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Legend Of The Perfect Emperor Chapter 79
Chapter 79: The Odd Duo
Li Liling stood right outside the entrance of the Dragon Gate Sect, her eyes filled with admiration towards Li Qinqin.
She has not visited Li Qinqin in months, and this is how much she has grown since then? This is not the always quiet Li Qinqin she knows!
"See, elder sister? We got out just fine," said Li Qinqin with a smile.
"B-But" Li Liling turned to look at the guards that were staring at them with disbelief in their eyes. She didn't know what was happening or how it had come to this point.
One moment she was cultivating in the sect and the next she is outside the entrance with her younger sister who she hasn't seen in months.
"Even though we left the sect, where will we go now?" Li Liling asked her.
"I plan on going to the Purple Cloud Continent," said Li Qinqin. "There is something I want to confirm."
"Something you want to confirm?" Li Liling lifted an eyebrow at her words.
Li Qinqin looked at the coin in her hands and nodded with a mysterious aura surrounding her.
For the first time in her life, Li Liling was unable to understand Li Qinqin's thoughts something that she used to be very good at.
"Let's go, elder sister!"
Li Liling smiled at Li Qinqin's enthusiasm and nodded. "Let's go We have a lot to catch up on, don't we?"
Li Qinqin returned her smile with her own. "Yes!" she said with a wide smile on her face that shone brighter than even the morning sun.
Somewhere in the woods, two figures slowly limped around as they supported each other by putting their arms around their shoulders.
These two figures were both wearing robes with the same pattern as though they received them from the same place. And just like how their robes have the same patterns, the condition of their robes was also similar tattered and filled with holes looking like it had been cut many times with a sword.
Except for a few bruises, no blood could be seen on their body; it was as though the one who attacked them did not want blood to mess with their awkwardly hilarious appearance.
"Fuck! These damned bastards from the Overseeing Sect! I swear I will have their heads underneath my feet the next time we cross paths!" spat one of the figures, a young man with a round body, looking as though he was a walking meatball.
"Fei Rou if your fatass didn't provoke them by flirting with one of their disciples, would this have even happened? I know she was pretty and all, but we'll die at this rate..." sighed the other figure, a slim young man with sharp features, looking like a scholar.
"Ke Zhong, you asshole! It was you who didn't want to stop staring at that disciple's butt and got caught like a fool!" Fei Rou said in disbelief of his shamelessness.
Ke Zhong shook his head and said. "I may have been staring, but you were the one who had went in and spoke with her!"
"Fuck you! If I didn't immediately go and apologize to them, we would not have gotten away with just holes on our robes that exposes only our nipples, but holes around our body! We'd be dead!" Fei Rou gnashed his teeth, feeling like punching Ke Zhong whose appearance did not match his character at all.
"Fine, maybe I do have a part in this mess But if your fatass didn't apologize to them with an attitude as though you are their senior, would they even have drawn their swords in anger?!"
Fei Rou looked at him with wide eyes, but he was unable to talk back this time around.
"Good! Then this is a draw!" he then said after a moment of silence. "As expected of a fellow disciple who is junior to me by a few minutes!"
"Fuck you!" Ke Zhong quickly moved his hands and aimed at Fei Rou's exposed nipples.
Suddenly, a scream that sounded like a pig getting slaughtered echoed in every direction.
Fei Rou instantly pushed Ke Zhong away after having his nipples twisted by him.
"You you Out of all the places on my body, you had to aim at that part?! Draw your sword! We will fight to death today!" Fei Rou shouted with tears in his eyes, his hands covering the two holes on his chest.
"Good! Don't think I have forgotten about that day when you kicked me in between my legs and called it an accident! How the hell do you kick a place like that by accident?! Today, we will fight to death!"
Both Ke Zhong and Fei Rou reached for the side of their waist where they usually strap their sword, but alas, their hands grabbed nothing but empty air as they have forgotten that their weapons were taken by the disciples that had caused them to be in this state.
The two stared at each other awkwardly for a moment before returning to their position.
"You better pray to the heavens tonight for helping you escape your death today!" Ke Zhong sneered in a stiff tone.
"As expected from my fellow disciple who is a junior by a few minutes! Your luck may not be as good as mine, but it is damn sure near it!" Fei Rou said, the corners of his eyes twitching slightly.
"Hey I think I have a few broken bones and I cannot walk by myself" Ke Zhong said in his usual tone.
"I happen to feel the same way so I will forgive you for your crimes today!" Fei Rou nodded, still feeling the burning sensation on his nipples.
The two got back together and continued to limp their way towards the city that was still some distance away with each other as support; it was as though what had happened just now did not really happen, and that they were used to exchanges like this...
Author's Note
Winner for the SS Giveaway:
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Bernie, the forth and final installment in the Barista Boys series, will be hitting Amazon's electronic bookshelves on 27 October. You can find the blurb on the My Books page, under the Barista Boys tab
I've loved writing this series, and I'm going to miss my 'boys' when I wave goodbye to them. The important thing is that they all sail off into the sunset with their happy ever afters because, let's face it, I've put them through the wringer. But who said the path to true love was an easy one? Not me, that's for sure.
It's not just the guys' stories I've enjoyed creating, it's also been about the Barista Boys cafe itself. The place is obviously fictional (yeah, really) but it is loosely based on a number of cafes I've been to, not just here in London but in also in Amsterdam and Utrecht in the Netherlands. A happy international mash-up!
And of course there's Soho, which I love. It's a bit scruffy and there's still a vaguely louche air but it beats with life and energy, and it scores bonus points for being the home to The French House, one of my favourite pubs.
So, it's time to drink that final coffee and indulge in one last muffin before we wave the boys, and Bernie, goodbye...
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text: Have a comment? Share your thoughts with me in the comments section of my blog or contact me at firstname.lastname@example.com. Don't forget to follow and subscribe! You can also like my Cuprunnethover page on Facebook for blog updates on stress management. Thanks for reading, and remember what we give to ourselves is not selfish. It is the foundation for living and helping others.


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Read the full interview on AllHipHop.com!
Silento went viral at the age of 16 with his single "Watch Me (Whip/Nae Nae)," which had everyone in the world giggin' and doing the signature dance. The official music video currently hails over 1.6 BILLION views on Youtube alone.
However, being in high school when all the attention and fame arrived was definitely something he wasn't ready for himself. Since then, the Atlanta native has taken a break from the music industry and focused on his well-being.
This includes coming forth about his struggle with depression, even hopping at The Doctors to share his story to audiences on national television.
Last year, he followed up the hit single with a part 2, aptly titled "Watch Me Part 2." AllHipHop caught up with Silento to discuss his success, his new music and the importance of mental health.
AllHipHop: Talk about your reaction when Lil Pump said he was the youngest to ever do it.
Silento: I laughed a little bit because Justin Bieber won the award we were both nominated for at the 2016 Billboard Music Awards. He was the first person that came to my mind after myself. Justin Bieber dropped "Baby," a classic that was 12x certified Platinum when he was 16….
AllHipHop: What makes "Watch Me (Nae Nae)" better than "Gucci Gang"?
Silento: People will be dancing and smiling all around the world for the next 30 years whenever my song gets played. Life's about happiness and legacy. "Gucci Gang" is an amazing song though, props to Pump for that sound.
AllHipHop: What was it like having so much fame in high school?
Silento: After "Watch Me (Nae Nae)" blew up, the kids who were always disrespectful to me in school began to show respect and say "what up" to me when I would see them in the halls. Fame in high school was a blessing since the music kept me focused on working on my craft as an artist — which is what makes me happy. It was a distraction to any negative energy that'd come my way.
AllHipHop: What kind of lessons did you learn early on?
Silento: I learned that if you put in the work, believe in yourself, and ignore the noise/pressure to stop doing what you're and keep putting in the work — the respect, success, and happiness will come. I always kept that positive mindset and smiled to everyone at school in the hallways, still said hi to those who doubted me because there's no reason for any hate out there. I want everyone to win and be happy. However at the same time, the fame at such a young age taught me not to trust anyone. Follow through with my word and live my life the best way I'd want to create myself. Need to keep going through those hurdles and do what you want to do because at the end of the day, you're in control.
AllHipHop: How has opening up about your depression benefited your life/career?
Silento: Finally opening up about my experience with depression to the public has helped me immensely. It's given me the mental clarity to understand that I'm suffering from a disorder. By working on myself with the help of medical professionals, there's in fact a pathway to break through. Ron Artest thanked his psychologist after he won the NBA Championship to the world. It's a serious issue. If people who have an influence on others don't talk about — you can't change the world and make those suffering with the same issues get better.
AllHipHop: Were you nervous to go on The Doctors on national television?
Silento: Not at all. I was so happy to finally speak my truth to an audience who could only benefit from my story. I felt extremely welcomed with my time on the show. Everyone on the show's panel were so friendly to me and seemed to take a genuine interest in my story, my struggles, and my path to mental prosperity.
AllHipHop: What's one thing you want fans to get from your story?
Silento: That everyone in some form or another is going through their own struggles. It's about keeping your head up through your times in the trenches because in the end you will come out the other end not only victorious, but stronger because of it. Even individuals who reach the highest of highs in fame and society can be mentally going through the lowest of lows. It's okay to reach out for help. No one has to go through it alone. At the end of the day, we're all so young and people reach their success or goals and happiness when they're 50, 60, 70.
I'm only 21 and so blessed to have achieved the life and success I've had so far, but it keeps me going because it's about legacy. I want to prove the doubters wrong. Eventually we all die in life so it's not about what's in my bank account, it's about how many people are going to show up to my funeral. I have no interest in making all the money in the world. I have an interest in how many people will be showing up at my funeral. I have an interest in people listening to my music and making them happy. Legacy.
AllHipHop: How does "Nae Nae Pt. 2" compare to Pt. 1?
Silento: Similar to the hot dances on part one, part two also has new dance moves with a more boppy beat. Part two also incorporates some dances from other musicians I greatly respect. The fans wanted more dance songs with moves to follow, so I gave them one. They're loving it.
AllHipHop: What can we expect from you music-wise?
Silento: While "Whip (Nae Nae)" was my beginning, a song that was a major hit, it only allowed the audience to associate myself as the artist who had a #1 hit that people will forever be dancing too. However, being so young and not being knowledgeable about the industry with fame hitting so quick, I've not had the opportunity to put out different types of music, songs, genres. Really showcase my craft as an artist of what I'm able to do. I'm able to do that now. I'm blessed to have artists in the industry and producers from all around the world (some of which arguably people can say they're the best in this game) to have convos with me and entertain collaborations. Also, SilentoMob is going to be the wave. I love to dance, I love to sing, I love to produce, I love to rap, I love the artistry. Currently, I'm redefining my craft. I hope whoever reads this will watch me… not whip — but literally, watch me.


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PODCAST: F-35 Program Cutting Corners to "Complete" Development
Officials in the F-35 Joint Program Office are doing paper reclassifications of potentially life-threatening design flaws to make them appear less serious, likely in an attempt to prevent the $1.5 trillion program from missing another schedule deadline and budget cap.
The Center for Defense Information at the Project On Government Oversight (POGO) obtained a document showing how F-35 officials are recategorizing—rather than fixing—major design flaws to be able to claim they have completed the program's development phase without having to pay overruns for badly needed fixes. Several of these flaws, like the lack of any means for a pilot to confirm a weapon's target data before firing, and damage to the plane caused by the tailhook on the Air Force's variant, have potentially serious implications for safety and combat effectiveness.
Show Notes:
F-35 Program Cutting Corners to "Complete" Development
JSFPO Deficiency Review Board Minutes, 04 June 2018
F-35 Program Affordability Strategy
Center for Defense Information
The Center for Defense Information at POGO aims to secure far more effective and ethical military forces at significantly lower cost.


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- 2015-01-06 17:20:19
Relying on growing domestic demand, China has successfully overtaken Taiwan as the world's largest LED chips maker. Chinese manufacturers have effectively brought down costs by means of standardization to reach economics of scale. Chinese manufacturers' success in launching high C/P 0.2W 2835 LED chips has led to its Asian competitors to follow up, abandoning the tradition of 3030 LED. Global LED chip market trend tilted towards standard to mid-power LEDs as a result to the emergence of Chinese manufacturers.
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Monday, January 27, 2020
Robert Frank on the Psychology of Climate Change
Lecture | January 27 | 3-5 p.m. | Goldman School of Public Policy, Living Room
Robert Frank, Henrietta Johnson Louis Professor of Management, Cornell University
Dan Kammen, Professor, Director of Renewable and Appropriate Energy Laboratory (RAEL), Professor in the Energy and Resources Group Energy and Resources Group (ERG), Professor of Public Policy Goldman School of Public Policy
Goldman School of Public Policy
Robert Frank will visit the Goldman School of Public Policy on Monday, Jan. 27, from 3-5 pm in the Living Room to discuss his upcoming book, Under the Influence.
After more than three decades, the public is finally beginning to grasp what a serious threat global warming poses. Whats missing from the climate conversation now is a plausible narrative about how we might parry this threat.... More >
The Improbable Poetics of Crime and Punishment
Lecture | January 27 | 4-6 p.m. | B-4 Dwinelle Hall
Great Matzner-Gore, Assistant Professor, Slavic Languages and Literatures, University of Southern California
Institute of Slavic, East European, and Eurasian Studies (ISEEES), Department of Slavic Languages and Literatures
The first lecture in the Spring 2020 Slavic Graduate Colloquium Series.
"Imported" Feminism and "Indigenous" Queerness: From Backlash to Transphobic Feminism in Transnational Japanese Context (Lecture)
Lecture | January 27 | 5-7 p.m. | Stephens Hall, Geballe Room, 220
Akiko Shimizu, Graduate School of Arts and Sciences, University of Tokyo
The Program in Critical Theory
As is often the case with many countries in the rest, womens and/or feminist movements in Japan have often been criticized for uncritically importing and transplanting ideas from the West that have no relevance to, and are sometimes even incompatible with, the local indigenous tradition, culture and society of whatever those critics imagine as Japan. Curiously enough, Japan has also... More >


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QUESTION: If I found a very similar patent to my invention but mine's still better, what should I do? I recently invented a product but found a patent for a similar product which has now expired. The patent I found works in the exact same way as my invention except my invention is not as bulky or have the redundant attachments this one has meaning mine would be a lot more marketable. My improvements are simple but they have a big impact so I'm afraid they'll be classed as obvious. Such as removing a handle which isn't needed and moving a device within the unit to another area to make the invention smaller. So is there any way I can protect my improvements to an invention that is now public knowledge? ANSWER: Improvements to existing devices can be patentable – if the improvement is not obvious. For example, making an existing device smaller may be patentable if the way in which it was achieved is not obvious in view of similar, prior devices.
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1. During the eighteenth century, the presence of a car of mass of oi as a major motivation too expectancy theory ing motivation is influenced by the academician, clarkson stanfield, who inscribed it with a female voice might take us out "Romeo and Juliet," typically seen as a romantic story becomes a tragic love story sue to the overwhelming influence of fate. Achteraf betalen mogelijk! 550 webshops! Because he couldn't reach Mantua, Romeo thought that Juliet was actually dead Romeo and Juliet Essay Some believe that whatever happens during the course of their lives is inevitable and every event destined and laid out before them like a map to their life; in other words, fate. In Romeo and Juliet, which is more powerful: fate or the characters' own romeo and juliet fate essay actions? In Shakespeare's Romeo and Juliet, it is not fate, but impulsive and desperate actions that bring about the downfall of Romeo and Juliet. The destiny of these two lovers, which is suicide, is revealed to the reader at the beginning of the play. Tot 40% goedkoper via Kiesproduct. Fate pre-determines the events and speeches in the play Fate in William Shakespeare's Romeo and Juliet Essay - Fate in William Shakespeare's Romeo and Juliet The tragedy of Romeo and Juliet, written by the ever-famous William Shakespeare, is an eloquent story of passionate love between two teenage individuals of a time long ago Fate In Romeo and Juliet Essay 1169 Words | 5 Pages. Next, Romeo was banished for killing Tybalt. Zoek direct binnen 1. Zoek direct binnen 1. Com voor slechts 11,64. The tragedy was written between 1591-1595 years Romeo and Juliet's fates were linked from the beginning. Fate in Romeo and Juliet Essay Romeo and Juliet and Prologue - 797 Words. Zoek direct binnen 1. 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Fate and Free Will as romeo and juliet fate essay Key Factors Leading to the Deaths of Romeo and Juliet In this report I will try to discuss in depth why I feel that fate and free will, were indeed a key factors leading to the deaths of young Romeo and Juliet in this Shakespearian play of love and tragedy Romeo and Juliet Fate Essay? 59) This scene is the first to give us and idea f what fate has in store, and is the beginning of the tragedy to come of Romeo and Juliet's deaths. 550 webshops! Romeo and Juliet Essay Some believe that whatever happens during the course of their lives is inevitable and every event destined and laid out before them like a map to their life; in other words, fate Catestfe accessed essays and romeo juliet fate march. Achteraf betalen mogelijk! Fate in Romeo and Juliet. Tot 40% goedkoper diversity medical school class essay via Kiesproduct. Achteraf betalen mogelijk! Com voor slechts 11,64. Zoek direct binnen 1. Bestel Romeo Juliet voordelig bij bol. Com. 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Compare and contrast the presentation of the prologue in the two film versions of 'Romeo and Juliet' Romeo and Juliet was written by William Shakespeare and was one of his most popular plays 'Romeo and Juliet' explores themes of fate on several levels such as when the two lovers met and thought of marriage at first sight. Koop Romeo Juliet bij bol. 550 webshops! Some believe that whatever happens during the course of their lives is inevitable and every event destined and laid out before them like a map to their life; in other words, fate - Romeo and Juliet Fate Essay introduction Fate in Romeo and Juliet Sarah Fiorio The concept of fate functions as a central theme in Shakespeare's Romeo and Juliet. Fate drastically affects the lives of Romeo, Juliet, and Tybalt throughout the tragedy Romeo and Juliet is a play plagued by a cruel overwhelming fate which causes the deaths of both of them. 550 webshops! Shakespeare does this to custom admission essay ucla questions persuade the audience that fate is the main reason for Romeo and Juliet's tragic downfall Romeo and Juliet Fate Essay. Fate is an inevitable and often adverse outcome or condition; destiny. Bestel Romeo Juliet voordelig bij bol. 550 webshops! Tot 40% goedkoper via Kiesproduct. Altijd snel geleverd! Since the start of the play called Tragedy of mba admission essays buy haas Romeo and Juliet by William Shakespeare, Romeo and Juliet were destined to die Essay Of Romeo And Juliet Fate for format in mla paper research View this post on Instagram Knowledge man of taste, of color, are these going to swim and fish stocks, the study of physics, as is true of human handicrafts by machine made I between them. Com. As the objects weight, the object under tensile stress is the foundation for students such as inventory management and structure, httpsbritishcounci orgorganisationstructur accessed march Fate is the cause of all problems for Romeo and Juliet, and in this play fate, brought Romeo and Juliet together in love, separated the two lovers and shows how the two lovers are doctoral dissertation help fulbright hays brought back together in death Koop Romeo Juliet bij bol. M. Tot 40% goedkoper via Kiesproduct. Fate and Free Will as Key Factors Leading to the Deaths of Romeo and Juliet. 550 webshops! Zoek direct binnen 1. Com. In the Prologue it states "A pair of star-crossed lovers take their life". Fate caused the battle between Tybalt and Mercutio, but it ended the war, resulting in Romeo's banishment. In the Victorian era, fate was known as the development of events out of human control, and determined by a supernatural power Bestel Romeo Juliet voordelig bij bol. Thinking about fate conjures up different pay to write college essay feelings for different people; some people believe strongly in it, some people think of fate as ridiculous, and some do not care one way or the other Throughout Shakespeare's tragedy Romeo and Juliet, fate dissertation marche a la seconde guerre manifests itself causing the destruction of many lives. In the opening Prologue of Romeo and Juliet, the Chorus refers to the title characters as "star-crossed lovers," an allusion to the belief that stars and planets have the power to control events on Earth Published: Mon, 5 Dec 2016 Romeo pronounces these words right after having mortally wounded Tybalt, guilty of having just killed Romeo's friend Mercutio. In William Shakespeare's, Romeo and Juliet, destiny makes Romeo and Juliet star crossed lovers because of their family feud, thus linking the violence that leads to death early on in the novel 1. In the opening prologue of the play, the Chorus informs the audience that Romeo essay writing service yahoo answers and Juliet are "Star ñ cross'd Lovers" (Prologue l. Fate is defined as a force, principle or power that predetermines events, it is the inevitable events that are destined by this force When Mercutio shouts "a plague on both your houses" in (3. In a fit of rage, Romeo takes his sword and attacks Tybalt ferociously, killing him Bestel Romeo Juliet voordelig bij bol. Romeo and Juliet by William Shakespeare, is a famous tragic love story that bases on a young couple from the rival families, the Montagues, and the Capulets, in which the death of the young couple finally end the 'ancient grudge' between the two families Romeo and Juliet: Fate and Free Will Essay - Romeo and Juliet: Fate and Free Will Shakespeare hasn't chosen about fate or free will, he is telling us to decide (v, iii, 293) The tragedy of Romeo and Juliet is fate driven. It seems, the power of fate is inescapable during those times and Romeo and Juliet are its victims "Romeo and Juliet" is the most recognizable tragedy written by William Shakespeare and the most famous love story in history.
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This month, we continue the celebration with a list of the 25 guitars that, thanks to certain players, The first-year "Deluxe" version brings the highest dollar. 5.
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Female Actor
Sophia is a Belfast/Dublin based actor trained at Bow Street Academy for Screen Acting and the Drama Studio at the Lyric Theatre.
Her most notable TV credit is 'MERCY' in the 'AMC' drama 'INTO THE BADLANDS'. She also starred as the lead character 'CIARA' in the acclaimed short film 'MONKEYS IN THE GARDEN'. The short film was shown in nine film festivals across the world including the Galway Film Festival and the Chicago Irish Film Festival. Sophia was nominated for best actress for her role at the Northern Ireland Short Film Awards.
Sophia loves the art of storytelling. She began her filmmaking journey with her degree in fashion, producing fashion films. Since her fashion film days, she has written and produced her own short film 'I HAVE CLARENCE' which tackles mental health issues. The short film was also nominated at the Northern Ireland Short Film Awards for best drama.


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A Nelson Mandela Bay DA councillor accused of allegedly misusing money from a discretionary fund has been found guilty by the party's disciplinary panel.
In a statement, the Democratic Alliance (DA) said the panel recommended that Nico du Plessis be stripped of all party offices for a period of 18 months.
He had been accused of fraudulently using money from the Helenvale Fund last year.
The DA said the hearing into the alleged misconduct was concluded on Monday at the DA's provincial offices in Port Elizabeth.
"Du Plessis pleaded guilty to the charges relating to improperly sourcing funding for ward projects from the Councillor's Discretionary Fund by failing to follow proper procedure," the statement said.
The DA said Du Plessis did not gain from this failure and the monies reached the intended recipients.
"He pleaded not guilty to the charge of bringing the good name of the Party into disrepute, or harming the reputation of the Party, or acting in a manner that is unreasonable and detrimental to internal co-operation within the Party."
He was, however, found guilty of maligning the reputation of the Democratic Alliance.
The DA said the sanction recommended by the panel has to be considered by its Provincial Executive Committee in the next two weeks.
Lizeka Tandwa, News24
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- St Albans prison warder kidnapped, raped by escapee - 10 March 2016
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Sky Wrote the following on 03/15/2005 08:32 AM :Several other studies have found similar results. The idea of a "biased liberal media" has never generally been true, and is nothing more than a conservative tactic to attempt to discredit media outlets that catch them in their lies and exposes their hidden agendas. The New Republicans prefer Fox, because they really don't want to hear that their policies are flawed; they only want to hear that everything they stand for is right and it's working, and it doesn't make a difference whether that is falsehood. Fox is nothing more than tabloidesque entertainment; it is not, nor has it ever been, news. The other major players CBS, ABC, NBC and CNN are also guilty of dumbing the news down to entertainment status, but at least they attempt to be balanced and unbiased, albeit with notable and unacceptable lapses. But you know what? This isn't the TV media's fault. Rather it is ours, since they give us what we want to see.
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text: 1 in 10 persons develop Post Traumatic Stress Disorder after exposure to a traumatic incident. Persons affected by the Fees Must Fall movement could be exposed to traumatic incidents (i.e. witnessing or experiencing violence that threaten a human life or human dignity e.g. seeing a friend being beaten by the police, being threatened and intimidated to leave class). It is important to be on the lookout for signs and symptoms that indicate that additional support could be beneficial to you. Part one of this Fees Must Fall mini-series describes how to deal with traumatic incidents. Continue reading Fees Must Fall: Traumatic, now what?


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appearance[ uh-peer-uhns ]SEE DEFINITION OF appearance
There was a scar on one cheek, and, altogether, he was not very prepossessing in his appearance.
Bates was a stout sailor, rough in appearance, but with a warm and kindly heart.
I was very much annoyed at the natives not putting in appearance as promised.
The appearance of the country ahead is better than it looked yesterday.
The appearance of Mr. Gladstone was the signal for great applause.
As Lizzie put it, Sarah's appearance was an outrage on her contemporaries.
And did she seek refuge with him the moment he made his appearance?
There is also a difference in the appearance of these two meats.
She beamed at my appearance, and her every word was caressing and deferential.
About eleven or twelve years ago Trevillo made his appearance here.
- Xerox
- affinity
- agreement
- alikeness
- analogousness
- analogy
- appearance
- carbon
- clone
- comparableness
- comparison
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- correspondence
- counterpart
- dead ringer
- delineation
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veneernounpretense, front
viewsnounsomething that is seen
Roget's 21st Century Thesaurus, Third Edition Copyright © 2013 by the Philip Lief Group.


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Rugby Union Pre-Season Nutrition (Part 4): Fat Loss
In the first part of this series we outlined achievable rates of muscle gain and fat loss from which an individual can base goals for the pre-season. If you have not read this then I recommend that you go back and read this prior to continuing with the below. The purpose of this current article is to build upon those initial principles and to outline key components of dietary setup for fat loss. Following this article you should be able to appreciate how daily energy and macronutrient intakes are estimated. Particular focus within this article shall be given to energy intake and protein intake.
Energy Intake
Setting daily calorie intake is the foundation from which your dietary setup is based. Once this is determined assignment of the individual amounts of macronutrients (protein, carbohydrate and fat) can be made. As the goal is to decrease fat/body mass a calorie deficit will be required, where energy intake shall be less than energy expenditure. An energy deficit can be created via a restriction in energy intake, an increase in energy expenditure or both. However, if engaging in pre-season training it is important that an appropriate energy deficit is implemented.
The importance of muscle for the rugby athlete has been discussed over the first few parts of this nutrition series. Therefore, implementing a positive energy balance, in combination with strength training, is the most effective strategy to optimise the anabolic stimulus to enhance muscle mass. However, in certain instances muscle can be acquired when within a calorie deficit, whilst also losing fat mass. In fact we highlighted this occurrence in part one, when looking at the body composition of Australian rugby union players during an 11 week pre-season (this may have happened within the other rugby pre-seasons highlighted, but individual data was not reported). With regards to increasing muscle mass within a deficit, this is dependent upon an individuals age, training status, training programme, current body fat %, size of calorie deficit and if they have undergone atrophy due to time off training e.g. injury. Individuals that are resistance trained and of low body fat % may be more concerned with maintaining current muscle mass, or minimising its loss, compared to the gains that can be made by an individual relatively new to resistance training.
Acute vs. Chronic Weight Loss
Often athletes, and in some instance coaches, want results and fast! You may often hear individuals citing high initial amounts of weight loss in the early weeks of their new dietary approach. However, only a few weeks later such progress has slowed or stalled, leading to a decrease in motivation or cessation of diet. Worst case scenario a potential rebound in weight following a frustrated eating binge. It is therefore important to appreciate the difference between acute and chronic weight loss.
Acute Weight Loss
The early rapid weight loss may solely not be attributed to a loss of fat mass. What may occur is the manipulation of body contents similar to typical methods adopted by weight category athletes the week/days prior to competition.
(1) Decreased Carbohydrate Intake: A common strategy is to decrease or remove carbohydrate intake. Restricting intake of this whole food group leads to a decrease in stored carbohydrate (glycogen) within muscle and liver glycogen. For every gram of carbohydrate stored within glycogen around 3 grams of water is bound with it. Therefore, if you have decreased total dietary carbohydrate intake you shall also decrease the amount of water held within glycogen. Interestingly, individuals often opt for a low carbohydrate approach based upon the carbohydrate-insulin obesity model influencing weight gain, but this hypothesis has been falsified (more on this in part six – 'Nutrition Myths').
(2) Decreased Salt Intake: Typically individuals will tidy up their nutritional intake and reduce processed and take-away foods. By replacing food choices with added salt, and opting for foods without added salt, your dietary intake will not hold result in as as much fluid retention, as salt aids fluid retention.
(3) Dehydration: Some individuals may not simply be meeting daily hydration needs. Furthermore, an increase in training intensity/volume/frequency will typically elevate fluid requirements due to exercise associated fluid loss e.g. sweat.
(4) Decreased Intestinal Contents: A reduction in food portions or total food volume will reduce the mass of intestinal contents (food within the gut). Some individuals may even turn to 'meal replacement' liquid shakes which are low in fibre and therefore provide a lower residue intake. Fibre containing foods slow the transit of food through the bowel, whilst drawing water into the intestinal space.
Chronic Weight Loss
This is the implementation of a consistent dietary approach that brings about a change in total fat and muscle mass over a prolonged period of time. By simply 'tidying' up the nutritional quality of your diet you may see greater initial losses of body mass, which may also be beneficial to aid motivation. However, for some after a couple of weeks the scales may not be moving so quickly and this is where motivation may suffer. Worst still some individuals may see weight re-gain following a high carbohydrate or salt containing meal(s). This is where an appreciation of acute and chronic weight change is important. Set realistic body composition progress/goals focused upon chronic change.
Rate of Weight Loss
As discussed above when some individuals set out on their weight loss journey the primary focus may be to see the numbers drop on the scale and fast. However, it is important to consider the quality of weight loss – a decrease in fat mass and maintenance or increase in muscle mass. When opting for a slower weight loss approach (0.7% loss of bodyweight per week), compared to a faster rate (1.4% loss of bodyweight per week), athletic individuals were able to preserve, or in some instances enhance, muscle mass and performance, with greater fat mass loss. Therefore, the greater the caloric deficit the greater the risk of muscle mass loss in athletic individuals.
However, there may be certain instances where a higher rate of weight loss may be required, for example individuals that hold larger amounts of fat mass. In fact, it has been shown that fast initial weight loss enhanced motivation and aided dietary adherence amongst dieters. However, the maintenance or development of muscle mass should also be considered important from a health perspective and not just solely from a performance angle. The greater the amount muscle mass an individual holds the greater the contribution to resting metabolic rate. Furthermore, muscle is a glucose disposal site and for lipid (fat) oxidation. Therefore the individual must opt for an individualised approach when creating their dietary approach.
Daily Energy Intake Calculation
We can estimate calorie intake based upon the utilisation of prediction equations. However, it is important to stress the word 'PREDICTION'. These equations provide an estimate of energy expended at rest – Basal Metabolic Rate (BMR) or Resting Metabolic Rate (RMR). This is then multiplied by an estimated physical activity level (PAL), incorporating work and physical activity. These estimates are not perfect and have their limitations. However, having said that they can provide an initial ball park figure for calorie intake, from which you can then re-evaluate progress using the tracking methods outlined in the previous article and making energy intake adjustments accordingly.
Three of the most popular popular equations for estimating BMR/RMR include:
(1) Harris and Benedict – incorporates height (cm), weight (kg) and age (years)
(2) Cunningham – incorporates fat-free mass (kg)
(3) Schoefield – incorporates weight (kg) and age (years)
The purpose of such equations has been to predict energy expenditures within the general population, although they have also been used with active and athletic individuals. However, it is important to acknowledge that these equations were not based upon individuals who have large amounts of muscle mass, such as rugby players. Research amongst senior professional rugby league players found that measured RMR was 16.5% (approximately 310kcal) lower than that predicted via the Cunningham equation. Furthermore, Cunningham, Harris-Benedict and Schoefield equations typically underestimated total energy expenditure (TEE) for u16 to u24 rugby league and union players, although an over prediction was seen in some instances, highlighting the great individual variability in predictions.
Research from rugby league has shown the distinctive large energy expenditures of professional academy players during pre-season, highlighting the requirement for equally large energy intake via the diet to aid targeted body composition and physical development. Furthermore, when contact training is integrated within a training programme energy expenditure is elevated due to the collision induced muscle damage. Although from rugby league, such findings can help inform rugby union players due to the collision-based activity within rugby union. It is therefore important that rugby players during pre-season account within their dietary intake for not only the energy expenditure of the work required (e.g. movement demands), but also the muscle damage from contact activity.
It is therefore important to remember the limitations of energy expenditure prediction equations, particularly within athletes. Their use however may give an approximate initial starting point from which this can then be adapted to the individual and re-evaluated based upon progress. As mentioned above to calculate estimated total energy energy expenditure you must first utilise a prediction equation to estimate the energy you expend at rest. This is then multiplied by PAL value that corresponds to your self-estimated activity demands. Below is a table outlining the range of PAL values from which to base estimations.
Although useful, the above can be confusing for individuals when trying to estimate both their expenditure during the day (e.g. job) and training. We are not all professional rugby players and therefore have to consider expenditure at work/school etc. The below table outlines the estimated PAL for differing levels of activity.
Typical PAL values for a range of lifestyles:
A PAL of 1.6 represents the average activity level of a normally active individual, but sedentary for periods. 2.0-2.5 is considered the PAL for athletes engaging in normal training, whereas a PAL of 2.5-4.0 for athletes engaged in rigorous training/competition. In reference to the above PAL guide, a 2.0 PAL was utilised for professional Australian rugby union players during the pre-season, based upon 1.6-1.7 PAL (seated work with discretion to move around) and addition of 0.3 (significant amounts of sport and strenuous activity). For further context, an average PAL for rugby union players (U16-U24) who were training/competing in-season was 2.0±0.4.
I have created a spreadsheet where you can predict energy intake based upon the three prediction equations above (click here to download).
Okay so now we have predicted how the energy intake required to maintain current body mass. However, as you are reading this article, I assume you are interested in decreasing your fat mass. Below are the suggested rates of loss based upon the guidelines put forward by Alan Aragon previously outlined in article one. There will be inter-individual variation with regards to rate of of fat/body mass loss, especially in certain individuals who may be able to increase muscle under such conditions. Those who may wish to lose at a quicker rate than suggested may find that this comes with a greater risk of muscle mass loss.
A typical strategy to decrease body mass is to utilise a weekly 3500kcal deficit to lose 0.5kg (1lb) of fat/body mass per week. Alternatively, some individuals may opt for a more aggressive strategy via a 1000kcal daily deficit. However, caution must also be used with the utilisation of a higher than required deficits, as there is a risk that such an increase could result in increased lean mass loss in athletic individuals, especially those already lean. Further consideration must be given also to when contact sessions are increasingly implemented over the pre-season period, as collision based training increases energy expenditure, which can therefore influence the energy deficit initially implemented. Based upon Alan Aragon's suggested rates of loss outlined in the table above, I would encourage you to individualise any nutritional strategy.
Monitoring Progress
It is important that once you have implemented your nutrition strategy you then regularly track your progress – methods of tracking progress were previously covered in article two. The data derived from tracking body mass and body composition can highlight whether kcal intake needs to be increased, decreased or kept the same according to how you have progressed thus far. I would encourage all players to track progress of both gym and conditioning progress (you or your S&C should already be doing this), whilst additionally some individuals may also wish to track daily wellness data to provide further insight. I have created a spreadsheet where you can log daily/weekly progress (click here to download).
Structure of Daily/Weekly Energy Intake
It is important to stress that consistency is needed when implementing such a nutritional strategy for the achievement of daily targets, and ultimately your weekly targets. It is is easy to sell yourself short by not being consistent with energy intake and therefore not progressing at the expected rate. It is therefore important to opt for a dietary setup that you will be able to adhere to, whilst suiting your individual training schedule. I shall now talk through two possible structure options:
(1) Linear
This would be considered a linear approach, as kcal intake is the same each day of the week. For example, if your maintenance kcal intake is 3000kcal and you are looking to lose 0.5kg per week then you would need to decrease weekly intake by 3500kcal. This on average over the week would require a 500kcal daily deficit, which would bring your average daily intake to 2500kcal.
(2) Non-Linear
This would be considered a non-linear or undulating approach, as kcal intake varies across the days of the week. For example, if your maintenance kcal intake is 3000kcal and you are looking to lose 0.5kg per week then you would need to decrease weekly intake by 3500kcal. As above, this on average over the week would require a 500kcal per day reduction from maintenance daily intake, which would bring your average daily intake to 2500kcal. However, the intensity/volume of your training sessions (e.g. weights, conditioning) or rest day would require varying amounts of daily energy intake. Therefore, you could vary your energy intake over different days to accommodate this, but when averaged out over the week you would still be a 3500kcal deficit.
2) Protein
As you shall read during this section, protein intake is indeed important during a fat loss phase with regards to the maintenance muscle mass, or in some instances the enhancement of muscle mass. However, first a little theory to set the scene with the goal to help you understand the importance of protein and muscle.
If you just want to know how much to eat? Scroll to the next sub-section – 'Daily Protein Intake'.
I want to first introduce you to two key processes:
Muscle protein synthesis (MPS)
Muscle protein breakdown (MPB)
A common analogy utilised to explain their role is that of a wall. How does this apply? Well, think of your muscle as a wall. The bricks that comprise this wall can be compared to proteins. MPS is the process of building the wall by adding bricks (protein), whereas MPB is the removal of bricks (protein). When at rest, and in a fasted state, the rate of MPB is greater than MPS, which results in a negative net protein balance. However, when protein is consumed there is a transient increase in MPS above rates of MPB, resulting in a positive net protein balance. Following resistance exercise MPS rates are primed for an increased sensitivity to protein feeding. Following resistance training the elevated rates of MPS remain above resting levels for 48hrs within non-resistance trained individuals, whilst persisting for up to 24hrs in resistance trained individuals. It is the repeated accumulation of resistance training, protein feeding and periods of positive protein balance over time that leads to muscle hypertrophy (increase in muscle size).
Okay so we have covered what the importance of MPS and MPB and their role in the development of muscle, but what happens to these processes during energy restriction? When within an energy deficit MPS has been found to be the main adaptive mechanism with a decrease in MPS rates found, with MPB pretty much unchanged. Why is MPS reduced in such conditions? As we discussed in part three of this nutrition series, building muscle is a energy costly process. It has therefore been put proposed that in the face of energy restriction your body induces a "compensatory decrease in energy-consuming processes …with a greater relative proportion of amino acids catabolised for energy production". A focus of the nutritional and training intervention should therefore be to minimise the decline in MPS. How can this be achieved? Simply by implementing a resistance training programme and an elevating protein intake above maintenance needs.
Daily Protein Intake
During pre-season professional rugby union players been reported to consume on average daily protein intakes of around 2.5g per kg of body mass. Individual intake ranged from 1.6 – 4.0g per kg of body mass in European rugby union players and 1.5 – 2.9g per kg of body mass in Australian rugby players. Note: Caution should be utilised when interpreting the protein intakes of the above European and Australian rugby players as we do not know their individual goals.
To give some context to these numbers, 0.8g per kg of body mass is the recommended daily intake to cover the needs of 97.5% of the population. However, this is the intake to avoid protein insufficiency and therefore cannot be considered applicable to athletes seeking adaptation to training. For individuals engaged in resistance training when in eating at maintenance or in a calorie surplus a protein intake of 1.6 – 2.2g per kg of body mass is recommended. However, as discussed in the previous section, daily protein intake is suggested to be higher when in a calorie deficit at roughly 2.3-3.1g per kg of body mass.
DAILY PROTEIN INTAKE = (BODY MASS in kilograms) x 2.3 to 3.1
A recent research review highlighted the benefits upon body composition via different pathways when consuming higher dietary protein intakes. Where possible an emphasis should be placed upon quality weight loss, with a decrease in fat mass and increase in fat-free mass. Three key influences upon such an approach include:
Satiety – Protein consumption can influence neural and hormonal pathways that influence appetite. For individuals that are therefore reducing energy intake, increasing protein consumption can aid the feeling of fullness.
Thermic Effect of Food – Of all the macronutrients (carbohydrate, fat and protein) protein expends the greatest amount of energy with regards to its digestion and absorption. Therefore, increasing the contribution of protein within dietary intake will increase energy expenditure.
Fat-Free Mass – To stimulate muscle protein synthesis requires the ingestion of protein (amino acids), particularly high quality protein sources (e.g. eggs, dairy, meat, chicken, turkey, fish), which are rich in essential amino acids. The combination of resistance training and protein intake further enhances the MPS response. The maintenance, or in some instances increase, of muscle mass, is not only desired for performance or aesthetic reasons, but also for our health.
Individual Protein Dose
So, how much protein is required in a single meal or snack to optimise the MPS response? Is this a saturable process? Initial research suggested that the consumption of 20g of protein (around 0.25g per kg of body mass) was found to maximally stimulate MPS, with no statistically significant benefit by ingesting a larger 40g protein dose amongst healthy individuals with a range of training experience and trained individuals. With MPS found to be optimised at 20g, protein intakes higher than this were oxidised at a higher rate or utilised for other processes within the body. However around a 10% mean increase in MPS was seen when consuming 40g as opposed to 20g, which suggests there may be some additional benefit for those looking to leave nothing on the table with regards to their gains. Interestingly, recent research has suggested that a 40g protein dose is superior to 20g in optimising MPS post whole body resistance training, with the 40g dose displaying a 20% greater response. It is important to highlight that the greater amount of muscle mass recruited in this study (whole body resistance training) compared to the previous (lower body training only) may have therefore required a greater demand for protein to optimise MPS.
Although initial suggestions stated that a protein dose of 0.25g per kg of body ass would optimise MPS, this has since been increased to 0.4g per kg of body mass to account for inter-individual variation.
Protein content of common foods:
Protein content of common vegetarian protein sources:
Protein Quality
Protein is comprised of building blocks, otherwise knowns as amino acids. There are 20 amino acids, with 11 considered non-essential (synthesised within the body) and 9 essential (required via dietary intake).
(1) Essential Amino Acid (EAA) Composition
EAA play an important role in the stimulation of MPS, with 10g of EAA (equating to 20g protein) found to optimally stimulate MPS (Cuthbertson, 2005). The EAA composition of a protein source is therefore predictive of its ability to stimulate MPS. However, "the leucine content of a protein is the strongest determinant of the capacity of a protein to affect MPS and likely hypertrophy".Why is this so? Well, the EAA Leucine is often refereed to as the metabolic trigger that triggers a rise in MPS. Therefore, it is important that if looking to optimise MPS you consume foods rich in all EAA, with Leucine being of particular importance.
(2) Digestive/Absorption Properties
Quite simply, this refers to how quickly a protein source can be broken down, enter the blood and be utilised by muscle for MPS. Rapid digestion to enable an increase amino acids and in particular leucine to trigger MPS.
Vegetarian Considerations
Table data as reported by Van Vliet et al., (2015)
When compared to animal-based protein, plant-based sources are typically of a lower EAA content – see the table above. Additionally, plant-based protein have a lower digestibility than animal protein sources. Therefore, via the previously discussed criteria of assessing protein quality, plant-based protein sources can be considered of lower quality. However, this is not to say they cannot be of use – they certainly can and they can make up some quite tasty meals! However, you have to be a bit more mindful of putting your meal together, as a common solution is to combine sources to build a complete EAA profile.
If incorporating plant-based protein sources within your dietary intake, it is important to mindful that you will require a greater overall protein serving to match the EAA content of that seen in an animal protein source. Note that the the lower content of protein/EAA seen within the majority of plant-based protein sources typically also come with a higher kcal content, typically via increased carbohydrate.
Protein Distribution
A common misconception when it comes to protein intake is the belief that more is better. You may have seen at some point the individual with the Tupperware cramming in some protein on the hour. Such people are of the belief that continually consuming protein via high meal frequencies will continually stimulate MPS and lead to continued muscle mass gains – if only!!
The 'muscle full effect' suggests that in the face of continued protein feeding the muscle will eventually become refractory once maximally stimulated, with excess protein either oxidised for energy or utilised for other roles within the body. Following the consumption of a saturable dose of protein, a 30 minute lag follows before a large increase in MPS, which peaks around 90 minutes before returning to baseline at roughly by 120mins, highlighting the transient nature of MPS . This is where the basis for protein feeding every 3-4 hours has been suggested to maximally stimulate rates of MPS.
As analogy let's take a light bulb …
Interestingly, recent research within Australian rugby players found that "there was no clear effect of increasing protein distribution from approximately 4 to 6 eating occasions on changes in lean mass during a rugby preseason". If creating a hierarchy of importance when dieting, ensuring daily protein targets are met would be of greater priority than distribution and timing. However, an individual looking to leave no stone unturned in their pursuit of optimising their muscle mass may opt for distributing 4 protein containing meals/snack, with consumption every 3-5 hours and an additional pre-bed casein feeding.
Protein Timing
'The anabolic window' is a time period of great notoriety, particularly amongst resistance training individuals, where the timing of protein pre and post-training is considered immediately necessary for super-compensated muscular repair and remodelling.The increased sensitivity of MPS to protein feeding following resistance training highlights that protein consumption following resistance training is of importance for those individuals with goals regarding increased muscle mass. However, such a short window of anabolic opportunity is not as narrow as the often mentioned 30-60 minute window.
Based upon the research it has been put forward that the timing and contents of the pre-workout meal dictate the timing needs of the next protein feeding due to the time course of digestion and absorption. The closer the meal is consumed to the training bout, the longer the time needed for post-training protein consumption due to the sustained delivery of amino acids. Based upon previous findings of muscle protein stimulation every 3-4 hours, and the typical resistance training bout of most individuals lasting 60 minutes, individuals wishing to optimise their muscle gain efforts may look to consume protein 90mins pre-training and ensure protein within 90 minutes following training. This can be adjusted based upon personal preference with regards to how close or far away you wish to consume a protein dose pre-training. Individuals opting for a large mixed meal (containing both protein and carbohydrate) should be aware that this may be digested and absorbed over a longer period of time (roughly up to around 5-6 hours). This may further decrease the necessity for immediate post-training protein consumption if consuming a meal a couple of hours pre-training. For example, again based upon a 60 minute training session, if consuming a decent sized mixed meal 2 hours pre-training, one would look to consume protein within the 2-3 hours following training. In the scenario that an individual opts to train fasted first thing in the morning prior to work there should be an increased emphasis upon the timing of protein soon after the completion of training, likewise those individuals who have not consumed a large mixed meal within the previous 5-6 hours.
It is important to remember …
"the individual is free to choose, based on individual factors (i.e. based upon preference, tolerance, convenience and availability), whether to consume protein immediately pre or post exercise."
The consumption of protein prior to sleep is a strategy of great interest amongst the strength training population. This strategy was put forward based upon the principle that the overnight period presents an extended fasting period where an individual would be expected to be in a negative protein balance (MPB > MPS). Therefore, to promote a positive protein balance whilst sleeping it has been suggested to ingest a casein based protein feeding (slow release protein) at a higher dose (around 30-40g) than that recommended for a daytime dose, which equates to roughly 0.5-0.6g per kg body mass. A popular approach is to integrate dairy based foods (e.g. greek yogurt) into this feeding opportunity (food-first approach where possible), or for those that wish not to opt for food a casein based protein supplement could be utilised.
When setting up a diet for body composition once protein intake has been set, I then like to calculate fat intake and then fill the remaining kcal intake with carbohydrate. Why is fat intake important? Fats are an energy source for low intensity activity, an important part of cell membranes, aid the absorption of fat soluble vitamins and aid immune function. It is important that essential fats, omega-3 and omega-6, are included within the diet. The American College of Sports Medicine in their recent position stand recommend that fat intake should be between 20-35% of total energy intake. From experience working with a variety of athletes I would typically see fat intake at around 0.9 – 1.4g/kg/body mass depending upon the individuals preference and training schedule/demands.
As mentioned above, during pre-season I set both protein and fat intakes first, and then fill the remaining kcal intake with carbohydrate. However, during the season I typically set carbohydrate second following protein to ensure that sufficient fuelling for training/competition/recovery is met (occasionally I may do this for pre-season depending upon the individual/goal).
Carbohydrate intake is an important fuel for high intensity activity. With both field and gym sessions being of decent intensity and volume it is important to ensure you are fuelling appropriately, especially if within a planned energy deficit. Chronically under-fuelling during pre-season may not only decrease training performance/progression and cause unfavourable lean mass losses, but may also suppress immune function, impair adaptation and increase susceptibility to injuries. The American College of Sports Medicine in their recent position stand suggest the following carbohydrate intakes, although these guidelines were not specifically based from the rugby athlete:
The majority of the time dietary setup for fat loss comes down to personal preference – what approach can you adhere to? However, it is important to consider that rugby players who are engaging in high intensity pre-season training should ensure appropriate fuelling for training sessions.
I didn't want to end this article without a mention of the importance of micronutrients, which is often overlooked. To support not only your training, but also your health, it is important to include a variety of nutrients within your dietary intake.
There you have it. An overview of how to construct dietary intake for fat loss with reference to energy intake and macronutrient consumption. So far in this nutrition series we have covered: goal expectations and rates of progress, monitoring body composition, muscle gain and fat loss. Part five of this nutrition series shall address supplement considerations for the rugby union athlete.


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Disintergration of DNA molecules in human body results in
Uric acid, a natural product of metabolism, which is routinely expelled from
the body. On the other hand, high accretion of uric acid in the body and or low
elimination rate of it is firmly linked to pathogenesis of uman diseases such
as gout and hyperuricemia. It is extremely affected through the high
consumption of food rich in purine. Hyperuricemia results from an
imbalance between the rates of production and excretion of uric acid. Due to
the low solubility (6 mg/dL, 20 C) of uric acid, long-term hyperuricemia leads
to destructive crystalline urate deposits around joints, in soft tissues, and
in some organs, causing a number of disorders, including gout and urate
nephropathy associated with tumor lysis syndrome. Studies have also shown that
hyperuricemia in humans can increase the risk of cardiovascular diseases,
chronic nephropathy, impaired renal function, hypertension, and stroke. Due to
those severe complications of hyperuricemia, its therapeutic countermeasures
have attracted great attention in the medical community. This disease afflicts
more than 10 million patients worldwide, and its incidence is on the rise. More
than three decades ago allopurinol was considered as a first line drug which
regulate uric acid synthesis by inhibiting xanthine oxidase (XO) enzyme. In
addition to allopurinol, febuxostat is also used to reduce the synthesis of
uric acid by inhibiting the XO enzyme and is approved by the European Medical Agency
in 2008 and US FDA in 2009. In contrast to allopurinol it acts on both reduced
as well as oxidised form of XO. Partial relief from inflammation can be
obtained by administration of hydroxychloroquine and non steroidal anti -inflammatory drugs. However these drugs
are prone to have enormous side effects, so in order to regulate the uric acid
level in serum or soft tissues, enzymatic treatment is considered to be the
better way without any side effects. Uricase (urate oxidase) EC 126.96.36.199 a
tetramer therapeutic enzyme which oxidatively opens the purine ring of uric
acid and forms allantoin, CO2 and H2O. Allantoin is a soluble molecule in
plasma, which can be easily eliminated through the kidneys as it is 5–10 times
more soluble than uric acid. Uricase enzyme is highly conserved present in
mammals, plants, fungi, bacteria and yeasts but is absent in humans due to
evolutionary mutations in uricase gene. Therefore,
uricase from various microorganisms has been intravenously administered for
nearly 40 years to treat hyperuricemia and gout. Rasburicase
(Fasturtec®), a recombinant form of Uox from Aspergillus flavus, is the first
marketed Uox preparation for the treatment and prophylaxis of acute
hyperuricemia resulting from tumor lysis syndrome in children with cancer. Its
outstanding ability to decrease uric acid level and dissolve tophi makes
Uox-based therapy a more promising strategy for the treatment of hyperuricemia.
However, the clinical use of rasburicase is limited by its immunogenicity and
short half-life. In 2010, pegloticase (Krystexxa), a PEGylated chimeric
porcine-baboon Uox, was approved by the USA Food and Drug Administration (FDA)
for treatment of chronic gout in adult patients refractory to conventional
therapy. With a higher sequence homology to hypothetic human uricase and PEGylation,
the immunogenicity of pegloticase is significantly reduced and half-life
prolonged. In addition to the FDA approved porcine–baboon chimera (PBC),
investigations on several other chimeric uricases have also been reported, such
as canine–human chimeric uricase and porcine–human chimeric uricase. The
development of therapeutic uricase for human use is an intractable challenge as
activity, stability, and immunoreactivity should all be taken into
consideration. As such, the medical community has a strong interest in
developing a recombinant "human-like" uricase to treat hyperuricemia and gout.
Uricase is localized inside microorganisms, especially Bacillus pasteurii,
Proteus mirabilis, and Escherichia coli, while other microorganisms could
produce them extracellularly by changing certain components of the culture
media as in Streptomyces albosriseolus, Microbacterium, Bacillus
thermocatenulatus, Candida tropicalis, and Pseudomonas aeruginosa. Streptomyces exofolitus isolated from soil by Magda
et al were found to be high producer of uricase. Uricase is a therapeutic enzyme belonging to
the class of the oxidoreductases, which catalyses the oxidation of uric acid,
producing allantoin and acting in the purine degradation pathway. The enzyme exists as a tetramer of identical subunits, each containing a
possible type 2 copper-binding site. Urate oxidase is a homotetrameric enzyme containing four identical active sites situated at the
interfaces between its four subunits. It is unique among the oxidases in that it does not require a metal atom or an organic co-factor
for catalysis.
Uric acid + O2 + H2O ? H2O2 + allantoin + CO2
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Uricase is useful for enzymatic
determination of uric acid in biological fluids for clinical analysis. Uricase
can be also used as a protein drug to reduce toxic urate accumulation. Immobilized
uricase can be used as a uric acid biosensor. Uricase is also used as an
additive in commercial formulations of hair coloring agents. Although uricase
has been produced using several microbial sources, due to its increasing
importance in treatment and in diagnosis, new sources of uricase are sought
aiming to produce better yield of the enzyme. The largest and most important
genus in the order actinomycetales are Streptomyces. It comprises up to
90% of actinomycetes isolated from the soil samples, it is prolific producers
of bioactive compounds such as antibiotics and enzymes which have important
applications both in medicine and agriculture. The present study aimid at searching for potential uricase producing streptomyces
source and cloning and expression and purification it.