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Systemic C is an enhanced, supercharged Vitamin C that provides superior antioxidant protection. The beneficial effects of Vitamin C are energized in this unique formula, which is designed to make more Vitamin C available for immune support, heart health, healthy aging, and free radical scavenging.
The benefits of Vitamin C are well known and clinically proven. It is one of the most important nutrients ever discovered for our health. It affects our energy, joints, aging, brain, skin, bones, and the body's detoxification process. It also helps support our heart and cardiovascular system due to its powerful antioxidant properties. It is an unparalleled key to health and well-being.
The evidence and scientific research continue to mount in worldwide clinical exams and tests: Vitamin C is one of the most important benefits to your health available today. Vitamin C can:
For decades, the conventional means of obtaining sufficient Vitamin C was through ascorbic acid. Now, however, because of the importance of Vitamin C, scientists may have discovered a means of helping the body obtain more Vitamin C for increased antioxidant protection. Nutrients added to Vitamin C may "recycle" Vitamin C that has been spent (oxidized) after neutralizing free radicals, by putting it back to work protecting your cells.
Systemic C from Source Naturals is an enhanced, non-acidic Vitamin C designed for superior antioxidant protection. After Vitamin C carries out its antioxidant function of neutralizing free radicals, it is usually oxidized and spent. However, the unique blends of other antioxidant compounds in Systemic C help to reactivate and recycle the Vitamin C in the body. Reactivating Vitamin C allows this important vitamin to continue its vital functions, providing the body with increased antioxidant activity.
Systemic C combines Vitamin C from calcium ascorbate with highly powerful anti-oxidant compounds that are intended to heighten its benefits:
Alpha-Lipoic Acid - a renowned antioxidant, scavenges hydroxyl radicals, nitric oxide, peroxynitrite, hydrogen peroxide, hypochlorite, superoxide and peroxyl free radicals.
N-Acetyl Cysteine - replenishes intracellular glutathione depleted by free radicals such as hydrogen peroxide and hydroxyl radicals.
Quercetin - has metal-chelating and stabilizing properties. It is also a strong inhibitor of lipid peroxidation through scavenging of peroxide radicals, which strongly oxidize and deplete ascorbic acid.
Grape Seed Extract - The proanthocyanidins in grape seed extract inhibit the production of free radicals, thereby increasing the function of the cells' own antioxidants, such as glutathione and ascorbic acid.
Modern life, with our constant bombardment by pollutants, excess sunlight, unnatural foods, chemical toxins, and electromagnetic radiation, produces damaging free radicals. Free radicals are also natural byproducts of such physical processes as respiration and mitochondrial energy generation.
Free radicals are unstable molecules. Because they seek stability, they damage cells and tissues as they steal electrons from other, healthy molecules. As more molecules damage others, a cascade effect begins. This can result in signs of aging, cellular imbalances and many other health concerns. According to an article in the Journal of Rheumatology, "increased free radical levels may be responsible for the development of fibromyalgia", a condition that affects an estimated 10 million people in the U.S alone.
Antioxidants combat free radials, directing the body toward greater health. Taking antioxidants like those included in Systemic C can help boost immune function, while protecting our cells and DNA from free radical damage. These antioxidants can also serve as a preventative measure against many different health issues from colds and flu to more serious disorders like cardiovascular disease.
There seem to be new strains of colds and flu each year. Additionally, stress can take a huge toll on our immune systems. Taking Systemic C along with other immune enhancing nutrients can help you and your family stay healthy. Should you begin to experience the onset of a cold or flu, you can also take a colloidal silver such as Colloidal Defense to help prevent or simply shorten the severity and duration of either one that you may have been exposed to. Please read our article "Staying Healthy for the Holidays" to learn more.
Johns Hopkins Study on Vitamin C
Capsules per Container: 120 Capsules
Serving Size: Two (2) Capsules
Servings per Container: 60
|Amount per Serving
|% Daily Value
|Total Carboyhydrates
|1 g
|Vitamin C (as calcium ascorbate)
|1000 mg
|Calcium (as calcium ascorbate)
|110 mg
|N-Acetyl Cysteine
|120 mg
|alpha-Lipoic Acid
|100 mg
|50 mg
|40 mg
|Grape Seed Extract (Proanthodyn)
|30 mg
*Daily value not established.
Other Ingredients: gelatin (capsule), colloidal silicon dioxide, and magnesium stearate.
Free of: Yeast, dairy, egg, gluten, wheat, sugar, starch, salt, preservatives, or artificial color, fragrance, or flavor.
Suggested Use: Two capsules, one to two times daily, or as directed by your healthcare professional.
Warning: If you are are pregnant, may become pregnant, breastfeeding, or taking any prescription drug, consult your healthcare professional before using this product.
Manufactured by Source Naturals.
† These statements have not been evaluated by the U.S. Food and Drug Administration (FDA) or EnergeticNutrition.com. This product is not intended to diagnose, treat, cure or prevent any disease. The information provided on this site is for informational purposes only and is not intended as a substitute for advice from your physician or other health care professional or any information contained on or in any product label or packaging. You should not use the information on this site for diagnosis or treatment of any health problem or for prescription of any medication or other treatment. You should consult with a health care professional before starting any diet, exercise or supplementation program, before taking any medication, or if you have or suspect you might have a health problem, or are pregnant. When you receive an item, carefully read all labels, warnings, and directions before use. Actual product packaging and materials may contain more and/or different information than shown on this website.


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Uniforms of the Waffen-SS: Vol 3: Armored Personnel - Camouflage - Concentration Camp Personnel - SD - SS Female Auxiliaries
272 pages. This three-volume set is unquestionably the best reference on German SS military uniforms ever produced. This spectacular work is a heavily documented record of all major clothing articles of the Waffen-SS. Hundreds of unpublished bw photos were used in production. Original and extremely rare SS uniforms of various types are carefully photographed and presented here.
Author: Michael D. Beaver. Hard cover.

subset: commoncrawl


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Strengthening Relationships
12 ways to be present for your child
Quote from the article:
"I think about how things are both easier and harder than ever for those of us raising children. Sure, we have all the modern-day amenities that spoil many of us rotten. But, at the same time, things are harder. The distractions, the too-much to do epidemic and the burn out from so much work and so little breaks...Many mamas are married but their spouses work away from the home for weeks or several months at a time. This is married single parenting. In my own family, my husband is gone 11 hours a day with zero flexibility to change that or his two-hour commute each day. There are single parents parenting 24/7 more than ever. And the expectations of parents and how we raise our kids are greater than ever. Add to that that many of us live several hundred miles away from any relatives so that sweet little safety net families used to rely on years ago is now non-existent for so many, which only contributes to the burnout and exhaustion parents are feeling all over. Perhaps we can blame this on how our schools are not functioning at the highest level they could. And so there's no wonder so many families feel disconnected. None of this is easy stuff to cope with and none of it makes raising children easier. Which is why I am so passionate about my work here helping you actually be more connected despite all the struggles — and the chaos. We all have struggles. We all suffer. We are all tired — some more than others. So, I'm offering this list 12 ways to be a present for your child because I am confident I will need these reminders as much as you tomorrow or the day after that or the week after that"
Connection begins with YOU
Quote from the article:
"What we know today is that children who do well in life have parents who have taken some time to make sense out of their own lives. As a parent, exploring where you come from and finding answers to why you are the way you are arms you with the understanding, emotional sensibility, and healing we all need to mature into the individuals we want to be, and, into the kind of parents we promised ourselves we'd be.
Being a Mindful Parent does require some time looking through the family microscope. But I assure you, this is not an examination of assigning blame, but of gathering information. The more you know about you, the more attuned and empathic a parent you will be. This process will help you understand why you repeatedly do the very things you swore you would never do, just as it will allow you to see all the things that you want to pass on. I call these family patterns, 'family heirlooms.' What we experience as children: observe, hear, and sense, becomes wired into our central nervous system in our earliest years, and "Ta Da!" we have wiring we have no intention of repeating, and yet we do." - (Pilar Placone, Ph.D)
Repairing the Connection
Quote from the article
"A parent and child can experience several conflicts a day and there are times when all parents become overly stressed, impatient and annoyed towards their child. In situations where the parent's anger explodes and they vent their anger at their child, this can be very traumatic for children; they can feel attacked, overwhelmed, trapped and confused. They will likely feel the full force of that anger and it definitely needs a lot of conscious repairing. If not repaired, the child will unconsciously develop defense mechanisms that help them cope at the time, yet they pay a high price for in other areas of their life, their their learning and ability to relate patiently in relationships in general. For more detailed support in learning to avoid exploding in anger, this teleseminar audio has been immensely beneficial to so many parents."
Understanding your child's love language
Quote from the article:
"These last two weeks, have been amazing to witness. I've watched her blossom. She's more confident, more daring, more independent than ever before. The secret you ask? We found our common love language. Quality Time. Nothing says, "I love you", like full, undivided attention.
I didn't even know love had a language 'til Zina mentioned the concept to me during one of our weekly phone chats. It didn't occur to me I could apply it like this until we stumbled on to the answer unknowingly. Once we made the connection, it was obvious. We had found our way to communicate our love for one another in a way we both could feel. My primary love language is touch. I let my children know how much I care by showering them with hugs and kisses, cuddling on the couch, and tickling their back at night before bed. My daughter's first love language, though, is all about positive affirmations. Neither of those things come naturally to the other one of us, and as a result we kept missing our chance to connect on a meaningful level. Sure, I praise her often, and she hugs me, at times, but it takes effort on both our parts to make it happen, and on days when it doesn't go as well as we would have hoped – we both end up feeling hurt and rejected. Now I can see and understand why." - (Stacy Teet of Kids Stuff World)
10 Ways to communicate with a Toddler
Quote from the article:
"Did you know that toddlers and preschoolers can solve their own problems? Yes, it is true! With the help and guidance of us grown ups, who often are too quick to offer solutions without giving children the opportunity to come up with a solution on their own, they just might be able to do it. They just might surprise us. The best part is that they can develop important life skills of problem solving and decision making. Not to mention, they will feel capable and independent."


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Fitness Tests
From VO2 Max testing (Cardio-respiratory fitness) to strength, and resting metabolic rate (RMR) we have a range of scientifically validated fitness assessments.
Fitness Tests – comprehensive analysis
Fitness assessments in East Dulwich to give you an accurate, pinpointed evaluation of your body's strength, agility, and recovery. To train effectively, whether as an endurance athlete, sportsperson, or someone committed to a healthy lifestyle means you need to know your fitness levels. And that means using a scientific approach, with a full data analysis of your fitness.
ESPH Fitness Assessments – East Dulwich, South London
Body composition analysis*, including fat percentage
Using the Tanita MC780, our test provides a measurement of body fat & muscle mass indices, a far better way to analyse fitness and health than weight or body mass index (BMI). From: £29
VO2 MAX test (Cardio-respiratory fitness) – London
The VO2 MAX analysis is the gold standard indicator of cardio fitness and aerobic endurance. The test is conducted sub-maximally on the treadmill or bike, whilst wearing a mask, to gain your maximal heart rate. We can perform a maximal test on a bike but for elite athletes only. From: £89
Resting metabolic rate (RMR) test
If you have had difficulty losing weight but are physically active, you may have a lower than normal metabolism. RMR is the definitive test to show you what your energy baseline is to help you lose weight effectively and keep it off. From: £89
Fitness Test
We also offer a full test to cover your VO2 MAX analysis, Strength and Body Composition. From: £109
Diet & nutrition screening
We can assess your nutrition food diary if you are a fitness enthusiast or can highlight if you are deficient in any micronutrient or macronutrient and advise suitable foods or supplements for optimum health.From: £85
All our assessments come with a fully detailed report. You can also have a full list of recommendations so you know what you need to successfully improve. Maximise your health and fitness.
Learn more… read our QUESTIONS QUESTIONS Fitness Tests opposite
Need more info? Call the number at the top of the page, pop in to visit or CONTACT US
Locations at: East Dulwich
* Exclusions apply to the body composition check. If you have particular requirements please call to discuss.


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Undergraduate Education
In order to practice homeopathic medicine you really need to be trained at an institution recognized by the Australian or New Zealand Government departments of education. That goes for wherever you are. That college also needs to be accredited with the professional bodies in either country to ensure you can then practice, be insured and be registered. Wherever you study has also got to be committed to quality, and have great teachers and administration.
Homeopathic Medicine is a mixture of the study of philosophy, medicines (hundreds of them), therapeutics, clinical training and medical sciences. It's a big deal. It's a lot of work. It's unbelievably interesting and fulfilling. It's not for everybody.
Endeavour College of Natural Health
New Zealand
College of Health and Homeopathy
Endeavour College of Natural Health
'I have been educating undergraduate homeopathic students for 20 years. I love it. I travel all over the world to do it. The UK, Ireland, Belgium, Spain, the US, Canada, India, Thailand and Malaysia, Australia and New Zealand.
I know what is required to practice homeopathy. I know how to deliver that information so that it is palatable, fun, solid, substantial and useable. I acknowledge the process, celebrate the milestones. We study philosophy in depth, study materia medica in a multi-dimensional way.'


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As actress Julie Hesmondhalgh prepares for an emotional exit from Coronation Street next week, she tells Susan Griffin about her memorable moments on the cobbles as transgender character Hayley Cropper
TODAY is the last day for Julie Hesmondhalgh on Coronation Street and it's only lunchtime but there have already been tears. "Jennie McAlpine (who plays Fiz) got me going with her card," says Accrington-born Julie, who has played Hayley Cropper for the last 16 years.
Her departing storyline, set to be shown on Monday, has been one of the most talked about in soap history, not only because she and husband Roy (David Neilson) are such well-loved characters, but because of the controversial decision to end her own life after being diagnosed with cancer.
Julie, 43, knew the moment she told the show's producer Stuart Blackburn she wanted to leave it wasn't going to be in a taxi.
"It wasn't necessarily what I wanted, but I knew it had to be that way for Hayley and Roy," explains the actress.
"It wouldn't have made sense to do anything else. Had I gone to Africa [as the character did in 2007 when Julie had a break], I would have been ringing Roy all the time."
Roy and Hayley were introduced by Alma Baldwin back in 1998, when Hayley was a shy shop assistant at Firman's Freezers. A friendship ensued until, on one of their dates, Hayley revealed she was a pre-operative transgender formerly called Harold (she later underwent sex change surgery).
"The trans community weren't happy with the storyline because, to be fair, it was a bit of a joke," says Julie, who's wearing a bright green Sixties-style dress, her peroxide crop messily styled.
"But I never saw it as a joke. I read up on it, talked to people and tried to do it properly, and when David and I started working together, we both took it on.
I think the trans community saw it and thought it was actually changing attitudes."
The fictional pair had a blessing ceremony in 1999, which is where Julie met her real life husband-to-be Ian Kershaw, the writer and actor who played "a baddie journalist" in the episode.
Then in 2010, Roy and Hayley got married to reflect the change in law. "Roy gave a speech, which remains one of my favourite lines ever," says Julie. "He said, 'We've remained the same, the world has turned to meet us'. And that's exactly what happened. They've just carried on their path and the world has adjusted."
The star's initial interest had been social work. She decided later to focus on acting, studying at LAMDA before joining an independent theatre company. Roles in The Bill, Dalziel And Pascoe and The Dwelling Place followed, before Coronation Street came calling.
Just as Hayley's arrival proved thought-provoking, so does her departure — in choosing to end her life before cancer treatment renders her incapable of doing so. "She wouldn't ever want Roy to assist her, and she doesn't want to get to a point where she isn't able to do anything," she says.
A member of the British Humanist Association, Julie has "quite strong views" in favour of pro-choice. For that reason, she's always understood Hayley's decision, while David hasn't.
"We have very different views, so he's played what he feels and I've played what I feel."
She describes the two of them as "like an old married couple". "My relationship with David's been the absolute best," she adds. "He's completely different from Roy. He's very dry, he doesn't suffer fools. He's loved and respected."
Viewers won't actually see Hayley die but they will witness her drinking a lethal concoction, which Roy tries to dissuade her from doing right up until the end.
The last scene was done in one take and the atmosphere on set was unlike anything she'd known before. "There's usually a lot of banter and fun, even when you're doing extremely dramatic scenes, but everything was silent, almost holy and church-like," she says.
As to her own future, she's set to star in two plays at Manchester's Royal Exchange.
Blindsided, opening on January 23, is written by Simon Stephens and tells the story of a girl growing up in Stockport who falls in love with a man who breaks her heart.
After that Julie will also be reprising her award-winning role as Sylvia Lancaster in Simon Armitage's moving play Black Roses, about the murdered Bacup teenager Sophie Lancaster, which opens at the end of February.
"That takes me right up until April. After that, who knows? I think I'll probably be ready for maybe a little bit of comedy," she says, laughing.
"Or a holiday!"


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Virtual Trainer: Hey PJ, thanks for taking my call this afternoon. Let's start out by talking about your first three Nationals as a professional. How did they go for you from a training and fitness point of view?
Seiji always talks about limiters that hold you back like skill or fitness. Do you think that your conditioning had anything to do with your limits in your first three Nationals or was it just a case of getting a feel for the speed?
I think that in general if you are not in shape going in then you won't be able to do very well. But like I said I think I was as ready as I could be going in. It's just something you have to learn; the fitness the speed and the other things that you need to do well.
I know that Coach Seiji has been your trainer throughout your amateur career. Tell me a little about your setup and how he trains you?
Well, I have been with Seiji pretty much my entire amateur career as an online client. We have spent weeks together periodically whenever I was in Texas but he has never been a full time trainer that I see everyday. Everything we do is pretty much online except for the few times we are together. Like we spent the week before Millville together to get ready for that race. Whenever I am in Texas we train together though.
Did Seiji travel with you when you raced as an amateur?
Yes. He was at every one of my races. I can't really remember a race that he wasn't at.
That has to be a little different now that he isn't traveling with you to all of your pro races. I mean he has been like a big brother to you and now all of the sudden you not only have to deal with the start of your career but you have to do it without a guy who has always been there with you at the races.
Yea, for sure it's different not having him there. He always made our food and got our drinks and made sure we were ready for our motos with warm-ups and stretching and all that. It is definitely different but it isn't too bad. I think sometimes you have to do things on your own for a while to really learn how to do it.
Yea, I'm sure you could have used him at Steel City after you banged your arm up after the second practice. Seiji is pretty famous for his "power" massages.
(laughs) Yea, he is good with his messages but I try to stay away from those things when I can. He wants to hurt you too bad during it but it does feel better afterwards.
You said you are primarily an online client of Seiji's. The coaching packages you use online are the very same packages anyone can buy. Tell me a little about how that has worked out for you.
Do you think that you will continue with the online coaching as your career progresses or do you think you will seek out someone to be with you on a full time basis?
Well, you never know what is going to happen in the future. I just know that I was happy with it throughout my amateur career so there is always a possibility that I will just keep things the way they are.
Cycling is a big part of a lot of pro riders training and Seiji likes to cycle as well. How long have you been cycling and do you like it?
I have been cycling for a couple of years now and yea, I really like it. I was doing some stationary biking and borrowing other people's bikes for a while. But I was never very seriously into it until a few years ago when I got my own bike and started cycling a good bit then.
Have you ever entered any road cycling races?
No. I have not done that yet. I just ride every Wednesday night with a group of guys from around my house. They are a really good group of guys. A few of them say that I would do pretty good if I entered a race but it's nothing I am currently considering. Maybe sometime in the future.
Your Motosport Xtreme Green contract requires you to move to California. That is a pretty big move. What do you think about that?
Yea, I'll actually be leaving home here in the next couple of weeks. I'm renting a house with Kyle Cunningham and Hunter Hewitt. We are actually going to rent Andrew McFarland's house. It'll just be the three of us living in the house out in Menifee.
Very cool. That sounds like a lot of fun plus you will have two other guys that are in the same situation as you. Will you guys train together as well?
Oh yea, for sure. I think it will be good. We have three good guys that want to go out and train and cycle and do the things that are necessary to get better.
JGR [Gibbs Racing] uses a team concept to train their riders. The trainer works for the team and is responsible for all of the riders. What do you think about this way of training?
You know it's really a toss up. It could be really good but it all depends on the guys that are on the team because they are the ones you have to train with. If you can have a whole team of guys who want to train hard and get better and all the other things it takes to get it right then it could be really good. But if you get a team with a couple of guys who could really just care less and not want to be training or doing any of the other stuff then that could really bring you down. Realistically, I think it all comes down to who is a part of the team and who the trainer is.
Now that the outdoors are over and Supercross is next, what will you concentrate on most to get ready for the season?
Well, for me I think I am in great condition and Supercross won't be a problem. I think for the most part I need to work on my timing and stay on the bike. I'll be putting in my 20 lap motos for sure to get ready for some of the longer tracks like Daytona and a few others that they stretch out. I think for the most part I just need to get used to Supercross and the tight conditions, the hard pack, and working on my starts. The physical conditioning side of it is all good. I just need to be able to put in 20 solid laps.
Well, I have never raced a Supercross but I have practiced on them quite a bit. I used to play around on Supercross tracks when I was on 80's and stuff like that. I go out with some of my buddies now and do some motos and stuff like that.
Are you going to race the US Open?
We aren't sure yet what we are going to do with the US Open. I would actually prefer not to race it since I won't have very much time on the new bike. But if I get on the new bike and adapt really quickly to it, then yes there is a possibility if that is what the team wants to do. It all depends on how the testing goes just before the Open and if the team wants me to.
What do enjoy more; outdoor racing or Supercross?
That's really a toss-up. Some days I feel like I want to go ride Supercross and other times I feel like I want to ride outdoors. It just really depends on the mood I'm in at the time and what I have been doing. Like right now I am really looking forward to Supercross and I enjoy riding Supercross a lot and it might actually fit me a little bit better. I am more of a smooth type rider than one to really let it hang out there. So I think Supercross will be good for me. I just need to be able to get some good starts and run up front.
I know this may be difficult to answer since you haven't actually race Supercross, but do you feel there is a big difference fitness wise between MX and SX?
For me it really just depends on the particular day and what I have been doing that week. I know sometimes I can go out and put in 20 lap motos on a Supercross track and feel great and some days I can't even do 10. It just really depends on the day and the atmosphere. But I think that a 15 lap Supercross main and a 30 minute moto are fairly close because on an outdoor track you get a good bit of time to relax and on a Supercross track you are pretty busy the whole time.
Let's talk a little bit about one of my favorite topics and that is performance enhancing drugs (PED's). Do you think there should be drug testing for PED's?
Get the Same Coaching Package PJ Uses Right Here!
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Thursday, 10 August 2017
Ride the Storm by Karen Chance
There are perhaps one too many action scenes, too many moments that are almost repetitive, which then takes the pleasure out of the story, because you know what is coming.
One of the aspects I particularly enjoyed was the way Chance brought in facts and story-lines from older books and even from the Basarab series. It is no secret that I think the Dorina Basarab series is superior to the Cassie Palmer one, so I was really excited to see her appear in this book. It was more of a secondary and explanatory role, but it was still a nice touch. I would love to see a Basarab and Palmer novel with both receiving the same amount of attention.
Mircea spent a certain amount of time clearing up certain things about Dory, and in doing so also reveals a secret that connects all of the Pythias throughout the centuries. It is also a betrayal on a certain level, as far as I am concerned, because he admits to planning ahead and wanting to use the Pythia, ergo Cassie, to achieve his greatest desire.
Now she is armed with this information will Cassie finally stand by her attraction to Pritkin and accept that perhaps she was nothing more than a means to an end for Mircea.
I find that Karen Chance manages to create more complex plot-lines and scenarios as the series goes on, as opposed to less interesting as the ideas dwindle. The trick is keeping the element alive that keeps the readers coming back for more. Chance is one of the more underrated stars of the urban fantasy genre, especially when it comes to the Basarab series, and definitely an author whose books I look forward to.
Buy Ride the Storm at AmazonUk or go to Goodreads for any other retailer.
Read Reap the Wind by Karen Chance
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Filed under: Addiction - Children, Complex PTSD, Exemplary Children of Divorce, Relationship with Father
Just heard comedian Sarah Colonna's special called I Can't Feel My Legs on Amazon Prime. Hope it's ok to quote or paraphrase a part of her skit. I can't write that fast and can't read some of it. Awesome that the audience claps when she cracks a joke about having 2 dads. It might be safe to assume that she is a Child of Divorce but can't say for sure. You don't hear very many jokes about extended family stuff. A long time ago I was at a Comedy night and the comedian took a poll of the audience looking for Children of Divorce to see if it was worth it to crack Divorce jokes. He decided against. Only one girl raised her hand.
"I hate single grocery shopping because I'm not home a lot. I'm not in town a lot so I only buy a quick few things when I'm out at the grocery store.
I guess I need to make a list. Because if you make a list then you can look at what all your items altogether might look like to someone else when they're in the basket.
Here's an example. I was grocery shopping this (unknown word, can't read my writing) in June which will make sense in a second. I was grocery shopping and had everything on the conveyor belt and there were two guys, early 30s and they were staring at me. 'Oh here we go. Can't even go to the grocery store anymore.' I was bending over. Then I realize they're not looking at me. They're looking at the conveyor belt.
And that's when I became painfully aware of what was on there. I had 2 bottles of vodka, you know, with the handle, the kind with the handle, tampons, skittles. I was hungry. And two Father's Day cards (laughter & clapping). It's like, O My God! Look at that girl with her period, her drinking problem, and all those Dads!!!"


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Last Thursday, 27 United States senators called for a Medicare-like option in the state health exchanges. Senators Bernie Sanders, Jeff Merkley, Patty Murray, Dick Durbin and Chuck Schumer, along with 22 other senators introduced a resolution that complements Secretary Hillary Clinton's proposal for a "public option"–a government plan like Medicare–that would be available to every American. Since then, six more senators have signed onto the resolution.
The senators want to give everyone in America the choice between commercial health insurance and government health insurance, the public health insurance option. A public option would not only give people more choice, but it would drive competition in the marketplace, bringing down health care costs.
A public option would also guarantee people the continuity of care they value. Right now, people in the state health insurance exchanges and people with employer coverage cannot rely on their commercial health plan coverage from one year to the next. Insurers enter and leave markets as they please. Recently, Aetna announced it is pulling out of many health care markets, claiming that it doesn't view them as profitable.
During the debates over the Affordable Care Act, the public option was introduced and ultimately quashed for lack of support in the Senate. At that time, the Congressional Budget Office estimated that it could drive down health insurance premiums as much as seven percent and reduce the deficit $68 billions over six years. But, doctors and hospitals were generally opposed to it for fear that it would bring down their reimbursement rates.
If you support the Senators' resolution and want a public option, click here and sign on to a petition at WeWantaPublicOption.com. The resolution currently has support from a range of advocacy organizations, including the Progressive Change Campaign Committee(PCCC), Presente.org, UltraViolet, Working Families Party, MoveOn.org, Democracy for America, Daily Kos, and the AFL-CIO.
Here's more from Just Care:


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Bank of England (BoE) governor Mark Carney said that interest rates will rise in the "relatively near term." He told the BBC Radio Four's Today Programme this morning: If the economy continues on the track that it's been on, and all indications are that it is, in the relatively near term we can expect that interest rates will
UK unemployment fell by 75,000 in the three months to July to the lowest level in 42 years, but workers continue to feel the squeeze as real wages remain stagnant.
It has been more than a year since The Bank of England unveiled the new and improved £5 note.
UK consumer spending fell for the third month in a row in July, the longest-running slump since February 2013, according to research from credit card giant Visa and financial services company IHS Markit.
The Bank of England's Super Thursday meeting takes place today.
Sir Dave Ramsden, a senior civil servant, who has been at the Treasury for 29 years, was appointed the Bank of England's deputy governor on Thursday.
British companies increased their marketing budgets in the second quarter despite economic uncertainty resulting from the UK's decision to leave the European Union (EU), according to a survey out today.
The Bank of England's Prudential Regulation Authority (PRA) has told lenders to demonstrate they are properly managing risks — including consumer credit.
The UK's inflation rate fell back in June, as many economists had predicted.
The Bank of England's semi-annual financial stability report, out today, has warned if the UK leaves the European Union without a bespoke trade agreement the consequences would be bad for all involved.
After cancelling the event last week in the wake of the Grenfell Tower disaster, Bank of England (BoE) governor Mark Carney and the UK chancellor Philip Hammond discussed Brexit and interest rates at Mansion House earlier today.
Silvana Tenreyro, 43, was appointed member of the Bank of England's (BoE) Monetary Policy Committee (MPC) on Monday.
The UK unemployment rate has fallen to its lowest level since 1975 at just 4.6 percent, with a total of 31.95m people now in work.
These three things will change the world today.
The Bank of England's (BoE) chief operating officer and deputy governor for markets and banking Charlotte Hogg resigned today amid allegations about a possible conflict of interest.
Donald Trump's reign as US president has been good for the UK economy, according to one the the UK's top central bankers.
Today, the Bank of England (BoE) upgraded growth forecasts for the next three years.
Today, the Bank of England announced its latest decision on interest rates and unveiled its quarterly inflation report.
These three things will change the world today.


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I saw this photo on Facebook the other day, I shared it and now it is going viral. A cold water wash is obviously best, and I do believe she didn't check herself out in her mirror before she left her home.
I just can't stress this enough, Do not wash wool in hot water.
One thing always do before I leave the house that I'm sure a lot of people out there don't do and that is look at my entire outfit in the mirror.
When I look at my outfit before I leave the house, I check all views INCLUDING my rear end.
I look at my outfit as though I am looking from someone else's perspective, for example, is it too short, too tight, does it ride up my backside, is it too see through, do my boobs bulge, etc.
I bend over slightly if it is a short dress or top and if you can see my underwear, I get changed, if my bust is popping out, the top is too small, I change it.
The one piece of advice I can offer all women, please, please look in the mirror before you leave the house, and don't just look, act if it looks like you wouldn't like your daughter or you mother going out looking like that.


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The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.
Peptidoglycan is a macromolecule of interlinked glycan chains and peptide bridges which forms the rigid structural component of the eubacterial cell wall, giving cells osmotic stability and imparting to them their shapes. Many of the final periplasmic steps in the synthesis and maturation of peptidoglycan are performed by the penicillin binding proteins (PBPs), enzymes to which β-lactam antibiotics bind covalently. The seven classic PBPs ofEscherichia coli were first observed in polyacrylamide gels by Spratt (33, 34), who named the proteins in order of their decreasing molecular mass: PBPs 1a, 1b, 2, 3, 4, 5, and 6. Recently, five additional PBPs have been identified and authenticated by genetic and biochemical means, including PBP 7 and its proteolytic artifact PBP 8 (11, 12), DacD (1), AmpC and AmpH (13), and PBP 1c (29), thus bringing to 12 the number of PBPs in E. coli.
The physiological importance of four PBPs has been established by examining temperature-sensitive mutants or by inactivating individual proteins by specific β-lactam antibiotics. E. colisurvives the loss of either PBP 1a or 1b, but inactivation of both proteins results in cell lysis (16, 37, 46). Because each of these proteins is a transglycosylase and transpeptidase, the implication is that these PBPs are required for initiating or continuing the elongation and cross-linking of glycan chains. PBP 2 helps govern cell shape; inactivating this protein causes cells to lose their rod shape and grow as enlarged spheres that eventually die unless compensatory mutations or conditions exist (26, 33, 35). Loss of PBP 3 inhibits cell septation, causing cells to grow as elongated filaments and establishing a central role for PBP 3 in this step of cell division (3, 33).
Seven low-molecular-weight PBPs are now known: PBPs 4, 5, 6, and 7, as well as DacD, AmpC, and AmpH. (Although AmpC and AmpH belong to the family of class C β-lactamases, we will also refer to these two proteins as PBPs because both enzymes bind covalently to at least one radioactively labeled β-lactam, which is the classical definition of a PBP [13].) Although the in vitro biochemical capabilities of these PBPs have been established and studied to various extents, the in vivo functions of these proteins remain mysterious. Any one of these PBPs can be deleted from the chromosome and E. coli will grow normally (1, 4, 9, 12, 13, 23, 24, 32), so that such PBPs are generally described as nonessential. However, many of these proteins have similar or identical enzymatic activities, making it possible that the loss of any one PBP could be masked by the presence of a surrogate PBP. For example, PBPs 4, 5, and 6 and DacD are carboxypeptidases, and PBPs 4 and 7 are endopeptidases (14, 27). Thus, within a particular enzymatic category, one PBP might substitute for the absence of another. If this were the case, then deletion of an individual PBP would not be lethal even though the biochemical activity that it represented might be essential for bacterial survival.
The question of whether several PBPs can substitute for one another can be addressed by creating E. coli strains in which multiple PBP genes have been mutated or deleted. However, only few such strains have been constructed. One of the earliest reports by Suzuki et al. (37) described five multiple mutants that contained combinations of temperature-sensitive (ts) and inactivating mutations (mutated PBP genes are listed in brackets): a double mutant [4 5]; three triple mutants [1a(ts) 4 5], [1b(ts) 4 5], and [3(ts) 4 5]; and one quadruple mutant [1a(ts) 1b(ts) 4 5]. Later, this same laboratory constructed three additional multiple mutants: [1a(ts) 3(ts) 4 5]; [1b(ts)3(ts) 4 5]; and [1a(ts) 1b(ts) 4 5] (40). All the triple and quadruple mutants grew normally at 30°C but died, elongated, or lysed at 42°C, depending on which combination of the three high-molecular-weight PBPs was inactivated. In contrast, the mutant lacking PBPs 4 and 5 was viable at any temperature (37). Other laboratories have also constructed a few multiple mutants: a double mutant lacking PBPs 5 and 6 is viable (4), as is a triple mutant lacking PBPs 4, 5, and 6 (9) and a quadruple mutant lacking PBPs 4, 5, and 6 and DacD (1). In none of these cases was an obvious growth or morphological phenotype associated with the loss of the low-molecular-weight PBPs 4, 5, and 6 and DacD, either individually or in combination.
We have chosen to address the questions of PBP function and substitution in the most direct manner possible, by constructing a set of 192 E. coli strains containing every possible combination of deletions of the following eight PBPs: PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH. The results confirm that from among these proteins, PBPs 2 and 3 plus either PBP 1a or 1b are the only PBPs required for laboratory growth of regularly dividing rod-shaped cells. In addition, several mutants did not lyse when PBPs 2 and 3 were inhibited by specific β-lactam antibiotics, suggesting that the loss of some combination of low-molecular-weight PBPs produces this new phenotype. Finally, this set of E. coli strains supplies genetic backgrounds in which the physiological roles of nonessential PBPs and other peptidoglycan-reactive enzymes may be observed and investigated in greater detail.
Bacteria, plasmids, phage, media, genetic techniques, and gel electrophoresis.Individual PBP genes were deleted initially fromE. coli JC9387 [thr-1 ara-14 leuB6Δ(gpt-proA)62 lacY1 sbcC201 tsx-33 qsr galK2 rac sbcB15 hisG4(Oc) rfbD1 recB21 recC22 rpsL31(Sm)kdgK51 xylA5 mtl-1 argE3(Oc) thi-1) (strain 6613 from the E. coli Genetic Stock Center, Yale University). The parent from which individual and multiple PBP genes were deleted wasE. coli CS109 (W1485 glnV rpoS rph) (from C. Schnaitman). E. coli S17-1λpir (recA thi pro hsdR [res−mod+][RP4::2-Tc::Mu-Km::Tn7] λpir phage lysogen) (20) was the donor for introducing parA-containing plasmids to mutated recipients. Plasmids pJMSB8 (containing a cloned parA gene) and pCK155 (the source of the res-npt-res kanamycin resistance cassette) were obtained from Claus Sternberg (20). The plasmid vector pBCSK− was from Stratagene (La Jolla, Calif.). Bacteriophages λ116, λ142, λ168, λ209, λ349, λ364, λ521, λ522, λ622, and λ650 were from the Kohara E. coli genomic library (18); all were supplied to us by Y. Kohara except for corrected versions of phages λ521 and λ522, provided by F. Blattner. Phage P1 was from laboratory stock, and P1 transductions were performed as described elsewhere (25).
Classical methods were followed for restriction enzyme digestion, ligation, and general cloning procedures (28), and restriction enzymes were from a variety of commercial sources. Southern blot hybridizations were performed as described elsewhere (31). Labeling, separation, and visualization of PBPs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as described previously (11). Bacteria were grown in Luria-Bertani (LB) medium or in M9-minimal glucose medium (25) containing the appropriate antibiotics: ampicillin (100 μg/ml), kanamycin (50 μg/ml), or chloramphenicol (50 μg/ml). Ampicillin was never used in growth media when PBPs were to be assayed. Aztreonam was from E. R. Squibb & Sons (Princeton, N.J.), and mecillinam was from Leo Laboratories Ltd. (Dublin, Ireland). Each of the latter antibiotics was added to log-phase cultures of E. coli to a final concentration of 10 μg/ml. Yeast extract and tryptone were from Difco (Detroit, Mich.). Other chemicals were from Sigma Chemical Co. (St. Louis, Mo.).
Construction of PBP deletion mutants.For each PBP, with the exception of PBPs 5 and 6, a DNA fragment containing the gene of interest was isolated from the appropriate λ phage in the Kohara genomic library and subcloned into the vector pBCSK− (Fig.1). The plasmids were transformed intoE. coli CS109, and overexpression of the PBP encoded by each subclone was confirmed by SDS-PAGE and autoradiography of125I-penicillin X-labeled PBPs. A DNA fragment internal to each gene was removed by cutting the plasmid with selected restriction enzymes, the ends of the plasmid were blunted by incubation with DNA polymerase I Klenow fragment, HindIII linker oligonucleotides were ligated to each end, and the ends were cut withHindIII and ligated to one another to create a cloned sequence containing a deletion in the PBP gene (Fig. 1). In some cases,HindIII and other enzyme recognition sites were removed from plasmids prior to construction of the deletions (not shown). In all cases, enough flanking DNA was present for recombination to occur between the plasmid and chromosomal copies of the genes (described below). Loss of PBP expression from the derived plasmids was confirmed by SDS-PAGE of labeled PBPs.
The cloning scheme described above was not used for creating deletions in the genes for PBP 5 (dacA) and PBP 6 (dacC) because overexpression of these proteins is lethal to E. coli. Therefore, an alternate method was used to construct plasmid-borne deletions of these two genes. For dacA, a 3.2-kb HindIII-SalI DNA fragment from Kohara phage λ168 was subcloned into pBCSK−. TheHindIII site was deleted by opening, blunting, and religating, and a new HindIII site was inserted at theSalI position by opening, blunting, and inserting the appropriate linkers (pSAD488-2). This cloned fragment included the amino terminus of dacA and sequences immediately upstream of the gene. Next, from the same phage, a 2.5-kbKpnI-SmaI DNA fragment was cloned into pBCSK−, and HindIII linkers were inserted into the SmaI site (pSAD485-1). This cloned fragment included the carboxyl terminus of dacA and sequences immediately downstream of the gene. The 2.5-kbHindIII-KpnI carboxyl fragment from pSAD485-1 was moved into the same sites in pSAD488-2 to create, in effect, adacA gene with an internal deletion of theSalI-SmaI DNA fragment (pSAD494-1) (Fig. 1). A deletion in the PBP 6 gene, dacC, was created in the same manner by combining two DNA fragments representing the amino and carboxyl termini of the gene into a single plasmid (pSAD506-6) (Fig.1).
For marking the PBP gene deletions, a kanamycin resistance gene cassette flanked by two res sites was isolated as a 2.0-kbEcoRI DNA fragment from plasmid pCK155 (20). The ends of this DNA were blunt ended with E. coli DNA polymerase Klenow fragment, HindIII linker oligonucleotides were ligated to each end, and the ends were cut withHindIII. This cassette was ligated into theHindIII site of each PBP deletion plasmid, thereby replacing the internal gene sequences denoted by the heavy rectangles in Fig. 1.
PBP deletions were transferred from each plasmid to the chromosome ofE. coli JC9387 by the λ transduction method of Kulakauskas et al. (21). Briefly, E. coli CS109 containing a plasmid with one of the kanamycin-marked gene deletions was infected with the Kohara λ phage which contained a wild-type copy of that particular PBP gene, and the plasmid-borne deletion and resistance marker were transferred by recombination to some of the phage in the resulting lysate. The lysate was used to transfer the PBP gene deletion to the chromosome of E. coli JC9387 by transduction and selection for kanamycin-resistant colonies. Deletion of the appropriate PBPs was confirmed by SDS-PAGE and autoradiography.
Nine mutants were constructed, each of which was missing the gene for one of PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, AmpH, and DacD. In addition, the mgt gene, which encodes monofunctional glycosyltransferase, was deleted from a 10th strain. These individual JC9387-derived mutants were infected with phage P1, and the resulting lysates were used to move each of the kanamycin-marked PBP gene deletions into E. coli CS109 by P1 transduction (25), thus creating a set of 10 single-deletion mutants. Once again, the loss of PBPs from all strains was confirmed by SDS-PAGE and autoradiography or by Southern blotting to detect the loss ofdacD and mgt. In addition, the strains were screened to certify that no auxotrophies were transduced from E. coli JC9387 to E. coli CS109.
We wished to create strains that would eventually contain eight or more gene deletions, but it was not possible to mark each individual gene with a separate antibiotic resistance cassette to produce such combinations. This problem was avoided by combining the resolvase method described by Kristensen et al. (20) with the λ transduction method of Kulakauskas et al. (21). The generalized procedure is outlined schematically in Fig.2. Briefly, a kanamycin-marked PBP gene deletion was moved into the chromosome of E. coli CS109 by P1 transduction, and the kanamycin resistance cassette was excised by transient expression of the RP4 ParA resolvase protein. Transient expression of resolvase was induced by conjugational mating of E. coli S17-1λpir(pJMSB8) to each kanamycin-marked strain. The transmissible plasmid pJMSB8 carries the parAgene under control of the lac promoter and is a suicide plasmid that replicates only in the presence of a λpirlysogen (20). Therefore, resolvase is expressed when the plasmid is transferred by conjugation into an E. colirecipient, but since the plasmid cannot replicate in this host, it is quickly lost as the recipient cell divides (20). In some of the recipients, resolvase excises the kanamycin marker from the site of the PBP deletion by acting on the two res sites that flank the gene cassette (20), leaving a single 8-bp ressequence at the site of the original deletion in the PBP gene. The mating mixture was plated onto M9-minimal glucose medium without kanamycin to select against the multiply auxotrophic donor, E. coli S17-1. E. coli CS109 colonies from these matings were replica plated onto minimal medium with and without kanamycin to identify those colonies that had become kanamycin sensitive, indicating that the resistance cassette had been removed from the chromosome. As before, the PBP profiles of kanamycin-sensitive deletion mutants were confirmed by SDS-PAGE and autoradiography.
Multiple PBP gene deletions were assembled into individual strains by repeated transduction and excision cycles: a kanamycin-marked PBP gene deletion was moved by P1 transduction into a previously constructed kanamycin-sensitive mutant, the newly added kanamycin resistance cassette was excised by resolvase, and the resulting strain was used as a kanamycin-sensitive recipient to which additional PBP gene deletions could be added. These two steps were repeated until the desired combinations of mutations were assembled in individual strains.
Construction of the set of PBP deletion mutants.We constructed a total of 192 different strains that contained every possible combination of deletions involving the following eight PBPs: PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH. The only combinations that could not be produced were those that included deletions in both PBPs 1a and 1b, consistent with previous observations that strains lacking these two PBPs are inviable (16, 37, 46). In addition, thedacD gene was deleted from two strains lacking seven PBPs, and these constructs were confirmed by Southern blotting (data not shown).
Table 1 lists the complete set of mutants and the PBPs that were deleted from each strain. The pathway by which each mutant was derived is displayed as a pedigree chart in Fig.3. The PBP profile of every strain was observed at each step during construction of the mutant set, to confirm that those PBPs that should have been deleted were indeed absent. Figure 4 displays the PBP profiles of eight mutant strains from which a single PBP was deleted (Fig. 4A) and the profiles of a representative subset of mutants from which additional PBPs were deleted, culminating in the two mutants from which seven PBPs were removed (Fig. 4B).
Complications associated with the strain construction procedure.To our knowledge, this set of PBP mutants is the largest group of isogenic bacterial strains containing multiple mutations in a single enzyme family. Because the resolvase procedure of Kristensen et al. (20) promises to be extremely useful for creating multiply mutated strains, it is important to point out two problems that arose during application of the method. The first problem was the perpetuation of a phenotype unrelated to the genes being deleted. In our initial constructions, the ampH gene was deleted from CS109 to create the mutant CS15-1, and this strain served as the parent for over 50 multiply mutated strains. However, we later discovered that CS15-1 had become resistant to infection by phage λ and had passed this characteristic to all of its descendants (data not shown). Therefore, the entire branch of mutants was reconstructed by different routes: ampH was deleted from CS109 to create the new strain CS15-3, and the descendants of CS15-1 were reconstructed by deleting PBP genes from different parental mutants (the corrected lineage is reflected in Fig. 3). Resistance to phage λ disappeared in these new mutants (data not shown). However, there does exist a phage resistance phenotype that appears to be related to PBP genotype and which is spread among many branches of the pedigree tree (45). Thus, it is important that the results of any phenotypic test be compared to the pedigree to help determine if the phenomenon is real or possibly an artifact of the construction pathway.
The second problem that arose during this method of strain construction was that a few mutants acquired an unselected resistance to ampicillin. Although we avoided cloning into vectors containing β-lactamase genes (because expression of the enzyme prevents labeling PBPs with radioactive penicillin-X), six mutants were discovered to be resistant to ampicillin at 100 μg/ml. By using PCR and appropriate primers, we detected a chromosomal copy of the gene encoding TEM β-lactamase in these mutants (data not shown). The gene was most likely acquired during the removal of the res-npt-res cassette, when the ParA resolvase carried by the suicide plasmid pJMSB8 was introduced into the mutants by conjugation. Although the plasmid does not replicate in the absence of the λpir lysogen and is therefore usually lost from the recipients, portions of the plasmid evidently integrated into the chromosome of these six PBP mutants, thereby imparting ampicillin resistance. These strains were replaced by repeating the curing step to eliminate the res-npt-rescassette from the original mutants and by screening to avoid ampicillin-resistant colonies. Fortunately, none of the six mutants had served as the parent for other deletion mutations, and so only these strains needed to be reconstructed. Although ampicillin resistance arose at a low rate (less than 3% of the strains constructed), mutants produced by this procedure should be confirmed to be ampicillin sensitive.
Finally, there are 5,040 different possible pathways by which seven genes can be deleted from a single strain and, therefore, 10,080 pathways by which the two seven-deletion mutants could have been constructed. It is theoretically possible that the order in which genes are deleted can affect the phenotype of the descendent strains. As a preliminary test of this possibility, we constructed six sets of mutants lacking three or four PBPs, the strains in each set differing only in the order in which the same three or four PBPs were deleted (data not shown). We examined these strains for morphological differences, growth characteristics, temperature sensitivity, and antibiotic sensitivity to two β-lactams. There were no obvious phenotypic differences between the duplicated strains in each mutant subset. Nonetheless, it should be remembered that it is possible that deletion order effects exist for traits not yet tested.
Mutants lacking multiple PBPs are viable.The most obvious result issuing from these mutants is that all were viable. Growth curves of mutants in LB medium at 37°C, including those of mutants from which seven or eight PBPs were deleted (representative data is shown in Fig. 5and6), were similar to the wild-type growth curves. Thus, the growth kinetics of E. coli were nearly normal if the following high-molecular-weight PBPs were present: either PBP 1a or 1b, and both of PBPs 2 and 3. Thus, for this particular set of proteins, the low-molecular-weight PBPs 4, 5, 6, and 7, AmpC, and AmpH, either individually or in any combination, were nonessential for growth of E. coli in a rich laboratory medium. In addition, the dacD gene was deleted from strains CS701-1 and CS702-1 to create two mutants that lacked eight different PBP genes (strains CS801-4 and CS802-2, respectively) (Table 1 and Fig. 3). These three octuple mutants were also viable, indicating that DacD was not essential even in the absence of all other low-molecular-weight PBPs.
Inactivation of PBP 2 induces lysis in mutants lacking PBP 1b.The β-lactam mecillinam binds to and inactivates PBP 2, inhibiting cell wall elongation so that each E. coli cell grows as a continually enlarging sphere (10, 39). The absorbance of a mecillinam-treated culture continues to increase at a logarithmic rate, but the cells eventually stop growing unless there are compensatory mutations in aroK or cya (26, 43) or unless ppGpp or the FtsZ protein are overexpressed (42, 44). Thus, in the absence of PBP 2, peptidoglycan synthesis continues at approximately the wild-type rate but overall cell shape is altered.
We tested to see if any low-molecular-weight PBPs were required for peptidoglycan synthesis in the absence of PBP 2 by adding mecillinam to log-phase cultures of each of the members of the PBP deletion set. Wild-type E. coli and all mutants that retained active PBP 1b continued to enlarge normally, without lysis, in the presence of mecillinam (data not shown). The extreme example was that of E. coli CS801-4, which exhibited a normal response to mecillinam even though eight PBPs were deleted from this mutant (Table 1), leaving only four active PBPs (1b, 1c, 2, and 3). Thus, when PBP 2 was inactivated in these multiple mutants, the three remaining active PBPs (1b, 1c, and 3) could still synthesize osmotically stable peptidoglycan in the shape of a sphere.
In contrast, deletion of PBP 1b alone was sufficient to make E. coli sensitive to lysis by mecillinam (Fig. 5A), as del Portillo and de Pedro reported for a single mutant (6, 7). In addition, mecillinam induced lysis in each of the 64 mutants from which PBP 1b had been deleted (see Fig. 5 for examples of this effect). In most of these Δ1b mutants, lysis began ∼20 min after addition of the antibiotic and proceeded at a rate (having a negative slope) that was approximately equal to or slightly slower than the original growth rate. The results establish that the synthetic activity of PBP 1b does not require the aid of any of the low-molecular-weight PBPs and that in the absence of PBP 1b, PBP 2 becomes essential for synthesis of intact, osmotically protective peptidoglycan, regardless of the activity of any low-molecular-weight PBPs.
Deletion of PBP 5 sensitizes Δ1b mutants to inactivation of PBP 2.Although all Δ1b mutants lysed when exposed to mecillinam, the time of lysis onset and its rate were accelerated in strains from which PBP 5 was also deleted. For example, the onset of mecillinam-induced lysis was decreased from 20 min in CS16-1 (Δ1b) to about 10 min in CS224-2 (Δ1b 5), and the ensuing lysis rate was more rapid in the double mutant (Fig. 5A). This trend was also exhibited by mutants from which multiple PBPs had been deleted. As a general rule, mutants lacking both PBPs 1b and 5 lysed more quickly and faster (e.g., Fig. 5B). However, some mutants lacking four or more PBPs lysed so quickly that removing PBP 5 reduced the time of lysis onset only slightly or not at all (e.g., Fig. 5C). Even so, in general, Δ[1b 5] mutant combinations lysed earlier and more rapidly than did their isogenic Δ1b relatives containing wild-type PBP 5. These observations indicate that active PBP 5 moderates the mecillinam sensitivity of Δ1b mutants.
Inactivation of PBP 3 induces lysis in mutants lacking PBP 1b.Aztreonam is a β-lactam that inactivates PBP 3, thereby inhibiting septation so that each E. coli cell grows as a continually elongating, nonsegmented filament (38). The absorbance, but not cell number, of an aztreonam-treated culture continues to increase at a logarithmic rate. Schmidt et al. (30) and del Portillo and de Pedro (6) reported that inactivation of PBP 3 lyses an E. coli strain which contains a temperature-sensitive mutation in PBP 1b. We confirmed and extended these results by adding aztreonam to the entire set of PBP mutants. The only strains that lysed were those 64 from which PBP 1b had been deleted; all other strains in which PBP 1b remained active continued to grow and form filaments when PBP 3 was inactivated. Once again, the extreme example was that ofE. coli CS801-4, which exhibited a normal response to aztreonam even though eight PBPs were missing, leaving PBPs 1b, 1c, 2, and 3 active. Thus, even when PBP 3 was inactivated, PBPs 1b, 1c, and 2 could synthesize osmotically stable peptidoglycan in the shape of an elongated filament. As with the experiments in which PBP 2 was inactivated, these results establish that the synthetic activity of PBP 1b does not require the aid of any of the low-molecular-weight PBPs. In addition, in the absence of PBP 1b, PBP 3 becomes essential for synthesis of intact, osmotically protective peptidoglycan, regardless of the presence or absence of any of the low-molecular-weight PBPs.
Synthesis of osmotically stable peptidoglycan when PBPs 2 and 3 are inactivated.Simultaneous inactivation of PBPs 2 and 3 lysesE. coli (10, 30). This result has been interpreted as evidence that at least one or the other of PBPs 2 and 3 is required for continued peptidoglycan synthesis (10). To see if this observation was true for the set of PBP mutants, we grew individual strains in 2-ml aliquots of LB broth and tested for lysis by adding mecillinam and aztreonam to inactivate PBPs 2 and 3, respectively. All mutants lacking PBP 1b lysed (data not shown), consistent with previous observations that these strains were lysed by either antibiotic alone.
Fifteen strains that demonstrated resistance to lysis in 2-ml cultures were tested by growing them in a large volume of medium in well-aerated flasks, followed by addition of the two antibiotics. Several of these mutants did not lyse. Instead, one of three reactions occurred: growth of some strains leveled off without lysis; in other strains, lysis was delayed significantly and its rate was reduced; and the absorbance of a few strains continued to increase after addition of the two antibiotics (data not shown). When mecillinam or aztreonam was added individually to these strains, the cells became spherical or filamentous due to inactivation of PBP 2 or 3 (data not shown). This result proved that when administered individually, the antibiotics inactivated their respective targets. Nonetheless, some of the PBP mutants had become resistant to the lytic effect that normally accompanies exposure to both antibiotics.
Two examples of mutants that did not lyse after inactivation of PBPs 2 and 3 were strains CS403-3 (lacking PBPs 1a, 4, and 7, and AmpH) and CS801-4 (lacking PBPs 1a, 4, 5, 6, and 7, AmpC, AmpH, and DacD) (Fig.6). These strains lacked PBP 1a and three or seven other low-molecular-weight PBPs, respectively. Thus, after PBPs 2 and 3 were inactivated in strain CS801-4, peptidoglycan was synthesized in the absence of 10 PBPs, and the structure of this peptidoglycan was sufficiently intact to prevent osmotic lysis in LB medium.
The microscopic appearance of strain CS403-3 was typical of the morphological responses of mutants that were resistant to the lytic effects of simultaneous addition of mecillinam and aztreonam (Fig.7). Log-phase cells looked quite normal before exposure to the antibiotics (Fig. 7, time zero). However, as soon as 30 min after addition of both antibiotics, the mutants ceased dividing and began to expand (Fig. 7, 30 min). Cells at this early stage often had a ring or bulge around their midpoints, suggesting an inability to produce a normal septum (Fig. 7B, 30 and 60 min). After 120 min, almost all cells were spherical or nearly so, and these spheres continued to grow and enlarge until the diameter of individual cells reached 5 to 7 μm (Fig. 7, 120 and 200 min). Many of these huge cells contained phase-lucent vesicles of unknown origin and composition (Fig. 7, 200 min). In addition, the enlarging spherical cells often retained pairs of short protuberances, probably representing the original cell poles which are known to be inert to insertion of new peptidoglycan. Also observed in increasing numbers over time were ghost sacculi (peptidoglycan shells emptied of their cytoplasmic contents) (Fig. 7, 200 min). This latter observation is consistent with the fact that these mutants eventually stopped growing and did lyse, given enough time, even though the strains were resistant to the immediate lytic effect of losing PBPs 2 and 3.
In summary, some mutants lacking multiple PBPs did not lyse in the absence of functional PBPs 2 and 3. Instead, individual cells continued to metabolize and enlarge, although they were unable to divide.
In 1978, Suzuki et al. suggested that it "might be the right time to construct a series of relevant mutants by combining these [PBP] mutations to dissect the roles of the binding proteins on cell growth, division, and penicillin-sensitive enzyme systems" (37). Their suggestion notwithstanding, only a handful of multiple mutants were constructed in the ensuing 20 years, and we believe that the absence of such a comprehensive set of mutants has delayed our understanding of the biological roles of individual PBPs. The mutants described in this work supply the genetic backgrounds in which the functions of this set of enzymes may be defined more exactly. Furthermore, preliminary examination of the phenotypes exhibited by these mutants places new constraints on current theories of peptidoglycan biosynthesis and suggests some novel ideas about howE. coli synthesizes its protective shell.
Preliminary caveats.Before considering the ramifications of these results, we wish to emphasize some cautionary considerations. The foremost of these is that in the 192 strains lacking from one to seven PBPs, several peptidoglycan-specific enzymes remain active: DacD, a recently identified carboxypeptidase (1); PBP 1c, a newly discovered high-molecular-weight PBP (29); MepA, an endopeptidase similar to PBPs 4 and 7 (17); Mgt, a monofunctional transglycosylase with similarities to portions of PBPs 1a and 1b (8); all of the lytic transglycosylases (14); a penicillin-insensitiveld-carboxypeptidase (41); and a putativeld-transpeptidase (5). It is theoretically possible that one or more of these active enzymes can compensate for the loss of the inactived PBPs. However, the results reported here seem to decrease the likelihood that E. coli remains viable by a simple substitution of one PBP for another.
A second caveat is that during strain construction, some PBP mutants may compensate for the loss of one or more PBPs by accumulating cryptic adaptations. Such compensations could take the form of secondary mutations that relieve deleterious effects created by the absence of PBPs. We have no evidence of such mutations, and the high frequency of successful P1 transductions from one strain to another argues against the occurrence of such mutations, but the theoretical possibility should be kept in mind.
A third consideration is that during construction of the mutants, a strain might develop a phenotype unrelated to PBP loss. So far, we have observed only one case of this: one mutant became spontaneously resistant to phage λ and passed this resistance to all strains derived from it. We eliminated this particular problem by reconstructing the mutants from unaffected parental strains. However, other unknown mutations may have accumulated, affecting traits that we did not measure. Those who use these strains in the future should be aware that the best method for detecting such unrelated hereditary mutations is to compare newly measured phenotypes to the family tree and treating with some suspicion those characteristics exhibited by all descendants of a single strain.
Nonessential PBPs.The most surprising result was that all the PBP mutants, including those mutants from which eight PBPs were deleted, survived and grew nearly as well as the parental E. coli strain. A long-standing argument is that inactivation or loss of one of the low-molecular-weight PBPs results in no phenotype because several proteins have similar functions and the remaining enzymes can substitute for one another. Based on this assumption, our original hypothesis was that sequential deletion of these PBPs would eventually yield a mutant in which one or more of the enzymes became essential for viability. The expectation was that at some point a multiply mutated strain would possess only one member of a particular substitution family. Instead, at least 8 of the 12 known PBPs could be eliminated with only slight morphological effects to untreated E. coli. Among these strains are mutants which grew in the absence of every known carboxypeptidase (PBPs 4, 5, and 6, and DacD), in the absence of two of the three known endopeptidases (PBPs 4 and 7), in the absence of both class C β-lactamases (AmpC and AmpH), and in the absence of all seven of these low-molecular-weight PBPs.
The fact that E. coli remains viable in the absence of so many PBPs raises the question of the normal physiological roles of these enzymes. There are at least three possible explanations for why no phenotype is associated with multiple mutations in the low-molecular-weight PBPs. First, these proteins may be completely dispensable for bacterial viability; second, another peptidoglycan-reactive enzyme may compensate for the loss of these PBPs; and third, the-low-molecular weight PBPs may not be essential for laboratory growth of E. coli but might, instead, affect bacterial viability or physiology under conditions not yet tested. We are currently screening the mutant set for phenotypes that might be expected to arise should PBP loss affect some aspect of the structure or function of peptidoglycan.
Interactions between high- and low-molecular-weight PBPs.One of the unanswered questions about cell wall growth involves how the PBPs work together to synthesize functional peptidoglycan. We would like to know not only which PBPs are essential but which ones interact and how. In particular, one unknown is how the low-molecular-weight PBPs might influence interactions among the high-molecular-weight PBPs. To determine if there existed any such relationships, we inactivated PBPs 2 and/or 3 in the set of PBP mutants.
First, we reconfirmed the observations of del Portillo and de Pedro (6, 7) and of Schmidt et al. (30) that inactivation of either PBP 2 or PBP 3 lyses Δ1b mutants but not Δ1a mutants. Furthermore, we extended these observations to include 64 different Δ1a mutants and 64 different Δ1b mutants, in which up to six other PBPs were deleted in all possible combinations. The results establish that neither the lysis susceptibility of Δ1b mutants nor the lysis resistance of Δ1a mutants depends on the activity of the low-molecular-weight PBPs. Functional peptidoglycan is synthesized if PBPs 1b and 2 are active (in Δ1a mutants treated with aztreonam) or if PBPs 1b and 3 are active (in Δ1a mutants treated with mecillinam), regardless of the presence or absence of up to seven other PBPs. Thus, peptidoglycan synthesis by PBP 1b (possibly aided by PBP 1c) requires either one or the other of PBPs 2 and 3, and neither PBP 1a nor PBP 1b requires the low-molecular-weight PBPs.
Even though the low-molecular-weight PBPs did not affect the growth of cells with active high-molecular-weight PBPs, the smaller PBPs may play a role in cell lysis induced by inactivation of PBPs 2 and 3. A form of osmotically stable peptidoglycan was synthesized by some Δ1a PBP mutants lacking up to eight PBPs and in which PBPs 2 and 3 were inactivated by antibiotics. In one mutant, a form of peptidoglycan was synthesized in the absence of 10 of the 12 known PBPs. In making this statement, we draw a distinction between the synthesis of peptidoglycan that has the size and shape of a normal bacterium and the synthesis of peptidoglycan that has an abnormal size or shape but which serves to protect the cytoplasm from rupture by osmotic pressure in LB medium. Clearly, PBPs 2 and 3 are essential in the sense that without themE. coli grows incorrectly and fails to divide normally. Nonetheless, in some genetic backgrounds, PBPs 2 and 3 are not essential for synthesizing peptidoglycan that is at least partially osmotically resistant, in that cells continue to grow and enlarge in a rich medium. In the extreme case of E. coli CS801-4, from which eight PBPs are missing, the only known peptidoglycan synthetic enzymes that remain are PBP 1b, PBP 1c, and Mgt. The conclusion is that protective, presumably adequately cross-linked peptidoglycan can be synthesized either by PBP 1b alone or by PBP 1b in conjunction with PBP 1c and/or Mgt. Little is known about PBP 1c (29) or Mgt (8, 36), but because PBP 1b is the major peptidoglycan synthetic enzyme in E. coli (19), PBP 1b by itself may synthesize an osmotically stable sacculus.
Implications for the multienzyme complex hypothesis of peptidoglycan synthesis.The results reported here place constraints on models of peptidoglycan synthesis. For example, the multienzyme hypothesis predicts that a complex of several different proteins executes a set of tightly orchestrated reactions to synthesize and insert new peptidoglycan strands into the older peptidoglycan of the intact sacculus (14, 15). In this scheme, an individual complex would include, at the very least, a synthetic transglycosylase (PBP 1a, 1b, or 1c), a synthetic transpeptidase (PBP 1a, 1b, 1c, 2, or 3), a lytic transglycosylase, and an endopeptidase (either PBP 4 or 7 or MepA) (14, 15). However, some of the mutants described here can synthesize a partially functional form of peptidoglycan in the absence of up to eight PBPs and when PBPs 2 and/or 3 are inactive. This means that some form of peptidoglycan, perhaps simplified in structure, can be synthesized by some combination of PBP 1b, PBP 1c, and Mgt (supplying transglycosylase and transpeptidase functions), MepA (the only known endopeptidase still remaining), any of the lytic transglycosylases, and, perhaps, a penicillin-insensitiveld-carboxypeptidase or ld-transpeptidase. A mutant lacking three of the five known lytic transglycosylases is viable (22), leaving only two other possible enzymes to fulfill this hypothesized role in the multienzyme complex. Thus, if a multienzyme complex is responsible for peptidoglycan synthesis, then in the mutants lacking eight PBPs there are only a limited number of potential combinations that remain. Perhaps a multienzyme complex provides specialized functions beyond the most basic requirements for peptidoglycan synthesis. In any case, the multiply mutated strains described in this report should make it easier to ask questions regarding these possibilities.
Implications for the dd-carboxypeptidase-dependent hypothesis.Mutants lacking PBP 1b lyse when PBP 2 is inactivated by mecillinam, meaning that cells containing only PBPs 1a and 3 (of the classic PBPs) are unable to survive. We found that if PBP 5 is also deleted, then such Δ[1b 5] double mutants lyse even more rapidly. The conclusion is that in the absence of PBP 5, PBPs 1a and 3 cope even less well after inactivation of PBP 2. This result is consistent with the hypothesis that dd-carboxypeptidase activity may be required to supply PBP 3 with substrate (2). This "dd--carboxypeptidase-dependent" hypothesis would predict that when PBP 5 is absent trimers are less available as substrates for PBP 3, resulting in poorer survival (quicker lysis) when PBP 2 is inactivated. However, if a dd-carboxypeptidase is absolutely essential for the activity of PBP 2 or 3, it is not at all clear how E. coli can survive and septate in the absence of all the known dd-carboxypeptidases (as in CS801-4 and CS802-2, which lack PBPs 4, 5, 6, and 7 and DacD). Therefore, although some of the results presented here are consistent with thedd-carboxypeptidase-dependent hypothesis (2), other results call into question the necessity of having anydd-carboxypeptidases at all. (It should be noted that our results say nothing about the contribution of anyld-carboxypeptidases.)
Implications for evolution of the peptidoglycan sacculus.It is difficult to envision a free-living bacterium without a cell wall because the peptidoglycan sacculus protects these cells from osmotic lysis in hypotonic environments. Therefore, protobacteria must have acquired, very rapidly, some means of osmotic protection. It is impossible to believe that the first cell walls required the organized activity of multiple enzymes. A more likely possibility is that a single enzyme evolved to fill this function and then, over time, additional enzymes were added to the wall-producing pathway. In this scenario, something as complex as a multienzyme peptidoglycan synthesis apparatus would have to be of relatively recent origin.
If peptidoglycan or some protopeptidoglycan structure were synthesized by a single enzyme, then there might be remnants of that mechanism in present-day bacteria. By removing or inactivating 10 of 12 PBPs fromE. coli, we have approached a situation in which a single enzyme might be synthesizing osmotically resistant peptidoglycan. The best candidate for an enzyme with this capability is PBP 1b. As discussed earlier, we cannot rule out the possibility that PBP 1b is still participating in a multienzyme complex, and there is no evidence that E. coli itself should retain such a primordial enzyme. Nonetheless, we should seriously entertain the idea that the basic requirements for peptidoglycan synthesis are simpler than usually believed.
Implications for physiological studies based on completed genomic sequences.We have entered an era in which we have more genetic information about many organisms than we have physiological information. For the organisms whose genomes are now completely sequenced, including even such a well-studied organism as E. coli, large numbers of sequences have no known genetic counterparts and many that do have counterparts have no established function. The most popular approach suggested for study of these myriad unidentified open reading frames is to mutate them individually and screen the resulting mutants for measurable phenotypes. It is likely that functions will be assigned to at least some unknown genes by using this approach. However, the results presented in this report suggest caution about what to expect from a strategy that uses mutation of individual genes.
We created a set of mutants in a group of eight genes that belong to a single related family. Despite knowing the general purpose and biochemical capabilities of members of the family, and therefore knowing what to look for, we observed no obvious phenotypes for most mutants. If this is the situation for a family of genes for which we have so much physiological and biochemical data, then it is likely to be true for other gene families for which we have much less information. Creation of strain sets lacking many genes, though tedious, may need to be the strategy of choice to yield clues about the functions of unknown genes.
Potential uses for the set of PBP mutants.Finally, among many potential applications of the set of PBP mutants are three that should be particularly useful. First, selected mutants can be used as starting points for asking questions about the physiological functions of other enzymes, such as the lytic transglycosylases or non-PBP endopeptidases. In particular, questions about specific theories of peptidoglycan synthesis can be approached which would otherwise be difficult or impossible to address. Second, the physiological or biochemical functions of any two of the eight enzymes can be compared by examining individual strains from which competing PBPs have been deleted and which differ only in possession of the two PBPs of interest. This should be valuable in testing the roles of the various subfamilies of PBPs. Finally, some of the mutants may make good hosts from which to purify cloned PBPs. This will be especially useful for purifying PBPs that require an affinity chromatography step which might otherwise retain contaminating PBPs.
The construction of some of the mutants described in this report was supported by a grant from SmithKline Beecham Pharmaceuticals.
We are deeply grateful to David Knowles and David Payne for their support and interest in the work. We especially thank Joachim-Volker Höltje for helpful discussions and comments on a previous version of the manuscript and for bringing to our attention the presence of TEM β-lactamase sequences in one of the original mutants. Finally, we thank Heather Skarhus and Hugh Nguyen for technical help, Bernadette Meberg for reformatting Fig. 1, and Victoria Swift for graphics assistance.
- Received 25 February 1999.
- Accepted 27 April 1999.
- Copyright © 1999 American Society for Microbiology


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I'm really impressed by how chef and Momofuku restauranteur David Chang has been able to build not just a restaurant empire but an audience to push his unique point of view.
That audience has gotten him a show on Netflix, Ugly Delicious. It's launching later this February. Check out the trailer.


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|Handbasket at a diner - lineart
I've procrastinated enough to put this up, although I drew this months ago. Around February or March. Never got around coloring it (actually started, and hated it and sank in to a slump). I draw these guys every now and then, mostly out of nostalgia.
These teenage-jerk-bags found a high school band called the Handbasket. If I ever made a comic of them, it'd be pretty much be about their retarded teenage-adventures. And the other two band-members (Sylvian and Leo) putting up with their megalomaniac pseudo-feminine vocalist (Deeca) who wants to make it big in the music business some day.
Not sure if I'll ever get around to making a comic about them. Problems being I'm lazy and suck at writing scripts.
|First rough sketch of Deeca's face for the "Diner" picture
|Expression sheet: Deeca
|Expression sheet: Sylvian
Unfortunately my slump hit me around when I was going to do an expression sheet of Leo. OH WELL.


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National Jr. Honor Society
The National Junior Honor Society chapter of Highland Park Middle School is a chartered and affiliated chapter of the national organization. Selection to the HPMS chapter is based on five criteria: scholarship (GPA of 3.60 or higher), leadership, service, citizenship, and character.
The deadline for the NJHS application was 3/21/14
We need the induction form (this is also the indication that the student has bee accepted into the NJHS) returned by 4/14/14
Induction ceremony will held on 4/17/14 6:30 in the auditorium after teacher conferences.


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C is for Characters!
What do you like to see most in characters that you read? For me, I like characters who do things. I don't have much patience for those character-types that spend all their time weighing options and never making any decisions. And I think, unfortunately, that this is probably my number one issue with a lot of books I read and try to like, but end up being annoyed with.
I also think this is one of the big reasons I enjoy Meg Rosoff's writing so much. Her characters are thrown into situations where they have to make choices and get things done. And they do. Daisy, in How I Live Now, for instance, could have spent an entire book series debating whether she should actually love Edmond. But she doesn't. She loves him, lets the audience know it, and then gets on with more important matters. And I love her because of it!
So has this influenced my writing? Definitely. I can't stand flip-floppers, so I don't write them. That's not to say that I haven't written characters who have trouble making decisions, or that I haven't written characters that annoy me(!), but I certainly keep in mind the thing that turns me away from reading a book, and make sure I avoid it as much as possible with my own characters.
What about you?


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Tooth Extractions
Get Out Of Pain & Get Back to Smiling
A tooth extraction is a restorative dental procedure that removes a tooth from its socket in the bone. Island Coast Dentistry takes every precaution to ensure that our patients' extraction process is as painless and pleasant as possible.
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If a tooth has been broken or damaged by decay, Dr. Skupny will try to fix it with a filling, crown or other treatment. Sometimes, however, there may be too much damage for the tooth to be repaired. This is the most common reason for removing a tooth.
Other Reasons for Extractions
The wisdom teeth, or third molars, are often extracted either before or after they come in, typically during the late teens or early 20's. These teeth often get stuck in the jaw (impacted) and do not grow in properly. They also need to be removed if they are decayed or cause pain. Some wisdom teeth can become blocked by other teeth or may not have enough room to come in completely. This can irritate the gums, causing pain and swelling.
People getting braces may need teeth extracted to create room for the teeth that are being moved into place.
Contact our Naples dentist at Island Coast Dentistry to schedule an appointment so we can take care of you or your child's problem tooth.


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Your Taxes: Next Steps for Forms 1095-A and 1095-B
If you were enrolled in health coverage through Maryland Health Connection during 2017, you will be filing your federal taxes using form 1095-A or 1095-B.
Both 1095-A and 1095-B forms will be mailed by Jan. 31. Only form 1095-A will be available in online accounts.
What is Form 1095-A?
Form 1095-A is an IRS tax form that serves as proof that you and members of your family met the requirement under the Affordable Care Act to have health insurance coverage. You will receive Form 1095-A if you were enrolled in a qualified health plan through Maryland Health Connection.
Do I use Form 1095-A if I received financial help?
If you received financial help with your plan, you will use this form to complete IRS Form 8962 (Premium Tax Credit). The information from these forms helps make sure you received the right amount of tax credit for your health insurance plan. Important: if your income changed during 2017, report this information to Maryland Health Connection immediately. If you received more credits than your household was eligible for, you may be required to pay back some of the credits you received. If you received less, this amount may be refunded to you or used to reduce any other income tax you might owe. Remember, you must reconcile your tax credits each year to be eligible to receive tax credits the following year.
What is Form 1095-B?
Form 1095-B is an IRS tax form that proves you or a member of your household received Medicaid or Maryland Children's Health Program (MCHP) health coverage during the previous year. In this case, your form will reflect year 2017.
What do I do with these forms?
Validate the information on the form. Check the names of household members enrolled, their information and dates they had health coverage. If you think the information shown on your form is wrong (or if you need another copy of the form), call Maryland Health Connection at 1-855-642-8572 (Deaf and hard of hearing use Relay service).
Someone in my household had coverage through an employer.
If anyone in your household was enrolled in health coverage through their employer, you will receive a separate Form 1095-C with instructions.
I want to see if I can get free help filing my taxes.
For more information about the Volunteer Income Tax Assistance Program (VITA), visit https://www.irs.gov/individuals/free-tax-return-preparation-for-you-by-volunteers. From this website, you can enter your zip code to find a VITA office near you. You can also find more information, FAQs and helpful guides now at https://www.marylandhealthconnection.gov/get-answers/health-coverage-and-your-taxes/
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