Thinking about the world today and some of the recent calls and emails to the NWTRCC office, the expression "la lucha continua" came to mind. For the heck of it I typed the expression into my search engine, and the first entry turned out to be worth mentioning: La Lucha Continua: a talking mural in San Francisco. Online you can click on each face to read about or listen to a statement by that activist. Many of those depicted will be familiar from the last 100 years of struggles for freedom, justice, and peace.
The mural does not include war tax resistance, but in this time of so much war and killing, the stories of so many of you can be on our own, imagined talking mural. Often the subjects are not flashy, but each represents a struggle on the continuum — a little more pressure on the arc bending toward justice that Martin Luther King Jr. envisioned. The callers below may not mind being public, but it's a random sample so we'll keep it anonymous. If you would like to support any of them with your relevant experience, contact the NWTRCC office to be connected.
Colorado: A freelance resister called to talk about the ins and outs of paying or not paying into social security. Many war tax resisters (WTRs) are struggling with this, knowing the money helps support the Pentagon through loans and investments but wondering what they will live on later in life. Talking it out based on individual motivations and circumstances can be helpful.
Wisconsin: A WTR who makes a living off a small business has managed to live outside the system for decades, not filing or paying. Now the state tax office (not the IRS) is closing in, and he's feeling the pressure. Along with federal taxes to the Pentagon, he knows that some of his state taxes are going to the National Guard and is adamant that he will never cooperate, never pay. "Just look at the world! How can I pay for that?" He's calculating whether to move or restructure his business somehow.
Ohio: A WTR put a war tax credit on his tax form to zero out any taxes due and received a warning letter to refile or risk a frivolous penalty of $5,000. He's choosing to refile and hopes to avoid the penalty.
Massachusetts: A longtime resister faces a levy on one of his part-time jobs. He's working on calculations to get his pay below the levy amount.
Oregon: A mother of a 4-year-old has chosen not to give her child a social security number. "Let him make that decision when he's old enough," she says. Meanwhile, a small, monthly inheritance payment has pushed her income to a taxable level, which she wanted to avoid. She can't lower her income with the Affordable Care Act subsidy and can't take child credits unless she files and shows a social security number for the child. She's looking for ideas from other parents with a similar quandary and is considering community living options.
New York: A telephone tax resister has called a few times in recent months about trying to get his company, Frontier, to honor his resistance and credit his account for past due excise tax. He has used information from our hanguponwar.org website in his many calls to "customer care," but they have no idea about excise tax refusal or get it confused with tax exemptions. One Frontier agent finally sent him a form that indicates they have a policy for refusal to pay the federal tax, so he'll try sending that in to see if it resolves the situation.
Montana: A nonfiler since 2001 found a card on her door from an IRS agent asking her to call the agent. Letters and demands from the IRS — and a good amount of stress — followed. Because she is low income now, she has decided to go on an installment plan to clear her account and hopefully stay below taxable levels in the future.
Washington: A WTR for the past few years got her first notice from the IRS for 2013 taxes just a couple weeks after filing. After another few weeks a notice of levy was sent for a bank account she had closed some time back. IRS notices came quickly but they got no revenue in return.
Wisconsin: A long time WTR who works as an independent contractor found out the IRS is demanding that the agencies that hire her treat the levy as a continuous wage levy. Contractor levies should be one-time only, but the IRS power to bend its rules and to intimidate employers is great. With the help of a lawyer she plans to fight this interpretation of the levy.
These individual struggles do add up, but it can be hard to feel like you are part of something bigger when an envelope from the IRS arrives. In these times when we desperately need to "make haste" toward peace (see p. 1, right column), one might question if fighting a phone company over a small amount of phone tax pushes us any closer to our goal. Maybe it helps to remember a favorite Pete Seeger story about teaspoons of sand that will add up and tip the balance. For WTRs that 50 cents of phone tax withdrawn from the system is our grain of sand. As antiwar organizers we are open to all ideas to make those grains add up a lot faster though.
Attorney Peter Goldberger has been an advisor to war tax resisters for decades, since his work with conscientious objectors during the Vietnam War. We are grateful for his help and guidance and take this opportunity to acknowledge his contribution to our network over the years — at the same time his work as a criminal appeals attorney keeps him very busy.
The more difficult aspect of our friend's message is how do we "make haste" toward peace?
More and more creative outreach has been suggested. We've invested in online outreach and social media. NWTRCC's May meeting approved $4,000 to produce and give away more literature and send people to speak or table at conferences and gatherings of all types. Call if you can help with this. We also have flyers, including for Gaza demonstrations and Hiroshima-Nagasaki days, you can download and use at nwtrcc.org/downloadables.php.
Thanks to volunteers in our network, we are in the process of updating the "Practical War Tax Resistance" series this summer. New versions will show up on the website first; #5, "Low Income/Simple Living As War Tax Resistance," was posted some weeks ago, and #1, "Controlling Federal Income Tax Withholding," was just updated at nwtrcc.org/publications.php.
We can still make changes if you notice something that was overlooked. The next step is to make the booklets available for e-readers to order off our website. We will produce new print versions, but generally NWTRCC's print publications are not selling very rapidly anymore, in large part because our groups are less active than in the past. Your input on which print resources are most important to you is always useful.
A nonfiling resister asked recently whether receiving unemployment would trigger new IRS interest in him. Looking into this question it came to our attention that if you have a tax debt the IRS can take 15% off the top of your unemployment benefit. Until a few years ago, unemployment was exempt from levy, and we apologize for not noticing that sooner.
If you have an experience where receiving unemployment triggered new interest from the IRS, please let us know.
My gosh, the intricacies of it and the shenanigans about it, and about Medicare — how dare they say if you don't start buying Part B every month as soon as you're 65 it will cost you double for the rest of your life??!! How dare they DECIDE FOR ME when it's appropriate that I retire, and base payments on that? Especially the part THAT'S GIVING ME MY OWN MONEY BACK! How dare they stop payments when someone is in jail — what if they're proven not guilty, what if they're in jail to protest illegal actions of the IRS? or the federal government?
I'm going to keep track of how long it takes me to get my actual dollars back in social security payments, and after that the rest is "gravy." The person on the phone was mostly interested if my years without any social security contributions could be explained logically. She used that word a number of times, asking me and assuring herself, it seemed, that those zeroes, and no numbers at all for the past three years, were "logical." Some health problems plus caregiving for my folks were all the reasons she needed. I did not mention additional reasons. She said I was a textbook example of some of the kinds of tax records she had to explain to her students!
While our focus remains on refusing to pay the IRS, we do hear from a number of people who have decided for one reason or another that they have to settle their debt (you can always start resistance again one day). Steev Hise wrote a good piece on Offers in Compromise in our December 2012 newsletter. It's still online (go to our newsletter page and find that issue on the "past issues" list) and good to read if you or someone you know is considering that option. Some people call and want a referral to an accountant to help them deal with the IRS. We do not keep a national list and suggest that people ask for referrals from their friends or progressive organizations in their community. One resister found the accountant to be very expensive and recommends dealing directly with the IRS; he figured he ended up making the same deal with the IRS and saved the cost of an accountant. Another was relieved to have the accountant handling the negotiation because she found it very stressful. Generally we recommend that if you decide to make a deal with the IRS it is best to keep to it or problems can get bigger. We just heard from someone who is choosing to stop paying on his agreed plan, so perhaps in a future issue we will report on how that's going.
To everyone who has donated recently and especially those who responded to our July "Celebrate Thoreau" online appeal!
Roy Prockter died on June 21 in England. He was a Quaker and longtime tax resister, and last year took on the task of establishing Conscience and Peace Tax International in England.
Over the last thirty years, as wages have stagnated across the country, average household debt has more than doubled. Increasingly, we are forced to take on debt to meet our needs — from housing, to education, to medical care. The results — wrecked lives, devastated communities, and an increasing reliance on credit to maintain our basic living standards — reveal an economic system that enriches the few at the expense of the many.
Originally published in September 2012, the Debt Resisters' Operations Manual was written and edited by a network of activists, writers, and academics from Occupy Wall Street. The 2014 edition includes a chapter on "Tax Debt: The Certainty of Debt and Taxes," that will interest NWTRCC readers. The section on "Resistance Strategies" references NWTRCC publications and information.
The Manual is for debtors everywhere to understand how the system really works, while providing practical tools for fighting debt in its most exploitative forms. In this little paperback book, you'll find detailed strategies, resources, and insider tips for dealing with some of the most common kinds of debt, including credit card debt, medical debt, student debt, and housing debt. The book also contains tactics for navigating the pitfalls of personal bankruptcy, and information to help protect yourself from credit reporting agencies, debt collectors, payday lenders, check cashing outlets, rent-to-own stores, and more.
In addition to tax debt, new chapters in the updated edition cover sovereign debt, the relationship between debt and climate, and an expanded vision for a movement of mass debt resistance.
Elizabeth Boardman is on NWTRCC's Administrative Committee and lives in Davis, California.
Having this book available a few years ago would have been helpful to me. Instead I learned the hard way about the use and abuse of credit cards. Some of these lessons are in the Manual , some add to its helpful information.
If you need a credit card and have one that doesn't charge annual fees and limits charges like late or over limit charges, stick with it to make purchases that you can pay in full three weeks later when you get your statement.
Keep track of your credit card's usage online or by calling in to check on recent charges.
Be aware that companies like Chase offer multiple credit cards. I was told that the creditors wouldn't attempt collection on debts under $5,000, but when I settled with Chase on the largest debt, they attempted to get me to pay more to settle the smaller debt as well since they owned all the cards.
Don't authorize any other users! Creditors hold the primary cardholder responsible for any and all debt incurred — even if the other user is using your card deceitfully.
Don't pay if you can't. Your credit score will eventually suffer, but you need to keep money to live on. Some creditors offer a protection service that suspends your payments if you are in a crisis or cancels your debt if you become disabled. This protection may be worth your money. After a couple months of not paying, you'll start getting lots of calls and can negotiate to pay off a low percentage of the debt. Avoid letting your debt go to a collection agency if possible since you may risk court charges in addition to everything else.
Income tax consequences: The "forgiven" debt written off by a settlement becomes regular taxable income, and you will get a 1099 for the tax year in which the creditor recorded your "phantom" income.
This chapter discusses the commodification of higher education and informs us that the overall costs of a college education have risen by over 1100 percent over the last three decades. The authors explore how these increased costs and the resultant student debt were created, who the profiteers are, and how debtors can survive. Underemployment, demographic analysis of debtors (81% of Black students leave school with debt, while 64% of White students do), consequences of defaulting on student loans, strategies to avoid default, and resistance tactics from both at home and abroad are also discussed. Readers will benefit from plainly written explanations of the student loan industry and its manifestations in academia, in state and federal government, on Wall Street, and in other contexts.
This chapter gives practical tips for people who "live at the margins of the credit and banking systems." It contains specific examples of how people can get basic needs met (food, clothing, shelter, healthcare, education, and transportation) without much money or access to credit. The chapter covers a range of tactics: from dumpster diving and squatting, to applying for government aid, to finding freebies on-line, to avoiding a credit check when trying to rent housing. These tips would be useful to review for anyone embarking on (or deepening) a practice of simple living as war tax resistance (see also NWTRCC's "Practical Series #5" on that topic).
When we think about debt, we have to think about what debts are counted and what debts aren't counted. As we criticize them, we count the debts the IMF and World Bank impose on the third world. Yet there is no legal system to count or repay the debts incurred by first world countries who exported so much of the third world's resources without compensation. Climate debt is the costs of dealing with climate change that are disproportionately borne by third world countries, such as the incredible costs of the typhoons in the Philippines (also the site of a lot of U.S.-funded colonization and military spending). Carbon emissions are one way that we can assess the amount of this climate debt, but we also need to generally adopt a more harmonious relationship with our planet and its limited resources.
The Debt Resisters' Operations Manual can be read for free online at strikedebt.org/drom. The paperback version (published by PM Press/Common Notions; 256 pages) is available from the NWTRCC office for $15 plus $3 media mail postage ($5.50 first class).
At this writing tax day and cooler weather seem like a distant memory, so we will spark yours by continuing with some new reports or those that did not fit in our previous newsletter.
New England War Tax Resistance solicits grant applications each spring and gathers to make their distributions decisions after tax day. This year they redirected $6,250 to seven groups: Prison Birth Project, the Beantown Society, Project Hip-Hop, and WUAPL (Worcester Unemployed etc.), to Grace Ross's anti-forceclosure group, to the City School, and to the Youth Justice and Power Union.
It was cold (about 40 degrees) in Chicago on April 15, but despite that we had a spirited war tax resistance demonstration in Federal Building Plaza. The annual event with speeches, singing, leafleting with WRL pie chart, penny poll, signs and banners was joined by several Chicago-area peace organizations and a north side Unitarian Universalist group.
John Boles, Anita LaFollett, and George Houser were part of a team of ten who passed out flyers at the Santa Rosa main post office on tax day. They also set up a Penny Poll a few days later at their retirement center's 30th birthday celebration.
Photo by Ruth Hyde Paine.
Brandywine Peace Community and the Delaware County Peace Center were out on tax day leafleting and carrying signs with the message "Taxes for Need Not War and Greed." The Delaware County Times featured the protest on April 16 with a photo of WTR Paul Sheldon looking determined but a bit cold and damp. On Good Friday they took their signs to the Lockheed Martin facility in King of Prussia (photo) to protest the largest war profiteer in the United States and especially Lockheed's business in drone technology.
Photo by Steve Tawa, CBSPhilly.com.
Paula Rogge and Jerry Chernow of the Madison (Wisconsin) Area Alternative Fund are working on a film project that NWTRCC agreed to sponsor at our May 2013 meeting. Here's their latest update and request for help.
We have attended and filmed at the Veterans for Peace Conference in the summer of 2013, the New England Regional WTR conference in the fall of 2013, and the NWTRCC gathering in May 2014. We have conducted approximately 20 interviews of WTRs, including those who have redirected taxes and faced seizures of homes and property and those who have chosen to live below taxable income.
As for our goals, we plan to edit these interviews so that excerpts can be viewed on-line this fall on the NWTRCC website. We plan to go on the road this fall to conduct more interviews and get footage of WTRs providing hospitality in Catholic Worker houses, living in homes they built, eating from gardens they planted and relying on sustainable energy sources. We also want to film resisters doing counter recruitment in the high schools and participating in anti-nuclear weapon, anti-drone and antiwar protests. We hope to interview the Peace Tax Fund bill director, one of the bill's congressional sponsors, and experts on the history and legal aspects of war tax resistance. We plan to interview an IRS official (if they are willing).
With this additional footage, we would like to edit a 60 — 90 minute film. We are planning to apply to Indiegogo for crowd-funding to help pay for our road trip and film editing costs.
What we need: We are looking for one or more volunteers to travel with us this fall to help set up lights, microphones, and camera, ask the prepared interview questions and keep track of interviewee releases. We would love to get help setting up a website. Donations of lodging and food when we are on the road would be greatly appreciated.
Please give us a holler if you can help or have questions, suggestions or advice!
29th annual New England Gathering of War Tax Resisters and Supporters, October 3–5, 2014, The New School, Kennebunk, Maine. Contact Jason Rawn, rawnjason@gmail.com.
NWTRCC's first ever live, online discussion, called "War Tax Resistance for a Better World," was recorded in March. Three panelists talked about how and why they do war tax resistance and how their actions contribute to creating the world they want to live in. Now we have a complete podcast version for your listening pleasure and to start off our occasional podcast series, "War Tax Talk." To listen, find the link at nwtrcc.org (right column). Subscribe to the podcast feed by adding feeds.feedburner.com/WarTaxTalkPodcast to your RSS reader. Send suggestions for podcast/Google Hangout topics or promotion ideas to Erica Weiland, wartaxresister@nwtrcc.org.
Please add $3 media mail postage or $5.50 first class per book.
Five bookmarks adorned with photos of Maurice McCrackin, Wally Nelson, Ralph DiGia, Eroseanna Robinson, and Juanita Nelson and an inspiring quote. Printed on recycled stock with soy ink. (1.5″ × 7″) Set of 5 — $3, 2 sets for $5 (includes postage and sold by set only).
To order send a check made out to NWTRCC to PO Box 150553, Brooklyn, NY 11215, or pay online through Paypal (use the comment section to list your order or send an email). Call (800) 269‒7464 with questions or for a resource list by mail.
The gathering officially begins with registration at 5 pm and dinner at 6 pm in the School of Religion dining room. The evening speaker, Joanna Swanger, will present on "How Social Change Happens." Workshops during the weekend include beginner and advanced war tax resistance; sessions with Jack Payden-Travers of the Peace Tax Fund Campaign and Maria Santelli of the Center on Conscience and War; David Gross presenting stories from his book "American Quaker War Tax Resistance"; and "Economic Disobedience" with a focus on student debt.
Those planning to fly should arrive at the Dayton (Ohio) airport. We will arrange for transport to Richmond; please ask NWTRCC about arrival times so that we can do group pick-ups. Greyhound serves Richmond. Housing will be in a guest house near the campus or with local hosts.
More information and a registration form are online at nwtrcc.org/meetings.php, or contact the NWTRCC office, 800-269-7464, for a form and details by mail. See you there!
That child version of me searched for different ways to bring that story to life in my own daily living. In high school, I came across The Long Loneliness , the autobiography of Dorothy Day, who co-founded the Catholic Worker movement. Before reading that book I can remember questioning American military policy, but lacking any pacifist role models, my thought lacked concrete form. Dorothy's strong connection between her faith and active nonviolence was the example I needed. In fall 1998, during my senior year in college, I moved into Casa Maria, the Milwaukee Catholic Worker. I had read about war tax resistance from Dorothy Day, but now I was living with people who practiced it. Now I had the theological foundation and the resources with which to implement war tax resistance. So beginning in spring 1999, I refused to pay the balance of my federal income taxes, started engaging in W-4 resistance, and began to redirect those taxes.
From 1999 until 2007, there were years when I was below the taxable limit and years that I owed some money but refused to pay. In 2007, I married Laura. We have a mixed marriage of sorts. We have always filed jointly, but only I employ W-4 resistance. In other words, she pays but has permitted me to file our forms without my portion of our federal income tax. As such, she is still legally liable, but this is a reality with which she is comfortable. As Laura is fond of saying: "If I ever become uncomfortable with the collection process, I can always write a check." For the time being, each time we read about a drone attack that kills a family on the other side of the planet, we both feel good about our current strategy. To this point, we have only had our Bush tax rebate and state income tax refunds seized, but it is likely that at some point we will have our wages garnished.
Since I have been resisting since 1998, I have had the pleasure of watching some my debt due to the IRS dissolve when it hit the ten year statute of limitations. For war tax resisters who file, the IRS has ten years to collect or begin court proceedings, or the tax is no longer collectable. I must admit, there was a sense of accomplishment in passing the ten year mark. Having the taxes due from my first year of resistance rescinded in 2009 was like receiving a reward for my continued commitment.
Each year, Casa Maria sponsors a tax day demonstration in Milwaukee. We often risk arrest, though I have not been arrested in a tax day demonstration for about three years. Early on in my involvement, we always protested in front of the IRS office in downtown Milwaukee, but a few years ago they moved to a more discreet location. Since then we have tried to become more creative. On tax day 2011, I was arrested with four others at the office of then recently elected U.S. Senator Ron Johnson. As a tea party candidate, he has a passion for reducing the federal budget. He states that all options are on the table, but he refuses to promote any cuts to the military. How can the federal deficit be seriously addressed if the largest portion of the deficit is left unchecked? This past April we held a vigil outside a local Army Reserve base in Milwaukee. Our message: "Fly Kites! Not Drones!" It was a little too windy to actually fly kites, but we prominently displayed a kite and one of our group was interviewed by a local NPR affiliate.
After almost sixteen years of war tax resistance, I have no regrets and many fond memories. I not only feel good every time I see a paycheck without any federal income tax withholding, but I know that that second grader who wanted to live out the story of the Good Samaritan would be very proud.


text:
Z Nation, Season 4, Episode 7: Warren's Wedding
Hey, you know how this season has been surprisingly mature and sensible for Z Nation generally sticking to the plot and not involving completely out there random episodes which just cause headaches?
Zombie Juggalos.
Yup, back into the weird stuff.
So after starting all mature and moving with Lucy's funeral the gang move son - only we can see Murphy is clearly traumatised and angry. He is attacking zombies, when normally he's the first to hide at the back of a crowd, and quick to intervene, spoiling for a fight. He's not listening to Warren (and has a lot of seething anger towards her)
This all leads to them rescuing a woman tied to a ferris wheel just out of reach of zombies. Warren doesn't want to intervene, but Murphy insists
On releasing her the woman talks about, well, lots of random stuff but mainly that her son is the house from which loud music is playing. Murphy is determined to charge in - rescue, revenge - whatever they're bad people so he's justified in hitting them and killing them
Warren desperately tries to stop him - it's the zombie apocalypse, there are few humans left, you can't just kill people. But lilley points out he's acting out of trauma - and 10k and Doc as well - and they can't be easily stopped. But she refuses to join them; she won't be part of it, she won't enable it. So she stays with the released woman who talks about fraygo, partying and pretty much all the drugs ever.
While Murphy & co go inside and get captured by juggalos. Or post apocalyptic zuggalos.
If you don't know what a juggalo is, join me in joyful confusion of this apparent subculture and wonder in a kind of desperate lostness why I am watching this or why this is here.
So being captured involves some circus style torture and Warren having a drinking game/quiz with king juggalo and holding her own because she knows a surprising amount about Insane Clown Posse apparently. This impresses him enough for this to turn into a rap battle, thena wedding to make Warren Queen Zugalo because why not
Which is when rescued lady returns with Lilley and she's king Zugalo's mother and she's all pissed off and then they both argue about zombie daddy who they keep locked in the cupboard because why not. Then they fight, Murphy decides to have a long rant about parenthood and how he wanted to be better for Lucy etc etc and then people die, there are zombies and it all gets messy.
Honestly the episode was the far side of bizarre but everyone dies except the gang
One thing that does come out of it is that Warren accepts responsibility for Lucy's death - since they were running after her vision quest and she realises she simply can't drag everyone else on this any more. Instead she's going to take them all to Newmerica first and then she's going to follow her visions alone
Labels: 2 fangs, dystopian, Syfy, television, z nation, Zombies
Supernatural, Season 13, Episode 7: War of the Worlds
Flame in the Dark (Soulwood #3) by Faith Hunter
Chaos Choreography (Incryptid #5) by Seanan McGuire
The Walking Dead, Season 8, Episode 5: Big Scary U
Lucifer, Season 3, Episode 8: Chloe Does Lucifer
Z Nation, Season 4, Episode 8: A Crisis of Faith
Superstition, Season 1, Episode 5: Tangled Web
Once Upon a Time, Season 7, Episode 8: Pretty in Blue
Once Upon a Time, Season 7, Episode 7: Eloise Gard...

redpajama_set_name: RedPajamaCommonCrawl


text:
Qatar international reserves may scale up to $38.1bn in 2023: FocusEconomics
May 09 2019 09:38 PM
Ooredoo showcases Qatar's digital business future in IoT conference
Qatar, UK sign bilateral agreements on occasion of London's hosting of Qatar Day
Qatar shares edge down on weakened buying interest
QIIB receives 'highest certificate' in protection of banking cards data
Bedaya concludes its 3-day Innovation Seminar
QATAR reserves international FocusEconomics
Qatar's economic growth is set accelerate this year, buttressed by higher hydrocarbon production, higher oil prices and infrastructure projects related to the 2022 World Cup, according to FocusEconomics. PICTURE: Ram Chand
Pratap John
Qatar's international reserves will scale up to $38.1bn in 2023 from $33.8bn this year, FocusEconomics has said in its latest country report.
The international reserves will cover 9.6 months of imports in 2023 as against 11.7 months this year, the researcher said.
Qatar's fiscal balance as a percentage of GDP is set to rise to 4.5% in 2023 from an estimated 0.5% this year, FocusEconomics said.
The country's public debt will fall gradually until 2023, the researcher said and estimated it to be at 53% this year, 50.8% (2020), 48% (2021), 46.4% (2022) and 44.9% (2023).
The current account balance (as a percentage of the country's GDP) will be 7.1% in 2023 compared with 6.7% in 2019.
Qatar's merchandise trade balance, FocusEconomics said, will be $56bn in 2023. This year, it will account for $49.4bn.
Qatar's gross domestic product is expected to reach $242bn by 2023, it said. By the year-end, Qatar's GDP may total $199bn.
Qatar's economic growth in terms of nominal GDP will reach 5% in 2023 from 3.6% by the year-end.
The country's inflation, the report noted, will be 2.1% in 2023 and 1% this year.
Qatar's unemployment rate (as a percentage of active population) will remain a meagre 0.2% in 2023, unchanged from this year.
Qatar's economy is likely to have accelerated in the first quarter of the year, following the "weakest" expansion in nearly a decade in fourth quarter (Q4) 2018.
The PMI for the non-energy private sector averaged higher in Q1 than in the previous quarter, with new business orders driving the expansion.
Moreover, industrial production "performed well" above Q4's average in year-on-year terms in the first two months of the year on the back of robust dynamics in the mining and quarrying sector. Despite remaining at "relatively low" levels, Qatar's real estate index grew throughout Q1, suggesting the real estate market is slowly stabilising.
In addition, private sector credit growth "grew steadily" in year-on-year terms in Q1, which, coupled with subdued price pressures, should have supported household spending.
The country's economic growth should accelerate this year, buttressed by higher hydrocarbon production, higher oil prices and infrastructure projects related to the 2022 World Cup.
FocusEconomics panellists see growth of 2.6% in 2019, which is down 0.1 percentage points from last month's estimate, and 2.8% in 2020.
Consumer prices fell 1.2% year-on-year in March (-1.4% in February). Low food prices and a flailing housing market have muted price pressures in recent months.
"Going forward, price pressures will likely pick up on stronger economic activity. The possible introduction of VAT presents an upside risk to the inflation outlook," FocusEconomics said.
FocusEconomics panellists expect inflation to average 1% in 2019 and 2.3% in 2020.
New report to explore Qatar's evolving tax regime
Goldman says oil price could drop $3 if virus plays out like Sars
New finance chief says Lebanon to decide on March bond next week
Turkish bank probe 'casts wide net over trading in financial sector'
Australian consumer gloom worsens amid bushfires
Investors look for insurance as US bull market runs: Fund
Fallen UBS trader Adoboli seeks redemption in bond market deals
Qatari climber reaches summit of highest mountain in Antarctica 1/22/2020 10:55:01 PM
Cold spell expected from Sunday: Met 1/22/2020 12:37:10 PM
Cold spell expected from Sunday: Met
Qatar takes preventive steps against 'coronavirus'


Jackson Morgan Southern Cream in peaches and cream tastes like biting into a freshly picked peach. This whiskey cream flavor is delicious in milkshakes, over ice cream, or simply sipped neat. Best served chilled.


text:
Snowshoe är en kattras från USA som härstammar från siamesen och vitfläckiga amerikanskt korthår.

Historia
Rasens historia går tillbaka till 1960-talet då Dorothy Hinds-Daugherty, en amerikansk uppfödare av siameser, upptäckte tre stycken kattungar med ett ovanligt men tilltalande utseende i en kull. Hon påbörjade ett arbete med att avla fram katter med detta utseende, men den första rasstandaren skrevs av en annan kattuppfödare, Vikki Olander, på 1970-talet. Idag är rasen registrerad av TICA och det internationella kattrasförbundet FIFe godkände rasen 2004.

Utseende
Snowshoe har en muskulös och långsträckt kropp med medelhöga ben och runda, stora tassar. Det medelstora huvudet är tämligen runt med breda kinder och kraftig haka. Öronen är breda vid basen, medelstora och med lätt rundade spetsar. De intensivt blå ögonen är valnötsformade och snett placerade. En snowshoe ska vara en välbalanserad katt med alla kroppsdelar välproportionerade, ingen del bör vara mer extrem än den andra. Pälsen är kort, silkig och tätliggande utan underull.

De färger som förekommer hos snowshoe är de samma som hos siamesen, olika standarder godkänner något olika färger, och rasen förekommer i många olika varianter, exempelvis brun, blå eller choklad med mer eller mindre vitt inslag. Färgteckningen är en kombination av colorpoint och vitfläckighet. Den är maskad, det vill säga ansiktet, öronen, benen och svansen har en mörkare färg än kroppen. Tassarna är vita och katten har också vitt runt nosen.

Den mest önskvärda teckningen är att framtassarna ska vara vita upp till handloven, "sockor", baktassar upp till hasen, "stövlar", och att det ska finnas en vit teckning i ansiktet, som ett introvert V över nosen. Om katten har mer vitt än detta räknas den som tvåfärgad (bicolourtecknad). Snowshoe kan också vara tabbymaskad eller sköldpaddstecknad (trefärgad).

Karaktär
Snowshoe är en livlig, nyfiken och intelligent katt och som husdjur är den ofta lätthanterlig, tillgiven och sällskaplig. Det är ofta bra att ha två katter som håller varandra sällskap. Dess intelligens gör att den ofta har lätt att lära sig olika lekar och grundläggande lydnadsträning.

Externa länkar

Kattraser från USA

redpajama_set_name: RedPajamaWikipedia


Missionary from Brooklyn, NY dropped a pretty solid demo a year or two ago. Following that demo is the debut EP, "American Strike," put out on Warthog Speak. Classic USHC on display here, with the slightest of Oi! influence in there for good measure. This EP is definitely a step up from the demo in my opinion. Clear vinyl that comes with a massive fold-out insert. Really solid record/packaging all around. Looking forward to hearing more from this band and label.
Damnit, I was gonna post this but you beat me to it.


The Clarissa Hulse Dill duvet cover graduates from the deepest teal to a pale aqua as silhouettes of flowering dill repeat in contrasting colours and the firework-shaped clusters evocative of British summer time. When turned down, the reverse is patterned by what appear to be rows of flowers, but are actually looped shapes created from stamens. Crafted from a beautifully soft 100% cotton and machine washable for ease of care. Matching oxford pillowcase sold separately.


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Chalets / Houses
Car Transfers / Taxi
Activities in St. Moritz ›
Olympic Bob Run
Activities in St. Moritz
Information Olympic Bob Run
Bobsleigh is a very important sport for St. Moritz, as it was here that this fascinating winter olympic sport was invented, at the end of the nineteenth century.
Olympic bobsleigh run in St. Moritz / Celerina was inaugurated in the year 1904, and it is the world's only ice-natural bobsleigh run. All other runs of Europe, North America or Japan have to be artificially frozen.
Olympic bobsleigh run (simply called bob run) is 1722m. long, it takes just 75 seconds to cover. In the track 135 km / h maximum speed and a power of up to 4.5 G can be achieved.
It has been the venue of 2 Olympic competitions and numerous sport events. Every year the athletes test their skills in the skeleton (simple sled) and luge (lightweight sled). The annual competitions on sledding (sleds are made of horns) and figure-skating in the bob run are very popular every year.
Book Olympic Bob Run
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in St. Moritz:
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Three property experts fail to get there message right, while Trump is out on his own.
So a bit of a mixed bag this week. The things that stuck out for me this week weren't so much in the data, but in the stories people are telling.
Propaganda is a fine art, even in property, and here are three complete fails and one total home run.
The pace of growth in property prices will slow down but not stop. Property prices will keep growing but at a lesser rate per year.
… If you are in the market to buy, don't put it off because you think prices will plunge – it never happens that way. Take a sensible measured approach, set a budget and buy the right property for you.
If you are thinking of selling, now's the time. It feels to me like we're at, or close, to the peak of this cycle, so if you want to fully capitalise on this boom, now is the time to sell.
Ok, so in short, don't believe the hype. The market is not going to crash, and now is the perfect time to buy, and the perfect time to sell.
Only a real estate agent could pull that off with a straight face. I'd agree that the negative press is overblown (again!), but I'm not convinced I see prices falling or even stalling.
This "peak of the cycle" business is classic real estate fodder. What better way to convince people to sell than tell them that the market has peaked?
But you also need buyers, who will just sit on the sidelines if prices are falling, and so we have the logical double-twist with half-pike that says prices will fall, but only a little, creating the perfect time to buy, the perfect time to sell, and the perfect time to pay a real estate agent a nice fat commission.
Pull the other one John.
The second fail is from Macquarie Bank. They reckon the Bank Levy will have a "disproportionate and unintended consequences" on their operations and they may have to move offshore.
I think the "I'm taking my bat and ball and going home" argument can pack some weight if you're a well-loved Aussie icon, like Holden or Vegemite. But who is going to cry for Macquarie?
All it says it that your only interest in Australia and Australians is purely financial, and you've got a bit of a glass jaw.
Don't let the door hit you on the arse on the way out.
"This policy could decrease foreign investment in NSW new property by as much as 15 per cent.
With every misguided new policy proposal, foreign investment will decrease along with new housing construction, housing affordability and construction employment.
I'm not sure about that last stat. Sounds a little suss to me. But one thing I could tell you is that if I owned a highrise apartment in Sydney, I'd be praying for a 15% fall in foreign demand, since, by law, that demand must go into new dwellings.
And adding more dwellings to an already oversupplied market can only lead to more downward pressure on prices.
I think there is real anger in the community about the way some shoddy highrises have been thrown together, have flooded the market, and have neglected the needs of locals just to serve foreign investors.
In most people's minds, Ms Jong just said the government's policy was a good idea.
Lastly, and just because I couldn't resist having a go at it, was Trump's 'Covfefe' gaffe.
In case you missed it, apparently, Donald Trump forgot to lock his phone, and arse-tweeted the free-world.
The internet, of course, went into melt-down.
The narrative, of course, is that Trump is unhinged and unpredictable, and can't manage basic things, like his phone.
But am I the only one who thinks it was deliberate?
The tweet was left up for hours, even though the White House would have known within minutes what had happened.
And what does it say about Trump? It says Trump isn't filtering himself. He's not passing his messages threw focus groups, before spewing up the most banal version possible to the listening public.
For Trump's base, he's just reinforcing the message that he's "authentic" and "not a politician." They go nuts for that stuff.
And what was the complete sentence there? "Despite all the negative press…" That will mean different things depending on who you are. If you're part of his base, he's talking about a vindictive press that's out to slander the President. If you're not, then it has been a pretty gruesome month in the news.
So no matter who you are, here's a funny word to break things up a bit: covfefe.
If only he'd use his talents for good, not evil.


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Denise strikes gold at Brighton casino relaunch
Monday 20th Oct 2014
Hove resident, Denise Harris, is celebrating having scooped £1,000 worth of prize money at the launch party of the new look Genting Casino Brighton.
The Preston Street casino has just received a £600,000 makeover and Denise, 52, won the ultimate Golden Envelope prize draw, taking home £1,000 in Virgin Experience Vouchers.
Denise who has been a regular at the casino with husband Rob for five years and is still deciding how to spend her vouchers, said: "My daughter Amanda and I went along to the VIP opening night and couldn't believe our luck! Not only did I win the prize draw but we both had a successful night on the tables.
"I don't drink or smoke, but take great delight from dressing up and socialising with friends at the casino. The new look is wonderful, having been brought bang up to date and beautifully decorated – you certainly feel very special."
Genting Casino Brighton employs over 30 part-time and full-time staff and the makeover has transformed the casino into a boutique style, contemporary destination, complete with new entrance and reception area and the addition of a stylish bar serving premium drinks and cocktails.
The major refurbishment scheme also includes a complete redecoration of the venue, new toilets, audio visual equipment and enhanced lighting.
General Manager of Genting Casino Brighton, Will Cotton, said: "The feedback to our new look casino has been fantastic. We have always delivered the very best customer service and we now have an environment to match."
At the heart of the casino is the gaming floor where all the popular games are available from American Roulette, Blackjack, Baccarat and 3 Card Poker together with Electronic Roulette and Slot machines with jackpots up to £10,000.
For those new to gaming, the casino offers pre-bookable and tailored learn to play sessions where customers can have fun, whilst being shown how to play Blackjack, Roulette or Texas Hold'em Poker.
Genting Casino Brighton plays a key role in the community, regularly supporting local events and charities including The OS&B Society benevolent fund, which is backed by local businesses and helps those in need. The venue is a responsible, professional gaming destination that works closely with GamCare and other organisations to ensure that its customers have a great experience.


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China moves to take full control over Hong Kong with a proposed national security law in China's National People's Congress
Posted on May 21, 2020 by Caleb Chen
Hong Kong will soon have a new national security law implemented without a single vote by a Hong Kongese politician. If that doesn't sound democratic, that's because it isn't. The National People's Congress (NPC) in China is starting its bimonthly meetings and at the top of the agenda is a new national security law for Hong Kong. Specifically, the agenda mentions a bill "on establishing and improving the legal system and enforcement mechanisms for the Hong Kong Special Administrative Region to safeguard national security." According to the South China Morning Post (SCMP), such safeguards would include forbidding the following:
"Secessionist and subversive activity, foreign interference and terrorism in the city."
These are new national security laws that will be largely aimed at pro-democracy protestors – which the world has seen is a large portion of Hong Kong's citizenry. A source that the SCMP spoke to in Beijing commented
"We can no longer allow acts like desecrating national flags or defacing of the national emblem in Hong Kong."
The National People's Congress (NPC) is a rubber stamp political organ that does the Communist Party's bidding
When one hears National People's Congress, one might be forgiven for assuming that such a political organ is analogous to the Congresses in democracies around the world; however, that would be a mistake. The NPC is more like an NPC (non-playable character) in a video game that only does what its developer/creator programs it to do. In this case, that creator is none other than the Chinese Communist Party (CCP). All important legislation that the NPC passes is first drafted and approved by the CCP and 70% of the NPC is officially part of the CCP while everyone else is directly under the CCP's thumb – meaning that legislation like this always goes through.
According to The Guardian, Zhang Yesui, a spokesman for the National People's Congress, said at the start of the NPC's meeting:
"Because of new circumstances and need, the NPC is exercising the power enshrined in the constitution to establish and improve a legal framework and mechanism for safeguarding national security and upholding the institutional framework for 'one country, two systems'."
China is no longer pretending to let Hong Kong be democratic
The circumstances are last year's protests by pro-democracy protesters in Hong Kong that resisted political action within Hong Kong's own congress that would have severely lessened democratic rights within the region. Now, instead of trying to push such controversial issues through Hong Kong's own congress that still attempts to keep democracy alive, China will pass national security laws for Hong Kong over a thousand miles away directly from Beijing without any input from Hong Kong's citizenry. An unnamed source in Beijing confirmed to the SCMP:
"The new law will be introduced in Hong Kong through promulgation, without the need for local legislation."
Beijing had previously had a policy of at least pretending that Hong Kong would be able to draft its own legislation and govern itself – but that farce is now completely over. After all these years, China is showing its true colors with regards to Hong Kong and breaking the illusion that a "one country, two systems" system is anything but absolute control from China's ruling cadre.
This is but the first step in China taking full control of Hong Kong
A pro-democracy lawmaker in Hong Kong, Dennis Kwok, told The Telegraph:
"I'm speechless. This is a complete and total surprise and I think it means the end of one country, two systems. […] When the world is not watching they are killing Hong Kong, killing one country, two systems, and using social distancing rules to keep people from coming out to protest. […] This is the most devastating thing to happen to Hong Kong since the handover."
While the move from China's government to pass this national security law in China for Hong Kong – as opposed to in Hong Kong for Hong Kong – is a clear change to the status quo and way of doing things, it's as of yet unclear how Hong Kong's citizenry will respond to this fundamental promise-breaking by the Chinese government. Ming Sing, a political scientist from the Hong Kong University of Science and Technology, commented to The Guardian:
"If Beijing passes the law … how [far] will civil society resist repressive laws? How much impact will it unleash on to Hong Kong as an international financial centre?"
When, not if, this law passes in China – and there is absolutely zero indication that it won't – it's likely that there will be reeling effects for Hong Kong's economic status as a world financial center. That status is preserved by special economic treatment designations from countries like the United States – a designation that could be in jeopardy due to this very law. The US Secretary of State, Mike Pompeo, recently delayed a report about Hong Kong pending legislation like this from the NPC. Amid this shocking political turn, pro-democracy protestors in Hong Kong have already started taking to the streets. As China flexes over Hong Kong, the whole world watches on with bated breath.


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What is Opoyi?
Home Latest Trending History Notifications
1 month ago . Mumbai, Maharashtra, India
Nearly 290 samples of foreign travellers sent for genome sequencing: Mumbai mayor
The administration is on constant alert about people returning from the so-called "at-risk" countries. (Photo Credit: Representative Image/PTI)
#Omicron#Coronavirus#Pandemic
After getting the list, "its staff will contact the passengers to inform them to remain home quarantined"
The Omicron strain has been classified as a 'variant of concern' by WHO
The first cases of the Omicron variant in India have been confirmed in Karnataka
ByAjay Joseph
Mumbai mayor Kishori Pednekar on Saturday informed that nearly 290 samples of international travellers were sent for genome sequencing in Mumbai, Maharashtra. The administration is on constant alert about people returning from the so-called "at-risk" countries.
Quoting Pednekar, ANI said that it "has prepared a complete action plan for it."
Also read: Karnataka to investigate South African national who tested negative with Omicron in 3 days
After getting the list, "its staff will contact the passengers to inform them to remain home quarantined for the next seven days. Adherence to the home quarantine order is mandatory and the passengers will be shifted to government-run institutions if they are found violating it," the civic body stated in the order.
"An RT-PCR test will be conducted after seven days and necessary steps will be taken accordingly," the order added.
Also read: Singapore says no data suggests Omicron different from other COVID variants
The Omicron strain, first detected in South Africa, has been classified as a 'variant of concern' by the World Health Organization (WHO). The first cases of the Omicron variant in India have been confirmed in two individuals in Karnataka.
However, the state government has launched an investigation into the test report of a 66-year-old South African man who was one of India's first Omicron cases. All of his primary and secondary contacts have tested negative, but it's unclear how he was able to fly out of the country while the results of his sample's genome sequencing were being awaited.
Trending on Opoyi
5 must-have food items to boost your immunity
India: Revised COVID-19 guidelines do not recommend masks for kids below 5
Goa reports 3,390 COVID-19 cases, 9 deaths; positivity rate at 41 pc


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<?php
namespace Tecnotek\Bundle\AsiloBundle\Util\Enum;

/**
 *
 */
abstract class Item6Enum extends BasicEnum {
    const ALONE = 1;
    const NEEDS_SUPERVISION = 2;
    const NEEDS_PARTIAL_HELP = 3;
    const NEEDS_FULL_HELP = 4;
}
?>

redpajama_set_name: RedPajamaGithub


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USTelecom Media
WC 16-143, 05-25 & RM-10593 et al: USTelecom Ex Parte on Business Data Services in with Pai
Download: ExParte_re_Pai_BDSMtg_04.12.17_jb.krs_.pdf
On April 10, 2017, Jonathan Spalter, Diane Holland and I met with Chairman Pai and his advisor Jay Schwarz to discuss business data services. The proposed Order, if put into place, will drive additional investment in building more fiber connections to more businesses, increasing competition, and expanding and strengthening the fiber networks that we all depend on.
We noted that the reduction in the FCC's detailed price setting role proposed in the Order is important, but relatively constrained. In the seven years between 2000 when the first petitions for pricing flexibility were granted, and 2007, the Commission returned broad pricing flexibility to markets covering roughly one-third of the country.1 Ten years later, the current draft Order would extend that flexibility to cover an additional one-third of the country. This expansion is at a slower pace than over the 2000-2007 period, and simply reflects growth in competition and the fact that the Commission refused to act to keep its regulatory scheme up-to-date for almost ten years. And, the Commission appears to be retaining authority to ensure that prices for traditional BDS remain "just and reasonable" under the Communications Act.
We also discussed the potential benefits of broader relief. We urged the Commission to adopt the non-dominance approach set out in filings from CenturyLink and Frontier. The Commission has acted broadly in other areas to recognize competition on a national level,2 and should do so here.
Finally, we pointed out several areas where the Commission's competitive market test (CMT) may be too conservative, and not reflect the amount and strength of competition in today's market. Initially, the data from 2012 understates the current levels of competition, as cable companies and CLECs have continued to build and upgrade facilities and to take share in business data services. We noted that the CMT in the draft Order at paragraph 126 appears to be overly narrow because it applies the half-mile test from only locations served by CLECs rather than from competitive fiber facilities. This will understate competition because it is standard engineering practice to build splice points into intra-city fiber networks so that customers can be connected to fiber facilities.3 In addition, the Commission's careful analysis at paragraph 135 of the draft Order of the tradeoffs between over and under regulation suggests that a competitive threshold for the CMT should be between 32% and 48%. The Commission proposes setting the threshold at the very highest end of this range, and rounding up from there. However, setting the threshold no higher than the mid-point of the range identified by the Commission — 40% — would better balance regulatory risks and better reflect competitive dynamics and the growth in competition and competitive facilities since 2012.
We concluded by emphasizing that the draft Order's proposal to take a measured step to better reflecting the dynamic growth that has been occurring over the last decade to upgrade networks that serve business customers will incent all competitors wanting to serve businesses to invest in more fiber to serve more locations. More fiber infrastructure will produce benefits throughout the country in both business and residential markets.
USTelecom President Talks Broadband Infrastructure
by Jonathan Spalter
Brought to you by Broadband: Techin' Out the Fourth
by Chloe Sanchez
USTelecom Welcomes Broadband Deployment Advisory Committee


I have good news. Abide is a verb!!!
Do you know what that means? That means that when we are abiding we are doing something! When we decide to ignore the To Do list and turn off all electronic devices and sit on the carpet and think about Jesus we are doing something!
When we choose to say no to the committee, and let someone else bake the 500 cupcakes for the school fundraiser so we can spend time soaking in God's word… we are doing something!
We are not being unproductive when we abide!!!
We are not being unproductive when we sit in silence longing for Him to speak to us.
We are not being unproductive when we turn up the worship music and dance like crazy.
We are not being unproductive when we fall on the floor and sob our eyes out.
We are not being unproductive when we drive the long way home to steal a glimpse of His creation.
As a matter of fact… abiding might very well be the most productive thing we will ever do.
But abiding requires us to reposition ourselves. We cannot go full steam ahead every waking moment and abide. Abiding requires us to say no to things that would distract us. Abiding requires us to come to grips with the fact that God doesn't want our accomplishments… He just wants us! He wants us to come, to sit, to rest, to love.
Mary discovered it—the one thing worth being concerned about.
Lord, let me discover it too!


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100WS Partners
Long-Term Initiatives
Community School Initiative
Addiction and Recovery Initiative
Grants by Year
Grants Photo Gallery
In addition to our yearly competitive grants, 100WomenStrong works closely with the community to create new programs that have made a lasting impact to Loudoun County residents.
We work as a group to learn as much as we can about the most pressing areas of need in Loudoun County and then reach out to community leaders, businesses and nonprofit organizations to create long-term initiatives designed to accomplish the following:
Identify and promote synergistic and collaborative opportunities between non-profits
Engage County government, private corporations and public/private organizations to focus on the solutions to community challenges
Incubate and encourage the formation of new non-profits and philanthropic groups
Promote public awareness of the needs of the community
Assist non-profits in being more effective (training, operational reviews, etc.)
Two of note are our Community School Initiative and the LCPS Backpack Coalition, both of which have proven successful since inception.
100WomenStrong began working closely with Loudoun County Public Schools, Loudoun Education Fund, SunTrust Foundation, Pactolus, Telos and others to change the face of education through its Community School Initiative (CSI) at Sterling Elementary School. Read more about it and how it has expanded since 2015 here.
LCPS Backpack Coalition
In 2013, 100WomenStrong and Loudoun County Public Schools (LCPS) began a program called the Backpack Coalition in support of an existing BackPack Buddies Program. At the time, only 40 percent of the students who were experiencing food insecurity were receiving support through BackPack Buddies. By partnering with the existing programs, as well as with the Loudoun Chamber of Commerce, Loudoun Education Foundation, Loudoun Insurance Group, Madison Wealth Management, Blue Ridge Area Food Bank and Loudoun Hunger Relief, the Backpack Coalition was able to serve thousands of additional students.
Over a two-year period, 100WomenStrong matched 100 percent of each dollar donated up to $100,000 to Loudoun Education Foundation in support of the Backpack Coalition, exceeding that goal to raise more than $200,000 to help children in Loudoun County schools.
Past Long-Term Initiative News Archives
100WomenStrong is a proud fund of Community Foundation for Loudoun and Northern Fauquier Counties
© Copyright 2016. 100WomenStrong. All rights reserved.
Website designed by KME.Digital


Be sure to review and update your company contact information, descriptions, and product categories for the Online Directory, Meeting App, and Printed Official Program. Meeting attendees will be encouraged to reference these resources pre-meeting, onsite and post-meeting.
Updated company information to include a written description, product categories, address, show specials, etc. are requested at the time your booth is assigned. Updates to company listings are required no later than August 2, 2018.
Updates submitted after this date will not be reflected in the printed Official Program.
Company listings not updated/submitted by August 2, may result in information from the last exhibiting year (2017 or prior) being printed in the Official Program.
Make your listing stand out by purchasing an enhanced or premium listing. The additional features are outlined here.


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Schedule and Fixtures
Liverpool have Leicester in sight, but must solve key puzzle to ensure victory
The Reds have the tools to emerge victorious in key clash, but must change a frustrating trait to do so.
Liverpool's attack will need to take chances against Leicester. (Image: Photo by Clive Brunskill/Getty Images)
Brendan Rodgers is a pragmatist.
He plays percentages better than you or I think. Think back to his time at Liverpool and you will see more than enough evidence.
Rodgers is a manager who can summon momentum unlike many others. But the Northern Irishman also readily accepts when factors are against him. His admittance that he knew Liverpool would not win the Premier League in 2014 when Jordan Henderson was sent off against Manchester City was a sobering one.
Rodgers will know something else going into today's game against Liverpool. While the coach will be working his players into a frenzy, he will be acutely aware that his Leicester team concede chances.
The Foxes currently stand side-by-side with Liverpool on 14 goals against this season, the joint lowest in the league. And the stats statistics also show a similar trend for both teams when it comes to defending.
Jürgen Klopp is right to support Man City over fixture mess favouring Liverpool
Both have a non-penalty expected goal (xG) against of 0.99 per game. They also have an identical xG per shot (0.10) and very similar shots per game against average (10.33 of Leicester compared to 9.82 Liverpool).
Yet Liverpool hold the upper hand in both penalty boxes, and this is where the game could be won or lost. Man for man, Jürgen Klopp will back his players over that of the opponent and rightly so.
The Reds need to find a way to stop Jamie Vardy. (Image: Photo by Plumb Images/Leicester City FC via Getty Image)
That will allow the Reds to take more chances in the game and look to force the issue. Of course they will need to prepare for the threat and pace of Jamie Vardy, however preparation is currently one of the key factors in making this team prosper.
Therefore, it will be down to Liverpool's attack to ensure they are not wasteful in the game, something that can be reasonably levelled against them in recent fixtures, especially in the early moments.
Chamberlain and Lovren injury updates, Milner back? Predicted Liverpool lineup vs Leicester
In both FIFA Club World Cup games, and going back to the recent Champions League decider against Salzburg, Klopp's team had what felt like a plethora of chances in the opening ten minutes.
The conversion rate can do with changing for the better as soon as possible, and this is the perfect place to start. If the Reds can lay a marker down early in the game, Leicester will have to put more energy and output into the opening stages, but also that they are reminded of their recent blip of one point in two games and a lack of confidence is reinforced.
Liverpool will back themselves in any situation, and today will be no different. A winning result, no matter how they achieve it, will further reinforce the notion that first place in the Premier League is already a forgone conclusion.
Trent Alexander-ArnoldA moment of adversity built Trent Alexander-Arnold into defensive warrior despite rivals' mythRivals always get it wrong about Trent Alexander-Arnold at Liverpool, and this is the proof.
Jurgen KloppLiverpool have one more land left to conquer to edge them closer to title gloryThis Liverpool side have done everything bar one thing...
Premier LeagueLiverpool are set for huge fixture dilemma, but wouldn't have it any other wayThe Reds embark on another intense round of games in the coming fortnight, but have made their peace with it.


The full-length heat shock protein 88 (HSP88) complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening the Paecilomyces tenuipes ( P. tenuipes ) Jocheon-1 Uni-Zap cDNA library and performing 5' RACE polymerase chain reaction (PCR). The P. tenuipes Jocheon-1 HSP88 cDNA contained an open reading frame (ORF) of 2,139-basepair encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipes Jocheon-1 HSP88 cDNA showed 77% identity to Nectria haematococca HSP88 and 45-76% identity to other fungal homologous HSP88s. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade. The P. tenuipes Jocheon-1 HSP88 also contained the conserved ATPase domain at the N-terminal region. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as an 88 kilodalton (kDa) polypeptide in baculovirus-infected insect Sf9 cells. Under higher temperature conditions for the growth of the entomopathogenic fungus, mRNA expression of P. tenuipes Jocheon-1 HSP88 was quantified by real time PCR (qPCR). The results showed that heat shock stress induced a higher level of mRNA expression compared to normal growth conditions.
Heat shock proteins (HSPs) are a specific group of proteins that can be induced by heat shock (or stress) in a wide range of organisms including bacteria, plants, insects, mammals, and humans (Lemaux ., 1978 ; Yamamori ., 1978 ; Kelley and Schlesinger, 1978 ; Miller ., 1979 ; McAlister and Finkelstein, 1980 ; Johnston ., 1980 ; Manjunatha ., 2010 ; Routray ., 2013) . As the name suggests, HSPs are induced in cells, tissues, or individuals exposed to sublethal heat shock. HSPs were first discovered in 1962 by Ritossa . It was reported that when Drosophila salivary gland cells were exposed to 37℃ for 30 min then returned to their normal temperature of 25℃ for recovery, "puffing" of the corresponding genes was induced in the chromosomes of the recovering cells (Ritossa, 1962) . In the 1970s, researchers found that this phenomenon was accompanied by an increase in the expression of proteins with molecular masses of 26 and 70 kilodalton (kDa) (Tissieres ., 1974) . These proteins were named "heat shock proteins". Since these discoveries were made, a large number of proteins, referred to as HSPs, have been reported.
In addition to heat shock, the synthesis of HSPs can be induced by a variety of chemical and environmental stimuli. Exposure of cells to heavy metals (Levinson ., 1980) , amino acid analogs (Kelley and Schlesinger, 1978 ; Li and Laszlo, 1985) , glucose analogs (Pouyssegur ., 1977) , Ca 2+ increasing agents (Ding ., 1996) , ischemia, sodium arsenite (Johnson ., 1980) , microbial infections, nitric oxide, hormones, and antibiotics also induce the expression of HSPs. HSPs have been shown to perform multiple functions in cellular processes. Notably, HSPs function as molecular chaperones (Ellis, 1987) to facilitate proper synthesis and folding of proteins throughout the cell (Walter and Buchner, 2002 ; Martin and Ulrich, 1997) . They also stabilize and assist in the trafficking of nascent peptides during normal growth (Hartl ., 1992) . Under stressful conditions such as heat shock or hypoxia (Hammerer, 2001) , increased expression of HSPs protects the cell by stabilizing unfolded or misfolded peptides and by allowing the cell time to repair or re-synthesize damaged proteins (Saibil, 2000) . HSPs have been shown to participate in protein assembly (Welch, 1991) , secretion (Wild ., 1992) , and degradation as well as cellular homeostasis (Mallouk ., 1999) , and the regulation of transcription factors and protein kinases (Sonya ., 2003) . Additionally, HSPs have appeared to serve a protective role in cardiovascular health by aiding (Benjamin and McMillan, 1998) smooth muscle relaxation (McLemore ., 2005) , preventing platelet aggregation, improving cardiac myocyte function, and preventing apoptosis after ischemic injury (Fan, 2005) . Extracellular and membrane bound HSPs are involved in binding antigens and presenting them to the immune system (Nishikawa ., 2008) ; therefore, HSPs are useful as immunologic adjuvants in boosting the response to a vaccine (Bendz ., 2007) . It has been speculated that HSPs may be useful for increasing the effectiveness of cancer vaccines (Binder, 2008) .
Recent studies on HSPs have implicated their role in physiological, biochemical, molecular, biological, ecological, evolutionary, and clinical research. These studies have led to cloning and characterization of genes encoding HSPs in many organisms including the entomopathogenic fungus in our present study. Until now, a great number of variable species of HSPs including small HSPs with molecular weights ranging from 15-40 kDa and many types of HSPs with molecular weights over 40 kDa have been studied; however, HSP88 is rarely investigated (Schlesinger, 1990 ; Sorensen ., 2003 ; Davis, 2004 ; Burnie ., 2006 ; Bendz ., 2007 ; Tsan and Gao, 2009 ; Manjunatha ., 2010 ; Shim ., 2012 ; Routray ., 2013) . Although the amino acid sequence of the HSP88 has been reported from a fungal species (Plesofsky-Vig and Brambl, 1998) , there are few reports regarding the full-length cDNA sequence. This is the first report, to our knowledge, about the HSP88 from Paecilomyces tenuipes Jocheon-1 ( P. tenuipes Jocheon-1), an entomopathogenic fungus also known as the vegetable wasp, and plant worm. P. tenuipes Jocheon-1 is an ecotype of the corresponding species. Dongchoonghacho, a kind of silkworm mushroom produced from the entomopathogenic fungus of P. tenuipes , has been known as a mysterious ancient medicine in Asian countries, particularly in China, Korea, and Japan. This paper describes the molecular cloning of HSP88 cDNA from P. tenuipes Jocheon-1, the expression of cDNA, the deduced amino acid sequence of the protein, and the influence of the heat shock treatment on the corresponding mRNA expression levels.
The entomopathogenic fungal strain, P. tenuipes Jocheon-1, was used in this study. This strain was collected from the mountainous region of Miryang, South Korea and was artificially cultured in egg yolk medium as described previously (Lee ., 2006) .
Total RNA was isolated from the fruiting body of P. tenuipes Jocheon-1 using the SV Total RNA Isolation System (Promega ). mRNA was isolated using the oligotex mRNA midi kit (Qiagen, Korea Ltd). cDNA was synthesized using the Uni-ZAP cDNA synthesis kit (Stratagene) according to the manufacturer's instruction. cDNA was ligated into the EcoR I and Xho I sites of the Uni-ZAP XR vector and packaged in the high efficiency system GigapackIII Gold packaging extract (Stratagene).
The Uni-ZAP XR lambda cDNA library clones were converted to the pBluescript SK(-) phagemid by in vivo excision as described by the manufacturer. Escherichia coli ( E. coli ) SOLR cells were infected with the phagemid, plated onto LB ampicillin agar plates, and incubated at 37℃ overnight. Colonies appearing on the plate with the cDNA insert were selected for plasmid isolation. Plasmid DNA isolation was extracted using Accuprep plasmid mini extraction kit (Bioneer). Plasmids were then subjected to sequence analysis. P. tenuipes Jocheon-1 ESTs were compared with the BLASTX of the NCBI database to identify the homologous genes.
According to the P. tenuipes Jocheon-1 UniZAP cDNA library sequencing results, one clone with the insert of 1,444 basepairs (bp) showed higher similarity to the previously reported fungal HSP88. This clone contained the complete 3′-end with a poly(A) tail, but it was incomplete at the 5′-end. In order to obtain the full-length sequence of this predicted HSP88 cDNA, 5′ RACE PCR was performed using the Marathon cDNA amplification kit (Clontech) according to the manufacturer's protocol. In order to guarantee the specificity of the PCR product, Nested PCR was performed using the following primers: Gene specific primer (GSP), nested gene specific primer (NGSP), adaptor primer 1 (AP1), and adaptor primer 2 (AP2) were supplied with the marathon cDNA amplification kit (Clontech). Amplifications were performed with a MyCycler Thermal Cycler (BioRad). The two specific primers were designed based on this partial sequence information. In the 5′ RACE reaction, adaptor-ligated cDNA was generated from poly(A) RNA isolated from the fruiting body of P. tenuipes Jocheon-1. The first PCR amplification was carried out using lower GSP1 (5′-TGAATGTTAACACGGGTCTTAAGA-3′) and AP1 (5′-CCATCCTAATACGACTCAC-TATAGGGC-3′). PCR cycling conditions were as follows: 33 cycles at 94℃ for 1 min, 60℃ for 1 min, and 72℃ for 2.5 min followed by a terminal extension cycle at 72℃ for 7 min. The second PCR amplification was carried out using upper GSP2 (5′-TGAATGTTAACACGGGTCTTAAGA-3′) and AP2 (5′-ACTCACTATAGGGCTCGAGCGGC-3′). The PCR cycling conditions were the same as with the first PCR cycling conditions except that the annealing temperature was 63℃. The PCR product was analyzed with a 1% agarose gel electrophoresis, and the band of approximately 1.4 kilobasepairs (kbp ) was excised from the gel. The band was then purified with the AccuPrep Gel purification kit (Bioneer) and cloned to the pGEM-T Easy vector (Promega, USA) following the manufacturer's instruction. Blue-white screening of HIT competent DH5a cells was used to identify potential transformants. Positive clones were selected and sequenced with an Applied Biosystem 3730XI DNA Analyzer (USA). The 5′ region sequencing result was combined with the poly(A) tail connected to the 3′ region sequence to make the predicted full-length cDNA sequence.
To isolate the full-length clone, the Marathon cDNA library of P. tenuipes Jocheon-1 was screened with the HSP forward primer (PtHSP-F 5′-AGCAACCATGAGTGTCGTCGGTG-3′) and the HSP reverse primer (PtHSP-R 5′-CGTGATATCTATTTGGAATCGGC-3′). These were designed based on the combined sequence information of the 5′-end sequence and the previously obtained partial sequence. Samples were run on a thermal cycle (BioRad) using PCR PreMix (Bioneer) under the following conditions: 35 cycles at 94℃C for 1 min, 53℃ for 1 min, and 72℃ for 2.5 min. The amplified fragment of approximately 2.1 kbp was purified and cloned to the pGEM-T Easy Vector that was then transformed to HIT competent DH5a cells (RH617, RBC). The plasmid was isolated using a plasmid mini extraction kit and sequenced with an Applied Biosystems 3730xl DNA Analyzer (USA).
The amino acid sequences of other reported HSP88s were retrieved from the GenBank database provided by the NCBI. The sequences were aligned in clustal W (Thompson ., 1994) . Molecular evolutionary genetics analysis (MEGA) software was used for statistical and phylogenetic analysis (Kumar ., 1994) . The phylogenetic tree was constructed with the neighbor joining method (Saitou and Nei, 1987) .
The 2,139-bp open reading frame (ORF) of HSP88 cDNA from P. tenuipes Jocheon-1 was subcloned between the Sbf I and Bgl II sites of pBacPAK9 (Clontech) to produce a baculovirus transfer vector pBacPAK9-PtHSP88. In the transfer vector, the P. tenuipes Jocheon-1 HSP88 cDNA was under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV).
Spodoptera fruigiperda IPLB Sf21-AE (Vaughn ., 1977) clone9 (Sf9) cells were maintained at 27℃ in TC100 medium (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma). Cells were cultured as described by standard methods (O'Reilly ., 1992) . Wild type AcNPV and recombinant AcNPV were propagated in Sf9 cells. The titer was expressed as plaque forming units (PFU) per mL (O'Reilly ., 1992) .
Thirty-five milliliter cell culture dishes were seeded with 1.0-1.5 × 10 6 cells and incubated at 27℃ for 1 h to allow cell attachment. One microgram of BacPAK6 viral DNA (Clontech), 5 μg of pBacPAK9-PtHSP88 in 20 mM HEPES buffer, and sterile water were mixed in a polystyrene tube for a total volume of 50 μL. Fifty μL of 100 μg/mL Lipofectin (Gibco Brl Life Technologies) were gently mixed with the DNA solution and the mixture was incubated at the room temperature for 30 min. The cells were washed twice with 2 mL serum free TC100 medium and fed with 1.5 mL serum free TC100 medium. The Lipofectin-DNA complexes were added in drops to the medium covering the cells while each dish was gently swirled. After incubation at 27℃ for 5 h, TC100 medium containing antibiotics and 10% FBS was added to each dish and returned to incubation at 27℃. After 3 d, the supernatant was harvested, clarified by centrifugation at 2,000 rpm for 5 min, and stored at 4℃ until use. Recombinant AcNPV was plaque purified as described by O'Reilly . (1992) .
Sf9 insect cells were mock-infected, infected with wild type Autographa californica nuclear polyhedrosis virus (AcNPV), or recombinant AcNPVs at a multiplicity of infection of 10 6 in 35 mm dishes. After incubation at 27°C for 3 d, cells were harvested. For SDS-PAGE of cell lysates, uninfected cells and virus infected Sf9 cells were washed twice with phosphate buffered saline (PBS), mixed with protein sample buffer, and boiled . Total cellular lysates were then subjected to 10% SDS-PAGE.
The normal temperature for cultivation of P. tenuipes Jocheon-1 in the developmental stage of fruit body is approximately 20℃. In order to investigate the effects of heat shock treatments on the expression level of HSP88 mRNA in the early fruit body developmental stage, P. tenuipes Jocheon-1 were kept at 25℃ and 30℃ for different amounts of time, 8 h, 16 h, or 24 h.
qPCR was used to measure the relative expression levels of HSP88 during the developmental stages of mycelium, early fruit body, middle fruit body, and late fruit body, respectively. qPCR has also previously been performed to analyze the effect of heat treatment on the relative expression level of HSP88 when P. tenuipes Jocheon-1 was in early fruit body development. One μg of total RNA was used for cDNA synthesis. cDNA was synthesized using the iScript cDNA synthesis kit (Biorad). qPCR was performed using an iQ5™ multicolor real time PCR detection system (BioRad) and sensimix SYBR kit (Bioline, according to the manufacturer's instructions.
Two HSP88 specific primers (RT-HSP-F ; 5′-GCCAGTGAAGAGGAGAAG-3′ and RT-HSP-R; 5′-GCGATGTAGACACCCTTT-3′) were used to amplify a PCR product of 101 bp. Two GAPDH primers (GAPDH-F; 5′-CACTCCTACACCGCTACC-3′, GAPDG-R; 5′-GAATGACCTTGCCGACAG-3′) were used to amplify a GAPDH gene fragment of 130 bp. GAPDH primers were based on sequences from GenBank Accession number GU997101. GAPDH was used as an internal control for qPCR. The qPCR amplifications were carried out in total volume of 20 μL containing 10 μL of SYBR, 1 μL of cDNA, 0.5 μL of each forward and reverse primer (5 pmol final concentration), and 8 μL of DEPC water.
Reactions were initiated with a denaturation step at 95℃ for 10 min, followed by 40 cycles at 95℃ for 15 s, 55℃ for 30 s, 72℃ for 20 s, and a dissociation curve at 95℃ for 60 s, 55℃ for 60 s, and 60℃ for 10 s. Two independent biological replicates were carried out . Melting curve analysis of amplification products was performed at the end of each PCR to confirm that only one product was amplified and detected. The comparative cycle threshold (Ct) method was used to analyze the expression level of HSP88 .
A cDNA library was constructed using fruiting bodies of P. tenuipes Jocheon-1. One thousand clones were randomly selected. Clones that were of low quality, too short (<700 bp), or had vector contamination were not included. Seven hundred and forty one ESTs were generated by a partial sequencing from the cDNA library. Of the 741 ESTs, 305 showed homology to genes identified and deposited in the NCBI database, 261 matched to unknown or unclassified functional genes, and 175 displayed similarity with an E-value that was lower than the adopted standard value of 10-5 in the process of functional annotation. According to the EST results of the cDNA library, one clone with an insert of 1,444 bp showed high homology to the previously reported HSP88 gene. The sequence contained the complete 3′-end with a poly(A) tail, but it was not complete at the 5′-end. Accordingly, the marathon 5′ RACE PCR amplification method was used to determine the unknown 5′-end sequence as described previously. The PCR product was excised from the gel, purified with a gel extraction kit (Promega), and subcloned into the pGEM-T Easy vector for sequence analysis.
The DNA fragment of approximately 2.1 kbp that amplified using the primer pairs PtHSP-F and PtHSP-R was excised and subcloned into the pGEM-T Easy vector for sequencing. The nucleotide and deduced amino acid sequences of HSP88 cDNA are represented in Fig. 1 . The cDNA contained an ORF of 2,139-bp encoding 713 amino acid residues. From the nucleotide and deduced amino acid sequence of the cDNA, five sequence motifs named phosphate1, connect1, phosphate2, adenosine, and connect2, which involved in ATP binding and interdomain coupling (Buchberger ., 1994) , have been identified ( Fig. 1 ). Alignment of deduced amino acid sequences of P. tenuipes Jocheon-1 HSP88 cDNA with other fungal HSP88s is represented in Fig. 2 . This data demonstrates that there are variable identities in the amino acid sequences of the homologous genes ranging from 45-77% ( Fig. 2 and Table 1 ). Further comparison of the P. tenuipes Jocheon-1 HSP88 s equence with the ATPase domain of HSP70 , the ATP-binding cell division protein FtsA of E. coli (Bork ., 1992) , and the fungal HSP88 of Neurospora crassa ( N. crassa ) (Plesofsky-Vig and Brambl, 1998) revealed that P. tenuipes Jocheon-1 HSP88 also contains the conserved ATPase domain at the N-terminal region ( Fig. 1 ).
The nucleotide sequence, deduced amino acid sequence, and ATPase domain of the P. tenuipes Jocheon-1 HSP88 cDNA. The start codon, ATG, is boxed, and the termination codon is denoted with the asterisk. The poly(A) signal sequence is shown with the shadow mark. P1, phosphate1; P2, phosphate2; C1, connect1 (interdomain hinge); C2, connect2 (interdomain hinge); A, adenosine. This cDNA sequence has been provided with the accession number GU983958 from GenBank.
Alignment of the amino acid sequences of P. tenuipes Jocheon-1 HSP88 with other known fungal HSP88s. GenBank accession numbers are: P. tenuipes Jocheon-1 (GU983958), G. graminicola (EFQ27583), N. haematococca (EEU34379), M. oryzae (EDJ96308), N. crassa (EDN96960), S. Sclerotio (EEP78775), B. fuckeliana (EDN30753), T. stipitatu (EED21621), P. marneffei (EEA25736), A. dermatitid (EEQ88845), A. benhamiae (EDN02919), Cn var. neoformans (AAW46676), and L. bicolor (EDR11689).
A phylogenetic analysis using the deduced amino acid sequences of known fungal HSP88 genes revealed that it is divided into two separate clades, ascomycetes and basidiomycetes ( Fig. 3 ). Within the ascomycetes fungal clade, P. tenuipes Jocheon-1 and Nectria haematococca ( N. haematococca ) formed a subgroup, P. tenuipes Jocheon-1 HSP88 showed the highest protein sequence identity (77%) to N. haematococca . The identity of P. tenuipes Jochoen-1 to other known HSP88s was 45-76% ( Table1 ).
Phylogenetic tree of aligned amino acid sequences of the P. tenuipes Jocheon-1 HSP88 and the other known fungal HSP88 sequences. The accession numbers of the sequences in the GenBank are as follows: P. tenuipes Jocheon-1 (GU983958), N. hamematococca (EEU34379), G. graminicola (EFQ27583), N. crassa (AAC23862), M. oryzae (EDJ96308), B. fuckeliana (EDN30735), S. sclerotiorum (EDN96960), A. dermatitidis (EEQ88845), A. benhamiae (EFQ98775), T. stipitatus (EED21621), P. marneffei (EEA25763), Cn var. neoformans (AAW46676), and L. bicolor (EDR11689).
To assess the P. tenuipes Jocheon-1 HSP88 gene, the ORF of HSP88 cDNA, which was comprised of 2,139 nucleotides, was inserted into a baculovirus transfer vector. The transfer vector pBacPAK9-PtHSP88 was constructed by inserting P. tenuipes Jocheon-1 HSP88 cDNA under the control of the AcNPV polyhedrin promoter of pBacPAK9. In order to confirm the correct insertion of the HSP88 ORF fragment, the transfer vector pBacPAK9-PtHSP88 was double digested with restriction enzymes Sbf I and Bgl II. The baculovirus transfer vector was used to generate a recombinant virus expressing P. tenuipes Jocheon-1 HSP88. The recombinant AcNPV (AcNPV-PtHSP88) was produced in insect Sf9 cells by co-transfection with wild type AcNPV DNA and the transfer vector. To examine expression, the protein synthesized in Sf9 cells infected with the recombinant virus was analyzed by SDS-PAGE. The recombinant P. tenuipes Jocheon-1 HSP88 expressed by the P. tenuipes Jocheon-1 HSP88 cDNA was approximately 88 kDa as determined by the gel from the cells infected with the recombinant virus but was not detected from the cells infected with the wild type AcNPV or mock-infected cells ( Fig. 4 ).
Expression of the P. tenuipes Jocheon-1 HSP88 cDNA in recombinant baculovirus-infected insect Sf9 cells. Lane1, size marker; Lane2, Sf9 cells that were mock-infected; Lane3, Sf9 cells that were infected with wild type AcNPV; and Lane4, Sf9 cells that infected with AcNPV recombined with PtHSP88 cDNA. The total cellular lysates were subjected to 10% SDS-PAGE. The proteins on each lane were stained with Coomassie Brilliant Blue R250.
The relative expression level of P. tenuipes Jocheon-1 HSP88 mRNA during the four different developmental stages, mycelium, early fruit body, middle fruit body, and late fruit body, was detected by qPCR. The mycelium stage was used as the control for comparative purposes in normal growth conditions. The highest expression was observed in early fruit body development with expression approximately 2.5 fold higher than that in the mycelium stage and 3.6 fold higher than that in late fruit stage ( Fig. 5A ).
Relative expression level of P. tenuipes Jocheon-1 HSP88 gene by real-time PCR. (A) Relative expression level of P. tenuipes Jocheon-1 HSP88 gene during different developing stages, (B) Relative expression level of P. tenuipes Jocheon-1 HSP88 after heat shock treatment.
To ascertain the potential of heat induced expression of P. tenuipes Jocheon-1 HSP88 cDNA, the fruit body of P. tenuipes Jocheon-1 was subjected to heat shock at various temperatures and times. The relative expression level of the P. tenuipes Jocheon-1 HSP88 gene after heat treatment is shown in Figure 5B . During the heat shock treatment of 25℃, the expression level was highest at 16 h, which was 3.36 fold higher than that of the control at the starting point (0 h at 20℃ in the fruit body). During the heat shock treatment at 30°C, the expression level of P. tenuipes Jocheon-1 HSP88 cDNA reached its highest at 16 h, which was 2.55 fold higher than that of the control at the starting point. After 24 h, expression had decreased to levels similar to the control at 24 h.
In this study, the full-length cDNA sequence of HSP88 was cloned from P. tenuipes Jochoen-1. The full-length cDNA of P. tenuipes Jocheon-1 is 2,139 bp and encodes a polypeptide of 713 amino acids. We had previously reported the genomic structure of the HSP88 genes in four entomopathogenic fungi, P. tenuipes Jochoen-1, P. tenuipes, Cordyceps militaris ( C. militaris ), and Cordyceps pruinosa ( C. pruinosa ), which are industrialized in Korea (Liu ., 2012) . Among them the three entomopathogenic fungal strains, P. tenuipes Jochoen-1, P. tenuipes , and C. militaris showed identical genomic structure, namely 5 introns and 6 exons with 13, 62, 1,438, 306, and 288 nucleotides encoding 713 amino acid residues. C. pruinosa revealed a different species because it contained 4 introns and 5 exons with the length of 13, 62, 32, 1,744, and 288 nucleotides encoding 713 amino acid residues (Liu ., 2012) . It is very interesting that almost same number of deduced amino acid residues for HSP88 in even different genera or species was identified. Pairwise comparison of P. tenuipes Jocheon-1 HSP88 with those of other fungal strains revealed a wide range of identity ranging from 45-77%. These results demonstrate less identity than in the fruiting body forming insect mushroom species studied previously in 2012 (Liu .) This reveals that the HSP genes compared in this study are homologous (even variable in similarity of identity) to the ancestral HSP gene, but there are many mutations in the corresponding sequences that have occurred through long periods of evolutionary time.
The ATPase domains of HSP70, actin, and hexokinase have a common three-dimensional structure (Flaherty ., 1991) . Five sequence motifs named phosphate1, connect1, phosphate2, adenosine, and connect2, which are involved in ATP binding and interdomain coupling (Buchberger ., 1994) , have been identified within these structurally similar domains (Bork ., 1992) . In this study, we found that these motifs are also present in the HSP88 of P. tenuipes Jocheon-1 and other fungal HSP88s, such as the HSP88 of N. crassa (Plesofsky-Vig and Brambl, 1998) . Analysis of the amino acid sequence of this gene revealed a considerable identity with the fungal HSP88s of N. haematococca , N. gramincola , and N. crassa . Sequence conservation was highest at the N-terminus and decreased towards the C-terminus. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as an 88-kDa polypeptide in baculovirus-infected insect Sf9 cells. Future research will continue on the construction of antibodies against HSP88 expressed from the recombinant baculovirus infected with PtHSP88 cDNA and identification of the protein with western blot analysis.
qPCR was performed to detect the relative expression level of P. tenuipes Jocheon-1 HSP88 during the developing stages of mycelium, early fruit body, middle fruit body, and late fruit body, respectively. Results showed that P. tenuipes Jocheon-1 HSP88 has the highest relative expression level during the early fruit body developmental stage. We conducted our experiments at 35℃, but sampling was difficult because of mycerial death at the treated temperature. In organisms that can grow over a broad range of temperatures, the maximum response is usually achieved at about 10℃ above the optimum growth temperature. In organisms that grow over a more restricted range, the maximum response occurs at about 5℃ above the optimum growth temperature. (Lindquist, 1986) . The optimum temperature for the growth of P. tenuipes Jocheon-1 during fruit body development is approximately 20℃. Heat shock treatments at 25℃ and 30℃ have been performed and qPCR has been used to analyze the relative expression level of HSPs after heat shock. Results demonstrated that the relative expression level of P. tenuipes Jocheon-1 HSP88 is greatest at 25℃ after 16 h, which is approximately 3.5 times higher than that of control. The relative expression level of P. tenuipes Jocheon-1 HSP88 also increased during heat shock treatment at 30℃ and reached its highest level after 16 h, and then levels reduced similar to those in the control group after 24 h. These results indicate that HSP88 production is probably dependent on both cellular differentiation and heat stress. The results obtained in this research will be useful to further investigate the molecular, biological, physiological, and developmental roles of the HSP88 in entomopathogenic fungi, which are very important species in the industrialization of insect mushrooms.
This work was supported by a 2 year Research Grant (2012-2013) from Pusan National University.
 YQ Liu , NS Park , YG Kim , KK Kim , HC Park , HJ Son , CH Hong , and SM Lee , "Molecular Cloning of the cDNA of Heat Shock Protein 88 Gene from the Entomopathogenic Fungus, Paecilomyces tenuipes Jocheon-1", International Journal of Industrial Entomology, vol. 2, no. 2, Jun 2014.


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