phages (1.5×1011 pfus) from Ph.D.-12 Phage Display Peptide Library (New England Biolabs, Beijing, China) were pre-absorbed by immobilized mouse IgG. The phages, diluted in Tris-buffered saline, were incubated at 4°C for 1 h with immobilized mAbs. The wells were washed for five times with TBST (0.1% Tween 20/Tris-buffered saline). Then, the phages that bound with mAbs were amplified by direct infection with E. coli ER2738. The amplified phages were purified by precipitation with 20% PEG8000, 2.5 M NaCl and used in the next cycle. Three rounds of selection were routinely performed.
The indirect binding assay to test the reactivity of anti-BoNT/B Hc mAbs with synthetic peptides was performed in 96-well plates as described previously [36] with minor modifications. The plates were coated with KLH-peptide 1, 2, 3 respectively. Following 2 washings and blockade of free protein-binding sites, mAbs were added to each well respectively. Mabs binding to peptide were detected by sequential addition of an appropriate dilution of HRP-conjugated goat anti-mouse IgG. To confirm the specific of the epitopes identified, anti-BoNT/B mAbs were incubated with synthetic peptides (P1, P2, P3) respectively, and then the mixture was respectively added to plates precoated with BoNT/B Hc. After 2 h incubation, the wells were washed and detected with an appropriate dilution of HRP-conjugated secondary antibody. After the addition of TMB and stop solution, absorbance was read at 450 nm by a microplate reader.
9. Flow cytometry assay
The inhibition of mAbs for cell binding activity of BoNT/B was examined by performing flow cytometry [37]. BoNT/B Hc was labeled with the FITC labeling kit according to the instruction (Pierce Biotechnology, Rockford, IL).The single or pairs of mAbs were mixed with FITC-BoNT/B Hc at 37°C for 1 h. Ganglioside-treated or Syt II transfected (Syt II+) or double positive PC12 cells were labeled by overnight incubation with the mixture at 4°C and washed with cold PBS, and examined by flow cytometry (BD Biosciences, San Jose, CA). The PC12 cells alone and FITC-labeled Hc fragment of BoNT/B were used as systemic control and negative control, respectively.
10. Confocal microscopy
FITC labeled BoNT/B Hc (1 µg) was incubated with control medium, monoclonal antibody (100 µg) or pairs of mAbs (50 µg of each Ab) for one hour at room temperature. Sub-confluent Ganglioside-treated SytII+-PC12 cells, plated on glass cover slips, were incubated with the BoNT/B Hc-mAbs mixtures at 4°C for one hour and then for 2 hours at 37°C. Cells were then washed with PBS for 15 minutes, fixed with 4% paraformaldehyde for 15 mins, and washed again before mounting. Confocal laser scanning was performed on a Olympus Fluoview 500 system using the 40×objective, and Fluoview software was used for image analysis.
11. Neutralization of BoNT/B activity in vivo
For the initial screening of the positive hybridoma that produced the neutralizing antibody, 0.5 ml of culture supernatant from hybridomas grown in a 24-well tissue culture flask for 3–5 days, was pre-incubated with 4 LD50 of BoNT/B for 1 h at room temperature, and the reaction mixtures intraperitoneally (i.p.) injected to each of six mice. More than four survivals in four days were considered as positive. In order to define the activity of the positive neutralizing mAbs and synergistic effect of pairs of mAbs, 100 µg of the positive neutralizing mAbs were added to the indicated number of mouse LD50 of BoNT/B neurotoxin in a total volume of 0.5 ml of gelatin phosphate buffer and incubated at RT for 60 min. For pairs of mAbs, 50 µg of each mAb was added. The mixture was then injected i.p. into female BALB/c mice (16–22 g). Mice were studied in groups of six and were observed at least daily. The morbidity and mortality of mice were determined over 30 days. For the cross-protection assays, 100 µg of 8E10 was pre-incubated with 20 LD50s of BoNT/A, B, E, F for 1 hour respectively. The mixtures were injected into mice, and the final death tally was determined 4 days after injection.
Supporting Information
Figure S1.
The effect of BoNT/B-specific antibodies on binding of BoNT/B Hc with ganglioside-treated Syt II+PC12 cells. Ganglioside-treated Syt II+PC12 cells were cultured with FITC-labeled BoNT/B Hc (green), with or without mAbs as indicated and visualized by confocal microscopy.
Figure S2.
Binding affinity of mAb 8E10 and BoNT A/B/E/F Hc. The 8E10 was immobilized on the surface of a Protein A cross-linked CM5 chip. Purified BoNT/A/B/E/F Hc at concentrations of 22.2 nM, 14.8 nM, 9.9 nM, 6.6 nM, 4.4 nM were directly injected over the target and the reference surface. The signal obtained from the reference surface was subtracted to avoid the binding of non-specificity.
Author Contributions
Conceived and designed the experiments: JXD YJG CCC HJW SHW. Performed the experiments: CCC SHW HJW XYM GHJ. Analyzed the data: JXD CCC HJW SHW YJG TCZ. Contributed reagents/materials/analysis tools: CCC SHW XYM TCZ GHJ XS TX WJL DPZ. Wrote the paper: JXD YJG CCC.
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West M (2005) DATE OF ISSUE 02/02/2005 REVIEW INTERVAL Five Yearly AUTHORISED BY Malcolm West.
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- 5. Rossetto O, Montecucco C (2008) Presynaptic neurotoxins with enzymatic activities. Pharmacology of Neurotransmitter Release 129–170.
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- 8. Blasi J, Chapman ER, Link E, Binz T, Yamasaki S, et al. (1993) Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365: 160–163.
- 9. Schiavo G, Santucci A, Dasgupta BR, Mehta PP, Jontes J, et al. (1993) Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBS letters 335: 99–103.
- 10. Darios F, Niranjan D, Ferrari E, Zhang F, Soloviev M, et al. (2010) SNARE tagging allows stepwise assembly of a multimodular medicinal toxin. Proceedings of the National Academy of Sciences 107: 18197–18201.
- 11. Lacy DB, Tepp W, Cohen AC, DasGupta BR, Stevens RC (1998) Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nature Structural & Molecular Biology 5: 898–902.
- 12. Simpson L, Rapport M (1971) The binding of botulinum toxin to membrane lipids: sphingolipids, steroids and fatty acids. Journal of neurochemistry 18: 1751–1759.
- 13. Dong M, Yeh F, Tepp WH, Dean C, Johnson EA, et al. (2006) SV2 is the protein receptor for botulinum neurotoxin A. Science 312: 592.
- 14. Dong M, Liu H, Tepp WH, Johnson EA, Janz R, et al. (2008) Glycosylated SV2A and SV2B mediate the entry of botulinum neurotoxin E into neurons. Molecular biology of the cell 19: 5226–5237.
- 15. Fu Z, Chen C, Barbieri JT, Kim JJP, Baldwin MR (2009) Glycosylated SV2 and gangliosides as dual receptors for botulinum neurotoxin serotype F. Biochemistry 48: 5631–5641.
- 16. Nishiki T, Tokuyama Y, Kamata Y, Nemoto Y, Yoshida A, et al. (1996) The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a. FEBS letters 378: 253–257.
- 17. Amersdorfer P, Wong C, Smith T, Chen S, Deshpande S, et al. (2002) Genetic and immunological comparison of anti-botulinum type A antibodies from immune and non-immune human phage libraries. Vaccine 20: 1640–1648.
- 18. Nowakowski A, Wang C, Powers D, Amersdorfer P, Smith T, et al. (2002) Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody. Proceedings of the National Academy of Sciences 99: 11346.
- 19. Dong M, Tepp WH, Liu H, Johnson EA, Chapman ER (2007) Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons. The Journal of cell biology 179: 1511–1522.
- 20. Lomneth R, Martin TFJ, DasGupta BR (1991) Botulinum neurotoxin light chain inhibits norepinephrine secretion in PC12 cells at an intracellular membranous or cytoskeletal site. Journal of neurochemistry 57: 1413–1421.
- 21. Jin R, Rummel A, Binz T, Brunger AT (2006) Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity. Nature 444: 1092–1095.
- 22. Swaminathan S, Eswaramoorthy S (2000) Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B. Nature Structural & Molecular Biology 7: 693–699.
- 23. Montecucco C (1986) How do tetanus and botulinum toxins bind to neuronal membranes? Trends in Biochemical Sciences 11: 314–317.
- 24. Rummel A, Eichner T, Weil T, Karnath T, Gutcaits A, et al. (2007) Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept. Proceedings of the National Academy of Sciences 104: 359.
- 25. Muraro L, Tosatto S, Motterlini L, Rossetto O, Montecucco C (2009) The N-terminal half of the receptor domain of botulinum neurotoxin A binds to microdomains of the plasma membrane. Biochemical and biophysical research communications 380: 76–80.
- 26. Levy R, Forsyth C, LaPorte S, Geren I, Smith L, et al. (2007) Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display. Journal of molecular biology 365: 196–210.
- 27. Garcia-Rodriguez C, Geren I, Lou J, Conrad F, Forsyth C, et al. (2011) Neutralizing human monoclonal antibodies binding multiple serotypes of botulinum neurotoxin. Protein Engineering Design and Selection 24: 321–331.
- 28. Kumaran D, Eswaramoorthy S, Furey W, Navaza J, Sax M, et al. (2009) Domain organization in Clostridium botulinum neurotoxin type E is unique: its implication in faster translocation. Journal of molecular biology 386: 233–245.
- 29. Stenmark P, Dupuy J, Imamura A, Kiso M, Stevens RC (2008) Crystal Structure of Botulinum Neurotoxin Type A in Complex with the Cell Surface Co-Receptor GT1b—Insight into the Toxin–Neuron Interaction. PLoS pathogens 4: e1000129.
- 30. Rummel A, Mahrhold S, Bigalke H, Binz T (2004) The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction. Molecular microbiology 51: 631–643.
- 31. Sharma S, Zhou Y, Singh BR (2006) Cloning, expression, and purification of C-terminal quarter of the heavy chain of botulinum neurotoxin type A. Protein expression and purification 45: 288–295.
- 32. Chapman ER, An S, Edwardson JM, Jahn R (1996) A novel function for the second C2 domain of synaptotagmin. Journal of Biological Chemistry 271: 5844.
- 33. Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495–497.
- 34. Jin C, Hantusch B, Hemmer W, Stadlmann J, Altmann F (2008) Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteins. Journal of Allergy and Clinical Immunology 121: 185–190. e182.
- 35. Kienle E, Buschmann H (1989) Specificity, cross-reactivity and competition profile of monoclonal antibodies to staphylococcal enterotoxins B and C 1 detected by indirect enzyme-linked immunosorbent assays. Medical Microbiology and Immunology 178: 127–133.
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- 37. Wang H, Li T, Shi J, Cai K, Hou X, et al. (2010) A new neutralizing antibody against botulinum neurotoxin B recognizes the protein receptor binding sites for synaptotagmins II. Microbes and Infection 12: 1012–1018.


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Press Releases
Bipartisanship Brings About Needed Reforms to FDA
Congressman Morgan Griffith (R-VA) issued the following statement today after joining his colleagues to unanimously pass the Food and Drug Administration (FDA) Reform Act of 2012:
Congressman Griffith was also pleased to have a chance to vote on The Creating Hope Act (CHA), which is included in the FDA Reform Act. Earlier this year, Congressman Griffith met with Brianna Commerford, a key advocate for this legislation. At just nine years old, Brianna was diagnosed with stage IV Hodgkin's lymphoma, a cancer in the lymph nodes. After about a year of chemotherapy, eight surgeries, and a deeply committed spirit, she beat this horrible cancer and has now been in remission for more than three years.
The FDA's review process is often very lengthy. If, however, a drug or biologics company develops a new treatment for particular diseases, like blinding trachoma or new forms of tuberculosis, they qualify for the FDA's priority review voucher program. The CHA, a bipartisan bill, would expand the FDA's priority review voucher program to allow pharmaceutical companies to expedite FDA review of more profitable drugs or biologics in return for developing treatments for rare pediatric diseases.
Click here to read a legislative summary of the FDA Reform Act of 2012.


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Residents were allowed back inside their homes after police say a suspicious device was found inside a building in the Queen Village section of the city.
PGW workers were called to a home on the 700 block of South 4th Street Monday night for a report of a gas odor. When they arrived, they found a milk jug with an unidentified liquid as well as wires inside. The wires were attached to a timer that was plugged into a wall outlet of a home.
Several houses as well as an apartment building in the area were evacuated and police as well as the bomb squad were called to the scene.
Police told NBC10 the device was set to go off at 11 p.m. and that it could have caused a fire inside the building. However, a PGW worker managed to pull the wire from the milk bottle which prevented anything from happening.
Investigators say the device is still inside the building but that the area is safe.
Police are now searching for the person who created the device and investigators are still at the scene.
Several homes on the street, including the one where the device was found, are currently under construction.
The device was located in the same area where Philadelphia Fire captain Michael Goodwin died almost a year ago.
On April 6, 2013, Goodwin was on the roof of Jack B. Fabrics at 4th and Fitzwater, when the roof of the building collapsed, trapping him inside. Goodwin was a 29-year veteran of the Philadelphia Fire Department.


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Release Date: 2004 |
incorporate huge uses of melodies compared to previous releases.
You get brownie points for reviewing one of the few good nu-metal bands.
"Through The Eyes" is a surprisingly good album, but I have yet to hear this one.
I'm in a good mood so pos all around...except for Crysis, he gets neg's
except for Crysis, he gets neg's
I guess people didn't like my latest one, oh well, it happens.
No I like your BFMV review. And while I disagree with your Demon Hunter one, it's still a good freakin review. I just like to break balls.
Breaking balls is the name of the fucking game here at Sputnik. It's everyone's favorite past time.
I know, I tell ya what, I'll love you forever if you rate Periphery a 5 ;)
Your offer is tempting....
I'm going to review 'Through the Eyes' next... it needs a review that isnt a TBT
I'm diggin the other review for this album, no offence Maniac, but that guy has got a way with words.
wow, that's weird, even when I click on this from your profile page it takes me to the spam review. take a pos man, I'm not a fan of these guys (never was) but your review's good.
Digging: Desolate Shrine - Deliverance From The Godless Void
Album Rating: 4.5I loved these guys back in the day. Decide and Worlds Divide are some of their best songs.
Album is terrible, review isn't though
"Nu Metal is one of those genres that Sputnik loves to hate"
sputnik and everyone else!
good job, pos
nice review (sorry for the late response)
Flaw are one of those nu metal bands I never checked out, but when I find the time, I'll give this a go as it sounds like it could be pretty good.
Album Rating: 3.4
Album is terrible
Digging: Adimiron - Et Liber Eris
Album Rating: 3.0Not bad even if i prefer Through the eyes to this. it's catchier.
Album Rating: 3.5good but through the eyes rules harder
Album Rating: 3.5This album is awesome. The lyrics make me feel really inspired and optimistic.
Bands: A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


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startx problem
hi to all
please help ,
i m using redhat4 enterprise server and when i typed startx from tty3
then my server goes shutdown.please tell me what problem can be and how to solve it
Your server shuts down or the screen just goes blank?
If X is not configured correctly and startx gets you a black screen
ctrl alt backspace
will kill the x server and put you back to cli. Post the error message.
Then configure your xorg.conf
If startx is actually causing a shutdown then post your xinit file
What windows mgr or desktop environment are you using?
Also if you are using redhat4 enterprise why not ask redhat for help?
and to add
is xorg and gnome or xfce even installed on that rhel 4 server ?
it might not be
|All times are GMT -5. The time now is 05:09 AM.


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Download Body Mass Index - Test apk free for Android smartphone
Posted by BS-Teknik in Health & Fitness | Feb. 24, 2011 | 0 Comments
Apk file size: 350.0 KB
Get easy, fast and accurate information about your weigh. The program can measure your Body Mass Index. So you can find out if your weight matches your height.
The program can be switch between kg, cm and Inch, Pound
Disclaimer: The program is indicative and may not be the basis for case escalation of BS-Teknik.
Whats new
1.0 First version
BS-Teknik part of our Health & Fitness and have average installs from 1 to 5. Last Update Feb. 24, 2011. Google play rating is 100.0. Current verison is . Actual size 350.0 KB.
Similar applications
Body Calculator (BMI, TDEE...)163 Comments
Body Composition Monitor (Pro)6 Comments


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I know there is a dedicated Twitter thread, but this is a biggie and deserves it's own thread. Jeremy just tweeted this...
And Hammond...Tonight, when you are asleep, a pan European Top Gear race begins. Me in a car. Hammond and May on public transport. Wish them bad luck.
And more...@MrJamesMay what in God's name are you rambling about? Please tell me you haven't been drinking: We're racing tomorrow. #StuckWithDrunkenMay
And to be clear, our races are not fixed. We start at 3.30 am. No-one knows who'll win. But this time, the odds are stacked in their favour


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Types of Student Clubs & Orgs
Are you looking to expand your academic engagement and knowledge outside of the classroom? We have over 30 organizations to choose from! A few include Collegiate DECA, Society for HR Management, Society for Women Engineers, and many more!
Are you interested in either educating others or yourself about global and/or environmental issues and politics happening in the world today? We have 20 organizations for you to choose from! A few include Red Cross Club, Speak Up!, Students for Freedom, and more!
Are you looking to grow professionally, enhance your resume, or to find your niche in the professional world? We have 14 organizations that can help prepare you for your career. A few include Beta Alpha Psi, Formula Zag, Alpha Kappa Psi, and more!
Over 700 Gonzaga University students participate in Club Sports annually, each with a variety of skill levels and interests.
If you like getting involved and giving back to the community, we have 11 organizations that can help you do just that! A few include Circle K, Man's Best Friend, Setons, and more!
Want to reconnect with your roots or learn more about other cultures? We have over 15 organizations to choose from! A few include Hawaii Pacific Islanders Club, Chinese Club, La Raza Latina, and many more!
Gonzaga University does not recognize any social fraternities or sororities. However, we do have some academic and honor societies with Greek letter names, but they are not social fraternities.
Are you interested in performing, designing, or writing? We have 14 organizations for you to choose from! A few include Dance Team, Bulldog Band, Writers Block, Game Design, and more!
Do you find yourself looking for clubs that help you develop hands on knowledge outside of the classroom? There are over 30 organizations for you to choose from! A few include Human Physiology Club, Collegiate DECA, Materials Advantage Club, and many more!
Find fellow passionate religious peers! We have 5 organizations that might tickle your fancy. A few include Jewish Bulldog, THIRST, and more!


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Orioles cut eight players from roster
Orioles cut eight players from roster
Although Tillman's demotion was the news of the day -- giving the fifth starter spot to David Hernandez -- manager Dave Trembley also raved about the strides made by the left-handed Castillo.
"Castillo had a great camp," Trembley said. "I put Castillo in the category with a lot of the other guys who have shown a tremendous amount of improvement."
"It was very difficult to tell him [of the cut] because he feels like he can get people out, whether they are right-handed or left-handed, and I really can't argue with him."
A Cuban defect, Castillo was believed to be in direction contention with non-roster invitee Will Ohman for the team's lefty specialist role. In nine games, he allowed two earned runs, pitching to a 2.08 ERA with four strikeouts and no walks.
Catcher Adam Donachie and outfielder Jeff Salazar were also reassigned to Minor League camp. Baltimore will hang on to infielder Scott Moore and right-handed pitchers Alfredo Simon and Ross Wolf, although they have also been reassigned to Minor League camp. The trio will stay in Major League camp to provide depth.
"Simon betrayed common medicine," Trembley said. "He came back from Tommy John surgery [performed May 5, 2009] and was ready to go, Day One, in camp."
The 28-year-old Simon will get the start in Friday's game in Tampa, Fla., against the Yankees, and will report to Triple-A camp after that. Simon allowed five earned runs in five innings -- three of which came in his last appearance March 20 -- and strung together three straight scoreless outings prior to that.
"I think he will be up here at some point in time this year in some capacity," Trembley said of Simon.
The moves leave the Orioles with 33 players left in camp.


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Commencement DVD Ships -- Copies Still Available
July 2, 2002
July 2, 2002, Greencastle, Ind. - "I think that our graduates and their loved ones will be very pleased," DePauw University director of media relations Ken Owen says of the DVD of Commencement 2002, which is being shipped today. The disc, which was produced and mastered by DePauw, was professionally replicated by Sony Digital Disc Manufacturing. It preserves an event that participants will always remember and cherish in today's highest-quality home video format, one that offers features tape cannot.
The DVD's menu allows users to go directly to a specific segment of the program. If you want to access the speech by US Secretary of Labor Elaine Chao or hear A Toast to DePauw, for instance, all you have to do is click on the appropriate chapter. Perhaps most importantly, the technology allows you to access a particular letter of the alphabet as diplomas are being distributed. If you want to find someone whose last name is Johnson, simply click on the menu's "J" button and you can go immediately to the first student whose last name begins with that letter and eliminate the guesswork involved in fast-forwarding a tape. Further, while tape deteriorates over time, the DVD and its pristine picture and digital sound should provide years of enjoyment.
Those who have ordered DVDs should be seeing them in their mailboxes soon as DePauw is shipping the discs today. You can still obtain copies of the limited-edition DVD online for $25 for the first disc, and $10 for each additional copy sent to the same address, by clicking here.
Also available is DePauw's first-ever DVD, which captures the excitement of the 2001 Monon Bell game. The Indianapolis Star's Steve Slosarek reviewed the disc, crediting DePauw with creating "a dual-sided disc (one for each half), complete with a menu that enables viewers to jump to a particular play. The game is small-college football at its best." The platter includes every play of the game, and offers several different angles on the thrilling final play of the contest.
The Monon Bell DVD is $15, and can be ordered online by clicking here.Back


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Basic SEO Techniques Rettendon :
A few basic SEO techniques:
- View Google's cache to see your page the way a search engine sees it. If you can't see an element of the page in the cache, the search engines can't see it either! This means that content isn't gaining you any search traffic.
- Be sure your page is reachable. Crawlers don't perform searches, they travel by links. This means your page has to be viewable by clicking on a link on another page or crawlers won't see it and it won't be searchable.
- Be sure you don't have metaframes preventing your page from being crawled. Many Rettendon webmasters use metaframes to prevent rogue bots from crawling their page and don't realize that these metaframes will also prevent search bots from crawling their pages!
- Don't let your links get lost in a sea of links. Pages with hundreds or thousands of links may not get crawled thoroughly in order to prevent spam and skewed rankings.
- Use specific keywords. General keywords will have lots of competition. By using more specific keywords, a webmaster can reduce competition and increase rankings.
- Don't abuse keywords. Use your keywords in natural speech. Optimize your page for one or two specific keywords that searchers might use when looking for information available on your page. Be sure your keywords are relevant to your content.
- Don't use keywords in link anchor text pointing to other pages on your site. (This is known as commiting keyword cannibalization.)
- Be sure your keywords are plain text HTML. Add ALT text for images, transcripts for videos and audio clips, and put captions with java or flash plugins or images. Although crawlers are improving, many are unable to process anything besides plain text.
Be Aware of User Experience
Increasing the usability, accuracy, and visual design of the website will also increase your search metrics. Search engines use search metrics and backlinks to determine the popularity and user-friendliness of your page. These factors play an important role in SEO. Always seek to create content that is pleasing for your reader.
Search Engines use Engagement Metrics to determine user satisfaction. Time spent on a search result means the user found the result much more helpful than a user who immediately hits the back button to look for another search.
The Panda update allows google to use machine learning to rank websites on quality and user friendliness. In 2011, human evaluators ranked thousands of websites based on quality and then implemented machine learning that mimicked those evaluators. This update changed more than 20% of search results.
Selecting Keywords:
Things to consider when selecting keywords:
- The Keyword's relevance to your page. Will the people using that keyword in searches be satisfied with the content on your page?
- The Keyword's specificity. Would a narrower keyword or keyphrase attract an audience that is more interested in your content than the audience of the broader term? On the other hand, is your keyword so specific it won't be searched?
- The Competition on that keyword. Can you compete with the current top ranked websites for your keyword?
Where to Put Keywords:
- In the Title: as close to the beginning as you can put it
- At the Top of the Page
- Several times throughout the body of the page
- At least once in the ALT text
- In the URL
- In the Summary or Meta description tag
Optimizing Title Tags:
- Titles should be between 45 and 55 characters long. Titles that exceed 55 characters in length may not show properly in google searches.
- Keywords should be placed near the beginning of the title
- Consider putting your brandname at the end of the title
- Don't sacrifice readability and emotional impact for keyword optimization
Visit https://moz.com/learn/seo/title-tag to see how the title and summary of your page would show up in a google search.
Constructing URLS:
- Keep them short
- Be sure your reader has an idea of what he will find on the page when reads the URL
- Use plain text links
- Use hyphens to separate words
- Use keywords
How Links to your Page Effect your SEO
- Are a wide variety of websites created by a wide variety of webmasters on a wide variety of topics linking to your site?
- Are many sites on similar topics linking to your site?
- Are the ranking sites for your keywords linking to your site?
- Are sites linking to your page using your keywords in anchor text to link to your site? (Anchor text is the blue text that shows on a webpage instead of the URL to the link)
- Are you getting links from trusted sources?
- Have you refrained from linking to spammy or poor quality websites and websites that link to your page?
- Have you been accruing new links?
- Is your page being shared on social media?
If you can answer yes to the eight questions above, links to your content are doing their job and helping increase your rankings. If you can't answer yes to each question, devise strategies to remedy your answer.
Creating good, quality content that related pages will naturally link to is one of the best ways to get links. Another great way, is through Manual Outreach. Don't under estimate the traffic that a well placed link can generate itself. Target specific websites who cater to similar audiences, preferably websites with few links, high credibility and high rankings for your keywords, and invite them to link to your page. Be sure to explain how linking to your page is a benefit for them, and not just an act of charity for you. You may also use this tool to see your competitors' backlinks. Finding sites who have linked to the top ranked sites for your keyword gives you a list of sites that whose links would be of value to you. Avoid self-created links on lower quality websites. While they may help rankings a little bit, they may also hurt your rankings. If you pursue this option, proceed with caution.
Search Engine Webmaster SEO advice
SEO advice from the major search engine webmasters includes:
- Be sure all pages can be reached by static, plain-text links. (Links of the form: , as opposed to links buried in images, java or flash.) These links are easier for crawlers to find.
- Don't "cloak" your work, or try to present different content to the search engine than you present to the user. Make sure your page is optimized to be found by a search engine when a user searches a relevant query. Don't try to cheat the crawler's and their index.
- Use accurate, descriptive, clear language with appropriate keywords in your titles, subtitles and ALT text. (ALT text is text that appears when an image cannot be displayed.) On hubpages, photo captions are ALT text and are therefore an extremely important part of search engine optimization.
- Create content filled with relevant keywords.
Check for Errors
To check for crawl errors see Google's Webmaster Tools.
What is The Future of Search
A company blog can be a valuable asset, if done the right way. Not only does it provide more online content to increase search engine optimization, it provides fodder for your social media campaigns and can position your brand as an industry expert and resource. But readers won't just magically materialize. That's according to Gene Richardson, COO of Experts Exchange, an online community of 500,000 IT professionals where they can get online tech assistance from each other, hire freelancers and take online courses. Here are his words about how to be intentional with your blog strategy.
1. Use your community to source content.
Do you have an engaged, interactive community? Capitalize on this by sourcing user-generated content for your site. Foster relationships with your most engaged visitors, members, and consumers to encourage them to develop content, feature them in member or consumer spotlights, and use them as a sounding board to gauge what content is and isn't enticing readers. Involve your community in your content to make them feel even more invested in your brand or product, positioning your blog as a great place of reference for further information on the who, what, when, where, and why of your industry.
2. Give your company experts a voice.
Utilize the people on staff whose opinions matter to your audience. Don't just reference them in press or gated content. Move their voice into the company blog to make them an accessible reference.
3. Prioritize quality over quantity.
It's great if you can produce one blog post per day, but make sure to track whether or not those daily posts are receiving any traffic. Don't publish content for its own sake. Quality content that provides obvious and immediate value--not only to your target audience but to other consumers--will pay off in the long run.
4. Encourage engagement through targeted campaigns.
Drive traffic to your blog through social engagement tactics, such as polls, Q&As, and consumer interaction prompts. Ask readers to submit success stories, product reviews, or insights into best practices. People love the opportunity to be heard, so hear them out and give them a platform.
For this to be successful, you first need to understand where your audience lives online. Attempt social pushes through channels where your consumer base isn't engaged online and you'll see no improvement in traffic. These channels are also a great place for sourcing content if you don't have a community of your own.
5. Participate in a guest blog exchange.
Do you have a great rapport with an industry partner who has different or higher blog traffic than you? Establish a plan to provide them with a current, quality blog post on a relevant industry topic in exchange for them to post on your site. This exchange will mix up the authored content and subject matter on your blog to entice your readers to check out content by a well-known source, and your content on their blog will diversify your exposure and result in an influx of traffic.
SEO is Not Hard — A step-by-step SEO Tutorial for beginners that will get you ranked every single time
As a small business, you may think it's impossible to get the word out about what you do. That's no excuse. And you don't need fads or gimmicks. Follow the proven, timeless tips and techniques of these entrepreneurs to help get the word out about your business and watch it grow.
1. Give Your Stuff Away
Ari Fleischer and Aly Moler of Frozen Pints have grown their craft beer ice cream business by leaps and bounds by attending craft beer shows and farmers markets to do one thing--give their product away. Once customers taste this unexpected combination (which happens to be delicious) for free, they line up at their local store to buy it or even request that the store carry it.
2. Attend Networking Events...
Desiree Scales of Bella Web Design is a master networker. She attends and presents at almost every event in town. Her contribution to the overall community makes her one of the first people that come to mind when anyone looks for an expert in her area of concentration: small business websites and drip marketing.
3. ...Or, Create Your Own Event
If you don't like the events you are attending, invent your own! Darrah Brustein has created one of the most successful networking events in Atlanta: Atlanta Under 40. The event, which Darrah created to connect with other young entrepreneurs in her city, is now being franchised to other cities.
4. Volunteer to Lead an Organization
The secret to getting the most out of a group or organization is not just to attend but to lead. Take Lisa Calhoun of Write2Market. She served as the president of Entrepreneurs' Organization, allowing her to rub elbows and connect with the brightest minds of the fastest growing companies in the Atlanta market.
5. Start a Podcast
Todd Schnick of Dreamland Interactive is the first person I saw create his own podcast--he interviews other business owners. People love to tell their story, and by highlighting them on a podcast you make an instant and meaningful connection. It's also a great way to get an education on a topic you are interested in.
6. Be Helpful
Most small business owners struggle to get their finances in line, especially when they move from an Excel spreadsheet to something as sophisticated as QuickBooks. Cathy Iconis of Iconis Group hosts a Quickbook Chat on Twitter every Thursday night at 7:00 EST to answer small business owners' questions--and potentially find some clients.
7. Send a Weekly E-mail
If you want to stay in relationship with your customers, there is nothing simpler than creating a weekly e-mail that provides something of value. Rick Houcek of Soar With Eagles sends one out every Monday that he calls the 2-Minute Monday Motivator. I look forward to getting it every week and often forward his advice to others.
8. Support a Cause
Mary Hester of LAN Systems throws an annual cookout with purpose every Earth Day. Party-goers are encouraged to bring their "e-waste"--old computer monitors and CPUs. At their most recent event they collected more than two tons of IT equipment, keeping it out of the landfills and creating goodwill with their customers, current and potential.
9. Sponsor an Organization
Many local organizations are not that expensive to sponsor for a year if you consider the so-called per meeting cost. If your product or service is a good fit with their audience, you will get exposure every time the organization sends out an e-mail and a mention every time they meet. Attendees always remember and appreciate companies who sponsor their favorite organizations.
10. Create a Cool Giveaway
When thinking through what your company will give away make sure it's something they won't want to throw away or easily lose in their desk or bag (think pen).


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Vance Memorial Library is located in the Collins Learning
Center. It operates within a Christian environment and provides
access to services and information resources to support the
educational and research needs of the Dallas Baptist University
community. In
addition to its core collection of circulating and reference
materials, the library also houses many interesting special
collections and important archives.
The mission of Vance Memorial Library at Dallas Baptist University
is to provide faith-integrated, quality service and access
to the information resources and services necessary to support
the various colleges in the achievement of their educational
goals. The Library seeks to equip students, faculty and staff
with research skills to enable them to communicate and function
effectively in a technology-oriented, information-rich society.
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"My purpose
is that they may be encouraged in heart and united in love,
so that they may have the full riches of complete understanding,
in order that they may know the mystery of God, namely, Christ,
in whom are hidden all the treasures of wisdom and knowledge."
Colossians 2:2-3
2003 Dallas Baptist University
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