Anti-gas protesters will need development approval for camp
ANTI-CSG protesters have been warned they will have to lodge a formal development application with Richmond Valley Council if they want to set up camp at Bentley again.
Any application to the council could take to up to six weeks to process and require a vote by councillors, according to Richmond Valley Council general manager John Walker.
Mr Walker emphasised the council was not opposed to the camp as "a matter of philosophy" but needed to process it properly under planning law.
"If it's going to be there we may as well get it done right, we want to make sure there's water, toilets, and garbage," Mr Walker said.
Last year's camp was "basically unlawful for a long time" after it grew beyond its 200-person limit, he said.
He advised protesters this time to "start earlier rather than later" in making applications to the council.
A spokesman for Energy Minister Anthony Roberts said the Minister was still assessing options following last Friday's Supreme Court decision and had not ruled out an appeal yet.


Watchdog group alleges conflicts of interest on high-speed rail board
Critics of California’s high-speed rail project Tuesday urged the state attorney general to investigate whether two prominent officials in Los Angeles County and Anaheim have conflicts of interest because they sit on the bullet train’s board while holding other public offices.
A Palo Alto group known as Californians Advocating Responsible Rail Design asserts that Anaheim Mayor Curt Pringle and Richard Katz, a board member for the Los Angeles County Metropolitan Transportation Authority, might be violating a state law that forbids public officials from simultaneously sitting on a board, commission or other government body whose interests are likely to clash with their public duties.
The group cited an opinion from the Legislature’s legal office, which concluded in April that a court would probably rule that Katz and Pringle hold incompatible offices — a finding that could force them to resign one or more of their positions.
Anaheim City Council members and the MTA board make decisions related to segments of the high-speed rail project planned for Los Angeles and Orange counties. A leg of the system would end in Anaheim.
Pringle, who chairs the High-Speed Rail Authority board, also is a member of the Orange County Transportation Authority board. Katz sits on the boards of the MTA and Metrolink, the commuter rail line that serves six counties. Those agencies are involved in the high-speed rail project as well.
“Local office and service on the high-speed rail board just don’t mix,” said Elizabeth Alexis, a co-founder of the watchdog group. “The biggest problem is maintaining public confidence in the integrity of the process. The project needs to be not just clean. It needs to be squeaky clean.”
Alexis pointed to recent concerns expressed by state Sen. Alan Lowenthal (D-Long Beach), who chairs the Senate’s transportation committee, which is overseeing the $42-billion project that would run from Anaheim to Los Angeles to San Francisco.
Last spring, Lowenthal’s committee looked into a proposed deal — backed by Pringle — to use $200 million in high-speed rail money to complete a huge, canopied transportation center next to Angel Stadium at the southern terminus of the bullet train’s first phase.
Lowenthal said it looked like parochial interests were getting priority over statewide interests. After the committee hearing, the High-Speed Rail Authority dropped the idea.
Quentin Kopp, a former judge who sits on the high-speed rail board, has also questioned the positions held by Katz and Pringle.
Pringle, who will be termed out as Anaheim mayor by the end of the year, said the incompatible office question “is a gray area.” He says there is only speculation at this point of a disqualifying conflict between the interests of the local agencies he represents and the High-Speed Rail Authority.
Katz, a former state legislator and experienced transportation official, said that the Legislative Counsel’s opinion was merely the advice of one legal office and that he was seeking a review by other attorneys. He stressed that Gov. Arnold Schwarzenegger appointed him to the high-speed rail board “to bring a local transportation agency viewpoint to it.”
Before he and Pringle joined the panel, Katz said, the bullet train authority “operated in this vacuum without a lot of concern for local issues and community concerns.” He said it “could doom the project” if local officials who could help integrate the line with other projects had to resign.
The watchdog group says the attorney general’s office has an obligation to investigate the matter, but has failed to do so.
In a July letter, the attorney general’s office notified the High-Speed Rail Authority that some members of its board held other public offices. The letter said it was unclear whether any of the positions were incompatible.
“We believe that the board members are taking our letter into serious consideration, and we are making ourselves available to answer their questions about the law,” said Michelle Quinn, a spokeswoman for the attorney general’s office.
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Andersen Partnership opens Hong Kong office
Based in World Wide House, Central Hong Kong and managed by recruitment veteran Michael Doherty, the business intends to hire in excess of 30 consultants over the coming months.
Commenting on the opening, The Andersen Partnership's CEO Brian Schroder commended the achievements of Doherty over the past fifteen years, particularly his start up experience and success in Hong Kong, but also highlighted the huge opportunities for the brand and staff in the future.
"Our accounting and insurance clients are truly global by nature and we’re already seeing huge demand and movement throughout the region.
"Hong Kong is a significant milestone for us being our tenth office in just five years and our sixth office outside of Australia."
“This is our fourth office in Asia this year for The Andersen Partnership,” said Chairman Niall Hamill, he also confirmed that they were at advanced stages with several more and that the group would open in Dublin, Beijing and Kuala Lumpur in mid-2015.

domain: geography


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Changes in financial aid mix since 2008
Golden, Colo., Dec. 14 – Associated Bodywork & Massage Professionals (ABMP) has released results of its biennial survey of massage school operations, providing benchmarking data massage therapy schools and programs can use to evaluate their operations. ABMP has conducted the research since 1998 as a service to the profession.
In September, ABMP e-mailed a confidential survey to 1,229 massage, bodywork and somatic therapy schools in the U.S. for which ABMP had e-mail addresses on file. ABMP received 253 responses, a 20.6 percent response rate. The survey results were tabulated and analyzed in October.[i]
Of the respondents, nearly half were career schools, a quarter were proprietary, 20 percent were colleges and the remaining 5 percent were public institutions. Nearly 70 percent of the responding schools reported being accredited.
Tuition and attrition
Financial aid was reported as available to students in 150 of the schools surveyed, representing 73.9 percent of the 203 respondents who answered this question. The percentage of students receiving financial aid varied widely. The financial aid entities used by massage programs were as follows.
- Title IV–83.2 percent.
- Personal loan–66.4 percent.
- Sallie Mae–48.2 percent.
These figures represent a dramatic change from two years ago in that Sallie Mae declined over the two-year period from 65.5 percent to 48.2 percent, while Title IV financial aid jumped by 36.4 points from 46.8 percent in 2008 to 83.2 percent. Personal loans rocketed from 3.8 percent in 2008 to 66.4 percent this year.
Fifteen schools (7.4 percent) offer payment plans for students, while 10 schools rely on state aid (4.9 percent) and 13 schools (6.4 percent) offer scholarships, Veterans Administration funding and work-study programs.
School attrition rates dropped slightly from the 14 percent reported in 2008 to 12.6 percent reported this year. In 2006, the average attrition rate was 9.9 percent and in 2004 it was 11 percent. This year, schools identified the top 10 reasons students say they do not complete school. Family or personal reasons and financial problems were the most common reasons students gave for leaving school.
Reflecting a trend over the last six years, schools report their most successful marketing efforts are word-of-mouth, websites and pay-per-click advertising. Of those answering the question, 92.4 percent named word-of-mouth (146 respondents) and their websites (145 respondents) as their most effective marketing tools. Social media are beginning to be used more often by schools for communication and marketing to prospects and students. More than 95 percent identified Facebook as their preferred medium for social networking efforts.
ABMP serves massage, bodywork and somatic therapy professionals, and is devoted to promoting ethical practices, fostering acceptance of the profession and protecting the rights of legitimate massage and bodywork practitioners. Representing more than 75,000 members, ABMP is headquartered in Golden, Colorado. ABMP is employee owned and is the largest massage therapy membership association in the nation.
[i] The questionnaire was e-mailed to state-approved massage therapy programs. Not all respondents answered every question. The same methodology has been used in the surveys since 1998.

domain: news


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Capstone Invest Advsrs Llc has invested 0.23% in Wal-Mart Stores Inc (NYSE: WMT ). Among 5 analysts covering TransEnterix ( AMEX:TRXC ), 3 have Buy rating, 0 Sell and 2 Hold. Examining Institutional Ownership at Wal-Mart Stores, Inc. ( NYSE WMT ) opened at 87.31 on Wednesday. Wal-Mart Stores, Inc. has a 50 day moving average of 82.57 and a 200 day moving average of 78.86.
The 48-year-old Miami-bound woman and the 28-year-old Nashville-bound man then got friendlier than expected. A couple who had never met before are facing a federal investigation after they indulged in a sexual act mid-flight to the horror of fellow passengers.
It closed at $50.1 lastly. It has outperformed by 441.54% the S&P500. Lord Abbett & Ltd Com reported 0.25% stake. The third largest holder is Primecap Management Co/Ca/, which now holds $225.52 million worth of this stock and that ownership represents almost 3.57% of its market capitalization.
Porter said the two-month lull in activity reinforces the point that "the frothy growth of the past year is over and done". Even considering the possibility of a return to positive territory in September, the third quarter will be hard to result with a growth of more than 2 %, " commented Benoit P.
Enterprise Financial Services Corp Raises Position in Cognizant Technology Solutions Corporation (CTSH)
On 11/1/2017 announced a quarterly dividend of $0.15 with an ex dividend date of which will be payable on 11/20/2017. Currently, 589 million total shares are owned by the public and among those 587.43 million shares have been available to trade.
PNC Financial Services Group Inc. grew its holdings in shares of Patterson-UTI Energy by 7.9% during the 1st quarter. Before trading, trader, investor or shareholder must have an eye on stock's historical performance. The ex-dividend date is Wednesday, December 6th. It last reported revenues of $413 million and EPS of -$0.14 for the June 2017, representing 199% top-line growth and -0.28 EPS growth.
The sales number for the automakers in the month of October stood at 146,446 units for Maruti , against 133,793 in the same month year-ago; Toyota reported sales of 12,403 units, as compared to 11,651 units last year. In fact, sales of Ciaz that comes under mid-size segment also declined 35.4 percent. The company sold 32,490 units in October 2017 compared to the 33,929 units sold in October 2016.
State Street Corp raised its position in shares of Coca-Cola by 0.9% in the first quarter. Vanguard Group Inc. now owns 273,897,295 shares of the company's stock worth $12,415,764,000 after buying an additional 5,478,675 shares during the period.
Earnings per share serves as an indicator of a company's profitability. Angled up and price is moving up (or was recently) overall, angled down and price is moving down overall, moving sideways and the price is likely in a range. Its P/Cash is valued at 9.87. Endo International PLC (NASDAQ: ENDP ) (TSE:ENL) has received a consensus recommendation of "Hold" from the twenty-four analysts that are now covering the firm, Marketbeat Ratings reports.
The comments came as Nationwide said United Kingdom house price growth had edged up to an annual rate of 2.5% last month though the outlook for the market remained subdued. However, we have found that most of the people we talk to have already taken into account the possibility of a rise in interest rates as it has been talked about for so long.
It has outperformed by 97.03% the S&P500. Jpmorgan Chase & Co now has $355.75 billion valuation. The hedge fund run by Ric Dillon held 4.67M shares of the major banks company at the end of 2017Q2, valued at $426.69 million, up from 4.13M at the end of the previous reported quarter.
Of course, she had to make a classic pumpkin costume for one of the newborns, as seen on baby Angela. "You can just kinda lay it over them", she said. Parents had a variety of costumes to choose from, including a Lions jersey, taco, superheroes, and JIF peanut butter. Others are dressed as a Hersey kiss, a donut, or a superhero.
This in turn contributed to a further increase in outstanding business, which rose solidly. That said, the rate of deterioration in vendor performance was only marginal. Chinese manufacturing employment fell again in October, thereby extending the current sequence of job shedding to four years. Notably, the rate of accumulation was the joint-steepest since March 2011 (on par with July 2016).
Underlying replacement cost profit for the third quarter was $1.9 billion, compared with $684 million in previous quarter and $933 million in the third quarter of a year ago. The company set aside an additional $206 million in the quarter to cover claims and finance costs related to the 2010 Deepwater Horizon oil spill in the Gulf of Mexico, bringing total charges for the disaster to $63.4 billion.
The Boring Company is still a long way from fulfilling Musk's vision of underground tunnel networks connecting major cities in the USA, however the firm has received provisional approval to start digging in Maryland, as reported by Tech Crunch .
Columbia Partners L L C Investment Management now owns 82,187 shares of the basic materials company's stock valued at $539,000 after purchasing an additional 15,693 shares in the last quarter. United States Steel Corporation (NYSE:X) posted its latest quarterly earnings results after hours Tuesday. AK Steel Holding Corp. shares are now trading off about 21.7% on the day.
It has underperformed by 33.32% the S&P500. The portion of a company's profit allocated to each outstanding share of common stock was $3.15 a share in the trailing twelve months. Independent Consultants holds 0% or 202 shares in its portfolio. Shares of Spirit Airlines, Inc. California State Teachers Retirement System reported 292,486 shares stake.
According to the Recommendation Trends of the stock polled by Finviz, the company has a consensus recommendation of 2.9 out of the scale from 1 to 5, where 1 stands for Buy and 5 means Sell. Iv Conger bought 36,180 shares of Freeport-McMoran stock in a transaction on Thursday, September 7th. The shares were acquired at an average price of $15.23 per share, for a total transaction of $551,021.40.
The firm has "Buy" rating given on Thursday, February 9 by Berenberg. Also, insider Flemming Ornskov acquired 8,400 shares of the firm's stock in a transaction that occurred on Tuesday, August 22nd. Therefore 88% are positive. Saga has GBX 255 highest and GBX 190 lowest target. This target by FinnCap means the possibile upside of 152.63 % from the stock close price.
EPS serves as a pointer of a company's profitability/success. These ratios are important while doing valuation of the company or the shares of the company. Its P/Cash is valued at 4.7. A number of equities analysts recently commented on the stock. According to the Recommendation Trends of the stock polled by Zacks Investment Research for this month, the company has a consensus recommendation of 2.42 out of the scale from 1 to 5 where 1 stands for Strong Buy and 5 means Strong Sell.
The company now has an average rating of Hold and an average target price of $47.63. First Solar Inc. has a 12-month low of $30.85 and a 12-month high of $74.29. Institutional investors and hedge funds own 53.07% of the company's stock. Schroder Investment Management Group now owns 1,679,286 shares of the solar cell manufacturer's stock worth $67,306,000 after purchasing an additional 1,666,064 shares in the last quarter.
The Cabaret Law was passed in 1926 during the Prohibition and policed musicians and dancing. That means there is a high likelihood that if a person has danced in New York City, they have broken a law. In recent years, proponents of repealing the law have organized their efforts through various advocacy groups such as the Dance Liberation Network , started by Discwoman co-founder Frankie Decaiza Hutchinson.
The tight inventory situation, particularly amongst existing units, is putting upward pressure on home prices. The 20-City Composite posted a 5.9% year-over-year gain, up from 5.8% the previous month. After seasonal adjustment, the national index rose 0.5% month-over-month, while the 10-city and 20-city indexes also both rose 0.5%. Blitzer said that over the a year ago, the consumer price index's 2.2 percent rise was driven largely by energy costs.
Sanders Capital LLC now owns 29,212,930 shares of the oil and gas exploration company's stock worth $839,952,000 after acquiring an additional 1,951,907 shares in the last quarter. ILLEGAL ACTIVITY WARNING: "Toronto Dominion Bank Has $3.96 Million Position in Cabot Oil & Gas Corporation (NYSE: COG )" was originally posted by StockNewsTimes and is the sole property of of StockNewsTimes.
In July this year, the suspect made the decision to detonate an explosive device in Germany "in order to kill and injure as many people as possible", the statement read. Yamen A. had been known to authorities since October 2014, according to Kohler. Interior Minister Thomas de Maiziere has praised coordination among German security agencies, the BfV and Federal Criminal Police Office.
At least eight people are dead near the West Side Highway in New York City after they were purposefully struck by a truck; the Federal Bureau of Investigation is now investigating the incident as a possible act of terror, ABC News reports.
Smoke breaks are particularly time-consuming at the company-about 15 minutes each-because smokers must travel from the office on the 29th floor to the basement. "Our CEO saw the comment and agreed, so we are giving non-smokers some extra time off to compensate", Mr Matsushma added. He spoke about how some Japanese companies are now offering non-smoking staff extra days off and he believes that Ireland should follow suit.
The SI to Dolby Laboratories Incclass A's float is 2.84%. The relative volume of the stock is 0.88. About shares traded. Evolving Systems Inc (NASDAQ:EVOL) has declined 3.74% since October 31, 2016 and is downtrending. The entertainment giant reported $1.58 earnings per share for the quarter, topping the consensus estimate of $1.53 by $0.05. The co's performance for 6 months was slashed of -10.80%, 1-year performance stands at 2.19% and year to date showing slashed performance of -2.97%.
The Stephen King renaissance is on full blast following the success of IT, so it's no surprise that Paramount is moving full speed ahead on its new adaptation of his 1983 horror novel Pet Sematary . After the death of their cat Church, their kindly neighbor introduces Louis to an ancient Indian burial ground behind the local pet cemetery, which has the power to bring the dead back to life.
This would potentially reduce users large losses on the machines and the risk of harm to both the player and wider communities in which these machines are located, such as the increased health costs associated with problem gambling . Proposals to launch a responsible gambling campaign and new advertising guidelines are among a raft of suggestions created to help minimise the risk to vulnerable people and children.
The areas of assistance will include the construction of the first subway in Metro Manila through a proposed 600 billion yen ($5.2 billion) loan, the development of the North-South Commuter Railway connecting Clark, Pampanga and Los Baños, Laguna, and energy, information and communications technology, environment, agriculture and disaster reduction projects.
Analysts giving shares a rating of 1 or 2 would be indicating a Buy. Shopify Inc (US ) notes that this is larger than its operating loss of $9.5 million from the same time previous year, which was likely behind some of the drop for SHOP stock today.
Mastercard Inc posted a quarterly profit on Tuesday that was higher than Wall Street's expectations, fuelled by increased consumer spending globally and maintaining market share over other payment channels. Perhaps, that suggests something about why 86.74% of the outstanding share supply is held by institutional investors. Covering sell-side analysts are estimating company growth for next year to be 19.03%.
Lukaku has scored three goals in as many Champions League appearances so far this season, while he has also impressed with his link-up play as he has created four chances. Do you think Mourinho and the players deserved boos on Saturday? Martial is well off that category, but that's where he's got to be looking. "He takes big moments on, so come on; let's see it every single week".
On average, equities analysts predict that Cisco Systems Inc . will post $2.43 EPS for the current year. It has underperformed by 3.50% the S&P500. ENDP's profit will be $140.11M for 2.37 P/E if the $0.67 EPS becomes a reality. William Blair maintained the stock with "Buy" rating in Thursday, June 29 report. Exane Derivatives increased Cisco Systems ( CSCO ) stake by 31.07% reported in 2017Q2 SEC filing.


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Overworked and Overwhelmed. Welcome to HR Today.
TLNT: The Business of HR
DECEMBER 1, 2021
Earlier this year, Lattice surveyed over 700 HR professionals to better understand their challenges and priorities. The resulting 2021 State of People Strategy Report found that 42% of HR teams cited emotional exhaustion and burnout as their top challenges.


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Difference between investment and gambling ppt
Investment vs. Speculation vs. Gambling - IndusWealth
What is the difference between insurance and gambling? What is the difference between insurance and gambling? Distinguish between insurance and gambling. ... loss might never occur while for gambling, ... Investment vs Speculation | Top 7 Differences You Must Know! Investment vs Speculation | Top 7 Differences You ... Gambling involves putting in money on ... Let us understand some of the differences between an Investment vs ...
Going All-In: Comparing Investing And Gambling - Investopedia
The Difference Between Saving, Investing, and Speculating ... www.pinnacleadvisory.com Every successful investor must begin by understanding the difference between saving, investing, and speculating. If you get those ... Investment, Speculation and Gambling: How are they ... Investment, Speculation and Gambling: ... So it is very important to understand the difference between Investment, speculation and gambling. ... Whereas, investment ...
Let's understand some of the basic differences between Investment vs ... Many may consider speculators as dangerous gamblers though they provide the ...
What this definition lacks is the real difference between investing and gambling…research.While gambling involves random chance, investing requires some research. Lest anyone get intimidated, this type of research doesn't require a PhD, and in some cases can be done in a short amount of time. Warren Buffett: The Difference between Investing... -… Investing. According to Buffett, an investment operation is “one where you look to the asset itself to determine yourSo, what is the difference between speculating and gambling?Perhaps that makes it as good as gambling, which can be easily picked up, and has a strong behavioral aspect to it. Is There a Difference between Investing and Gambling? ::… Keywords: Investment, Zero Sum Game, Gambling. JEL Classification: G10. Suggested Citation: Suggested Citation. Saunders, Kent T., Is There a Difference between Investing and Gambling? A Christian Perspective (January 1, 2016). Journal of Biblical Integration in Business, Vol. 19, p. 57-71... Difference between investment speculation and gambling -…
4 Jan 2019 ... Investing and gambling both involve risk and choice. Interestingly, both the gambler ... This is a key difference between investing and gambling.
Difference Between Investment and Gambling. However, there is a huge difference between investing and gambling when it comes to stopping losses. In gambling, particularly in sports gambling, there are no loss-mitigation strategies due to the activity being so speculative. Top 10 Difference between Stock Investing and Gambling Top 10 Difference between Stock Investing and Gambling Most people, especially none investors and even investors believe that investing is gambling. That is why there are always highs and lows in the stock market, because majority of stock investors believe in throwing a dice, picking a stock based on intuition and hoping that it goes up.
Investment- Sacrifice of certain present value for the uncertain future reward Financial Investment-An exchange of financial claims/securities-Focused on real investment concept of businessman “The employment of funds with the objective of realising additonal income or growth in value of investment at a future date” 3.
Structuring Your Business for Investment and Growth. Presented by Teri G. Rasmussen Capital Crash Course 2008 December 2007. Teri G. Rasmussen Partner and Vice Chair, Business Law Practice Group Lane, Alton & Horst, LLC trasmussen@lanealton … Information Technology and Blind People in a
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domain: finance


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Henry Winkler Happy Days Fonzie Costume Guide for Cosplayers
Fonzie’s costume is inspired from the famous movie, Happy Days. It is worn by Henry Winkler. He plays a leading role in the movie and gained much appreciation and admiration from the audience. The costume contains a dark brown leather jacket having rib-knitted cuffs, giving you a slim and sleek look.
Henry Wrinklerr Happy Days Costume has a round neck white t-shirt down the jacket. Blue denim looks extra ravishing as a bottom. To get a hairstyle like him, you will need a classic comb along with styling pomade. Black combat boots make your attire more charming.

domain: entertainment


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Gastrointestinal (GI) complaints comprise a majority of chronic health concerns. The most common symptoms include diarrhea, constipation, bloating, abdominal pain, sudden urgency, excessive flatulence, halitosis, and fatigue. The most diagnoses include parasites, candida overgrowth, SIBO, and IgG food intolerances. Other related conditions that produce the same symptoms include Crohn’s disease, Ulcerative Colitis, IBS, and diverticulitis, and slow motility.The GI complaints are nearly always curable by discovering through proper testing the presence of parasites, bacteria overgrowth, yeast, and IgG food allergies or intolerances.
IgG Food Intolerances
This idea throws many people off since most patients have struggled to notice any allergic reactions to the food when they eat. This is because immune reactions to food also produce other antibodies such as IgG, IgM, and even IgA that differ in their immune response compared to IgE antibodies. IgE food reactions causes more obvious and generally immediate allergic reactions when consumed and thus easier to associate reactions with a specific food. The IgG antibodies produced, which are linked to many illnesses including in the GI tract, are of the IgG class, which are actually also known as delayed or slow onset allergic reactions. In other words, the reaction occurs hours and even days following the exposure. These become extremely difficult to determine when using symptoms as the only diagnostic assessment. With an IgG food allergy test a patient is able to view upwards of a hundred or more foods ranging from grains, animal proteins, fruits, vegetable, legumes, and even yeast and candida. These tests can be invaluable to help determine a toxic underlying cause of GI diseases.
In previous articles we have stated, that yeast has a major side effect leading to localized inflammation and irritation in the gastrointestinal tract. Many studies in the last few years have also shed light on the additional contribution from proteins that were once deemed healthy. For example, gluten, contained in wheat, barley, and rye, produces a protein called Zonulin that causes severe irritation of GI cells and leads to leaky gut. This leaky gut, in turn, causes the GI tract and immune system to react to any and all proteins that are introduced in the GI tract from the diet. In time we become reactive to everything that we eat. Just like yeast, normal or even healthy foods, become major immune distractors.
With the immune system being overwhelmed by these food proteins, healing from normal “wear and tear” is reduced. The cure is in discovering the food allergies through testing and removing those foods to allow the GI tract to heal. In time with proper dietary protocols and with additional support of an anti-yeast diet symptoms improve. Food allergies along with yeast are the most often overlooked causes of gastrointestinal complaints. Testing is key and success is sure as we begin to heal the gut. (A negative celiac panel is insufficient to rule out gluten IgG reactivity.) Consider that the leading food allergy is also the leading health food, wheat. A blessing to some and a curse for so many others. IgG food sensitivity testing is the only way to know for sure. Once a patient begins to treat two of the most common causes, which are candida and food sensitivities, the immune system starts to rebound to full activity.
Testing begins with a simple blood draw. Once the results are in, the lab generates a comprehensive report detailing your IgG toxic foods. Sometimes people find that, while they may only have a few food toxicities, these make up foods that they eat regularly. Therefore, if you are allergic to pasta and you eat it four or five times per week, you will be poisoning yourself four or five times per week.
The most notable point to make here is how much better you’ll feel. Some people rid themselves of lifelong chronic conditions like migraines which are almost always caused by a food toxicity. Some people feel a tremendous boost in energy, and see a general increase in mental acuity. Others notice that their constant irritability is gone.
A Journal of American Medical Association (JAMA) article revealed that more than 75% of the money spent on health care is spent on chronic conditions. Our research indicates that a great deal of these chronic conditions are the direct result of food toxicities; easily treatable by simply removing the foods from your diet.
Besides GI benefits the following conditions have also been reported to improve with food IgG testing and elimination: Attention Deficit Disorder (ADHD), Arthritis, Autism, Asthma, Candidiasis, Celiac, Chronic Fatigue Syndrome (CFS), Depression, Diabetes, Digestive Disorders, Ear Infections, Eczema, Fibromyalgia, GERD, G.I. Disorders, Headaches & Migraines, Heartburn, Irritable Bowel Syndrome (IBS), Joint & Muscle Pain, Leaky Gut, Mental Fatigue, Obesity, Poor Memory, Psoriasis, Sinusitis, Skin Disorders, Weight Loss, and Yeast Infections. Click here to view a IgG Food Allergy Sample Report.
STUDIES ON IgG FOOD INTOLERANCE TESTING

domain: health


[Law] Utah may be slipping as America's No. 1 nanny-government state—at least if this bill manages to pass. It seeks to make the use of profanity a criminal offense in South Carolina. (It also places the crime of saying "shitdamnfuckhell" in the presence of a 17-year-old on par with child prostitution.)
The most shocking thing about it is not that an elected state senator believes that such a measure could pass constitutional muster—but that a Utah legislator didn't think of it first.
(But keep it on the QT—we don't want to be giving them any big ideas.)


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Anyone who is looking to quickly level their characters within Grand Theft Auto V's online multiplayer mode will be happy to learn that Rockstar Games is hosting an in-game event this weekend that gives players the chance to earn RP twice as fast. The developer announced on their Rockstar Newswire blog that a special GTA V weekend event is now underway. Throughout the remainder of weekend, fans will be able to enjoy double RP payouts while playing select missions that were added to the verified playlist earlier this week.
"Starting today and lasting through Sunday, February 8th - get 2X RP in the GTA Online Event Playlist of new Verified Jobs. To jump right in and start earning Double RP, just fire up GTAV and hit the 'Event Playlist' button on the launch screen, or access it via the 'Playlists' section of the GTA Online Start Menu."While this week's new addition of Rockstar Verified missions for GTA V added 10 new player-crafted missions to the title's official online playlist, only five of those jobs will qualify for this weekend's Double RP bonus. A recent Inquisitr report detailed many of the new verified missions, which include one fan-made job that challenges GTA V to survive a map filled with undead clowns.
Rockstar Games released informative descriptions of every one of the new Grand Theft Auto V verified jobs on Tuesday. The developer selected the online missions from content that was built by fans using GTA Online's in-game creator tools.
"We've got ten exemplary additions to the pool of Rockstar Verified GTA Online Jobs, including the first batch of creations utilizing the Last Team Standing mode. There are also five additional new Verified Jobs, including a fun and frightful Capture that's among the best we've seen to date."In order to make the missions playable across all platforms, each one of the new GTA V jobs has been locked to only allow a maximum of 16 players. This allows those playing on last-gen consoles to be able to still enjoy the content. However, GTA V users on the Xbox One and PlayStation 4 are also still bound to the lower player count, despite the fact that the newer consoles can actually handle twice that many players in a single match.
This weekend's Double RP event for Grand Theft Auto V is now live on Xbox One, Xbox 360, PS4, and PS3. GTA V fans will also soon receive more reasons to log onto the game's online servers, as co-op heists have been confirmed to finally release in the coming weeks.
[Images via Rockstar Games]


CNC Plasma Cutters use a computer and a special coding language called G-code to program projects. The cutter then carries out the cuts you desire. This type of machine can increase your productivity by 1000% or more. So, what are you waiting for? Oh right! You want to know what to look for when you are buying one.
Recent improvements in technology and manufacturing have created an excellent buying environment. Plasma cutters are cheaper than they ever were and they are also more advanced than ever before.
First, you will want to be familiar with the plasma cutting process, since this will help you understand the different ways to classify plasma cutters. If you know your way around a plasma cutter already, feel free to skip the next paragraph.
In order for plasma cutting to work, plasma has to be created. Inside a plasma cutting torch, plasma is created by firing an electric spark into the torch nozzle while pressurized gas (often air) surges out of the nozzle onto the metal to be cut. The spark heats the gas to 15,000 degrees Celsius at which point the gas turns into plasma. Plasma is the fourth state of matter; it is both a liquid and a gas at the same time (this is analogous to how visible light is a wave and a particle at the same time). The electrical spark is created when the plasma cutting torch is touched to the cutting surface. This closes an electric circuit between the plasma cutting machine and the work piece.
Automated plasma cutting machines come in two flavors: conventional and precision. These classifications are based on the properties of the cutting flame. Conventional plasma cutting systems are slower than precision systems and they produce more kerf (the volume of metal cut out) and bevel. Being the superior system, precision plasma cutting equipment costs considerably more.
Buyer beware, manufacturers often mismatch power sources and machines. The machines come in two parts: the base machine (with the table, computer terminal and bridge or gantry) and the plasma cutter (precision or conventional). The power sources for those two components are separate and they have to be matched properly. The mismatches are often the manufacturer's fault, but you the buyer have to keep your eyes open to make sure that it does not happen. In the event that you do have mismatched hardware, do not start running it because what will ruin the machines. Contact the company where you bought the equipment and figure out a solution to the problem.
Today's automated plasma cutter market is dominated by two different models of machine, bridge style and gantry style machines. And the machines are generally made from two types of metal, steel (fabricated) or aluminum (extruded). The extruded aluminum machines are "light duty" and they are found in the realm of the artist and the hobbyist. They are best for doing limited amounts of cutting on thinner metals. The industry-strength machines are the ones made from fabricated steel; they can cut continuously all day as well as devour thick metals.
You will want to decide what sort of drive system you need for your projects. If you are a hobbyist or artist, using a conventional machine, a single-side drive system should be all you need. You may also consider a single-motor, dual-side drive system. The higher level drives increase precision which is not what conventional plasma cutters are built for so you would be throwing your money away if you bought higher-level drives. A precision plasma project will require a dual-side two-motor drive system to reach optimum performance.
You will have to think about what size of motor and gear box you require. If the mass of the motor and gear box are too small compared to the mass of the machine then the motor will not be able to effectively change the direction of cutting. This is important when the cuts go back and forth at high speeds. If the masses are not matched properly it will lead to unimpressive cut results and increase wear on the machinery, which may lead to premature failure.


by Katie Gorka
Today marks the third day in ISIS’ latest offensive against a string of Christian towns and villages in northeast Syria. According to Aziz Mirza, with the Syriac Cultural Association, speaking from Qamishli by phone today, ISIS is continuing to push forward in trying to take control of this predominantly Assyrian Christian region.
Mirza said an estimated 350-400 people are missing from 12 different villages, but it has been very difficult to confirm those numbers because all cell phones appear to be confiscated. When relatives have tried calling, members of ISIS answer the phones, Mirza said.
Kurdish and Christian fighters have been working together for the past year and half to protect this area from ISIS. The Christian fighters, who operate as the Syriac Military Council, had forces in 3 villages: Tel Hormizd, at the southern end of the Khabur valley, Tel Shamiram, at the northern end, and Tel Tawil on the northeast flank. The SMC and Kurdish (YPG) forces had been there since Feb 7, but with the pre-dawn attack that was launched on February 23rd, ISIS has now retaken control. So far, the Syriac Military Council has lost four fighters with another 12 deaths suspected but not yet confirmed.
ISIS is currently focusing its efforts on the town of Tel Tamar, which is the regional center. Yesterday, ISIS set off three car bombs just outside the barricades protecting Tel Tamar, near where the Syriac Military Council forces and Kurdish forces are headquartered. There were no casualties. Mirza also said that fighting ebbed somewhat on Tuesday because of heavy rains, but today, February 25, it was back at full force.
In a separate phone call, Sewerios Malke from the Syriac Military Council confirmed that ISIS is still trying to take control of the region and that they are trying to cross the Khabur river. The Kurdish and Syriac forces have been able to hold them back so far but it is uncertain how much longer that can last. He estimates the number of ISIS fighters at several thousand.
According to a Combined Joint Task Force Operation Inherent Resolve News Release, U.S. and coalition military forces have carried out airstrikes in the Khabur River region where this fighting is going on. Between 8 a.m. yesterday (Feb 24) and 8 a.m. today (Feb 25), local time, fighter and remotely piloted aircraft conducted nine airstrikes in Syria:
- Near Hasakah, three airstrikes struck an ISIL vehicle.
- Near Kobani, six airstrikes struck three ISIL tactical units, an ISIL fighting position and destroyed six ISIL fighting positions.
However, both Malke and Mirza asserted that there were no airstrikes in the Khabur region, only further south, 22 kilometers below Qamishli. The stark contrast between the stories of hundreds or even thousands of ISIS fighters attacking villages, taking as many as 400 hostages, and three U.S. airstrikes in the same region that struck only one vehicle, suggests the U.S. may be missing the target.
Some have argued that without troops, or at least advisors, on the ground, there is insufficient intelligence to guide airstrikes. In addition, representatives of the Syriac forces have been making the case in Washington for the past several years that their location in Syria made them particularly vulnerable to attack by ISIS and they could be key players in the fight against ISIS. They were therefore lobbying to be included in the equipping and training of opposition forces in Syria. The State Department has confirmed that the Syriac forces were not included in the first round of training.
Katie Gorka is the president of the Council on Global Security. @katharinegorka.


text:
Mock disaster exercise tests preparedness at Grand Forks AirportAn emergency disaster exercise, required by the FAA, was conducted at the Grand Forks Airport Wednesday morning.
By: Michael Yoshida, WDAZ
An emergency disaster exercise, required by the FAA, was conducted at the Grand Forks Airport Wednesday morning.
The event started with emergency personnel responding to an emergency landing at the airport. The training exercise also involved medical and terrorist scenarios. More than 100 people were involved in Monday's event, with around 50 of those being volunteers who acted as the plane's passengers and crew.
"When you have multi-agencies coming together, that coordination, that communication is important and you don't really get to test that until you have a scenario like this," said Lt. Mike Ferguson with the Grand Forks Police Department.
During the next week the agencies involved will debrief and review the exercise.


text:
The march ended at Miller Park, where (former) State Senator Ernie Chambers took to the stage. The Omaha World Herald described part of Chambers’s statement as follows:
(Chambers) asked black voters to be wary of white politicians who use them during campaigns and then disappear afterward.
But a recent article in The Reader fleshes out what Chambers said. It went like this:
Chambers attacked politicians who pledged support for the event but did not march. He had especially scathing words for Jim Esch, the Democratic candidate for the House from the Omaha-dominated 2nd District.
“I could get an axe murderer, I could get a serial rapist and put him up here and say ‘this is Jim Esch, vote for him, he’s here with you.’ and everybody would clap,” said Chambers. “They don’t know what he looks like. Is Jim Esch here now? How are you going to vote for him? They sent a flunkie because black people are so dumb and so easily tricked that the white man sends his flunkie.”
Esch was present at the initial gathering but left before the march began.
Esch has tried to make himself out to be a man concerned about North Omaha, but Chambers calls him to the mat here.
And this after some strange rumblings about Chambers thinking of running in the 2nd Congressional District race as an Independent. With Chambers having nearly 100% name ID, think how that would shake up the race.
Our guess is that Chambers has other fish to fry, but whenever you toss in Ernie’s name, things get much more interesting.
And speaking of Esch, Jim’s campaign came forward with a counter to the Lee Terry campaign’s “Truth Squad”, led by Esch primary opponent Richard Carter.
But the name of Esch’s group? The “Dare Squad”. Something about daring Terry to talk about energy policy (presumably about Esch’s recent endorsement of Terry’s energy plan ).
Of course, the Esch campaign clearly missed the irony of calling it the “Dare Squad”, as opposed to the D.A.R.E squad — you know, the one that teaches school age kids about the dangers of drugs and alcohol…
We’re still taking guesses here on what Fahey will announce tomorrow. KMTV‘s Joe Jordan says he “can’t find anyone” who thinks Fahey will run for re-elect. The OWH seems to think so too. And frankly that’s the way we’ve felt for awhile.
But all that being said, we’ve heard that Fahey has told numerous people he IS going to run. So we don’t know what to think.
Maybe he’s just going to pull a Hagel. “I’m here to announce that I’ll make my announcement at a later time. Feel free to enjoy some cake and punch.”


text:
Thousands of consumers who sent their old mobile phones to recyling site Cash4phones.co.uk have been left out of pocket after the company filed for insolvency.
The website offered cash to consumers who wanted to get rid of an old phone after an upgrade, but payments were only made once handsets were received and users complained they were ultimately offered far less than originally quoted online. In some cases they reported the company failed to pay up at all.
Attempts by the Guardian to contact Cash4phones via an email address listed on the site failed, and the website has now been taken down.
Capital Books has been appointed to liquidate the company. A spokesman said any affected customers should lodge a claim as a creditor, but the chances of getting the cash value promised was slim. It said it has so far been handed 750 old handsets.
Company director Nearchos Chacholiades filed for bankrupcy after setting up Yemonia Limited as the holding company for Cash4phones. The last known company address was a mail box at a newsagent's in Paddington, London.
"There is a meeting of creditors on 13 January where questions can be raised directly with the director, and they can also lodge their complaint so we will investigate the director's conduct more rigously," a spokesman for Capital Books said, who estimated that at least 1,000 people are owed money. Trading Standards is also investigating the company.
Rachel Smalley is one customer who faces a fight for her cash. She recently upgraded her mobile and decided to send her old one to Cash4phones. "After entering my details I was quoted £182 for my iPhone 4S," she said. "I was rather pleased with this so sent the phone off to them, but then I got a revised offer via email stating they would only pay £148 as it failed their tests – the reason being cosmetic wear and tear, despite the phone being in great condition."
She decided to take the revised sum. If she wanted the phone back she was told she would face a postage charge. "I got an email saying payment had been made and would be transferred into my account in two to three working days, so I waited and still got nothing – I have not heard anything since."
Consumers who have lost money through Cash4phones can contact the liquidator on 01622 754 927 or by email at firstname.lastname@example.org. A meeting of creditors is scheduled for Monday 13 January at Hither Green in London.


text:
the heavens, the stars came out bright and clear, but nothing unusual occurred. Saunders informed us the same phenomenon never appeared twice. Patiently we waited and watched till near midnight, then, disappointed and angry over the delay, hurriedly pushed on, when we were startled by the sudden appearance of four great globes of light flaming just above us.
"Hoo-ray! hoo-ray!" yelled Sheldon. "Whatever it is, it signals to us. We sent up four rockets and they respond with four balls of fire. What'll we do with 'em?"
Saxe. rushed to the car, returning with rockets and sent them up himself, but their small light faded in the flood of fire that burst from those brilliant globes. The stars vanished as the sky tinged to a fiery sea, flames forked and twisted, seeming to gather volume, and in a second turned to a thousand different hues. It was magnificent!
Gradually the fire dimmed, the stars twinkled richly in the pinkish glow and the four globes above swayed gently. Then they descended nearer the earth while a greenish, blue flame darted from each, floating upon the air like a great ribbon, the color deepening as the four ends joined, then formed into loops and circles, and in the second a word blazed across the sky.
"Centauri!" I gasped.
"Centauri!" my three friends exclaimed.
My head grew light. "Centauri," I murmured. "I saw it."
"I should think you did," Saxe. cried. "We all

domain: literature


Frozen is coming to Broadway! Disney taps veteran director to helm live theatrical version of hit animated film
Disney has tapped one of Broadway's freshest talents to direct one of its most anticipated musicals, Frozen.
According to a Disney Theatrical Productions announcement on Tuesday on Twitter, Alex Timbers will helm the show, which is expected to land on Broadway in 2018 alongside its hits Aladdin and The Lion King.
Timbers is a two-time Tony Award nominee who directed Peter and the Starcatcher, Bloody Bloody Andrew Jackson, The Pee-Wee Herman Show and Rocky.
Coming to Broadway! Disney has tapped one of Broadway's freshest talents to direct one of its most anticipated musicals, Frozen, following the success of the animated film
He also is a co-creator and co-writer of the music-themed Amazon series Mozart in the Jungle.
The show will have sets by seven-time Tony winner Bob Crowley, choreography by Peter Darling, lighting by five-time Tony winner Natasha Katz and sound design by four-time Tony nominee Peter Hylenski.
Two-time Tony winner Stephen Oremus is the music supervisor and will create vocal and incidental arrangements.
The show will have sets by seven-time Tony winner Bob Crowley, choreography by Peter Darling, lighting by five-time Tony winner Natasha Katz and sound design by four-time Tony nominee Peter Hylenski
It had been previously revealed that the husband-and-wife team of Robert Lopez and Kristen Anderson-Lopez are working on the show and Jennifer Lee, co-director and screenwriter of the film, is writing the story.
Frozen has earned Disney more than $1.27 billion at the box office worldwide, becoming the most successful animated movie of all time. Its signature song, Let It Go, won an Oscar. Apple said the animated film's soundtrack was the year's top-selling album on iTunes in 2014.
The musical Frozen will have an out-of-town tryout in summer 2017 at a theatre to be announced later.
Helming the project: Alex Timbers, pictured at the Broadway opening night for Therese Raquin in New York in October, will helm the musical, Frozen, which is expected to land on Broadway in 2018
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text:
It has been proposed that environmental stimuli can activate transposable elements (TEs), whereas few substantial mechanisms have been shown so far. The class-II element Tam3 from Antirrhinum majus exhibits a unique property of low-temperature-dependent transposition (LTDT). LTDT has proved invaluable in developing the gene isolation technologies that have underpinned much of modern plant developmental biology. Here, we reveal that LTDT involves differential subcellular localization of the Tam3 transposase (TPase) in cells grown at low (15°C) and high (25°C) temperatures. The mechanism is associated with the nuclear import of Tam3 TPase in Antirrhinum cells. At high temperature, the nuclear import of Tam3 TPase is severely restricted in Antirrhinum cells, whereas at low temperature, the nuclear localization of Tam3 TPase is observed in about 20% of the cells. However, in tobacco BY-2 and Allium cepa (onion) cells, Tam3 TPase is transported into most nuclei. In addition to three nuclear localization signals (NLSs), the Tam3 TPase is equipped with a nuclear localization inhibitory domain (NLID), which functions to abolish nuclear import of the TPase at high temperature in Antirrhinum. NLID in Tam3 TPase is considered to interact with Antirrhinum-specific factor(s). The host-specific regulation of the nuclear localization of transposase represents a new repertoire controlling class-II TEs.
Under unfavorable environmental conditions, plants immediately respond in a physiological way to avoid fatal consequences, but plants also alter the genome per se to produce potential progenies adapted to the given environmental changes. Mechanisms by which the physiological responses in plants follow the environmental changes have been intensively studied in various aspects. It is known that the structural changes of the plant genomes are occasionally induced by transposable elements (TEs) activated by environmental stresses (Grandbastien, 1998; Kalendar et al., 2000), whereas the mechanisms regarding the activation processes of TEs are limited in our knowledge. To maintain genome integrity, plants may adopt diverse strategies to regulate the activities of transposable elements.
Tam3 from Antirrhinum majus is a class-II transposon belonging to the hAT superfamily (Calvi et al., 1991). Tam3 excision and, by implication, transposition, exhibit the unusual features of activation at low growth temperatures (15°C) and inhibition at high temperatures above 25°C (Harrison and Fincham, 1964; Carpenter et al., 1987) (Figure 1a). Activation of Tam3 can occur as a rapid and reversible response to a temperature change during the lifetime of a single plant (Hashida et al., 2003). Such low-temperature-dependent transposition (LTDT) of Tam3 represents a response of transposon to environmental stimuli. Because the activity of Tam3 can be controlled by temperature, this transposon system has been used for the generation of mutations and a gene tagging system that have made remarkable contributions to the study of developmental and metabolic systems in plants (Coen et al., 1989; Schwarz-Sommer et al., 2003).
Unlike known mechanisms in the stress responses of other TEs, LTDT is not regulated at the level of transcription of the TPase gene (Hashida et al., 2003). Although a reversible change of DNA methylation state in Tam3 that occurred in somatic tissues was positively correlated with growth temperature (Hashida et al., 2003), methylation was not a cause of LTDT of Tam3 (Hashida et al., 2005). Instead, nuclear extracts from plants grown at 15°C were capable of binding to the Tam3 sequence, but the extract from plants grown at 25°C did not bind, suggesting that the methylation change in Tam3 is affected by the binding ability of its transposase (TPase) to Tam3, which is dependent on the growth temperature (Hashida et al., 2006). Previous results in vivo led to a conclusion that Tam3 TPase exhibits differential binding abilities to Tam3 between 15° and 25°C that might be related to LTDT.
Here, it is reported that the nuclear localization of Tam3 TPase in Antirrhinum is arrested at temperatures at which Tam3 transposition is inhibited. The Tam3 TPase reporter constructs fused with green fluorescence protein (GFP) and the luciferase (LUC) assay demonstrated that the nuclear localization of Tam3 TPase in Antirrhinum cells is severely restricted at high temperature, and is permitted in about 20% of the cells at low temperature. The results are provided to demonstrate that LTDT of Tam3 is controlled by the nuclear localization of TPase, and evidence is shown for specific interactions between Tam3 TPase and host factor(s) to establish LTDT.
Presence of Tam3 TPase in Antirrhinum cells
The LTDT of Tam3 is not regulated by transcription of the Tam3 TPase gene or the DNA methylation state of Tam3 (Hashida et al., 2003, 2005). To test whether LTDT is correlated with the level of the Tam3 TPase in Antirrhinum cells, immunoblot analysis was performed using an anti-peptide antibody, pN1 (Uchiyama et al., 2008) (see Figure S1). pN1 detected a polypeptide of about 100 kDa corresponding to Tam3 TPase in young leaf proteins extracted from Antirrhinum plants grown at 15° and 25°C (Figure 1b). This result demonstrated that the processes prior to translation of TPase are not responsible for LTDT.
Because TPases should be transported into the nucleus in order to catalyze the transposition reaction (Heinlein et al., 1994; Ono et al., 2002), it was considered that LTDT might reflect different accessibility of the TPase to the Tam3 DNA sequences at the two temperatures: the low temperature could enhance the TPase accessibility to the Tam3 sequences, whereas the high temperature could abolish the accessibility. In previous results, proteins in a nuclear extract from plants grown at 15°C, but not at 25°C, bind to the Tam3 sequence (Hashida et al., 2006). Differential nuclear localization of the TPase at high and low temperatures is thus a possible mechanism to explain LTDT.
Nuclear localization of Tam3 TPase
To confirm the above assumption, transient expression analyses were conducted using a plasmid, pTam3TPase-GFP, containing the Tam3 TPase gene fused to the GFP gene. The plasmid was introduced into young petal epidermal cells of Antirrhinum plants grown at 15° or 25°C using a particle bombardment method, and the bombarded petals were further incubated for 24 h at 15° or 25°C (Figure 2). The continuous low temperature (Ll) or low temperature after bombardment (Hl) conditions did not allow the plasmid to express TPase-GFP in the Antirrhinum petals, because the transient expression of the plasmid required relatively high temperatures. Continuous high temperature (Hh) conditions allowed the cells to express GFP, and most expressed GFP in the cytoplasm, whereas only 1% of the cells expressed GFP in their nuclei. After bombardment, in high-temperature (Lh) conditions, GFP was detected in the nuclei of about 18% of the cells with green fluorescence (Figure 2). These results showed that the TPase-GFP was not directed to the nucleus in most cells of the Antirrhinum plants grown at high temperature, whereas about 20% of the cells localized TPase-GFP to the nuclei at low temperature. When pTam3TPase-GFP was delivered into tobacco BY-2 cells and onion epidermal cells, GFP signals were observed in the nuclei of nearly 100% of the cells (see Figure 5 and Figure S2), suggesting that LTDT of Tam3 is an Antirrhinum-specific phenomenon.
Previously, Antirrhinum callus showed LTDT of Tam3 similar to the plants (Hashida et al., 2005). Then, we examined transgenic calli of Antirrhinum leaf that produced a chimera protein of Tam3 transposase and GFP. The plasmid pGWB5-Tam3 that contains the Tam3 TPase gene in the pGWB5 construct consisting of 35S promoter and GFP gene (Figure 3a) was introduced into the Antirrhinum genome by Agrobacterium-mediated transformation. In contrast to the transient expression of GFP, the transgenic calli stably expressed the fluorescence even at a continuous low temperature of 15°C (Figure 3b). The nucleus-to-cytoplasm GFP (NCG) ratio was calculated based on the GFP fluorescent intensities of the nucleus and cytoplasm in the cell. The Antirrhinum calli transformed with the plasmid pGWB5 encoding merely GFP did not exhibit any difference in distributions of the NCG ratio between cells grown under low and high temperatures (Figure 3c). In the calli expressing GFP-fused TPase, however, the NCG ratios of the cells grown under low temperatures were clearly distributed in a higher ratio compared with that of cells grown under high temperatures, which was similar to the findings observed in calli transformed with pGWB5 (Figure 3c). Therefore, the transgenic Antirrhinum calli verified that the low temperature induces the GFP-fused TPase to accumulate in the nuclei.
Quantitative measurements of nuclear localization of Tam3 TPase
For quantitative measurement of the level of Tam3 TPase in nuclei, the experiments using the GAL4 system were adopted (Aoyama et al., 1995; Igarashi et al., 2001). This system uses the effector protein containing GAL4 DNA-binding domain (GAL4 BD) and VP16 activator domain (VP16), both of which are required for the transcriptional activation of the reporter gene carrying GAL4 binding sites in its promoter sequence. We constructed two effector plasmids, pGATam3 and pGAGUS, where the Tam3 TPase or GUS proteins were fused to GAL4 BD and VP16 activator domains (Figure 4a). The reporter plasmid, pGBL, contained two operons for Renilla luciferase and firefly luciferase genes, as shown in Figure 4a: the Renilla luciferase gene driven by the 35S promoter is a reference to standardize differences in efficiency of the bombardment delivery, and the transcription of the firefly luciferase gene can be induced by the binding of the effector protein to the GAL4 binding sites (six tandem copies). The firefly luciferase is produced by nuclear import of the effector proteins containing GAL4 BD and VP16. The constructs were introduced into young petal epidermal cells of Antirrhinum plants grown at 25°C by the bombardment method, and the bombarded petals were incubated for 16 h at 15° or 25°C, and then the firefly and Renilla luciferase activities were measured. The outcome for the level of nuclear import of Tam3 TPase or GUS protein was indicated by the ratio of the firefly luciferase activity to the Renilla luciferase activity (LUC ratio). When pGATam3 was bombarded along with pGBL, the incubation at 15°C of the petal cells showed as much as five times higher LUC ratio relative to the incubation at 25°C (Figure 4b). The delivery of pGAGUS and pGBL into the petal cells resulted in the higher LUC ratio at 15°C relative to that at 25°C, but it was not statistically significant (Figure 4b). In the case where pGBL alone was introduced, the LUC ratios were much reduced in both of the temperature conditions (Figure 4b). It is noteworthy that GAL4 BD in the effector protein contains an NLS, and so is the effector protein directed to the nucleus. Accordingly, the GUS fusion protein from the pGAGUS construct should be fully imported into nuclei, as pGAGUS induced high LUC ratios. The LUC ratio in the pGATam3-introduced cells incubated at 15°C were about one-fifth of that of pGAGUS-introduced cells incubated at 15°C (Figure 4b). This was the same proportion with that estimated with pTam3TPase-GFP: about 20% of the cells with Tam3 TPase-GFP had the signal in nuclei at low temperature (Figure 2). Therefore, although low temperature facilitated nuclear localization of Tam3 TPase in Antirrhinum, about 80% of Tam3 TPase might not be permitted to be present in Antirrhinum nuclei. At high temperature, nuclear localization of Tam3 TPase was even more strictly restricted. The quantitative measurements using the luciferase gene constructs verified the results in the analyses using the GFP fusion proteins.
Arrest of nuclear import of Tam3 TPase at high temperature
As described in the previous sections, two mechanisms for the temperature-dependent differential nuclear localization of TPase were considered: (i) TPase is imported into the nucleus only at low temperatures; (ii) TPase is exported from the nucleus into the cytoplasm at high temperatures. To test these possibilities, leptomycin B (LMB), which inhibits the export of nuclear proteins into the cytoplasm by targeting exportin 1, was applied to protoplasts prepared continuously at 25°C. The effect of LMB was verified using the plasmid pJ4-RSG-GFP, which encodes a GFP-fusion protein containing an NLS along with a nuclear export signal (NES) (Igarashi et al., 2001). The RSG-GFP fusion protein was predominantly localized to the cytoplasm of both Antirrhinum and BY-2 protoplasts (Figure 5c,g,j). In protoplasts treated with 20 ng ml−1 LMB, the ratios of cells that accumulated RSG-GFP protein in the nucleus to the number of total cells expressing GFP increased in both Antirrhinum (from 28 to 88%) and BY-2 (from 15 to 53%) protoplasts (Figure 5d,h,j). In the same protoplasts, LMB failed to alter the ratios for the nuclear localization of GFP from pTam3TPase-EGFP, which was mostly observed in the cytoplasm of Antirrhinum and in the nuclei of BY-2 cells (Figure 5f,j). Therefore, the temperature-dependent differential nuclear localization of TPase must result from the inhibition of TPase import into the nucleus in cells grown at 25°C, and not from the inhibition of export. The control of nuclear import of TPase underlies the LTDT of Tam3 in Antirrhinum. Even under low temperature conditions, the nuclear translocation of Tam3 TPase-fusion protein was limited to about 20% of the total cells expressing GFP or LUC in Antirrhinum (Figures 2 and 3). This may be a mechanism to suppress transposon activity and maintain genome stability that has evolved in Antirrhinum.
Nuclear localization signals in Tam3 TPase
Nuclear localization signals are intrinsic for the entry of nuclear proteins to the nucleus after translation in the cytoplasm. Based on the amino acid sequence of the Tam3 TPase, three short stretches (approximately 35 amino acids) were predicted as NLSs, which contain six or seven basic amino acid residues (K/R) (Figure 6 and Figure S1). Each of the three regions was fused to a GFP plus GUS protein (to prevent GFP from autonomously entering the nucleus). Bombardment with every construct resulted in green fluorescence that was mostly detected in the nuclei in both Antirrhinum petals and onion epidermal cells incubated at 25°C (Figure 6). The results revealed that Tam3 TPase possesses at least three NLSs, which facilitated entry into the nucleus. These findings were supported by other data obtained using onion cells bombarded with deletion mutants of the Tam3 TPase (Figure S2). These results suggest that the Tam3 TPase is equipped with a domain for the arrest of nuclear translocation of the TPase at high temperatures.
Nuclear localization inhibitory domain in Tam3 TPase
To test whether Tam3 TPase contains a domain required for the arrest of nuclear translocation at high temperatures, further transient assays were carried out. One intact and four truncated sequences of the Tam3 TPase gene fused to the GFP gene were introduced into the young Antirrhinum petals of plants grown at 15° or 25°C through the bombardment method (Figure 7, left). The bombarded tissues were incubated in Hh or Lh conditions as described in Figure 2. The truncated constructs contained at least one NLS, so that the GFP fusion proteins potentially entered the nuclei, as depicted in Figure 7. When the two plasmids pTP231–577E and pTP578–803E were introduced into the petals of plants grown at 15° and 25°C, most of all the cells (87.5–100% of cells with GFP signal) expressed green fluorescence in the nuclei under either of the temperature conditions (Figure 7, right). The GFP fusion protein from pTam3TPase-GFP was delivered into the nuclei in 1 and 18% of the cells observed in Hh and Lh conditions, respectively (Figures 2 and 5). Similarly, low frequencies of nuclear localization of GFP signal were observed in the assays with pTP1–577E and pTPΔBE (Figure 7). These three constructs, pTam3TPase-GFP, pTP1–577E and pTPΔBE, all encoded the amino acid sequences 55–244 of the Tam3 TPase (Figure 7). This 55–244 amino acid sequence should be associated with the arrest of nuclear translocation of Tam3 TPase itself. Therefore, this region was designated as the nuclear localization inhibitory domain (NLID), which is potentially capable of suppressing the translocation of Tam3 TPase to the nuclei in Antirrhinum cells. Compared with pTP231–577E and pTP578–803E, these three constructs had even lower frequencies of nuclear localization of the GFP signal in both the low and high temperature conditions. The NLID of Tam3 may not only function at high temperature to strictly prevent nuclear translocation of the TPase, but also functions at low temperature to confine nuclear translocation to around 20% of the total TPase yield.
These five plasmids used in the above analysis were delivered into onion epidermal cells by the bombardment method, and most of the cells with GFP clearly exhibited green fluorescence in nuclei (Figure S2). The NLID may interact directly with Antirrhinum-specific factor(s) to prevent nuclear translocation of the TPase itself. The NLID is considered as a Tam3-unique region; no homology was detected in the N-terminal 170 amino acids of Tam3 TPase, which partly comprises the NLID, although the rest of the TPase amino acid sequence was homologous with rice putative TPases (Dart) (AB206820) (Fujino et al., 2005; Tsugane et al., 2006).
This study elucidated the temperature-dependent differential nuclear localization of Tam3 TPase: low temperatures allowed the TPase to enter the nuclei, but high temperatures did not allow entry. Control of the nuclear translocation of the TPase thus underlies the LTDT of Tam3 in Antirrhinum. The regulatory systems of transposon activities identified so far are known to have specific limitations: the germ line-specific transposition of the P element in Drosophila because of splicing being limited to the germ cells (Laski et al., 1986); the inactivation of Tc1 elements in the germ line of Caenorhabditis elegans attributable to RNA interference (Ketting et al., 1999); and the developmentally regulated transposition of Zea mays (maize) transposons (Levy and Walbot, 1990). Repressor genes have also been identified against P (Black et al., 1987), Spm (Cuypers et al., 1988) and Mu (Slotkin et al., 2005) transpositions, which result from aberrant transposase genes or the rearrangements of their derivative elements. In addition, non-specific regulation of transposon activity has been observed because of DNA methylation and/or RNA silencing (Martienssen, 1998; Colot and Rossignol, 1999; McDonald et al., 2005; Buchon and Vaury, 2006). LTDT of Tam3 occurring at a post-translational stage is considered as a unique regulatory mechanism relative to the above regulation mechanisms of transposon activity.
For Tam3 TPase, the differential subcellular localization regulated by temperature is unlikely to be accomplished by itself, because the same phenomenon did not arise when pTam3TPase-GFP was introduced into tobacco BY-2 and onion cells, where green fluorescence was preferentially detected in the nuclei at high temperature. The control of nuclear localization of Tam3 TPase should be established by cooperation with Antirrhinum host factor(s). Martin et al. (1989) and Haring et al. (1991) also showed no enhancement of Tam3 transposition at low temperatures in transgenic tobacco plants. These previous results support the prediction that LTDT requires Antirrhinum-specific factor(s).
In general, TPases are transported from the cytoplasm into the nucleus after translation, because of the presence of NLS-related domains, to allow TPases to bind to nuclear DNA for the cut-and-paste processes. Thus, NLSs seem to be essential for TPases. The two transposons, Ac (Boehm et al., 1995) and Mu (Ono et al., 2002), in maize have at least one major transcript in their autonomous elements encoding proteins containing NLS signals, which are capable of directing the proteins to the nuclei in petunia protoplasts and onion epidermal cells, respectively. Tag1 in Arabidopsis was also inferred to possess an NLS from the amino acid sequence (Liu and Crawford, 1998). At least three monopartite NLSs were identified in Tam3 TPase. When GFP fusion proteins carrying each of the NLSs were introduced into Antirrhinum or onion cells, the green fluorescence was localized to the nuclei at both temperatures. In addition to these monopartite NLSs, Tam3 TPase harbors an NLID that was identified as a region to inhibit nuclear localization of the TPase. The NLID consisted of an amino acid sequence unique to Tam3 TPase, and is a possible region to regulate LTDT of Tam3 and to interact with Antirrhinum-specific factor(s). The NLID might also play a role to limit nuclear translocation up to approximately 20–30% of the total Tam3 TPase production, even at low temperatures. These results seem to reflect the complexity of the host mechanism involved in LTDT.
Tam3 is classified into the hAT superfamily (Calvi et al., 1991), of which related elements have been widely found in multicellular organisms, implying that the hAT superfamily probably arose before the separation of plants, animals and fungi. These elements are considered to have co-evolved vertically with individual host organisms, because there is no evidence for their horizontal transfer (Rubin et al., 2001). The distinct behavior of Tam3 in Antirrhinum might reflect coordinate evolution between host-specific factor(s) and the transposon.
We used the Antirrhinum majus line HAM5 (nivearecurrens: Tam3), which gives rise to variegated petals at low temperatures of around 15°C because of Tam3 transposition. This line was initially grown for 2 months at 25°C and subsequently transferred to a 15°C growth chamber or grown continuously at 25°C. The tobacco BY-2 cell line was maintained in the liquid MS medium and subcultured every week. Onions were used within 1 week of purchase.
Antibody production and purification
Antibody production was performed by the Molecular Biology Laboratory (Nagoya, Japan). An oligopeptide, GCGTGTLTRHLTAKHKNRD, which corresponds to the amino acid residues 212–230 of Tam3 TPase (see Figure S1) was synthesized and used to immunize a rabbit. To purify the antibody, the serum obtained was incubated with the resin-immobilized oligopeptide (FMP-activated Cellulofine; Seikagaku Kogyo, http://www.seikagaku.co.jp/)) overnight at room temperature (23°C). The resin was washed extensively with wash buffer containing 20 mm Tris–HCl (pH 7.5), 1 m NaCl and 1% Triton X-100, and then with Tris-buffered saline (TBS). Absorbed antibody molecules were eluted with 0.1 m glycine-HCl (pH 2.5) and neutralized immediately with 1/20 volume of 1 m Tris at 4°C. The purified antibody was designated as pN1.
Total protein was extracted from the young leaves or flower petals of Antirrhinum plants. About 0.5 g of tissue was homogenized with 1 ml of protein sample buffer containing 60 mm Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, 5 μg of aprotinin and 35 μg of PMSF, and then supplemented with 50 μl of 2-mercaptoethanol. The homogenate was centrifuged for 5 min at 20 400 g. The resulting supernatant was separated from the cell debris. The protein concentrations of the extracts were measured using a Protein Assay kit (Bio-Rad, http://www.bio-rad.com). About 20 μg of protein was separated by 10% (w/v) SDS-PAGE and transferred to a poly-vinylidenedifluoride membrane. After blocking with 4% (w/v) skimmed milk, the membrane was incubated overnight at 4°C with 2500-fold-diluted primary antibody. The membrane was incubated for 1 h with 5000-fold-diluted secondary antibody (anti-rabbit immunoglobulin antibody conjugated to horseradish peroxidase), and then stained with ECL Western Blotting Detection Reagent (RPN2106; Amersham Bioscience, now part of GE Healthcare, http://www.gelifesciences.com) according to the manufacturer’s recommendations. Signals were detected using Fuji X-ray films.
To construct plasmids able to express GFP, modifications were made to the basic construct pJB1414, which contains the CaMV 35S promoter, EGFP coding sequence and the terminal sequence of nopaline synthase (TNOS) in a cistron, and the ampicillin resistance gene. For the TPase gene-fusion plasmid, pTam3TPase-GFP, the full-length Tam3 TPase coding sequence (2409 bp in AB038406) was inserted between the 35S promoter and EGFP sequences in pJB1414 (Uchiyama et al., 2008). This insertion process was mediated by the Gateway reaction (Invitrogen, http://www.invitrogen.com) and the primers used in GATEWAY cloning are listed in Table S1. Similarly, the plasmid, pGWB5-Tam3, for Agrobacterium-mediated transformation, was constructed, based on pGWB5 (Nakagawa et al., 2007), by the GATEWAY cloning system. The plasmid, pGWB5-Tam3, locates the Tam3 TPase gene upstream of the synthetic green fluorescent protein with the S65T mutation (sGFP gene), which is present between the 35S promoter and TNOS in pGWB5 (Figure 3a). Deletions of parts of the Tam3 TPase coding sequence for pTP1-577E, pTP231-577E, pTP578-803E and pTP1-351E were produced by PCR, and the deletion of pTPΔBE was performed by self-ligation after BspT1071 digestion with the PCR segment of the Tam3 TPase gene (the sequence corresponding to Tam3 TPase amino acid positions 55–803), which can take away the DNA sequence between amino acid positions 245 and 588 of Tam3 TPase. These truncated sequences were also introduced between the 35S promoter and EGFP coding sequence in pJB1414 through the GATEWAY method, as described above. To survey the NLS sequences, each of the candidate NLSs (Table S2) with a basic amino acid cluster was inserted between the 35S promoter and EGFP sequences in pJB1414. These plasmids also contained the GUS gene, which was inserted between the EGFP coding and TNOS sequences of pJB1414, to prevent autonomous translocation to the nucleus (the EGFP molecule by itself tends to enter the nucleus).
Transient expression assay
Transient expression of GFP in Antirrhinum petal and onion epidermal cells was carried out using the Helium Biolistic gene transformation system (Bio-Rad). The epidermis of each tissue was peeled and placed on half-strength MS medium. Plasmid DNA (approximately 2 μg) was precipitated onto gold particles (1.6 μm in diameter) as described previously (Hashida et al., 2003). The bombardment delivery into intact tissue was performed according to the method of Uchiyama et al. (2008). The subcellular localization of GFP fusion proteins was observed using a UV fluorescence microscope (Olympus, http://www.olympus-global.com) equipped with a UV-A fluorescence filter set (330- to 380-nm excitation filter; 400-nm dichroic mirror; 420-nm barrier filter). Bombardments were repeated three or more times for each plot.
Agrobacterium-mediated transformation and GFP microscopic observation
Leaves from in vitro-grown plants were used for callus induction in MS medium containing 5 mg L−1 Picloram (Sigma-Aldrich, http://www.sigmaaldrich.com). Two weeks after callus induction, 5-mm square pieces of leaf laminae were inoculated with freshly grown Agrobacterium tumefaciens strain LBA4404, transformed with pGWB5-Tam3 or pGWB5 (Figure 3a). Inoculated leaf segments were co-cultivated on 0.2% Gellan gum (Wako, http://www.wako-chem.co.jp) solidified MS medium containing 30 g L−1 sucrose. A total of 150 leaf explants were treated with Agrobacterium and cultured in plastic dishes. After 5 days of co-cultivation, the leaf segments were transferred to the same medium with the addition of 300 mg L−1 cefotaxime (Hoechst AG, now part of Sanofi-Aventis, http://www.sanofi-aventis.de) to eliminate Agrobacterium and the additions of 25 mg L−1 Hygromycine and 50 mg L−1 Kanamycine were added for the selection of transgenic cells. The induced calli were subcultured every 2 weeks on the same medium. The proliferated calli grown for 1 week after the second subculture at 25° or 15°C were excised and examined for GFP observation using a UV fluorescence microscope (Nikon, http://www.nikon.com) equipped with B-2A (450- to 490-nm excitation filter) for EGFP and UV-1A (360-nm excitation filter) for DAPI. In order to estimate the nucleus-to-cytoplasm GFP (NCG) ratio, GFP fluorescence intensities were measured by scanning of the microscopic images with imagej v1.43 (http://rsb.info.nih.gov./ij). An average of GFP fluorescence intensities at points with an equal distance within the nucleus or cytoplasm was used to estimate the NCG ratio. In each plot, 50 cells collected from at least three independent transgenic lines were randomly selected from the microscopic images and were analyzed to determine the NCG ratio.
Quantitative transient assay using GAL4 system
The plasmid pGBL has two operons, as shown in Figure 4a: one consists of the firefly luciferase gene (pGL4.12; Promega, http://www.promega.com) with TNOS sequences driven by the six tandem copies of the GAL4 binding site upstream of the TATA box; and the other, which is a reference to standardize differences in the efficiency of the bombardment delivery, carries the 35S promoter plus the Renilla luciferase gene (pRL-null; TOYO B-Net CO., LTD, http://www.toyo-b-net.co.jp/) with TNOS sequences. The segment containing the GAL4 binding sites and TATA box was adopted from pTA7001 (Aoyama et al., 1995) as a PCR segment with 5′-GACGTTGTAAAACGACGGCCAC-3′ and 5′-AGGCCAGCGTGTCCTCTCC-3′. pTA7001 was provided by Dr N.H. Chua (The Rockefeller University, New York, USA). In the two effector plasmids, pGATam3 and pGAGUS, GAL4 DNA-binding domain (amino acid positions 1–74) and activator of VP16 (positions 75–154) were inserted following a viral translation enhancer (Ω) present at the 3′ end of the 35S promoter sequence. The segments of GAL4 BD and activator were also amplified from pTA7001 with 5′-ATGAAGCTACTGTCTTCTATCGAAC-3′ and 5′-CCCACCGTACTCGTCAATTCC-3′. As shown in Figure 4, each of pGATam3 and pGAGUS contains Tam3 TPase gene or GUS gene, respectively, followed by the TNOS sequence.
The introduction of the plasmids into the flower petals (HAM5 line) was performed by the bombardment method. A mixture of pGBL (approximately 2.0 μg) and pGATam3 or pGAGUS (approximately 0.8 μg) was precipitated onto 1.2 mg of gold particles (1.6 μm in diameter) with ethanol, and 0.12 mg of the plasmid DNA-coated gold particles was delivered into intact tissue as described above. The bombarded buds were incubated for 16 h at 25° or 15°C. As the luciferase detection system is very sensitive, the pre-incubation of the bombarded buds at 25°C was not necessary. Firefly luciferase and Renilla luciferase activities were assayed using a Dual-luciferase Assay Kit (Promega), in accordance with the manufacturer’s instructions. Chemical luminescence was measured using a luminometer (Mithras, LB 940; Berthold Technology, http://www.berthold.com).
PEG transformation and microscopy observation
Protoplasts were isolated from young leaves of Antirrhinum plants (line HAM5) and tobacco BY-2 cells. Incubation of the protoplasts in isolation buffer (1.5% Cellulase Onozuka R10, 0.05% Pectolyase Y-23, 0.55 m mannitol) was performed for 3 h at a temperature at 15° or 25°C. For polyethylene glycol (PEG) transformation, purified protoplasts were washed in 0.55 m mannitol, 0.5% 2-(N-morpholine)-ethanesulphonic acid (MES), pH 5.6, and carrier salmon sperm DNA and plasmid DNA were added to 100 μl of protoplast suspension per tube. Subsequently, the same volume of 40% PEG 4000 was added. These protoplasts were washed once and incubated in W5 solution (154 mm NaCl, 125 mm CaCl2, 5 mm KCl and 5 mm glucose, pH 5.8) for 24 h at 25°C with 20 nm LMB until the observation. The protoplasts expressing GFP fusion proteins were observed using an UV fluorescence microscope (Olympus) equipped with the U-MNIBA filter set (470- to 490-nm excitation filter; 505-nm dichroic mirror; 515- to 550-nm barrier filter) for enhanced GFP.
We are especially grateful to Yoshio Sano (Laboratory of Plant Breeding) and Cathie Martin (Department of Cell and Developmental Biology, John Innes Center) for comments on the manuscript, and Yohsuke Takahashi (Graduate School of Science, Hiroshima University) for providing the plasmid pJ4-RSG-GFP. This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) and Grant-in-Aid for Young Scientists from MEXT (no. 02J00614 to S.-N.H).

domain: biology


A great many people complain that they can't transfer ISO file to USB. Why can’t put ISO file on USB drive? How to fix the problem on Windows 10/11? Now, this post from MiniTool Partition Wizard discusses these questions together with you in detail.
As it’s well known to us all, USB flash drives are widely used as removable storage media in our daily work. People use it to burn ISO files, store videos, create bootable installation media, and more. At the same time, lots of issues also appear while using USB drives, such as “USB drive greyed out”, “USB flash drive unable to play videos”, and “can’t burn ISO file to USB”.
Here’s a heatedly discussed topic about “can’t put ISO file on USB drive” from the answers.microsoft.com forum:
Can't put ISO file on USB drive. I downloaded a Win10_20H2_English_x32.iso file and it's 4.4GB. The drive on which I want to store it is a Fujifilm USB is 128GB and formerly a PNY flash drive with 16 GB. But when I put the ISO in both of the drives, I get a message that says that "it's too large for the volume's format. It's only 4.4 GB but it can't fit in a 16 and 32 GB flash drive? This makes no sense at all.https://answers.microsoft.com/en-us/windows/forum/all/cant-put-iso-file-on-usb-drive/b5c64413-63cb-4bf6-8671-78c52ef371a2
Why You Can’t Transfer ISO File to USB
According to user reports, the problem often occurs when putting an ISO file, like Windows 10/11 and Ubuntu into a USB flash drive. Why can’t copy ISO file to USB drives? According to a survey, there are several possible reasons for ISO file not copied to USB:
- The ISO file is too big for the file system of the USB drive: If your USB drive is formatted to FAT32, an ISO file larger than 4GB cannot be copied to the drive due to the FAT32 partition size limit.
- The transferring process is interrupted: If the process of copying is interrupted by a power outage or other factors, the ISO file could be corrupted, and you can’t burn ISO file to USB.
- Insufficient space on the USB drive: The flash drive itself is not big enough to hold the ISO file.
- Lack of a professional ISO burn tool: If you want to create a bootable installation USB using ISO, you can’t burn ISO file to USB without a burning tool like Windows Media Creation Tool or Rufus.
- The ISO file structure is not compatible with the USB: If you want to create a Windows bootable drive using the ISO file, the USB drive must be formatted as FAT32. Otherwise, you can’t copy ISO file to USB.
Now, you should have an overall understanding of the ISO file not copied to USB issue. Let’s keep reading to see how to solve it.
How to Fix It If You Can’t Transfer ISO File to USB on Windows 11/10
How to fix it if you can’t move ISO file to USB on Windows 11/10? Here we summarize 5 simple troubleshooting methods after analyzing extensive user reports from different forums and communities. You can try them in order until the problem gets solved.
# 1. Re-download the ISO File Properly
First of all, you can try re-downloading the correct version of the ISO file from its official website and make sure that the transferring process is not interrupted by any factors. Here we take the Windows 11 ISO file for example.
Step 1. Visit the official Microsoft website and scroll down to the Download Windows 11 Disk Image (ISO) section.
Step 2. Select Windows 11 (multi-edition ISO) > Download, select a language, confirm these options, and tap on 64-bit Download.
Step 3. This process may take you some time to complete. So, wait patiently. Once downloaded, you can copy or burn the ISO file to the USB drive.
# 2. Reformat the USB Drive
A lot of users from the Microsoft forum reported that the “can’t copy ISO to USB” issue can be solved by reformatting the USB to a compatible file system. If the ISO file is larger than 4GB, you need to format the drive to NTFS or exFAT (some people found that the ISO file can be put on an exFAT USB drive). However, if you are creating a Windows bootable media, the USB must be formatted as FAT32.
How to format a USB flash drive on Windows 11/10? You can use the Windows built-in tool – Disk Management, or use a free USB drive formatter – MiniTool Partition Wizard. Choose one according to your situation.
Way 1. Format the USB Using Disk Management:
If your USB flash drive is smaller than 32GB, you can use the Windows tool to format the drive to FAT32/NTFS/exFAT according to your ISO file. Here’s a tutorial for you:
Step 1. Press the Win + R keys to open the Run dialog box, and then type diskmgmt.msc in the box and hit Enter to open the Disk Management window.
Step 2. Right-click the USB drive and select Format.
Step 3. In the pop-up window, select a compatible file system from the drop-down menu and click on OK to confirm the change. Note that if the ISO file is larger than 4GB, you should select NTFS or exFAT.
Way 2. Format the USB Using MiniTool Partition Wizard:
However, the Windows tool has limitations when formatting a USB larger than 32GB to FAT32 or exFAT. Sometimes you may encounter some issues, like “all options are greyed out in Disk Management”. So, we strongly recommend you use a professional exFAT and FAT32 formatter – MiniTool Partition Wizard.
Besides, it is a feature-rich and free partition manager that can extend/resize/move/copy/align partitions, convert FAT32 to NTFS, change serial number, check drive performance, recover lost data/partitions, migrate OS, and more.
Step 1. Launch the MiniTool software to enter its main interface, and then select the USB drive from the disk map and click on Format Partition from the left panel.
Step 2. Select FAT32 or exFAT from the File System drop-down menu according to your ISO file and click on OK. Besides, you can change the cluster size and partition label from here according to your needs.
Step 3. Click on Apply to execute the formatting process.
# 3. Convert the File System of USB to NTFS
Some users from the techguy community reported that the “can’t transfer ISO file to USB” issue can be solved by converting FAT32 to NTFS. This is because FAT32 doesn’t support a single file larger than 4GB. How to convert FAT32 to NTFS without data loss? MiniTool Partition Wizard can help you do that easily. For that:
Step 1. In the main interface, select the USB from the disk map and click on Convert FAT to NTFS from the left action panel. Also, you can right-click the USB and select the conversion.
Step 2. Click on Start and wait for the process to finish. Once done, click on Close, and you should find the USB partition has been converted to NTFS without data loss.
# 4. Burn the ISO File Using a Professional Tool
If you can’t copy ISO to USB directly, you can try using a professional ISO burning tool like Rufus. It is an open-source program that can help you create a bootable USB drive by burning the ISO file into the drive. Here’s how to use the tool:
Step 1. Download the Windows ISO file from Microsoft properly as we showed in the first solution.
Step 2. Click here to get Rufus on your PC and run the program to enter its main interface. It will automatically detect the USB flash drive.
Step 3. Click on the Select button under the Boot selection tab, and then select the Windows ISO file from its directory.
Step 4. Then you can specify other parameters, including Partition scheme, Target system, File system, and Cluster size according to your needs.
Step 5. Click on the START button to begin burning the ISO file into USB. When the process is complete, click Close.
# 5. Check the USB Drive for Errors
If you still can’t move ISO file to USB after trying all the above methods, it may indicate that the flash drive has been damaged. In this case, you should check if there are bad sectors or file system corruption on the USB. Here’s how to check them using CHKDSK.
Step 1. Type cmd in the search box, and then right-click Command Prompt and select the Run as administrator option. Then click on Yes to further confirm it.
Step 2. Type the following command and press Enter. Here you need to replace L with the drive letter of your USB. Once repaired, reconnect the USB and see if it can copy the ISO smoothly.
chkdsk L:/f /r
Sometimes, the CHKDSK tool may get stuck into different errors, such as CHKDSK write protected, CHKDSK taking forever, etc. Under these situations, MiniTool Partition Wizard is a better alternative. It cannot only check bad sections but also repair file system corruption on Windows easily.
I can’t transfer ISO file to USB when creating a Windows 11 bootable media. Fortunately, MiniTool Partition Wizard helped fix the problem by converting FAT32 to NTFS. You may also need this tool.Click to Tweet
What’s Your Opinion
Here comes the end of this post. It provides 5 effective ways to fix the “can’t transfer ISO file to USB” issue on Windows 10/11. Have you any other opinions about the problem? We are glad to see them in the following comment area.
In addition, you can contact us via [email protected] if you have any questions about MiniTool Partition Wizard. We will reply to you as soon as possible.


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domain: psychology