Anker's New Alexa Speaker Is Like an Echo Dot With Better Audio - Get It For $40

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If Anker’s Eufy Genie is basically an Echo Dot, but cheaper, then Anker’s new ZOLO Halo is like an Echo Dot, but upgraded. Oh, and cheaper too.

Joining a few true wireless earbuds under Anker’s new ZOLO smart audio umbrella, the Halo is a 5W speaker with microphones and Alexa built right in. It won’t be as loud or clear as a full-sized Echo with its 15W speakers, but it’ll be a huge upgrade from the likes of the Dot or the Genie, which boasts only a 2W speaker.

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We expect the Halo to typically retail for $60, but to celebrate its launch, Anker’s offering it for $40 right now, or $10 cheaper than the typical price of the Echo Dot.




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What Happens When Doctors Only Take Cash (2017)

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When Art Villa found out, after one too many boating accidents, that he needed a total knee replacement, he began asking around to see how much it would cost. The hospital near his home in Helena, Mont., would charge $40,000 for the procedure, he says. But that didn't include the anesthesiologist's fee, physical therapy or a stay at a rehabilitation center afterward. A 2015 Blue Cross Blue Shield study found that one hospital in Dallas billed $16,772 for a knee replacement while another in the same area charged $61,585.

It was in the midst of this confounding research that Villa, who's 68, heard about the Surgery Center of Oklahoma, whose business model is different from that of most hospitals. There, the all-inclusive price for every operation is listed on the website. A rotator-cuff repair for the shoulder costs $8,260. A surgical procedure for carpal tunnel syndrome is $2,750. Setting and casting a basic broken leg: $1,925.

The catch is that the whole facility is cash-based. It doesn't take insurance of any kind. Not Aetna. Not Cigna. Not Medicare or Medicaid. Patients or their employers pay whatever price is listed online, period. There are no negotiated rates, no third-party reimbursements and almost no paperwork. "We say, 'Here's the price. Here's what you're getting. Here's your bill,'" says Keith Smith, who co-founded the Surgery Center in 1997 with fellow anesthesiologist Steven Lantier. "It's as simple as that."

To Villa, the model seemed refreshingly subversive. The Surgery Center would charge $19,000 for his whole-knee replacement, a discount of nearly 50% on what Villa expected to be charged at his local hospital. And that price would include everything from airfare to the organization's only facility, in Oklahoma City, to medications and physical therapy. If unforeseen complications arose during or after the procedure, the Surgery Center would cover those costs. Villa wouldn't see another bill.

Sometimes called direct pay, and closely related to concierge care, this sort of business model was once seen as the perquisite of rich folks and medical tourists from foreign lands. But nowadays many of the people seeking cash-based care are middle-class Americans with high-deductible insurance plans. For a patient with an $11,000 family deductible, for example, it might make more sense to seek out a cash-based center like the Premier Medical Imaging facility in Minneapolis, which offers a basic MRI for $499, than to cough up the several thousand dollars that the same procedure generally costs at a traditional hospital. Cash payments don't count toward a patient's deductible, but for some it's worth the gamble.

Self-insured companies, like the trucking and storage firm where Villa is the chief administrative officer, are also fueling the trend. Because such companies pay their employees' medical bills out of their operating budget, it's in their interest to steer everybody to the cheapest option. Villa, for example, says his decision to go to the Surgery Center saved his company money, since his $19,000 bill is less than it would have been charged, even with a negotiated discount, by a traditional hospital. The Oklahoma state public employees' insurance fund, which covers 183,000 people, recently did similar math. In 2015 it announced a new rule: If patients go to a traditional hospital, they pay their deductible and co-payment. If they go to a cash-based provider that meets the fund's criteria, including the Surgery Center of Oklahoma, they pay nothing at all.

While no organization keeps track of how many cash-based medical centers have cropped up nationwide in recent years, Smith and Lantier say they've witnessed an explosion. In Oklahoma City alone there are roughly three dozen centers that are all or partly cash based, specializing in everything from radiology to oncology. Texas has two dozen such facilities, and in Torrance, Calif., the Ocean Surgery Center posts many of its prices online. Thousands of cash-based primary-care practices have also sprung up across the country.

This trend may accelerate nationally. With the Affordable Care Act on the chopping block, many experts expect the free-market model to take off. While congressional Republicans have yet to produce a viable replacement for the Obama Administration's health care law, almost any change is likely to result in more Americans' choosing high-deductible insurance plans, which would help fuel the cash-based marketplace for years to come.

A few days after Villa's knee-replacement operation was completed on Jan. 17, his daughter captured a video of the happy patient, in headphones, "boogying down the hallway," as he put it, of the Marriott Residence Inn in Oklahoma City, where he stayed for a week and a half to recover. After the surgery a physical therapist and a nurse visited Villa in his hotel room, bearing gifts: an ice machine, pain medication, a thermometer and detailed, hands-on instructions for his recovery, all of which were included in his original bill. "I've really never experienced this quality of care," Villa says.

There is good reason to think Villa's experience could be the shape of things to come. Since taking office, President Donald Trump has signed an Executive Order instructing the Department of Health and Human Services to begin weakening Obamacare, while standing by his previous promise that any replacement plan will allow Americans with pre-existing conditions to access affordable insurance.

To meet these goals, Republican leaders have been targeting a series of reforms that President Obama opposed. They will likely allow insurers to sell across state lines, resulting in the sale of more plans with limited coverage of basic health care or prescription drugs. They will also likely scrap the prohibition on bare-bones, high-deductible "catastrophic" plans and eliminate deductible limits entirely. If the final draft includes all or any combination of those provisions, the result will be many more Americans' signing up for low-premium, high-deductible plans--precisely the type of insurance that has driven the rise of cash-based medicine over the past six years.

But even without a new Republican system, cash-based care has been growing under Obamacare, which required insurers to provide more-comprehensive coverage and to offer plans to anyone who wanted one. Insurers made up for having to cover a more expensive patient population by getting customers to contribute more out of pocket with higher deductibles, co-payments and co-insurance. While Obamacare imposed limits on how high deductibles could be--$7,150 for an individual and $14,300 for a family--the out-of-pocket contributions rose for many Americans, turning more patients into price hounds. If you're paying cash for that mole removal anyway, why not find the cheapest dermatologist in town? The Surgery Center of Oklahoma, among the first in the country to post its prices online in 2008, saw an uptick in business after Obamacare. "I guess it's ironic that Obamacare created this market for us," Smith says, with a laugh.

In the health care world, the Surgery Center of Oklahoma's business model is considered radical, in part because the industry, as it's structured now, doesn't lend itself to price transparency. Providers charge different insurance companies different amounts for the same procedures, and in many cases, insurers' contracts explicitly bar hospitals from publicly disclosing their reimbursement rates. That many regions of the U.S. are now dominated by one hospital chain also creates a monopoly problem: if an insurer wants to offer plans in that area, it's got to accept the hospital's rates. Some providers say it's not possible to set prices in the first place, since medical procedures aren't normal consumer products.

In arriving at their price list, Smith and Lantier did an end run around the whole system. They asked their fellow doctors how much compensation was expected per procedure, factored in necessary expenses like surgical equipment and medical implants, then tacked on a 10% to 15% profit margin. Since their surgery center does not employ the army of administrators that is often required to haggle with insurers and follow up on Medicare reimbursements, their overhead is smaller. The whole operation is 41 people. "Finding an average price doesn't require complicated math," Smith says. "It's arithmetic." Since posting the price list eight years ago, they've adjusted it twice, both times to lower rates.

One problem with a free-market, cash-based system of health care is that it promises to work really well for people like Villa, whose companies stand to save money by avoiding traditional hospitals, but less well for others. Without safeguards, it threatens to marginalize the poorest and sickest among us, who could not possibly afford, say, a $19,000 knee replacement without help from an employer, the government or a charity. While Americans tend to accept certain inequities as a reality of capitalism--there are always going to be people who succeed and those who fall behind--we are less comfortable with them when it comes to health care. We don't like the idea of families going bankrupt after a cancer diagnosis or losing coverage after a parent loses a job. In poll after poll Americans of both political parties say they support provisions ensuring that people with pre-existing conditions can access health care.

Twila Brase, president of the conservative Citizens' Council for Health Freedom and one of the most energetic advocates of free-market-based health care, acknowledges the problem. For a direct-pay system to work, she says, providers must be willing to give away care. "Charity has always been part of the medical practice," she says. (Smith and Lantier say they perform charitable operations, although to avoid being inundated with requests, they don't report details.) Another way to make a free-market-based system work is to increase government safeguards: expand Medicare and Medicaid, compel states to create "high-risk" pools to underwrite coverage for those with pre-existing conditions, or require insurance companies to cover everyone. Which explains in part why replacing Obamacare is so vexing for the GOP: all that sounds a lot like Obamacare.

Villa, meanwhile, remains a convert. When he returns to work, he says, he's considering helping his company create financial incentives to steer employees to the Surgery Center of Oklahoma. "Even with airfare and hotel stays," he says, "the savings could be huge."



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Townsquare Media - An Ignored Value Play

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Background:

Townsquare Media (NYSE:TSQ) operates in two segments - Local marketing solutions (radio) and live events. It owns and operates 317 radio stations in the US as of last 10-Q, which generate around 65% of the company's revenue YTD. What makes TSQ unique is that it focuses on small and mid-size markets for its radio business where it can operate with less competition. Its largest market is Albany with a population of roughly 500k, but most of its stations operate in markets with populations in the 20-50k range. Their radio station business is highly cash generative and has yielded consistent and steady growth (up 3.5% YTD ex-political).

Why does this opportunity exist?

Townsquare Media's heavy debt load steers away many risk-averse investors. With a market cap of 214 million (assuming diluted shares), the company has long-term debt of 631 million, including operating leases. Though management has spoken about reducing its debt load in the past, it has been slow to deliver.

Live events business has struggled YTD with revenue down 7 percent and will most likely end up struggling in 4th quarter as well due to customer fears after the Las Vegas tragedy. On top of this, Congress's actions restricting H-2b visa program hindered margins for live events business, as TSQ is reliant on foreign workers for some of their operations. This resulted in management having to find some shorter term solutions so they could staff their events.

So what's the good news?

The risks of heavy debt load may be somewhat overblown. TSQ is highly cash generative and pays a reasonable weighted average interest rate of 5.4% on its debt. Moreover, the company is targeting a net leverage position of around 4.0x Adjusted EBITDA, which is more than achievable should EBITDA rise back to its previous levels and beyond in 2018 and 2019. The slow paydown of the debt load is very much due to management finding lucrative acquisition opportunities in the past, which it seems it is easing up on for the moment.

The company is cheap - subtracting capex, rent expense, cash interest, and cash taxes from Adjusted EBITDA results in a trailing FCFE yield of ~20% at current levels and though Congress's visa actions may remain in place, much of the margin decline was due to the company having to find shorter term solutions to the new visa restrictions. With more time to prepare, we can expect the company to boast slightly higher margins than before, but not at the same level as prior to the H-2b visa restrictions.

In recent calls, management has discussed possible divestitures from the live events business. Contrary to what seems to be popular opinion, I believe this is a positive step for the company. While radio stations are steady cash generating machines, the lower margin live events business is seasonal and can be adversely affected by things like weather and changes in customer preferences and trends. The company has even gone as far as to name two Co-CEOs (one in charge of each segment), thus increasing the gap between the live events and radio business even further. However, seeing as TSQ runs over 550 live events, they will most likely be strategic about which ones to divest from. I highly suggest listening to the last earnings call, as it even looks like some analysts and funds are waiting for more details on these divestitures before taking a position.

According to the last earnings call, management may also start paying dividends in the back half of 2018. Though this makes little difference to me, many investors simply expect dividends from companies operating in mature industries like radio. However, this will be dependent on the company meeting its leverage target, which, with strategic divestitures, I believe is very likely.

Valuation

Note: All references to EV or Adj. EBITDA assume the capitalization of operating leases and the addition of rent expense to EBITDA. Also, all ratios and valuation assume diluted shares of 28.221 million.

The company currently trades at an EV/Adj. EBITDA multiple of 6.1. This is hardly justified for a highly cash generative company. Though I believe this is because of the riskiness of their live events business as well as their debt load. Assuming TSQ ends 2017 with a 1% decline in revenue growth and 21% Adj. EBITDA Margins (which is well below the average of around 25%), I believe the company will end 2017 with Adjusted EBITDA of around 107 million (adding back rent expense) and economic FCFE of around 26 million. This leads to a FCFE yield of around 12% for 2017. Using an 8x EBITDA multiple, we get a valuation of $10.5 per share - 41% upside from current levels. TSQ has significant NOLs, so free cash flow may be a better valuation method.

However, I believe the real magic happens in 2018/2019 with either the divestiture or turn-around some of the company's live events. I believe Adjusted EBITDA margins can get back up to at least 23% even without many divestitures and zero sales growth. This can lead to Adjusted EBITDA of over 117 million in 2018-2019 along with 35 million in FCFE (forward yield of 16%). If we assume a very modest reduction of net debt to 530 million (vs. 578 million, including operating leases) and an 8x EBITDA multiple gives us a share value of $14.20 - a 91% upside.

Be aware that due to its high leverage, the share price is very sensitive to the EBITDA multiple chosen. Choosing a 7.5x multiple leads to a $12.20 valuation. However, I still believe that there is a significant margin of safety at these levels.

Catalysts

  • Smarter capital allocation, dividend payments and divestitures of underperforming live events.
  • Turnaround of live events business
  • Debt paid down to more manageable level

Risks

  • Further delay in debt pay down will also delay the initiation dividend payments
  • Further short-term deterioration of live events attendance due to gun violence and/or terrorism
  • Stock is volatile and low volume so it's easily knocked down by small sales

Thank you for reading this Seeking Alpha PRO article. PRO members received early access to this article and get exclusive access to Seeking Alpha's best ideas. Sign up or learn more about PRO here.

Disclosure: I am/we are long TSQ.

I wrote this article myself, and it expresses my own opinions. I am not receiving compensation for it (other than from Seeking Alpha). I have no business relationship with any company whose stock is mentioned in this article.

SeekingAlpha


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Seattle's Minimum Wage Killed the 'Five-Dollar Footlong'

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  • Fist of Etiquette|1.10.18 @ 9:33AM|

    Hey, at least Subway didn't go to a touchscreen menu system.

  • Fist of Etiquette|1.10.18 @ 9:33AM|

  • A Cynic's Guide to Zen|1.10.18 @ 9:49AM|

  • kevino|1.10.18 @ 12:38PM|

    Economics: it's only a matter of time before businesses look to cut costs.
    The true minimum wage is zero.

  • Qualitarian|1.10.18 @ 2:52PM|

    Already been in place in Perryville, MO franchise since 2016. At the drive-thru.

  • I am the 0.000000013%|1.10.18 @ 4:00PM|

    There pretty common around here. I think the slogan at participating franchises is "Come touch yourself a good six incher".

  • TrickyVic (old school)|1.10.18 @ 5:29PM|

    Carlos Danger has a franchise?

  • Paper Wasp|1.10.18 @ 6:43PM|

    "We call this sandwich the Harvey Weinstein."

  • The_Hoser|1.10.18 @ 10:46PM|

    Because you think you're getting a role, but instead you get the meat.

  • albo|1.10.18 @ 10:08AM|

    Convenience store food counters in my area (Sheetz, Rutter's) do this already and it's so easy and makes so much sense. It's coming to fast food sooner than later.

  • Brandybuck|1.10.18 @ 10:59AM|

    it's always made sense once the techology was available. The problem was that unskilled workers were still cheaper than the system plus its maintenance and social costs. But drive up minimum wage past that point and now it's cheaper to dump the workers.

  • NoVaNick|1.10.18 @ 12:10PM|

    Sheetz has been doing this for 10 years at least-though it didn't work too well the time I tried it.

  • crufus|1.10.18 @ 10:29AM|

    Wawa (in PA) already uses touchscreens for food orders with a much larger menu of custom sandwiches.

  • kevino|1.10.18 @ 12:42PM|

    Wawa's system is fantastic: many other convenience stores should copy what they do. They provide fresh sandwiches with terrific ingredients at a low price, and their ordering system is great. As it guides the user through a long list of choices, it helps the user. Anyone except my blind mother can do it. And it has options to add double mustard, extra pickle, or applewood bacon.

    Anyone in the sandwich business needs to go to Wawa and look at their system. They are best in class.

  • Loss of Reason|1.11.18 @ 11:21AM|

    Buckee's here in Texas uses a Touchscreen too. Anything from cold and hot sandwiches to bakery and fudge.

  • Citizen X - #6|1.10.18 @ 10:55AM|

    Touchscreens and mysteriously, disturbingly wet sandwich meats do not mix well.

  • operagost|1.10.18 @ 11:20AM|

    Don't know what you're talking about, dude. The touchscreens are for the customers to use.

  • Citizen X - #6|1.10.18 @ 1:16PM|

  • TrickyVic (old school)|1.10.18 @ 5:30PM|

    Wouldn't that would be called a fondlescreen.

  • IceTrey|1.10.18 @ 3:14PM|

    Just bought Subway with drive through touchscreen.

  • GeneralWeygand|1.11.18 @ 5:52PM|

  • GeneralWeygand|1.11.18 @ 5:52PM|

  • mylene|1.11.18 @ 2:03AM|

    You can earn more than $15,000 each month from you home, and most special thing is much interesting that the job is to just check some websites and nothing else. Enjoy full time and money freedome, also an awesome career in you life....
    just click the link given belowHERE..... http://ift.tt/2DlClTU

  • Diane Reynolds (Paul.)|1.10.18 @ 9:39AM|

    Five-dollar footlong is my nickname... right now.

  • BestUsedCarSales|1.10.18 @ 12:26PM|

  • I am the 0.000000013%|1.11.18 @ 9:27AM|

  • Domestic Dissident|1.10.18 @ 9:44AM|

    The true minimum wage is always zero dollars.

    But I wouldn't eat one of Subway's shitty-ass sandwiches even if it was just one dollar. The last time I made the mistake of going there it tasted like I was eating a rubber mattress.

  • Incomprehensible Bitching|1.10.18 @ 9:49AM|

    That's what welfare is for: if you can't make enough money to keep people from feeling sorry for you, then you get to make other people's money. That's social justice.

  • albo|1.10.18 @ 10:09AM|

    Subway's bread is fluff and its cold cuts have had all the flavor removed. It's beige food.

  • operagost|1.10.18 @ 11:21AM|

    That's why I only get the sweet onion teriyaki chicken.

  • CE|1.10.18 @ 4:23PM|

    And the meat is portioned out one molecule-thick layer at a time, but at least they give you a whole 3 molecule-thin slices to cover all the fluffy bread.

  • Paper Wasp|1.10.18 @ 6:46PM|

    Jersey Mike's and Jimmy John's are way better. No reason to go to Subway.

  • Radioactive|1.10.18 @ 11:07AM|

    did you pay extra for the rubber?

  • |1.10.18 @ 11:58AM|

    You ate a rubber mattress?

  • Citizen X - #6|1.10.18 @ 2:10PM|

  • IceTrey|1.10.18 @ 3:17PM|

  • Rhywun|1.10.18 @ 9:56AM|

    "unintended consequences"

    Doubtful. The people who put these policies in place know exactly what the consequences are and they don't care. Hell, they cheer them on. Anything that rewards friends and punishes enemies is progress.

  • Radioactive|1.10.18 @ 11:09AM|

    all a ploy to make Seattle a subway free zone...

  • BestUsedCarSales|1.10.18 @ 12:26PM|

  • My Dog Bites Better Than Yours|1.10.18 @ 3:05PM|

    A predictable outcome from an action is not "unintended". It is possible bad outweighs the good, but that is called a "trade off".

  • Fuck you, Shikha (Nunya)|1.10.18 @ 11:38PM|

    And it is possible they don't understand the irony of actually causing or worsening the very problem they sought to resolve. That the answer in their mind can never be answered as just let it work itself out is the real outcome.

    I mean, I always appreciate someone telling telling me that only they know what is best not only just me but everyone. To that point, I actually use sugary drinks to help maintain weight. When I stop drinking them I drop ten pounds to 150lbs at 5'11". And I'm over 40. But at least I'll have the privilege of spending like I'm going to GNC for weight gainer while only buying Dr Pepper. (And before you give me shit for those drinks, at least they didn't give me esophagitis like weight gainer did.)

  • Curt|1.10.18 @ 10:00AM|

    What part of this do these greedy corporate assholes not understand? They need to embrace these changes as a reduction to their profit instead of trying to push them off on the little guy. They already make more money than they need. They should appreciate the opportunity to improve people's lives instead of increasing costs on normal people while they fly their corporate jets.

  • Jerryskids|1.10.18 @ 10:03AM|

  • Sevo|1.10.18 @ 10:14AM|

    Curt|1.10.18 @ 10:00AM|#
    "...They need to embrace these changes as a reduction to their profit instead of trying to push them off on the little guy...."

    I have spoken with people who imagine businesses will do this, or if not, should be forced to do this. Those sorts of people exist, sadly.

  • loveconstitution1789|1.10.18 @ 12:41PM|

    When someone says something like that, it becomes immediately apparent that that person has zero idea how business works or economics.

    I say zero I mean absolutely no idea.

    It makes it easy to sort those people out though. On the down side, they tend to go into government jobs.

  • TrickyVic (old school)|1.10.18 @ 5:33PM|

    ""I say zero I mean absolutely no idea.""

    Yep, they are also the ones deeply in debt because they don't understand how credit works.

  • Galane|1.10.18 @ 6:20PM|

    Oh they know how it works. They just think that if they believe in it *really hard* that an economic system can have one part (such as wages or taxes) fiddled with, without having any effect on the rest of the system.

    They're the ones that claim "trickle down" is utter bollocks. The effects don't just go down, they up, sideways and in every other direction.

  • Fuck you, Shikha (Nunya)|1.10.18 @ 11:41PM|

    Yeah, that's always the funny part. The magic hand apparently only works through more regulation and more taxation. Apparently the hand must first be shackled.

  • Elias Fakaname|1.11.18 @ 1:30AM|

    And everyone acts repulsed when I say how we're going to have to get rid of the progs at some point. They will never ever stop, unless they are stopped. Complaining and handwringing are not solutions to the progressive problem.

  • Nuwanda|1.10.18 @ 5:02PM|

    "I have spoken with people who imagine businesses will do this, or if not, should be forced to do this."

    That's the hidden agenda, of course. Lots of useful idiots holding up signs demanding these things, but the true statists know the endgame is to control how business sets prices and realises profits. Eventually they will demand oversight of these aspects.

  • Paloma|1.10.18 @ 7:16PM|

    How much do you think the greedy corporate assholes need, Curt?

  • cc2|1.10.18 @ 8:29PM|

    In many cases, the owner of a couple of sandwich shops is not some big company but a guy or family and the "profit" is his salary. If he can't make enough money off the shops (and he has lots of cash tied up in them) he will close them and do something else. It is that simple.

  • Loss of Reason|1.11.18 @ 11:24AM|

    People don't understand at least in cases like these that Subway doesn't own the restaurant, a franchise owner does. They operator on tight budgets and small small profits. Most (I'm sure some excepts) aren't living high on the hog.

    We are from the government and we are here to help. Worse sentence ever!

  • Jordan|1.10.18 @ 10:35AM|

    Um, you realize that Subway shops are franchises, right?

    According to a report on food franchising by Franchise Business Review, 51.5 percent of food franchises earn profits of less than $50,000 a year; roughly 7 percent top $250,000, with the average profit for all restaurants coming in at $82,033. That doesn't sound too bad, until you factor in the initial investment.
  • Curt|1.10.18 @ 10:45AM|

    Profits of $50,000 a year is a lot more than the cook making minimum wage gets. So the franchisee should be excited about the opportunity to share that with his co-workers. But, yes, I agree that evil corporations like Subway should definitely accept less profit through lower franchise fees so that they can boost opportunities for the people who actually do real work.

    Initial investment... It's a write-off for them. They just write it off. Jerry, all these big companies, they write off everything.

  • Brandybuck|1.10.18 @ 11:18AM|

    For the owner of a single Subway franchise, that $50,000 profit their ENTIRE FUCKING INCOME!

    It's like progressives have never worked in an actual business before. Back in college I once worked in a shop where my part time minimum wage paycheck brought home more dollars than the flipping owner took home. It's not unusual. I've had bosses go slowly bankrupt out from underneath me. But to progressives all employers are evil fat cats. Fuck progressives. They couldn't manage a lemonade stand if their lives depended on it.

  • Ron|1.10.18 @ 12:31PM|

    Many employers make less than their employees, I know a few years mIne made more than I did so now I no longer have employees

  • Gaear Grimsrud|1.10.18 @ 4:21PM|

    Been down that same road myself.

  • Sevo|1.10.18 @ 3:49PM|

    "For the owner of a single Subway franchise, that $50,000 profit their ENTIRE FUCKING INCOME!"

    Pretty sure "Curt" is a troll. Stupidity of that level is rare indeed.

  • My Dog Bites Better Than Yours|1.10.18 @ 4:28PM|

    I took Curt's comments as sarcasm.

  • Curt|1.10.18 @ 4:30PM|

    It certainly wasn't intended to be trolling. It was parody. The troll intends to make people angry and provoke responses like most of the ones it got. Parody is intended to make people laugh at the obvious stupidity of the comment.

    When you said, "those sorts of people exist, sadly," I figured that you saw it for parody. Jerryskids, DesigNate, and MJGreen also got it. The others... not so much.

    I thought it was sufficiently ridiculous to be obvious. Certainly wouldn't pass the ideological Turing test. Then again, I guess lots of people can't see the difference between their opponents and their strawmen.

  • Nuwanda|1.10.18 @ 5:03PM|

    "I thought it was sufficiently ridiculous to be obvious."

    It was.

  • Nuwanda|1.10.18 @ 5:08PM|

    "I thought it was sufficiently ridiculous to be obvious."

    It was BUT when we live in a world where it's regarded as sane to promote bathroom policies that allow men dressed as women to use the same facilities as young girls, well, the irony, satire or sarcasm can never be assumed.

    It is however a fun game to play with Leftists since they tend to agree with enthusiasm.

  • The_Hoser|1.10.18 @ 5:46PM|

    Agreed.

    Also, after you said "in a world," I read the rest of your comment in the voice of the movie trailer guy.

  • Brandybuck|1.10.18 @ 5:19PM|

    ""I thought it was sufficiently ridiculous to be obvious.""

    We now live in an age where such stuff is not obvious. No matter how ridiculous you make it sound, there will be a progressive out there somewhere who would utter it.

  • Paloma|1.10.18 @ 7:22PM|

    I've heard people make similar statements. In person, you can tell sarcasm by the tone of voice. On the internet not so much.

  • Ragoftag|1.11.18 @ 12:52AM|

    You might start ending with either sarc/ or /sarc, to make sure folks catch your drift. Too many Leftists troll these boards and leave idiot remarks.

  • Loss of Reason|1.11.18 @ 11:28AM|

    Haha, it's sad that I couldn't tell anymore. I was hoping it was sarc but it's so hard to tell anymore!

    To many people like Tony these days. Damn it, I was promised a zombie apocalypse to thin this people other.

  • Finrod|1.10.18 @ 11:27AM|

    Wow, someone's challenging Tony for Most Idiotic Poster.

  • Rhywun|1.10.18 @ 11:31AM|

    I suspect another parody account. Yay.

  • Unlabelable MJGreen|1.10.18 @ 11:37AM|

    I guess the Seinfeld quote wasn't blatant enough.

  • Curt|1.10.18 @ 2:51PM|

    Yeah, I thought that including the Seinfeld quote in the second comment would really put it over the top. And the funny thing was that everyone started responding after DesigNate suggested that the first person needed to check his sarc-o-meter.

  • |1.10.18 @ 12:00PM|

    Are this stupid, daft and ignorant?

    Go take a business class. Learn about something and layoff the lefty playbook.

    Jesus I can take people being uninformed but this is getting ridiculous.

  • DarrenM|1.10.18 @ 12:20PM|

    Ignorance is freedom. You can say any dumb crap and think you're clever for doing so.

  • Paper Wasp|1.10.18 @ 6:56PM|

    So the franchisee should be excited about the opportunity to share that with his co-workers.

    ROFLcopter. Because the franchisee doesn't have rent and bills to pay of his own? Because the franchisee is running a charity that throws unearned money at people who couldn't be bothered to take advantage of their free public education to improve themselves?

    Yeah, um...no. Capitalism works. Pro tip: it works really well for people who get off their dead asses, go to class when they're given the opportunity, apply for scholarships, and make goals and work towards them. Try it sometime.

  • Paloma|1.10.18 @ 7:19PM|

    loveconstitution is right. Curt really DOES have zero idea.

  • Elias Fakaname|1.11.18 @ 1:32AM|

    Curt, this is some kind of performance art, right? You're not really dumber than Tony, are you?

  • DesigNate|1.10.18 @ 10:50AM|

  • Radioactive|1.10.18 @ 11:11AM|

    how the fuck do you know how much money people need? fucking asshole

  • Bob Straub|1.10.18 @ 1:05PM|

    Curt, please define "need." Then please review your personal financial status *objectively* and decide whether you earn more or less than what you *need*, as opposed to what you *want*. If you earn more than you need, I guess you wouldn't mind if your governments taxed the rest of your income away. That's essentially what you want to happen to businesses.
    Or did I just swallow a trolled hook?

  • Telcontar the Wanderer|1.10.18 @ 8:58PM|

    Everyone who didn't immediately realize that Curt's comment was a parody is an idiot.

    Every. Single. One.

  • Tionico|1.10.18 @ 9:33PM|

    any idea the margins for a franchise like Subway? If so, come out with it together with your source of information. Any idea the actual COSTS involved in opening and running a Subway franchise?

  • Elias Fakaname|1.11.18 @ 1:34AM|

    I've financed enough restaurants, which involves seeing their books, to know I will never fucking open or invest in one.

  • albo|1.10.18 @ 10:07AM|

    These horrid small business people are ruining Seattle's noble effort to create a workplace progressive utopia. They should be called out and be forced to attend re-education camps.

  • |1.10.18 @ 12:01PM|

    Or in this case, force him to sell the foot-long.

  • BestUsedCarSales|1.10.18 @ 12:33PM|

    No, Seattle is spearhead of the anti prostitution movement as well

  • |1.10.18 @ 1:14PM|

  • shawn_dude|1.11.18 @ 7:16PM|

    Or, instead of taxing residents to subsidize Subway's under-paid employees, they could just tax the employers to fund food stamps, welfare, and medicaid.

    Walmart is the same way. Tax payers have to make up the difference between what Walmart pays full time employees and what it costs for them to live (and poorly at that.)

  • Unlabelable MJGreen|1.10.18 @ 10:12AM|

    Seattle could subsidize trips to the farmers market... and roll back "white-privileged, institutional racism."

    lol

  • Zeb|1.10.18 @ 12:16PM|

    Fight racism by subsidizing stuff white people like. Seems legit.

  • Crusty Juggler|1.10.18 @ 1:05PM|

    I shouldn't have to pay for Netflix.

  • Eric Bana|1.10.18 @ 1:52PM|

    Netflix is a human right.

  • The_Hoser|1.10.18 @ 5:48PM|

    If they keep subsidizing the farmer's markets, they'll NEVER get the white people to leave.

  • zazoo|1.11.18 @ 5:27PM|

    And no white bread at Subway either, only brown.

  • |1.10.18 @ 10:17AM|

    Racism has yet to disappear in Seattle

    Citation needed: Seattle is woke, yo.

  • BYODB|1.10.18 @ 12:06PM|

    Insanely high minimum wages directly harm minorities on purpose? Am I doing it right?

  • shawn_dude|1.11.18 @ 7:17PM|

    "Insanely high minimum wages" -- citation needed. Even at $15/hour, minimum wages are dramatically lower (when adjusted for inflation) than they were decades ago.

  • GroundTruth|1.10.18 @ 10:26AM|

    The net outcome: In 2016, the "higher" minimum wage actually lowered low-wage workers' earnings by an average of $125 a month.

    That can't be true. I just won't believe it. (Comment made by some random economics-denier).

  • Radioactive|1.10.18 @ 11:09AM|

    MATHS>FEELZ...just sayin.

  • Ron|1.10.18 @ 12:33PM|

    Higher wages means higher taxes which means lower take home pay and thats before your hours are reduced

  • MarkLastname|1.10.18 @ 5:11PM|

    Tony would just accuse the economists who found minimum wage increases to be causing disemployment of fucking their cousins.

  • Paloma|1.10.18 @ 7:32PM|

    That's what happens when you get fired because your employer doesn't want to pay you a living wage. You go home and fuck your cousin.

  • Elias Fakaname|1.11.18 @ 1:36AM|

    Tony should go drink some Drano. Best thing for hm really, his progtardedness is going nowhere.

  • shawn_dude|1.11.18 @ 7:23PM|

    Good. You shouldn't believe it.

    The UW study contradicts other studies along the same lines. It has a sample issue as well. It may be right, but so far it's in the minority when it comes to showing losses. Other studies done at UW show no increase in food costs as a result of the 2016 minimum wage increase. So even UW studies aren't self-consistent.

  • VinniUSMC|1.10.18 @ 10:37AM|

    "Sadly, the consequences of high minimum wages, excessive taxation, and mandate-happy public policy are not limited to the death of cheap sandwiches. The cost of doing business in Seattle is higher than the Space Needle, and the unintended consequences of those policies are piling up too.

    ....

    the number of hours worked in low-wage jobs has declined by around 9 percent since the start of 2016 "while hourly wages in such jobs increased by around 3 percent." The net outcome: In 2016, the "higher" minimum wage actually lowered low-wage workers' earnings by an average of $125 a month."

    I wonder, who would've ever guessed this? Surely nobody was smart enough to predict this outcome... Totally.

  • Radioactive|1.10.18 @ 11:08AM|

    someone with the powers of the MATHS and who wasn't afraid to use demm...

  • Finrod|1.10.18 @ 11:24AM|

    Didn't you know? Meritocracy in mathematics is a 'tool of whiteness', according to some idiot that can't count:

    http://ift.tt/2CTlD0v

  • TrickyVic (old school)|1.10.18 @ 5:40PM|

    So is that teacher saying people of color are such poor performers that they can't compete in a world that looks at quality of performance?

  • RPGuy16|1.10.18 @ 11:47AM|

    It doesn't feel true, so it must be false.

  • Brandybuck|1.10.18 @ 11:11AM|

    Damn, progressives are fucking ignorant as to basic first semester economics. It's like they actually believe in the tooth fairy or something. They think that just because they have a placard then magical things will happen.

    There's a tax on soda, but no tax on equally unhealthy coffee drinks. Guess who drinks more soda than fatty sugary coffee drinks? The poor! This is a tax on the poor! It's not even a proper pigouvian tax to "nudge" people into healthier diets, otherwise they would have taxed the hipster drinks too! The sole point of this tax is that the Seattle elite don't like the poor drinking corporate sodas.

    And then they think if the impose a price floor on labor there won't be a labor surplus (ei. unemployment). Fucktards! it doesn't mean businesses will go and lay everyone off, but it does mean they're going to reduce the labor they use. They'll let attrition get rid of the excess, reduce new hires, install automation, etc. They drop sales and raise prices to make up for it.

    Progressives are stupid because they think their good feelz are sufficient for their magical hand waving to result in their wishes coming true.

  • Diane Reynolds (Paul.)|1.10.18 @ 11:31AM|

    Love is love, science is real. I read it on a sign in pretty much everyone's front yard.

  • Rhywun|1.10.18 @ 11:39AM|

    no tax on equally unhealthy coffee drinks

    The excuse is that those drinks are "milk-based". That's clever.

    And never mind that equally woke progressives in my town have been demonizing milk for years in favor of water and (sugary!) fruit juice.

  • IceTrey|1.10.18 @ 3:22PM|

    Starbucks isn't in the coffee business they are in the coffee flavored milk business.

  • sarcasmic|1.10.18 @ 11:46AM|

    Intentions. Raising minimum wage is intended to help the poor by forcing greedy employers to dip into their unlimited profits and pay a living wage, and if you disagree then you have bad intentions. Taxing tobacco is intended to discourage people from buying deadly products, and if you disagree then you have bad intentions.

    Economic laws can be changed with legislation backed by good intentions.

    Anyone who disagrees is motivated by bad intentions, and must be ignored.

    See? Intentions are magic.

  • DarrenM|1.10.18 @ 12:28PM|

    There is a very simple solution to all this. Since employers make obscene profits on the backs of their slave labor (as any good Lefty knows), instead of workers protesting they should just become employers themselves then they can make obscene profits, too. In fact, the government can pass a law mandating that everyone become an employer. This way we can eliminate welfare and other entitlements.

  • GeneralWeygand|1.10.18 @ 1:32PM|

    O, that is good. Tres bien!

  • Conchfritters|1.10.18 @ 5:27PM|

    If everyone became a business owner and had to pay estimated taxes every three months, you would see real tax reform occur pretty quickly.

  • Paloma|1.10.18 @ 7:38PM|

    Experts with good intentions are the driving force and greatest good for progressives.

  • Paper Wasp|1.10.18 @ 7:17PM|

    Oh, but they did tax the hipster drinks...but at only a cent per ounce, not 1.75 cents like they added for teh eeeebil corporate sodas. You wouldn't want to have to see the insufferable hipster tech fags that drink Rachel's Ginger Beer ugly cry at a city council meeting, would you?

  • GeneralWeygand|1.11.18 @ 6:00PM|

    Slake my thirst for hipster tech fag derision

  • Diane Reynolds (Paul.)|1.10.18 @ 11:30AM|

    By tacking another 1.75 cents onto every ounce of sweetened beverage purchased within the city, then-mayor Ed Murray promised, Seattle could subsidize trips to the farmers market, pay for free community college, and roll back "white-privileged, institutional racism."

    I made the mistake of passing by NPR and heard a segment on the awful state of Baltimore's public schools. The woman being interviewed said there was one reason, and one reason only for the sorry state of Ballmer's pub education: Institutional Racism.

    All these places have all this institutional racism-- and the people running the fucking joints are the ones screaming it. Funny that.

  • DesigNate|1.10.18 @ 11:49AM|

    Well, she's not wrong. Democrats are HUGE fucking racists.

  • DarrenM|1.10.18 @ 12:29PM|

    Democrats are HUGE fucking racists.

    Then they need to go on a diet.

  • ByteRot|1.10.18 @ 4:14PM|

    That's what the soda tax is for.

  • |1.10.18 @ 12:30PM|

    I made the mistake of passing by NPR and heard a segment on the awful state of Baltimore's public schools.

    After the hubbub about Gillespie's "Why don't they just move?" story, I couldn't help but laugh when NPR ran a story about how, in New York, 1% of the tax base pays 50% of the taxes (you mean OWS didn't fix that?) and, because of Trump's Tax Cuts they won't be able to deduct it from their federal taxes. The prevailing feeling is that (this time, it's different, and...) they may be forced to move. They'll have to see what DeBlasio does with his tax plan (which is touted as taxing more/raising money). It was just hilarious.

  • shawn_dude|1.11.18 @ 7:29PM|

    Or, you know, it could be related to the way the state and counties fund the schools. You're assuming the "institution" in "institutional racism" is the school itself. It might be the way more tax monies are sent to richer (and whiter) districts to pay for computers and other equipment while poorer (and non-white) schools are under-funded and don't have the same access to modern equipment or highly qualified teachers.

    In your leap to reinforce your pre-existing belief you appear to have discarded both empathy and imagination.

  • BYODB|1.10.18 @ 12:02PM|


    The cost of doing business in Seattle is higher than the Space Needle, and the unintended consequences of those policies are piling up too.


    Well you know what they say; foreseeable consequences aren't unintended consequences.

  • Telcontar the Wanderer|1.10.18 @ 9:12PM|

    T-34s doing donuts in front of the Reichstag was a foreseeable consequence of Operation Barbarossa, but I'm pretty sure Adolf wasn't foreseeing that when he gave the order to invade.

    "Foreseeable" is a contraction of "foreseeable by non-morons".

  • NoVaNick|1.10.18 @ 12:14PM|

    I'm sure the progs in Seattle, SF, and NYC, who claim they are trying to help poor people with $15 minimum wage don't really give a rats ass about Subway or any fast food, which they scoff at. They want "those people" to be paying $15 for a fair trade, organic, gluten-free tofu flatbread just like they do.

  • Paper Wasp|1.10.18 @ 7:25PM|

    Listen to them long enough, the snotty locavore progs will say something that is basically a variant of "if we got all those fatassed poors to eat tiny portions of expensive organic tofu and line-caught wild salmon and kale and quinoa and wash it down with Evian like we do, they'd be thin and almost acceptable."

  • |1.10.18 @ 12:18PM|

    If Seattle Americans can't keep up with the cost of living wages, why don't they just move?

  • loveconstitution1789|1.10.18 @ 12:45PM|

  • NoVaNick|1.10.18 @ 12:46PM|

    I would prefer that they stay where they are and suffer the consequences, or better yet, come to their senses and vote these idiots out. Unfortunately, they have the habit of bringing their bad voting habits with them wherever they move, and drive up rents and cost of living for everyone else...

  • Paper Wasp|1.10.18 @ 7:27PM|

  • Longtobefree|1.10.18 @ 12:28PM|

    The cost of doing business in Seattle is higher than the Space Needle, and the unintended consequences of those policies are piling up too.

    The consequences are fully intended. Not a single one was unknown at the time of passage, so they are intentional.
    Democrats hate the poor except on election day.

  • DarrenM|1.10.18 @ 12:30PM|

    Some needs to spellcheck that sign.

  • CE|1.10.18 @ 12:50PM|

    the tax has had the more immediate effect of hiking the price of a can of Coke by 20 cents, making a typical 36-can case of soft drink now $7.56 more expensive.

    Pretty sure that's 21 cents per can.

  • The_Hoser|1.10.18 @ 5:54PM|

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  • nicmart|1.10.18 @ 12:55PM|

    There have been reports that Subway franchisees all over the country are up in arm at the return of the 5 buck foot-long. They claim that they will lose money on it.

  • Crusty Juggler|1.10.18 @ 1:04PM|

    Here's that sign, courtesy of Krisitin Ellis, an executive assistant at the Washington Policy Center, who tells Reason she spotted the dismaying sign yesterday afternoon while picking up a veggie footlong in the SoDo neighborhood:

    Not only did you spell her name wrong, you reported the disgusting sandwich she ordered.

    Holy hell.

  • GeneralWeygand|1.10.18 @ 1:35PM|

    I, too, was revolted by the revelation of her "sandwich" selection.

    No Gusto!

  • Juice|1.10.18 @ 1:38PM|

    SoDo neighborhood

    Welcome Home.

  • Eric Bana|1.10.18 @ 1:49PM|

    "If they can't pay a living wage, then they shouldn't be in business in the first place!"

    Business shuts down because revenue is less than expenditures.

    "There aren't enough jobs! Free market dogma has failed again!"

  • Mickey Rat|1.10.18 @ 5:26PM|

    And complaints about "food deserts" as well.

  • Paper Wasp|1.10.18 @ 7:31PM|

    "If they can't pay a living wage, then they shouldn't be in business in the first place!"

    Subway is replaced by trendy restaurant that charges $16 for an organic, farm-to-table sandwich and $5 for a local craft soda.

    "Food is too expensive! Low-income people cannot afford meals out!"

  • Grooveman|1.10.18 @ 2:03PM|

    The first law of economics is scarcity. The first law of politics is to ignore the first law of economics.

  • Enjoy Every Sandwich|1.10.18 @ 2:46PM|

    and the unintended consequences of those policies are piling up too

    Saying that the consequences are unintended is too generous by half. I think the assholes who imposed these policies know damn well what the consequences are but don't give a fuck. They won't have to suffer the consequences themselves. And the sickest of the fuckers look forward to "fixing" the consequences with yet more interventions.

  • Social Justice is neither|1.10.18 @ 2:57PM|

    What is dismaying about that sign? Not woke enough? bad intentions from the owner?

  • IceTrey|1.10.18 @ 3:41PM|

  • Get To Da Chippah|1.10.18 @ 4:11PM|

    It it costs $6 to make a sandwich and you can't sell it for $5 and make a profit then you shouldn't be in business to begin with!

    /progderp

  • Otis B. Driftwood|1.10.18 @ 7:48PM|

    Alright! BIG win for Tony! You guys cut that greedy capitalist pig down, didn't you Tony?!

    Now if we could just get that minimum wage up to $20/hour......

  • cc2|1.10.18 @ 8:26PM|

    $15/hr is starting wage in Chicago for college graduates (in general). The idea that someone taking your order at a fast food place should make the same is looney toons. Why stop at $15? Why not mandate that everyone make $50/hr? Or $100/hr?

  • Tionico|1.10.18 @ 9:29PM|

    Leave it to Costco to put the toll of the new tax front and centre, and unavoidably seen. Remember back when Costco helped bring about the end of the state's liquor control board's monopoly on all liquor sales, mandating tht they be able to be sold by grocery stores and privete retailers? Walked into a Costco not long after, and their huge price signs above the product listed four items:
    cost of the bottle of booze, cost of the "litre tax", cost of the exise or liquore board's special booze tax, then the state retail sales tax on the total of the earlier three. I had just returned from California and had bought a bottle of their Kirkland brand 1.75 litre vodka... for $16. That same product and package, in fact, same "item number" in the Washington Costco cost a whopping $38. So whenever I'm south of the COlumbia River, wanting some hootch, and have some cash to melt, I stock up. Let's see now, how many cases would I have to bring back home to refund the cost of the fuel burned on the trip? Not many.. and I'd a whole lot rather see my money going up in tailpipe smoke than feed the greedy trolls in the Marble Zoo on the hill in Olympia.

  • DrZ|1.10.18 @ 9:34PM|

    Wait, the left wants to help the poor. By artificially raising wages, you can create more poor people and you will then have more people to help.

    It all makes sense if you see it through the eyes of a liberal.

  • Tionico|1.10.18 @ 9:39PM|

    remember that Seattle is the same city tht imposed a hefty tax on all gunsales within city limits, and another hefty one on all ammunition sales within city limits. Never mind such a tax is illegel per state law, they did it anyway and said MAKE ME>

    They had predicted huge revenue numbers to come in from the tax..... but the city's largest gun store, an independent, told the city hooh hahs if you pass that tax I WILL move my business outside the city limits. Council thought he was bluffing, so the passed it. As soon as they did,, he went real estate shopping, secured a new location just outside Seattle, closed his FFL at the former location and got a new one for the new place. Other stores dropped all firearms and ammunition sales lines, and the huge volume of predicted revenue never happened... in fact, since ALL the tax monies that used to go to Seattle from all these businesses ended, Seattle has enjoyed a signficant overall loss in revenue. Not only did they not get the new tax, they also lost all the existing tax they used to get. Looks good on them. Someone forgot to read about the Laffer Curve.......

  • tommhan|1.11.18 @ 12:03AM|

    Yeah, nobody saw this coming huh?

  • Mark22|1.11.18 @ 1:57AM|

    Seattle's $15 Minimum Wage is Hurting the Workers It's Intending to Help

    No, it's not. The $15 minimum wage is supposed to help union bosses and Democratic donors and politicians, and it's doing exactly that.

  • Craig Johnston|1.11.18 @ 2:06AM|

    This is a win-win for progressive politicians: they get to be seen as giving stuff to their constituents while letting the economic realities make those same constituents' lives' worse, fueling class warfare and motivating their base. Is it more disrespectful to think they didn't understand this or that they did?

  • Liberty Lover|1.11.18 @ 5:51AM|

    Every deal eventually goes away. It is called inflation. Inflation is a government policy to tax us to death. It is a hidden tax. I am old enough to remember the 10 cent beer. Try to find that now days.

  • Butler T. Reynolds|1.11.18 @ 7:29AM|

    JB Stoner and George Wallace were morons.

    THIS is how you get the riff-raff out of your city.

  • vek|1.11.18 @ 11:48AM|

    I live in Seattle... But not for long. I'm taking me, and my business, out of this city. My main business hasn't been effected much by the specific bad laws they've passed here, but a newer side business that has the potential to grow a lot would be KILLED and basically made unprofitable if I were to scale it up here. So I'm definitely bailing the city, but probably the whole state.

    It rarely gets mentioned but they passed a $13.50 STATE minimum wage a bit back that will be phasing in over the next few years too. Hence bailing the state. I'm sure I'm not the only one. All these laws don't hurt a company like Amazon because of the types of employees they have etc. But they absolutely make it impossible for many other types of businesses to be here.

    Living on even $15 an hour would be brutal in the city proper, but it's a starting wage... Now they're going to lose all the upper level guys at types of businesses that need SOME minimum wage type employees, but also have better paid people too. If you can't make it work here because of the minimum wage no $20 an hour shift leads, or $30 an hour managers or accountants, etc. They'll all be gone.

    It'll be just like my home state of California. When I was a kid there was a lot of diverse types of businesses there. Now everything except a few high wage industries is mostly gone, and then the service workers to make the lattes for the techies. No more middle middle class jobs left compared to when I was a kid 20-30 years ago.

  • GeneralWeygand|1.11.18 @ 6:09PM|

    Tow truck engineers, waste retrieval technicians, spare change consultants, turd unclogging warrant technicians and methodists all agree

  • shawn_dude|1.11.18 @ 7:12PM|

    The UW study has some issue and isn't even consistent with other studies done at UW on the pay raise. So I would put to much weight on it. A similar study done by Berkeley found no detectable change in hours worked for food services.

    UW vs Berkeley studies

    Something to bear in mind is that under our current safety net system, taxpayers essentially make up for the extremely low income of minimum wage workers through food stamps, welfare, medicaid, and other programs. If the employer has to provide things like health care and a living wage, the tax payers don't need to subsidize low wage employment to the same degree. Subway could only offer those deals because Joe Taxpayer was subsidizing Subway's cost of employment.



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With ‘Downsized’ DNA, Flowering Plants Took Over the World

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When people consider evolutionary events related to the origin and diversification of new species and groups, they tend to emphasize novel adaptations — specific genes giving rise to new, beneficial traits. But a growing body of research suggests that in some cases, that deciding factor may be something much more fundamental: size. In a paper published today in PLOS Biology, a pair of researchers studying the angiosperms, or flowering plants, has named genome size as the limiting constraint in their evolution.

The success of flowering plants, a group that includes everything from orchids and tulips to grasses and wheat, represents a long-standing puzzle for biologists. (In an 1879 letter to the renowned botanist Joseph Dalton Hooker, Charles Darwin called it an “abominable mystery.”) Terrestrial plants first appeared nearly half a billion years ago, but flowering plants arose only in the past 100 million years, beginning in the Cretaceous period. Yet, once angiosperms emerged, their structural and functional diversity exploded — far outpacing the diversification and spread of the other major plant groups, the gymnosperms (including conifers) and ferns.

Today, the 350,000 flowering-plant species, which have flourished in the vast majority of environments on Earth, constitute 90 percent of all plants on land. Since Darwin’s time, biologists pursuing the answer to that abominable mystery have sought to explain how the flowering plants could possibly have achieved this level of dominance in such a relatively short time.

Perhaps the answer has been so elusive because those scientists have usually focused on the physiological traits that set the angiosperms apart from their relatives. In the PLOS Biology paper, however, Kevin Simonin, a plant biologist at San Francisco State University, and Adam Roddy, a postdoctoral fellow at Yale University, argue that it’s the genome sizes underlying those individual adaptations that really matter.

The Advantages of Genome Downsizing

Species diverge hugely in genome size, without respect to the organisms’ complexity; in an oft-cited example, the onion has five times as much DNA as humans do. In the new study, Simonin and Roddy demonstrated what that variability in genome size means for large-scale biodiversity. They compiled vast amounts of data on genome size, cell size, cell density and photosynthetic rate for hundreds of angiosperms, ferns and gymnosperms, then traced the correlations among those traits back through time to weave a cohesive evolutionary narrative.

The emergence of angiosperms was marked by many events in which lineages of plants duplicated their whole genome. This process opened a door for greater diversification because the extra copies of genes could evolve and take on new functions. But because carrying so much genetic material can also be physiologically taxing, natural selection typically followed up these duplication events by aggressively pruning unneeded sequences. This “genome downsizing” often left flowering plants with less DNA than their parent species had. In fact, by following the family trees of the flowering plants back to their base, researchers have determined that the very first angiosperms had small genomes. “We now know that this not only contributed to their diversity, but may have given angiosperms the metabolic advantage to outcompete the other plant groups,” Simonin said.

He and Roddy posited that angiosperms’ small genomes set off a cascade of effects that over time flowed from their physiology into their structure and ultimately into their ecological role. Less DNA made it possible for the flowering plants to build their leaves from smaller cells, which in turn allowed them to pack more of certain cell types into the same volume. They could therefore have a higher density of stomata — the pores that facilitate the intake of carbon dioxide from the air and the release of water vapor — and a higher density of veins to provide enough water to keep those pores open. And the flowering plants did not have to sacrifice a high density of photosynthetic cells to achieve these benefits.

As a result, flowering plants could turn sunlight into sugars through photosynthesis much more efficiently. The rise of their superior capacity for hydration and gas exchange also coincided with falling levels of atmospheric carbon dioxide during the Cretaceous period, which contributed further to the angiosperms’ competitive edge over their green-plant peers. According to Simonin and Roddy, phylogenetic evidence shows that changes in genome size and the relevant physiological traits occurred together.



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MacBook Pro? No

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Let me count the way my latest MacBook Pro is not suitable for professional use, but before I do that, you should know that I’ve been buying and recommending Macs since 2001. I’ve spent a fortune on them. I love them, but I only like my latest MacBook Pro (a 2016 model with the Radeon Pro 460). I write this with a heavy heart and a malfunctioning keyboard.  This is a story about unrequited hardware love.

Typing

The keyboard on my MBP has begun to fail, with the ‘b’ key sometimes giving me doubled characters and sometimes none.

The cursor keys are in that awful cluster configuration where the up and down arrow keys, the ones I use the most, have half the space they should have and consequently make it next to impossible to navigate using them. Not so much cluster as clusterfuck.

They also feel different to the other keys, less responsive, if that were possible.

This £2800 laptop has a distinctly unprofessional keyboard. It’s not a whole lot better than the membrane keyboard on the first computer I owned, the Atari 400 back in 1982. I wouldn’t be surprised if the travel was shorter than that on the rubber keyboard of the ZX Spectrum. I don’t mind the feel, but that’s not good enough for a £2800 laptop. I should adore the keyboard. It doesn’t need much more travel, but it does need it. I don’t expect a Matias Tactile Pro, but I could do with a Magic Keyboard and I’ll take the extra thickness in return for a usable primary input method.

Finally, where’s my damned Esc key? I go for that so often, and miss. Putting it on the Touch Bar was a stupid idea.

Touch Bar

Why? The only thing of any use is the TouchID sensor, but you don’t need a whole keyboard-level touch screen for that!

For a touch typist, if you’re going to have a touchable surface, it might as well be the screen. I don’t want to look at the keyboard, ever; I want to look at the screen. Looking down at that pointless strip that offers me no feedback when I interact with it and which has no delineated edges is an extra cognitive burden that I just don’t need and therefore, as a professional, never use, unless I’m forced to. You know that a design has failed when you’re forced into using it. The Touch Bar is Apple’s Clippy. An embarrassment.

Track Pad

It’s too big. I never use all of that space, and I keep accidentally triggering it with my wrists when I don’t want to. Why not give the keyboard some extra height so I can have some proper cursor keys and while you’re at it, make those keys tactile and replaceable? It’s not a Wacom for crying out loud.

Ports

I like USB Type C, I really do, but like many people with a MacBook Pro, I have a camera and like most decent cameras, it has an SD card slot, except that Apple saw fit to remove this slot. Professional photographers have used the MacBook Pro for as long as they’ve been made. Only now, they need an accursed dongle.

I’d understand and forgive this on a lesser machine, like one without the “Pro” moniker, but not having an SD card on this £2800 machine strikes me as totally stupid. Rather than use another expensive adapter, I just use the iMac, which makes that the professional machine, and this one just a designer’s wet dream.

Power supply

I have a professional laptop, but I can’t tell it’s charging unless I open the lid, gain entry and look at the tiny power indicator in the menu bar. Which genius thought this would be a Pro touch? I know it makes a sound when you plug it in; fine, genius, but what if I didn’t hear the sound? How do I tell when it’s fully charged? You took off the MagSafe, which was a genuinely useful innovation and gave us USB C charging — fine, but add a little adapter that has a charge indicator, and yes, if you must, charge for it, though you really shouldn’t if you want to call this a “Pro”.

While we’re on the subject of lights, I miss the glowing Apple logo at the back of the machine. Bring that back please. Why are you cutting costs on a £2800 machine?

Also, why don’t we get a power extension cable like we used to? Don’t know about you, but I find it hard to find power sockets near me all the time and this is just another tight move.

Battery Life

You claim five hours. I can offer you some more realistic benchmarks. Not five. Sometimes not four. It’s a Pro machine, right? Don’t go backwards then. Give me 8 hours or give me death.

Conclusion

If Apple wants to make a MacBook Pro, they should quit with the design fundamentalism on a machine costing £2800 (£2800 is a ton of money for an ordinary laptop, which apart from the display, this is) and quit with what seem like cost-cutting measures in the name of power efficiency. This machine is no doubt powerful. It never struggles with software, everything runs at a decent clip (when the power is plugged in) and it’s stable, but it’s not a Pro machine — just about any decent PC laptop at not much more than half the price of my MacBook Pro will give me “Pro” functionality.

I say make it faster, make the battery bigger, make the laptop slightly thicker, make the keyboard decent (heck just make it like the Magic Keyboard), get rid of the Touch Bar, make the display a 16” 4K HDR OLED, bring back some kind of MagSafe, bring the lit Apple logo back, bring back the SD card slot, add three USB 3 slots, make the trackpad smaller, beef up the GPU so that it can handle VR and games, and make it £4000. I’ll buy it. In the meantime, drop the price on this experiment and stop calling it a Pro. It doesn’t feel any more Pro than the standard MacBook.



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White House personal cell ban starts next week

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Story highlights

  • White House chief of staff John Kelly sent an internal memo to staffers Wednesday
  • It's a decision that has been met with widespread frustration

The new policy includes "all portable electronic devices" not issued or authorized by the White House from "being carried into or otherwise possessed" in the West Wing from 6 a.m. to 8 p.m. Monday through Friday, regardless of whether they are turned off. The memo, a copy of which was viewed by CNN, advises staffers to leave their phones at home, in their vehicles or inside lockers near the West Wing lobby.

"This policy is to protect White House information technology infrastructure from compromise and sensitive or classified information from unauthorized access or dissemination," according to the memo. 

"Any person entering or working in the West Wing must comply with this policy, regardless of whether he or she is a federal employee, intern, volunteer or visitor," the memo reads. "Violations of this policy by [Executive Office of the President] staff are security incidents that may indicate knowing, willful, and negligent conduct in violation of security policy and may therefore result in disciplinary action and, for other Federal employees and visitors, may include being indefinitely prohibited from entering the White House complex."

The decision to ban personal cellphones is one Kelly had floated since July but not executed until now. The move has prompted widespread frustration among staffers, many of whom are questioning how they are expected to stay in touch with their families and spouses while often working 12-hour days.

White House press secretary Sanders defended the decision in recent weeks, saying it has been in the works for the past six months. During that period, the White House has looked for ways to increase White House officials' ability to use "other applications" on their phones and to ensure compliance with the Presidential Records Act, Sanders said.

The standard is expected to apply to everyone -- including top officials such as communications director Hope Hicks -- except the President, one person familiar with the decision told CNN.



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Ibuprofen alters human testicular physiology

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Ibuprofen alters human testicular physiology to produce a state of compensated hypogonadism

  1. David Møbjerg Kristensena,b,1,2,
  2. Christèle Desdoits-Lethimonierb,1,
  3. Abigail L. Mackeyc,d,
  4. Marlene Danner Dalgaarde,
  5. Federico De Masie,
  6. Cecilie Hurup Munkbølf,
  7. Bjarne Styrishavef,
  8. Jean-Philippe Antignacg,
  9. Bruno Le Bizecg,
  10. Christian Platelh,
  11. Anders Hay-Schmidti,
  12. Tina Kold Jensenj,
  13. Laurianne Lesnéb,
  14. Séverine Mazaud-Guittotb,
  15. Karsten Kristiansenk,l,
  16. Søren Brunaka,
  17. Michael Kjaerc,d,
  18. Anders Juuli, and
  19. Bernard Jégoub,2
  1. aDanish Headache Center, Department of Neurology, Rigshospitalet, University of Copenhagen, 1165 Copenhagen, Denmark;
  2. bUniversité de Rennes I, Inserm, EHESP-School of Public Health, Irset (Institut de Recherche en Santé, Environnement et Travail) - UMR_S 1085, F-35000 Rennes, France;
  3. cInstitute of Sports Medicine, Department of Orthopaedic Surgery M, Bispebjerg Hospital, 2400 Copenhagen NV, Denmark;
  4. dCenter for Healthy Aging, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1165 Copenhagen, Denmark;
  5. eDTU Bio and Health Informatics, Technical University of Denmark, 2800 Kongens Lyngby, Denmark;
  6. fDepartment of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 1165 Copenhagen, Denmark;
  7. gL'Université Nantes Angers Le Mans (LUNAM), Oniris, UMR 1329 INRA Laboratoire d'Etude des Résidus et Contaminants dans les Aliments (LABERCA), F-44307 Nantes, France;
  8. hUnité de coordination hospitalière des prélèvements d’organes et de tissus, Centre Hospitalier Universitaire de Rennes, 35000 Rennes, France;
  9. iDepartment of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, 1165 Copenhagen, Denmark;
  10. jDepartment of Environmental Medicine, University of Southern Denmark, 5000 Odense, Denmark;
  11. kDepartment of Biology, Faculty of Science, University of Copenhagen, 2100 Copenhagen, Denmark;
  12. lInstitute of Metagenomics, BGI-Shenzhen, Shenzhen 518083, China
  1. Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved December 1, 2017 (received for review August 31, 2017)

Significance

Concern has been raised over declining male reproductive health in humans. Our study addresses this issue by extending data showing antiandrogen effects of analgesics and suggests that such compounds may be involved in adult male reproductive problems. Using a unique combination of a randomized, controlled clinical trial and ex vivo and in vitro approaches, we report a univocal depression of important aspects of testicular function, including testosterone production, after use of over-the-counter ibuprofen. The study shows that ibuprofen use results in selective transcriptional repression of endocrine cells in the human testis. This repression results in the elevation of the stimulatory pituitary hormones, resulting in a state of compensated hypogonadism, a disorder associated with adverse reproductive and physical health disorders.

Abstract

Concern has been raised over increased male reproductive disorders in the Western world, and the disruption of male endocrinology has been suggested to play a central role. Several studies have shown that mild analgesics exposure during fetal life is associated with antiandrogenic effects and congenital malformations, but the effects on the adult man remain largely unknown. Through a clinical trial with young men exposed to ibuprofen, we show that the analgesic resulted in the clinical condition named “compensated hypogonadism," a condition prevalent among elderly men and associated with reproductive and physical disorders. In the men, luteinizing hormone (LH) and ibuprofen plasma levels were positively correlated, and the testosterone/LH ratio decreased. Using adult testis explants exposed or not exposed to ibuprofen, we demonstrate that the endocrine capabilities from testicular Leydig and Sertoli cells, including testosterone production, were suppressed through transcriptional repression. This effect was also observed in a human steroidogenic cell line. Our data demonstrate that ibuprofen alters the endocrine system via selective transcriptional repression in the human testes, thereby inducing compensated hypogonadism.

Much concern has been raised over declining male reproductive health, and the disruption of male endocrinology has been suggested to play a central role (1, 2). Male reproduction and general health rely on androgens, as well as on other hormones, which are mainly produced by testicular Leydig and Sertoli cells. In addition to the testis, the androgens act in many somatic organs, e.g., producing anabolic effects on muscle mass and influencing cognitive functions (3). Luteinizing hormone (LH) produced by the pituitary is the primary stimulator of testosterone production, and the testosterone/LH ratio is routinely used as a clinical marker of Leydig cell function. When Leydig cell function is compromised, normal or nearly normal testosterone levels can often be sustained by augmented LH levels, as observed in the clinical entity termed “compensated hypogonadism” (4). The essential importance of the pituitary–gonadal axis is emphasized by the recent association of hypogonadism with a wide range of risk factors and all-cause mortality in men (4, 5).

The so-called “over-the-counter” mild analgesics (hereafter simply called “analgesics”), such as acetaminophen/paracetamol, acetylsalicylic acid/aspirin, and ibuprofen, are among the most commonly used pharmaceutical compounds worldwide (6, 7). Increasing evidence from recent years shows that exposure to analgesics can generate negative endocrine and reproductive effects during fetal life (6). Nonetheless, no in-depth studies have analyzed the effect of mild analgesics on the human pituitary–gonadal axis. In this context, ibuprofen is especially interesting because of its increasing use in the general population and in particular by elite athletes (812).

Therefore in this study we focused on how ibuprofen, used in the general population for aches, pains, fever, and arthritis and heavily used by athletes (13), affects the pituitary–testis axis. Because of the intrinsic great challenge in identifying endocrine-disrupting effects of chemicals in the adult human, we performed a unique combination of three interconnected approaches: (i) a randomized, controlled clinical trial; (ii) an ex vivo organ model using adult human testis explants; and (iii) a standardized in vitro model system based on a steroidogenic cell line of human origin to complement the ex vivo approach. The complementary results revealed that ibuprofen induces a state of compensated hypogonadism by modifying hormonal profiles through selective repression of gene expression.

Results

Ibuprofen Affects the Hormonal Balance in Adult Men.

We conducted a randomized, controlled clinical trial of ibuprofen administration to identify its possible effects on pituitary–gonadal feedback in young men. During administration, ibuprofen levels in plasma ranged on average from 25 to 35 µg/mL (∼1.2–1.7 × 10−4 M); the highest level measured was 100 µg/mL (4.85 × 10−4 M). The mean of this ibuprofen concentration was in the range measured after administration of 600 mg of ibuprofen to healthy volunteers (14). Samples drawn before administration showed that there were no initial differences between the placebo and ibuprofen groups for hormones or sex hormone-binding globulin (SHBG), a liver protein that specifically binds a substantial part of circulating testosterone (Fig. S1).

We investigated the levels of total testosterone and its direct downstream metabolic product, 17β-estradiol. Administration of ibuprofen did not result in any significant changes in the levels of these two steroid hormones after 14 d or at the last day of administration at 44 d (Fig. 1 A and C). The levels of free testosterone were subsequently analyzed by using the SHBG levels. Neither free testosterone nor SHBG levels were affected by ibuprofen during the administration (Fig. 1 A and C).

The pituitary gonadotropin hormones LH and FSH regulate the production of testosterone and peptide hormones by acting on Leydig and Sertoli cells, respectively. The levels of LH in the ibuprofen group had increased by 23% after 14 d of administration (P = 0.05) (Fig. 1A). This increase was even more pronounced at 44 d, at 33% (P = 0.01) (Fig. 1C). While a slight, nonsignificant increase in the average FSH concentration was observed at 14 d (+5%) (Fig. 1B), no difference in the average value of this hormone was seen at 44 d (Fig. 1D). These data suggested a link between pituitary LH levels and ibuprofen exposure. This assumption was confirmed when we investigated ibuprofen and LH levels in plasma and found they were significantly and positively correlated at 14 d (r = 0.73; P = 0.003) (Fig. 1E). We next calculated the free testosterone/LH ratio in the men. We found an 18% decrease (P = 0.056) in the ibuprofen group compared with the placebo group after 14 d (Fig. 1A) and a 23% decrease (P = 0.02) after 44 d (Fig. 1C). Taken together, these in vivo data suggest that ibuprofen induced a state of compensated hypogonadism during the trial, which occurred as early as 14 d and was maintained until the end of the trial at 44 d.

Inhibin B and anti-Müllerian hormone (AMH) are peptide hormones secreted to the blood from the Sertoli cells. The administration of ibuprofen did not change mean inhibin B levels (Fig. 1 B and D). To examine the stimulatory action of FSH on the Sertoli cells, we next examined the inhibin B/FSH ratio and found that it decreased by 4% after 14 d (Fig. 1B) and by 12% at the end administration at 44 d (Fig. 1D). Importantly, further investigation of Sertoli cell activity showed that AMH levels decreased significantly with ibuprofen administration, by 9% (P = 0.02) after 14 d (Fig. 1B) and by 7% (P = 0.05) after 44 d compared with the placebo group (Fig. 1D). The AMH data show that the hypogonadism affected not only Leydig cells but also Sertoli cells and also occurred as early as 14 d of administration.

Ibuprofen Inhibits Steroidogenesis ex Vivo and in Vitro.

To determine the direct effect of ibuprofen on the testis, we next exposed adult testis explants from donors to doses of 10−9–10−4 M, which corresponded to the oral doses producing mean plasma levels of 1.2–1.7 × 10−4 M used among the men in the trial (see above). We first investigated testosterone production after 24 and 48 h of ibuprofen exposure to assess its effects on Leydig cell steroidogenesis. Inhibition of testosterone levels was significant and dose-dependent (β = −0.405, P = 0.01 at 24 h and β = −0.664, P < 0.0001 at 48 h) (Fig. 2A) and was augmented over time (10−4 M at 24 h and 10−5–10−4 M at 48 h, −40%) (Fig. 2A). Examination of the effect of ibuprofen exposure on both the ∆4 and ∆5 steroid pathways (Fig. 2B) showed that it generally inhibited all steroids from pregnenolone down to testosterone and 17β-estradiol; the production of each steroid measured decreased at doses of 10−5–10−4 M. Under control conditions, production of androstenediol and dehydroepiandrosterone (DHEA) was below the limit of detection except in one experiment with DHEA (Fig. 2B).

Fig. 2.
Fig. 2.

Ibuprofen inhibits Leydig cell hormone production in human testicular explants. (A) Dose effect of ibuprofen for 24 and 48 h on testosterone production by the adult human testis. Values are means ± SEM of five independent experiments from different donors. Dose responses were analyzed for significance with the Mann–Whitney U test. The slopes and P values of these results were calculated with Spearman correlation. (B) Effect of 10−5 and 10−4 M ibuprofen on the Δ4 and Δ5 steroidogenic pathways. Steroid precursors were measured by GC-MS/MS. Values are means ± SEM of three independent experiments from different donors. Dose effects were analyzed for significance with the Mann–Whitney U test. DHEA, dehydroepiandrosterone. (C) Effect of 10−5 and 10−4 M ibuprofen on the gene expression of steroidogenic enzymes after 48-h exposure. Values are means ± SEM of five independent experiments from different donors. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Dose responses were analyzed for significance with the Mann–Whitney U test. (D) Dose effect of ibuprofen for 24 and 48 h on INSL3 production by the adult human testis. Values are means ± SEM of three independent experiments from different donors. Dose responses were analyzed for significance with the Mann–Whitney U test. (E) Specific Leydig cell hormone gene expression. Values are means ± SEM of five independent experiments from different donors. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Dose responses were analyzed for significance with the Mann–Whitney U test. BZW1, basic leucine zipper and W2 domains 1; CYP11A1, cytochrome P450 family 11 subfamily A member 1; CYP17A1, cytochrome P450 family 17 subfamily A member 1; CYP19A1, cytochrome P450 family 19 subfamily A member 1; GUSB, β-glucuronidase; INSL3, insulin-like factor 3; HSD17B3, hydroxysteroid 17-β dehydrogenase 3; HSD3B2, hydroxy-δ-5-steroid dehydrogenase 3 β- and steroid δ-isomerase 2; LHCGR, luteinizing hormone/choriogonadotropin receptor; StAR, steroidogenic acute regulatory protein; TSPO: translocation protein. *P ≤ 0.05, **P ≤ 0.01.

We next examined the gene expression involved in testicular steroidogenesis ex vivo and found that levels of expression of every gene that we studied except CYP19A1 decreased after exposure for 48 h compared with controls (Fig. 2C). Suppression of gene expression concerned the initial conversion of cholesterol to the final testosterone synthesis. Hence, expression of genes involved in cholesterol transport to the Leydig cell mitochondria was impaired: Compared with controls, TSPO/BZRP fell significantly after exposure to 10−4 M; StAR expression was suppressed at both doses of 10−5 and 10−4 M (Fig. 2C); CYP11A1, CYP17A1, and HSD17B3 were suppressed by 40–50% at doses of 10−5–10−4 M; and HSD3B2 was reduced by about 90%.

The data from the ex vivo testis model showed inhibition of both the ∆4 and ∆5 steroid pathways. A previous study reported androsterone levels decreased by 63% among men receiving 400 mg of ibuprofen every 6 h for 4 wk (15), suggesting a possible inhibitory effect on CYP17A1. We therefore exposed the NCI-H295R cells of human origin [which, although derived from the adrenal gland (instead of testis) and from a cancer originating in the cortex, are considered the best in vitro model of human steroidogenesis according to the Organization for Economic Cooperation and Development (OECD) (16)] to ibuprofen and, as a positive control, to the CYP17A1 antagonist abiraterone. The latter compound is clinically used as an androgen biosynthesis inhibitor (17). Before testing ibuprofen’s action on steroidogenesis, we verified that none of the doses used had a toxic effect on the NCI-H295R cell line (Fig. S2 A and B). This in vitro work confirmed the global antagonistic effect of abiraterone on CYP17A1 (Fig. 3A). In contrast, ibuprofen significantly decreased androstenedione and other sex steroid levels downstream from the CYP17A1 lyase activity, including testosterone (Fig. 3 and Table S1), complementing the results in the ex vivo testis model. These data therefore reveal that ibuprofen, unlike abiraterone, is not a general CYP17A1 antagonist. It is noteworthy that the NCI-295R cell line was less sensitive to ibuprofen than were the ex vivo cells in the organ model, likely due to its different origin (6, 18).

Fig. 3.
Fig. 3.

The steroid screen identifies ibuprofen as an inhibitor of steroidogenesis in the human NCI-H295R cell line. (A) Effects of ibuprofen (red trace; n = 9–18) and abiraterone (blue trace; n = 615) exposure on the relative steroidogenic hormone production in the dose ranges of 10−6–10−3 M and 3.15 × 10−11–3.15 × 10−6 M (x axis), respectively, in the human NCI-H295R cell line, according to OECD standards. The position of each graph corresponds to the position of that particular steroid hormone in steroidogenesis. Steroid concentrations (y axis; % of control) are depicted as mean ± SEM with key enzymes shown in blue boxes. For statistics see Table S1. (BD) Quantitative RT-PCR screen of steroidogenic gene expression in NCI-H295R cells after 48 h of culture with 10−7–10−3 M ibuprofen. Values are means ± SEM of three independent experiments analyzed with one-way ANOVA followed by a post hoc Dunnett’s test. CYP11A1, cytochrome P450 family 11 subfamily A member 1; CYP17A1, cytochrome P450 family 17 subfamily A member 1; CYP19A1, cytochrome P450 family 19 subfamily A member 1; HSD17B3, hydroxysteroid 17-β dehydrogenase 3; HSD3B2, hydroxy-δ-5-steroid dehydrogenase 3 β- and steroid δ-isomerase 2; StAR, steroidogenic acute regulatory protein; TSPO, translocation protein. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Measuring the mRNA expression of genes involved in steroidogenesis in vitro showed that ibuprofen had a profound inhibitory effect on the expression of these genes (Fig. 3 BD), consistent with that seen above in our ex vivo organ model. Taken together, these data examining effects on the endocrine cells confirm that ibuprofen-induced changes in the transcriptional machinery were the likely reason for the inhibition of steroidogenesis.

Ibuprofen Inhibits Leydig Cell Insulin-like Factor 3 in the Adult Human Testis ex Vivo.

In addition to steroids, Leydig cells also produce insulin-like factor 3 (INSL3) (19). Its production increased at 24 h at the 10−9 M dose and subsequently decreased at the 10−7 M dose (Fig. 2D). These variations were transient, with no significant effect of ibuprofen observed after 48 h. INSL3 expression decreased by 50% at a dose of 10−4 M, but expression of the LH receptor, luteinizing hormone/choriogonadotropin receptor (LHCGR), which is also Leydig cell specific, was not repressed (Fig. 2E). Nonetheless, overall the changes in gene expression indicate that the transcriptional machinery behind the endocrine action of Leydig cells was most likely impaired by ibuprofen exposure.

Ibuprofen Impairs Sertoli Cell Function in the Adult Human Testis ex Vivo.

Data from the trial showed that ibuprofen affected Sertoli cells, inhibiting AMH and decreasing the inhibin B/FSH ratio. Ex vivo, increasing doses of ibuprofen resulted in an inverse correlation with inhibin B after 24 h (β = −0.467; P = 0.01), although none of the individual ibuprofen concentrations significantly inhibited this hormone (Fig. 4A). After 48 h of exposure, however, ibuprofen doses of 10−7–10−4 M significantly decreased inhibin B production, resulting in a further significant negative association between ibuprofen concentrations and inhibin B (β = −0.739, P < 0.0001). AMH production was also negatively correlated with increasing ibuprofen concentration (β = −0.451; P = 0.01) (Fig. 4B). Accordingly, a dose of 10−4 M repressed gene expression of AMH and INHBB by ∼35% (Fig. 4C). Together, these data show that ibuprofen also directly impairs Sertoli cell function ex vivo by inhibiting transcription. Of note, no significant changes were found in the gene expression of the Sertoli cell-specific FSH receptor (FSH-R) or of LAMA5 (Fig. 4C).

Fig. 4.
Fig. 4.

Ibuprofen affects Sertoli cell activity in human testicular explants. (A and B) Dose effect of ibuprofen on the production of inhibin B after 24 and 48 h (A) and anti-Müllerian hormone (AMH) after 48 h (B) by the adult human testis. Values are means ± SEM of three independent experiments from different donors. Slopes and P values of Spearman correlation are indicated. (C) Quantitative RT-PCR performed after 48 h of culture treated with 10−5 and 10−4 M ibuprofen for specific Sertoli cell gene expression. Values are means ± SEM of five independent experiments from different donors. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Dose responses were analyzed for significance with the Mann–Whitney U test. AMH, anti-Müllerian hormone; BZW1, basic leucine zipper and W2 domains 1; FSHR, follicle-stimulating hormone receptor; GUSB, β-glucuronidase; INHBB, inhibin B subunit B; LAMA5, laminin subunit α5. *P ≤ 0.05, **P ≤ 0.01.

Ibuprofen Selectively Affects Peritubular Cells’ Gene Expression in the Adult Human Testis ex Vivo.

Peritubular cells lining the seminiferous wall play an important role in sustaining seminiferous tubule function (20). The peritubular cells are not broadly characterized in terms of specific markers. Nevertheless, we investigated the expression of a few genes that are assigned to these cells. We found that ibuprofen selectively repressed ACTA2 and MYH-11 by 50%, but two other peritubular cell markers, THY1 and KCNIP4, did not change significantly (Fig. 5A).

Fig. 5.
Fig. 5.

Ibuprofen decreases gene expression in peritubular cells but does not affect germ cells or morphology in human testicular explants. (A and B) Quantitative RT-PCR performed after 48 h of culture treated with 10−5 and 10−4 M ibuprofen for gene expression in peritubular cells (A) and germ cells (B). Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Values are means ± SEM of five independent experiments from different donors. A Mann–Whitney U test was performed. (C) Number of apoptotic germ cells. Values are means ± SEM of caspase+ cells in three independent experiments from different donors. (D) Immunostaining of apoptotic germ cells in testis explants cultured for 48 h in the presence of DMSO (control) or 10−5 or 10−4 M ibuprofen. Each micrograph shows representative areas of ibuprofen-induced morphology compared with corresponding area. (Scale bars: 50 µm.) ACTA2, actin α2 smooth muscle aorta; ALPP, alkaline phosphatase, placental; BZW1, basic leucine zipper and W2 domains 1; GUSB, β-glucuronidase; KCNIP4, potassium voltage-gated channel interacting protein 4; MYH11, myosin heavy polypeptide 11, smooth muscle; PGK2, phosphoglycerate kinase 2; PRM2, protamine 2; THY1, Thy-1 cell-surface antigen. **P ≤ 0.01.

Ibuprofen Spares the Spermatogenic Cells in the Adult Human Testis ex Vivo.

Turning our attention to germ cells in the explants, we found no significant changes in the expression of genes involved specifically with spermatogenesis (Fig. 5B). The absence of a change in the germ cell complement by ibuprofen was confirmed by staining for caspase 3 after 48 h of exposure: Apoptosis did not increase significantly in the testis after exposure (Fig. 5 C and D), and the histopathology of the testis at the highest doses did not differ from that of controls (Fig. 5D).

Ibuprofen Suppresses Prostaglandin Production in the Adult Human Testis ex Vivo and in Vitro.

As ibuprofen acts specifically on COX sites of prostaglandin H2 synthase (prostaglandin endoperoxide synthase or prostaglandin G/H synthase and cyclooxygenase, PTGS), and because prostaglandin receptors and synthesizing enzymes are widely distributed within the testis (21), we investigated prostaglandin D2 (PGD2) and E2 (PGE2) in our culture system. Ibuprofen produced a significant dose-dependent reduction of PGD2 (β = −0.781; P < 0.0001 at 24 h and β = −0.797; P < 0.0001 at 48 h) (Fig. 6A) and of PGE2 (β = −0.707; P < 0.0001 and β = −0.627; P < 0.0001, respectively) (Fig. 6B). PTGS1 and PTGS2 gene expression decreased similarly: PTGS1 mRNA levels fell significantly by 24 and 48 h (Fig. 6C), and PTGS2 mRNA was significantly repressed after 48 h of exposure. These ex vivo data show that ibuprofen suppressed both PTGS enzyme activity and PTGS gene expression in the adult testis.

Fig. 6.

Ibuprofen decreases PGE2 and PGD2 production and PTGS gene expression in human testicular explants. (A and B) Dose effect of ibuprofen exposure after 24 and 48 h on PGD2 (A) and PGE2 (B) production by adult human testicular explants. Values are means ± SEM of five independent experiments from different donors. Dose responses were analyzed for significance with the Mann–Whitney U test. Slopes and P values of Spearman correlation are indicated. (C) Quantitative RT-PCR was performed after 48 h of culture treated with 10−5 and 10−4 M ibuprofen. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Values are means ± SEM of five independent experiments from different donors. Differences in gene expression were analyzed with a Mann–Whitney U test. BZW1, basic leucine zipper and W2 domains 1; GUSB, β-glucuronidase; PTGS, prostaglandin-endoperoxide synthase. *P ≤ 0.05, **P ≤ 0.01.

To complement our ex vivo model system, we next screened the human NCI-H295R cell line for prostaglandin production. This screen showed that the NCI-H295R cells produced detectable levels of prostaglandins, which were decreased dose dependently with ibuprofen (Fig. 7A). As also shown in the experiments presented above, ibuprofen decreased the expression of PTGS1 and PTGS2 in the NCI-H295R cell line (Fig. 7B).

Fig. 7.

Ibuprofen dose-dependently reduces prostaglandin levels and mRNA expression in human endocrine NCI-H295R cells. (A) Effects of ibuprofen on general prostaglandin production from NCI-H295R cells after 24 h. Values are means ± SEM of three independent experiments analyzed with one-way ANOVA followed by a post hoc Dunnett’s test. (B) Quantitative RT-PCR screen of steroidogenic and PTGS gene expression in NCI-H295R cells after 48 h of culture with 10−7–10−3 M ibuprofen. Values are means ± SEM of three independent experiments analyzed with one-way ANOVA followed by a post hoc Dunnett’s test. PTGS, prostaglandin-endoperoxide synthase. *P ≤ 0.05, **P ≤ 0.01.

Discussion

The pituitary–gonadal axis plays key roles in growth, sex development, metabolism, musculoskeletal build-up, strength, mood, energy, immune system, libido, and reproduction (2224). Fluctuations in or impaired fine-tuning of the axis can result in a wide range of endocrine disorders that may be local but severe, e.g., infertility (25), or affect the entire body, as seen with adverse outcomes involving this axis such as sexual symptoms (4), depression (26), coronary heart disease/heart attack (27), autoimmune diseases such as arthritis (28), and diabetes (29, 30). Testosterone forms a negative feedback loop that inhibits the production of both LH and gonadotropin-releasing hormone (GnRH) in the hypothalamus (31). While testosterone plays multiple roles outside the testes, the extratesticular actions of inhibin B are more subtle, working primarily to decrease FSH (32). Nonetheless, inhibin B is a key clinical marker of reproductive health (32). The function of AMH, also secreted by Sertoli cells, and its regulation through FSH remain unclear in men (33). It has, however, been shown that the AMH concentrations are lower in seminal plasma from patients with azoospermia than from men with normal sperm levels (32).

Our trial showed that ibuprofen use in men led to (i) elevation of LH; (ii) a decreased testosterone/LH ratio and, to a lesser degree, a decreased inhibin B/FSH ratio; and (iii) a reduction in the levels of the Sertoli cell hormone AMH. The decrease in the free testosterone/LH ratio resulted primarily from the increased LH levels, revealing that testicular responsiveness to gonadotropins likely declined during the ibuprofen exposure. Our data from the ex vivo experiments support this notion, indicating that the observed elevation in LH resulted from ibuprofen’s direct antiandrogenic action. Accordingly, in the trial the average inhibin B levels did not differ significantly in the ibuprofen-treated men and the control group. This is consistent with a previous report that men who volunteered to take another nonsteroidal antiinflammatory drug (NSAID), acetylsalicylic acid, coadministered with human chorionic gonadotropin (hCG), which mimics LH, had lower levels of steroidal hormones than controls exposed to hCG but not to the analgesic (34).

AMH levels were consistently suppressed by ibuprofen both in vivo and ex vivo, indicating that this hormone is uncoupled from gonadotropins in adult men. The ibuprofen suppression of AMH further demonstrated that the analgesic targeted not only the Leydig cells but also the Sertoli cells, a feature encountered not only in the human adult testis but also in the fetal testis (35). It is noteworthy that ibuprofen repressed the expression of both AMH and INHBB as well as genes encoding essential proteins and enzymes involved in both cholesterol transport and steroidogenesis. Thus, ibuprofen displayed broad transcription-repression abilities involving steroidogenesis, peptide hormones, and prostaglandin synthesis. However, these repressive abilities were selective, as a number of gene-expression patterns were spared by ibuprofen, namely prostaglandin inhibition in Leydig cells (CYP19A1 and LHCGR), Sertoli cells (LAMA5 and FSHR), peritubular cells (KCNIP4 and THY1), and all those investigated in germ cells. Of note, the absence of an effect of ibuprofen on the expression levels of gonadotropin receptor genes (LHCGR and FSHR) indicates that the responsiveness of Leydig cells and Sertoli cells to the action of LH and FSH is likely not affected by ibuprofen. However, more investigation is required at this level.

Several compounds have been found to have unintentional antiandrogenic effects, and these are normally investigated in connection with fetal male development using rodent models (36, 37). Our approach, using ibuprofen as an example, demonstrates how a chemical compound, through its effects on the signaling compounds, can result in changes in the testis at gene level, resulting in perturbations at higher physiological levels in the adult human. The analgesics acetaminophen/paracetamol and ibuprofen have previously been shown to inhibit the postexercise response in muscles by repressing transcription (3840). However, the striking dual effect of ibuprofen observed here on both Leydig and Sertoli cells makes this NSAID the chemical compound, of all the chemical classes considered, with the broadest endocrine-disturbing properties identified so far in men. Previous ex vivo studies on adult testis have indeed pointed to an antiandrogenicity, only on Leydig cells, of phthalates (41), aspirin, indomethacin (42), and bisphenol A (BPA) and its analogs (43). However, ibuprofen’s effects were not restricted to Leydig and Sertoli cells, as data showed that the expression of genes in peritubular cells was also affected. Previous studies have shown that long-term fetal exposure to acetaminophen and acetylsalicylic acid in mice and rats targets primordial germ cell proliferation by blocking RNA synthesis and thus leads to reduced follicle reservoir and subsequent decreased fertility in adulthood (4446). By contrast, in the present study using human testes, germ cells were the only cell category not altered by this analgesic in our ex vivo culture conditions. However, it must be noted that our ex vivo model systems can be used only for short-term exposure. Therefore, determining the effect on men that sustained exposure to ibuprofen would generate in terms of sperm production and fertility would require designing specific and challenging experiment(s). It is noteworthy that exposure to analgesics in men has been associated with increased time to pregnancy (47).

An important question is the exact relationship between the prostaglandin-inhibitory actions of ibuprofen and its effects on testosterone and gene expression. This has been investigated previously in studies on rodent and human testicular development, which showed no correlation between the endocrine-disruptive effects of analgesics and their prostaglandin-inhibitory actions (6, 48, 49). However, in the present study using testes from adult men, the suppression of androgens and prostaglandins occurred in parallel, and, because for several decades prostaglandins have been known to be involved in male reproduction (50), a link between the endocrine-disruptive properties of ibuprofen and the prostaglandin-inhibitory action of NSAIDs in the adult testis cannot be excluded.

In the clinical setting, compromised Leydig cell function resulting in increased insensitivity to LH is defined as compensated hypogonadism (4), an entity associated with all-cause mortality (5). Therefore, investigating ibuprofen-induced compensatory hypogonadism is crucial, as this clinical state is generally associated with smoking and aging (4, 51). Moreover, compensated hypogonadic men present with an increased likelihood of reproductive, cognitive, and physical symptoms (4, 5254). Further characterizations of the state of compensated hypogonadism induced by ibuprofen, which was already established after 14 d of ibuprofen administration, are therefore important in determining the potential effects on healthy young men. Several reports have stressed the high level of long-term analgesic use among both amateur (55) and professional athletes; ibuprofen has been favored in this use and abuse (5659). Of note, an inverse relationship was recently reported between endurance exercise training and male sexual libido, but the possibility that medication uptake might interfere in this observation could not be totally excluded (22). Moreover, ibuprofen appears to be the preferred pharmaceutical analgesic for long-term chronic pain and arthritis (60). Therefore it is also of concern that men with compensated hypogonadism may eventually progress to overt primary hypogonadism, which is characterized by low circulating testosterone and prevalent symptoms including reduced libido, reduced muscle mass and strength, and depressed mood and fatigue (4, 60, 61).

Materials and Methods

In Vivo Intervention Trial.

Design and participants.

The in vivo study was designed as a double-blinded, placebo-controlled, randomized intervention trial in which ibuprofen or placebo was administered to subjects for 2 wk before and 30 d after a single exercise session. Staff not involved in the project prepared and distributed the medication in boxes weekly. Study personnel and participants were blinded to treatment, and all later analyses were performed blinded to the treatment type, participant, and time point. The study was part of a broader investigation also focusing on muscle biopsies, collected on days 0, 2, 7, and 30 postexercise, a subset of which is described elsewhere (62).

The study protocol was in compliance with the Helsinki Declaration, was approved by the Regional Scientific Ethical Committees of Copenhagen in Denmark (Ref: HD-2008-074), and was registered at ClinicalTrials.gov (no. NCT00832663). The study recruited 31 healthy white men, age 18–35 y. Subjects were included after an interview, a questionnaire assessing physical activity status, and the results of a screening blood sample. Exclusion criteria included body mass index above 30, knee injuries, peptic ulcers, signs of liver or kidney dysfunction, and participation in regular physical activity (especially strength training) apart from cycling as a means of transport. All individuals provided written informed consent to participate in the study. Subsequently, the subjects were assigned to either a placebo (17 subjects) or ibuprofen (14 subjects) group; the groups were matched for age, height, and weight.

Supplementation.

One group of subjects received ibuprofen, 2 × 600 mg/d, (Ibumetin; Nycomed Denmark Aps) for a period of 6 wk, from 14 d before to 4 wk after the electrical stimulation exercise. Ibuprofen was detected only in participants to whom ibuprofen was distributed and only after administration began. The second group received placebo pills (which were visually indistinguishable from the ibuprofen pills) over the same period. Subjects received the medication in Medidos No. 1 boxes (KiBodan A/S), which were refilled every week. To verify compliance, ibuprofen levels in the blood were determined by HPLC at every blood-sampling time point (see below). Additionally, to monitor liver and kidney function, blood samples were analyzed for creatine, C-reactive protein, alkaline phosphates, and total cholesterol during the study. No subjects reported any adverse signs of taking the medication, nor did any blood parameters indicate or suggest adverse effects.

Blood samples and hormonal analysis.

A 40-mL blood sample was collected from the antecubital vein of the nondominant arm when subjects arrived at the laboratory for screening. Samples were also taken at 3 and 2 wk before exercise (days −21 and −14), on the day of exercise before the exercise (day 0), after exercise (+2 h; day 0.1), and subsequently at 2, 4, 7, and 30 d after exercise. Hence, ibuprofen and placebo administration was ongoing for samples drawn on day 0, 0+2 h, 2, 4, 7, and 30. In the present study, we focused on samples drawn on day 0 after 14 d of supplementation. Plasma samples were stored at −80 °C until being assayed. The samples were analyzed for LH, FSH, testosterone, 17β-estradiol, AMH, inhibin B, and SHBG, as previously described in Aksglaede et al. (63), and ibuprofen as previously described in Farrar et al. (64). Of note, the LH measurements of one subject in the placebo group were aberrant and were discarded, thus decreasing the n value from 17 to 16.

Ex Vivo Organ Model: Testis Explant Assay.

To determine the direct effect of ibuprofen on adult human testis physiology, we used a validated organ model assay (Testis Explant Assay) (65). Testes were obtained from prostate cancer patients who had not undergone any antihormonal treatment or from multiorgan donors (average age 46.9 ± 4.4 y). The protocol was approved by the local ethics committee (Agence de la Biomédine; authorization no. PFS09-015), and informed consent was obtained from all donors or their next of kin.

After collection of each testis, the organ was placed at 4 °C and rapidly processed for experimentation. Observation by transillumination allowed us to discard the rare testes not displaying spermatogenesis. Four 3-mm3 testis explants were placed onto a polyethylene terephthalate insert (Falcon Labware) at the interface of air in 1 mL of DMEM (Sigma-Aldrich) in a well of a 12-well plate. The medium for exposure experiments contained 0.1% DMSO as a control and ibuprofen at different concentrations (purity >99%) (Sigma-Aldrich) in 0.1% DMSO. Four wells were analyzed for each condition. Exposures lasted 24 or 48 h, with a total medium change at 24 h, in a humidified atmosphere containing 5% CO2 at 34 °C. The culture medium was then collected and stored at −80 °C. On the day of collection, three testis explants for each culture condition were randomly selected and fixed in neutral buffer, 4% formaldehyde, or Bouin’s fixative for 2 h at 4 °C, embedded in paraffin, sliced into 5.0-µm-thick sections, and stored at 4 °C until immunostaining.

Hormone levels were assayed in duplicate in the culture medium. Testosterone levels were assayed with a specific RIA (Beckman Coulter). The intra- and interassay coefficients of variation were ≤8.6 and 11.9%, respectively. Control testis explants produced an average of 10.26 ± 2.50 ng testosterone per milliliter of explant after 24 h of culture and 8.47 ± 2.16 ng testosterone per milliliter of explant after 48 h of culture. INSL3 production was assayed with a commercial RIA (Phoenix France). Each sample was diluted twofold in RIA buffer before RIA. The intra- and interassay coefficients of variation were ≤15 and 7%, respectively, and the lower limit of detection was 20.17 pg/mL. Control testis explants produced an average of 0.29 ± 0.25 ng INSL3 per milliliter of explant after 24 h of culture and 0.20 ± 0.21 ng INSL3 per milliliter of explant after 48 h of culture. Inhibin B was assayed with a commercial ELISA kit (Beckman Coulter) according to the manufacturer’s instructions. Each sample was diluted twofold in sample diluent solution before reactions. The intra- and interassay coefficients of variation for serum samples were ≤5.6 and 7.6%, respectively. Control testis explants produced an average of 570.43 ± 78.50 ng inhibin B per milliliter of explant after 24 h of culture and 529.44 ± 79.17 ng inhibin B per milliliter of explant after 48 h of culture. PGD2 and prostaglandin E2 (PGE2) were assayed by an ELISA method (Cayman Chemical Company), as was AMH (Immunotech).

RNA was extracted from testes with the NucleoSpin RNA II kit (Macherey-Nagel) according to the manufacturer’s instructions and then was precipitated. Each total RNA sample (250 ng) was reverse transcribed with the Iscript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed according to the manufacturer’s instructions with iTaq Universal SYBR Green Supermix (Bio-Rad) and a 2.5-µL cDNA template in a CFX384 Touch Real-Time PCR Detection System (Bio-Rad). The amplification program was as follows: an initial denaturation of 3 min at 95 °C; 40 cycles of 10-s denaturation at 95 °C; and 30 s at 62 °C for annealing and extension. Dissociation curves were produced with the thermal melting profile performed after the last PCR cycle. To avoid amplification of contaminating genomic DNA, primer pairs were selected on two different exons. Bzw1 and GusB mRNA were used as internal controls for normalization. Results were calculated by the ΔΔCT method as n-fold differences in target gene expression with respect to the reference gene and the calibration sample.

To analyze steroidogenesis in the explants, we performed solid-phase extraction (SPE) with C18 cartridges, reagents, and solvents from Solvent Documentation Synthesis. Standard reference steroids were from Sigma. Deuterated internal standards were from Steraloids. Quantification was performed by isotopic dilution. Samples were spiked with 400 pg of internal standards (etiocholanolone-d5, 17a-testosterone-d3, dihydrotestosterone-d3, 19-androstenedione-d3, progesterone-d9, 17a-methyltestosterone-d3, and 17b-estradiol-d3). Samples were applied to a C18 SPE column (2 g stationary phase) previously conditioned with 10 mL methanol and 10 mL water. The column was washed with water (5 mL) and then with cyclohexane (5 mL), and the steroids were eluted with methanol (10 mL). The extracts were dried (N2, 458C), and 400 pg of external standard (norgestrel) was added. Derivatization procedures and measurements in GC-MS/MS in electronization mode were performed as previously described (66, 67).

Cells were labeled with the primary rabbit antibody directed against cleaved caspase-3 (1/100; Cell Signaling) (41, 42) to enable detection of cells undergoing apoptosis in the explants. Slides were then scanned with a NanoZoomer slide scanner (Hamamatsu Photonics). Caspase-3+ cells were counted by ImageJ software, and the results were expressed as percentages of the control values.

In Vitro: NCI-H295R Cell Line.

The NCI-H295R human adrenocortical carcinoma cell line was obtained from ATCC (CRL-2128), and experiments aiming at completing the ex vivo experiments using human adult testis explants were conducted in accordance with OECD guidelines (16). Steroids were analyzed after exposure to ibuprofen, abiraterone, and PGD2 (Sigma-Aldrich). Toxicity was evaluated with the Alamar Blue assay (Sigma-Aldrich). Deuterated steroid analogs were obtained for analysis from CDN Isotopes and Toronto Research Chemicals, and derivatization quality-control standards (7b-17β-estradiol-17-acetate), instrument control standards (estrone-3-methyl ether), and derivatization reagents (N-trimethylsilylimidazole, N-methyl-N-trimethylsilyl-trifluoroacetamide, and 1,4-dithioerythritol) were obtained from Sigma-Aldrich. Steroid extraction and the subsequent quantification of steroid hormones were performed according to Holm et al. (68).

For prostaglandin secretion analysis, the NIC-H295R cells were plated in 12-well plates and were exposed overnight; the global prostaglandin synthesis was evaluated immediately afterward with a Prostaglandin Screening ELISA Kit (Cayman Chemical Company).

For transcriptional analysis with RT-qPCR, RNA was isolated from cells with the All Prep DNA/RNA kit (Qiagen). RNA integrity was measured on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific), and only 260/280 and 260/230 ratios >2.0 were accepted for further processing. Quantitative PCR was performed as previously described.

Statistical Analysis.

For the trial, each individual’s samples were normalized by division by the mean of the baseline samples drawn before the intervention. Hence, samples from each volunteer were normalized with the individual’s own baseline values before the administration. Unpaired Student t tests were used to compare the placebo and ibuprofen groups after 14 and 44 d of administration. For the ex vivo experiments, data were compared using the Mann–Whitney U test and slopes with P values and Spearman correlation when indicated. For in vitro cell experiments, analysis was performed with one-way ANOVA followed by a post hoc Dunnett’s multiple comparison test. All data are expressed as mean ± SEM, and differences were considered statistically significant when P ≤ 0.05.

Acknowledgments

We thank the staff at the Pontchaillou Hospital (Rennes, France) for help with organ donation and the men who participated in the intervention trial (Copenhagen). Funding for this study was provided by a Nordea Foundation Healthy Ageing Grant, The Lundbeck Foundation, The Danish Council for Independent Research (Medical Sciences), INSERM, University of Rennes 1, the School of Public Health, and Agence Nationale de Sécurité du Médicament et des Produits de Santé Grant AAP-2012-037. No funding bodies had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

  • Author contributions: D.M.K., C.D.-L., A.L.M., M.D.D., S.M.-G., M.K., A.J., and B.J. designed research; D.M.K., C.D.-L., A.L.M., M.D.D., C.H.H., B.S., J.-P.A., B.L.B., C.P., L.L., S.M.-G., M.K., and A.J. performed research; D.M.K., S.B., and B.J. contributed new reagents/analytic tools; D.M.K., C.D.-L., F.D.M., C.H.H., B.S., J.-P.A., B.L.B., C.P., A.H.-S., T.K.J., L.L., S.M.-G., K.K., S.B., M.K., A.J., and B.J. analyzed data; and D.M.K., C.D.-L., and B.J. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at http://ift.tt/2D7KGuv.



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