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Road Map
Going down the HEALTH ROUTE (Children)_ - first steps
ROUTE MAP or maybe ROUTE MARCH!
For those of you who are not good with directions (I am one of them) and those of you who are familiar with Spaghetti Junction – you will be grimacing now for sure! Get your strongest shoes on, grab a flask of tea (maybe something stronger!) and let's get started. Please don't expect miracles or quick fix solutions, the wheels turn VERY slowly. This article is intended for use with children. For those of you who are adults or adolescents who are in search of a diagnosis, please read the article entitled 'ADULT NEEDING DIAGNOSIS, HELP!
The minute a parent 'flags' up an issue with their child's behaviour, eyebrows appear to become raised and fingers of blame are pointed. It is sadly a sign of the times. Parents by enlarge get blamed for most things, including the behaviour of their little sweetie. It's a tough decision for many to seek help, but for those that do here are a few hints to prepare you. Please note for international readers this information is applicable to the UK.
Generally parents head for their family doctor or if the child is school age it could be the teacher heading for the parents!
But given the choice which way is the best?
Let's explore both routes and find out who everyone is. You may come into contact with a few people. Let's take the mystery out of it. You may not have to see everyone on the list beneath but it is likely that you will have to make more than one visit for accurate assessment and diagnosis. Be prepared to be handed from one to the other like pass the parcel! Latest research suggests that for every ONE child suspected of being on the Autism Spectrum – families will need to see 16 professionals. For those families with multiple children – well you do the maths!
General Practitioner/Family doctor
This is a fully qualified doctor who has trained separately to become a general community doctor looking after the needs of people within their community. These doctors are generally consultant level. It will take approximately 10yrs to reach this position and involves graduate/doctoral level at university/medical school.
Consultant Paediatrician or Psychiatrist
These are expert doctors specialising in children and mental health. It will take approximately 13yrs reach this position at both graduate and post graduate/doctoral level at university/medical school.
Community Paediatrician
Community paediatric doctors are a team of specialist children's doctors with skills and expertise in child development, immunisation, social and educational paediatrics. They work within a number of multidisciplinary teams to assess and meet the health needs of children and young people with disabilities or special needs, those who are 'Looked After' or where there are concerns regarding a child's safety. It will take approximately 8 – 10yrs to reach consultant level at doctoral level.
Consultant Neurologist
A Consultant Neurologist is a doctor specialising in diseases and illnesses of the nervous system and brain. He/She will deal with epilepsy, multiple scleroses, headache, brain injury, neck and back injury, stroke, nerve damage, neuro-degenerative disorders, neurodevelopmental conditions and Parkinson's disease. It will take minimum of 13yrs to reach this level. There are only a very small number of Paediatric Neurologists in the UK.
There are a variety of these; Educational, Clinical, Chartered being the ones you will more than likely come into contact with. These professionals will use specific tests to establish a diagnosis and to advise further. Some are able to diagnose as well as assess. You may see more than one! A specialist Psychologist will have undertaken minimum 6yrs training at graduate and post graduate level at university.
Health Visitor
Health visitors are Registered Nurses (min 4yrs training at university/hospital clinical setting) who have undertaken further training to work as part of a primary health care team. They specialise in child development and will see children from birth through to adolescent. It has to be said though that predominantly they work with the under 5's and pre schoolers. As their name suggests, their role is to promote mental, physical and social well-being in the community by giving advice and support to families in all age groups. Health visitors will train for approximately 6yrs to become qualified.
Specialist Autism Nurse
These are Registered Nurses nurses who have post graduate qualification and experience in Autism and associated conditions. Nurses with this experience can take medical histories, train and advise on a one to one level or clinic based. It will take 4yrs of general nursing training followed by post graduate 1 – 5yrs courses at university.
Speech and Language Therapists
A specialist in speech and language difficulties Communication includes speech (articulation, intonation, rate, intensity, voice, resonance, and fluency), language (phonology, morphology, syntax, semantics, and pragmatics), both receptive and expressive language (including reading and writing). Speech and Language Therapists train for 3yrs at graduate level.
Occupational Therapist
Occupational Therapists provide services to improve the patient's ability to accomplish everyday tasks associated with a maximum level of safe independence.
Occupational therapists assess functioning in activities of everyday living, including dressing, bathing, grooming, meal preparation, writing, and driving, which are essential for independent living. Occupational Therapists train for a minimum of 2yrs at graduate level but can take up to 6yrs depending on speciality.
Social Worker
A trained specialist in the social, emotional and financial needs of families and patients. Social workers often help families and patients obtain the services they have been prescribed. It is usual for a family in need to be appointed a Social Worker who can co-ordinate other services. Social Workers will have trained for minimum 4yrs at graduate and post graduate level.
Psychotherapists work with individuals, couples, families and groups to help them overcome a range of psychological and emotional concerns. With the client as an active participant, psychotherapists use personal treatment plans and a variety of non-medical-based treatments the clinical training to become a Psychotherapist takes a minimum of 4 years (part time) and is the responsibility of each training institution to decide when a trainee is fit to qualify. In general, to work as psychotherapists, individuals will need to undertake training recognized/accredited by the Association of Psychotherapists or equivalent. It should be noted that anyone can call themselves a 'psychotherapist'. We recommend you check they are qualified and recognised by an official organisation.
An audiologist is a healthcare professional specializing in identifying, diagnosing, treating and monitoring disorders of the auditory and vestibular system portions of the ear. Audiologists are trained to diagnose, manage and/or treat hearing or balance problems. It takes approximately 7yrs to train at doctoral level.
An orthoptist investigates diagnoses and treats sight-related problems and abnormalities of eye movement and eye position. Orthoptists carry out tests to diagnose problems and to determine appropriate management. They work with patients of all ages, from the very young to the elderly, and use specialist skills to help improve their vision. It takes approximately 4yrs at graduate and post graduate level.
Play therapists, art therapists, music therapy.
Creative therapy refers to a group of techniques that are expressive and creative in nature. It enables clients to find another form of expression beyond words. This provides limitless modes of expression. Therapies can include sand play, clay, movement workshops, and role play with music therapy. It can take anything from a year to 3yrs to train depending on the level of course entry.
Outreach workers
Outreach workers are, by definition, employed to reach out to the community in a direct way and much of their work will focus on family homes or education and play centres used by the Early Years Development and Childcare Partnerships. Training can be ongoing with a good educational background and qualifications to support it.
So as you can see the list is long and we haven't included EVERYONE here, this is just a taster of the most likely! Everyone is different so no one follows exactly the same path.
As you can probably understand the path is a long difficult and arduous one. It is no wonder parents are constantly feeling overwhelmed.
Jelibean aims to offer a 'wraparound' approach putting everything under one roof. One day we will achieve our aim of 'one stop Jelishop' everywhere in the country.
For the other route, please click into – Going the Education Route, find out what to expect!


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What is Search Engine Optimization (SEO)?
Search Engine Optimization (SEO) is the practice of optimizing one's website to improve its rankings on major search engines (primarily Google and Bing). The traffic resulting from effective SEO is often referred to as "organic" or "natural" traffic, as opposed to the "paid" traffic generated from buying clicks through Pay-Per-Click advertising. While PPC advertising can drive instant results at a cost, SEO pays great dividends in the long-term in the form of free traffic.
How Does SEO Work?
Anyone with a website can relate to the need to drive more visitors to their website, without paying for every click. While the investment for SEO may not provide an instant return, having a well optimized site can significantly reduce your marketing investment over time. From lead generation and eCommerce, to driving general brand awareness, great SEO helps Google and Bing connect the right searches to your content.
Get a Free SEO Service Consultation
The Value of SEO
Organic / Free Traffic
Unlike paid advertising, the visits resulting from SEO efforts are free. The costs associated with SEO are driven by labor put into improving the website versus paying for the traffic itself.
High Quality Visitors
Search engines are judged by how effectively they tie website content to user searches. The visitors that result from organic searches are generally seeking the content the search engine has provided – hopefully your content.
Trackable ROI
Just as with PPC, effective SEO involves tracking many web metrics and systematically improving them over time. Associated costs are very traceable and return on investment is fairly easy to get to.

subset: commoncrawl


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"Well…erm…well, you know why you're here. Erm…well, Harry here had the idea – I mean" – Harry had thrown her a sharp look – "I had the idea – that it might be good if people who wanted to study Defense Against the Dark Arts – and I mean, really study it, you know, not the rubbish that Umbridge is doing with us" – (Hermione's voice became suddenly much stronger and more confident) – "because nobody could call that Defense Against the Dark Arts" – "Hear, hear," said Anthony Goldstein, and Hermione looked heartened – "well, I thought it would be good if we, well, took matters into our own hands." (p. 339 in Harry Potter and the Order of the Phoenix, 2003)
In the movie version, this is simplified to the line, "We need a teacher, a proper teacher."
The students of Hogwarts realized that they were not going to learn the real spells and magic they would need if they just accepted the status quo being delivered by Dolores Umbridge and her dull and useless textbook. Hermione was intelligent enough to recognize that her friend Harry had experience and knowledge that he could teach them. The group was formed, a place for secret practice was found, and "Dumbledore's Army" was born.
Maybe the analogy of preparing for war in the wizarding world seems extreme for those returning to school here in New York State, but as the nation debates the possible consequences of engaging in real war in Syria, Governor Cuomo ramped up the hostile rhetoric today by saying failing schools deserve the "death penalty." Yes, you read that correctly, it's a quote. I could hardly believe it when I saw it. What's really failing is the leadership in Albany that has accepted the federal bribe money through Race to the Top and is creating havoc with lousy tests, inappropriate measures of teacher performance and school quality, and a privatization plan that is unproven and as scary as the return of Lord Voldemort.
So teachers and students, don't despair. You don't have to start this year in fear – of failing, of being unfairly judged, of seeing your school closed or resources cut to the bone. It's time to learn how to defend yourself against these dark times. Here are some tips for starting the year off strong:
1. Read and get informed. That means not just the news, which is sorely lacking in investigative journalists, at least in education. Find the blogs, the active Twitter users, the Facebook groups, the organizations of people who are actively working for positive change and supporting the public schools that are under assault. A good place to start is Diane Ravitch's new book Reign of Error:The Hoax of the Privatization Movement and the Danger to America's PublicSchools, out in mid-September.
2. Practice your talking points. You will need these not just for staff meetings, informal lunch conversations, PTA meetings, or school board testimonies. You need to start talking at dinner parties, after lectures and book talks, at the gym, in the taxi, anywhere you find people willing to engage in conversation so they too can be informed and involved.
3. Get inspired. Learn from history, from role model activists like Bayard Rustin, who just was awarded the Presidential Medal of Freedom posthumously (you can see a beautiful documentary about him called Brother Outsider: The Life ofBayard Rustin).
4. Show off and be proud of your many accomplishments. You have bulletin boards, windows and walls, and strings to hang things from across your classrooms, and websites and email newsletters, and concert and play performances, and charitable actions and good deeds. Let the world know that you are proud of what you do.
5. Find strength in numbers. Join up with others, make a regular working lunch date to talk and strategize, organize pot lucks, ask for volunteers, help people who want to get involved but don't know how to begin.
6. Make time for play, and joy, and being creative. If that doesn't come naturally, maybe you can start by organizing a cardboard challenge on October 8th for some kids and let them teach you.
7. Ask for help and support when you need it. It's normal to have bad days, to feel depressed and overwhelmed. Reach out to your friends and let them know you need their encouragement and kind words.
8. Sometimes you just need to go on a march. If that seems old-fashioned or ineffective, read about the 50th anniversary of the 1963 March for Jobs and Freedom and listen to the speeches of Martin Luther King Jr.'s family, Rep. John Lewis, President Obama, and others who commemorated the anniversary on August 28th.
Most of all, have a good year. Do good, and be well.


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Ranch dressing is great for salads and dipping vegetables. Store bought ranch dressing and dips are loaded with fats and preservatives and are definitely not vegan. Here's a great recipe that's super quick and easy to whip up!
vegan mayo (I use vegenaise)
italian spices
vegan milk (soy, almond, rice, etc)
salt and pepper, to taste
Start off with adding some mayo into a bowl (it will be the main base of the dressing). Add some milk, how much you use depends on how thick you want your ranch. If you want it like a dip, just add a little bit but if you want it for a dressing on salads add more. Use a fork to blend the mayo and milk. Add more milk or mayo to get desired consistency. Then go crazy with spices. I use the Italian grinders you can find which have rosemary, oregano and basil. You can also use fresh herbs or whichever dry herbs you enjoy the most. The fresh herbs won't keep as long so keep that in mind. Add salt and pepper to taste and you're done!
This dip would also be a good dip for the fried okra recipe that I just posted!


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Ready to ship gift!
This unique handmade knit hooded scarf would make a great winter accessory for a man or a woman.
This hooded-Scarf is many tones of soft blue yarn.
The scarf is a mobius strip.
Really Nice for fall and winter warmth.
You can wear the hood up or down and it will stay right around your neck ,like a blue cowl or regular scarf, til
you are ready to take it off.
This is a gorgeous one of a kind blue tones hooded scarf .
The infinity mobius scarf can be wrapped around your neck a second time, or just lay in a single loop around your neck.
It offers unique style as well as warmth and would make an excellent gift for him or her this christmas or hannakah .
It is extra soft and fuzzy.
However, this hooded-scarf could be worn most of the year if you wish .
Be the Brightest most unique person on any occasion!
Or give this hat as a stockng stuffer, would make a great gift for your girlfriend or wife.
This scarf hood would make a great gift for almost anyone, be it adults men or women, teens or kids.
This high quality unique and cozy brilliant hooded scarf would look great on any occasion.
It will keep you warm and whimsical.
Ask me about custom color ideas/ options, and wholesale or boutique orders.
Thanks for looking!
Check out my other scarves and hooded scarves here-
shop home- https://www.etsy.com/shop/LeafLee?ref=si_shop
1-3 business days
Buyers are responsible for any customs and import taxes that may apply. I'm not responsible for delays due to customs.
Just contact me within: 3 days of delivery
Ship items back to me within: 7 days of delivery
But please contact me if you have any problems with your order.
Because of the nature of these items, unless they arrive damaged or defective, I can't accept returns for:
Buyers are responsible for return shipping costs. If the item is not returned in its original condition, the buyer is responsible for any loss in value.


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Chocolate Cake in a Mug
Total TimePrep/Total Time: 10 min.
Makes1 serving
- 1/4 cup sugar
- 1/4 cup all-purpose flour
- 2 tablespoons baking cocoa
- 1/8 teaspoon salt
- 1 egg
- 3 tablespoons canola oil
- 3 tablespoons 2% milk
- 1/8 teaspoon vanilla extract
- 3 tablespoons semisweet chocolate chips
- Optional toppings: coffee ice cream and caramel ice cream topping
- In a small bowl, combine the sugar, flour, cocoa and salt. In another bowl, whisk the egg, oil, milk and vanilla. Stir into dry ingredients just until moistened. Stir in chocolate chips.
- Transfer to a 14-oz. microwave-safe mug coated with cooking spray. Microwave, uncovered, on high for 1-1/4 to 1-1/2 minutes or until top appears dry. Cool for 1 minute. Serve with toppings of your choice.
Editor's NoteThis recipe was tested in a 1,100-watt microwave.
Nutrition Facts1 cake (calculated without optional toppings): 955 calories, 59g fat (11g saturated fat), 215mg cholesterol, 392mg sodium, 102g carbohydrate (70g sugars, 5g fiber), 14g protein.
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Mar 4, 2020
Subtly sweet but just the right size when you don't want to make a whole cake. I would say this is definitely more than one serving, though. It's enough for two people to share. I made it in an 8 cup pyrex baking dish and it fit perfectly.
Apr 11, 2015
It was okay....quick fix for something sweet
Jan 31, 2014
Wonderful chocolate fix - quick and easy! Works great in a taller coffee mug and 1 1/2 min seems right on the time. I topped ours with a scoop of ice cream and drizzle of homemade chocolate sauce.
Feb 17, 2013
Made this into 3 seperate servings and poured into ramekins not mugs. Added a dollop of whipped cream and my family fell in love with this recipe!Little mess and a quiçk dessert for company!
Oct 20, 2012
The flavor was not a rich as I would have liked. This also made a huge mess in my coffee cups. If I make it again, I will have to spray the cups with A LOT of no-stick.
Oct 7, 2012
Fun and easy for a quick chocolate fix, even without the chips! I can't believe this is 955 calories, though. I hope it's a mistake.
Aug 2, 2012
Made this for my wife & I. This was excellent! One mug is enough for 2 people, unless you really need a "big" chocolate fix! I fixed one mug with 2 tbls of mini-chocolate chips and 1 with just 1 tbls of chips and thought that 1 tbls was enough. You decide how rich you want it. Also, go to the bottom of cup with each spoonful to get the rich gooey stuff. It was great with either vanilla or coffee ice cream...we tried both.
Jun 29, 2012
I've already made is several times. It really makes 2 to 4 pieces. I even take a tablespoon of butter and cocoa and mix in 3 to 6 tablespoons of powdered sugar. Add a little vanilla and milk to make frosting. Yummy! Try adding cinnamon to your taste. That's good too!


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Interview with Antonio Mora – Surreal & Lucid Artist
Imagine walking to the deep end of your subconscious and learn about your true desires. Imagine reaching your highest level of awareness. Imagine an extraordinary experience that explores your resilience which flows through the self-imposed boundaries of the intellect. Feel the sensation of the current of your own agency to revive your strength and the superhero … More Interview with Antonio Mora – Surreal & Lucid Artist


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A journal about the Third Chapter, my life as a widow. Cooking-for-one, Entertaining, Travel, Grief, Family, Friends, Ageing, Photography, Living in San Miguel de Allende, GTO, Mexico and Living in the time of the Coronavirus
Friday, January 31, 2014
I Never Thought I Would See the Day.....
I've had my house for 12 years and I never thought I would see the day when I could go on line and order from Amazon and have it show up at my door but it has happened.
I needed a travel tripod that would fit in my suitcase and not weigh too much. I have a tripod I love but it is too long and too heavy for the current luggage restrictions on airplanes. I was on line looking at tripods, thinking that I'd have to have it shipped to one of the services that brings stuff across the border when I noticed that Amazon said that the tripod was available for global shipping. Whoa! Would this be cheaper and/or faster than the local services I had used in the past? I decided to give it a try.
I placed my order on the afternoon of January 26th
The tripod shipped on January 27
January 28 it arrived in Mexico City
January 29 it left Mexico City
January 30 at 2:30 PM UPS rang my doorbell.
I paid about 10% of the cost of the item for shipping and handling but that will vary based on the size and weight of what you are ordering.
I did not have to pay any sales tax since it was being shipped outside of the USA. That saved me about 8.75% of Texas sales tax which just about did away with the cost of shipping and handling.
And then the cost to clear customs was about 18%. A little more than the usual 16 or 17% but maybe there are brokers involved.
I am amazed. I never thought I would be able to order from Amazon and have it show up at my front door in FOUR days. Besides that I really like this colorful 15" tall tripod. Red is a good color for me.
12 comments:
YOU are blazing trails I never even considered. My neighbor in the guest house is having things sent from the USA to this address frequently and each time they arrive I stand with an open mouth in amazement. Who knew?ReplyDelete
RED is YOUR color for sure!Replies
What was really shocking to me was how fast it arrived. FOUR days.Delete
Red is a power color. I'm a powerful woman! LOL Need a tripod that reflects my personality. Besides a thief might think twice about trying to walk off with a red trimmed tripod.
Oh I love this! Please tell us what kind it is! How perfect!ReplyDelete
Thank you in advance.
Barbara, It is MeFoto and is called a travel tripod. It comes with a variety of colors. It is 15" tall and weighs a little more than 3 pounds. Also comes with a nice padded case. It will be a little more trouble to open up because it has more leg sections but at least it will fit in a suitcase.Delete
You have thrown down the blog gauntlet. I feel a test shipping coming in the future. Of course, with my current travel schedule, I could probably be my own international shipper.ReplyDeleteReplies
Steve, I would have brought it back with me in December and paid no duty but I didn't have room in my suitcase. So if you can bring stuff back when you return from the USA, it will be at least 16-18% cheaper. However it is nice to know that we do have an alternative if we need it.Delete
- AnonymousFebruary 1, 2014 at 9:49 AM
I'm always amazed that they allow tripods in checked luggage. In my opinion, a tripod would make a reasonably decent offensive weapon, but I guess TSA doesn't agree. But seriously, would you rather be attacked with a butter knife (banned) or a tripod?ReplyDelete
I think I'd go for the butter knife any day.
Kim G
Boston, MA
Where we have one, frequently-used travel tripod, and one sturdy, seldom-used, heavy-duty tripod.Replies
Kim, You are talking about carry-on luggage? I totally agree with you about the butter knife vs. tripod. Do you carry your tripod on or put it in checked luggage. I'm hoping that I can get it in my checked baggage just to keep my carry-on lighter.Delete
I have a heavier tripod in Houston and also one here but I think I'll find that this lighter one sees more action than either of the other two unless I'm using my LARGE camera. However the weight limit for this small tripod should hold the large camera as well. So who knows. Besides, this one is just so darn cute with the red trip. Very stylish! Always important to look good as a photographer. LOL
- AnonymousFebruary 9, 2014 at 9:59 AM
Sorry, I mis-wrote. I mean allowing tripods as carry-ons. And yes, I've carried on my lighter Velbon "ultra maxi S" tripod, but it expands to about 5' high, so it's not small.Delete
There are a lot of people who think you need a heavy-duty tripod to get clear, vibration-free shots, but I've had pretty good luck with my lighter tripod using a standard DSLR (Canon 30D). To avoid vibrations, I'll often use the self-timer so I'm not jiggling the tripod when I shoot. That, combined with lengthy exposures (>1/2 second) seem to quell any vibration problem.
Of course the self-timer is both my favorite and most hated feature. I love it for the use above, but then ALWAYS forget to turn it off, and of course on the next shot, it drives me nuts.
Kim G
Leaving the self timer on....Delete
Been there done that! LOL
Last July picked-up a Velbon Ultra-Maxi in like new condition at a thrift store in Las Vegas for $2 usd. Seemed like a good deal ;-)Delete
Bragging rights on that transaction.
I love thrift stores and re-sale shops. But that was a bargain. Go ahead and brag. LOLDelete


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We have good news for fans of the movie, Beaches (is there anyone who hasn't cried over it?).
Iris Rainer Dart, author of the book, has now turned her beloved story into a musical; currently in previews outside Washington, D.C. She talks with her longtime friend, WHOA! co-founder Darryle Pollack about her love of Broadway musicals, Bette Midler, and some stories and surprises behind Beaches.
Click here for Darryle's perspective on Iris.
Click here for the inspiring story of how Iris went from Beaches to Broadway.
Click here for more on Beaches the Musical.


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WASHINGTON, DC – Today the House Armed Services Committee voted down a Republican amendment that attempted to use the sacrifices of our veterans and their families to score political points.
The following is a joint statement from Armed Services Committee Chairman Ike Skelton (D-MO), Budget Committee Chairman John Spratt (D-SC), Military Personnel Subcommittee Chairwoman Susan Davis (D-CA) and Readiness Subcommittee Chairman Solomon Ortiz (D-TX):
"Our committee has never accepted amendments
that require sequential referral to another committee,
without first obtaining a letter of consent from that
committee. While we all support finding a resolution to the
underlying issue, we cannot allow our bill to be subjected to
a point of order on the floor.
"The authors of this amendment know these rules and choose to ignore them, hoping no one would notice their lack of sincerity at truly solving this problem. This amendment requires viable offsets from mandatory spending accounts in other committees, and no attempt has been made by the authors to identify these offsets or obtain the approval of the committees of jurisdiction.
"In our committee, we don't play games. We don't accept legislation that we can't pay for ourselves, and we don't try and gain political points by raising the hopes of the men and women who sacrificed so much for our country."
*** All provisions are subject to change pending final passage of H.R.2647 ***
Fact Sheet on the Use of Mandatory Spending
Prepared by the HASC Staff
Since taking control of Congress, Democrats have made great strides in providing for our retirees and their families. Since 2007, the first year Democrats were in control of Congress, we have greatly advanced the ball for both SBP/DIC and concurrent receipt.
Please see the attached fact sheet for additional legislation passed by the Democratic Congress.
• In 2007, we authorized a monthly payment of $50 to surviving spouses who are denied the full amount of their SBP benefit because of concurrent receipt of DIC. The amount will be increased to $100 by 2014. This has since been updated in the recently-passed Tobacco Bill to increase that amount to $150/month in 2014, and increasing it to $310/month in 2017.
• In 2007, we also authorized disabled retirees, who are considered 100% disabled by virtue of being unemployable, to receive full concurrent receipt of military retired pay and disability compensation effective back to January 1, 2005.
When Republicans controlled the House, the Senate and the White House, they chose not to resolve these important issues. During that time, there were budget surpluses that could have been used to benefit our military retirees and their families, but instead, the Republicans chose tax cuts for the wealthy. They included reserve funds in their budgets, but chose not to use that flexibility to help our veterans.
Republicans have asked for programs totaling an estimated $36 billion over the next ten years. They have asked the Budget Committee Chairman to use his authority to tap into the "Reserve Funds" in the budget to pay for these mandatory spending programs.
The Republicans are demanding use of an authority that does not exist. The reserve funds they refer to are "deficit-neutral", meaning they can only be activated when mandatory spending cuts are identified by one committee and made available for use by that committee or another committee, through this reserve fund.
The Chairman of the Budget Committee cannot simply invent resources. In the event that offsets are identified for use through the reserve fund, he can use his limited authority to direct those resources towards increasing a committee's budget allocation. Until that occurs, the resources do not exist, and the Chairman cannot make a reserve fund adjustment.
A proposed across-the-board cut to all mandatory spending programs is simply smoke and mirrors. It is not a viable solution because it proposes indiscriminately cutting programs that are funded through formulaic calculations. In order to decrease the amount spent, you must change the basic formula used to determine costs, and Republicans have no intention of undertaking this effort.
The Republican proposal is fiscally irresponsible. They have not identified viable offsets that can be used to pay for these programs. They have offered $36 billion worth of spending without offering a realistic means of paying for it.
Democrats favor fiscally responsible solutions. We will continue to make improvement in a fiscally responsible manner that does not jeopardize the military retirement and health care benefits our TRICARE for Life beneficiaries have earned.
Supporting our veterans takes more than lip service. For 12 years, the Republicans were in control of Congress and never attempted to fully resolve this problem, and it is irresponsible of them to raise it now without proposing a viable solution. They are using our veterans to gain political points by creating unrealistic expectations among our wounded warriors, survivors and reservists who are bravely serving our country.
By putting forward unworkable and overly simplistic proposals, Republicans are creating unrealistic expectations. They know the committee cannot pay the $36 billion required to fix these programs without reducing military retirement benefits or health care benefits for our Medicare-eligible military retirees.
Republicans are hypocritical for criticizing deficits while pushing for a new $36 billion initiative. To knowingly offer this program without viable offsets, while criticizing Democrats for increasing federal deficits, is the very definition of hypocrisy.


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Small Group Training
Get Fast, Efficient Results with our unique training program
Aquafit's Small Group Personal Training Programs take small group personal training to a whole new level. Take on the challenge of our unique programs designed to keep you motivated, provide variety in your training, introduce you to new techniques and help you on your way to achieving your fitness goals! With small group personal training you can experience the benefits of working with a personal trainer, without the additional cost.
Our Small Group Training Program allows you to choose how many sessions you would like to attend over a four week cycle. Register with Reception and then participate in any session that takes your fancy.
The Sessions
Our sessions are conducted either indoors in our Queenax functional training zone or outdoors utilising our specialised outdoor training area. This is functional training at its best where you will push, pull, squat, lunge, bend and twist your way to increased strength, tone and overall fitness, delivering maximum results with a workout that will challenge all fitness levels. All sessions have a maximum of 15 participants.
Non Aquafit Members are welcome to participate in any of our sessions, both indoors and outdoors.
All sessions are 60 Minutes
Challenge and strengthen all muscles in the 8 major parts of your body – Shoulders, Arms, Chest, Abdomen, Back, Glutes, Thighs and Calves. Our LIFT sessions focus on building strong bodies. Run within our Queenax Functional training zone, you will learn the right techniques and exercises, with extra motivation and support to ensure you take your strength, fitness and body shape to a new level.
Using Aquafit's unique outdoor area, as well as a variety of grounds in and around the local area, this program delivers a whole body workout that is sure to get the heart pumping. Kettlebells, adjustable weights, power bags, medicine balls, ropes, agility ladders and hurdles, boxing equipment and more… OUTDOOR combines the latest functional training tools to keep your body guessing with a cardio/ strength combination that is extremely effective.
HIIT is High Intensity Interval Training - great for maximizing caloric burn and jump-starting your metabolism – this is true lung busting cardiovascular training. Utilising only bodyweight exercises in a variety of training protocols and activities, you will be guaranteed to improve fitness throughout the cycle, and burn a lot of calories in every workout.
HIRT - High Intensity Resistance Training - one of the most effective ways to build muscle, improve muscular endurance and burn calories. HIRT incorporates a variety of different pieces of equipment & tools to mix things up a lot.
This is a high intensity metabolic conditioning workout. This session involves functional movement patterns that are interspersed with high intensity cardio intervals, designed to challenge all energy pathways. Designed to boost your metabolism, reduce body fat, improve strength & conditioning as well as improve your ability to perform every day activities. Session will use a range of functional equipment, including kettlebells, powerbags, slamballs, suspension trainers & rowers.
Small Group Training Program is designed to be flexible, letting you choose how many sessions you would like to attend each week. Non Members are now welcome to join in the full program.
|program cost:
|1 session/wk
|2 sessions/wk
|3 sessions/wk
You can also refer to our Information Sheet for further information.
With so many programs now on offer, our Small Group Training Program Timetable will assist you in choosing the most suitable sessions for you. The timetable is versatile and changes over time to meet demand.
Timetable Starting 13 January - 9 Feburary 2020
Online Session Bookings
Bookings for all sessions need to be made online via the Aquafit App. Please note that bookings are based on a first come first served basis for capped sessions.
To access your account outside of the Aquafit App or sign up follow this link to My Wellness.
You can also refer to our Online Booking Procedures for further information and guidance.
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Speak to one of our Personal Trainers or Reception Team on 4629 7297 or send us an email at email@example.com


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Keep study 🙂
This is the /etc/config/wireless from old version vocore2(Linux 3.18) which is working well with uci system. Thanks to its contributors. It is from Linkit.
config wifi-device radio0 option type ralink option variant mt7628 option country CN option hwmode 11g option htmode HT40 option channel auto option disabled 0 config wifi-iface ap option device radio0 option mode ap option network lan option ifname ra0 option ssid VoCore2 option encryption none config wifi-iface sta option device radio0 option mode sta option network wwan option ifname apcli0 option led mediatek:orange:wifi option ssid UplinkAp option key SecretKey option encryption psk option disabled 1
Note: this file is generated by /lib/wifi/ralink.sh:detect_ralink
Now we can add some log to /lib/netifd/wireless/ralink.sh, check how it read the file and how it call the driver to setup the connection.
It is a pretty long script…
If my last blog is correct, it will call "enable_ralink" … what? no such function…
OK, let's add some log to it. In every function head, we can add "echo xxxxxx_function >> /tmp/vocore2.log", it will show the called function name into vocore2.log.
But nothing show in the file…so it is not used at all!
How it works? just a mystery.
Emm, it won't help, get rid of it, let's check openwrt with mt76 script, that one works without hack.
No luck today, try tomorrow.
To be continue…


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After reading on Facebook the other day that the grandmother of our neighbors was going over to their house for lunch made by her grandkids, I was excited when they posted a photo of their lunch made by 8 year old Samuel and thrilled when he agreed to come over and allow me to interview him for his recipe for "Taco Dogs".
Apparently, entrepreneurally-minded Samuel has been busy this summer running a business he calls "The R Family Cafe". He charges $2.00/5 year membership for regular lunches and an extra $1 per "romantic meal". Gene and I will have to take him up on that one! Wonder what he charges to cook for a dinner party of 4-6? We might have to contract him for one of our Dexter dinners, although with the violence and Debra's cursing, we'll probably have to send the Chef home to bed prior to showtime!
When I asked what his little sister was doing while he was slaving away over a hot microwave, Samuel informed me that she "played on the computer" but after lunch provided an impromptu ballet recital for the entertainment.
When asked what he served with his taco dogs, Samuel stated that his mom steamed broccoli from their backyard garden "you know, on top of the stove where the fire is" and then put cheese on it to form a tasty sauce. He also said that since they had to pick off a few bugs who were trying to munch for free on their garden broccoli, that meant it was extra good. I agree. That's how you know your garden is successful, when you have to fight the critters and bugs for your produce!
All I have to say in closing is "Watch Out!" to all my local chef friends: Dave Ponder, Steve Young, and Brad Lyons, Samuel is on his way up and he's going to be a force to be reckoned with!
Samuel's Taco Dogs
Recipe By Samuel R.
- 4 All Beef Hot Dogs
- 4 American Cheese slices
- 4 Taco sized Tortillas
For each taco dog: Lay out one tortilla. Place slice of cheese on it, then one hot dog. Roll them up tightly, and place on a microwave safe dish, seam side down.
Microwave on high for 45 seconds per taco dog or until heated through.
Sam, grinning to show me his plan for supplemental income for the cafe from the tooth fairy.


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Bighorn 50 - Racers
Runners are listed in alphabetical order according to Last Name:
Racer: Katie Oglesby
Year raced event: 2013
Division: 40-49
Blog: runlongkatie.com
2013 Race: All year I have trepidatiously dubbed this race my "Do-Over." It's not uncommon for me to have a poor race performance. (If every race were great, they'd all be average.) But Big Horn last year was... Read Mor


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With so many fad diets currently being promoted online, it is natural to find yourself in some trouble when trying to put together a health meal plan. Finding what works for you and what does not can take a lot of time, and this often makes people go to sites that have pre-designed menus and help you select a healthy meal plan as per your requirements. These plans certainly have their benefits, but you need to understand that you need to pay attention to three elements to make any diet plan work. These three elements are food choices, portion control, and hydration. Let's find out more about it.
Food Choices
In today's world, you have so many food choices. So many people are now interested in microwavable meals, but the truth is that a wrong choice here can make you pay through the nose. It may not take long to prepare these foods but they provide you with empty calories, and athletes can certainly do without such foods. Athletes should focus on eating food that would help repair muscle tissues and improve overall gains. You will be better off putting your money on organic and all-natural foods to maintain good health and achieve your fitness goals.
Similarly, it is important to ensure that the food you eat contains enough carbs and protein. Foods like beef, lean chicken, and fish are great sources of protein, whereas you can get carbs from whole grain foods, quinoa, and other such foods. Including fruits and vegetables in your diet will also help fuel your body properly. So, even when you are using an online healthy meal plan, ensure it contains enough carbs and protein.
Portion Control
Even if you are following a healthy diet, eating too much of it can cause problems. Even the healthiest food contains calories and eating too much of such food can make you eat excessive calories. It can make weight loss a real struggle. Therefore, you should be very careful about how much you eat. Be sure to include more fruits and veggies in your diet because the fiber they contain can keep you full for long.
Never underestimate the importance of keeping your body hydrated. No matter how healthy your diet plan is, improper hydration can lead to serious health issues. Athletes should drink more frequently – it is better to drink some water every 15 minutes to keep your body hydrated. You may also consider adding lemon to your water to replenish electrolytes you lose during training. Avoid soda and fruit drinks as much as possible and stick to water as much as you can.
The fact of the matter is that you are not going to get your fitness goals if you do not consider these three elements while selecting a healthy meal plan. Not making right food choices or eating more than required can put you in trouble. Similarly, inadequate hydration can lead to several complications. Therefore, you should always consider these elements before selecting a diet plan for you.


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Active Duty Dental Program (ADDP)
The Active Duty Dental Program is a program which focuses on dental care only for members of the military. Dental care is provided to service members who are in duty stations and residences which are more than 50 miles away from a military dental facility. ADDP is a program that is also provided to The National Guard members and Reservists who have been active for more than 30 days and reside over 50 miles away from a military dental facility.
This program allows for dental related prescriptions to be filled for free through your treatment facility.


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Nantucket band Miss Fairchild�s song �Vanilla Place� will play on tonight�s episode of �Real World: Hollywood,� the MTV reality show that spawned the genre. Bunim-Murray Productions has chosen �Vanilla Place� off Miss Fairchild�s �Ooh La La Sha Sha� album to appear as background music in one of the scenes in this weeks� episode, the second of the season.
�The Real World� will air at 10 p.m. tonight on Comcast Cable channel 28. The MTV website ��www.mtv.com � also hosts a Real World page. After the episode airs, the page will have information about Miss Fairchild, the scene in which �Vanilla Place� was used, and links to Miss Fairchild�s online resources.


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NBA News
Longtime Jazz coach Jerry Sloan dies at age 78
Sloan, who coached the Jazz for 23 seasons as part of a Hall of Fame career, died Friday from complications from Parkinson's disease and Lewy body dementia.
Source Link
Leave a Reply
NBA News
N.Y. governor: Pro teams can return to facilities
New York Gov. Andrew Cuomo says teams in his state can return to their facilities for training after a pause of more than two months.
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NBA News
The games that made Tracy McGrady a Hall of Famer
Whether it was putting up triple-doubles, dropping 62 points or scoring 13 in 33 seconds, Tracy McGrady racked up his fair share of memorable performances in his Hall of Fame career.
Source Link
NBA News
The NBA in a bubble: The blueprint for how the league could finish the 2019-20 season
Hope perseveres to eventually declare a 2019-20 NBA champion. ESPN spoke to a range of stakeholders studying the "bubble" concept and compared their concerns with the league's thinking.
Source Link
Nash Drops 27 PTS & 16 AST To Guide Suns | #NBATogetherLive Classic Game
The 10 BEST Ankle Breakers & Ball HANDLERS In High School 2020 Edition! Sharife, Dior, Zion & More
N.Y. governor: Pro teams can return to facilities
LA Clippers basically traded for Kawhi after they got Paul George – Rachel Nichols | SportsCenter
Giannis Antetokounmpo double-double leads Bucks to Game 2 win vs. Pistons | NBA Highlights
The NBA should be commended for its quick action on coronavirus – Rachel Nichols | SportsCenter
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The Clippers are set to do a giant flex on the Lakers by buying The Forum – Clinton Yates | The Jump
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First Take discusses the evolution of Michael Jordan's legacy
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Steph Curry Drops 23 In Return!
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Paige Bueckers Goes CRAZY In 2nd PLAYOFF Game!! FLASHY Handles & Dimes For The #1 Ranked Player!!


text:
ata1: exception Emask 0x10 SAct 0x0 SErr 0xd0000 action 0xe frozen
ata1: irq_stat 0x00400000, PHY RDY changed
ata1: SError: { PHYRdyChg CommWake 10B8B }
ata1: hard resetting link
So how does one interpret the cryptic SErr magic? Pages 269-270 of the SATA ATA spec serialata10a.zip explains the SError register fields in fine detail.
Well, "SErr 0xd0000" relates to the SError bits 16, 18 and 19 which are the SERR_PHYRDY_CHG, SERR_COMM_WAKE and SERR_10B_8B_ERR bits as defined in include/linux/ata.h. The kernel decodes these and dumps out the error state in the "ata1: SError: { PHYRdyChg CommWake 10B8B }" line above - so that's helpful.
Anyhow, the spec describes these error codes in the DIAG field (page 270) - they are the top 16 bits of the SError register. Armed with the spec one can then decode these error bits. It's not rocket science, one just needs to know where to look this information up.


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Kathy Holeton
- Administrative Services
Kathy began her University of Iowa career as a Clerk Typist III at the Institute of Hydraulic Research in 1982. From 1986-2000, she worked as a Secretary IV at the Office of Statewide Clinical Education Programs, a unit of the UI College of Medicine Dean's office. In February 2000, she joined the Public Policy administration staff as an Administrative Assistant I, and was promoted to Administrative Associate in 2005. Due to the PPC administration restructuring in 2014, she now oversees expenses for Transportation & Vehicle Safety Policy grants and produces comprehensive, financial reports and graphics for researchers in this division. Kathy also is the conference coordinator of the Driving Assessment conferences, a highly successful bi-annual event with international recognition and an outstanding reputation.
Educational Background
- B.L.S., Bachelor of Liberal Studies, University of Iowa, 1996
- A.A.S., Retail/Fashion Merchandising, Kirkwood Community College, 1974


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Fianna Fáil has expressed serious concerns at reports that the cabinet has yet to hold a special meeting to discuss the budget with just two and a half weeks left to budget day.
Commenting on reports in today's Sunday Independent that the cabinet has yet to hold a special meeting to discuss the budget, Fianna Fáil Finance Spokesperson Michael McGrath stated: "It is deeply worrying and nothing short of extraordinary that the cabinet has yet to hold a special meeting on Budget 2013 with a little over 2 weeks left to budget day. The Economic Management Council is not the government and the government's budgetary strategy must be underpinned by the principle of collective cabinet responsibility.
"Each cabinet Minister is collectively responsible for the entirety of the budget package and not just budget decisions affecting their own department. Under no circumstances can the Economic Management Council be allowed to usurp the constitutional role of cabinet. The cabinet is not just a forum for rubber-stamping decisions made by the Taoiseach, Tánaiste, Minister for Finance and Minister for Public Expenditure & Reform.
"The absence of any cabinet meetings to discuss the budget is a measure of the complete lack of trust between the government partners. However, the approach of keeping the cabinet in the dark has not stemmed the flow of pre-budget kite flying with a litany of proposed cuts and tax increases already making their way into the public domain.
"The essential point here is that no select group within cabinet can take on the role of making decisions for the full cabinet. Ultimately every member of cabinet will have to take full responsibility for all measures announced on December 5th. All Ministers would be well advised now to ensure they have a meaningful input into all elements of the budget currently being prepared."


text:
Sometimes, installing new software, we are rushing through
the installation process and clicking on all "OK" buttons without reading fine
print. And later we note with surprise that our home page has changed, new
toolbars installed, and for some reason the speed of the browser loading and
general operations substantially declined.
You can definitely go to the Settings page and start
recovering from the unwanted changes step by step. But if you do not feel
comfortable with such operations, you may get assistance from Auslogics Browser
Care freeware.
Auslogics Browser Care lets you sort through all add-ons and
toolbars installed in your web browsers, remove unsafe or simply unneeded ones
and choose useful items you want to keep.
With Browser Care you can view and manage all your installed
browsers in one place. This includes the list of all plugins for each browser
along with their status and safety rating, as well as the webpage and search
engine that are set as default. If your home page has been hijacked, this tool
lets you change it to the page you'd like to start your browsing sessions with.
If you've never used the search window in your browser because it is linked to
a search engine you don't like, Browser Care lets you set your favorite search
engine as default in all your browsers for faster and more convenient searches.
Never find the time to clear browser cache? This tool lets you easily sweep out
temporary internet files from all your installed browsers.
Browsers currently supported: Internet Explorer, Mozilla
Firefox, Google Chrome.
Fonte: http://feedproxy.google.com/~r/FreeUtilitiesEvaluationAndReviews/~3/imhiX3M5nCo/browse-without-annoying-toolbars.html


text:
Your institution must subscribe to JoVE's Medicine section to access this content.
Fill out the form below to receive a free trial or learn more about access:
Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method—adapted from a coculture model of synaptogenesis—for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.
Cite this ArticleCopy Citation | Download Citations | Reprints and Permissions
Zhang, C., Suckow, A. T., Chessler, S. D. Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells. J. Vis. Exp. (76), e50365, doi:10.3791/50365 (2013).
Translate text to:
Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.
We describe here a method to facilitate investigations of how the extracellular domains of specific transmembrane proteins affect insulin secretion. The method probes the effects of interactions of the protein of interest with proteins (or possibly other molecules) on the pancreatic beta-cell surface. The method allows investigations of how cell-surface proteins expressed by beta cells or by other neighboring cells (e.g. endothelial cells, neurons, pancreatic alpha cells) affect beta-cell function through transcellular interactions (i.e. through interactions with interaction partners on the surface of adjacent beta cells).
The cellular plasma membrane contains a complex array of structural and functional proteins serving as bridges to the extracellular environment. By formation of transcellular connections or by initiation of plastic signaling events, interactions between cell-surface proteins can help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within the pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis1. As revealed, for example, by the importance of extracellular EphA-ephrinA and neuroligin-2 interactions in the regulation of glucose-stimulated insulin secretion, it is becoming ever more clear that increased knowledge of the extracellular interactions occurring between proteins on the surfaces of adjacent beta cells will be of great importance for gaining a full understanding of insulin secretion, beta cell functional maturation and the maintenance of glucose homeostasis1-3. The goal of the method described here is to enable investigations of the effects on beta cell function of transcellular interactions involving specific transmembrane or otherwise-cell-surface-associated proteins. By co-culturing beta cells with HEK293 cells transfected with different expression constructs, the effects on beta cell function of different cell-surface proteins or mutated variants thereof can be efficiently probed. This is accomplished without having to transfect the beta cells themselves.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of beta-cell mRNA and protein expression, potentially affecting beta cell health and/or function in ways that could confound analyses of the effects of specific extracellular interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and, further, do not allow effects due to interactions between proteins on or in the same cell to be distinguished from the effects of transcellular interactions. Here, a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is described. This method is applicable to insulin-secreting beta-cell lines, such as INS-1 cells4, and to dissociated primary rodent or human beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers could identify proteins that drive synapse formation5,6. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell function and functional maturation might be driven by similar transcellular interactions7-9. In order to probe these interactions, we developed the system described herein in which beta cells are cocultured on a layer of HEK293 cells expressing a protein of interest10. This system allows the beta-cell cytoplasm to remain untouched while extracellular protein-protein interactions are manipulated.
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1. Transfection of HEK293 Layer
- Prepare HEK293 cell medium by adding to 500 ml bottles of DMEM (with 4.5 g/ml glucose and phenol red and without glutamine): 50 ml FBS, 5 ml 100X penicillin/streptomycin solution, 5 ml 100x L-glutamine solution and 500 μl amphotericin B.
- Plate out HEK293 cells in a 24-well plate using 0.5 ml of HEK293 media per well. Ensure that the cells are spread evenly across the bottom of the plate.
- When HEK293 cells reach 100% confluency, transfect with 0.8 μg of plasmid coding the protein of interest (inserted in a mammalian expression vector) and 2 μl of Lipofectamine 2000 according to the Lipofectamine protocol. We have opted to use the pcDNA 3.3 backbone designed for expression in mammalian cells.
- Optionally, after 6 hr of incubation, exchange the transfection media described in the Lipofectamine protocol with normal HEK293 media.
- To evaluate expression of the transfected protein, a subset of the transfected tissue culture wells can be used for immunofluorescent detection of the expressed protein or for western blot analysis of protein expression using standard techniques.
2. Optional Fixation of HEK293 Cells Expressing Transfected Protein
Transfected HEK293 cells can be gently fixed in order to facilitate coculture in media that might be harmful to living HEK293 cells (e.g. step 3.9 below) or to allow the efficient preparation in advance of plates for several experiments all at once (see also Discussion).
- Conduct pilot studies to determine the time course of the expression of the protein in the HEK293 cells.
- After transfecting the HEK293 cell layer, aspirate the media from the cells at the post-transfection time point associated with highest level of expression. If this occurs within the first 24 hr, do not aspirate until 24 hr after transfection.
- Wash HEK293 cells gently with D-PBS then add 500 μl 4% paraformaldehyde (PFA) and incubate at RT for 30 min. (To make 4% PFA solution, start with 16% solution in 1X PBS and dilute 1:4 with 1X PBS.)
- Using sterile PBS, gently wash the cells 3 times and incubate in PBS O/N at 4 °C.
- Wash cells 3 times with sterile PBS, then add PBS and leave at 4 °C until needed (maximum storage time may vary depending on protein expressed. We store HEK293 cells transfected to express neuroligin isoforms for up to 2 weeks without significant changes in observed effects on insulin secretion).
3. Co-culturing of INS-1 Beta Cells with HEK293 Cells
- Prepare INS-1 media by adding to 500 ml RPMI 1640 (with glucose and phenol red and without glutamine): 50 ml FBS, 5 ml 100X penicillin/streptomycin, 5 ml 100X L-glutamine solution, 5 ml sodium pyruvate, 500 μl amphotericin B, 500 μl beta-mercaptoethanol (this is nearly identical to the medium typically used for primary islet culture except for the addition of beta-mercaptoethanol).
- Using Cell-stripper (or another non-enzymatic cell remover), harvest INS-1 cells at approximately 70-80% confluency off the bottom of a T75 culture flask. Each flask will provide roughly enough cells for 48 wells.
- After spinning down cells in the centrifuge for 3 min at 1,200 rpm, resuspend in a 50/50 mix of INS-1 and HEK media. For a 70-80% confluent flask of INS-1 cells, resuspend in approximately 25 ml of mixed media.
- If using HEK293 cells that have been fixed in paraformaldehyde rather than live HEK293 cells, resuspend the INS-1 cells in 100% INS-1 media rather than mixed media.
- Using a 10 ml pipette, dislodge the cells from the pellet to make a homogenous cell suspension.
- Aspirate to remove the media on the HEK293 cells.
- Using a p1000 pipette, gently add 500 μl of INS-1 cell suspension onto the HEK cell layer along the sides of the well. After every 6 wells, use a 10 ml pipette to resuspend the INS-1 cells again to ensure a homogenous cell suspension. The overall scheme of the coculture set-up is depicted in Figure 1.
- Incubate the cells for 24-48 hr depending on the experimental protocol.
- If more than 48 hr coculture period is required, use optional steps in section 2 above to fix the HEK293 cells.
- If using primary dissociated rodent or human islets [e.g. see prior protocols in JoVE11-15], ensure that an appropriate media such as RPMI with 5 ml pen/strep, 5 ml L-Glut, 0.5 ml amphotericin-B is used instead of INS-1 media. Although one can use dissociated islet cells in a 24 well plate, in practice because of the large number of islets potentially needed, it is recommended that consideration be given to scaling down to a 48 well plate (necessitating fewer islet cells per well). On a 48 well plate, add approximately 100 islets worth of dissociated cells per plate to start. Depending on the efficacy of dissociation and the method used, fewer islets may be required.
4. Glucose Stimulated Insulin Secretion
- Preincubate cells in 250 μl of 2.5 mM glucose in KRB (Krebs-Ringer bicarbonate buffer) for a minimum of 1 hr. To preincubate, remove coculture medium and immediately replace with KRB. This exchange should be done one well at a time to minimize exposure of INS-1 cells to environmental oxygen. Aspirate with one hand while pipetting the low (2.5 mM) glucose KRB with the other.
- Exchange preincubation KRB to 250 μl of either 2.5 mM or 20 mM glucose in KRB buffer one well at a time.
- If appropriate for the specific experiment, additional secretagogues such as 100 μM IBMX may be added to produce a more robust stimulated insulin secretory response. Dissociated primary islet beta cells in particular are poorly responsive to increased glucose concentrations alone, so here especially addition of IBMX or other secretagogues should be considered16,17 .
- After 1 hr of incubation, transfer media to microfuge tubes and spin 1,500 x g for 5 min.
- Remove 200 μl of the supernatant and use this for analysis by insulin RIA or ELISA.
- To prepare cell lysate, add 100 μl of RIPA cell lysis buffer (150 mM NaCl, 1.0% IGEPALCA-630 or Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with protease inhibitors to the plate immediately after removing media and incubate at 4 °C for 20 min.
- Spin down lysate for 15 min at 10,000 x g and remove 50 μl of supernatant for analysis by insulin RIA or ELISA.
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Representative Results
Using the method described here, we have tested the effect of different variants of the protein neuroligin on insulin secretion. This complements our published work investigating the effect of neuroligin-2 on beta-cell function10. Figure 2, for example, depicts results obtained from coculturing INS-1 beta cells with HEK293 cells transfected to express a neuroligin isoform referred to here as NL-X. This experiment was designed to test the hypothesis that NL-X engages in transcellular interactions that increase insulin secretion. In Figure 2A, it can be seen that expression of NL-X increased basal and stimulated insulin secretion by cocultured INS-1 cells. The approach of transfecting HEK293 cells so that they express NL-X allows us to know that it is the extracellular domain-which is the region displayed on the HEK293 cell surface and thus able to come into contact with cocultured INS-1 cells-that brings about the observed increase in secretion. The NL-X extracellular domain must be interacting with another protein (or perhaps some other molecule) on the INS-1 cell surface to cause the INS-1 cells to secrete more insulin. If desired, secreted insulin could also be expressed as a percent of cellular insulin content, which can be measured after lysing the cells.
In Figures 2B and 2C, results from other types of analyses using the coculture system are depicted: specifically, analyses of the effect of transcellular interactions involving neuroligin variant NL-Y on cellular insulin content and on PDX-1 and insulin mRNA levels. NL-Y increased the insulin content of the cocultured INS-1 cells (Figure 2B). Further testing would be necessary to determine whether this was due to an increase in the amount of insulin per cell or due to increased INS-1 cell proliferation. In Figure 2C, RNA was harvested from the cell layer and analyzed by RT-qPCR. Since the HEK293 cells are of human origin, for transcripts that might be present in both HEK293 and INS-1 cells, use of rat-specific PCR primers (INS-1 cells are of rat origin) ensures that only mRNA of INS-1 origin is measured. Here it can be seen that coculture with NL-Y increased insulin and PDX-1 mRNA levels (normalization was to 18S RNA). The primers and methodology used for this RT-qPCR analysis were previously described10, online supplement.
Figure 1. Coculture set-up. HEK293 (HEK) cells are added to 24-well tissue culture plates and then transfected. A bright field image of a representative HEK293 cell layer is shown on the top, right. The HEK cells are transfected to express an epitope-tagged transmembrane protein. Here, immunofluorescent staining is used to reveal the transfected cells (green). Next, INS-1 cells are added. Immunofluorescent staining for insulin was performed to allow visualization of the cocultured INS-1 cells (red).
Figure 2. Results obtained from coculturing INS-1 beta cells with HEK293 cells transfected to express the neuroligin protein variants NL-X or NL-Y (filled-in, black columns). Control HEK293 cells were transfected with the same plasmid constructs altered so as not to express protein (white columns). A, insulin secretion by cocultured INS-1 cells under low (2.5 nM) and high (20 mM) glucose conditions and also after stimulation by high glucose together with IBMX. B, INS-1 cell insulin content after coculture with HEK293 cells expressing NL-Y. C, analysis of insulin and PDX-1 transcript levels by RT-qPCR using RNA isolated from INS-1 cells cocultured with either HEK293 cells transfected with NL-Y or with control-transfected HEK293 cells. (*, P<0.05; **, P<0.01; ***, P<0.001).
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The coculture method described here provides an effective way to determine the physiological importance of specific beta-cell-surface, transmembrane proteins, and specifically of their extracellular domains. By culturing beta cells or insulinoma cells (such as the INS-1 cells employed here) in contact with HEK293 cells displaying a protein of interest on the cell surface, experiments can be designed to determine the effects of extracellular protein-protein interactions without directly disturbing the intracellular milieu of the cells. Since the proteins of interest are expressed by HEK293 cells, any effects on β-cell function that could be mediated by the intracellular domains of the transfected proteins would be absent and therefore would not complicate analysis of the effects of extracellular interactions.
Effects on insulin secretion can be readily determined by comparing secretion from beta cells cocultured with HEK293 cells expressing a protein of interest to secretion from beta cells cocultured in contact with control HEK293 cells. The control HEK293 cells can be mock-transfected or, alternatively, transfected with a variant of the experimental plasmid construct mutated to produce a non-functional variant of the protein of interest. In our hands, both types of controls have yielded identical results. Since the HEK293 cell surface inherently contains a great variety of proteins, either type of control allows the function of beta cells in contact with many different cell-surface molecules, including the protein of interest, to be compared to the function of beta cells in contact with the same set of cell surface molecules with the exception of the protein of interest (or including a mutated version of the protein of interest).
INS-1 cells (or other beta cells) are added to the HEK293 layers at low density so that there is minimal or no contact of INS-1 cells with other INS-1 cells. This allows the effects of transcellular interactions between the protein of interest expressed on the HEK293 cell surface and proteins on the INS-1 cell surface to be observed in the absence of potential background noise caused by transcellular interactions involving the same protein expressed endogenously on the INS-1 cell surface.
After transfection, the HEK293 cell layer can be gently fixed. This optional fixation can be utilized to allow multiple coculture plates to be prepared at once for subsequent addition of beta cells at a later date. Fixing the HEK293 cells would also be advantageous if there were concerns that HEK293 metabolism or secretory function would somehow interfere with analysis of beta cell function. Lastly, the fixation protocol can be employed to facilitate prolonged coculture experiments. Here, the chief advantage of using fixed HEK293 cells is that the optimal culture medium for the beta cells can be employed rather than using a mixed media (e.g. see step 3.3) necessary for maintaining cocultured HEK293 and beta cells simultaneously. In prolonged coculture experiments, use of the mixed media-which is not optimal for either cell type-can impair beta cell viability.
HEK293 cells are employed here because they are highly amenable to transfection: they can be transfected with high efficiency using a variety of different transfection reagents18,19. HEK293 is one of two cell types-along with COS cells--that are routinely employed in similar neuronal coculture systems5. Lipofectamine 2000 is a proven transfection reagent very commonly used with HEK293 cells and routinely achieving transfection efficiencies (percent of cells transfected) of greater than 95% in published studies18-20. Other reagents can achieve similar efficiencies of transfection19,20. With our neuroligin-2 plasmid construct, transfection efficiency is generally ~80%. Although we focus here on studies of glucose-stimulated insulin secretion, other modes of analysis are possible, for example by FACS, immunohistochemistry, western blotting and/or, as seen in Figure 2C, qPCR.
Despite being a very useful tool in helping uncover the function of particular extracellular protein interactions, the method described here is not without its limits. Because the beta cells need to be in direct contact with the HEK293 cell layer, insulinoma cell lines such as INS-1 cells are particularly well-suited for this coculture system4. Translating the results to primary islet cells requires the dispersion of isolated islets. Since the islet structure itself is integral to the proper signaling and response to glucose, the stimulated insulin response is blunted in dispersed primary beta cells relative to intact islets1,3,17. Furthermore, because transient transfection does not result in uniform expression of the protein across the HEK lawn, when performing imaging-based (immunohistochemical) experiments, a method to normalize to protein expression levels may be required to interpret results. An experimental approach that takes advantage of this non-uniform expression to analyze the effect of the transfected protein on the secretory machinery of cocultured INS-1 cells is depicted in Figure 3 in Suckow et al., 201210.
It should also be noted that the extent of contact between any given beta cell and neighboring cells expressing the protein of interest is likely to be less in this system than in intact islets in vivo, in which beta cells might be surrounded on all sides by cells expressing the protein. This relatively reduced cell-to-cell contact could reduce the magnitude of changes in insulin secretion to below what would be observed if the relevant transcellular interactions were somehow eliminated from native islets.
Use of a clonal population of stably-transfected HEK293 cells rather than transient transfection would result in more uniform expression and possibly simplify experimental set-up, but would require substantially more up-front time and effort in order to generate the stably-expressing cells. Future studies will determine whether, by transfecting two or more transmembrane proteins into the HEK293 cell layer, possible synergistic effects could be observed.
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We have nothing to disclose.
This work was supported by National Institutes of Health grant R01DK080971 and Juvenile Diabetes Research Foundation grant 37-2009-44. We also appreciate support received from the UCSD Pediatric Diabetes Research Center (PDRC).
|Catalog Number
|pcDNA 3.3 vector/backbone
|Lipofectamine 2000
|Pen/strep solution
|Amphotericin B
|Sodium Pyruvate
|Cell stripper
|T75 Flask
|16% Paraformaldehyde
|Electron Microscopy Sciences
|10x PBS
|Fetal bovine serum
|RIPA lysis buffer
- Kelly, C., McClenaghan, N. H., Flatt, P. R. Role of islet structure and cellular interactions in the control of insulin secretion. Islets. 3, 41-47 (2011).
- Konstantinova, I., et al. EphA-Ephrin-A-mediated beta cell communication regulates insulin secretion from pancreatic islets. Cell. 129, 359-370 (2007).
- Jain, R., Lammert, E. Cell-cell interactions in the endocrine pancreas. Diabetes Obes. Metab. 11, Suppl 4. 159-167 (2009).
- Poitout, V., Olson, L. K., Robertson, R. P. Insulin-secreting cell lines: classification, characteristics and potential applications. Diabetes Metab. 22, 7-14 (1996).
- Craig, A. M., Graf, E. R., Linhoff, M. W. How to build a central synapse: clues from cell culture. Trends in Neurosciences. 29, 8-20 (2006).
- Scheiffele, P., Fan, J., Choih, J., Fetter, R., Serafini, T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell. 101, 657-669 (2000).
- Abderrahmani, A., et al. Neuronal traits are required for glucose-induced insulin secretion. FEBS Lett. 565, 133-138 (2004).
- Lowe, A. W., Madeddu, L., Kelly, R. B. Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common. The Journal of Cell Biology. 106, 51-59 (1988).
- Suckow, A. T., et al. Expression of neurexin, neuroligin, and their cytoplasmic binding partners in the pancreatic beta-cells and the involvement of neuroligin in insulin secretion. Endocrinology. 149, 6006-6017 (2008).
- Suckow, A. T., et al. Transcellular neuroligin-2 interactions enhance insulin secretion and are integral to pancreatic beta cell function. The Journal of Biological Chemistry. 287, 19816-19826 (2012).
- Kohnert, K. D., Hehmke, B. Preparation of suspensions of pancreatic islet cells: a comparison of methods. J. Biochem. Biophys. Methods. 12, 81-88 (1986).
- Qi, M., et al. Human pancreatic islet isolation: Part I: digestion and collection of pancreatic tissue. J. Vis. Exp. (27), e1125 (2009).
- Qi, M., et al. Human pancreatic islet isolation: Part II: purification and culture of human islets. J. Vis. Exp. (27), e1343 (2009).
- Szot, G. L., Koudria, P., Bluestone, J. A. Murine pancreatic islet isolation. J. Vis. Exp. (7), e255 (2007).
- Zmuda, E. J., Powell, C. A., Hai, T. A method for murine islet isolation and subcapsular kidney transplantation. J. Vis. Exp. (50), e2096 (2011).
- Hauge-Evans, A. C., Squires, P. E., Persaud, S. J., Jones, P. M. Pancreatic beta-cell-to-beta-cell interactions are required for integrated responses to nutrient stimuli: enhanced Ca2+ and insulin secretory responses of MIN6 pseudoislets. Diabetes. 48, 1402-1408 (1999).
- Spiess, Y., Smith, M. A., Vale, W. Superfusion of dissociated pancreatic islet cells attached to Cytodex beads. Diabetes. 31, 189-193 (1982).
- Pham, P. L., et al. Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis. Biotechnol. Bioeng. 90, 332-344 (2005).
- Thomas, P., Smart, T. G. HEK293 cell line: a vehicle for the expression of recombinant proteins. J. Pharmacol. Toxicol. Methods. 51, 187-200 (2005).
- Maurisse, R., et al. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnol. 10, 9 (2010).
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The Ultimate Behemoth is a type of RV similar to that of a double-decker bus, but the steering wheel is on the top floor. It features a big screen TV, its own satellite, an elevator, 4 deep fryers (one for each part of the chicken), along with various other features (including a horn sounding very similar to a ship's foghorn). It was being sold at Bob's RV Round-Up. Homer and the children were impressed, but Marge worried the expensive price on the RV.
Homer wanted to buy the impressive RV in an attempt to stay ahead of Flanders, despite Marge's warning not to because the family can't afford it. While working out a price deal with Cowboy Bob in his office, a dismayed Homer finds out that he had been rejected due to his poor credit rating. It turns out that the Ultimate Behemoth is way out of Homer's price range and he's only qualified for a dilapidated RV which further disgusts the rest of the Simpsons, hoping he'd have the common sense to walk away without one.


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Ted Bundy was definitely no exception. He was a man of many fetish. Voyeurism, pornography, bondage, rape, murder, necrophilia. He was a Ted of all trades. However, the one fetish I find fascinating, to say the least, is Ted's sock fetish.
"This is for real. I mean, I've got a sock fetish. No question about it. I must have six or seven pairs right here with me in my cell. I am really sick when it comes to socks. These are some of the things for people who really want to know what makes Ted Bundy tick. They're parts of the combination to the deepest, most secret recesses of my mind. I'm very close to my feet. Right now. I'm lying on my back with my foot propped up on the bars. And I'm studying my toes. For a good portion of the night. They're probably the most attractive feet you've ever seen.
Socks are such a serious part of my life. They're so very important to me. They kept reading the list of socks and all and I felt proud. Honestly, it didn't even begin to occur to me that people might wonder why I had all these socks. I just felt proud that I owned all those socks.
The only time I began to have a little bout of sheepishness was when he read about a white sock with a blue band and green stripe on the toe. Those are odor eaters – and that was getting too personal."
They all know me by my car
They all know I'm gone, but not how far
I'm heading straight down 101
I'm going to go and get me some
My lips are movin' but there's no sound
Someday somebody's gonna get run down
My lips anr movin' but there's no sound
Someday somebody's gonna get run down
– Faith No More


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Friday, March 17, 2017 - 20:30
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non·sen·si·cal [non-sen-si-kuhl] - (of behavior, conduct, actions, etc.) foolish, senseless, fatuous, or absurd
she·nan·i·gans [shuh-nan-i-guhnz] - Silly or high-spirited behavior; mischief.
Tuesday, January 31, 2012
Monday, January 30, 2012
Just a disclaimer
Listen. Just because I listened to something that you produced does not mean I'm in love with you. Yes, maybe I did come to a show that I wouldn't have normally come to, but NO it was not just because you were there. Well, maybe that was part of it. But I know you! And I had nothing else to do! So why not?! I'm not stalking you, or coming to your house and holding a radio over my head saying something along the lines of "OH MY GOSH WHY DON'T YOU LOVE ME?!" or "WHY WON'T YOU BE MY BOYFRIEND?!" One, I'm not that kind of girl, and two, I don't know where you live. Also this is not an 80's romcom and that shit doesn't work anymore. Or maybe it does, I don't know, it's never happened to me. I'll make sure to report my findings if/when that happens. Although my window faces an alleyway with huge garbage bins so, that's unlikely. Unless the guy likes garbage. In which case I'll have to change my scent...actually, no, I'll keep what I'm wearing. Smell garbage on your own time, buddy. I'm not that kind of girl!
So...now that we've gotten THAT out of the way...wanna make out?
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Carl W. Kenney II is an award winning columnist and novelist. He is committed to engaging readers into a meaningful discussion related to matters that impact faith and society. He grapples with pondering the impact faith has on public space while seeking to understand how public space both hinders and enhances the walk of faith.
Monday, March 12, 2012
Supporting the Right to be Married
Photo from Ms Magazine
On Saturday, March 17th, citizens of Durham will renew their vows. On last year, I officiated over the ceremony that hitched folks with the city we all love so much. I'm happy to report that my love affair with Durham has strengthened over the past year. I'm learning to love her even more.
As I prepare to lead the service of renewal, I'm saddened by what it means to be one able to take advantage of my position of privilege. It's a truth I never thought would part my lips. Never before have I considered myself one able to function with the advantages that come with being formed with the body given me at birth. I've come to take for granted being minimized due to my humanity. I've assumed disadvantages as a black man.
Living with this black skin framed in manhood has led me to assume the worst whenever I move in the presence of white privilege. I take for granted not getting interviews despite my massive credentials and talent. I take for granted that people will deny me opportunity simply because of how I look. I also understand that this is a truth that those who function in privilege simply can't understand.
What does that have to do with marrying Durham? Everything. It draws attention to the assumptions that come with heterosexism. We heterosexuals reap the advantages of all that privilege related to being able to go to the court house, pick up a marriage license and marry another willing heterosexual at will. We have the right to express our love legally. It's a privilege that we uphold based on our contention that those unlike us are unworthy of the same privileges we take for granted.
So, imagine how it must feel for people to marry the city when they can't get married to their partner? They are allowed to come out and make vows to a community while being forced to accept they can't do the same with the person they love so much. That is hurting me deep.
I'm thinking about it more this year due to my intent to get married soon. Yes, I've come to the conclusion that it's time to throw away bachelorhood and tie the old knock with Ms. Connie. Given all I feel for her, and all it means for me spiritually to jump on the marriage wagon, it hurts that doing so pits me as one willing to take advantage of my position of privilege while those I care about are unable to do the same.
By now a line of conservative minded Christians are ready to throw stones my way. I can hear the pages turning from that Bible they use to fight. I'm certain they are preparing to use that Bible to prove how God hates homosexuality, and that I'm standing on the side that supports people in maintaining those sinful ways. Others will talk about how allowing "those people" to get married will destroy the fabric of society and lead America even further from being what the crafters of the Constitution intended.
Isn't America supposed to be a nation that offers freedom for all citizens? Shouldn't we honor the separation of Church and State? Shouldn't we want for all our citizens the same privileges that those with power take for granted?
Given my personal internal struggle, it has become difficult for me to embrace marriage at this time. How can I when others I care about can't. What does it mean for me to walk within that privilege and hold my bond of love before them as a reminder of what they can't have due to the laws of this land? Is that love? Is that right? Is that being the compassionate person we all claim to be?
Some may regard that as a cop out. Go ahead and say it – you don't want to get married! Not true. I do, but how can I cheerfully participate within a system that uses my heterosexuality as the privilege that validates my decision to walk in that space?
Would it be different if only white people could marry? Maybe we could see it if we limited marriage for those within a certain economic bracket. I hear my critics again. It's not the same. Gay folks are an admonition against God! Okay, since you go there, is that a theological claim? If so, isn't that in opposition to the Constitution we claim is used to keep America the land of the free?
Those critics will argue that those white dudes who signed the Constitution had a Christian nation in mind when they spoke of one nation under God. Give me a break! We are more than a Christian nation. If we allow our views of God to fuel our political decisions, we become what America was before those who signed the dotted line left England to find religious freedom.
Yes, I will renew my vows to Durham on Saturday. I want to get married, but what does it say given the struggles of my friends to do the same?
Slap me for caring about those I love. It isn't right.
All they want is to say I do.
Posted by REV-elution at 11:38 AM
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