text:
I'm going to admit to a terrible truth. I have spent the past week watching every last episode of The Real Housewives of New Jersey and DC. It started innocently enough. I was surfing the web late one night when, crippled with insomnia, I started craving a dose of crap Internet television to aid me in my quest to slumber. Soon what started as a minor curiosity spiraled into a majorly obsessive thirty-hour binge fiesta.
Thirty hours, people! That's nearly a full-time job.
What happens to an insomniac working mother of a toddler after she's spent that amount of time with the Housewives? She begins to evaluate her own conflict management skills.
I've wanted to write a post about how to fight fair when your toddler is looking on for quite some time. The problem is, my husband and I can be equally hopeless when it comes to re-enacting the sort of positive communication we've both read so much about. Rather, we occasionally reenact scenarios more likely to be seen on Bravo.
Not the sort of example I want to set for my 21-month-old son. In existential terms, he gets more or less the same couch potato treatment from watching us mime "communication" that I get from witnessing "reality" on television. Therefore, the best way for me to justify my television sabbatical with the Salahis and Menzos is to think of it as an intensive communication effectiveness seminar.
Here are my top three takeaway lessons.
Lesson 1: Whenever Possible, Restrict Battles to Banter
Let's face it, there are times that we find it … challenging … to communicate effectively with our partner or husband, particularly when he or she does things that annoy you. As such, when your audience (that would be your toddler) is in view, do your very best to battle out marital issues with wit, dignity and, whenever possible, British sarcasm, like Cat.
Lesson 2: Before You Deflect, Use Caution
Deflecting emotions before being fully self-expressed can result in a catastrophe of Danielle Staub like proportions. We all need to let off emotional steam every now and then. Show your kid that it's normal to vent. After that, deflect at will with something lighthearted, like ham throwing.
Lesson 3: If Everything is Not Okay, Don't Smile Maniacally Like It Is
Famed party crasher Michaele Salahi could smile her way through a nuclear war. It's weird, and sends the wrong message to everyone around you. If you're trying to gracefully bow out of an argument, sensitive legal situation, or publicly humiliating grocery store meltdown, smiling like you're on candid camera is sadistic, strange, and confusing for partners, viewers, and toddlers alike.
What other lessons might reality television teach us when it comes to fighting fair in front of toddlers? Maybe no matter how bad things get, never resort to this:
All images via Bravo


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More Interesting Facts
Almond Milk
Basmati Rice
Brewers Yeast
Brown Rice
Celiac Disease
Flax Seed
Gluten Free Diet
Lactose Intolerance
Meat Substitutes
Nutritional Yeast
Organic Foods
Raw Food Diet
Soy Milk
Soy Protein Isolate
Sticky Rice
Wild Rice
Horchata or
orxata is the name for several kinds of vegetable
beverages, made of ground almonds, rice, barley or
tigernuts (chufas).
The name
comes from Valencian orxata, probably from ordiata, made
from ordi (barley) (Latin *hordeata/hordeum). The French
and English 'orgeat', the Italian 'orzata', and the
Surinamese Dutch orgeade have the same origin, though
the beverages themselves have diverged, and none of them
are typically made from barley anymore.
to a folk etymology, James I of Aragon was offered a
glass of the beverage by an Arab girl after his conquest
of Valencia, and exclaimed, Aixo es or, xata! (This is
gold, girl!).
In Spain, it usually
refers to orxata de xufes (horchata de chufas), made
from tigernuts, water and sugar. Originally from
Valencia, it is served ice cold as a refreshment. It has
a regulating council[2] to ensure the quality of the
product and the villages where it can come from, with
the Denomination of Origin. The village of Alboraia is
well known for the quality of their horchatas. The idea
of making horchata from tigernuts comes from the period
of Muslim presence in Valencia (from the 8th to 13th
Latin America
In Mexican cuisine,
horchata is a rice based beverage. While the drink is
usually white and "milky" it can be made dairy-free
through the use of blanched almonds, though some recipes
call for milk. Other ingredients often include sugar,
cinnamon, and vanilla, orange or lime. Though horchata
was once typically homemade, it is now available in both
ready-to-drink (shelf-stable or refrigerated) and
powdered form in grocery stores. In the US, rice-based
horchata is served in some Mexican restaurants, and the
horchata de chufas is virtually unknown.
The horchata
found in Ecuador is similar to the Mexican kind, but
sesame seeds are used instead of almonds. In El
Salvador, horchata is typically flavored with ground
cocoa and cinnamon as well as sesame seeds, and in some
cases is strained; this style is served in Salvadorean
restaurants, particularly in the Washington, D.C. metro
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Medical questions & health advice by board certified doctors
"What does it mean if your hair hurts?"
I'm 22 years old and male and my hair hurts. It hurts like right by where it comes out of my scalp. I do wear a hat a lot, but never had this problem before. What would make someone's hair hurt like this?
It is really common to have sensitivity in the hair shafts from wearing a hat all of the time. This is because the pressure from the hat tends to bend down the hair shafts, creating tension at the base which can lead to sensitivity and pain. I would suggest that first you stop wearing the hat for a few weeks to see if that clears up the problem. If so, then there is no need to worry further. Another common cause of pain in this area is exposure to chemicals, especially hair bleaches and dyes. If you use these products, you may also wish to discontinue them to see if it makes a difference. On the other hand, if the pain continues, then you may want to see your doctor. There are several other conditions of the scalp that can lead to pain and discomfort or sensitivity. One of the most common of these is seborrheic dermatitis. This is an inflammatory condition of the scalp that leads to redness, dryness, itching, and flaking or scaling (dandruff is a very mild form of seborrheic dermatitis). The condition can be easily diagnosed by your primary care doctor and, if present, there are very effective treatments that are available.
Need more info?See a primary care-doctor today
Zocdoc Answers is for general informational purposes only and is not a substitute for professional medical advice. If you think you may have a medical emergency, call your doctor (in the United States) 911 immediately. Always seek the advice of your doctor before starting or changing treatment. Medical professionals who provide responses to health-related questions are intended third party beneficiaries with certain rights under Zocdoc's Terms of Service.


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Every week there will be three new Rush Hour puzzles.
This Rush Hour applet was written by William Shubert. The original Rush Hour puzzle is published by Binary Arts. Please visit their web site for more information!
This site is featured in Puzzles.com. Puzzles.com is a premier puzzle website about puzzles and optical illusions, magic and origami, scientific toys and math diversions, other things that involve intellectual recreations.
If you can't use the applet, but want the three new puzzles every week, they are available in an all text format also.


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So that is what this process is all about, the tearing down of everything old and misconstructed, and the rebuilding of the most solid foundation of them all. Namely that fact that you are indeed sovereign beings, complete and perfect, and ready to start to go out in the world as such. And when you do, you will start to interact with all of these other, new rock-solid beings that surround you, in the shape of your fellow men and women. And together, you will build the world of your dreams. But again, that dream must start at home as it were, with the complete dismantling of anything old and substandard that may be present inside, for then, to put the good pieces back together and build it into this rock-solid and immortal being you are all starting to emerge as. For what we have talked about today is not news to any of you, for you are not novices in this. For you have been in this construction business for a very long time now, and now, the time has come to survey this magnificent structure you are about to complete. It can be difficult at times in midst of all of the seeming chaos to see just how powerful a structure this really is, and just as it is on a regular construction site, without surveying the blueprint, it can be very difficult to get a clear image of what is under way in this confusing mass of busy workers. But we have the blueprints, dear ones, and we can see just how faithful you all are in following them, and so we can see clearly just how close you are to putting that final capstone into place, and declare this, the work of re-constructing yourselves, as finished.
It may not be all of the comfort you need at times like this, when tempers fly and irritation can become high in this, the final flurry of finishing touches before the unveiling. But trust us when we say that even if you might feel a bit stressed out now and then, and you feel as distant from the finished product as ever, know that this is not the case. For you are not lagging behind, not in any way, and you will all make the deadline you have set for yourselves for this process. So fret not, even if you at times may feel like your whole structure is tethering on the brink of collapse. For your building is indeed build on solid ground this time, not on the loose and treacherous sands of yesteryear, and as such, it can weather anything that maybe waiting ahead. We do not say this to imply any sort of cataclysmic event, we just say this in order to remind you that you are build of sturdy stuff now, and so come rain or shine, storm or quiet, you will be standing tall and proud and rightly so.
For what you have done, is to build something that will last and last, and when you get to know this new you better, you will also see the bright future that you have in store. Not just as brilliant individuals, but as this whole beautiful conglomerate of magnificent structures, all linked together in a way that will make everything else pale in comparison. For this is no fata morgana, this is the true paradise you are building. For as you struggle and push to get those final pieces in place in your own personal setup, you are also simultaneously doing the same to this whole planet. For this is what dreams are made of. Not mere fiction, but the facts that you have already started to make it come true by making yourselves become true. The true humans, based on their true conviction that what you have become, will never be torn down again, and as such, paradise is already rising from the ashes of the old. And you are the ones making this happen, by doing the hard work of rebuilding your own personal being from the ground up. And now, you can all feel dizzy at times as you have started to raise this new you to such lofty heights, you are already towering above the old renamnts of the one you used to be. There is no need to look down now, dear ones, just keep your focus up, and you will have a better chance to finish this whole process off without being too overwhelmed by vertigo.
For remember, you are not yet used to moving in these let us call them elevated spheres, and as such, you will feel a little bit insecure and unsteady on your feet. So just stay focused on where you are going, and do not spend too much time mulling about where you actually came from. That way, your heart will follow your eyes, and you will find yourself striving towards your own completion in a way that will help you to attain it faster than if you spend too much time looking back over your shoulder. For you are not going back, you are simply to keep moving ahead, and that is what you do each and every day, even at the times where you feel more like you are holding on for dear life, on the brink of being tossed back down onto the ground again. But trust us when we say that this is not the case, it is simply a signal that you have not yet gotten used to the purer air at this height. So just take your time and go slow whenever you feel the need to do so. As we said, there is no risk that you will miss your stipulated time of completion. So again we say that all is well, and even if your whole structure seems to be swaying considerably at times, it is more solid than you think and it can withstand it all. For you are made of the same stuff as the stars, indeed you are all stars, and you shine brightly even in the brightest of daylight. Not just today, but forever.


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My sweet A&M friends (I call them that even though technically only half of them went to A&M) threw Peter and me a little gender reveal party a couple weeks ago and it was so cute and fun! Below are the cute hostesses... Emily, Gina, Berkeley, Nicole, Kiley, and Jennifer. All of our husbands went to A&M together which is how they got the title "A&M friends".
**the quality, pretty pictures below are taken by my friend Jason Kindig of Jason Kindig Photography, but please note that the not so quality ones are not by him.. they are by my iphone! :)
|Cute cupcakes made by the talenteddd Amanda Buccola of Red Raspberry Cakes
|And cute bow and mustache cookies made by my mom -- her website is here
|water bottle labels for baby boynton!
|the chalkboard where all the guests could vote on whether it was a boy or girl...
and the cute bows and mustaches that people pinned on their shirts!
|Maybe these old wives tales are more accurate than we think!
|Cute balloons and food table
|Baby pics of me and Peter.. such a good idea!
|The hostesses also had everyone secretly bring a book for Baby Boynton!
Now for the REVEEAALLL..... whats it gonna be!?
IT'S A BOYnton!!! Peter was clearly very excited.... :)
|Showing off the blue filling in my cupcake
|Happy parents-to-be!
And here's a video in case you want to watch Peter throw his arms in the air and bang the countertop with overwhelming joy...its pretty priceless. Sorry the video is little and blurry!
Can't wait to meet our little baby boy... thanks again hostesses for the super fun partay!!


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Random thoughts from an older perspective, writing, politics, spirituality, climate change, movies,knitting, writing, books, refinishing furniture, getting off the grid, writing, plotting and planning an organic garden, writing. I MUST STAY DRUNK ON WRITING SO REALITY DOES NOT DESTROY ME.
Thursday, March 16, 2017
In the Beginning....Part 1.
When I bought this place over 13 years ago, I knew my relationship with my darling house and land would all end some day. I'm very conscious of the ticking of my own clock. I stay in moments and cherish them and reflect on my privilege and gratitude to have this house overlooking the ocean, surrounded by trees and hills, with views right out of some exotic magazine. With its own off the grid artist's cabin tucked up on the hill overlooking the bay.
We are so finite in this world and sometimes a tap on the shoulder comes, intuition if you will, and we must pay attention.
My friend Helen's death was a huge tap for me. Then a whole series of friends fell to the wayside shortly thereafter way before their time. I say way before their time when, really, what is human time? Three score plus ten? I've been losing friends since I was six when Geraldine died of meningitis and at eight Eithne was burned in a house fire and at fifteen Rosario had brain cancer. So death walks along beside me even though many of us behave as if we have two hundred years to live. And to live with full mental and physical functions intact. Not so. Take a look around at your Zimmer frames, oxygen tanks and wheelchairs and bewilderments in the supermarkets. I do. Not morbidly but noddingly, know what I mean? Constantly aware too that most health impacted seniors don't shop for themselves so we don't see the Alzheimer's, the dementia, the legless and blind and stroke victims.
I thought to take charge then, back in 2015. I live alone. Have a fierce streak of independence, turned down potential partnerships here, 3 or 4 at last count to offer an example, and wish to be proactive rather than reactive to any future challenges I might face.
I remember a dear blog friend, I was her role model of aging well for some reason, saying at one point: "Well it's a good job I have ten more years to catch up to you and loads of time to live creatively" but sadly she didn't. She died Christmas 2013 rather quickly, from cancer.
So the power of now became a mantra for me long before it was fashionable.
To be continued.....
26 comments:
What a meaningful post. This is my first visit to your site, but I am committed to discover more about you as I read on.ReplyDeleteReplies
Ah Toni thanks for your words!Delete
I hope your move goes smoothly...ReplyDeleteReplies
So t be happening for a while yet dearest E. More to follow in this.....Delete
I appreciate how you may feel, especially as we become older and so many family and friends leave us behind -- their numbers dwindling at a faster and faster rate. The past ten to twenty years have seen that be true for me. Recognizing when to make changes matters, so will look forward to hearing more from you when settled.ReplyDeleteReplies
I echo you back Joared, I've lost so many dearies in the past wee while, it breaks my heart over when I ponder the enormous gaps they've left.Delete
Not sure where you're going with this. I can only say that I used to value my independence a lot more than I do now. What's wrong with sharing your life, and leaning on someone else once in a while, as they lean on you?ReplyDeleteReplies
I share my life with many Tom, but not in sharing all of it. I am allergic to live-in relationships as I am far too independent.Delete
But having said that, there are many who dwell in my heart.
What has happened to you suddenly that you are beginning to sound so morbid? It is totally unlike the WWW that I have known so far!ReplyDeleteReplies
What? Not at all. Reflective perhaps. Conscious of the speed of time.Delete
I continue to follow you closely and with great interest! Love that photo of your house; how beautiful your location is. I've been imagining my own possible future with some anxiety; it's hard not to, at times. I insist on finding the value and desirability of aging, but it sure as hell isn't always easy, is it! -KateReplyDeleteReplies
I think taking charge is the key, Kate. Being the captain so to speak. I've seen too many reaction scenarios and burdens on the children. It's time.Delete
Dunno what to say, m'dear. Think I'm a few older than you, but for sure not wiser. I know the dilemma, the what, choices, you write.ReplyDelete
No solutions here, nor are there any, really. Some afternoons I'd like not to face another day of this, then reality sets in. Still have kids, grandkids, they need to know that grandpa had the will to see them as long as I can.
So there's that.
No words of wisdom here, Mike.Replies
I often think of you Mike as I know you're up against the bell. Don't know about myself, I keep on keeping on. I got two good guys doing the assessments of my conditions.Delete
Just taking a few steps now with all that in mind.
I have had two friends die suddenly just this week. One had her affairs in order; the other didn't.ReplyDelete
I'm making the best use I can of my remaining time and hope to leave some worthwhile things behind me.
Best to you, and I hope you won't get too overburdened by your move. I will probably be able to stay in my house, that I love, so I count myself lucky.Replies
I am so sorry Hattie, that's really rough. I think a partner changes situations and as I don't have one I'm simplifying everything and will focus on what I can do, healthwise in a more relaxed environment.Delete
Your words once again strike home for this one.ReplyDelete
I also chose to live solo even though many chances for a mate.
I am also independent (you know this) but so many have passed
on and wonder of my future. Mind is fine but do not like the limitations of arthritis. Ashamed to say this as could be worse. Now another shameful remark - that I may live
longer then I want too. So a challenge of doing what I can
at a slower pace, love this cottage surrounded by nature
and now is time just to enjoy and let a lot I have done in the past stop. Miss family so much as they are all so busy
but son here - Oh it is wonderful for a few more weeks.Replies
I love that your son is home from Thailand, Ernestine. I know you're dilemma and your health issues. Where I am going has trees and a gorgeous lake and the ocean close by and a lot of things I love like theatre and movies and opera and symphony and writers and artists groups.Delete
I am actually looking forward now.
Sorry to hear you think the days at your lovely house are numbered. But 13 years is a good long stay to look back on, whatever happens next.ReplyDeleteReplies
Yes, I was on a waiting list and thought I'd have another couple of years here but I moved up rapidly and grabbed it while I could as it is ideal.Delete
Downsizing or moving late in life is ALWAYS difficult. What to keep....what to discard...not to mention just the physical aspect of the darn thing. Don't know if this post means that is in your near future....but with the little I know of you....you appear to be strong....this storm shall be weathered .......also.ReplyDelete
I shall call in the helpers, Jo as I'm not fully up for the heavy stuff and challenging decisions of what to take.Delete
It's happened a little quickly.
You certainly began losing friends through their dying at an early age. You have had more practice in this than many of us. I didn't find this post morbid, but very thoughtful. I look forward to part 2. I'm glad you called it In the Beginning rather than At the End!ReplyDeleteReplies
Thanks Carolyn. It surely is. I love winding up in a city again. I adore rural living but city living is better suited, I feel, to elders.Delete
It's good that you wish to be pro-active, WWW - and hopefully by being so it'll mean you need not have been!ReplyDelete
A bit like having an insurance, I guess.
I and husband are both much older than you - he's 80 this week, I'm a couple of years behind him. Thankfully, so far we're good (good as can be expected anyway) but we do realise that things can change quite suddenly.
We try not to spoil what we have left by worrying ourselves too much, something that might hasten us to the brink. Not easy, but doable. Sometimes.
I sense that you'll feel better once you've made the move, WWW - a new page to complete in your adventurous life story. There'll be many more chapters!Replies
Thanks T. I'm not much behind you age wise being in my seventies also. I think a partnership can defer the process unless the single goes all peculiar and hides away with 10,000 newspapers and 85 cats. Fortunately, I fall into neither category so can take charge of my destiny before Daughter sends the cavalry in to dig me out!Delete
I'm ready and will keep posting on my journey.


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VITAMIN B COMPLEX: Necessary for carbohydrate, fat and protein metabolism. Helps function of the nervous system. Helps maintain muscular tone in the gastro-intestinal tract. Maintains healthy skin, hair, eyes, mouth and liver. The B-Complex vitamins are active in providing the body with energy.*
VITAMIN B-1 (Thiamine): Helps maintain healthy nervous system. Stabilizes the appetite. Stimulates growth and good muscle tone. Beneficial effect on mental attitude. Will help prevent undue accumulation of fatty deposits in the artery walls.*
VITAMIN B-2 (Riboflavin): Aids in formation of antibodies and red blood cells. Maintains cell respiration, allowing utilization of cell oxygen. It is also necessary for good vision, skin, nails and hair.*
VITAMIN B-3 (Niacin): Helps maintain health of skin, tongue, and digestive system. Effective in improving circulation and reducing the cholesterol level in the blood. Helpful for weight reduction. Also, research has shown that is can reduce the pain caused by arthritis.*
VITAMIN B-5 (Panthotenic Acid): Improves body's resistance to stress. Aids in the prevention of premature aging and wrinkles. Stimulates the adrenalin glands, which is important for healthy skin, hair and nerves. Important in the prevention of arthritis. It is probably the greatest defense against fatigue and also helps build antibodies for fighting infection.*
VITAMIN B-6 (Pyridoxine): Aids in the formation of antibodies. Helps maintain balance of sodium and potassium, which regulates body fluids and promotes the normal functioning of the nerves and muscular skeletal systems. There is evidence to suggest that it may be involved in cholesterol metabolism, aiding in the control of atherosclerosis. As a natural diuretic, it aids in the prevention of water build-up in the tissues.*
VITAMIN B-12 (Cobalamin): Essential for normal formation of blood cells. Maintains a healthy nervous system. Unique in being the first cobalt-containing substance found to be essential for longevity. Provides relief for fatigue. It also aids in the production of DNA and RNA, the body's genetic material.*
BIOTIN: Biotin is a water soluble B-Complex vitamin. It aids in the utilization of protein, folic acid, pantothenic acid and vitamin B-12. The use of Biotin has shown to be beneficial in treating dermatitis and baldness.*
CHOLINE BITARTRATE: Important in normal nerve transmission. Aids metabolism and transport of fats. It prevents fats from accumulating in the liver. Choline is also essential for the health of the myelin sheaths of the nerves, which are the principal component of the nerve fibers. It plays an important role in the transmission of nerve impulses. It also helps to regulate and improve liver and gall bladder functioning and aids in the prevention of gallstones. Reduces high blood pressure because it strengthens weak capillary walls. It is a fat and cholesterol dissolver and is used to treat atherosclerosis and hardening of the arteries.*
PARA-AMINO BENZOIC ACID (PABA): PABA is an anti-oxidant that is considered by some as a B complex vitamin, and sometimes called vitamin Bx. However it is not really a vitamin, but actually an amino acid that is part of folic acid. It helps in the utilization of Pantothenic Acid. It is important for healthy skin and hair. May prevent accumulation of abnormal fibrous tissue. Also shown to assist formation of red blood cells. Helps to maintain intestinal flora.*
FOLIC ACID (Folacin): Important in red blood cell formation. Necessary for growth and division of body cells and for proper brain function. Essential for mental and emotional health. Aids in the performance of the liver. Folic Acid may prevent the graying of hair when used with P.A.B.A. and Pantothenic Acid.*
INOSITOL: Recognized as part of the vitamin B-Complex and is closely associated with Choline and Biotin. It aids in the metabolism of fats and helps reduce blood cholesterol. In combination with Choline, it prevents the fatty hardening of arteries and protects the liver, kidneys, and heart. Also found to be helpful in brain cell nutrition. Vital for hair growth and may prevent thinning hair and baldness.*
VITAMIN C (Ascorbic Acid): Helps heal wounds, scar tissues and fractures. Gives strength to blood vessels. May provide resistance to infection. Aids in forming red blood cells and preventing hemorrhaging. Fights bacterial infections. Protects the brain and spinal cord from destruction of free radicals. Essential in the formation of adrenalin. Vitamin C is frequently used in the prevention and treatment of the common cold. Arthritic patients may find some relief of pain. Speeds up the healing process. It may lower blood cholesterol. Important in alleviating stressful conditions. It stimulates the production of interferon. It prevents the formation of carcinogenic nitrosamines from nitrites and nitrates found in some foods. Important for recovery from heart attacks by preventing free radical damage. Vitamin C is essential for stimulating the immune system, enabling the body to resist diseases including cancer. May slow the aging process and help build muscle tissues.*
CHROMIUM PICOLINATE (U.S. PATENT #4,315,927): a BREAKTHROUGH MINERAL NUTRIENT. Craving for sweets is greatly diminished. Body fat is reduced without the loss of lean muscle tissue. Blood glucose is stabilized and cholesterol is decreased. Appetite is diminished. Metabolism is more efficient. Research has also shown that with the addition of an exercise program, Chromium Picolinate can allow development of lean muscle tissue.*
SWEET ORANGE (CITRUS SINENSIS): The numerous health benefits are linked to the high concentration of antioxidants and other health promoting nutrients including vitamin C- (ascorbic acid), Hesperidin, Limonene, Narirutin and Beta-Cryptoxanthin. The health benefits of sweet oranges range from anti-cancer and anti-asthma to the ability to support healthy collagen formation. Collagen is what gives skin a smooth, wrinkle-free appearance. Sweet Orange contains Hesperidin, a health promoting flavonoid, which act as a strong antioxidant, protecting the skin from free radical assault. It is also been shown to provide protection against some cancers, including human breast cancer and androgen-dependent prostate cancer, by suppressing cancer cell proliferation. Vitamin C – (Ascorbic Acid) appears to reduce the risk of cardiovascular disease by raising the levels of HDL cholesterol while lowering blood pressure and reducing the conversion of fat into plaque in the arteries.*
PANAX GINSENG: Stimulates both mental and physical energy. Helps increase physical endurance and mental work capacity. Research has shown, it lowers blood pressure and cholesterol. Enhances liver metabolism and promotes the health function of the pituitary gland, the body's master gland.*
GUARANA (PAULINA CUPANA) (SEED): Long lasting caffeine source. Reduces mental and physical fatigue by increasing energy. Guarana also boosts your metabolism which activates your body's own lipotropic (fat-burning) processes. Helps headaches, joint and back pain. Increases urine flow which eliminates toxins from your body. Enhances athletic performance. Test on human's show Guarana can improve task performance and increase attention span for up to six hours which can help adults perform better at mental tasks and fight the fatigue associated with long periods of brainwork, like studying for finals.*
GINKGO BILOBA (LEAF) EXTRACT: Extensive research has shown that Ginkgo Biloba can help you nutritionally counteract the effects of aging as well as mental fatigue and lack of energy. It helps to reduce inflammation and protects capillaries from becoming brittle. Ginkgo Biloba increases oxygen to the brain, thereby inhibiting memory loss. It aids in the absorption of glucose and oxygen, which helps support healthy brain functions and mental activity. Nutritionally helps circulation throughout the entire body. It promotes elasticity and strength in blood vessels and capillary walls.*
SIBERIAN GINSENG: Increases stamina and endurance. Speeds recovery from workouts. Improves reflexes and concentration, particularly in longer endurance events.*
ROYAL JELLY: Has earned the title: NATURES SUPER FOOD. It is known to contain important antibiotic properties. Helps stimulate the immune system and nutritionally supports deep cellular health. Rich in proteins, trace minerals, Vitamins B-1, B-2, B-6, B-12, Niacin, Biotin, Folic Acid and Inositol; it is also an excellent source of Vitamin B-5, Pantothenic Acid. This important nutrient is vital for supporting the health of the adrenal glands, one of the primary organs utilized by the body for managing stress.*
CAYENNE PEPPER (Capsicum): Helps to increase blood circulation which allows more oxygen to be delivered to all the cells throughout the body.*


text:
Press Release - L.A. GOP Chairman: Jerry Brown, the Democrats' Frankenstein and Our New Politics of Scarcity
ENCINO, May 18, 2015 – Most of the time, politics is an exercise in getting the attention of people who are busy with living their lives and don't have the time to follow the events of the day – even if it is news they can use. Try as we might, we know for a fact that there are thousands of voters in Los Angeles County who would be interested and receptive to the GOP message … if only we were able to connect with them.
2014 proved that we can absolutely bring new voters to our Republican side and persuade them to take a look at our platform of economic growth, local control, public accountability and individual freedom. We have further to go, but we are making progress.
Still, it is a daily challenge to get the attention of the public, inform them of what is happening and encourage them to get involved in new and more effective ways. Too often, we can't seem to find a way to reach everyone.
And then the Democrats start rationing water. Think people might pay attention now?
A Summary of the Story
Let's review the bidding. California is currently in a drought. This has never happened before – well, unless you count the fact that much of the state is located in a dry, arid climate (or even the desert), and has been in a drought condition off and on for, literally, thousands of years.
For just about as long, Democrats have been thinking up ways to restrict water use by farmers, regulate excessive environmental rules and refuse to construct storage facilities and institute wise industrial planning. They prefer to tax and borrow to continue California's massive social spending and hope for – I guess – a rainy day.
Is our Democrat governor working with the Democrat president to ease the regulations that have diverted millions of gallons of water to protect a tiny fish? No.
Is he calling for an end of the policy that diverts massive amounts of usable water into the ocean for fish habitat purposes? No.
Is the Democrat Legislature green-lighting desalination plants to put them on line as soon as possible? No.
Has the Director of the Department of Water Resources – on whose watch this has all happened – been asked by anyone in Sacramento to resign? No.
What do you have to do to lose your job in state government, anyway?
Democrats vs. Greens? Don't Let it End Soon
Right now, I believe there is a sort of calm before the storm. The only people really protesting this issue are the environmentalist left which opposed Brown's idea of a water tunnel with such ferocity that he responded, "shut up, because you don't know what the hell you're talking about."
This is fascinating for a very big reason: Not because Brown's water tunnel is a bad or good idea … it's irrelevant to our present needs. The interesting part is that it is at least an idea about building and constructing a technological solution … which the environmentalists totally oppose.
So Brown got angry. But he should save his ire for his fellow Democrats – who created this radical movement and used it to win many elections and finance many more.
The Democrats' Frankenstein monster rising up to attack them? Grab a seat and break out the popcorn.
A Simple Plan and a Complicated Lack of Response
But the Democrats are on the job and they have a plan to deal with a crisis they have made dramatically worse: Unprecedented compulsory reductions in residential water use and/or fines of tens of thousands of dollars.
That's their plan. Shutting off your water or socking you with huge penalties. Or both.
So far, the water police haven't handcuffed anyone to their garden hose and stopped them from watering their lawn. And no one has yet received a fine perhaps 10 times their rent for washing their car by hand. Yet.
To date, our Republican leaders have done the responsible thing by engaging in bipartisan dialogue and offered innovative solutions to help with the crisis. I believe we have been handed a golden opportunity. Will we take it?


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The Community Grants Program supports not-for-profit organisations to present a variety of projects and activities which enhance the social, cultural, environmental and economic development of the community.
There are no information sessions scheduled at this time.
Council assesses grant applications twice a year, in March (Round 1) and August (Round 2).
Applications for Round 1 2019 will close on Friday, 8 March 2019.
The Community Grant Guidelines (PDF 296kb) explain how to apply.
Grant applications are only accepted using the SmartyGrants on-line system. You can download a paper copy of the application form (PDF 88kb) for draft purposes if you wish.
Potential not for profit applicant groups that are not legally incorporated are encouraged to consider being auspiced by a legally incorporated group to be able to apply for a community grant.
Legally incorporated groups are encouraged to consider their situation, interest, and ability, to auspice potential applicant groups that are not legally incorporated.
You can speak to Council's Grants Officer about auspicing or seek general auspicing information at Justice Connect www.nfplaw.org.au/auspicing
Auspiced applicants are required to complete a Community Grants Auspicing Form (PDF 218kb)
Successful applicants will be sent a Tax Invoice / Funding Agreement (PDF 290kb) for their project which must be completed and returned to Council before grant funds are paid.
Please contact Council's Grants Officer on 1300 BCOAST (226 278) or (03) 5671 2211 or email email@example.com.
FRRR and Gardiner Foundation - Grant Seeking Workshop and Writing Grants Presentation (PDF 2.3mb) - as presented at the Get Grant Ready Forum on Thursday, 16 February 2017
Get Grant Ready - Resources for Community Groups (PDF 112kb)
Writing great applications (external web page)
Community Tool Box (external web page)
Guidelines and checklist for installing Solar Panels on Council buildings information sheet (PDF 142kb)
Australian Sports Foundation - Grassroots Sports Fundraising Toolkit (external web page)
Fax (03) 5671 2222 | National Relay Service 13 36 77 | firstname.lastname@example.com


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Withdrawn: If an author's text is signed and marked with DoNotEdit, do not edit, move, or archive it.
Issues with DoNotEdit
Signing a text is inherently an indication not to edit.
But what about correcting spelling, updating moved links, or updating terminology?
A recent example of updating terminology is finding out which of "attribute" or "property" was more widely used, and going through all pages to change "property" to "attribute".
Refactoring is a benefit of a wiki, a definining characteristic.
We can choose to discourage that, which DoNotEdit does, but where's the rational?
The rational behind refactoring is to make a topic as concise, complete, clear, and simple as possible. Seperating out sub-topics from a main topic is factoring, not "archiving".
"Moving" isn't clear. It's common in ThreadMode to address a reply directly to the text in question, thus "moving" someone else's text "down".
Similarly, it's common to break up long threads into subthreads, and rearrange accordingly, seperating by a horizontal rule (----).
Re "ArchivedDiscussion", those discussions are not "archived", they're not mothballed and they are not "concluded" by indicating that discussion of a topic is a seperate page from the topic under discussion.
Redundant and Vague
This proposed policy is vague and redundant and should be dropped. There is already a policy in place, "Please don't delete each other's comments (portions of text identified with a person's name)." That covers it.
- DoNotEdit . . . . 3 matches
- ArchivedDiscussion . . . . 1 match
- WellFormedEntryByExample . . . . 1 match


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- Research article
- Open Access
Yeast karyopherin Kap95 is required for cell cycle progression at Start
- Francisco José Taberner1 and
- Juan Carlos Igual1Email author
© Taberner and Igual; licensee BioMed Central Ltd. 2010
- Received: 18 November 2009
- Accepted: 29 June 2010
- Published: 29 June 2010
The control of the subcellular localization of cell cycle regulators has emerged as a crucial mechanism in cell division regulation. The active transport of proteins between the nucleus and the cytoplasm is mediated by the transport receptors of the β-karyopherin family. In this work we characterized the terminal phenotype of a mutant strain in β-karyopherin Kap95, a component of the classical nuclear import pathway.
When KAP95 was inactivated, most cells arrested at the G2/M phase of the cell cycle, which is in agreement with the results observed in mutants in the other components of this pathway. However, a number of cells accumulate at G1, suggesting a novel role of Kap95 and the classical import pathway at Start. We investigated the localization of Start transcription factors. It is known that Swi6 contains a classical NLS that interacts with importin α. Here we show that the in vivo nuclear import of Swi6 depends on Kap95. For Swi4, we identified a functional NLS between amino acids 371 and 376 that is sufficient and necessary for Swi4 to enter the nucleus. The nuclear import driven by this NLS is mediated by karyopherins Kap95 and Srp1. Inactivation of Kap95 also produces a dramatic change in the localization of Mbp1 since the protein is mainly detected in the cytoplasm. Two functionally redundant Kap95- and Srp1-dependent NLSs were identified in Mbp1 between amino acids 27-30 and 166-181. Nuclear accumulation was not completely abolished in a kap95 mutant or in the Mbp1 mutated in the two NLSs, suggesting that alternative pathways might contribute to the Mbp1 nuclear import to a lesser extent.
Kap95 plays an essential role at the initiation of the cell cycle by driving the nuclear import of Swi4, Swi6 and Mbp1, the three transcription factors responsible for the gene expression at Start. This transport depends on the specific nuclear localization signals present in cargo proteins.
- Nuclear Localization Signal
- Nuclear Import
- Putative Nuclear Localization Signal
- Start Transition
- Functional Nuclear Localization Signal
In eukaryotic cells, the basic cellular functions occurring inside the nucleus, like transcription or DNA replication, require the transport of proteins across the nuclear envelope. The nucleocytoplasmic transport of most proteins is an energy-dependent process requiring the participation of the transport receptors of the β-karyopherin family [1–7]. β-karyopherins specifically recognize the nuclear localization signals (NLS) or the nuclear export signals (NES) in cargos to carry them through the nuclear pore complex. The binding and release of cargo proteins by karyopherins is controlled by the GTPase Ran cycle [2]. In the import process, karyopherins bind the cargo in the cytoplasm and, once translocated into the nucleus, the binding of Ran-GTP destabilizes the complex releasing the cargo. In the export process, the binding of the cargo by the karyopherins in the nucleus is stabilized by Ran-GTP and, once in the cytoplasm, hydrolysis of GTP to GDP triggers the dissociation of the cargo.
The most prevalent import pathway is the classical import pathway. This pathway transports those proteins carrying classical nuclear localization signals [8]. These signals consist in either a short cluster of basic amino acids (monopartite) or two short clusters of basic amino acids separated by 10-12 residues (bipartite). S. cerevisiae Kap95 (homologous to mammalian importin β) is the β-karyopherin involved in the nuclear import of proteins with classical NLS. However, Kap95 is unable to bind the cargo protein directly, but requires an adaptor protein, Srp1 or importin α, to recognize the classical NLS [9–11]. Cse1, another β-karyopherin involved in the classical import pathway, is needed to recycle Srp1 to the cytoplasm once the complex Kap95-Srp1-cargo has been dissociated in the nucleus [12, 13]. The KAP95, SRP1 and CSE1 genes are essential for cell viability. A mutation in the SRP1 gene arrests cells at the G2/M phase of the cell cycle [11]. This indicates that the classical import pathway plays an essential role in cell cycle progression in mitosis.
A major control point in cell cycle progression in eukaryotic cells occurs at the end of the G1 phase in a process called Start in Saccharomyces cerevisiae. In Start, yeast cells decide whether or not to initiate a new cell cycle depending on external (nutrient availability, presence of pheromones) and internal (protein synthesis/cell size, DNA integrity) cues [14]. Execution of Start consists in the activation of a transcriptional program that implies the coordinated expression of a large number of genes [15, 16]. A lot of those genes codify for the proteins involved in the events of budding, spindle pole body duplication and DNA replication; that is, the events that are activated at this initial step of the cell cycle.
The gene expression at Start depends on two transcription factors: SBF and MBF [17–19]. Both factors are heterodimeric complexes formed by a common regulatory subunit, Swi6, and two different DNA binding proteins, Swi4 for SBF and Mbp1 for MBF. SBF and MBF show a certain degree of redundancy [20]. Despite this, the SBF- and MBF-depending genes can be clustered into functional categories: the expression of growth, morphogenesis and spindle pole body-related genes, as well as CLN1 and CLN2 cyclin genes, depend mainly on SBF, while those genes involved in the control and execution of DNA replication and repair, including the CLB5 and CLB6 cyclin genes, are transcribed by MBF. SBF and MBF bind target promoters at the beginning of G1 [21], but the gene expression is restrained by the association of transcriptional repressor Whi5 [22, 23]. At the end of G1, the cyclin-dependent kinase Cdc28-Cln3 enters the nucleus [24] and phosphorylates Whi5, SBF and MBF, thus promoting the dissociation of the repressor and transcriptional activation [22, 23, 25]. As a result, kinases Cdc28-Cln1 and Cdc28-Cln2 accumulate and act on Whi5, SBF and MBF to establish a positive feedback loop that is important for the proper execution of Start [26]. Later in the cell cycle, this transcriptional wave is inactivated by the phosphorylation of SBF by kinase Cdc28-Clb2 [27] and the association of repressor Nmr1 to MBF [28].
Spatial regulation adds a new step to the control of the protein function. With regards to transcription factors, this spatial regulation necessarily implies a nuclear import mechanism and, in some cases, an export mechanism that could contribute to the control of transcription factor activity. As regards Start transcription factors, Swi4 shows a constant nuclear localization in all the cell cycle stages [29]. However, Swi6 and Whi5 shuttle between the nucleus and the cytoplasm along the cell cycle: they are nuclear from the end of mitosis to the S-G2 phases in the case of Swi6 [30–32] or to Start in the case of Whi5 [22]. Localization of Swi6 is controlled by an import mechanism mediated by a classical NLS [32, 33] and an export mechanism that depends on karyopherin Msn5 [31]. Changes in Swi6 localization are basically due to the cell cycle regulation of its nuclear import: phosphorylation of Ser160 by Cdc28-Clb6 blocks the nuclear import during the S-G2 phases until the end of mitosis when the Cdc14 phosphatase removes the phosphate group to once again allow the nuclear accumulation of the protein [30, 32, 33]. Localization of Whi5 is controlled by an import mechanism mediated by two functionally redundant classical NLS recognized by karyopherin Kap95 and an export mechanism that depends on karyopherin Msn5 [25, 34]. Conversely with Swi6, the nuclear import of Whi5 is not cell cycle-regulated, and changes in localization are due to a regulated nuclear export: multiple phosphorylation of specific residues drives the export until this phosphorylation is reverted by the Cdc14 phosphatase at the end of mitosis [34].
In this work we studied the role of Kap95 in cell cycle progression. We show that inactivation of Kap95 arrests cells at two critical cell cycle points: G2/M transition and G1/S transition. The SBF and MBF subunits Swi6, Swi4 and Mbp1 are delocalized in kap95 cells. As previously shown for Swi6, Srp1 is also required for the nuclear localization of Swi4 and Mbp1. Thus, the classical nuclear import pathway plays a key role in the control of Start regulators.
Kap95 is involved in the Start transition of the cell cycle
In conclusion, all these observations indicate that the inactivation of Kap95 causes a double arrest in cell cycle progression. Most cells accumulate with a large bud, duplicated DNA and a single nucleus, indicating that the absence of Kap95 generates a major arrest in the G2-M transition. This is in agreement with the results previously reported for the mutants in the other karyopherins of the classical import pathway [11, 35]. It is noticeable, however, that a minor yet significant number of kap95 cells (approximately 20%) accumulated as unbudded cells with unreplicated DNA and a single nucleus, indicating a G1 arrest.
In order to further characterize the defect in executing Start in the absence of Kap95, cells were synchronized at the telophase by means of a thermosensitive cdc15 mutant allele. After release from the arrest, progression through Start was monitored by the occurrence of rebudding (cdc15 cells show a defect in cytokinesis/cell separation after recovering from telophase arrest). As it can be observed in figure 1D, in the case of the control strain, the rebudded cells accumulated 60 min after release from the arrest, representing approximately 80% of the total cells after 90 min. Conversely, kap95 mutant cells failed to rebud after the release, even after 120 min. This was not due to a defect in the recovery from telophase arrest since, unlike what was observed with the telophase-arrested cells, digestion of the cell wall with zymoliase rendered most cells unbudded (Figure 1E). Thus, the lack of rebudding indeed reflects the inability to progress through Start in the absence of Kap95.
It is important to note that a similar result to that described for the tetO 7 :KAP95 mutant strain was obtained with a tetO 7 :CSE1 mutant strain (data not shown), indicating that the Start defect is not a specific phenotype of Kap95 inactivation, but must relate to the complete classical import pathway function. The arrest at the G1/S transition is a novel phenotype for the mutants in this pathway and demonstrates a role of the classical nuclear import pathway in the Start transition.
Start transcription is impaired in the absence of Kap95
It is known that the ectopic expression of CLN2 suppresses the lethality of the mutant strains in the Start transcriptional program. When a plasmid expressing CLN2 under the control of the S. pombe adh1 promoter was introduced into the tetO 7 :KAP95 mutant strain, the percentage of G1 cells significantly lowered: 24% compared to 38% for the control plasmid (Figure 2C). This suggests that the defect in Start transcription contributes to the blockage of kap95 mutant cells at G1. However, many G1 cells are still present, which suggest that other functions involved in the G1/S transition are still affected. A similar result was obtained in synchronized cdc15 tetO 7 :KAP95 cultures. As it can be observed in Figure 2D, the ectopic expression of CLN2 caused a significant increase in the percentage of rebudded cells 120 minutes after the release from the arrest; however, most of the cells were still unable to initiate a new cell cycle. In conclusion, these results indicate that the impaired Start gene expression contributes to the blockage of kap95 mutant cells at G1 but that other affected functions in addition to the transcriptional program cause the defect in cell cycle progression through the G1/S transition.
Role of Kap95 in Swi6 nuclear import
Characterization of Swi4 nuclear import
The high degree of homology between Swi4 NLS and the consensus classical NLS suggests that the classical import pathway could import the Swi4NLS-GFP4 protein into the nucleus. To test this dependency, we analyzed the distribution of the Swi4NLS-GFP4 protein in the absence of Kap95. As observed in Figure 4C, Kap95 inactivation resulted in a cytoplasmic localization of the chimera, which indicates that Kap95 may recognize the NLS from Swi4.
Next we examined the role of the identified NLS in the nuclear import of Swi4 by analyzing the effect of its inactivation on Swi4 localization. As endogenous levels of Swi4 are difficult to detect by indirect immunofluorescence, we expressed Swi4 under the control of the GAL1 promoter to increase the amount of protein. The overexpression of Swi4 did not alter the localization of the protein, which accumulated in the nucleus of all the cells (Figure 4D). Nevertheless, inactivation of the described NLS resulted in a cytoplasmic localization of Swi4. Therefore, this NLS is necessary to drive the nuclear localization of Swi4.
Finally, we investigated the localization of Swi4 in the doxycycline-treated tetO:KAP95 cells. Consistently with the previous result, inactivation of Kap95 caused a homogeneous Swi4 distribution through the cell (Figure 4E). The same result was observed in a mutant strain in the importin α Srp1 (Figure 4F). All the obtained results help establish the mechanism underlining the nuclear localization of Swi4. Swi4 has a unique NLS between amino acids 371 and 376 which is sufficient and necessary for its nuclear import, an import that is mediated by the classical import pathway involving karyopherins Kap95 and Srp1. Therefore, Swi4 is another Start regulator whose nuclear localization depends on Kap95-Srp1 activity.
Role of Kap95 in the nuclear import of Mbp1
Next we studied the karyopherin involved in the nuclear import of Mbp1. By considering that Mbp1 and Swi4 belong to the same family of transcription factors, and the fact that they share structural similarities, we envisaged the possibility of them possibly entering the nucleus assisted by the same import route. Therefore, we analyzed Mbp1 distribution in a mutant in karyopherin Kap95. The immunofluorescence assays show a cytoplasmic accumulation of Mbp1 when Kap95 was inactivated, unlike the strong nuclear signal observed in the wild-type cells (Figure 5). This result indicates that Mbp1 enters the nucleus assisted mainly by the classical import pathway. Nevertheless, a faint Mbp1 nuclear signal was observed in some kap95 cells, suggesting that other karyopherins might contribute to the Mbp1 nuclear import to a lesser extent.
The fact that the nuclear import depends on Kap95 led us to screen the Mbp1 protein for the putative classical NLS sequences. Two putative classical NLSs were identified in the N-terminus region of Mbp1: a monopartite one (NLS1) between amino acids 27 and 30 and a bipartite one (NLS2) located between amino acids 166 and 181 (Figure 5B). In order to test whether these putative NLSs were in fact functional, we fused a fragment of Mbp1 from amino acids 23 to 184 (containing both candidate NLSs) to four copies of GFP. Unlike the control GFP4 protein, the chimera Mbp123-184-GFP4 localized in the nucleus (Figure 5C), thus demonstrating the existence of at least one functional NLS in this region of the Mbp1 protein. As expected, the Mbp123-184-GFP4 protein became cytosolic when Kap95 was inactivated (Figure 5D). Importantly, the nuclear accumulation of Mbp123-184-GFP4 in a srp1 mutant strain was also eliminated (Figure 5E). These results confirm that the nuclear import activity in fragment 23-184 of Mbp1 requires an intact classical import pathway
Further characterization of Mbp1 NLS was carried out by a direct mutagenesis of the basic residues in both putative NLSs, as indicated in Figure 5B. The subcellular localization of the resulting Mbp123-184-NLS1i-GFP4 and Mbp123-184-NLS2i-GFP4 chimeras was analyzed. In both cases, inactivation of a single putative NLS did not significantly change the location of the proteins, which were clearly detected in the nucleus. Thus, neither of the two putative NLSs is essential for the nuclear import of Mbp1. Importantly, however, when both putative NLSs were simultaneously mutated, nuclear localization was avoided and the Mbp123-184-NLS1i2i-GFP4 protein was located mainly in the cytoplasm. These results demonstrate that Mbp1 contains two functional and redundant NLSs, and that either of them is able to mediate the nuclear import of Mbp1 by Srp1-Kap95 karyopherins.
Two of the first karyopherins to be connected to cell cycle regulation were Srp1 and Cse1, which together with Kap95, constitute the classical nuclear import system of proteins. The inactivation of Srp1, and to a lesser extent the inactivation of Cse1, causes an arrest in cell cycle progression at the G2/M phase [11, 35]. Consistently, in this work we describe how the inactivation of Kap95 triggers a blockage at the G2/M transition. However, the inactivation of Kap95 also originates a previously uncharacterized accumulation of cells in G1. This result could indicate that Kap95 may undertake a function in G1/S that is not shared with Srp1. To support this possibility, Kap95 has been recently described to contribute to the import of proteins without requiring Srp1 and by recognizing an import signal distinct from the classical NLS [36, 37]. However, the fact that the major cell cycle defect in the kap95 mutant also occurs in G2/M and, more importantly, the fact that we also observed G1 arrested cells in a tetO 7 :CSE1 mutant strain, suggest that the different phenotype we observed in the kap95 mutant cells, compared to the previously reported phenotype of mutations srp1-31 and cse1-1, is more likely caused by the different effect of the distinct mutant used in the classical nuclear import pathway activity. In fact, the nuclear localization of the key Start regulators described in this work depends on both Kap95 and Srp1. Thus, our results reveal a new function of the classical import pathway in the control of the cell cycle at the Start transition. Nevertheless, it cannot be ruled out that Kap95 might control other proteins involved in the G1/S transition independently of Srp1. The fact that only a minor proportion of kap95 mutant cells are blocked in G1 could simply reflect a greater sensitivity of the G2/M transition to the reduced activity of the pathway, so the blockage of cells at this cell cycle step precludes an accumulation of cells in the G1 phase. This is supported by the results obtained when cells were arrested at the telophase (after the major G2/M arrest point) before inactivating KAP95. In this case, cells mostly accumulate in the G1 phase, demonstrating that Kap95 is required for executing Start.
Despite the defect in the G2/M phase in the mutants in the classical import pathway having been characterized several years ago, there is no explanation for the causes of this arrest. As inactivation of Srp1 results in longer half lives of some mitosis regulators like cyclin Clb2, some authors have proposed that this import pathway could affect the protein degradation machinery required for progression through mitosis [11]. Alternatively, the inability to enter the nucleus of a key mitotic regulator required for mitosis progression could also explain the blockage at the G2/M transition. A good candidate is separase Esp1, the protease that triggers sister-chromatid segregation once in the nucleus [38, 39]. Failure to locate separase in the nucleus is expected to result in cell arrest at the metaphase, which is consistent with the terminal phenotype of the mutants in the classical import pathway. Interestingly, the analysis of the Esp1 sequence identified a consensus classical monopartite NLS between amino acids 1188 and 1192 (RRKRR), which strongly suggests that the classical nuclear import pathway may be responsible for separase nuclear accumulation.
It is noteworthy that additional defects have been observed in the G2/M kap95 cells. First, most G2/M cells contain a single nucleus, although this is not properly located near the bud neck in a fraction of the cells. Second, a small yet significant percentage of cells that escapes the blockage segregates chromosomes inside mother cells. A similar defect was observed in the cse1-1 mutant strain [35]. These observations suggest that the inactivation of the classical import pathway causes a dysfunction in the nucleus positioning and/or spindle orientation.
As previously mentioned, our results reveal a new function of karyopherin Kap95 and the classical import pathway in the control of the cell cycle in addition to its known connection with mitosis. Specifically, Kap95 is involved in the execution of Start at the initiation of a new round of division, which suggests that the classical import pathway may mediate the nuclear import of proteins controlling Start. Start consists in the activation of a transcriptional program, so the transcription factors involved in Start are good candidates to be regulated by Kap95. In fact, the expression of G1/S regulated genes is impaired in the absence of Kap95. Given the robustness of the Start transcriptional program conferred by the functional redundancy between its components, we suspected that the G1 arrest in the absence of Kap95 could reflect the delocalization of several Start regulators. This was indeed the case since transcription factors Swi6, Swi4 and Mbp1 enter the nucleus assisted by Kap95 and Srp1. It is important to note that the ectopic expression of CLN2 does not completely suppress the G1/S arrest of the kap95 mutant cells. This indicates that Kap95 must control the additional proteins besides the Start transcriptional activators, whose mislocalization in kap95 mutant cells could contribute to the blockage of the Start transition. As regards Swi6 and Swi4, Kap95 is the only importin responsible for their nuclear localization. Regarding Mbp1, Kap95 is the main import pathway; however, nuclear accumulation in the kap95 mutant strain is not completely abolished. This could be interpreted by the presence of a residual Kap95 activity in the cells that are able to import Mbp1, but not Swi4 or Swi6. However, the fact that the overexpression of an Mbp1 protein in which the two classical NLSs are inactivated still shows significant nuclear localization (data not shown), points to the existence of a secondary import pathway besides Kap95. To characterize this alternative pathway, we analyzed the distribution of Mbp1NLS1i2i in all the β-karyopherin mutants (except kap123), but the nuclear localization of Mbp1 was not completely abolished in any of the mutant cells (data not shown). This strongly suggests that several alternative minor import pathways are able to translocate Mbp1 into the nucleus. The presence of multiple import pathways for Mbp1 is not unique between yeast proteins. Several cases of proteins imported by several β-karyopherins have been described, such as Sas2, the histones H3 and H4, ribosomal proteins and ribosomal associated proteins, which enter the nucleus via Pse1 and Kap123 [40–43]. There is also the more dramatic case of the HO endonuclease [44] and the Asr1 protein [36], whose nuclear import could be assisted by four or five different importins, respectively.
Besides being involved in the import of Start transcription activators, we have recently shown that Kap95 is the importin of the Start transcriptional repressor Whi5 [34]. By taking into account that the nuclear import of Cln3, the cyclin that initiates the Start transcription program when it is translocated to the nucleus, depends on a canonical classical NLS [45, 46], it is conceivable that Cln3 might also be imported by the classical import pathway. Thus, it is striking that the nuclear import of the complete transcriptional machinery of the Start transition is mediated by the classical import pathway. The use of the same pathway should prove advantageous for the coordinated assembling of the transcriptional circuitry. This is particularly relevant when transcriptional activators and repressors restraining its activity exist. The fact that the components of SBF, MBF and Whi5 enter the nucleus by Kap95 facilitates the colocalization of activators with its repressor. If each regulator entered the nucleus by distinct import pathways, the system could be more prone to unscheduled transcription and a deregulation of the G1/S transition with the detrimental consequence to cell physiology.
Yeast strains and plasmids
Strains used in this study are W303-1a (MATa leu2 ade2 trp1 his3 ura3 can1 ssd1-d), cdc15 (MATa leu2 trp1 his3 ura3 cdc15-2), srp1 (srp1-31 in W303-1a), JCY635 (tetO 7 :KAP95-KanMX6 in W303-1a), JCY710 (SWI6-HA-TRP1 in W303-1a), JCY637 (SWI6-HA-TRP1 in JCY635), JCY816 (MBP1-HA-TRP1 in W303-1a), JCY817 (TRP1-GAL1:HA-SWI4 in W303-1a), JCY848 (MBP1-HA-TRP1 in JCY635) and JCY1543 (tetO 7 :KAP95-KanMX6, CMVp(tetR'-SSN6)::LEU2 in cdc15). The substitution of the KAP95 promoter by the tetO 7 promoter was obtained by integrating a DNA fragment amplified from plasmid pCM225 (a gift from E. Herrero). The susbtitution of the SWI4 pormotoer by the GAL1 promoter was obtained by integrating a DNA fragment amplified from plasmid pFA6a-TRP1-PGAL1-3HA (from Dr. J.R. Pringle). Tagging of the Swi6 and Mbp1 protein at the C-terminus with HA was achieved by integrating a DNA fragment amplified from plasmid pF6a-HA:TRP1 (from Dr. J.R. Pringle). Yeast cells were grown on standard yeast extract-peptone-dextrose (YPD) media or synthetic dextrose (SD) or galactose (SR) minimal media supplemented as required. To repress the tetO 7 promoter, doxycycline was added to a concentration of 5 μg/ml.
Plasmid pSWI4NLS-GFP4 derived from pNLSSV40-GFP4 [34], which contains, in order, the ADH1 promoter, the SV40 NLS, and four GFP gene copies). It was obtained by site directed mutagenesis resulting in the substitution of the SV40 NLS by the SWI4 NLS (KKRRKK). pSWI4NLSi-GFP4 plasmid was obtained from pSWI4NLS-GFP4 by site directed mutagenesis of the SWI4 NLS to an inactive NLS version (EKGRE).
Plasmid pMBP123-184-GFP4 was obtained removing the SV40 NLS coding region from pNLSSV40-GFP4 by KpnI-BamHI digestion and introducing an MBP1 gene fragment coding from amino acid 23 to 184 amplified from genome with a forward oligo containing a KpnI restriction site and a reverse oligo containing a BamHI site. pMBP123-184 NLS1i-GFP4 plasmid was obtained from pMBP123-184-GFP4 by site directed mutagenesis of Mbp1 NLS1 (KRKK) to an inactive NLS version (IGTT). pMBP123-184 NLS2i-GFP4 plasmid was obtained from pMBP123-184-GFP4 by site directed mutagenesis of Mbp1 NLS2 (RKR-X10-RRK) to an inactive NLS version (TIG-X10-GLE). pMBP123-184 NLS1i2i-GFP4 plasmid was obtained from pMBP123-184 NLS2i-GFP4 by site directed mutagenesis of Mbp1 NLS1 (KRKK) to an inactive NLS version (IGTT).
pGAL1:HA-SWI4 plasmid was obtained by cloning a DNA fragment containing the coding region for an HA N-terminal tagged Swi4 protein expressed under the control of GAL1 promoter, amplified from the genome of JCY817 using a forward oligo containing a SalI restriction site and a reverse oligo containing a KpnI site in SalI-KpnI digested YCplac33 vector. pGAL1:HA-SWI4NLS1i plasmid was obtained from pGAL1:HA-SWI4 by site directed mutagenesis of the SWI4 NLS (KKRRKK) to an inactive NLS version (EKGRE).
DNA content analysis
Approximately 107 cells were fixed in EtOH 70%. Fixed cells were treated with RNase A (1 mg/mL) in Tris-HCl 50 mM [pH 7.5] buffer for 3 hours at 37°C and pepsine 5 mg/mL for 5 minutes at room temperature. Finally cells were stained with propidium iodure (50 μg/mL). DNA content was analyzed in a EPICS XL (Coulter Inc.) cytometer.
Fluorescent microscopy
For indirect immunofluorescence assays, approximately 108 cells were fixed in growth medium with 37% formaldehyde (1:10 dilution) for 60 min with agitation. After mild sonication, cells were collected, washed with buffer A (1.2 M sorbitol, 0.1% β-mercaptoethanol, 50 mM KH2PO4 [pH 7.0]), and incubated at 37°C for 1 to 2 h in 0.5 ml of buffer A containing 100 μg of zimolyase 20-T/ml. Spheroplasts were collected, washed with buffer A, resuspended in 20 μl of a solution (8 μg/ml of rat anti-HA antibody 3F10 from Roche in phosphate-buffered saline, PBS, containing 0.1% bovine serum albumin), and incubated overnight at 4°C. After washing with PBS, PBS containing 0.1% NP-40, and PBS, cells were incubated with the secondary antibody Alexa 546-labeled anti-rat IgG antibody (Molecular Probes) as indicated for the primary antibody. DNA was stained at the end of the process by resuspending spheroplasts in PBS containing 1 μg/ml of DAPI (4,6-diamidino-2-phenylindole, Sigma Inc.). In the case of Mbp1-HA immunofluorescence assays, an additional step was included in order to amplify the signal: after incubation with the primary rat anti-HA antibody 3F10, cells were sequentially incubated with Alexa 546-labeled goat anti-rat IgG antibody and Alexa 546-labeled rabbit anti-goat IgG antibody (Molecular Probes).
GFP tagged proteins were visualized in living cells grown in synthetic media. Confirmation of GFP signal as a nuclear signal was achieved in control experiments by DAPI staining of absolute ethanol fixed cells. All microscope samples were analyzed in an Axioskop 2 microscope (Zeiss Inc.). Images were captured with a SPOT camera (Diagnostic Instruments Inc.).
This work was supported by Grants BFU2007-67929-C02-02 from Ministerio de Ciencia y Tecnología of the Spanish Governement and ACOMP07/086 from Generalitat Valenciana. F.T. was recipient of a F.P.I. Predoctoral Fellowship from the Spanish Governement.
Authors' Affiliations
- Chook YM, Blobel G: Karyopherins and nuclear import. Curr Opin Struct Biol. 2001, 11 (6): 703-715. 10.1016/S0959-440X(01)00264-0.View ArticlePubMedGoogle Scholar
- Kuersten S, Ohno M, Mattaj IW: Nucleocytoplasmic transport: Ran, beta and beyond. Trends Cell Biol. 2001, 11 (12): 497-503. 10.1016/S0962-8924(01)02144-4.View ArticlePubMedGoogle Scholar
- Mosammaparast N, Pemberton LF: Karyopherins: from nuclear-transport mediators to nuclear-function regulators. Trends Cell Biol. 2004, 14 (10): 547-556. 10.1016/j.tcb.2004.09.004.View ArticlePubMedGoogle Scholar
- Pemberton LF, Paschal BM: Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic. 2005, 6 (3): 187-198. 10.1111/j.1600-0854.2005.00270.x.View ArticlePubMedGoogle Scholar
- Strom AC, Weis K: Importin-beta-like nuclear transport receptors. Genome Biol. 2001, 2 (6): REVIEWS3008-10.1186/gb-2001-2-6-reviews3008.PubMed CentralView ArticlePubMedGoogle Scholar
- Fried H, Kutay U: Nucleocytoplasmic transport: taking an inventory. Cell Mol Life Sci. 2003, 60 (8): 1659-1688. 10.1007/s00018-003-3070-3.View ArticlePubMedGoogle Scholar
- Cook A, Bono F, Jinek M, Conti E: Structural biology of nucleocytoplasmic transport. Annu Rev Biochem. 2007, 76: 647-671. 10.1146/annurev.biochem.76.052705.161529.View ArticlePubMedGoogle Scholar
- Lange A, Mills RE, Lange CJ, Stewart M, Devine SE, Corbett AH: Classical nuclear localization signals: definition, function, and interaction with importin alpha. J Biol Chem. 2007, 282 (8): 5101-5105. 10.1074/jbc.R600026200.PubMed CentralView ArticlePubMedGoogle Scholar
- Enenkel C, Blobel G, Rexach M: Identification of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes. J Biol Chem. 1995, 270 (28): 16499-16502. 10.1074/jbc.270.28.16499.View ArticlePubMedGoogle Scholar
- Goldfarb DS, Corbett AH, Mason DA, Harreman MT, Adam SA: Importin alpha: a multipurpose nuclear-transport receptor. Trends Cell Biol. 2004, 14 (9): 505-514. 10.1016/j.tcb.2004.07.016.View ArticlePubMedGoogle Scholar
- Loeb JD, Schlenstedt G, Pellman D, Kornitzer D, Silver PA, Fink GR: The yeast nuclear import receptor is required for mitosis. Proc Natl Acad Sci USA. 1995, 92 (17): 7647-7651. 10.1073/pnas.92.17.7647.PubMed CentralView ArticlePubMedGoogle Scholar
- Hood JK, Silver PA: Cse1p is required for export of Srp1p/importin-alpha from the nucleus in Saccharomyces cerevisiae. J Biol Chem. 1998, 273 (52): 35142-35146. 10.1074/jbc.273.52.35142.View ArticlePubMedGoogle Scholar
- Solsbacher J, Maurer P, Bischoff FR, Schlenstedt G: Cse1p is involved in export of yeast importin alpha from the nucleus. Mol Cell Biol. 1998, 18 (11): 6805-6815.PubMed CentralView ArticlePubMedGoogle Scholar
- Mendenhall MD, Hodge AE: Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1998, 62 (4): 1191-1243.PubMed CentralPubMedGoogle Scholar
- Cho RJ, Campbell MJ, Winzeler EA, Steinmetz L, Conway A, Wodicka L, Wolfsberg TG, Gabrielian AE, Landsman D, Lockhart DJ: A genome-wide transcriptional analysis of the mitotic cell cycle. Mol Cell. 1998, 2 (1): 65-73. 10.1016/S1097-2765(00)80114-8.View ArticlePubMedGoogle Scholar
- Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Eisen MB, Brown PO, Botstein D, Futcher B: Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol Biol Cell. 1998, 9 (12): 3273-3297.PubMed CentralView ArticlePubMedGoogle Scholar
- Breeden L: Start-specific transcription in yeast. Curr Top Microbiol Immunol. 1996, 208: 95-127.PubMedGoogle Scholar
- Breeden LL: Periodic transcription: a cycle within a cycle. Curr Biol. 2003, 13 (1): R31-38. 10.1016/S0960-9822(02)01386-6.View ArticlePubMedGoogle Scholar
- Wittenberg C, Reed SI: Cell cycle-dependent transcription in yeast: promoters, transcription factors, and transcriptomes. Oncogene. 2005, 24 (17): 2746-2755. 10.1038/sj.onc.1208606.View ArticlePubMedGoogle Scholar
- Bean JM, Siggia ED, Cross FR: High functional overlap between MluI cell-cycle box binding factor and Swi4/6 cell-cycle box binding factor in the G1/S transcriptional program in Saccharomyces cerevisiae. Genetics. 2005, 171 (1): 49-61. 10.1534/genetics.105.044560.PubMed CentralView ArticlePubMedGoogle Scholar
- Cosma MP: Ordered recruitment: gene-specific mechanism of transcription activation. Mol Cell. 2002, 10 (2): 227-236. 10.1016/S1097-2765(02)00604-4.View ArticlePubMedGoogle Scholar
- Costanzo M, Nishikawa JL, Tang X, Millman JS, Schub O, Breitkreuz K, Dewar D, Rupes I, Andrews B, Tyers M: CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast. Cell. 2004, 117 (7): 899-913. 10.1016/j.cell.2004.05.024.View ArticlePubMedGoogle Scholar
- de Bruin RA, McDonald WH, Kalashnikova TI, Yates , Wittenberg C: Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5. Cell. 2004, 117 (7): 887-898. 10.1016/j.cell.2004.05.025.View ArticlePubMedGoogle Scholar
- Wang H, Gari E, Verges E, Gallego C, Aldea M: Recruitment of Cdc28 by Whi3 restricts nuclear accumulation of the G1 cyclin-Cdk complex to late G1. EMBO J. 2004, 23 (1): 180-190. 10.1038/sj.emboj.7600022.PubMed CentralView ArticlePubMedGoogle Scholar
- Wagner MV, Smolka MB, de Bruin RA, Zhou H, Wittenberg C, Dowdy SF: Whi5 regulation by site specific CDK-phosphorylation in Saccharomyces cerevisiae. PLoS One. 2009, 4 (1): e4300-10.1371/journal.pone.0004300.PubMed CentralView ArticlePubMedGoogle Scholar
- Skotheim JM, Di Talia S, Siggia ED, Cross FR: Positive feedback of G1 cyclins ensures coherent cell cycle entry. Nature. 2008, 454 (7202): 291-296. 10.1038/nature07118.PubMed CentralView ArticlePubMedGoogle Scholar
- Koch C, Schleiffer A, Ammerer G, Nasmyth K: Switching transcription on and off during the yeast cell cycle: Cln/Cdc28 kinases activate bound transcription factor SBF (Swi4/Swi6) at start, whereas Clb/Cdc28 kinases displace it from the promoter in G2. Genes Dev. 1996, 10 (2): 129-141. 10.1101/gad.10.2.129.View ArticlePubMedGoogle Scholar
- de Bruin RA, Kalashnikova TI, Chahwan C, McDonald WH, Wohlschlegel J, Yates J, Russell P, Wittenberg C: Constraining G1-specific transcription to late G1 phase: the MBF-associated corepressor Nrm1 acts via negative feedback. Mol Cell. 2006, 23 (4): 483-496. 10.1016/j.molcel.2006.06.025.View ArticlePubMedGoogle Scholar
- Baetz K, Andrews B: Regulation of cell cycle transcription factor Swi4 through auto-inhibition of DNA binding. Mol Cell Biol. 1999, 19 (10): 6729-6741.PubMed CentralPubMedGoogle Scholar
- Geymonat M, Spanos A, Wells GP, Smerdon SJ, Sedgwick SG: Clb6/Cdc28 and Cdc14 regulate phosphorylation status and cellular localization of Swi6. Mol Cell Biol. 2004, 24 (6): 2277-2285. 10.1128/MCB.24.6.2277-2285.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Queralt E, Igual JC: Cell cycle activation of the Swi6p transcription factor is linked to nucleocytoplasmic shuttling. Mol Cell Biol. 2003, 23 (9): 3126-3140. 10.1128/MCB.23.9.3126-3140.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Sidorova JM, Mikesell GE, Breeden LL: Cell cycle-regulated phosphorylation of Swi6 controls its nuclear localization. Mol Biol Cell. 1995, 6 (12): 1641-1658.PubMed CentralView ArticlePubMedGoogle Scholar
- Harreman MT, Kline TM, Milford HG, Harben MB, Hodel AE, Corbett AH: Regulation of nuclear import by phosphorylation adjacent to nuclear localization signals. J Biol Chem. 2004, 279 (20): 20613-20621. 10.1074/jbc.M401720200.View ArticlePubMedGoogle Scholar
- Taberner FJ, Quilis I, Igual JC: Spatial regulation of the Start repressor Whi5. Cell Cycle. 2009, 8 (18): 3013-3022.View ArticleGoogle Scholar
- Xiao Z, McGrew JT, Schroeder AJ, Fitzgerald-Hayes M: CSE1 and CSE2, two new genes required for accurate mitotic chromosome segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1993, 13 (8): 4691-4702.PubMed CentralView ArticlePubMedGoogle Scholar
- Fries T, Betz C, Sohn K, Caesar S, Schlenstedt G, Bailer SM: A novel conserved nuclear localization signal is recognized by a group of yeast importins. J Biol Chem. 2007, 282 (27): 19292-19301. 10.1074/jbc.M700217200.View ArticlePubMedGoogle Scholar
- Strahl T, Hama H, DeWald DB, Thorner J: Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus. J Cell Biol. 2005, 171 (6): 967-979. 10.1083/jcb.200504104.PubMed CentralView ArticlePubMedGoogle Scholar
- Nasmyth K: Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu Rev Genet. 2001, 35: 673-745. 10.1146/annurev.genet.35.102401.091334.View ArticlePubMedGoogle Scholar
- Uhlmann F: Chromosome cohesion and separation: from men and molecules. Curr Biol. 2003, 13 (3): R104-114. 10.1016/S0960-9822(03)00039-3.View ArticlePubMedGoogle Scholar
- Franke J, Reimann B, Hartmann E, Kohlerl M, Wiedmann B: Evidence for a nuclear passage of nascent polypeptide-associated complex subunits in yeast. J Cell Sci. 2001, 114 (Pt 14): 2641-2648.PubMedGoogle Scholar
- Mosammaparast N, Guo Y, Shabanowitz J, Hunt DF, Pemberton LF: Pathways mediating the nuclear import of histones H3 and H4 in yeast. J Biol Chem. 2002, 277 (1): 862-868. 10.1074/jbc.M106845200.View ArticlePubMedGoogle Scholar
- Rout MP, Blobel G, Aitchison JD: A distinct nuclear import pathway used by ribosomal proteins. Cell. 1997, 89 (5): 715-725. 10.1016/S0092-8674(00)80254-8.View ArticlePubMedGoogle Scholar
- Schaper S, Franke J, Meijsing SH, Ehrenhofer-Murray AE: Nuclear import of the histone acetyltransferase complex SAS-I in Saccharomyces cerevisiae. J Cell Sci. 2005, 118 (Pt 7): 1473-1484. 10.1242/jcs.01739.View ArticlePubMedGoogle Scholar
- Bakhrat A, Baranes K, Krichevsky O, Rom I, Schlenstedt G, Pietrokovski S, Raveh D: Nuclear import of ho endonuclease utilizes two nuclear localization signals and four importins of the ribosomal import system. J Biol Chem. 2006, 281 (18): 12218-12226. 10.1074/jbc.M600238200.View ArticlePubMedGoogle Scholar
- Miller ME, Cross FR: Mechanisms controlling subcellular localization of the G(1) cyclins Cln2p and Cln3p in budding yeast. Mol Cell Biol. 2001, 21 (18): 6292-6311. 10.1128/MCB.21.18.6292-6311.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Edgington NP, Futcher B: Relationship between the function and the location of G1 cyclins in S. cerevisiae. J Cell Sci. 2001, 114 (24): 4599-4611.PubMedGoogle Scholar
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AGM have signed a Joint Development Agreement with German based oil company PURAGLOBE GmbH. Since entering into a formal JDA, PURAGLOBE and AGM have been collaborating to understand how AGM's graphene can best be incorporated into PURAGLOBE's products based on SYNTAINICS® technology. This has progressed to a point where we are now forming trip-partite agreements, particularly with metalworking and cutting fluids but also adjacent market companies. A database of the performance of several of PURAGLOBE's products containing graphene has been generated by an OEM accredited and certified German test lab and will help speed up the acceptance as well as proof that the performance of these unique materials is ready to be used in the industry.
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|View from Cottage
You may have noticed this image is sketched on canvas and displays some fairly dynamic impasto/sgraffito strokes. Well, that is because I finally got round to testing my giant Sennelier oil pastels and I can honestly say they are absolutely amazing for sketching. This image was made in a 30 x 40cm canvas sketchbook using white, medium grey and black pastels with a craft knife for scratching and a blending stump for blending.
|Holyrood Raven
Pesky things never sit still so here we go again with the great photo debate - well I am firmly on the side of taking reference images, I love taking photos, I do it more than sketching - but as many say, it is often best to use reference as inspiration rather than slavishly copy. I mostly use photos for initial form and lighting info then stop looking and let the image grow by itself. Here I quite liked the slightly irksome distribution of tone and the way the contrast between bird and bleached out background makes my eyes dance in a jarring kind of way. Art shouldn't always be about balance and harmony you know, shake things up! Why should the viewer always get their creative nuggets spoon fed? :D
|Salisbury Crags
|Three Graces, Canova
|Wolf Head


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How many of you know that Cleveland is home to a huge salt mine? Cargill owns a mine 1800 feet under Lake Erie and four miles out from downtown. This Cleveland mine and another out by Fairport Harbor combine to produce a huge portion of the salt mined in North America.
And have any of you ever heard of the Salt Cathedral in Poland? I wouldn't know anything about this one [show image] but for my daughter having spent a college semester in the Czech Republic. While she was there, she was able to travel quite a bit, and on a trip to Poland she learned about this enormous salt mine, now closed, in which the workers left quite the record of their presence, carving out rooms and sculptures and an entire chapel, which is often in use for weddings! Imagine getting married in a salt mine! Even those chandeliers are made of salt, salt that's been treated until it is so fine that it looks like glass. And the sculptures, like this one of The Last Supper [shpw], look like granite – but they, too, are salt.
A salt mine, whether it remains in its original state, as the Cleveland mines do, or has been transformed by Polish miners, is a pretty spectacular sight. Huge deposits of salt carved into rooms so that miners can reach their goal – huge deposits far underground, in places most of us don't know exist. Huge deposits of something we take completely for granted – I'm sure there are several salt shakers in your homes – but which, until about 100 years ago, was a product of great value, difficult and expensive to attain.
I want you to think about the church for a moment. Think about the church, this church, as salt. At one time, it was a beacon of great value. A tremendous amount of money, and energy, and time, went into the building of this church, starting with this Foster Hall as its first sanctuary. Back in the 1960s, and in the 1860s, churches like ours were deemed to be of momentous importance. They popped up in neighborhoods all over the place, and were filled with people who were called to flavor their communities with the salt of God's love.
Have we now reached the point where churches like ours, like salt, are taken for granted? People expect to see them, and most of the time they walk right on by, just as we expect to find salt in our kitchens and on our tables without taking any notice of them. When folks think they need a church, say for a funeral or for a wedding, they expect us to be here, just as we expect to find a salt shaker when we reach for one at the dinner table.
What has happened to our sense that we are salt for the world? Have we become more like underground deposits of salt than like salt scattered across the globe?
Deposits of salt, by themselves, are not of much use to us. They go unnoticed – until just a few years ago, I had no idea that Lake Erie's waters covered salt mines – and even if they are transformed inside, as Poland's salt mine was, very few people realize that they are there. Salt has to be brought out into the open before it becomes a commodity useful in preserving and preparing food. Useful in favoring the world.
And once it's in the open, salt plays amazing roles in human relationships. Perhaps even in human relationships with God. That's what both history and the Bible tell us.
There's a wonderful book entitled SALT. The nonfiction story of SALT is a whirlwind tour through world history, through which the compound salt runs as a crucial, savory thread. Salt lies at the bottom of major world events: exploration, trade, currency, revolution, warfare. SALT could probably be turned into a smash hit television miniseries, filled with travel and adventure and danger and romance and royalty.
Salt, above ground and out in the open, creates transformation. Mine salt, and people start looking for more, and start to discover new possibilities. Sprinkle salt around, and revolutions start: people see that life can be different than it is.
What does that mean for us? If we stay as we are, we are like a salt mine under the lake – no one knows that we are here. If we create something beautiful here in our own church and keep it here, we're like the Polish salt mine – an intriguing place for tourists and brides, but not an exciting force in the world. BUT – if we mine our salt, if we dig deep into scripture and prayer and worship, we will start to discover possibilities that have never occurred to us. And if we start salting our neighborhood, perhaps a revolution will begin, will begin with a vision of new life and energy on Lake Shore Boulevard.
Salt was a valuable commodity long before Jesus' time, and a valuable commodity in Jesus' time. And it's a commodity with a specifically Biblical history. Did you know that in the Old Testament, in the Book of Numbers (18:19), God refers to temple sacrifices as a sign of God's covenant with them, as a sign of the "salt covenant?" And that in the Book of Leviticus (2:13), God instructs the people, through Moses, saying, "You shall not omit from your grain-offerings the salt of the covenant with your God; with all your offerings you shall offer salt." Salt was from the earliest times of the life of the Hebrew people a sign of God's promises to them and of their reverence for God; salt, that valuable and hard-won commodity, was a sign of the love of God and of relationship with God.
Those Biblical allusions would have been well known to the people to whom Jesus spoke, and the people to whom Matthew wrote. One of the important things to know about the writer whom we call Matthew is that he seems to have been a Jew intent upon convincing his largely Jewish audience that Jesus was indeed the messiah promised in scripture. Matthew spends a great deal of time on Jewish customs and scriptural passages, making all sorts of connections between Jesus and earlier writings. Some of these connections we might not recognize right away. We know, for instance, that salt is important. We know that while we take it for granted, not so long ago it could not be taken for granted at all. But we may not realize that in Jewish life, salt was a sign: a sign of God's covenantal relationship with God's people, a sign of God's presence and promise of love and care. The word "salt" and all that it meant was probably obvious to those who listened to Jesus, and to those who listened to or read the words of Matthew, but we ourselves have to give it some thought.
And what is it Jesus says about salt? In our reading today, he's still up on the mountain, preaching the words we know as the Sermon on the Mount, and in The Message translation, which we read this morning, he says, "You're here to be salt-seasoning." Interestingly, in the NRSV, the translation we usually used, he says, "You are the salt of the earth." You ARE. Or, "you ARE here TO BE." Present tense.[1] Not "you will be" or "you might be" or "you could be." But you ARE. Already.
Imagine what those words meant to Jesus' listeners. Matthew's listeners. They already knew what salt meant: God's presence. God's covenant. God's love. Now they know something else: They – we – are God's presence and covenant and love to others. We ARE the salt of the earth.
Notice something else that Jesus tells them: "I come not to demolish the law or the prophets, but to complete them." More evidence that Jesus is paying attention to Jewish life – the law, those commandments God gave Moses on another mountain long before – but expanding that law. Not throwing it out at all, but saying instead: You are the salt who will help the make the love which is the foundation of the law known. And Jesus is paying attention to the prophets, those who proclaim God's justice, those who clarify what we are to do: Jesus says: You are salt; bring out God-flavors in this world.
And how, I ask you again, will we do that? Not by leaving God's gift of love buried deep under the ground, like a salt mine is. Not by allowing it to remain beautiful but hidden, like the Salt Cathedral in Poland.
Not merely by saying, "we have all that's needed, here in this underground church mine that we run. We can store God's love safely right here." Not by saying, "We've got something beautiful here, and we'll wait for people to come and find out – as people traveling in Eastern Europe happen upon the Salt Cathedral."
No. Instead, we're going to start asking ourselves: Are we leaving the salt of God in a subterranean cavern today? Are we forgetting that subterranean salt is for sharing with the world?
Our charge comes from the God who made salt a sign of promise ro God's Old Testament people. Our charge comes from Jesus, who took that sign and said, "You are the ones! You are the salt!" Our charge comes from Matthew, who wrote a gospel so that Jesus' words would echo across the centuries. Our charge comes from the Spirit, who says to us:
Mine the salt! Bring it up to the surface and scatter it around a world in need of the taste of the Lord! Remember that salt glistens with the presence and promise of God – and so do you. For you ARE the salt of the earth and you ARE the light of the world. Amen
[1] Amy Oden. "Preaching This Week." workingpreacher.org, 2014.


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Wastewater Treatment Plants
Wastewater treatment plants remove contaminants from wastewater generated by water runoff and from household and commercial sewage. Runoff generally refers to rainfall that may pick up sediment, metals, organic compounds, animal wastes, oil and grease as it travels across the ground. There are still some open sewers that dump runoff into a river or stream, but more and more are being converted to closed sewers that are collected at a treatment plant.
Sewage – waste from toilets, baths, showers, kitchens and sinks – is created by residents, institutions and businesses. Wastewater is 80 percent soapy water from showers, dishwashers and laundry and only 20 percent by volume from toilets.
Wastewater treatment generally involves three stages: primary, secondary and tertiary treatment. Primary treatment consists of holding sewage in a basin where heavy solids settle to the bottom as oil, grease and lighter solids float to the surface. The settled and floating materials are removed and the remaining liquid can be discharged or subjected to secondary treatment. Secondary treatment removes dissolved and suspended biological matter, typically through the use of indigenous, water-borne micro-organisms in a managed habitat. Tertiary treatment refers to any treatment after the secondary phase. Treated wastewater can be discharged into bodies of water or used for irrigation, or it can be used for groundwater recharge or agricultural purposes. Most of today's tertiary treated water is cleaner than the stream or river into which it is discharged.
AMERICAN ductile iron pipe is a long-established choice for wastewater treatment plants. Its wide selection of joints and fittings can be fabricated for any application, including buried and exposed. In particular, the additional joint deflection offered by AMERICAN's exclusive boltless Flex-Ring joint is ideal for pipe and valve installations in and around structures and loaded tanks. AMERICAN provides a range of linings and coatings designed for different situations. In addition, AMERICAN's line of valves and spiral-welded steel pipe make the company a premier manufacturer of wastewater treatment plant products.