MAYBE he was tired of killing. Maybe he remembered, at the very end, that he was human being.
But whatever was going on in his murderous mind, Derrick Bird's last act before he turned his gun on himself was one of mercy.
It emerged yesterday that the monster cabbie spared the lives of a young family after slaughtering 12 innocent people on his rampage through Cumbria.
Zoe and Lee Turner were on holiday with their sons, aged three and four, when they saw Bird crash his taxi near the tiny Lake District hamlet of Boot.
The killer then lurched towards them, red in the face and clutching a rifle.
Zoe said: "I was terrified. My instinct was to get my boys safe.
"When I looked into his face, which was red and flustered, I felt sick with fear.
"But he saw my look of terror and his expression changed. He said, 'It's all right', and moved away. He didn't want me to be scared."
Moments before the family's chilling encounter with Bird, they watched him career across a bridge in his Xsara Picasso cab, forcing two walkers to leap out of the way, before crashing.
Lee said: "I went over to make sure that the driver was OK. He looked dazed and disorientated.
"The walkers asked if he was all right and he made it clear he didn't want any help.
"A rifle with large scope was on the passenger seat.
"I thought he might have been out hunting and had a few drinks. He seemed shifty and was anxious not to draw attention to himself."
After leaving the family, Bird walked off into a wood and killed himself.
It was only later that the Turners realised they had come face to face with a mass murderer.
Zoe said: "We didn't sleep well, knowing we might have been the last people to see him alive."
The Turners' young kids won't remember Bird, but nine-year-old Jordan Williams will never forget him.
Jordan was out with his mum in Seascale on Wednesday when he saw Bird gun down 64-yearold cyclist Michael Pike, the 10th to die in the massacre.
The youngster watched the shooting from just six feet away. And he told yesterday how the cabbie stared at him "like a hawk" after the killing.
Jordan said: "I saw a man just coming up the hill on his bike.
"Then a crazy, like, taxi just came up and shot his back wheel and then shot him in the cheek.
"There was blood everywhere.
It spurted all down the cyclist's top. It was horrible.'' "The crazy driver stared me in the eyes, and my mum. He looked a bit mad.
"He had his eyes like a hawk staring at something. His eyes were black.
"We were like that for seven seconds but it felt a lot longer. I didn't run - I don't know why.
"I was scared - not of him but of the gun he had. He'd just shot a man in front of me.
"He just kept on driving and he just zoomed off."
Moments later, Jordan and his mum heard another gunshot. Bird had just murdered his 11th victim, 66-year-old spinster Jane Robinson, as she delivered Betterware catalogues.
"She was nice," Jordan said.
"She used to tell me to watch out for adders in the grass at the bottom of the garden."
Jordan, mum Gill and sister Mia ran for their lives after the shootings.
He was sick three times that night. He said: "I dreamed that the man shot me and my sister and my mum and dad."
His mum Gill said: "I haven't stopped shaking since.
"Mia was screaming for her dad and I just told Jordan to run. We ran for our lives.
"We took him to the doctors and they prescribed a lot of love and hugs and encouraged him to talk about it. I'm so proud of the way he's handled it."
Jane Robinson lived in Seascale with her twin sister Barrie, who spoke for the first time yesterday about her loss.
"She was the best sister I could ever have," Barrie said. "As twins, we were especially close.
"Jane was such a caring, considerate and generous person in the local community. She always had time to help people.
"Her kindness to people also extended to animals and birds. She worked tirelessly for the PDSA and later devoted her time to caring for birds. She was especially fond of her pigeons.
"She was a real Seascale person and loved the area so much."
Michael Pike's wife and two children said he died "a contented man" as he enjoyed the sport he loved.
They added: "Mike was a kind, brave, generous person.
"He was enjoying his retirement and was happier than ever before. He loved this area and spent much of his time walking and cycling locally.
"It is sad that his enjoyment of his well-earned leisure was cut short, but he died a contented man and we are proud of him."
The family of Bird's fourth victim, Ken Fishburn, 71, told how he served his country as a soldier for 25 years.
Ken was gunned down near his home in Egremont, a few miles from Seascale. After hearing that an armed madman was on the loose, he had gone outside to check that his neighbours were all right.
His family said: "Ken was a brother, brother-in-law and uncle.
"He was a quiet, private man and liked nothing better than a few bets and a game of snooker.
"Ken was a lifetime Army man, having served 25 years in the forces. He served with the Durham Light Infantry which took him all over the world.
"A further five years were served as a reservist, serving six months each year with the UN peacekeeping force."
Ken lived quietly in retirement, but was a well-known and popular face at the local Conservative club and the town's branch of Ladbrokes.
Diane Lowery, who works at the bookies, said: "He was just a lovely man. We all loved him."
Bird killed his twin brother David, family lawyer Kevin Commons, 60, and fellow cabbie Darren Rewcastle, 43, before turning his gun on Ken.
He then murdered mum-of-two Susan Hughes, 57, in Egremont, retired couple Jim and Jennifer Jackson in nearby Wilton, Isaac Dixon at a roadside and farmer Garry Purdham, 31, in a field.
After shooting down Michael Pike and and Jane Robinson in Seascale, Bird claimed his final victim, 23-year-old estate agent Jamie Clark, who was driving through the town.
Jamie's parents, Richard and Jane, won £2.3million on the lottery in 2001. They said of their son: "He was the most wonderful, gentle, loving, considerate man."
Jen's last words of love
Kind-hearted pensioner Jennifer Jackson spent the last hours of her life thinking of others.
Just two hours before Bird killed Jennifer, 68, and her husband Jim, 67, she talked to her local vicar about the school bus crash that killed three local people last week.
The vicar, Richard Lee, said: "She was just saying how overwhelmingly sad it was. She said one should always look on the bright side of life.
"A couple of hours later a colleague texted me to say she had been killed, and her husband as well."
The couple had lived for years in the tiny hamlet of Wilton, and Jennifer was a pillar of the local church.
Their children, Chris and Kathryn, and the rest of the family said: "They were wonderful, quiet, loving people, right at the heart of the community.
"Everyone here is in shock."


Bundler is developed entirely by a team of volunteers. Support their work and help make Bundler better for everyone.
Development work on Bundler has been sponsored by many companies over the years. Here, we thank the companies that have directly contributed money or significant paid time to implement features or fix bugs.
Heroku employs Terence Lee to maintain the Ruby platform on Heroku, including ongoing contributions to Bundler.
Mavenlink employed André Arko to work full-time on open source software, including Bundler, for several months.
The Bundler dependency API provides dependency information to Bundler version 1.1 and higher. It speeds up `bundle install` by several times over, but requires server capacity beyond what RubyCentral is able to provide for rubygems.org. These sponsors have provided the resources that make hosting the Bundler API possible.
Heroku provides the Ruby and Postgres servers that the API runs on.
Honeybadger provides exception reporting, tracking, and alerting.
PagerDuty provides operational alerting, notifications, and on-call schedule rotation.
Papertrail provides hosted log management for all of the API servers.
Rollbar provides exception tracking and analysis.


Security vulnerability is found in the PHPUnit/Mailchimp library for Drupal
According to the Public Service Advisory PSA-2011-002 - External libraries and plugins Drupal Security team has released an advisory today with regard to an exploit found in the third party library.
Current PHPUnit/Mailchimp library exploit
A vulnerability that is being actively exploited on some Drupal sites is detected. The vulnerability is in PHPUnit and has a CVE# CVE-2017-9841. The exploit targets Drupal sites that currently or previously used the Mailchimp or Mailchimp commerce module and still have a vulnerable version of the file
[library-path]/eval-stdin.php. See below for details on whether a file is vulnerable or not. The vulnerable file might be at other paths on your individual site, but an automated attack exists that is looking for that specific path. This attack can execute PHP on the server.
Follow release announcements by the vendors of the external libraries and plugins you use.
In this specific case, check for the existence of a file named
eval-stdin.php and check its contents. If they match the new version in this commit then it is safe. If the file reads from php://input then the codebase is vulnerable. This is not an indication of a site being compromised, just of it being vulnerable. To fix this vulnerability, update your libraries. In particular you should ensure the Mailchimp and Mailchimp Ecommerce modules and their libraries are updated.
If your website is using the Mailchimp-related modules and libraries and you are not sure how to tackle this matter further, then please contact AltaGrade team and we will do our best to assist you promptly.


NYC Waterfront Development | Robert Khodadadian
NYC – There are several plans in place to boost the economy in the area around the city’s Metro-North stations including new waterfront development. The population in the neighborhood has been steadily increasing and the Department of City Planning is looking for new ways to bring even more people to the area. The department conducted a study that looked at all aspects of the current neighborhood and analyzed which areas weren’t being used and how existing areas could have more value added to them. One of the main conclusions was that waterfront properties needed to be increased and expanded upon.
One of the specific goals mentioned by the report was to redevelop the waterfront area near the stations so people could access the Harlem River easier. There are also several existing areas like parks and playgrounds that are hard to reach. Even though the neighborhood has a high population, these areas are typically deserted because no one can get to them. They are either blocked off by highways or other obstacles, so more waterfront access is ideal for the entire community.
Another feature that is already being planned is a pathway between Randall’s Island and South Bronx. This would be accessible by foot and by bike. In addition to greater access to parks and other recreational areas, the department hopes that businesses could see more success along the waterfront. While planning is in the early stages, the department hopes to transform the neighborhood in order to accommodate the people continually moving and being born in the region.
While it may be years until waterfront development gets well under way, for anyone interested in development projects around the Metro-North stations, there has never been a better time to get involved.


text:
The aim of the BEAM Awards © is to showcase and honour the most talented figures from BAME backgrounds that go unrecognised in the UK
The BEAM Awards © will offer sponsors, official partners and all contributors the opportunity to pioneer their company as a true advocate of diversity
The BEAM Awards © will be judged by leading and proactive figures from BAME backgrounds within the entertainment, sports, politics and media industries
The BEAM Awards Host
“I am delighted to have been asked to be a part of these Awards. They have become increasingly important in our society and will continue to be so.”
Sir Trevor McDonald, OBE
The BEAM Awards Nominees Include
Nominations for the BEAM Awards are now closed!
Andrew Lloyd Webber warns of diversity crisis in British theatreDecember 1, 2016
Sir Lenny Henry and Olympic hero Mo Farah among top 10 most influential Black BritonsNovember 16, 2016
Naomi Campbell speaks for the first time about what it means to be black and British and joins other famous stars who were told to limit their ambitions due to their skin colourNovember 14, 2016
FCA to triple ethnic minority staffNovember 14, 2016
Get in touch!
SHARING IS CARING!

domain: news


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What Is The Short-Term Lease Exemption Under IFRS 16 [With Example]
The new IFRS accounting standard for leases, ‘IFRS 16’, that was announced in early 2016, includes several exemptions which a lessee has the option to apply when implementing the new guidelines. Having already discussed the low-value asset exemption in detail here, we’d now like to examine the short-term lease exemption.
The lease accounting exemption for short-term leases is included in IFRS 16 to ease the financial burden of the new standard. It’s an accounting policy choice which a lessee may elect to apply, but they can only have that option if the lease meets certain criteria.
In short, a lease agreement can be exempted from inclusion on balance sheet, should a lessee choose to do so, if it:
- Does not extend beyond 12 months in duration
- Does not contain any purchase options
- All leases of the same underlying assets receive the same treatment (in regards to exemption).
Also, all items of a similar class must be treated equally. For example, if a lessee applies the exemption to the lease of a franking machine, using the above criteria, it must apply the exemption to all other franking machinery the business has on lease.
But this being the case and the agreement being shorter than 12 months in duration is not always enough.
Beyond all this, the lessee will have to compare the lease contract against the new standard, paying particular attention to any lease extension options or break clauses.
If there are either of these, the lessee will have to apply the reasonably certain assessment criteria to judge whether the respective option to extend or terminate the lease will be taken up. And this is similar to what exists under current IAS 17 standards.
This means, a lessee could not treat a lease as exempt simply because it had a break clause option inside 12 months, if that option would not pass the reasonably certain assessment criteria to be activated. For example, a major civil engineering contractor involved in a five-year motorway contract could not keep their heavy plant exempt just because it had a break clause after 11 months. It is clear they will need the plant for longer than this, so it cannot be exempt.
Why There Are Exemptions
The board decided to include two exemptions within the new standard in order to appease concerns, expressed in the feedback, that implementing the new standard would be a big financial burden.
Due to a combination of the redefining of what constitutes a lease and the ever growing size of lease portfolios, the work involved with becoming compliant with the new standard is huge.
In The End Less Than 12 Months Is Short-Term
The final IFRS 16 treatment of short-term lease exemption was widened to include those of which is not “reasonably certain” that the term will be more than 12 months, when the likelihood of taking up options of extensions and the severity of termination clauses are considered.
As a result, a lessee is not required to recognise an asset or liability with a lease term of 12 months or less, as long as the extension options and termination rules in the agreement make it likely the agreement will not extend beyond a year.
This may be a subtle consideration but it allows some accounting flexibility for the lessee, whilst also helping to achieve the wider aim of IASB; getting a fairer representation of lease portfolios in front of those who assess a company’s financial state.
This is despite concerns raised during the drafting process that, by putting a definite line in the sand of “short-term” being defined as 12 months or less, the opportunity for understatement of a lessee’s assets and liabilities could still arise due to, what participants in offering feedback called, “structuring opportunities”.
Tactically Using Repeat Short-Term Agreements Is Not Allowed
In the end, the IASB staff deemed that a clear 12 month “bright line” in the sand found the right balance between offering some flexibility for those who benefit from being able to keep short-term agreements being reported on balance sheet and still keeping the period short enough to be truly “short-term”. The reasoning was that there is a huge economic difference between entering into a one year lease versus a five-year lease, compared to entering a three-year lease versus a five-year lease.
In short, one year is short-term and that’s that.
If companies choose to use “structuring” in their lease portfolio management, the savings and potential benefits will be negligible in comparison to the effort put into achieving them.
At the same time, any unscrupulously engineered, repeatable short term agreements - Fictional Airlines leasing Boeing planes for 11 months and 30 days, time after time, for example - should be painfully clear and be reprimanded appropriately.
Small Value Ticket Price Explained
Measured by looking at individual leased items, if a lease meets the “low value” criteria it can be exempt from the general considerations of IFRS 16. If you want to read in more depth about small value ticket item exemptions check out this blog post: IFRS 16 exemptions in relation to being small value lease assets are explained in more depth here: What is the IFRS 16 Exemption for Low-Value Assets? (With IFRS 16 Example).
However, as both the small value and short term asset exemptions are optional, a lessee may choose to ignore them and recognise all their assets and liabilities in line with the general requirements in IFRS 16.
Make Sure You Are Compliant And Take The Work Out Of Doing So
We have created a completely free and no obligation 7 step guide to becoming compliant with IFRS 16. Inside it, we run through the global changes in a little more detail, show you the 7 step walkthrough to becoming fully compliant and give you some options on how to do it. Press the button below to open your copy.
Download it below.

domain: law


Students are required to engage in direct service assignments and other learning tasks/experiences that can provide them with the opportunity to develop as beginning level generalist social workers. Within each semester of the Practicum, assignments are designed to enable students to meet the specific expectations identified as the behavioral and educational objectives of that semester, culminating in the acquisition of the six generalist practice competencies expected of graduates of the Program. In this way, the primary objective of integration of classroom and field learning is achieved.
To allow sufficient opportunity for the student to achieve the expected educational objectives of the Program, the agency is expected to provide learning activities from among the following assignments and experiences:
- A variety of service providing assignments, with different size client systems representing diverse populations, that require:
- A range of interventive modes and worker roles.
- Interactions with other agencies and resource systems.
- Professional teamwork, which may be interdisciplinary in nature.
- Access to information about the agency system, including administrative auspices and funding sources, organizational policies and procedures, formal and informal organizational structure, decision making processes, and service delivery statistics and outcome data.
- Observation of or participation in staff meetings, board meetings, and community meetings related to agency purpose, goals, and function.
- Observation of or participation in committee meetings of board or staff.
- Exposure to and involvement in agency research.
- Observation of or participation in meetings with representatives of other community agencies.
- Field visits to other agencies.
- Access to decision making processes within the agency in order to report on data collection and assessment and to make proposals and recommendations for enhancing agency service delivery and effectiveness in relation to community needs.
- Exposure to and participation in preparation for testimony and reports for presentation to legislative committees, administrators and boards of resource systems, resource allocation committees, and others, in order to influence decision making and allocation processes.


The Dirty Shammy Towel
A Super Absorbent Microfiber Towel For Your Dog. Take the Dirty Dog Shammy Towel EVERYWHERE!!!
Take it to the Beach, or the Dog Park, and use it for Bath time, anywhere.
The Dirty Shammy Towel is perfect for active dogs. Made of super absorbent microfiber, it can soak up to 20x more water and mud than other towels, and dries dogs 8x faster than cotton or a blow dryer. The machine washable material is easy to transport to the beach, dog park, or for bath time. Get ready to take your pup everywhere and keep them dry with the Dirty Shammy Towel.
• A Super Absorbent Microfiber Towel For Your Dog - Great for Bath Time!
• TAKE THE DIRTY DOG SHAMMY Towel EVERYWHERE!!! - The Beach - The Dog Park - On Walks
• SUPER absorbent microfiber soaks up to 20X MORE water and mud than other Shammy Towels
• Dries dogs 8x FASTER than a cotton towel or blow dryer
• Easy to wash - Machine Washable!
• One Size Fits All


Every weekday, we bring you the Ad Age/iSpot Hot Spots, new TV commercials tracked by iSpot.tv, the real-time TV ad measurement company with attention and conversion analytics from more than seven million smart TVs. The ads here ran on national TV for the first time over the weekend.
A few highlights: Actor/rapper Daveed Diggs (best known for playing Marquis de Lafayette and Thomas Jefferson in "Hamilton" on Broadway) rhymes his way through an ad for digital-payment network Zelle. A distressed woman causes some pretty serious damage to an office building after hearing about Little Caesars' ExtraMostBestest pizza deal. And Apple hypes the "studio-quality portraits" you can take with the iPhone X by showing a series of studio-quality portraits of some awesome-looking people.


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Revolutionizing Oral Care: The Impact of Longmont Modern Dentistry
Innovative Treatments and Services
At Longmont Modern Dentistry, we understand that a radiant smile can transform your life. Our cosmetic dentist Longmont team is dedicated to providing treatments that not only improve the aesthetics of your smile but also enhance your overall dental health. From teeth whitening to veneers, we offer a range of services tailored to meet your individual needs.
Our cosmetic dentistry services include:
- Professional Teeth Whitening
- Porcelain Veneers
- Dental Bonding
- Smile Makeovers
Embracing the latest advancements in dental technology, Longmont Modern Dentistry ensures that every visit is comfortable, efficient, and delivers the best possible results. Whether you’re looking to brighten your smile or correct imperfections, our team is here to support you every step of the way.
As part of Longmont family dentistry, we believe in creating lasting relationships with our patients. We take the time to understand your goals and work with you to achieve the smile you’ve always wanted. Experience the difference at Longmont Modern Dentistry, where your smile is our top priority.
At Longmont Modern Dentistry, we understand the profound impact that a missing tooth can have on your quality of life. Dental implants Longmont offers a permanent and aesthetically pleasing solution to this problem, restoring both function and confidence.
- Durability: Dental implants are designed to last many years, often a lifetime with proper care.
- Functionality: They restore the ability to eat and speak without concern.
- Aesthetics: Implants provide a natural look, blending seamlessly with your existing teeth.
- Bone Health: By stimulating bone growth, implants help maintain facial structure.
Dental implants are not just a cosmetic fix but a crucial investment in your oral health. They prevent the shifting of teeth, address bite issues, and contribute to overall dental wellness.
Choosing the right dental implant provider is essential. Our team at Longmont Modern Dentistry is equipped with the latest technology and expertise to ensure your implant procedure is a success. We invite you to schedule a consultation to explore how dental implants can transform your smile and improve your life.
When dental emergencies strike, Longmont Modern Dentistry is your reliable partner for immediate care and relief. Our dedicated team is equipped to handle urgent dental situations with promptness and the highest level of expertise.
- Common Dental Emergencies:
- Severe toothache
- Chipped or broken teeth
- Lost fillings or crowns
- Abscesses or infections
- Trauma to the mouth or teeth
At Longmont Modern Dentistry, we understand that time is of the essence in an emergency. That’s why we offer same-day appointments and after-hours services to address your needs as quickly as possible.
For non-life-threatening dental emergencies, we recommend calling our office immediately to schedule an appointment. Our staff will guide how to manage your situation until you can be seen by one of our professionals.
At Longmont Modern Dentistry, we understand the critical importance of maintaining healthy gums for overall oral health. Periodontal treatment is essential for patients experiencing gum disease, which can lead to serious complications if left untreated.
Our periodontal services are designed to address various stages of gum disease, from gingivitis to advanced periodontitis. We offer a range of treatments tailored to individual needs, ensuring the best possible outcomes for our patients.
Our commitment to patient education and preventive care is at the heart of our periodontal treatment approach. We strive to provide our patients with the knowledge and tools necessary to maintain optimal gum health.
The following is a brief overview of our periodontal treatment options:
- Non-surgical treatments like scaling and root planing (deep cleaning)
- Surgical procedures for advanced cases, including flap surgery and bone grafts
- Maintenance programs to prevent the recurrence of gum disease
Our team is dedicated to providing a comfortable and effective treatment experience, utilizing the latest techniques and technologies in periodontal care.
At Longmont Modern Dentistry, we believe that prevention is better than cure. Preventive dentistry is the cornerstone of maintaining a healthy smile for life. Our dedicated team provides comprehensive care to prevent dental issues before they arise.
- Regular dental check-ups
- Professional teeth cleaning
- Dental sealants
- Fluoride treatments
Emphasizing patient education, we equip individuals with the knowledge and tools necessary for optimal oral hygiene at home.
Understanding the importance of early detection, we utilize the latest technology to monitor your oral health. Our goal is to ensure that every visit to our clinic contributes to your long-term dental well-being.
Restorative dentistry at Longmont Modern Dentistry is about more than just repairing teeth; it’s about restoring confidence and quality of life. Our expert team is dedicated to providing treatments that not only improve the function of your teeth but also enhance their appearance.
We offer a comprehensive range of restorative services to meet the unique needs of each patient. From fillings and crowns to more complex procedures like root canals and bridges, our goal is to ensure that every patient leaves with a smile that is both healthy and beautiful.
Our restorative treatments are designed with patient comfort and satisfaction in mind. We utilize the latest techniques and materials to achieve results that are both durable and aesthetically pleasing.
Here is a brief overview of some of the restorative services we provide:
- Tooth-colored fillings
- Dental crowns
- Root canal therapy
Each treatment is tailored to the individual, ensuring that patients receive the most effective care for their specific situation. At Longmont Modern Dentistry, we believe that everyone deserves a smile they can be proud of.
Meet Our Expert Team
At Longmont Modern Dentistry, our team is our pride. Each member brings a unique set of skills and a shared passion for delivering exceptional dental care. Our dedicated professionals work tirelessly to ensure that every patient feels comfortable and informed throughout their treatment journey.
- Dr. Jane Smith, DDS – Lead Dentist
- Dr. John Doe, DDS – Cosmetic Dentistry Specialist
- Dr. Emily White, DMD – Orthodontist
- Dr. Mark Brown, DDS – Periodontist
- Sarah Johnson – Dental Hygienist
- Rachel Green – Patient Coordinator
Our team’s commitment to continuing education and staying abreast of the latest dental technologies means that you are always receiving the most advanced care possible.
We believe in a collaborative approach to oral health, where dentists, specialists, and support staff work together to provide a seamless and comprehensive dental experience. Our team is not just about providing treatments; it’s about building relationships and crafting smiles that last a lifetime.
At Longmont Modern Dentistry, our story is deeply rooted in a commitment to providing top-notch dental care while fostering a warm and welcoming environment for our patients. Our journey began with a vision to revolutionize oral care by integrating the latest dental technologies with a patient-centric approach. Over the years, we’ve grown not just in size, but in our ability to offer a comprehensive range of services tailored to meet the unique needs of each individual who walks through our doors.
Our dedication to excellence is reflected in the continuous education of our team, ensuring that we remain at the forefront of dental advancements. We take pride in our ability to adapt and innovate, always keeping our patients’ well-being as our top priority.
Our milestones include:
- The introduction of cutting-edge dental equipment
- Expansion of our expert team to include specialists in various fields
- Development of a robust patient education program
- Implementation of community outreach initiatives
We invite you to become a part of our story, where every smile is a testament to our passion for dental health and patient satisfaction.
At Longmont Modern Dentistry, we understand that patients often have questions about their dental care. We’re here to provide clear, concise answers to help you make informed decisions about your oral health. Our FAQ section is designed to address common inquiries we receive, ensuring you have all the information you need at your fingertips.
- What should I expect during my first visit?
- How often should I schedule dental checkups?
- What payment options are available?
- Can I receive dental care in an emergency?
Our commitment to patient education is unwavering. We believe that a well-informed patient is better equipped to maintain optimal dental health.
For more detailed responses to these questions and others, please do not hesitate to reach out to our friendly staff or visit our website. We are dedicated to providing you with a seamless and informative experience.
Welcoming new patients to our practice is always a joy. Your first visit to Longmont Modern Dentistry sets the tone for a relationship centered on personalized care and mutual trust. We strive to make every aspect of your initial appointment as seamless and comfortable as possible.
To ensure a smooth transition into our patient family, we’ve outlined a few steps to prepare for your first appointment:
- Complete the new patient paperwork available on our website to save time during your visit.
- Bring a list of any medications you are currently taking, along with your dental insurance card if applicable.
- Arrive a few minutes early to acquaint yourself with our office and team.
We believe in transparency and open communication, so please feel free to ask any questions or express any concerns you may have. Our team is here to support you every step of the way.
Remember, our goal is to provide you with exceptional care from the moment you walk through our doors. We look forward to meeting you and being a part of your dental health journey.
Patient-Centric Care Experience
At Longmont Modern Dentistry, we believe in giving back to the community that has supported us throughout our journey. Our commitment to community involvement is unwavering, as we actively participate in local events, provide educational outreach, and support various charitable causes.
- Participation in local health fairs
- Educational seminars at schools
- Free dental check-up camps
- Fundraising for local charities
Our dedication to the community goes beyond oral care; it’s about building lasting relationships and contributing to the well-being of our neighbors.
We understand that our role in the community is not just as healthcare providers but also as responsible citizens. That’s why we’ve established programs that allow us to engage with people of all ages, promoting not only dental health but overall wellness.
Insurance & Financing
Understanding that dental care is an essential part of overall health, Longmont Modern Dentistry offers a variety of flexible payment plans to ensure that patients can receive the care they need without financial stress. Our plans are designed to fit different budgets, with options that include no-interest payments and monthly installments as low as $75.00.
We are committed to making dental care accessible. That’s why we provide additional discounts for those who travel more than 10 miles to visit our practice. Ask about our “Travel Discount” during your free orthodontic consultation.
For those concerned about insurance, our office accepts a wide range of providers and our knowledgeable staff is always available to help navigate your benefits. We believe that financial considerations should not be an obstacle to obtaining high-quality dental care.
Understanding that dental care is an essential part of overall health, Longmont Modern Dentistry offers a Discount Plan to ensure services are accessible to all. Our plan is designed to provide relief to those who may not have traditional dental insurance, making it easier to maintain oral health without financial stress.
We believe that everyone deserves access to quality dental care, which is why we’ve introduced flexible payment options tailored to fit your budget.
Our Discount Plan includes a variety of payment plans with no interest, and monthly payments can be as low as $75.00. This initiative is part of our commitment to patient-centric care, where the well-being of our community is our top priority.
For those traveling more than 10 miles to visit our practice, Dr. Gibson offers an additional ‘Travel Discount.’ Be sure to inquire about this special offer during your free orthodontic consultation. It’s just one of the ways we strive to accommodate our patients and show our appreciation for their commitment to Longmont Modern Dentistry.
Our patients’ experiences speak volumes about the quality of care we provide at Longmont Modern Dentistry. Their glowing testimonials reflect our commitment to excellence in every aspect of our practice.
- Jane Dunlavy appreciated the ahead-of-schedule treatment and the massage chairs, noting the friendly staff.
- Michele MacPherson praised Dr. Gibson’s work and was very pleased with the treatment outcome.
- Jo Piccari found the atmosphere pleasant and felt relaxed despite a busy life.
- Shannon Frenzel highlighted the great patient waiting area with massage chairs, creating an inviting environment.
At Longmont Modern Dentistry, we believe that a beautiful smile can make a lasting impression. Our goal is to ensure that every patient leaves our office with a sense of satisfaction and confidence in their smile.
Gloria Hale and Stephanie Nasso echoed the sentiment of an awesome staff and excellent results, while Dedi and Kayanne Klipka were thrilled with Kayanne’s beautiful teeth and the professional service they received. Steve Hammond’s feedback further underscores our dedication to friendly and professional care.
Advanced Orthodontic Solutions
Longmont Modern Dentistry is at the forefront of providing Longmont Invisalign solutions, offering a discreet and comfortable way to achieve a perfect smile. Invisalign aligners are custom-made, ensuring a precise fit and effective treatment. Our expert team, led by top provider Dr. Colin Gibson, has extensive experience in transforming smiles with this innovative technology.
- Benefits of Invisalign:
- Nearly invisible aligners
- Removable for easy cleaning
- No dietary restrictions
- Comfortable, custom fit
Embrace the confidence that comes with a beautifully aligned smile. Invisalign treatment is tailored to your unique dental structure, promising results that go beyond aesthetics to improve overall dental health.
Our competitive pricing and flexible payment plans make Invisalign accessible to all our patients. With prices starting as low as $1999 and monthly payments from just $75, we ensure that financial constraints do not stand in the way of your dream smile. Avoid the pitfalls of DIY orthodontic solutions and trust our professional team to guide you through a safe and effective treatment journey.
At Longmont Modern Dentistry, we understand the desire for a less noticeable orthodontic solution. Clear braces offer the perfect blend of traditional effectiveness and aesthetic appeal, making them a popular choice among patients of all ages. With the latest trends and advances in orthodontic treatment, clear braces have become a seamless part of many of our patients’ lives.
Clear braces not only straighten teeth but also allow for a more natural look during treatment, which can be a significant confidence booster.
Here are some benefits of choosing clear braces:
- Aesthetically pleasing as they blend with the natural color of your teeth
- Made of ceramic materials that are strong and less prone to staining
- Suitable for patients with metal allergies
- Provide the same effective treatment as traditional metal braces
For those considering clear braces, we offer a comprehensive consultation to discuss your specific needs and the expected outcomes. Our team is dedicated to providing personalized care that ensures the best results for your smile.
Braces For Kids and Teens
Understanding the importance of a confident smile, Longmont Modern Dentistry offers specialized orthodontic solutions for kids and teens. Our braces for kids and teens are designed to correct misalignments and improve dental health while considering the unique needs of younger patients.
At Longmont Modern Dentistry, we believe that early intervention can lead to better long-term outcomes. That’s why we encourage parents to consider orthodontic evaluations for their children by age 7.
Our treatment plans are tailored to each child’s developmental stage and dental needs, ensuring a comfortable and effective experience. Here’s a quick overview of what to expect:
- Initial Consultation: A thorough examination to assess your child’s orthodontic needs.
- Customized Treatment Plan: Development of a personalized plan that outlines the treatment duration and goals.
- Regular Adjustments: Scheduled visits to adjust braces and monitor progress.
- Post-Treatment Care: Guidance on maintaining a beautiful smile after braces are removed.
We strive to make orthodontic care accessible and affordable for all families. For more information on pricing and payment options, please contact our office.
Sports Mouth Guards
Ensuring the safety and protection of athletes’ smiles, Longmont Modern Dentistry offers custom-designed sports mouth guards. These guards are not only crucial for those engaged in contact sports but also for anyone looking to safeguard their teeth during physical activities.
Our sports mouth guards are tailored to provide a comfortable fit while offering maximum protection. This personalized approach helps in preventing dental injuries and contributes to the overall performance of the athlete.
The process of obtaining a custom mouth guard is simple:
- Schedule a consultation with our dental experts.
- Get a precise impression of your teeth taken.
- Receive your custom-fitted sports mouthguard.
Remember, a well-fitted mouth guard is an investment in your dental health and athletic performance. Don’t compromise on protection—choose the best for your smile.
Fix And Remove Braces
Understanding the right time to fix and remove braces is crucial for achieving the best orthodontic results. At Longmont Modern Dentistry, we ensure that the process is smooth and tailored to each patient’s unique dental needs.
Our commitment to excellence means that we not only focus on the aesthetic outcome but also on the long-term health of your teeth and gums.
When it comes to removal, our specialists are meticulous in ensuring that your teeth remain protected and that every step is as comfortable as possible. Here’s a brief overview of what to expect:
- Initial assessment to determine the readiness for brace removal
- Careful detachment of brackets and wires
- Thorough cleaning and polishing of the teeth
- Discussion of post-braces care, including retainers
Remember, every smile is unique, and so is every orthodontic journey. Trust us to guide you through each phase with expertise and compassion.
Promotions and Special Offers
New Patient – Special Offer!
At Longmont Modern Dentistry, we are excited to welcome new members to our dental family with an exclusive New Patient
Promotions and Special Offers. Our goal is to make your first visit a seamless and affordable experience, ensuring you receive the highest quality care from the start.
We believe that everyone deserves a healthy, radiant smile, which is why we’ve crafted special offers tailored for our new patients.
To express our gratitude for choosing us, we’re offering a comprehensive dental exam, including X-rays, for just $39
Promotions and Special Offers
- Valued at $189; Non-Insurance Guests Only
- Offer valid for first-time patients only
- Appointment required
Please contact our office to schedule your appointment and take advantage of this special offer. Our friendly staff is here to assist you with any questions and to help you set up your visit. Remember, this is more than just a promotion; it’s the start of a long-term relationship focused on your dental health.
At Longmont Modern Dentistry, we believe that quality dental care should be accessible and affordable for everyone. That’s why we’re excited to offer our patients exceptional value through our Current Specials. These promotions are designed to help you achieve the smile you’ve always wanted while respecting your budget.
- New Patient Bundle: Comprehensive exam, X-rays, and cleaning at a significantly reduced price.
- Whitening for Life: Receive custom whitening trays and gel with a qualifying treatment.
- Referral Rewards: Earn credits towards your treatments for every new patient you refer.
Our Current Specials are our way of saying thank you for choosing Longmont Modern Dentistry. We’re committed to providing you with the best care at the best value.
Remember, these offers are for a limited time and subject to change, so be sure to inquire during your next visit or call us to learn more about how you can take advantage of these amazing deals.
In conclusion, Longmont Modern Dentistry is at the forefront of revolutionizing oral care, offering a comprehensive suite of services that cater to the diverse needs of its patients. From cosmetic dentistry to emergency care, and advanced orthodontic solutions, the practice ensures that every patient receives personalized and state-of-the-art treatment. With a team of dedicated professionals and a commitment to community involvement, Longmont Modern Dentistry is not just transforming smiles, but also enriching lives. Their affordable and accessible care, underscored by special offers for new patients, demonstrates their dedication to making top-tier dental services available to all. As we’ve explored throughout this article, the impact of such a modern and patient-centric approach to dentistry is profound, promising a future where oral health is synonymous with overall well-being.

domain: medical


text:
Bicycle-Ride Video 9th October-2021 Episode-2. Ride around the historic town of Arundel.
After yesterdays ride, I don’t think I’ll be going very far at all in the next few days, if anywhere at all! I was wiped out and in a fair amount of discomfort, come the end of the day! But not enough that it’s stopped me from going out again today!
So seeing as today was such a lovely sunny day, I cycled to the pretty town of Arundel. A round trip of 18 miles.
Bicycle-Ride Video October-2021 Episode-2
Subscribe to Garry’s YouTube channel.

domain: geography


text:
Everton have been suffering with an injury crisis throughout this season, seeing many key players missing for large chunks of the season which has inevitably led to the side falling into 16th in the Premier League, and struggling to gain wins to climb out of a potential relegation battle.
But, new manager Frank Lampard has been able to guide the Toffees to their first win in the league since the beginning of December with a 3-0 victory over Leeds United, and momentum is now well and truly in their favour.
The task is still not complete and with further injuries racking up since the ex-Chelsea bosses arrival, it could be a good opportunity to look at the youth Everton have on offer.
Yerry Mina has now sustained his second serious injury of the season, with the player already missing 39 days for Everton over October and November last year, and will now be missing for at least 8-10 weeks as Lampard confirms that the thigh injury was more serious than first thought.
With that being said, Lampard should be capitalising on the opportunity to develop a younger player, something he was renowned for doing at Chelsea and Derby during his managerial career, giving Mason Mount and Reece James their Premier League debuts who have now gone on to be integral to their side, winning the Champions League following his departure from Stamford Bridge.
One player that has already proven he can make an impact on this Everton side is Jarrad Branthwaite, who was given his Premier League debut by Rafael Benitez when the side found themselves in an injury crisis at the end of last year ahead of their clash at Stamford Bridge.
The 19 year old not only put in a confident defensive performance under pressure against the Chelsea side with two tackles and one interception, but also managed to score the equalising goal that secured Everton a much needed point away from home.
Lampard did give an opportunity to Branthwaite against Newcastle United after Mina got injured late in the first half, with the side struggling to control the game it eventually led to a defeat of 3-1.
The £2.7m-rated gem who “has got everything” according to former manager Steven Pressley, could be Phil Jagielka 2.o if he can break into the first team squad and seizes the opportunity in Mina’s absence to gain a starting spot in the side.
Like Jagielka, Branthwaite did not come through the Everton youth academy but has developed at the club to become one of their own, especially after his phenomenal goal at Stamford Bridge, with the potential of building a stronger connection with the fans the more he is given the chance to play.
That could eventually lead to the young centre back following in the footsteps of Jagielka if he can build a career and emulate the loyalty shown by the ex-Everton defender.
The Toffees boss will have a challenge on his hands as he tries to establish a philosophy that will win Everton crucial games and will have to decide who he trusts to save Everton’s season in a short amount of time, especially with barely any room for error.
Branthwaite must make an impactful impression on his manager in the coming weeks that Mina is side-lined for.
In other news: Moshiri makes yet another mistake as Mohamed-Ali Cho thrives


text:
Below is the poem entitled Too Late which was written by poet
Fox. Please feel free to comment on this poem. However, please remember, PoetrySoup is a place of encouragement and growth.
Read Poems by
Poets fingers write but once complete
who can by deceit or wit
cancel a line or even a bit
No washing a word or meaning of it

domain: artistic


I know that much of this simply reiterates what I posted at 6:15 however first off I think this information deserves to be shared repeatedly, and second Rachel does an amazing job of sharing it in a way that is both informative and entertaining.
Which by the way has certainly gained her show some special notice.
Courtesy of The Hill:
MSNBC host Rachel Maddow dedicated 53 percent of her program over the past six weeks to Russia, according to a Thursday analysis from the left-leaning The Intercept.
“Maddow’s Russia coverage has dwarfed the time devoted to other top issues, including Trump’s escalating crackdown on undocumented immigrants (1.3 percent of coverage); Obamacare repeal (3.8 percent); the legal battle over Trump’s Muslim ban (5.6 percent), a surge of anti-GOP activism and town halls since Trump took office (5.8 percent), and Trump administration scandals and stumbles (11 percent)," the analysis reads.
As we have mentioned before this approach has garnered Maddow quite a number of new viewers and the ratings for show continue to climb.
And the fact that this is clearly driving the Trump supporters around the bend is easily measured by the number of angry comments we receive right here on IM, insulting Rachel and attacking the format of her show, which is apparently too factually driven for habitual viewers of Fox News and Alex Jones.
Let's face it good journalism is enjoying a renaissance right now, and information seekers are finally starting to recognize the incredible contribution made by folks like Rachel Maddow.


text:
Cullin E3 ligases are the largest family of ubiquitin ligases with diverse cellular functions. One of seven cullin proteins serves as a scaffold protein for the assembly of the multisubunit ubiquitin ligase complex. Cullin binds the RING domain protein Rbx1/Rbx2 via its C-terminus and a cullin-specific substrate adaptor protein via its N-terminus. In the Cul3 ubiquitin ligase complex, Cul3 substrate receptors contain a BTB/POZ domain. Several studies have established that Cul3-based E3 ubiquitin ligases exist in a dimeric state which is required for binding of a number of substrates and has been suggested to promote ubiquitin transfer. In two different models, Cul3 has been proposed to dimerize either via BTB/POZ domain dependent substrate receptor homodimerization or via direct interaction between two Cul3 proteins that is mediated by Nedd8 modification of one of the dimerization partners. In this study, we show that the majority of the Cul3 proteins in cells exist as dimers or multimers and that Cul3 self-association is mediated via the Cul3 N-terminus while the Cul3 C-terminus is not required. Furthermore, we show that Cul3 self-association is independent of its modification with Nedd8. Our results provide evidence for BTB substrate receptor dependent Cul3 dimerization which is likely to play an important role in promoting substrate ubiquitination.
Citation: Choo YY, Hagen T (2012) Mechanism of Cullin3 E3 Ubiquitin Ligase Dimerization. PLoS ONE 7(7): e41350. https://doi.org/10.1371/journal.pone.0041350
Editor: Karl-Wilhelm Koch, University of Oldenburg, Germany
Received: April 11, 2012; Accepted: June 20, 2012; Published: July 20, 2012
Copyright: © 2012 Choo, Hagen. 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 study was supported by a grant from the Singapore National Medical Research Council (grant number EDG09May050). The funder 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.
Cullin3 (Cul3) E3 ubiquitin ligases are involved in the recognition and recruitment of numerous important substrates for ubiquitination –. Cul3 dependent ubiquitination has emerged as a mechanism to control various critical cellular processes including the antioxidant response, cell migration, cell cycle progression and retrograde trafficking , –. It has been shown that lack of Cul3 is embryonically lethal in mice . Functionally, absence of Cul3 has been reported to cause inhibition of cell migration in human and drosophila cells . This is due to stabilization of the Cul3 substrate RhoA which controls actin cytoskeleton stress fiber development . As a consequence, cells with reduced Cul3 expression exhibit abnormal actin stress fibers and distorted cell morphology .
Cul3 is a member of the cullin protein family, which also includes Cul1, 2, 4A, 4B, 5 and 7 in mammalian cells –. Similar to other cullins, Cul3 serves as a scaffold protein for the assembly of the multisubunit ubiquitin ligase complex that contains a RING domain protein and a substrate adaptor protein , . All Cul3 substrate receptors contain a “Bric a brac, Tram-track and Broad Complex/Pox virus and Zinc finger” (BTB/POZ) domain, which binds to the Cul3 N-terminus. Cul3 recruits substrates via a variable substrate binding domain such as Zinc Finger, Kelch, MATH and Ras homology domains . Cul3 binds to the RING domain protein Rbx1 via its C- terminus. Rbx1 functions to recruit the ubiquitin-charged E2 conjugating enzyme –. The ubiquitination activity of all cullin E3 ligase complexes is activated by conjugation of the ubiquitin-like protein Nedd8 onto a conserved C-terminal lysine residue in the cullin protein . This neddylation process is mediated via the Nedd8-specific E1 APPBPA-Uba1 heterodimeric enzyme and the E2 enzyme Ubc12. Nedd8 is known to activate all Cullin ubiquitin ligases. The Nedd8 conjugation activates Cullin RING E3 ligases by inducing a conformational change in the cullin C-terminus/Rbx1 structure, thereby increasing the flexibility of the Rbx1 RING domain. Thus, this brings the ubiquitin charged E2-conjugating enzyme and the target substrate into a close proximity –.
The best-characterized BTB domain containing Cul3 substrate receptors are SPOP and Keap1. The substrate binding domain of SPOP comprises of a MATH domain whereas Keap1 contains a Kelch repeat domain , –. Both the MATH domain and the Kelch repeat domains play an essential role in recruiting Cul3 target substrates for degradation –. It is also known that Cul3 substrate receptor proteins form tightly bound homodimers via their BTB domains . Keap1 protein homodimerizes to recognize two separate motifs, the ETGE and DLG motifs, in its substrate Nrf2 –. The binding of the Nrf2 transcription factor via two binding sites to the Kelch domains of the Keap1 homodimer is important to position Nrf2 in an appropriate orientation to facilitate ubiquitin transfer from the ubiquitin-charged E2 enzyme –. The ETGE motif serves as a “hinge” to clutch on to Keap1 and mediates the high affinity binding between Nrf2 and Keap1 whereas the DLG motif in Nrf2 acts as a “latch” to mediate the low affinity binding , . This “hinge & latch” binding mode is essential for Nrf2 ubiquitination and Nrf2 ubiquitination is inhibited if binding of either the DLG or ETGE motif to Keap1 is disrupted . Under normal conditions, the Cul3/Rbx1-Keap1 ligase recruits Nrf2 constitutively for polyubiquitination and consequently targets it for degradation to maintain low basal levels of the transcription factor , . However, Nrf2 ubiquitination is inhibited upon exposure of cells to electrophiles and oxidative stress due to the suppression of the Cul3/Rbx1-Keap1 ligase activity. This leads to an increase in Nrf2 stability and hence the activation of the antioxidant transcriptional response , .
It has also been shown that a homodimeric SPOP substrate receptor complex facilitates the ubiquitination of Jun kinase phosphatase Puckered (PUC) and variant histone MacroH2A . PUC and MacroH2A are substrates for the Cul3/Rbx1-SPOP E3 ligase in Drosophila and mammalian cells, respectively , . These two substrates have multiple SPOP-binding sites to bind to the SPOP dimer . A model has been proposed according to which ubiquitination of PUC or MacroH2A is mediated by binding of a single substrate to two flexibly orientated substrate-binding sites in the MATH domains of the SPOP dimer .
Dimerization of the Skp1-Cul1-F-box(SCF)Cdc4 complex via the cdc4 substrate receptor has been shown to promote the efficiency of substrate ubiquitination . This is likely due to the dimeric Cullin ligase complex providing a bivalent geometry that has two docking sites for E2 ubiquitin enzyme and thus initiates optimal ubiquitin transfer and elongation of the target substrate . With regards to the dimerization of the Cul3 E3 ligase, two models have been proposed. Firstly, it has been suggested that the Cul3 dimer complex formation is indirect and mediated via BTB domain-containing substrate receptor homodimerization . Our previous study also provided support for the model that Cul3 dimer complex formation is mediated via the association with BTB domain-containing proteins . In contrast, Wimuttisuk and Singer, (2007) reported that two Cul3 proteins dimerize directly via interaction of the WH-B domain. According to this model, the BTB domain-containing proteins are not required for Cul3 dimerization. The dimer consists of one neddylated Cul3 and one unneddylated Cul3 molecule . In the current study, we studied the mechanism of Cul3 dimerization in mammalian cells in order to resolve this controversy. We also investigated the proportion of Cul3 that exists as a dimeric Cul3/Rbx1-BTB protein complex in vivo.
Mutant Cullin3 proteins that are unable to bind to BTB domain-containing proteins exhibit markedly reduced Cul3-Cul3 association
To test whether Cul3-Cul3 binding is mediated via the homodimerization of BTB proteins, we previously used a Cul3 S53A/F54A/E55A mutant (Cul3(SFE)) that lacks the ability to bind to BTB proteins including Keap1 . However, Wimuttisuk and Singer, (2007) used a different mutant of Cul3, L52AE55A (Cul3(LE)) , . We therefore initially compared the ability of the two different Cul3 mutants to associate with wild type Cul3 by using co-immunoprecipitation assays. As shown in Figure 1A, both Cul3 mutants exhibited a marked decrease in the binding to wild type Cul3 although Cul3(LE) mutant showed a slightly higher remaining binding affinity to wild type Cul3. Our results that Cul3 mutants that are unable to bind to BTB domain-containing proteins show markedly reduced Cul3-Cul3 association suggest that homodimerization of BTB proteins is important for Cul3-Cul3 binding. These Cul3-Cul3 associations most likely represent Cul3 dimerization mediated by BTB dimers.
A–C, HEK293 cells were transfected in 60-mm cell culture plates for 2 days with expression constructs for the proteins indicated at the top of each panel. The cells were lysed, and the lysates were subjected to V5 immunoprecipitation (IP), as described under “Materials and Methods.” Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies.
We next investigated the binding of these two Cul3 mutants to the BTB domain-containing proteins SPOP and Keap1. As shown in Figure 1B, both the Cul3(LE) and Cul3(SFE) mutants showed no significant interaction with the SPOP protein compared to strong binding between wild type Cul3 and SPOP. When measuring binding of Cul3 to Keap1, we also noted markedly reduced binding when using Cul3(SFE). In contrast, the interaction of Cul3(LE) with Keap1 was only moderately reduced compared to wild type Cul3, (Figure 1 C and D). Therefore, the Cul3(LE) and Cul3(SFE) mutants have different affinities to Keap1 and possibly to other BTB proteins. This may explain why association between Cul3 and Cul3(LE) was still observed in the study by Wimuttisuk and Singer, (2007).
Cul3-Cul3 binding is independent of the WH-B domain
Wimuttisuk and Singer, 2007 proposed that in the active Cul3 ubiquitin ligase complex, the dimerization of Cul3 proteins is mediated via the hydrophobic residues in a Winged-Helix B (WH-B) domain near the Cul3 C-terminus. Thus, according to this model, the Cul3 C-terminus but not the Cul3 N-terminus is involved in the dimerization. To test for the involvement of the WH-B domain, we generated a C-terminal deletion construct of Cul3, comprising of amino acids 1–427 which excludes the WH-B domain in human Cul3, and measured its interaction with wild type Cul3 by co-immunoprecipitation assays. As shown in Figure 2A, Cul3(1–427) could still bind to wild type Cul3. To further confirm that Cul3-Cul3 association is not mediated via the Cul3 C-terminal WH-B domain, we generated an N-terminal deletion of Cul3, comprising of amino acids 392–768 containing the WH-B domain. However, this truncated Cul3 protein was not well expressed. This is partially due to N-terminally deleted Cul3 being degraded by the 26S proteasome (Figure 2B). Therefore, we generated a fusion construct comprised of the C-terminus of Cul3 (amino acid 392–768) fused to the N-terminus of Cul2 (amino acid 1–394) and also carrying a C-terminal V5-tag for immunoprecipitation (Cul2(NT)-Cul3(CT)-V5). As shown in Figure 2B lane 5 and lane 6, Cul2(NT)-Cul3(CT)-V5 was expressed well compared to Cul3(CT)-V5 and not subject to proteasome dependent degradation. We then used the Cul2(NT)-Cul3(CT)-V5 fusion construct to determine whether the Cul3 C-terminus could mediate Cul3-Cul3 association. As shown in Figure 2C, wild type Cul3-V5 could interact with full length Cul3-HA. In contrast, no interaction between the Cul2(NT)-Cul3(CT)-V5 fusion protein and Cul3-HA could be observed. These results suggest that the Cul3 N-terminus is necessary for Cul3-Cul3 binding, which is likely due to the requirements of the BTB domain-containing protein (eg, Keap1 or SPOP) for Cul3 self-association. In contrast, the WH-B domain near the Cul3 C-terminus does not play a role in Cul3-Cul3 association.
A, The cells were cotransfected with Cul3-HA and Cul3(1–427)-V5 (ΔCT), as indicated at the top of each panel. The cells were lysed, and the lysates were subjected to V5 immunoprecipitation (IP), Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies. B, cells were transfected with wild type (wt) Cul3-V5, Cul3(CT)-V5 and Cul2(NT)-Cul3(CT)-V5, as indicated. Two days after transfection, the cells were treated with 25 ìM MG-132 for 6 h, where indicated, and then lysed, followed by Western blotting of cell lysates with V5 antibody. C, HEK293 cells were transfected in 60-mm cell culture plates for 2 days with expression constructs for the proteins as indicated at the top of each panel. After cell lysis, the cell lysates were subjected to V5-immunoprecipitation and immunoblotting with V5 and HA antibodies.
Inhibition of Cullin Neddylation Does Not Affect Cul3-Cul3 Binding
It has been reported that the Nedd8 molecule is essential for Cul3 dimerization and that Cul3 is a heterodimer consisting of one neddylated and one unneddylated Cul3 . To confirm this, we used a Cul3 K712R mutant in which the neddylation site is mutated. Therefore this mutant cannot be conjugated with Nedd8. However, this mutation does not affect the interaction between Cul3 and BTB proteins . We then co-transfected wild type or K712R mutant Cul3-V5 together with Cul3(K712R)-HA and performed V5 immunoprecipitation. As shown in Figure 3A, the binding affinity between two Cul3 K712R mutant proteins shows no difference compared to the binding affinity between K712R mutant and wild type Cul3. This result indicates that neddylation is not required for Cul3 dimerization.
A, The cells were cotransfected with Cul3(K712R)-HA and wild type Cul3-V5 or Cul3(K712R)-V5 as indicated at the top of the panel. The cells were lysed and the lysates subjected to V5 immunoprecipitation (IP). Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies. B, cells with stable expression of dnUbc12 under control of a tetracycline-inducible promoter were transfected in 60-mm cell culture plates for 2 days with expression constructs for the proteins indicated at the top of each panel, followed by 1 ìg/ml tetracycline treatment for 18 h, where indicated. The cell lysates were subjected to V5 immunoprecipitation (IP). Immunoprecipitates and aliquots of lysates were analyzed by Western blotting with the indicated antibodies. C, Tet-on dnUbc12 cells were transfected in 12-well cell culture plates as indicated at the bottom of each panel. Cells were treated with 1 µg/ml tetracycline for 24 h where indicated. The cells are fixed and were subjected to in situ proximity ligation assay, as described under “Materials and Methods” and then viewed by fluorescent microscopy. Aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies.
To provide further evidence that cullin neddylation does not play a role in Cul3 dimerization, we used a HEK293 cell line with tetracycline-inducible expression of dominant negative, C111S mutant Ubc12 (dnUbc12) . We co-transfected Cul3-V5 and Cul3-HA into dnUbc12 cells and induced the expression of dnUbc12 with tetracycline. Subsequently, we performed Cul3-V5 immunoprecipitation with the cell lysates to measure Cul3 self-association. As expected, induction with tetracycline resulted in loss of the neddylated form of Cul3 (Figure 3B). Consistent with our results in Figure 3A, there was no significant change in the Cul3-Cul3 binding between tetracycline induced and control cells (Figure 3B). In addition, we also visualized Cul3-Cul3 binding in the dnUbc12 cells by performing an in situ proximity ligation assay. In these experiments, Cul3-V5 and Cul3-Flag were cotransfected and the expression of dnUbc12 was induced with tetracycline. Two different primary and secondary antibodies directed against the V5 and Flag epitode tags were used and a ligation assay was performed. The signal from each ligated pair of Cul3-V5-Cul3-Flag dimers (or multimers) was detected by fluorescence microscopy. As shown in Figure 3C, the observed distinct fluorescent spots in control cells and in cells induced with tetracycline were qualitatively similar. Taken together, these results indicate that Nedd8 modification does not affect Cul3-Cul3 binding.
The Cul3-Keap1 and Cul3-SPOP Complexes Contain Multiple Cul3 Proteins
Small et al. (2010) showed that the recombinant Cul3/Rbx1-Keap1 complex is formed at a ratio of 1∶2 . Thus, they reported that Cul3/Rbx1-Keap1 is a complex consisting of one molecule of each Cul3 and Rbx1 and two molecules of Keap1. In contrast, Zhuang et al. (2009) revealed that the recombinant SPOP protein forms a 2∶2 complex with Cul3/Rbx1 . To determine the stoichoimetry of the Cul3 E3 ligase complexes in vivo, we co-transfected two differently tagged Cul3 proteins, Cul3-HA and Cul3-V5, together with Flag-SPOP or Flag-Keap1 into HEK293T cells. We then immunoprecipitated SPOP or Keap1 protein by using Flag agarose. The bound Cul3 complexes were eluted with Flag peptide. Subsequently, we performed V5-immunoprecipitation and probed the immunoprecipitates with HA antibody to detect tetrametric or multimeric Cul3-V5–Keap1–Keap1–Cul3-HA or Cul3-V5–SPOP–SPOP–Cul3-HA complexes. As shown in Figure 4A, both Cul3-V5 and Cul3-HA showed significant binding to Flag-SPOP. This result suggests that two or more Cul3 proteins are involved in the Cul3-SPOP complex in vivo. Similarly, both Cul3-V5 and Cul3-HA also exhibited significant binding to Flag-Keap1 (Figure 4B). These data therefore suggests that multiple Cul3/Rbx1 proteins are present in both the Cul3/Rbx1-Keap1 and the Cul3/Rbx1-SPOP complex in vivo. Thus, Cul3 E3 ligases exist at least partially in a dimeric or multimeric state containing substrate receptor dimers and multiple Cul3/Rbx1 proteins under in vivo conditions.
A–B, The cells were transfected as indicated at the top of each panel. The cells were lysed, and the lysates were subjected to Flag immunoprecipitation (IP) and eluted with Flag peptide. The complexes were then subjected to V5 immunoprecipitation (IP). Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies. NS and * denote a non-specific band.
Estimation of the proportion of Cul3 that exists in multimeric Cul3/Rbx1-BTB protein complexes in vivo
The experiments described above indicate that Cul3 E3 ligases exist at least partially as tetrameric Cul3/Rbx1-BTB protein complexes. However, it is currently not clear what proportion of Cul3/Rbx1 exists as a dimer or multimer in vivo. To quantify the percentage of Cul3 that exists in a self-associated form, we expressed two forms of Cul3 with different molecular weights to distinguish them by their mobility in SDS-PAGE gels. One form consists of full length Cul3 with an N-terminal Flag tag that was used for immunoprecipitation. The other form consists of full length Cul3 carrying a C-terminal EGFP tag. To directly compare the abundances of the two Cul3 forms, both proteins also contained an HA tag at the extreme C-terminus. In our experimental approach, we co-transfected cells with both Cul3 forms followed by immunoprecipitation of Flag-Cul3-HA using Flag agarose. We then compared the ratio of Cul3-EGFP-HA to Flag-Cul3-HA in the cell lysates (input) with the ratio in the immunoprecipitates (corresponding to dimeric or multimeric Cul3 complexes). In the representative experiment shown in Figure 5A, the ratio of Cul3-EGPF-HA to Flag-Cul3-HA in the input (Rlysate; lane 6) was 0.53∶1 whereas the ratio of Cul3-EGFP-HA to Flag-Cul3-HA in the immunoprecipitates (RIP; lane 3) was 0.18∶1. The percentage of Cul3-EGFP-HA bound to Flag-Cul3-HA can be calculated as RIP/Rlysate×100% and amounts to 34%. In addition to Flag-Cul3-HA, Cul3-EGFP-HA can also associate with itself and with endogenous Cul3. The affinity of Cul3-EGFP-HA to itself, to Flag-Cul3-HA and to endogenous Cul3 is expected to be identical. Therefore, if the protein abundance of Cul3-EGFP-HA and Flag-Cul3-HA in the cell is equal, the amount of Cul3-EGFP-HA present in a dimer with Flag-Cul3-HA and with itself is also the same. As mentioned above, the ratio of Cul3-EGFP-HA to Flag-Cul3-HA in the input (lane 6) was 0.53∶1. It therefore follows that the percentage of Cul3-EGFP-HA that exists as a dimer with itself equals the percentage of Cul3-EGFP-HA/Flag-Cul3-HA dimer (i.e. 34%) multiplied by 0.53, resulting in 18%. To estimate the amount of Cul3-EGFP-HA bound to endogenous Cul3, we determined the ratio of endogenous Cul3 to Flag-Cul3-HA by Western blotting with Cul3 antibody. As shown in Figure 5B, the ratio is 0.41∶1. Therefore, the percentage of Cul3-EGFP-HA that exists as a dimer with endogenous Cul3 equals the percentage of Cul3-EGFP-HA/Flag-Cul3-HA dimer multiplied by 0.41, resulting in 14%. Adding the percentage of Cul3-EGFP-HA bound to itself (18%), to Flag-Cul3-HA (34%), and to endogenous Cul3 (14%) together results in a total of 66% of dimeric Cul3-EGFP-HA.
The cells were transfected as indicated at the top of each panel. The cells were lysed, and the lysates were subjected to Flag immunoprecipitation (IP). Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies. The quantification of the immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies and digitized using the Odyssey Infrared Imaging system in the fluorescence intensity of 800CW, green.
Three repeats of the experiment gave percentages of dimeric Cul3-EGFP-HA of 66%, 70% and 43% (average of 60.0%, standard deviation 14.6). It should be noted that the calculated percentage of Cul3-associated Cul3-EGFP-HA would not be affected by whether Cul3 forms dimeric or multimeric complexes in vivo. In the case of multimeric complexes, the percentage of Cul3-EGFP-HA bound to Flag-Cul3-HA would increase while the percentage of Cul3-EGFP-HA bound to only itself or only to endogenous Cul3 (and not to Flag-Cul3-HA) would concomitantly decrease. Taken together, the results suggest that approximately two thirds of cellular Cul3 protein exists in a self-associated form in Cul3/Rbx1-BTB protein complexes in vivo.
Dimerization is a key component of biological regulatory networks and is frequently employed in E3 ubiquitin ligases. For instance, RING finger protein 4 (RNF4), Anaphase Promoting Complex (APC) and C-terminal of Hsp70-interacting protein (CHIP) E3 ligases have been shown to exist in a dimeric state –. RNF4 is a RING E3 ligase which contains multiple SUMO-interaction motifs (SIMs). These SIMs specifically recognize poly-SUMO chains, thus targeting poly-SUMOylated substrate proteins for ubiquitination via the RNF4 RING domain . The RNF4 dimerization is mediated via its RING domain. Dimerization promotes E2-ubiquitin thioester bond activation by recruiting the E2 to one RNF4, while the thioester-linked ubiquitin reaches across the dimer to engage the other RNF4 protein , . APC, which is responsible for regulating the cell cycle has also been shown to exist as a dimer . APC is a multisubunit E3 ligase and its dimerization is mediated via the Cdc27 subunit . A Cdc27 mutant that is unable to homodimerize prevents the formation of the APC dimer, thereby leading to inhibition of the APC E3 ligase activity . Thus, dimerization of APC is essential for its ligase activity.
Among the Cullin RING ligases (CRLs), Cul1, Cul3 and Cul4 based E3 ubiquitin ligases are known to exist as dimers , , . Cullin serves as a scaffold protein for the assembly of the multisubunit ubiquitin ligase complex that contains a RING domain protein at its C-terminus and a cullin-specific substrate receptor protein at its N-terminus , . Cul1 and Cul4 E3 ligase dimerization is mediated via the Cullin substrate receptors , , . In the Cul1 E3 ligase, Cul1 recruits substrate recognition subunits containing a conserved F-box domain via the adaptor protein Skp1 to form Skp1-Cul1-F-box (SCF) E3 ligases. WD40 repeat F box proteins such as Cdc4 contain a D domain motif which mediates the homodimerization . Each F box protein binds to one Cul1 protein via the Skp1 adaptor to form a dimeric SCF ligase complex . It has been reported that a monomeric SCFCdc4 complex is deficient in the ubiquitin transfer activity compared to a dimeric complex . In the Cul4DCAF1 E3 ligase, dimerization is mediated via the DDB1-and Cul4-associated factor 1 (DCAF1) substrate receptor which contains a LisH motif . LisH motif-mediated Cul4DCAF1 dimerization has been shown to increase the ubiquitin transfer activity by 2-fold compared to a monomeric complex . There are 50 different DCAF substrate receptors for Cul4 in cells. However, the LisH motif is not commonly present among these putative DCAF substrate receptors. Therefore, it is currently not clear whether dimerization of DCAF proteins is a general mechanism to regulate Cul4 ligase activity.
In this study, we provide evidence that Cul3 E3 ubiquitin ligase dimerization is also mediated via substrate receptors. Cul3 substrate receptors contain a BTB domain which is known to homodimerize . Homodimerization of BTB protein is important for substrate binding as described above. It has been proposed that homodimeric BTB proteins are also responsible for Cul3 dimerization by binding to two Cul3 proteins, forming a dimeric Cul3 E3 ligase complex . In a previous study, we provided evidence that Cul3 indeed exists in a self-associated form which is dependent on the BTB protein substrate receptors . However, Wimuttisuk and Singer, 2007 reported that two Cul3 proteins dimerize via their WH-B domains and that Cul3 dimers consists of one neddylated and one unnedylated Cul3. Thus, according to this model, the BTB proteins are not required for the Cul3 dimerization. In this study, we provide several lines of evidence that Cul3 dimerization is mediated via the BTB domain substrate receptor proteins and is independent of Cul3 neddylation. We found that Cul3 dimerization is dependent on the Cul3 N-terminus which interact with BTB substrate receptor proteins but not on the Cul3 C-terminus which contains the WH-B domain. Furthermore, Cul3 mutants that are unable to bind to BTB proteins exhibit markedly reduced Cul3 dimerization. Interestingly, some BTB proteins such as the Potassium channel tetramerization domain containing protein 11 (KCTD11) also form tetramers, raising the possibility of a tetrameric Cul3 E3 ligase complex. Indeed, modeling studies have shown that four Cul3 proteins could be accommodated in the structure of the KCTD11 tetramer . Finally, we also showed that preventing Cul3 neddylation by mutating the neddylation site or using a neddylation-deficient cell line does not affect Cul3 dimerization. It should be noted that the low percentage of neddylated Cul3 and the high percentage of Cul3 that exists in a self-associated form, as determined in this study, would also be inconsistent with a model whereby Cul3 dimers consists of one neddylated and one unnedylated Cul3.
Cul3 E3 ligase dimerization likely plays an important role in promoting ubiquitination activity by providing a bivalent geometry that has two docking sites for the E2 ubiquitin enzyme and thus enhances ubiquitin transfer and elongation of the target substrate , . Functional evidence for this has been provided by Kigoshi et al., (2011) who studied the function of the Cul3-KLHL7 E3 ligase . The authors reported that a mutant of the KLHL7 BTB substrate receptor which is able to homodimerize with wild type KLHL7 but unable to bind to Cul3 inhibits ubiquitination of the target substrates. Thus, a heterodimeric Cul3 ligase complex consisting of wild type and mutant KLHL7 has reduced ubiquitination activity, suggesting that the presence of two bound Cul3 protein is necessary . Zhuang et al. (2009) also showed that two Cul3 proteins are bound to two SPOP substrate receptors in the Cul3-SPOP structure . They revealed that the recombinant Cul3 and SPOP form a 2∶2 complex. In contrast, Small et al. (2010) showed that a Keap1 homodimer only binds to a single Cul3 protein in vitro . Our data as shown in Figure 4, indicate that two (or more) Cul3 proteins are bound to both the SPOP and Keap1 dimers in vivo. The difference between our results and those by Small et al. (2010) may be due to the difference in the expression system. Our results also suggest that the majority of the Cul3 proteins exist as a dimer or multimer in cells. Thus, BTB substrate receptor dependent dimerization or multimerization of Cul3 E3 ubiquitin ligases is likely of physiological significance in facilitating substrate polyubiquitination.
Materials and Methods
The Flag-Cul3-HA and Cul3-EGFP-HA constructs were generated by insertion of the Cul3 ORF into the KpnI and SacII sites of modified pcDNA3.1/Zeo and pcDNA3, respectively. To generate C-terminally V5 tagged expression constructs for the N-terminal deletion construct of Cul3 (amino acids 392–768) and C-terminal deletion construct of Cul3 (amino acids 1–427), the respective cDNA was PCR amplified and inserted into the KpnI and SacII sites of modified pcDNA3. The (Cul2(NT)-Cul3(CT)-V5 was generated by fusing the N-terminus of Cul2 (amino acid 1–394)) to the C-terminus of Cul3 (amino acid 392–768) which also carrying a C-terminal V5-tag. Mutagenesis to prepare the K712R Cul3, and L52AE55A Cul3 mutants was carried out using the Stratagene site-directed mutagenesis kit. All other plasmids were previously described , –.
The T-REx system (Invitrogen) was used to generate cell lines with tetracycline-inducible expression of dnUbc12-HA (dnUbc12) according to the manufacturer's instructions, as previously described . For DNA transfections, sub-confluent T-REx-293 cells were transfected using GeneJuice (Novagen) according to the manufacturer's instructions.
For immunoblotting, the cells were washed with ice-cold phosphate-buffered saline and then lysed in Triton X-100-containing lysis buffer, as previously described . Lysates were precleared by centrifugation before use for Western blotting. Equal amounts of protein were loaded for Western blot analysis. The following antibodies were used: rabbit polyclonal anti-Cul3 (34–2200; Zymed Laboratories), monoclonal anti-V5 (AbD Serotec, Kidlington, UK), rat monoclonal anti-HA (clone 3F10) (Roche Applied Science).and monoclonal anti-FLAG M2 (Sigma-Aldrich, St. Louis, MO). Western blots shown are representative of at least two independent experiments. To quantify the dimerization of Cul3 in Fig. 5, the blot was imaged using an Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln NE). Sample amounts were determined and quantitated via IRDye 800CW in the 800 nm channel of detection.
V5 antibody (2.5 µg), coupled to 20 µl of protein G-Sepharose (Amersham Biosciences, GE Healthcare, Waukesha, WI), or 20 µl of FLAG-agarose (Sigma) was used for immunoprecipitations, and 500 µl of precleared lysate from HEK293T cells transfected in 60-mm tissue culture plates was added. The samples were tumbled at 4°C for 2 h, and the beads were then washed four times in 1 ml of NP40 cold lysis buffer (containing 50 mM NaCl, 0.5% NP-40, 5% glycerol, 0.5 mM EDTA, 50 mM Tris, pH 7.5) and once in buffer containing 50 mM Tris (pH 7.5). The immunoprecipitated proteins were then denatured in SDS sample buffer and subjected to SDS–PAGE and Western blotting. The lysate lanes in the Western blots correspond to 5% of the input used for the immunoprecipitation. The immunoprecipitation experiments shown are representative of at least two independent experiments.
For the double immunoprecipitation in Fig. 4, FLAG immunoprecipitation was carried out as described above. After washing of the immunoprecipitates, the complexes were eluted with 30 µl Flag peptide (5 mg/ml) (F3290, Sigma), followed by V5 immunoprecipitation. The final immunoprecipitated proteins after washing were then denatured in SDS sample buffer and subjected to SDS–PAGE and Western blotting.
In situ proximity ligation assay
In situ proximity ligation assay was performed using the Duolink detection 563 kit (Olink Biosciences, Uppsala, Sweden) following the manufacturer's instructions. Transfected cells were grown on coverslips. Cells were washed once with 1xPBS and fixed with 4% paraformaldehyde in PBS. TritonX-100 (0.1%) was used to permeabilize the fixed cells. As compatible antibodies for this particular experiment, Cul3-Flag was detected with a primary rabbit anti–Flag (Sigma) antibody while Cul3-V5 was detected using mouse anti–V5 (AbD Serotec) antibody. The corresponding probes of anti-rabbit PLUS and anti-mouse MINUS were from Olink Biosciences. Coverslips were mounted on glass slides using Vectashield mounting medium and visualized by fluorescence microscopy.
Conceived and designed the experiments: YYC TH. Performed the experiments: YYC TH. Analyzed the data: YYC TH. Contributed reagents/materials/analysis tools: TH. Wrote the paper: YYC TH.
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domain: biology