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…to when you roll the dice and swear your love's for me
Song of the day: Finley Quaye with William Orbit: Dice
Well, I'll be the first to say it; I've been wholly MIA for the past week. And I don't just mean from the internet; I mean from life in general. I've been completely swamped and overwhelmed with my final days of practicum. Work and school completely took over. I didn't have time for even my most basic exercise regime or hanging out with friends. As a result, both my physical and mental health were shot out the window. I'm feeling tired, lazy, and just plain gross as a result. Starting today, I'm kicking my life into order. I have a little over a week to shape up and it's going to happen. These are the areas I'm going to be focusing on:
- Physical activity: I've done little more that a few yoga classes this past week, which just leave me feeling deprived. I'm one of those people who literally needs copious amounts of physical activity to function. Without it, I feel depressed, tired, and (yeah, I'll say it) just plain fat and lazy. Having not done enough cardio or having enough variety the past week, I'm really going to step it up and fit in as much as I can in my time off. That means at least two activities a day, but hopefully three. Obviously I'll be focusing on running and yoga, and I'll try to throw in some swimming and maybe kayaking or hiking. I want to get back into the physical and mental state I was in while focusing on marathon training. I know I'm not as commited just to running now that I've thrown a bunch of other basic activities into the mix, but I'm going to whip out my old book on running (thanks for all the advice, John; you're the best!) and start with a really basic plan, to keep my motivation up. I recieved the book a few years ago for Christmas and it really kept me on track (pun wholly intended, heheh) because I really wanted to hit my goals each day. not to mention it kept things interesting since I would mix things up between hill runs, slow runs, long runs, and so on. So we'll see how it pans out. I'll probably aim for a morning run to wake me up, or an afternoon run to soak up the summer sun.
- Detoxification: Yes, I'm one of those people who believes in detox and ridding the body of toxins and waste. And while I know all the arguments for and against things like colon cleanses (namely, that the body is already designed to deal with ridding itself of waste, so messing with it will just cause problems), I always feel significantly better after cleansing and notice it in many areas of my body (including better skin, better digestion, and far less bloating). Plus, having been wicked stressed out the past few weeks, I definitely went on a bender for the past two days and intook horrific amounts of toxins. Thankfully, bikram does a solid job of ridding quite a bit of toxins. I've tried my own "homemade" cleansers at home before (basically just water and sea salt), I've decided to go less drastic this time around. I don't want to get dehydrated since I'll also be doing yoga. I'm going to try the SlimQuick Cleanse. I feel comfortable with the ingredients, and it sounds less harsh than just (essentially) making yourself dehydrated. Of course, I'll need to ensure that I intake at least double the water that I've currently been intaking, so I'm going to aim for about 12 litres a day (yes, I drink a lot of water; it's necessary for the amount I exercise and the amount I sweat; not to mention, it gives me great skin). I haven't even gone out and bought it yet, since I've pretty much just woken up (thank God I barely get hangovers, because today would be brutal), but I'm going to pick it up today or tomorrow and I'll update with how I feel about it and how it works.
- Organization: Okay, I'll just admit it; I'm an organization freak. While other all my friends were reading Cosmopolitan and Elle, I was reading Style at Home, House and Home, and Home and Garden. I have every single Ikea catalogue since 1998 (and they're all neatly piled away in an Ikea folder on an Ikea shelve, I may add). These past few weeks, the condo has become a disaster. I've barely had time to bathe and clothe myself, let alone clean, do laundry, or put things away. As a result, things are piled everywhere. I haven't taken the recycling out in two weeks (this includes all paper products, metals, plastics and glass, as well as bottles and cans), I have a pile of clean laundry on my floor which has yet to be folded and put away, as well as a pile of dirty laundry threatening to overtake the hamper, and the kitchen sink is a disaster. Normally I'm a neat freak and on this stuff immediately (especially the kitchen, since I get really grossed out when anything that's touched food is out), but I have literally not had time. My roommate is pretty much the opposite of me when it comes to putting things away, so essentially the problem has arised from me not having the time to clean up after her all week. It's out of control and I'm going to attack it as soon as I finish this entry. I'll hit the kitchen first and do an all-over attack. Then I'll hit the dollar store and get a bunch of hangers, binders, baskets, etc. to organize all the things I've collected over the past few weeks of practicum and to get myself organized for classes starting next week. Then I'll hit up the bottle depot downtown sometime today or tomorrow to rid the place of all that nastiness and to make a solid couple of dollars (oooh yeeeeah). It's gonna be great. It's sad how excited I am.
- Nutrition: Again, my life has fallen to pieces lately. I've been coping with disgusting amounts of sugar, fat, and basically shit food that I normally avoid at all costs. I'm going to have to start again with my food/drink rules. I try to avoid rules and just stick with my basic intuition, since I normally eat pretty well anyway. but I've completely fallen off the wagon and I need to throw myself back on and that's going to take a huge amount of force. So I'm going to have to cut a lot of stuff out. The "no-go" foods are basic and obvious: anything refined (sugar, flour, fats), dairy, caffeine, soda, anything high in fat (basically nut butters, which is a personal issue I've talked about before), anything deep fried. Pretty much just unhealthy foods. I need to work on intaking a lot of water-based foods and lean protien. Think fish and veggies.
- Writing and reading: I want to really get a focus back on writing. I try to fit it in as often as possible when I'm in school and working, but it's difficult. As I've mentioned, I've recently begun writing a second novel, and I want to really focus on it. I began it a few weeks ago and have yet to make much process. I've literally got nothing more than a basic chapter-by-chapter outline and the first few paragraphs of the first chapter. I need to sit down and plan exactly how much I'm going to write each day. Last summer I did a great job of finishing my first novel and staying on task, but I also had a job that allowed me to work on it for 10 hours a day. This week, I'd essentially like to park myself at a coffee shop (or on a patio, weather allowing) for at least a couple of hours a day to just write.
So there you have it; my plan to get back on track. As of right now, my days are loosly set up as such: wake up, run, find a coffee shop and write, swim, find a coffee shop and write, yoga, clean and organize my house, sleep. It should be good. Lots to do, but all things that I love and enjoy and will feel really positive about. I'm feeling great; I've got a really good feeling about this and a great energy. I suppose I'd better get off my ass and actually get into gear right now. I'll be updating.
Much love!
PS: Can we talk about how much love I have for the summer time, even when I get horribly burnt because my sunscreen only lasts a few hours and I forgot to pack extra for an afternoon walk?


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T.I. wants everyone to know that he loves his life. After several stints in jail, T.I. is back (again) with his first official single off his eighth studio album, Trouble Man LP.
"Love This Life," produced by Mars of 1500 or Nothin, is a throwback to 2008's "Whatever You Like," when T.I. gave his lady anything she wanted in the world.
But this time around, T.I. isn't speaking to a stock music video character — he's directly addressing the one and only woman in his life, his wife Tameka "Tiny" Cottle. The married couple is living out their happily ever after for all the world to see on their reality TV show on VH1, "T.I. & Tiny: The Family Hustle."
In "Love This Life," T.I. goes beyond his usual materialistic references — bags, shoes and jewelry — and expresses his need for an understanding relationship.
"Mansions, jewelry all I need is for you and me to have some understanding / Have a couple candid conversations / Your frustrations I take in consideration."
But that's not to say the brand name-dropping rapper doesn't fill his quota of swag.
"Anything you could ask, do it for you / Drop Jags, Berkin bags, Louboutin heels / Summer homes in Miami, Hollywood Hills."
Listen to the entire song in the video below.
By Sara Gates

subset: commoncrawl


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Make in India - Andhra Pradesh - The Future____________________
About Andhra Pradesh
January 2017
VISAKHAPATNAM: To promote and strengthen the MSME sector in the district, the district administration has appointed a 12-member committee, including former chairmans and managing directors of RINL, BHPV and HSL Siva Sagara Rao, Venkateswarlu and RL Bhatiya.
The Union government's public procurement policy-2016, clearly advocates that every PSU should procure at least 20 per cent of its material from MSME sector.
The report would used as a State policy document to boost up the sector across the State, according to Ramalinga Raju, the convenor of the committee and general manager of the Industries department. The district collector is involved in the exercise.
November 2016
Action in respect of Re-organisation of Andhra Pradesh has been completed as the Conceptual Development Plan (COP) in respect of Vizag-Chennai Industrial Corridor (VCIC) was submitted by the Asian Development Bank (ADB) in December 2014 as part of ECEC. ADB has identified four nodes namely Vishakhapatnam, Kakinada, Gannavaram-Kankipadu and Srikalahasti-Yerpedu of Andhra Pradesh for development.
The Draft Final Report on Reginal Perspective Planning (RPP), was submitted to Government of Andhra Pradesh (GoAP) in February 2016 by ADB to solicit their feedback.
Master planning of all four nodes as identified by ADB in their COP commenced on 21st March, 2016 and is likely to be completed by March, 2017
May 2016
ITC will set its Agri-Business Headquaters at Guntur, A.P.
Jan 2016
A.P. - CII partnership Summit resulted in signing of memorandums for a total of Rs. 4,78,000 crore with private sector (331 MOUs) and total of Rs. 6.21 lakh crores including public sector.
Updated 21 January 2017, 13 November 2016, 1 May 2016, 14 Jan 2016


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Shaking sand out of our shoes and packing up a treasure trove of happy memories tonight.
Friday afternoon, we pulled the kids out of school at lunch time.
The school secretary played along and said "good luck at the doctor" as I walked out the door early with the kids grumbling that they were going to miss PE class; their favorite.
It wasnt until they got in the car and noticed the beach toys and boogie board packed that they unraveled the mysterious early pick-up from school.
A chorus of delighted squeals erupted from the back seat!
We have a friend who owns a hotel down in Myrtle Beach and has graciously made it available to us a number of times. We jumped at the chance for one more kiss of summer sun. What a blessing!
We've soaked up sun, laughed a lot, eaten junk food and just enjoyed togetherness as our little family. Long walks, shell collections, late nights, lazy mornings. Blissful vacation!
This was Gabriellas debut trip to the beach. My mother tells me I took my first steps at the beach. A love for this place runs deep. Im proud to say, another generation loves the seashore!
The guys dug trenches, rode waves and and threw the football for hours....
Mommy got to play "dress up" with her girls for a photo shoot I was asked to do for Dayspring Dresses and Nana Rose Designs. A fun new coordinating line that was super-fun to shoot with the beautiful backdrop of the beach.
Family photo shoot = Fail!
Why is it that I love photographing other families, but doing my own stresses. me. out!?
Oh yeah, because I belong BEHIND the camera!
This photo cracks me up. This is what we got after an hour and a half of getting everyone showered, dressed, hair combed, etc......a cranky baby and 5 tired kiddos after 6 hours straight on the beach.
Dont they look happy?:)
Oh well, there's always next year....
My gifts list for this week:
Sand between toes
The smell of Banana Boat lotion
Strong hand of my beloved in mine
Falling asleep to the sound of waves crashing on the shore
Little freckled noses (Izzy calls freckles "sprinkles")
Last kiss of summer sun on shoulders
Giggles from frolicking children carrying across the dunes
Salty sea spray
Last light of day reflecting off of tidepools
The pitter-patter of little feet that leave such profound imprints on my heart!


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| Welcome to my website. I am a Departmental Lecturer in Syntax and a Departmental Lecturer in Indo-Iranian Philology in the Faculty of Linguistics, Philology & Phonetics, University of Oxford. Prior to this I held a three-year postdoctoral research fellowship, funded by the Leverhulme Trust, also at Oxford.
Besides this website, I have a page on Academia, where you can find some additional talks and papers. If you are a member of Academia, you can also 'follow' me to keep up with my work. I also have a page on ResearchGate. This website is likely to be the most up to date.
I have recently finished a monograph on 'transitive' nouns and adjectives in Sanskrit and Pali, published by Oxford University Press in June 2017. My previous monograph, based on my D.Phil. research on the syntax and semantics of participles in Ṛgvedic Sanskrit, was published in 2015, also by Oxford University Press. I am also working more widely on syntax, semantics, and historical linguistics. You can download my CV here.
My research interests include: Sanskrit language and linguistics, syntactic theory, formal semantics, Indo-Iranian comparative and historical linguistics (Sanskrit, Prakrit, Avestan, Old Persian), Greek and Latin historical linguistics, Indo-European comparative philology, the Ancient Indian grammatical tradition, Germanic comparative and historical linguistics (especially Gothic, Old Norse).
I 'know' the following languages: Ancient: Vedic Sanskrit, Classical Sanskrit, Prakrit, Avestan, Old Persian, Middle Persian, Greek, Latin, Gothic, Old Norse, Old English, Aramaic. Modern: French & German; a bit of Russian and Hindi. I am currently learning Biblical Hebrew.
My departmental email address is of the form firstname.lastname@example.com.


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Mouse: Click and drag to create a box. everything in the box will be destroyed!
Help the garden gnome find his treasure! Gnome Sweet Gnome is an awesome puzzle game where you must use your physics knowledge to solve tough puzzles! Your objective is simple; destroy blocks with your mouse in order to solve the puzzle! THATS IT! Also, learn fun gnome facts along the way. Simple, addicting, and challenging. What more can you ask for?
Developer: Unknown
Published: 2014-07-07


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This is a condensed version of our Ultimate City Bootcamps talk on the stress hormone cortisol. We will cover what cortisol is, what it does, why we have it, some of its effects and also some action steps in how to reduce it.
We hope after reading this, which you may have to do in a few sittings as it is still quite comprehensive, you will feel empowered and aware of what may be the sticking point in your life (as it is in the lives of many).
This little hormone that is so essential to our well being is also a thorn in the side of optimal health, fat loss, lean muscle gain, a good nights sleep, and can be the cause of depression, anxiety, food cravings, reduced bone density and the onset of certain degenerative diseases, as well as many, many other factors.
Definition of STRESS
'What you feel when life's demands exceed your ability to meet those demands'
a) What Is Stress?
Cortisol is a highly tuned and efficient hormone that activates when the body comes under stress. This is the hormone that activates for the fight or flight reaction to stress.
If we look at an example of cortisol in action there is an initial brief increase in energy levels, hormone levels, and ability for forceful muscle contraction. The body is prepared to deal with the stressor or escape from it (fight or flight). The simple science is that the cortisol causes the body to release sugars and fats into the blood stream as these are the fuels for this fight or flight response. Think of an example of a caveman foraging for food when he comes face-to-face with a Saber Toothed tiger. On first contact the body will release the cortisol instantaneously to allow the man to run away fast, or to sharpen his senses in order to find a weapon nearby to fight the predator.
b) A Look At Cortisol
Cortisol is produced by the adrenal glands which are walnut sized glands located on the tops of the kidneys. These glands produce many crucial hormones and are vital for our well-being.
Normal cortisol levels help the body function properly. It helps convert fats and proteins to energy, keeps us alert, balances electrolytes, calibrates the hearts beat and pressure and counteracts inflammation.
Cortisol is a very important hormone in the body but we need it balanced, not too much and not too little. It is naturally higher at about 8am to give us some get up and go for the days start and reduces later in the day at about 4pm when we should naturally be starting to wind down for the day. This is the natural cycle of cortisol… If we think about how we evolved, we had no electricity, so would wake when the sun rose and get to bed when the sun had set.
Stress rapidly increases cortisol levels but after removing the stress, these elevated levels come back to normal. In other words our bodies are not designed to be stressed for long periods.
The problem is that modern day stresses are not always removed which causes our cortisol levels to remain high and cause long term health problems over time.
In the past we would have seen the Saber Toothed Tiger, ran away/fought it, or got killed, and used up the extra sugar and fat in the bloodstream as fuel to get away and then have our cortisol levels come back to normal. Once the stressor is removed the Cortisol is back to where it should be.
Studies show that cortisol levels spike if we do not get the magic eight hours sleep a day. Back in the 1910 working adults got on average nine hours a night, now it's at seven and this is just the average. As we stress more, we sleep less and so cortisol levels remain elevated… it's a vicious circle. In many ways sleeping less than eight hours a day is as bad for overall wellness as gorging on junk food or becoming a couch potato and if you do all three.
In the modern world we have late dinners, meetings, nights out, family pressures, stressful work, etc. and all the while we consume stimulants like caffeine, nicotine and alcohol amongst others.
Things that can also raise cortisol levels-
The job is long hours and high pressure, economic climate, money worries, family and relationship, public speaking, being late, illness, personality conflicts, kids, bills, chemicals at home and work, air pollution, bad diet, electromagnetic pollution (from electrical items at home and work), pollutants in the foods and drinks we consume, the list goes on…..
Is it any wonder that people are getting ill and have problems in their lives!
In terms of how stress relates to the workplace please read this report from back in 2003 (before the financial crash) where 1.5 million hours are said to be lost to this hormone every year. If you work in Human Resources then this has to be something that is targeted to ensure people are healthy, happy and in the office.
Also too much exercise can stress the body and lead to big health issues. Ever hear of the runners and athletes that drop dead from heart attack, that can be cortisol working it's way over years due to lack of recovery, as the body is in constant stress.
A coffee a day is shown to have health benefits, but once you approach three and more then your adrenal glands start to get stressed out and produce too much cortisol. Many people drink far more caffeinated drinks too, such as Red Bull, Coke, Pepsi, and a whole host of others.
Also look out for supposed wonder diet pills. They come in lots of different forms and with marketing attached saying how they will strip the fat off you, or it is something that Amazonial tribes have been taking for years and they're not fat…. right?
These products are guaranteed to give the adrenal system an unwelcome overload (often on top of other stressors), which will alter your bodies natural process. People may lose weight short term, but they are lowering their immune system and once they stop the pills, the weight will often return. Avoid them!
Not sure if you know the stats on cigarettes, but we won't go there…
Scientific research shows-
High levels of cortisol has a debilitating effect on long term health
1 – Increased appetite and food cravings
2 – increase body fat
3 – decreased muscle mass
4 – decreased bone density
5 – increased anxiety
6 – increased depression
7 – mood swings
8 – reduced libido
9 – impaired immune response
10 – memory and learning impairment
11 – increased symptoms of PMS
Lets look at the points –
1 – Increased appetite and food cravings
Primary role of cortisol after responding to a stressor is to encourage the body to refuel, because it assumes you have used the fuel (fats and sugars) you dropped into your blood stream to escape from the stressor.
However in modern day stress cases, the cortisol remains there, which makes you, feel hungry all the time and craving carbs or sugar as these are what supposedly need topping up.
Many people don't realise that losing that final few inches off their waists is down to removing stress from their lives and not the amount of time they spend exercising.
Stress induced appetite will cause us to gain body fat in the abdominal region, as this is the area that is supposed to be able to activate the fastest in the flight or fight response. In women the area may be more targeted towards the butt and legs.
No one wants a fat belly but more importantly belly fat is associated with heart disease, diabetes and cancer, so you don't just look bad, you are now at a higher risk of these diseases.
If we are stressed we will keep storing fat in this way until the stressor is removed or we throw in some stress reducing tactics
Cortisol is catabolic meaning it will make your muscles smaller and weaker – vicious circle if you are trying to "tone up" or lose some fat of your bum or make your stomach flat.
The muscles break down as the cortisol allows the release of certain amino acids (proteins to you and I) that are also used in the metabolic process of fight or flight for a burst of energy.
In order to get the best results from exercise we need to make sure we are not constantly doing long, cardio based workouts and focus more on Weight Training and Circuit based classes. Read this from the blog for more info >>>SPINNING<<<<
Bone density also takes a hit as the bones are robbed of vital nutrients and minerals due to the presence of cortisol. If the cortisol remains in the body then the bones will not be allowed to properly maintain themselves.
This, over time, will open the door for such things as osteoporosis and arthritis. In later life people are more susceptible to falls through slowing down (the wasting away of muscles through lack of use) and through stress response. Once they fall down many may break a hip, or bone due to bone weakness.
The body then gets even more stressed through more cortisol being released and so the bones never repair. Another vicious circle. Start thinking now about looking after your bones.
This problem can be compounded with women after menopause and is another reason that weight-bearing exercise is so important for anyone of any age!
The anxiety is there for a reason. We are on edge to think fast and take action against that nasty stressor. Every sense is on full alert. Have you ever walked into a dark room that you were not sure about and someone stepped out and shouted boo. You were on edge to spring into action and away.
However, have you ever been in a traffic jam, with no control to get where you want to be on time? You get agitated and god help anyone that is in the car with you. This is cortisol and this was developed by evolution over thousands of years, whereas traffic jams have developed in the last century.
This is why shouting, or banging the wheel can be a good short-term stress reliever. Even stop the car and have a quick sprint, or do some press-ups. Sure you'll look stupid, but it's a great way to lower stress levels.
Many people turn to a coffee, a cigarette or an alcoholic drink in times of stress, not realising that they are actually adding to their already heightened cortisol levels. Green Tea is a good alternative to Coffee as it contains theanine which is shown to reduce cortisol levels as well as a whole host of other things… >>>>CLICK HERE FOR THEANINE INFO<<<<
Depression can be set off by many factors that are far too broad to discuss here, but it is important to understand one of the reasons that we may be getting depressed in the first place… Cortisol!
Who likes to be stressed? Depressing thought isn't it? Who likes going up a trousers size, or a dress size? Who feels great after having a junk meal and who beats themselves up after being so stupid in eating that meal and then orders a desert to make them feel better?
Who enjoys the thought of being on a diet? It's actually been proven that one of the main factors that diets don't work is that the thought of starving ourselves and having to miss out on our favourite foods, releases cortisol and makes us crave even worse foods even more than usual. We may begrudgingly resist the foods for the duration of the diet, but as soon as we come off it then the body is now primed for fat storage as it needs to top up the stores for the cortisol to be able to act and boom we put all that weight back on.
Serotonin production, the bodies feel good hormone, is supressed when cortisol is high.
This ties in once again to the feeling of anxiety and also to how we feel as a result of eating foods that satisfy our cortisol spiked hunger, but not the well being functioning of our bodies.
We feel good after eating a balanced meal that is high in good fats and good protein. When we eat junk food we get sluggish and moody due to another hormone being worked overtime called insulin which as many of you will know, is a big factor in diabetes.
A good way to see how food is affecting your mood is to keep a food diary. Note down what you have eaten and then what your mood is thirty minutes and one hour later. It can make for very interesting reading! If you have children then keep a food diary for them too and you may see that they are playing up due to the food you are feeding them and not because they particularly mean to.
Increased cortisol decreases the sex hormones testosterone and estrogen. Testosterone is the major hormone for the males amongst us, but females need it too to feel frisky. Women also need estrogen, and a few other hormones.
Cortisol also supresses endorphin production which is the hormone that makes you feel up or down as mentioned when we're depressed.
Some men take supplementary forms of testosterone to try to increase their levels, but this may not work as the cortisol is still too high and overpowers them.
Cortisol is the enemy of Testosterone, so if you're stressed you're unlikely to want to get undressed!
This is one of the stranger effects of the release of cortisol, as it would seem to make sense that the body would not make a hormone that actually can cause self-damage.
However on closer inspection it is shown that the immune system actually gets a boost initially upon cortisol release and as we were programmed to only have occasional spikes in cortisol then this worked just fine. Remember the stressor would usually have disappeared in the past.
Now that cortisol is high day and night then the problems occur as the finely tuned bodies response system is now confused the response system is blunted and the high spike in immune response has to adjust, as what goes up must come down.
In certain cases the body may be open to attack from certain autoimmune diseases such as cold sores and mouth ulcers. However as time goes on and as stress is not combatted then the body will possibly become open to attack by certain degenerative diseases such as Heart disease, Cancer, Alzheimer's, Osteoporosis, and so on.
10 – Memory and learning impairment
I'm sure everyone now is getting the idea that once the body is stressed then priority is given to some areas over other areas all in the name of survival.
In much the same way that you would not go outside and paint the garage when a tornado is approaching, the body does not see memory as an important requirement when in a life or death predicament.
The area that is given low priority as brain function moves over to being in the moment and focused at first so to be able to escape or to know where to strike first in the case of a fight. Remembering what you have to do next week does not really matter if a predator eats you now!
Once the body has been exposed to the stress for too long though cortisol takes hold and starts to play more havoc with the brain chemicals called the neurotransmitters.
11 – Increased symptoms of PMS
Well if you have a quick look at what we have covered so far in the effects of cortisol on the effects of cortisol on irritability, mood swings, even anger and rage then you may be able to make the tactful connection as to why Cortisol can have drastic effects on PMS.
Couple this with the statistics by a physicist named Guy Abrahamson which discovered that PMS patients ate 62% more refined Carbs, 275% more refined sugar, 79% more dairy and a whole host of other percentages and we can see how PMS and cortisol combine.
c) What can be done to alleviate stress and reduce cortisol
if you're not just a little bit stressed out from listening to that, then you've not been listening very well.
However there are proven effective ways to help us counteract this very modern problem that most of us face without realising the harm it can cause.
If you are anything like us, then a light bulb has gone off in your head and you now realise there is a potential answer to a few questions of why we don't feel as 'on the ball' as we used to.
It is also important to note that some people have much higher thresholds to stress than others and seem to thrive in high-pressure situations. These people must be careful of burnout, however, and make sure they get a balance in life or cortisol will eventually take its toll.
Our Grandmothers had it right when they said get enough sleep, eat good food and get some exercise. If we look after our bodies then our bodies will look after us, after all there is a lot of sense in the words of the well-known nutritionist Michael Colgan; "If you don't look after your body then where are you going to live!"
Step one is to be aware of the factors that stress you out. This sounds simple but most people live in a world where they do not take the time to stand back and ponder why they are lacking energy, sleeping badly, overweight, down, etc. and put it down to being just how it is or the best excuse ever… that they are getting old.
You should now be aware of a few areas where you are stressed, such as lack of sleep, too much coffee, too much exercise, bad late night eating habits and maybe work pressures.
Naturally some stressors are easier to remedy than others with a strong mind-set. For example stopping to drink so much coffee is far easier that quitting your job.
Be aware in order to take care!
This is the one where the most of us can make a difference quite fast if we take the steps.
Ways to improve sleep. Cut stimulants such as alcohol, caffeine and nicotine after 17:00. Alcohol can have big effects on the quality of sleep, even though people assume it's relaxing, and caffeine and nicotine are no brainers. (please note the removal of all of these will make a big difference)
Cut junk food (processed, high sugar and saturated fat foods) completely and especially after 16:00.
Eat protein with every meal. Eat more good fats. Are you taking supplements? A good quality zinc and magnesium supplement before bed may be the thing you are missing. f you are missing just one millionth of a gram of a certain mineral every day, over a period of time then you now have an imbalance and that imbalance may effect a whole host of things. Sleep can be one of them.
Try to get a set time to go to bed every night in the week and stick to it, we should be able to reprogram our sleep habits within a week of consistent sleep timing. You have to stick with this, think of having jet lag and re-adjusting to the new timezone. If you can do it then, you can do it for anytime.
Try to do something relaxing before bedtime, like having a herbal bath or having a relaxing tea. Reading a book can help some too, as long as it's relaxing. (Notice the use of relaxing multiple times). If you watch a horror film that gets your pulse racing, don't be surprised if sleep is not in abundance.
Make sure you room is dark. Light pollution is a major problem in cities and we will not get the best sleep if even a little light is present. Black out blinds should be fitted to the windows.
Drink plenty of water. Once dehydrated the body will respond as if it is experiencing stress, thus increasing cortisol levels and increasing the likelihood of disrupting our sleep.
Be aware of excessive amounts of electrical equipment in your bedroom and especially around your bed as the electromagnetic pollution can affect sleep/wake cycles. Make sure any alarm clocks, mobile phones on charge, music systems, TV's etc. are turned off near the bed, or make sure they are placed well away from the bed. Any electric cable running under the bed, or close to it should also be powered off.
It's not a case of saying you have no time for exercise, it's a case of finding the time for exercise. Three times per week is ideal and if you see the exercise as part of your working day, a client if you will, then you will find the time to schedule this important activity into your life to improve your life. You are your most important client!
Exercise is proven to reduce cortisol levels, through hormonal changes and through using up the sugar and fat that is flowing about in the blood.
Serotonin and dopamine levels increase during and after exercise and exercise is thus shown to be more effective than anti-depressants over e 4 month period if exercising for 30 minutes a day three days per week.
Naturally good exercise will decrease body fat, increase muscle tone, gives bones better density, improve mental function. It also stimulates the immune response, reduces appetite and makes you look great naked…… I'd say that's a pretty good trade off for investing three hours a week keeping active.
Make sure you always take the stairs, your body will thank you for that one tip.
However exercise to most is about as interesting as watching paint dry, so that's why we are offering everyone here a free trial session at out indoor Ultimate City Bootcamps gym near Liverpool Street
Yes Hippocrates knew what he was talking way when he stated. "Let food be thy medicine and medicine be thy food". What an amazing quote and the more we get to know about what we do and don't eat, the more this rings true. Conversely a modern version could be; "let food be thy poison and poison be thy food."
So from this nutrition can be crudely split into two areas; what to eat and what not to eat. There are some crossovers between the two and we all want to have a life and one of the pleasures of life is a great meal! Through simple choices in the majority of the week we can easily enjoy those meals that in the past would have weighed down on us…. Literally!
We don't have much time today and our blog has lots of free information on food choice. Here are a few tips to reduce stress and lose fat in the process;
- Eat Breakfast!
- Do not drink calories
- Eat Natural Foods
- Read labels
- Eat protein with every meal
- Cut down on carbs
- Eat good fats
- Take top quality vitamins
- Eat Breakfast – Breakfast is without a doubt the most important meal of the day. It sets you up for the day ahead and what you eat will determine what kind of day your body has on a biological level.
Step one is to start to eat breakfast, and i know you may not be used to it. We understand it's a big step for some people, so even natural yoghurt with some oatmeal on it and a little something to sweeten it (say some berries, or cherries) is a good starting point. Maybe some nut butter on some brown bread. This is by no means an ideal breakfast, but it's all about conditioning the body for step two.
Once you have got your head around actually eating breakfast it is time for step two. If you do this modern nutrition science backs up that you are having the best start to a day that you can give yourself as you will be eating foods that will give you the clarity, alertness, good energy levels, better moods and a whole host of other things. It's hard for a lot of people to get their heads around, but then I imagine that was the case when people first heard that the world was round and not flat and that the earth orbits the Sun and not the other way around!
Read this article, take action and feel and see the difference >>>PERFECT BREKKIE<<<
- Do not drink calories – this is very simple, and will make a big difference to the vast majority of you. Stop drinking juice, eat the fruit instead. Juice is loaded with sugar, without the fiber to slow down it's digestion. That leads me nicely to any fizzy drinks that have sugar in, keep away from them. The sugar free ones are bad too, but better than their sugared cousins.
Do you have sugar in tea and coffee? If you have two, then start to drink it with one and a half for two weeks. Then go down to one for two weeks, and then half… you get the idea. I did this and can now actually taste what I am drinking instead of a sweet nothingness.
Do you like lattes? Ever thought of how many calories are in a latte? Many people like a skinny latte….. Listen if you take the fat out of milk, then you are left with pretty well just the lactase (the milk sugar). When the sugar is there with no fat to slow down it's absorption in the gut, then it gets digested far too quickly. This means you get fat storage from drinking a drink that you think will make you skinny, like the name. Do not be fooled. If you have milk in a coffee, switch to an Americano with a splash of milk. Far better for you.
Protein shakes post workout are the only times we would say to drink calories. This is the window of opportunity, when the body wants the nutrition in a form that it can digest quickly and provide the bodies cells with what they need to recover and build back stronger and leaner. Check out the blog and FB Page for post workout tips.
- Eat Natural Foods – when possible the lions share of the food you eat should have come from the following; It should have been grown on a tree, grown in the ground, or have had parents. For some tips on this type of eating then check out this blog post >>>>PALEO FOOD<<<<< Eat as much of your diet like this and see how you feel! Clue the word starts with a 'G' and sounds like weight.
- Read labels – no brainer here, just turn the product around and ignore all the marketing on the front. Read what the label says and that is where the truth is. Is the product market 'low fat' and yet loaded with sugar? Is the main ingredient fructose, or glucose, or both? Is flour one of the first ingredients? Is there lots of saturated fat? Is there lots of words and letter and numbers that you really don't understand? Best to steer clear then and look to a natural unprocessed, natural, and real food instead.
- Eat Protein with every meal – The best way to provide your body with the proper nutrition to function at an optimal level is proven to be to feed it little and often and to include a serving of protein with every meal.
Protein is essential to life and eating it with regularity helps to keep the metabolism fired up, to stop cravings for other fatty and sugary foods, to help properly maintain blood sugar levels and thus keep us energised and helps provide the body with the nutrients to combat cortisol, rather than to fuel it.
Naturally the addition of vegetables, occasional fruit, nuts, seeds and other natural foods help balance this out.
- Cut down on carbs – different people have different views on the subject of how many carbs to have on our diets. Needless to say the average western diet is loaded with too many of them, and of the wrong type.
If you have read above then you will know that we certainly say to steer away from any carbs for breakfast (that is after you have conditioned yourself with the protein and good fats breakfast).
Keep away from white carbs, like white bread, white rice, white pasta, breakfast cereals etc. Eat muffins for breakfast, then don't be surprised if you get a muffin top over your jeans.
If you are going to eat carbs, stick to starchy vegetables and other pulses such as lentils, or certain beans, no not baked beans (read the label of a can of them one day).
I will have organic steel cut oats occasionally for a change, but I can tolerate carbs better than most people.
The occasional piece of fruit is ok, but look to have it for a mid morning snack or early afternoon. Also the type of fruit is important. Best to go for is apples and pears, or cherries and certain berries. Keep away from too many grapes, or bananas, or pineapples if you want to keep the sugars low!
- Eat good fats – there are fats that are good for us and fats that are bad. The really bad ones are the hydrogenated ones we find in spreads and margarines, biscuits, pastries, and a million other processed foods. Remember when I said check labels, well if you see the word hydrogenated then put that item down right away and walk away!
Contrary to old beliefs, some saturated fat is good for us and is important for hormonal function, so don't get scared if there is a little saturated fat there. Butter is by far a better choice as a spread than margarine and other 'butter type' spreads.
For cooking use butter or my favourite extra virgin coconut oil. It has a high smoke point (so it does not go rancid) and tastes great. But it does not stop there, here's the down low on this amazing oil >>>>COCONUTS<<<<
Want to know what we feel is the best oil and what should be taking pride of place in each person's fridge???? If you get the right stuff then it will be the best nutritional step you have taken in years, think there is something fishy going on? Well check this link >>>>THE KING OF OILS<<<<
Not forgetting the other amazing oils that are found in nuts, seeds and avocados. When it comes to nuts, I do not include peanuts. They are actually a legume and can cause more bad than good. Stick to the super nuts, such as walnuts, almonds, cashew nuts, Brazil nuts, and pecans. Seeds such as sunflower, sesame, pumpkin, chia, and the big hitter flax. Flax is great milled such as the ones that Linwood's produce that are bought in Holland and Barrett's and can be sprinkled on most things.
- Take Top Quality Vitamins – we get all the micro nutrients (vitamins/minerals) we need from our diet don't we? Alas, no we don't. If you want to reduce the rate at which you age, and reduce cortisol levels through nutrition then you need to be providing your body with ALL the nutrients it needs. If you miss just a few millionths of a gram of just one micro-nutrient on a daily basis over a duration of time then you can be leaving yourself open to attack. Sleep, energy, metabolism, cell reproduction, joint health, vision, and a whole host of other things we take fore granted will be compromised if vitamin, mineral and antioxidant levels are not optimised. For more information, and why choosing the 'multivitamins' is vital for health then READ MORE HERE. If health is a priority then this is a must read!
Breath Deeply
I wanted to add this important part of the equation in.
Many of us have our own stress reduction steps, often without even realising it. For example some may reach for a glass or two of wine to 'wind down'. For others it is sitting in front of the TV and watching very little (and doing even less) and for other it is the comfort of comfort eating that cake. All of these can be considered as stress relief in the short term, but stress causing factors in the longer term, as you'll have already read.
It is important to find other healthy outlets to help us deal with the stress and deep breathing is one of those steps. Shallow breathing is common in the Western World and the more stressed many of us get, the more shallow we tend to breath. It's a vicious circle.
I could go on but I think it's much better to hand over the reigns to the amazing Dr Michael Murrey and his steps to reduce stress and relax by breathing properly. his site is a Aladdin's cave of amazing, health improving information.
Manage Stress With Diaphragmatic Breathing
"If you fail to plan, then you plan to fail!" This is one of my mantras and is so important in the subject of Stress relief and thus the regulating of cortisol.
You have hopefully gone through the steps of awareness and identified what stresses you out. Now you have that list, then plan on which stressors you will reduce first.
For example if the list was as such, need to sleep more, need to drink less than the four cups of coffee I drink daily, drink no alcohol on weekdays, need to get less stress at work, need to remove electrical items from close to my bed, need to black out my bedroom.
The easier ones there would be to get the bedroom blacked out and to remove the electrical items and place them elsewhere or turn them off (you'll also save on electricity – bonus). These two may also help sleeping more!
Next up would be coffee. Go from drinking four cups a day, down to three for a week. The next week you go down to two and the final week down to one. Swop a glass of water into its place or a green tea (I know many people don't like it, but drink it for it's benefits as the theanine will also help with de-stressing you and fat loss as well as all the good antioxidants in it!). Once you are down to one cup then remove it all together – just to prove to yourself you can. Now see how much more alert you feel, and guess what, maybe you can sleep ok again now too. Now when you have a coffee you'll get that buzz you used to get when you first started to drink it way back when and get the positive effects without the negative ones!
With the alcohol, just cut down from a few drinks a night to one, to none! You can do it, no doubt about it. You'll also notice some fat loss from doing this and from removing the other stressors!
Next would be sleep. Well hopefully you are now sleeping better as you have removed the other stressors and added the blackout blinds. See the section on sleep for tips on how to improve it.
Last stressor was work. This is one that is harder to change. However now you have changed the others, then your threshold for stress and the release of cortisol is now much higher! The little things that used to bother you and stress you will now not even raise a eyebrow and now that you are aware of the stressors and what stresses you, then you'll walk out of the office and hit the gym, or do some breathing exercises.
Recommended reading:
If you want to take your studies on Stress and Cortisol to the next level then here are two amazing reads.
Please check out our FACEBOOK page for the latest free info from us at Ultimate City Bootcamps. Thanks for reading and life's here to be enjoyed…. don't stress about it.
Train Hard, Live Easy
Tom @ UCF


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We are just like you, bunch of people which are very commend original idea from every one, with no exception. That is the reason we always keep the original images without changing anything including the watermark. Every pictures gallery we publish are be guaranteed carrying the owner link where it belongs to be below each photos. Many message came to us about the proper right related with the photos on our gallery. When you want to know your right, please contact the website on each photos, the reason is we cannot determine your true right. Do not forget, no watermark does not mean the images is able to freely used without permission.


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We are planning the Maine Comics Arts Festival for Spring of 2009. We will announce the exact location and date in the very near future. It will be held in Portland.
This event will be a celebration of the comics art form. We expect to have dozen's of guests. This will be similar to events such as SPX and APE that are creator driven. That means that there will be no "dealers" set up at this show. Writers, artists and publishers are welcome.
Anyone interested in volunteering to help can e-mail firstname.lastname@example.com . There will be plenty to do.
Much of the planning for this has been in the works for this for some time now. We know that the northeast has a very strong comics community, and Portland in particular has many creators. We plan on reaching out beyond just a regional show .
The to do list is growing, but hopefully we can get the registration information up soon. A new website for this is in the works. Keep posted to this blog for more information. Thanks!
Rick Lowell
Casablanca Comics
Portland, ME
Sunday, November 16, 2008
Maine Comics Arts Festival- 2009
Posted by Maine Comics Arts Festival at 6:40 PM
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- Research article
- Open Access
Identification and characterization of microRNAs involved in growth of blunt snout bream (Megalobrama amblycephala) by Solexa sequencing
© Yi et al.; licensee BioMed Central Ltd. 2013
- Received: 20 August 2013
- Accepted: 31 October 2013
- Published: 5 November 2013
Blunt snout bream (Megalobrama amblycephala) is an economically important fish species in the Chinese freshwater polyculture system for its delicacy and high economic value. MicroRNAs (miRNAs) play important roles in regulation of almost all biological processes in eukaryotes. Although previous studies have identified thousands of miRNAs from many species, little information is known for miRNAs of M. amblycephala. To investigate functions of miRNAs associated with growth of M. amblycephala, we adopted the Solexa sequencing technology to sequence two small RNA libraries prepared from four growth related tissues (brain, pituitary, liver and muscle) of M. amblycephala using individuals with relatively high and low growth rates.
In this study, we have identified 347 conserved miRNAs (belonging to 123 families) and 22 novel miRNAs in M. amblycephala. Moreover, we observed sequence variants and seed edits of the miRNAs. Of the 5,166 single nucleotide substitutions observed in two libraries, the most abundant were G-to-U (15.9%), followed by U-to-C (12.1%), G-to-A (11.2%), and A to G (11.2%). Subsequently, we compared the expression patterns of miRNAs in the two libraries (big-size group with high growth rate versus small-size group with low growth rate). Results indicated that 27 miRNAs displayed significant differential expressions between the two libraries (p < 0.05). Of these, 16 were significantly up-regulated and 11 were significantly down-regulated in the big-size group compared to the small-size group. Furthermore, stem-loop RT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in ten tissues, and the result revealed that the conserved miRNAs expressed at higher levels than the novel miRNAs, especially in brain, liver and muscle. Also, targets prediction of differentially expressed miRNAs and KEGG pathway analysis suggested that differentially expressed miRNAs are involved in growth and metabolism, signal transduction, cell cycle, neural development and functions.
The present study provides the first large-scale characterization of miRNAs in M. amblycephala and miRNA profile related to different growth performances. The discovery of miRNA resource from this study is expected to contribute to a better understanding of the miRNAs roles playing in regulating the growth biological processes and the study of miRNA function and phenotype-associated miRNA identification in fish.
- Mature miRNAs
- Single Nucleotide Substitution
- Zebrafish Genome
- Solexa Sequencing
- sRNA Library
Blunt snout bream (Megalobrama amblycephala Yih, 1955), which natural distribution is limited in the middle and lower reaches of the Yangtze River in China[1], has been widely favored for its delicacy and recognized as a main aquaculture species in the polyculture system of Chinese freshwater fish since 1960s[2]. Due to its high economic value, the total production of M. amblycephala is rapidly growing[3]. However, owing to overfishing of wild resources, artificial breeding and fast domestication, the germplasm resources of M. amblycephala are under threat of recession and admixture[4]. Unfortunately, the cultured population of M. amblycephala gradually exhibits growth depression, early sexual maturity, and disease susceptibility. At present, molecular techniques are still not widely used in the breeding of M. amblycephala due to a lack of genetic and genomic information. In recent years, several traits of M. amblycephala, including body shape, hypoxia resistance and disease resistance, have been studied through development of molecular markers[4–6]. Although it is an important economic trait, the growth rate of M. amblycephala has not been well defined by molecular markers or control mechanisms.
MicroRNAs (miRNAs) are a class of small (approximately 22 nucleotides; nt) endogenous noncoding RNAs in length[7], which are embedded within the stem regions of hairpin transcripts that exist in a wide range of invertebrates and vertebrates. miRNAs play a pivotal role in the regulation of gene expression at the post-transcriptional level, especially for signaling pathways involved in development, cellular differentiation, proliferation, apoptosis, and oncogenesis[8]. They negatively regulate gene expression through sequence-specific interactions with the 3′ untranslated regions (UTRs) of target mRNAs and thereby cause translational repression or mRNA destabilization[8, 9]. Since the discovery of the founding members of the miRNA family, lin-4 and let-7 in Caenorhabditis elegans in 1993[10–12], many endogenously encoded miRNAs have been identified in mammals, plants, insects, worms, and viruses through plasmid vector cloning, northern blotting, microarray assay and sequencing technology in recent years[13–15]. Currently, 21264 mature miRNAs from 193 species have been discovered and deposited in the public available miRNA database miRBase (Release 19.0, June 2013)[16].
Recently, next-generation sequencing technology has a substantial impact on a broad range of biological applications. Sequencing technology made it possible to precisely identify non-conserved or weakly expressed miRNAs, and been widely used to facilitate the identification and detection of miRNAs in multiple species, such as fish, chicken and silkworm[14, 17, 18]. Previous studies have identified many miRNAs from model fish species[19]; nonetheless, little attention has been given to the miRNAs' roles in non-model species. The identification of miRNAs in aquaculture fish species began with the study of cloning and characterization of miRNAs from the rainbow trout (Oncorhynchus mykiss) in 2008[20]. Subsequently, a large number of miRNAs has been identified in other aquaculture species, including the Japanese flounder (Paralichthys olivaceus)[21], bighead carp (Hypophthalmichthys nobilis), silver carp (H. molitrix)[22], common carp (Cyprinus carpio)[23] and channel catfish (Ictalurus punctatus)[24]. Unfortunately, little information is available in M. amblycephala. In this study, we constructed two small-RNA cDNA libraries from the growth related tissues (brain, pituitary, liver and muscle) of M. amblycephala using individuals with relatively high and low growth rates. Through high throughput sequencing of the small RNA library and subsequent bioinformatic analysis, miRNAs in two libraries of M. amblycephala were identified and the differentially expressed miRNAs were analyzed. The discovery of miRNA resource from this study will contribute to a better understanding of the miRNAs roles playing in regulating the growth biological processes in fish and the study of miRNA function and phenotype-associated miRNA identification in M. amblycephala.
Result and discussion
Solexa sequencing of small RNAs
Due to the lack of whole genome data for M. amblycephala, we aligned the selected small RNA sequences to the genome sequence of zebrafish, which is evolutionarily the closest related species with an available sequenced genome, to perform a distribution analysis on a genomic scale using SOAP. For the selection of the computing algorithm, we chose a tolerance of one mismatch for mapping, which resulted in a total of 16,461,503 reads (86.32%) that represent 26,984 unique sRNAs were mapped to the reference genome (Additional file2: Figure S1).
Conserved miRNAs in M. amblycephala
To identity the known miRNAs in M. amblycephala, we compared the data from two libraries to known miRNAs in miRBase 19.0 (http://www.mirbase.org/). According to the results, we analyzed the base bias on the first position of identified miRNAs with certain length and on each position of all identified miRNAs respectively. Among the 16,411,948 sequences screened in total, 2,711 unique sequences in big-size group were found to be similar to known miRNAs from other species that had previously been deposited in miRBase. Allowing no more than two mismatches between sequences, these miRNAs represented 332 known miRNAs, belonging to 8,171,600 sequences in total in the big-size group. Meanwhile, 2,695 unique sequences dated from small-size group were screened out in the same way, and we identified 343 conserved miRNAs (Additional file1: Table S1-4). Combining the data from two libraries, a total of 347 unique mature miRNAs were identified, which belong to 123 families, including 326 miRNAs that overlapped between the two libraries, 4 and 15 miRNAs that were detected only in the big-size and small-size libraries, respectively. The reads of these miRNAs were ranged from 1 to 2,649,630, indicating that not only highly expressed miRNAs but also weakly expressed miRNAs were identified by Solexa sequencing. From the 5,406 unique sequences, a total of 60 duplex-like miRNA: miRNA* pairs were obtained, in which the mature miRNAs and miRNA*s (miR-#-5p and miR-#-3p) align to the 5′ and 3′ end regions of the precursors, respectively (Additional file3: Table S2). Recently, some miRNA* sequences (miR-#-3p) were reported as mature functional miRNAs with abundant expression, and miRNA/miRNA* ratios may vary dramatically in different stages of development[25]. In M. amblycephala, it was found that most of the miR-#-3p (miRNA*s) were detected at the same or relatively low expression levels than miR-#-5p. It is suggested that the expression level of miR-#-3p mainly relied on degradation degree and degradation rate, because both strands of miRNA duplex were necessarily produced in equal amounts by transcription. However, some miR-#-3p showed relatively higher expression levels than miR-#-5p (such as miR-206-3p, miR-199-3p, miR-124-3p). The relatively high number of reads of these miRNA*s indicates that it may play a functional role in regulating gene expression. Such a phenomenon has also been described in several previous studies[22, 24].
For the two libraries, more than 8 million sequences corresponding to 85.86%, 86.26% of all clean reads were annotated to known miRNAs, respectively, showing that our sRNA libraries were highly enriched with mature miRNAs. The identified sequences being 18 ~ 26 nucleotides in length from two libraries showed a strong bias for U in the first nucleotide (Additional file4: Figure S2). For the miRNAs that have already been identified and validated, mam-let-7a-5p has the highest expression in both libraries, with 2,393,380 and 2,649,630 sequences in big-size and small-size libraries, respectively. These data were in agreement with other studies of miRNAs[22, 26, 27], showing that let-7a ranked among the highest expressed miRNAs in the muscle. The mam-miR-1-5p, a muscle specific miRNA, was also abundant in both libraries which has been implicated in the determination of the differentiated state of muscle cells and in myogenesis[28, 29]. The mam-miR-122 was also dominant in both libraries belongs to a liver specific miRNA family which is implicated in fatty acid and cholesterol metabolism[30, 31]. Previous studies have shown that miR-122 was characterized as the most frequent miRNA isolated in the adult liver, reaching around 70% of all cloned miRNAs[32]. This tissue-specific miRNA is also thought to establish patterns of gene expression and may be responsible for maintaining tissues differentiated states[33, 34]. In contrary to let-7a-5p, miR-122, the numbers of some miRNAs (such as miR-132b, miR-183, miR-218b and miR-430) was less than 5 reads in both libraries.
Novel miRNAs prediction
Since the genome data of M. amblycephala is unavailable, the unannotated small RNAs that could be mapped to the zebrafish genome sequences were subjected to novel miRNA prediction analysis of their secondary structure, the Dicer enzyme cleavage site and the minimum free energy using Mireap software (https://sourceforge.net/projects/mireap/). The approach was used to predict novel miRNAs relies on the phylogenetic conservation of the sequences, and this would cause unidentified novel miRNAs. According to the criteria for miRNAs used in this study, we finally obtained 22 putative novel miRNAs in M. amblycephala. Of these novel miRNAs, 14 novel miRNAs were found in both libraries, while 3 novel miRNAs were identified only in the big-size group and 5 novel miRNAs were identified only in the small-size group (Additional file5: Table S3). Intriguingly, the sequencing frequencies of these novel miRNAs (1,467 in big-size group, 2,130 in small-size group) were much lower than that of conserved miRNAs in the big-size and/or small-size groups' libraries. The same pattern has also been reported in other species[22, 24], which suggests that novel miRNAs are usually weakly expressed while conserved miRNA genes are highly expressed. In addition, the length of the novel miRNA sequences varied from 21 to 23 nt, with a distribution peak at 22 nt (63.89%). The novel miRNAs identified from the two libraries had the same characteristics with the conversed known miRNAs, which have a strong bias for U in the first nucleotide (Additional file6: Figure S3). Many studies had showed that one notable feature of miRNAs was an overwhelming bias for U at the first position[35, 36].
Differentially expressed miRNAs
These differentially expressed miRNAs were sequenced at varying frequencies. Some miRNAs such as mam-miR-462, mam-miR-92a, mam-miR-92, were detected with relatively high sequence counts both in the big-size and small-size libraries, although their abundance differed substantially. Subsequently, we compared the differentially expressed miRNAs that have the relatively high reads numbers in both libraries. The expression level of mam-miR-462 (7,665 reads), mam-miR-92a (1,882 reads), mam-miR-92 (2,083 reads) and mam-miR-23b (375 reads) was more than 2 fold higher in the small-size library than that in the big-size library. Remarkably, studies indicated that mam-miR-462 was up-regulated with viral haemorrhagic septicaemia virus (VHSV) infection in zebrafish[36] and rainbow trout[37]. Similarly, mam-miR-92a, mam-miR-92 and mam-miR-23b were associated with proliferation of myeloid cells and biosynthesis of interleukin[38, 39]. This result suggests that these miRNAs at high expression level in small-size group may be associated with immunity and disease of fish. In contrast, the sequencing frequencies of some miRNAs, including mam-miR-10b-5p, mam-miR-10d-5p, mam-miR-133b-5p, mam-miR-9b-3p, mam-miR-novel11, mam-miR-novel19, mam-miR-novel21, mam-miR-novel22, mam-miR-novel6, mam-miR-2187-5p, and mam-miR-551, were low in both of the libraries. It is possible that these miRNAs are expressed at low levels in certain cell types and/or under certain conditions.
Sequence variants and editing of bases in the seed region
Target prediction of differently expressed miRNAs and KEGG pathway
The identification of miRNA targets is an important step to describe the function of miRNAs well. Computational approaches have been used in many studies as an effective strategy to predict miRNA targets[22–24]. The conventional point of view is that miRNAs regulate target genes by binding to the 3′ UTRs of target mRNAs, and multiple binding sites for multiple miRNAs in 3′ UTRs can strongly enhance the degree of regulation. Recently, many studies have also demonstrated that a large number of miRNA binding sites reside in the 5′ UTR and the coding sequence of mRNAs[48]. A total of 1,901 targets (1,371 unique targets) included 154 EST sequences of M. amblycephala were predicted for the 24 highly significant differently expressed miRNAs (P < 0.01), which were identified in this study. The predicted targets for the differently expressed miRNAs were shown in Additional file8: Table S5. Some predicted targets were likely to be targeted by multiple miRNAs at multiple targeting sites. Typically, insulin-like growth factor binding protein 2a (NM_131458) can be targeted by five miRNAs, including mam-miR-462, mam-miR-1-1, mam-miR-122-2, mam-miR-novel 12 and mam-miR-novel 6.
The predicted target genes were classified according to KEGG functional annotations to identify pathways that were actively regulated by miRNA in M. amblycephala (Additional file9: Table S6). Intriguingly, the most over-represented miRNA targets belonged to the metabolic pathways, which is described as a set of complex metabolic networks[49], such as lipid metabolism, carbohydrate metabolism, amino acid metabolism, and energy metabolism. This complex network performs a variety of anabolic and catabolic tasks[50], which serve to transfer part of the chemical energy of the substrate to ATP (or its equivalents) or to NADPH for use in other cell functions, and convert precursor molecules into compounds from which macromolecules, including proteins, complex carbohydrates, nucleic acids, and lipids[51]. Another pathway targeted by the differentially miRNAs was the MAPK signaling pathway, which is a key signaling pathway in skeletal muscle of fish, where its activation is absolutely indispensable for muscle cell proliferation[52]. In fish, the MAPK/ERK can be activated by the insulin-like growth factor-I (IGF-I) in primary cultures of myosatellite cells[53, 54], which is a key regulatory hormone that controls growth in vertebrates[55]. Likewise, the insulin signaling pathway was also found to be enriched in our results. Insulin is the most potent anabolic hormone, mediating a wide spectrum of biological responses, including the synthesis and storage of carbohydrates, lipids and proteins and inhibiting their degradation and release back into the circulation[56].
Furthermore, pathways associated with phagosome, biosynthesis of secondary metabolites, tight junction, protein processing in endoplasmic reticulum, purine metabolism, endocytosis, adheren junctions, cell adhesion molecules, regulation of the actin cytoskeleton, focal adhesion, cell cycle and chemokine signaling pathway were all significantly enriched, indicating the role of the differentially expressed miRNAs in the regulation of cell motility, cell proliferation, the cytoskeleton, cell nutrition, communication between cells and the extracellular matrix. Moreover, enriched pathways for neuroactive ligand-receptor interaction, axon guidance and the neurotrophin signaling pathway, suggest that the differentially expressed miRNAs participate in nervous system development and function. On the whole, the results indicated that these differentially expressed miRNAs were involved in regulating growth and metabolism of M. amblycephala. It is, however, important for future studies to functionally validate the predictions of these differentially expressed miRNA targets.
In summary, we identified 347 distinct conserved miRNAs and 22 novel miRNAs from growth related tissues (brain, pituitary, liver and muscle) at different stages of M. amblycephala using Solexa sequencing. Our study provides the first large-scale characterization of miRNAs in M. amblycephala and miRNA expression in response to the high and low growth rates. The expression levels of these miRNAs displayed a large range, and many of these miRNAs showed differential expression between the big-size and small-size groups. Function annotation of the predicted target genes of the differentially expressed miRNAs revealed a broad range of the metabolic pathways and biosynthesis processes. These findings support the hypothesis that certain miRNAs along with their target genes might be essential in the intricate growth regulation networks, and it will be critical to develop new strategies for the molecular breeding of M. amblycephala.
Animals and tissue collection
All experimental animals were derived from offspring of M. amblycephala selective population, which were bred in the Ezhou Fish Breeding Base of College of Fisheries, Huazhong Agricultural University. All experimental procedures involving fish were approved by the institution animal care and use committee of the Huazhong Agricultural University. Tissue samples were collected from individuals in big-size group and small-size group, which were selected from the same families, including 3-month-old, 6-month-old, 12-month-old and 18-month-old stages, with 6 individuals from each stage, respectively. The fish were anaesthetized in well-aerated water containing the 100 mg/L concentration of tricaine methanesulfonate (MS-222) before tissue collection. Tissue samples including muscle, heart, liver, kidney, gill, spleen, gonad, intestines, pituitary and brain were immediately collected to extract total RNA. The samples were snap-frozen in liquid nitrogen and stored at −80°C.
Small RNA isolation and cDNA library construction
For Solexa sequencing, tissue samples including brain, pituitary, liver and muscle from the big-size and small-size groups were collected within the same population, respectively. Total RNA was isolated from each sample using Trizol reagent (TaKaRa, Dalian, China) according to the manufacturer's protocol. RNA quality and quantity was measured using the NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). All the samples were standardized to 500 ng/μL, and equal volumes of the tissue samples from different individuals in the same group were combined into one pool.
Two small RNA (sRNA) libraries were constructed for brain, pituitary, liver and muscle tissues from the big-size and small-size groups, respectively. Briefly, small RNAs of 16–30 nt in length were first isolated from the total RNA by size fractionation, and these small RNAs were ligated to with 5′-RNA and 3′-RNA adapters and then reverse transcription PCR using the RT primer was used to create cDNA. Subsequently, a PCR reaction was performed using primers complementary to the two adaptors. The amplified cDNA constructs were purified and sequenced by Illumina/Solexa technology (BGI, Shenzhen, China).
Sequence data analysis
Low quality reads were trimmed and reads containing poly A stretches, reads less than 18 nt and adaptor sequences were accurately clipped with the aid of a dynamic programming algorithm. Subsequently, the remaining clean reads were analyzed by BLAST against the Rfam (ftp://ftp.sanger.ac.uk/pub/databases/Rfam/) database and the GenBank noncoding RNA database (http://blast.ncbi.nlm.nih.gov/) to annotate rRNA, tRNA, snRNA, repeat associate sRNA, and other ncRNA sequences, and then aligned to exons and introns of mRNA to find the degraded fragments of mRNA in small RNA tags. The common and specific reads of two samples were summarized, including the summary of unique reads and total reads. The clean reads were mapped to the zebrafish genome with a tolerance of one mismatch in the seed sequence by SOAP[57] to analyze their expression and distribution on the genome.
In addition, miRNA identification was performed by comparing the data from two libraries with the known mature miRNAs and the miRNA precursor of all plants and animals in miRBase19.0 (http://www.mirbase.org/), showing the sequence and count of miRNA families which can be found. Subsequently, we analyzed the base bias on the first position of identified miRNAs with certain length and on each position of all identified miRNAs, respectively. The sequences that are not identical to the conserved miRNAs were used to BLAST against the zebrafish genome to identify potential novel miRNAs using the SOAP software. Sequences with a perfect match or with one mismatch were retained for further analysis. We used Mireap software (https://sourceforge.net/projects/mireap/) to predict novel miRNA by exploring the secondary structure, the Dicer cleavage site and the minimum free energy of the unannotated small RNA tags which could be mapped to the zebrafish genome. Then the prediction of novel miRNA candidates were summarized, including the base bias on the first position among small RNA candidates with certain length and on each position among all small RNA candidates. Based on this summary, the prediction accuracy could be assessed according to the base bias of known miRNAs.
Differential expression analysis
The N1 and x represent total count of clean reads and normalized expression level of a given miRNA in sRNA library of the big-size individuals' tissue samples, respectively. The N2 and y represent total count of clean reads and normalized expression level of a given miRNA in sRNA library of small-size' tissue samples, respectively. When |log2 (big-size group/small-size group) | oupll-size mall0.05, it was be seen as differential expression.
Quantitative stem-loop RT-PCR with SYBR Green PCR Master Mix (Applied Biosystems) was performed to profile the expression levels of the differential expressed miRNAs in 10 tissues. Total RNA from heart, liver, spleen, kidney, muscle, intestine, brain, gill, gonad and eye of M. amblycephala was isolated using Trizol reagent (Invitrogen) following the recommendations of the manufacturer, and real-time quantification of miRNAs was performed by stem-loop RT-PCR. Briefly, eight primers of differential expressed miRNAs for stem-loop RT-PCR were designed according to descriptions in prior study[58] (Additional file10: Table S7). Real-time PCR was carried out on a Rotor-Gene Q real-time PCR Detection System (QIAGEN, Germany) according to the manufacturer's instructions, and all real-time reactions were performed in triplicate. Relative expression levels of the novel miRNAs were measured in terms of threshold cycle value (Ct) and were normalized to 5S rRNA using the equation 2-△△Ct, in which ΔCt = Ct miRNA–Ct 5S.
MiRNA target prediction
In order to predict the target genes of miRNAs in M. amblycephala, a support vector machine (SVM) developed at BGI (Shenzhen, China) was trained to determine the optimal parameters to be use in RNAhybrid. The parameters included helix constraint (2–8), internal loop size (5), bulge loop size (5) and maximum target length (100,000). RNAhybrid predicts potential binding sites for miRNAs in large target RNAs using the principle of finding the most energetically favorable hybridization site between two sequences[59, 60]. Considering that the genome references of M. amblycephala are not available, we selected the sequences of zebrafish genome and EST sequences of M. amblycephala sequenced in our laboratory to predict the target genes with the strategy described in prior studies for target prediction[9, 61]. Briefly, the criteria were as follows: 1) no more than four mismatches between sRNA and target (G-U bases count as 0.5 mismatches), 2) no more than two adjacent mismatches in the miRNA/target duplex, 3) no adjacent mismatches in positions 2–12 of the miRNA/target duplex (5′ of miRNA), 4) no mismatches in positions 10–11 of miRNA/target duplex, 5) no more than 2.5 mismatches in positions 1–12 of the of the miRNA/target duplex (5′ of miRNA), and 6) minimum free energy (MFE) of the miRNA/target duplex should be > = 75% of the MFE of the miRNA bound to it's perfect complement.
Variants of the miRNAs in M. amblycephala
Variants of miRNAs, called isomiRs, are commonly reported in deep-sequencing studies[62]. The Solexa sequencing results in this study revealed that the majority of identified miRNAs showed length and sequence heterogeneity. The nucleotides at position 2–8 of a mature miRNA is known as the seed region (the so-called "miRNA seed") and this region is highly conserved[63]. The target of miRNA might be different with the change of nucleotides in this region. In our analysis pipeline, miRNAs which might have base edit can be detected by aligning unannotated sRNA tags with mature miRNAs from miRBase19, allowing one mismatch on certain position.
Availability of supporting data
All small RNA data are available in the NCBI Gene Expression Omnibus database under accession GSE51638. The other supporting data are included as additional files.
This study was funded by the National Natural Science Foundation of China (No. 31201988), the Modern Agriculture Industry Technology System Construction Projects of China titled as—Staple Freshwater Fishes Industry Technology System (No. CARS-46-05), National Ministry of Science and Technology Support Program (No. 2012BAD26B00), Excellent Youth Foundation of Hubei Scientific Committee (2013CFA032), New Teachers' Fund for Doctor Stations, Ministry of Education (20110146120021) and Fundamental Research Funds for the Central Universities (No. 2011PY023 and 2013PY066).
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Suicide bombers in cars hit checkpoint in eastern Libyan city of Benghazi, killing 5
CAIRO – A spokesman for the Libyan army says suicide bombers in two cars have attacked an army checkpoint in the eastern city of Benghazi, killing five people and wounding 20.
Col. Ahmed al-Mesmari said it was not immediately clear how many of the casualties were military or civilian. He spoke from Tobruk, where the internationally recognized government has been based since being expelled from Tripoli by Islamist-backed militias last year.
Libya has been mired in months of fighting between rival militias. Extremist groups, including affiliates of the Islamic State group, have exploited the turmoil since the 2011 overthrow and killing of Moammar Gadhafi.
Also Tuesday, officials in the Tripoli government mourned the death of Mohammed al-Aribi, a commander killed fighting against Tobruk forces in Benghazi alongside Islamic State and al-Qaida-linked militants.


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Ayr may have been league leaders before this match kicked off but little good it did them yesterday afternoon at Meggetland where they lost their first match of the season. The home team gave their illustrious visitors a lesson in accurate, high tempo, off-loading rugby and Ayr couldn't comprehend let alone cope with it.
The visitors held almost all the high cards and out-muscled their hosts across the field but Muir, by necessity as much as anything, played with pace, running the ball from all corners of the field and catching Ayr cold. The three tries and four points were no less than their adventure and ambition deserved.
Muir did enough to suggest that they can be competitive in the BT Premiership at least as long as the weather stays warm and Edinburgh offer them the use of breakaway Jamie Richie. The professional flanker put in a huge shift for the home team, carrying tirelessly and showing unexpected offloading skills.
The Muir backs were slick and adventurous and they combined, along with Richie, to score the best try of this match, a length-of-the-field effort in the first half. Just about everyone got their hands on the ball as Muir attacked left and then right with loosehead prop Ross Dunbar scooping up the loose ball and sprinting behind the posts.
For the visitors scrum-half David Armstong ran well and posed problems with the ball in hand but Ayr's backs were a little sloppy especially in the first half when their wingers fluffed scoring chances. Cammy Taylor was bundled into touch five yards out and Craig Gossman spilled a scoring pass on the opposite flank just a few minutes later.
Muir's four point lead at the break became an 11-point lead just a few short minutes into the second half. The Ayr defence parted like the Red Sea for Muir's number eight and skipper Craig Keddie who looked like he might go all the way only to be dragged down short of the line. So it was left to lock Jamie Scott to barrel over from short range after a simple pick and drive, Laidlaw converting expertly. Trailing by 17-6 it suddenly dawned on Ayr that they were in danger of losing this one and the men in pink duly adopted their best game face. Frasier Climo kicked a penalty into the corner and Armstrong thought he had scored after a clever lineout ploy only for the match officials to spot a foot in touch.
It wasn't until Scott was sent to the sin bin that Ayr's big pack began to press their foot firmly on Muir's throat. One lineout drive was held up before a set scrum marched their way over the line for number eight Blair MacPherson to score one of the easier tries of his career.
Just as it looked like Ayr might come off the canvas, Muir scored their third try of the afternoon. It was sparked by winger Jordan Edmunds who made far more metres than he should have up the middle of the park. Muir battered away at the Ayr line until eventually Keddie squeezed over by the posts and Laidlaw was never going to miss.
To add injury to insult Ayr's flanker Gregor Henry left the field on a stretcher in the closing minutes to top off a forgettable afternoon for the visitors.