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Dust Cyclone Monster
Remember Dr. Frankenstein when he had the mad look on his face and screamed, it’s alive! Well, when I hit the switch and started sucking up dust I’m sure I had the same look on my face and was screaming it works! I found that Electrolux vac. in a flea market and it was marked 3 bucks. I asked them if I could plug it in to see if it worked or not. It did and had pretty darn good suction and the bag was half full. I tripped off to WM and got some new bags and pretty much forgot about it. Well one day sitting around in the shop I noticed that old water jug that has been there for ever, never Know when something like that will come in handy. Anyway I started studying on it and even did a test fit of the hose stuck into the opening. To my surprise the hose went in and was a nice tight fit, almost collapsed the jug when I fired up the vac, so the wheels in my head really started turning. The barrel I picked up from the local used barrel dealer for 5 bucks and a couple of fittings from Lowe’s and I was off to the shop to see if it would all come together and work. It does a real good job with all but, the really fine dust, it’s makes a few hot laps around the jug and then off to the filter bag. All in all I’d call it a success! The old Electrolux does a lot better than I had hoped for. I did hook up the Shop Vac just to see and man-o-man did the dust really make hot laps around inside the jug! I’ve already started thinking about a rebuild on it but really hate to, it works so good! I did add a couple of 45 degree elbows to direct the dust downward but, it’s not as much fun to watch now!
I love it!It’s good to see other people re-purposing assorted junk to make useful stuff. It has long been a favourite passtime of mine.Just for fun, have a look at the DC I just put together. It should make you smile like yours did for me.http://lumberjocks.com/shipwright/blog/76186
Nice way to make a cyclone.I saw one of those jugs the other day and should have picked it up.I build a wood cyclone and it works great but it would be so much more fun if I could see it getting the job done.
It was fun putting it all together! I just love taking stuff I have laying around, adding a few new things that I can pick up on the cheap and turning it into something useful. And hopefully light the fire in someone else to come up with their own version. This one was inspired by other LJ’s version of a cyclone on the cheap. I’ve seen some made out of traffic cones and one made from a cardboard concrete form and htl’s version made of wood, which I might add was total craftsmanship on his part! I don’t think I could have build it the way he did. And then there is Paul’s dust control Monster, Paul I wish I had the space for a system that large! Great job! All in all it was just a fun learning project!!
My wife ”The Crazy Cat Lady” asked me today why I was so excited about making it into the daily top three and I had to think about the answer a little while. Well, what I came up with was, it’s having others look at and approve of what you have done. I told her that this site makes me think of the old time General Store where all the men folk would gather around the cracker barrel and cuss and discuss what was going on and what they were working on.
Really this has no deep craftsmanship to it and really doesn’t belong in the daily top three. There are much better projects then this, heck there’s not even that much wood in it. I only posted it as a project so maybe someone else would be inspired to build one from junk they have on hand!
Really this has no deep craftsmanship to it and really doesn t belong in the daily top three. There are much better projects then this, heck there s not even that much wood in it. I only posted it as a project so maybe someone else would be inspired to build one from junk they have on hand!
- OldGuysRule
The fact that is ended up in the Top 3 is proof that it belong there. That happened because what you did interested enough people that they wanted to look at it. We are all glad you shared it and it definitely makes us think out of box more. Personally, I oogle more over things like this than seeing it’s $10k counterpart. Seeing people get creative with “junk” just proves it doesn’t take moeny to do this hobby – just problem solving skills |
VEGF-targeted therapy and beyond: pharmacotherapy and emerging treatments in agerelated macular degeneration.
Treatment of age-related macular degeneration (AMD) has changed dramatically over the last decade. It has evolved from primarily destructive therapies (laser-based treatment strategies) to nondestructive therapies (intravitreal pharmacotherapies that target angiogenesis). Intermittent intravitreal ranibizumab, an inhibitor of VEGF, is currently the gold standard of care for neovascular AMD as it is the only treatment where a significant proportion of treated eyes have been shown to experience improvements in visual acuity. As our understanding of the pathological and molecular mechanisms involved in AMD increases, multiple new treatments are emerging. Distinct therapeutic approaches are being used, including inhibition of oxidative stress, interference with the visual cycle and immunomodulation. Targets within the complex cycle of angiogenesis include upstream pathways, such as mTOR and HIF-α, and downstream targets include integrins, PDGF and receptor tyrosine kinases. |
. Solve 2*f = p*h - 6, z = 5*f + 12 + 3 for h.
0
Let i = 48 - 94. Let h = i - -46. Solve d + 15 = -2*m - h*d, 25 = -4*m - d for m.
-5
Suppose 0 = 4*y - 92 - 20. Suppose 2*u + 1 + 3 = 0, u - y = -2*o. Solve 4*s - s - 12 = -4*a, -5*a = 5*s - o for s.
0
Suppose 5*o + 2*n - 20 = 0, 5*n = -4*o - 8 + 41. Solve 5*t - 5 = -6*f + o*f, -f - 2*t + 5 = 0 for f.
-5
Let m = -228 - -234. Solve h + 4*c + m = 0, -4 = 4*c - 0*c for h.
-2
Suppose 84*t = 62*t + 110. Solve x = -f + 2*x + 6, 5*f + t = -2*x for f.
1
Let u be (-3)/(-3)*(4 + 2). Let k be u/9 - (-368)/(-12). Let c be (30/(-25))/(4/k). Solve -3*x = -5*a + 13, 0*a - c = -x - a for x.
4
Let j = 5 - -13. Suppose -2*a + j + 24 = 0. Let r = -16 + a. Solve 4*c + c + r = -l, -4*c - 4 = 3*l for l.
0
Let l be 1/((11/(-30))/(-11)). Let d be 1250/l - 4/6. Suppose 3*h - 2*y = d, 5*y = -5*h + 83 + 2. Solve -3*i = 3*s + h, 0*i = i + 3*s + 13 for i.
-1
Suppose 0 = -0*b - 2*b. Solve 4*k - 3*w + 20 = b, -3*w = 5*k - 3 + 1 for k.
-2
Suppose -2*k - z + 39 = 0, 0*z + 94 = 5*k - z. Let q be (-3)/(-6)*-1*0/(-1). Suppose q = -y - 0*y + k. Solve 3*g - 4*b + y = 0, g = -4*g - b - 1 for g.
-1
Let o(d) = -d**2 - 5*d + 10. Let q be o(-6). Suppose -5*k + 93 = -b - 16, 0 = -q*k + b + 88. Solve 3*s + k = 3*y, 0 = -3*s + 4*y - 0*y - 23 for s.
-5
Let g = -261 + 265. Solve 5*u + 3*l = -28, 21 = -g*u - 2*l + l for u.
-5
Let p = -68 - -68. Solve 5*w + 3*k + 20 = p, -4*w - w - 5*k - 30 = 0 for w.
-1
Let q(s) = -s**2 + 20*s - 86. Let j be q(13). Solve j*n = -0*c - c + 15, 4*c - 2*n = -6 for c.
0
Let z(t) = t**3 - 3*t**2 + 6. Let o be z(3). Solve 0 = 3*y + 3*c - o, -8 = -3*y - 2*y - 4*c for y.
0
Let y(s) = -s**3 - 3*s**2 + 6. Let a be y(-3). Suppose -a*l + 3 = -21. Solve -2*p - 5*i + 24 = 0, -18 = p - l*i - i for p.
2
Suppose -18 = 5*x - 8. Let o(t) = -t**3 - 2*t**2 - t + 1. Let h be o(x). Solve 3*g + l + 15 = 0, 2*l - 4 - 2 = h*g for g.
-4
Let z(a) = 3*a - 3*a + 0*a - a - 27. Let r be z(0). Let g = -19 - r. Solve 3*j - g = 7, -22 = -3*m - 2*j for m.
4
Let s = 322 - 320. Solve -3*b + 3*r = -2*b, -4*r - 2 = -s*b for b.
3
Let z be 16/(-56) + 23/7. Let x = 6 - 1. Let i be (4 + -5)/((-1)/x). Solve -m = -z*g - 0*g - 9, -i*g - 14 = -2*m for g.
-4
Suppose -4*z = -3*m + 4, -3*m = -5*z + 4*z - 1. Solve m = -2*d - 4*s - 6, 0 = -15*d + 10*d - 2*s + 1 for d.
1
Suppose -8*k + 0*k = -32. Suppose k = 2*h + 2*d, 0 = -2*d - 0*d - 2. Solve h = 3*n - 4*g + 2, -2*g - 4 = 2*n for n.
-1
Let c(j) be the third derivative of j**6/120 + 2*j**5/15 + j**4/4 - j**3/2 - 2*j**2. Let p be c(-7). Solve z - 2 = -4*r + 12, p*r = -5*z + 22 for r.
3
Let q = -18 + 21. Suppose -b = 3*b - 16. Suppose -4*m = 3*h - m - q, 4*m - b = 4*h. Solve 4*y + y = -3*t, h = 3*y - 2*t - 19 for y.
3
Suppose -22*p = 5*p - 243. Solve -4*w + 3*d + 9 = 0, 0 = -7*w + 3*w - 3*d - p for w.
0
Let f = -1527 + 1533. Solve g + 4*m = -16, 3*g = -3*m - 18 + f for g.
0
Let n be -15*4/60*-5. Solve 3*q = -l + 13, -9 = -n*q + 2*l + 20 for q.
5
Suppose 3*d = -l + 15, -3*d + 5*l = 2*d - 45. Let j = d - 2. Solve 5*x + 3*w - 5*w + 14 = 0, -3*x + j*w - 14 = 0 for x.
-2
Let w = -50 + 12. Let l = 57 + w. Solve 3*y - 7*g = -3*g + l, 0 = 5*y + 3*g + 7 for y.
1
Let b(y) = -2*y + 1. Let p be b(3). Let h be ((-2)/p)/(4/20). Let t = 2 - h. Solve t = 5*v + 4*d - d - 14, -2 = v + 3*d for v.
4
Let o(v) = -4*v - 54. Let t be o(-14). Solve t*s - 6 = -2, -10 = 2*y - 2*s for y.
-3
Let t(n) = -8*n**2 + 127*n + 21. Let b be t(16). Solve -y - 4*o + 19 = 0, 0 = -b*y + 4*o - 1 - 0 for y.
3
Suppose -26*q + 2 = -27*q + 2*r, 2*q + 2*r = 14. Suppose 2*p = -5*j + 26, -p = -5*p + 2*j + 4. Solve -h + 14 = q*o, -p*h = 2*o - 8 - 4 for o.
3
Let r(o) = -4 + 2*o - 2*o**3 - 59*o**2 + 57*o**2 + 6*o**3 + 3. Let x be r(1). Solve -5*y = 5*b + 10, x*b + 2*y + 2*y = -3 for b.
-5
Let m(k) = -4*k**2 + 22*k + 13. Let q be m(6). Let s be -3 + (q - -2) + 0. Solve -5*x - g + 25 = 0, 4*x + s*g + 5*g = 20 for x.
5
Let m = 14 + -9. Let s(a) = -3*a**2 + 14*a - 3. Suppose 0*v = 3*v + 4*t - 20, -2*v - 2*t = -12. Let l be s(v). Solve m*q + 5*j = 20, -16 = -l*q - 2*j + j for q.
3
Suppose 0 = 4*k - 2*f - 2, 5*k + f + 6 = 26. Suppose 2*n + g + 0*g = 41, -2*n + 2*g + 32 = 0. Solve 5*o = 3*i - n, 2*i - i - k = 0 for o.
-2
Let m(i) = -22*i + 71. Let u be m(3). Solve 4*z = u*b - 9*b - 8, 2*b = 5*z + 24 for z.
-4
Let d = -2496 + 2501. Solve 5*t + 20 = 5*v, d*v + 3 = 2*v for t.
-5
Let a(x) = -x**2 + 9*x - 6. Let f be a(8). Solve -5*r = -5*s - f - 3, 0 = 5*r + 4*s + 31 for r.
-3
Let v = 588 - 580. Solve -v*l + 3*l + n = -17, 0 = l - 4*n + 8 for l.
4
Let c be -1*((-16)/(-2))/(-2). Suppose 0 = 3*m + 2*o + 82, 79 = -3*m - 4*o - o. Let x be 8/m*(-38 + 3). Solve -f = -5*t + x, -3*f - c*t = -f + 6 for f.
-5
Let x be ((-2)/5)/(4 + (-164)/40). Solve -3 = 2*r - 3*a, -r - a = x*a - 5 for r.
0
Let o be (-8)/(20/8 - (-3 - -6)). Solve -6 = 3*u, o = -5*z + z + 2*u for z.
-5
Suppose 63 = 2*i + 293. Let y = -112 - i. Solve -2*j + 3*j = -2*f, 0 = -y*j - 4*f for j.
0
Let r be ((-2)/(-4)*0/(-31))/(-1). Solve r = -3*h + x + 20, -5*h + 0 + 20 = x for h.
5
Let a = 19 - 17. Suppose -a*w + 3 = -3. Solve -q - 5 = -0*q, -j + 13 = -w*q for j.
-2
Let x(o) = -o**3 - 13*o**2 - 12*o + 9. Let b be x(-12). Let m(u) = u - 9. Let z be m(b). Solve z = l + s + 2, 0*l - 4*s = l - 4 for l.
-4
Let h(l) = -l**2 + 9*l - 10. Suppose -2*p + 4*p = 14. Let b be h(p). Solve 4*u - o + 20 = 0, -2*o + 2 = -b*u - 18 for u.
-5
Suppose -3*h - 3 = -21. Suppose 305 + 47 = -2*q. Let w = q - -181. Solve w*l - 4*x + 7*x = h, l + 2*x = -3 for l.
3
Let a = -27 - -44. Let r = 22 - a. Suppose -5*t = r*b, -2*t - 6 = 4*b - 5*b. Solve -3*d - 14 = 2*i + b*i, -4 = 2*i for d.
-2
Let n be ((-8)/(-6))/(18/(-27)). Let h be 1 + -1 + (n - -13). Solve -h + 3 = 4*v + 5*t, -3*t = -4*v + 24 for v.
3
Let a = -1455 - -1488. Solve 5*g - 4*r = a, 2*g + 13 = 5*g + r for g.
5
Let u = 56 - 57. Let o be u/(-4)*(-9)/(27/(-24)). Solve -2*a = -3*a + 3*q - 16, 0 = a - o*q + 11 for a.
-1
Let g be (0 - 34/(-10)) + 2/(-5). Solve -4*v - 4 = -4*z - 2*v, -3*z - g*v - 15 = 0 for z.
-1
Let t(f) = -4*f**3 - 2*f**2 - 2*f + 2. Let j be t(-2). Let n = -451 + 440. Let u = j + n. Solve 0 = -5*q - r + 21, 0*q + r + u = 3*q for q.
5
Suppose -3 + 2 = -i. Suppose -45*t = -43*t. Suppose 16 = 4*q - 4, t = k + q - 8. Solve -k*p = 3*z + 9, -3*z + 0*z = p + i for p.
-4
Suppose 5*t - 98 + 33 = 0. Let k = 13 - t. Solve -2*s - 5*r + 14 = k, r = -5*s - 14 + 3 for s.
-3
Let t be 1*(-10)/15 + 8/3. Solve -t*o - o - 5*w = -1, o - w = 3 for o.
2
Let q be ((-10)/(-3))/(-12 + 380/30). Solve 2*f + 5*a + 9 = -f, q*a = 0 for f.
-3
Suppose -3*z - 5*b + 19 = -4*b, -3*z = -3*b - 3. Solve -i - z = 4*a, -a + 16*i - 15*i = 0 for a.
-1
Suppose -28*f - 1 = -33*f - 3*v, 2*v = 5*f - 16. Solve h + 2*d = 0, -26*h + 4*d - f = -29*h for h.
2
Let h = 311 + -306. Solve h*u - 27 = -p + 3, -25 = -4*u - p for u.
5
Let l = -541 + 543. Solve 2*d = l*s + s + 7, s + 24 = 5*d for s.
1
Suppose -2*d + 4 = -0*d. Suppose -2*o - 4*y = -y - 15, -d*y = -o - 3. Solve 0 = -z - 2*s - 5, 5*s - o = 6*s for z.
1
Suppose -9*f + 40 = -7*f. Suppose 0*i = 4*i - f. Suppose 4*s + t - 16 = -0, -s + 5*t = -4. Solve s*g = -h - 11, 3*h - 2*g + 7*g = -i for h.
5
Suppose -4*p = -2*c - 18, -24*c + 22*c = -5*p + 21. Solve -5*i + 19 = -4*z + 9, 0 = p*i - z + 1 for i.
-2
Let k(t) = t**2 - 10*t + 20. Let r be k(8). Let w be 8/(r - 3) + -2. Suppose -w*j = -2*j - 5*s, 3*j - 2*s = 0. Solve m - 3*p + 5 = j, -3*p + 7 = -m + p for m.
1
Let q be 2 - 3/((-21)/14). Solve 0*h - z + 13 = q*h, 2 = -h + 5*z for h.
3
Let z be (5 - -1 - -3) + (-6 - -4). Suppose 3*h - z = -1. Solve -2*u = 3*p - 4*p + 5, -u = h*p - 5 for u.
-1
Suppose 0 = -3*s - 30 - 30. Let w be (-2)/(-8) + 5/s. Suppose m + 4*k - 8 - 32 = w, 0 = m + k - 34. Solve -4*u - 4*a = -m, 2*a + 35 = 5*u + 6*a for u.
3
Let d = -174 - -177. Solve j = -s + 1, s = j + d*s for j.
2
Suppose -10 = -j + 22. Suppose 10*w - 2*w - j = 0. Solve -x - w*x - 9 = -a, -x = 2*a + 4 for x.
-2
Let w be (-10)/(-4) - (-3)/(-6). Suppose -7*n - 29 = -57. Solve -3*q = -3, 0 = -w*g - 2*g - n*q + 16 for g.
3
Let y = -5 - -8. Let l be 3 - (2/1 + -3). Let s(c) = -c**3 + 5*c**2 - 4*c + 3. Let j be s(l). Solve -4*k - 16 - 15 = y*q, -q + 7 = -j*k for q.
-5
Suppose 4*b - 74 = -33*b. Solve -y - b*t = 4, -2 = -5*y + 4*t - 3*t for y.
0
Let h(c |
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Re: REAL HOME BASED WORK (Harlem / Morningside)
Here we go again! These people are scammers, do not fall for their lies:
What they’re not telling you is that once you earn $150, you either have to refer 40 people or buy your referrals in order to get your money. And you can only buy your referral …
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Craigslist is full of people having a discussion about various posts they come across, in the form of Re: posts. This site is dedicated to providing a continued discussion forum, as well as archiving those discussions. You decide who's the idiot ;) |
It’s ready. Whatever you have in mind for this weekend, our 35 ST can handle it. The 35 ST continues to dominate the offshore scene with aggressive lines that tout an elevated level of confidence.
Features include a one piece level deck from bow to stern, twin raised livewells, walk-through transom, self-bailing cockpit, and a walk-in step down console to name a few.
Whether it’s a kingfish tournament off North Carolina or billfish tournament off Miami, this is the boat that can put you in the winner’s circle. |
Perioperative management of the patient with cardiac disease.
The overall incidence of perioperative death is relatively low. However, patients with coronary artery disease are at higher than average risk of perioperative cardiac complications. Thus, preoperative testing for cardiac disease should be done in certain patients in an effort to reduce postoperative mortality and morbidity. Patients who require emergent orthopaedic surgery are at greater risk of perioperative cardiac events than are those who undergo elective procedures. Certain modalities, such as beta blockers, statins, and alpha-2 agonists, may be started or continued in the postoperative period to further enhance cardiac function. We review the current recommendations for preoperative cardiac testing in orthopaedic patients and for perioperative management of orthopaedic patients with known cardiac disease. |
DU cancels lecturer's appointment -The Asian Age
The appointment of a lecturer at Sociology Department of Dhaka University (DU) has been cancelled in allegations of biased recruitment. The university's highest policy-making board, the syndicate, also relieved two teachers as they did not join even after their PhD leave expired.
The decisions were taken at the syndicate meeting on Monday night, syndicate member and Dean of Faculty of Engineering and Technology of the university Dr Md Hasanuzzaman confirmed.
The sacked teachers are- Assistant Professor Masfiqur Rahman Khan and Lecturer Tamanna Afrin Rimi. Meanwhile, the appointment of SM Fazul Haque Isan at Sociology Department has been cancelled. Allegation ran rife that the department did partiality to make him a lecturer.
On 13th August, DU Pro-VC (Academic) Prof Dr Nasreen Ahmad held viva-voce for the appointment of three lecturers of Sociology Department at her office. Other candidates alleged that Isan was recommended bypassing more competent, higher CGPA holders, and even candidates with foreign degrees.
The syndicate cancelled the appointment upon observations of its members. The syndicate has also appointed two- Israt Jahan Yaman and Wasifa Tasnim Shamma - who were ahead with higher CGPA.
Among them, Yaman's Honors and Master's CGPA is 3.74 and 3.88 respectively, Wasafia Shammar CGPA is respectively 3.72 and 3 83. Both of them were first in Honors and Master's. On the other hand, SM Fazul Haque Isan was 9th in Honors and Master's result. |
Contents
Synopsis
While resting one night Ash and co. were awakened by a mysterious light in the night sky. The next day they heard about a man with connections to aliens named Professor Icarus, who lives isolated, except with an Elgyem he found. Team Rocket has plans for the Elgyem. Will Elgyem return home, or stay with the professor?
Episode Plot
During the night, the heroes sleep. Suddenly, they wake up, as a strong wind is blowing. A strong light appears and goes beyond a mountain. Cilan sees it is time for deduction. He knows most reports of these UFO-s are flawed, but sees it couldn't have been a satellite, nor a plane. Ash sees Cilan knows a lot, so Cilan explains he is also a science Connoisseur. Ash believes the UFO contained space aliens, but Cilan doubts that, for it is not based on science facts. Next day, the heroes are at a diner and learn they are at Area 28, the area Cilan remembers as the place with numerous sightings of UFO-s. A customer reports there is Prof. Icarus, which Cilan remembers as the one being an expert on UFO-s, but the others think he is a strange scientist.
The bartender remembers there was a mailman that received a headache and started seeing things, scenes from outer space. They all went to Prof. Icarus, thinking his research may affect things around. However, Icarus was very hostile and refused them to investigate his house. Both the bartender and the customer also felt a powerful headache. Cilan wonders about these reports, thinking a psychic power was used to transmit the images from outer space into the mailman's brain. Cilan sees they need to investigate; after all, the most logical conclusion is to explain the supernatural events. They cross a bridge, but a Pokémon from a house sees them. Suddenly, the heroes are struck by an image of Ash falling down the bridge. Ash knocks on the logs and they see the bridge is dangerous and the image presents the future.
The Pokémon teleports away, while Team Rocket saw that. Meowth plans to study its movements to catch that Pokémon. They know it is from outer space, making it rare. They come to Icarus' house, where Cilan is pleased to meet the Prof. himself. He asks Icarus if he saw the UFO last night, but Icarus replies they should leave. Cilan admits he read Icarus' book and wants to know the progress on Icarus' research. Icarus replies it is known UFO can be flown anywhere using dark matter. Cilan explains it is a substance immune to gravity. Icarus tells he found a way to manipulate the dark matter, but it needs dozens of years to prove the theory. Cilan knows through experimentation, even being ridiculed, can the theory be advanced, for these are the words Icarus wrote. Icarus invites them for a drink, which the heroes accept.
Cilan likes how Icarus can explain the science of UFO-s easily. Ash wants to read the book, but Iris doubts he could even read the first page. Cilan tells he hasn't seen Icarus on TV lately, but Icarus admits he is not much into that anymore. Suddenly, they are granted a vision of an explosion. The heroes follow Icarus to the lab and see a saucer, while Icarus tries to disable the machine that is about to blow up. The heroes try to disable the saucer, but a strange power unplugs it and prevents the explosion. The heroes see an Elgyem, the one who helped them unplug the saucer and send visions. Icarus repairs the saucer and admits he likes UFO research he became a professor on astrophysics. He quit the university to make his own saucer, but used propellers, knowing real saucers manipulate dark matter to travel around space.
He clarifies the sightings of UFO-s were actually of his saucer, but there were sightings even before he came to this area. About six months ago, his saucer crashed and he found an Elgyem, wounded. He brought it back to health. Though Elgyem was frightened of Icarus, they eventually became good friends. Later, however, Icarus received a vision of outer space. He thought Elgyem sent the vision because it wanted to return home, but Icarus was uncertain of that. Once Elgyem started using Telekinesis, Icarus started researching if it is related to dark matter. Soon after, the people around the lab started gaining visions Elgyem sent them. The heroes think Elgyem is lucky to meet a man with love for UFO-s. Suddenly, Team Rocket knock on the door and claim a new machine was invented to manipulate dark matter, gaining Icarus' attention.
Team Rocket uses a new device to capture Elgyem and freeze the heroes and Icarus. Team Rocket removes the disguises and flies off with Meowth. Cilan sends Dwebble, whose X-Scissor destroy the device that binds them. The heroes decide to help Icarus get Elgyem back using the saucer. Icarus powers the saucer up and flies off, catching up to Team Rocket. Jessie sends Woobat and Ash Tranquill. They both use Gust, but Tranquill manages to hit Woobat. James sends Yamask, who hits Tranquill with Shadow Ball. Axew uses Dragon Rage to hit Yamask back, while Cilan sends Pansage, who uses Bullet Seed to free Elgyem. Elgyem teleports to Icarus, while Pikachu uses Electro Ball, destroying Team Rocket's machine. Team Rocket uses jetpacks to fly off, while Yamask uses Nightshade, causing the saucer to malfunction. The saucer flies down a cliff, though Elgyem saves them using Telekinesis to levitate the saucer.
Later, Icarus thanks Elgyem, knowing he will repair the saucer and return Elgyem to outer space. Icarus claims even if he is wrong that Elgyem is from outer space, he will find it out on the trip. Suddenly, they see Elgyem's memories, so Icarus sees Elgyem wants to stay with him instead. The heroes bid the professor and Elgyem farewell and see a light in the sky moving. |
Pastor Rob Bell, the controversial former megachurch leader, has admitted in a new interview that he never felt comfortable in an evangelical environment, and accused evangelicals of showing their real values by largely supporting President Donald Trump.
“Even when I was a pastor in a local church, that seemed like a strange freak-show,” he said.
Tweet to always embed in articles
No matter who you are or what you have done, you are qualified to be a part of God’s family.❤❤
The former leader of Mars Hill Bible Church in Michigan, before attracting controversy for supporting same-sex marriage and for his 2011 book Love Wins, which challenged Christian beliefs on Hell, told The Church Times in an interview published on Thursday that the label of “heretic” has become somewhat of a badge of honor for him. |
African American-white differences in lipids, lipoproteins, and apolipoproteins, by educational attainment, among middle-aged adults: the Atherosclerosis Risk in Communities Study.
Measures of socioeconomic status have been shown to be related positively to levels of high density lipoprotein (HDL) cholesterol in white men and women and negatively in African American men. However, there is little information regarding the association between educational attainment and HDL fractions or apolipoproteins. The authors examined these associations in 9,407 white and 2,664 African American men and women aged 45-64 years who participated in the Atherosclerosis Risk in Communities Study baseline survey, and they found racial differences. A positive association for HDL cholesterol, its fractions HDL2 and HDL3 cholesterol, and its associated apolipoprotein A-I was found in white men and white women, but a negative association was found in African American men, and there was no association in African American women. In whites, there was also an inverse association of low density lipoprotein (LDL) cholesterol and apolipoprotein B with educational attainment. With the exception of African American men, advanced education was associated with a more favorable cardiovascular lipid profile, which was strongest in white women. Racial differences in total cholesterol (women only), plasma triglycerides, LDL cholesterol, apolipoprotein B (women only), HDL cholesterol, HDL2 and HDL3 cholesterol, and apolipoprotein A-I were reduced at higher levels of educational attainment. Apart from triglycerides in men and HDL3 cholesterol in women, these African American-white lipid differences associated with educational attainment remained statistically significant after multivariable adjustment for lifestyle factors. Lipoprotein(a) showed no association with educational attainment. These findings confirm African American-white differences in lipids, lipoproteins, and apolipoproteins across levels of educational attainment that were not explained by conventional nondietary lifestyle variables. Understanding these differences associated with educational attainment will assist in identifying measures aimed at prevention of cardiovascular disease. |
Respiratory impedance in children with cystic fibrosis using forced oscillations in clinic.
Measurement of lung function is an important component of clinical management in cystic fibrosis (CF), but has been difficult in young children. The present study aimed to characterise the utility of the forced oscillation technique for measurement of lung function in preschool-aged children with CF in a routine clinical setting. Lung function was assessed in 56 young children (aged 2-7 yrs) with CF. Respiratory system resistance (R(rs)) and reactance (X(rs)) at 6, 8 and 10 Hz were measured and expressed as Z-scores. Children were classified as asymptomatic or symptomatic based on an administered respiratory questionnaire and physical examination at the time of testing. Between-test repeatability was assessed in 25 children. Measurement of lung function using the forced oscillation technique was feasible in the CF clinic. The children with CF, as a group, had Z-scores for R(rs) at 6 Hz (R(rs,6)) R(rs,8), R(rs,10), X(rs) at 6 Hz (X(rs,6)) and X(rs,8) that were significantly different from zero. Children with current symptoms showed significantly decreased X(rs) and increased R(rs,6) compared with asymptomatic children. Measurement of lung function using the forced oscillation technique is feasible in young children with cystic fibrosis in a clinical setting. The technique has the potential to improve knowledge concerning early cystic fibrosis lung disease. |
Africa is Better Placed Than Ever for Investment
JOHANNESBURG, South Africa, Nov 18 2019 (IPS) - The Presidents Cyril Ramaphosa of South Africa, Paul Kagame of Rwanda, Nana Akufo-Addo of Ghana and Prime Minister Agostinho do Rosario of Mozambique engaged in a discussion titled, Invest in Africa’s Space: Conversation with African Heads of State, moderated by Dr. Victor Oladokun, African Development Bank Group Director of External Relations and Communications, at the Africa Investment Forum, Johannesburg, 11 November 2019.
President Cyril Ramaphosa identified infrastructure, energy, manufacturing and tourism as the sectors where the most investment opportunities exist in South Africa.
Rwanda’s President Paul Kagama said his country has created a conducive investment environment through good governance systems and security, and according to the World Bank, it is the second easiest African country with which to do business.
For Ghana’s President. Nana Akufo-Addo, the Africa Continental Free Trade Area remains a priority. He says his government is working to strengthen the country’s macro-economy. The country’s current priorities are infrastructure, agriculture and mineral resources.
Prime Minister Agostinho do Rosario representing the President of Mozambique, said his government is open to investment, fighting corruption and has improved transparency. |
Cervico-facial actinomycosis. A retrospective study.
A retrospective investigation of 25 cases of verified cervico-facial actinomycosis recorded in the period 1971--76 is presented. The results have been compared with the findings from a previous examination carried out by one of the authors during 1955--64. A marked increase of cervico-facial actinomycosis was noted. The clinical picture seems to be changing to a more alarming appearance, in agreement with the classical description of this entity. According to this agreement with the classical description of this entity. According to this a prolongation of the period of treatment was found. Other aspects of the disease are discussed based on the present results. The recommended treatment is still a combination of antibiotic medication and surgical removal of infectious foci. |
Download Download (last few seconds were clipped)Keith Olbermann lets the Secretary of Homeland Security, Michael Chertoff, know how insecure the homeland must be if we are to rely on his "gut."
So there are your choices: bureaucratic self-protection, political manipulation of the worst kind, the dropping of opaque hints, a gaffe backfilled by an "instant report," or the complete disintegration of our counter-terror effort. Even if there really is never another terror attempt in this country, we have already lost too much in these last six years, to now have to listen to Michael Chertoff's gut, no matter what its motivation.
John Amato: "I wrote about Chertoff's "gut" remark the other day which resonated throughout the blogosphere and said he should be fired for it. O'Reilly liked it because he's paid to say that about "Chertgut."....Olbermann had a few choice words of his own." Transcripts below the fold...
And now as promised, a Special Comment -- on Michael Chertoff's gut.
You have by now heard the remark -- instantly added to our through-the-looking glass lexicon of the 21st Century, a time when we suddenly started referring to this country as "the homeland" as if anybody here has used that term since Charles Lindbergh or the German-American Bund in 1940.
Michael Chertoff's "gut feeling."
Which, he took pains to emphasize, was based on no specific nor even vague intelligence -- that we are entering a period of increased risk of terrorism here.
He got as specific as saying that Al-Qaeda seems to like the summer, but as to the rest of it, he is perfectly content to let us sit and wait and worry - and to contemplate his gut.
His..... gut!!
We used to have John Ashcroft's major announcements.
We used to have David Paulison's breathless advisories about how to use duct tape against radiation attacks.
We used to have Tom Ridge's color-coded threat levels.
Now we have.... Michael Chertoff's gut!
Once, we thought we were tip-toeing along a Grand Canyon of possible and actual freedoms and civil liberties destroyed, as part of some kind of nauseating but ultimately necessary and intricately-designed plan to stop future 9/11's or even future Glasgow car bombers who wind up having to get out and push their failed weapons.
Now it turns out we are risking all of our rights and protections -- and risking the anger and hatred of the rest of the world -- for the sake of Michael Chertoff's gut.
I have pondered this supreme expression of diminished expectations, for parts of three days now. I have concluded that there are only five possible explanations for Mr. Chertoff's remarkable revelations about his transcendently important counter-terrorism stomach.
Firstly, Mr. Chertoff, you are as Richard Wolffe said here the other night, actually referencing not your gut but your backside -- as in, "covering it." C-Y-A.
Not only has there not been a terrorist attack stopped in this country, but your good old Homeland Security hasn't even unraveled a plausible terrorist plan.
And you and your folks there have a different kind of stomach pain, knowing that with a track record that consists largely of two accomplishments -- inconveniencing people at airports, and scaring them everywhere else -- your department doesn't know what the hell it's doing, and even you Mr. Chertoff, know it.
Secondly, of course, there is the explanation of choice for those millions of us who have heard the shrill and curiously timed cries of wolf over the past six years -- what we've called here "The Nexus of Politics and Terror" -- that there isn't anything cooking, and your "gut feeling" was actually that you'd better throw up a diversion soon on Mr. Bush's behalf, or... something real -- like the Republicans' revolt about Iraq, and the nauseating "gut feeling" that we have gotten 3,611 Americans killed there for no reason -- was actually going to seep into the American headlines and consciousness.
It's impossible to prove a negative, to guarantee that you and your predecessors deliberately scared the American public just for the political hell of it -- even though your predecessor Mr. Ridge admitted he had his suspicions about exactly that.
Suffice to say, Mr. Chertoff, if it ever can be proved, there will be a lot of people from Homeland Security, and other outposts of this remarkably corrupt Administration, who will be going to prison.
Thirdly, and most charitably, I guess, Mr. Chertoff, is the possibility that you have made some credible inference that we are really at greater risk right now, but that any detail might blow some sort of attempt at interruption. There is some silver lining in this one.
But the silver lining would have been a greater one if this National Counter-Terrorism Center Report hadn't leaked out the day after you introduced us to your gut; a report suggesting Al-Qaeda had re-built its operational capacity to pre-9/11 levels.
Not only did this latest hair-on-fire-missive remind us that Al-Qaeda's re-growth has been along the Pakistan/Afghanistan border...
Not only did it remind us that your boss let this happen by shifting his resources out of Afghanistan to Iraq for his own vain and foolish purposes, to say nothing of ignoring Pakistan...
Not only did it underscore the ominous truth that if this country is victimized again by Al-Qaeda, the personal responsibility for the failure of our misplaced defenses would belong to President Bush and President Bush alone...
But on top of all of it, Mr. Chertoff, it revealed you for the phony expert you are -- the kid who hears in confidence something smart from somebody smart, and then makes his prediction, that what the smart kid said confidentially, is about to happen.
It reads just as you revised the "gut" remark this morning, sir -- the "informed opinion." The kid telling stories out of school.
The fourth possibility is a simple reversal of the third, Mr. Chertoff.
You shot off your bazoo, and then this National Counter-Terrorism Center report was rushed out -- even created -- to cover you, to give you credibility, to cloud the reality that you actually intoned to the Chicago Tribune, the 21st Century equivalent of "by the pricking of my thumb, something wicked this way comes."
But the fifth possible explanation of your gut, Mr. Chertoff, is the real nightmare scenario.
And it is simple.
That you, the man who famously told us "Louisiana is a city that is largely under water," meant this, literally.
That we really have been reduced to listening to see if your gut will growl.
That your intestines are our best defense.
That your bowels are our listening devices, your digestive tract is full of augurs, your colon produces the results that the torture at Gitmo does not.
All hail the prophetic gut!
So there are your choices: bureaucratic self-protection, political manipulation of the worst kind, the dropping of opaque hints, a gaffe backfilled by an "instant report," or the complete disintegration of our counter-terror effort.
Even if there really is never another terror attempt in this country, we have already lost too much in these last six years, to now have to listen to Michael Chertoff's gut, no matter what its motivation.
We cannot and will not turn this country into a police state.
But even those of us who say that most loudly and insistently, acknowledge that some stricter measures, under the still-stricter supervision of as many watchdogs as we can summon, are appropriate.
But you're not even going to wring any of that from us, Mr. Chertoff, if we're going to hear remarks about your "gut feelings."
You have reduced yourself to the status of a hunch-driven clown, and it's probably time you turned your task over to somebody who represents the brain and not the gut...
Certainly to somebody who does not, as you do now, represent that other part of the anatomy -- the one through which the body disposes of what the stomach doesn't want. |
Cardiac Lesions in the Critical Care Setting.
The long-held belief that pregnancy is absolutely contraindicated in maternal cardiovascular disease is no longer justifiable using evidence-base medicine. There are some conditions in which pregnancy is contraindicated, and high maternal risk and poor fetal outcome can be predicted. However, in many women with heart disease, a more favorable maternal and fetal outcome is expected. This article focusses on the cardiac conditions that require more attention and have the potential to require observation in the intensive care unit setting. |
Cutaneous respiration by diving beetles from underground aquifers of Western Australia (Coleoptera: Dytiscidae).
Insects have a gas-filled respiratory system, which provides a challenge for those that have become aquatic secondarily. Diving beetles (Dytiscidae) use bubbles on the surface of their bodies to supply O2 for their dives and passively gain O2 from the water. However, these bubbles usually require replenishment at the water's surface. A highly diverse assemblage of subterranean dytiscids has evolved in isolated calcrete aquifers of Western Australia with limited/no access to an air-water interface, raising the question of how they are able to respire. We explored the hypothesis that they use cutaneous respiration by studying the mode of respiration in three subterranean dytiscid species from two isolated aquifers. The three beetle species consume O2 directly from the water, but they lack structures on their bodies that could have respiratory function. They also have a lower metabolic rate than other insects. O2 boundary layers surrounding the beetles are present, indicating that O2 diffuses into the surface of their bodies via cutaneous respiration. Cuticle thickness measurements and other experimental results were incorporated into a mathematical model to understand whether cutaneous respiration limits beetle size. The model indicates that the cuticle contributes considerably to resistance in the O2 cascade. As the beetles become larger, their metabolic scope narrows, potentially limiting their ability to allocate energy to mating, foraging and development at sizes above approximately 5 mg. However, the ability of these beetles to utilise cutaneous respiration has enabled the evolution of the largest assemblage of subterranean dytiscids in the world. |
Q:
Do you bother with markup formatting?
I am a front end guy who is getting more and more into scripting and that being the case, I like my regurgitated markup to kind of look nice.
I ran a loop over some database values for a list and while most sites would just show a big old concatenated slew of <LI> tags back to back, I kind of like them \r\n distanced with proper \t tabbing. Weird thing is, the first list member renders like LI> rather than <LI> about 1 out of 5 page serves.
Anyone seen this? Should I not bother? Am I formatting the loops badly? Here's an example:
while ($whatever = mysql_fetch_array($blah_query)){
echo "\t\t\t\t\t\t";
echo "<li>\n";
echo "\t\t\t\t\t\t";
echo '<a href="#'.$whatever['name'].'" id="category_id_'.$whatever['id'].'">';
echo ucfirst($whatever['name']);
echo "</a>\n\t\t\t\t\t\t</li>\n";
}
A:
this seems as if the goal is to output a page source that types out the proper indentions for you?
at least for right now to debug and be easier read?
while ($whatever = mysql_fetch_array($blah_query)){
echo "\t\t\t\t\t\t";
echo "<li>\n";
echo "\t\t\t\t\t\t";
echo '<a href="#'.$whatever['name'].'" id="category_id_'.$whatever['id'].'">';
echo ucfirst($whatever['name']);
echo "</a>\n\t\t\t\t\t\t</li>\n";
}
since you're using PHP to echo out those HTML codes, just type them as you would see them on the page source
while($whatever = mysql_fetch_array($blah_query)){
//When you want a new line, just hit enter. PHP will echo the carriage returns too
echo'
<li>
<a href="#'.$whatever['name'].'" id="category_id_'.$whatever['id'].'">ucfirst($whatever['name'])</a>
</li>
';
}
this is how I would do it so that it would line break every time including the first time incase I have a left over "</div>" or some other closing tag without a line break after it.
it will output a nicer clean list item that tabbed in with the breaks
|
Fort de Pré-Giroud
The Fort de Pré-Giroud, also known as the Fort de Vallorbe, is a 20th-century Swiss fortification located in the Jura Mountains near the Swiss border with France. The fort defended the Col de Jougne at Vallorbe, one of the few points in the Jura that could be easily traversable by an invading force. It was a component of the Swiss Border Line of defenses. Built between 1937 and 1939, the fort covers the Swiss end on the Mont d'Or railroad tunnel and the Joux valley. It is armed with three artillery blocks for 75mm guns and two machine gun blocks. All are camouflaged, the artillery blocks as rock formations, and the machine gun blocks as houses. Deactivated as a military post in the 1980s, it is operated as a museum.
Description
The Fort de Pré-Giroud is located to the east of Vallorbe, in a steep hillside facing north, towards the Jougnes gap. It is part of the Border Line defenses built by Switzerland in the late 1930s, prior to a shift in Swiss priorities to the National Redoubt in the Alps.
The fort is laid out in a roughly triangular shape, with the base of the triangle at the bottom of the steep slope. Three multi-level artillery casemates containing 75mm guns are located near the middle, flanked by machine gun blocks. To the rear, deeply buried in the hillside, are ammunition magazines, utility areas and underground barracks. The barracks are at a depth of . The main fort is surrounded by four smaller bunkers, unconnected to the main fort system.
Armament includes:
C1: casemate with one 75mm Model 1939 semi-automatic gun
C2: casemate with one 75mm gun, one 47mm semi-automatic anti-tank gun, observation post
C3: as C1
M1: machine gun casemate with one 7.5mm Model 1911 water-cooled gun, observation post and emergency exit
M2: machine gun casemate with two machine guns
M3: as M2
M4: as M1
The perimeter blockhouses are each armed with two machine guns. Infantry for exterior protection were further armed with 24, and 47mm anti-tank weapons, two 81mm mortars, four machine guns and eight automatic rifles. These were added in 1941, along with barbed-wire entanglements, stated to be lessons learned from the successful German assault on the Belgian Fort Eben-Emael.
The machine gun blocks are covered by wood superstructures in the shape of chalets, while the artillery blocks are covered in rough rock-shaped shells, with metal branch-like camouflage. Unlike contemporary French fortifications of the Maginot Line, which are sited to avoid prominent exposures and which are positioned to fire obliquely, along the defensive lines, the Pré-Giroud fort is relatively exposed and fires directly ahead.
The fort was manned by more than 200 men.
Present situation
The Fort de Pré-Giroud was decommissioned in the 1980s, on grounds that its levels of protection and armament were inadequate and obsolete. It was sold in 1988. It is now operated as a museum.
Notes
References
Kauffmann, J.E., Jurga, R., Fortress Europe: European Fortifications of World War II, Da Capo Press, USA, 2002, .
External links
Fort Pré-Giroud 39-45 official site
Category:Border Line fortifications of Switzerland
Category:Cold War museums in Switzerland
Category:World War II museums in Switzerland
Category:Museums in Vaud |
1. Field of the Invention
The present invention relates to the configuration of a housing of a printer.
2. Description of the Related Art
There exist portable printers in which, instead of performing printing by individually inserting sheets, continuous printing can be performed by mounting a plurality of sheets. In such a printer, a cover of a printer main body is sometimes utilized as a tray for mounting sheets (for example, refer to U.S. Pat. No. 6,507,408).
FIG. 11 is a perspective view illustrating a non-use state or a portable state of such a printer.
A sheet feeding cover 2 is provided so as to cover the upper surface of a printer main body 1 in a state of being rotatable around a rotation axis 2a. An I/F (interface) connector 3 for performing data exchange by being connected to an external apparatus is provided at a side 1b of the printer main body 1. A sheet discharge opening 1e where a sheet on which printing has been completed is discharged from the inside of the printer main body 1 is provided at a front 1a.
FIG. 12 is a perspective view illustrating a state of use of the printer shown in FIG. 11. By being opened by rotating around the rotation axis 2a, the sheet feeding cover 2 operates as a tray for mounting sheets 4. In the non-use state shown in FIG. 11, the sheet feeding cover 2 covers a sheet feeding opening 1g. The mounted sheets 4 are individually separated by a separation mechanism from the sheet feeding opening 1g provided at the upper surface 1f of the printer main body 1. The separated sheet is fed to a recording portion by a conveying mechanism. After a printing operation by a recording mechanism, the sheet is discharged by a conveying mechanism from the sheet discharge opening 1e provided at the front 1a (the above-described mechanisms are within the printer main body 1, and are not illustrated). An access cover 5 is provided immediately below the sheet feeding cover 2, so as to allow exchange of an ink tank, processing during a sheet jam, and the like. An operation unit 6 is also provided at the upper surface 1f of the printer main body 1 that is the same surface as a surface where the access cover 5 is provided, so that various operations for the printer can be performed and a state can be displayed. In this printer, the sheet feeding cover 2 is utilized as a sheet tray by opening the sheet feeding cover 2 during the use of the printer. Since the sheet feeding cover 2 is closed when the printer is carried, penetration of dust or foreign matter from the sheet feeding opening 1g into the printer main body 1 when the printer is carried is prevented.
FIG. 13 is a perspective view of the printer shown in FIG. 11, as seen from the back of the printer. FIG. 13 illustrates a state in which a connection cable 8 is connected to the I/F connector 3 provided at the side 1b of the printer main body 1, and a connection cable 9 from a commercial power supply (not shown) is connected to a DC jack 7 provided at a back 1d of the printer main body 1. When intending to secure a space on a desk by reducing the occupied area of the printer main body 1 (in this case, equal to the projected area of the sheet feeding cover 2) in a state in which cables and the like are inserted as in this case, the occupied area is reduced by installing the printer in a state in which, as shown in FIG. 14, the front 1a where the cables and the like are not inserted is made a surface of installation, or in a state in which, as shown in FIG. 15, another side 1c facing the side 1b is made a surface of installation.
FIG. 16 is a perspective view illustrating a conventional non-use state (or a portable state) of another printer.
A sheet feeding cover 2 is provided so as to cover the upper surface of a printer main body 1 in a state of being rotatable around a rotation axis 2a. A sheet-discharge-port cover 10 is provided at a front 1a so as to be rotatable around a rotation axis 10a. An I/F connector 3 for performing data exchange by being connected to an external apparatus is provided at a side 1b of the printer main body 1.
FIG. 17 is a perspective view illustrating a state of use of the printer shown in FIG. 16. By being opened by rotating around the rotation axis 2a, the sheet feeding cover 2 operates as a tray for mounting sheets 4. In the non-use state shown in FIG. 16, the sheet feeding cover 2 covers a sheet feeding opening 1g. By opening of the sheet-discharge-port cover 10 by rotating around the rotation axis 10a, a sheet discharge opening 1e is exposed. The mounted sheets 4 are individually separated by a separation mechanism from the sheet feeding opening 1g provided at an upper surface 1f of the printer main body 1. The separated sheet is fed to a recording portion by a conveying mechanism. After a printing operation by a recording mechanism, the sheet is discharged by a conveying mechanism from the sheet discharge opening 1e provided at the front (the above-described mechanisms are within the printer main body 1, and are not illustrated). An access cover 5 is provided immediately below the sheet feeding cover 2, so as to allow exchange of an ink tank, processing during a sheet jam, and the like. An operation unit 6 is also provided at the upper surface If of the printer main body 1 that is the same surface as a surface where the access cover 5 is provided, so that various operations for the printer can be performed and a state can be displayed. This printer is carried by closing the sheet feeding cover 2 and the sheet-discharge-port cover 10. When using the printer, the sheet feeding cover 2 is utilized as a sheet tray by opening the sheet feeding cover 2 and the sheet-discharge-port cover 10, and the sheet 4 on which printing has been completed is discharged from the inside of the printer by exposing the sheet discharge opening 1e. Thus, penetration of dust or foreign matter from the sheet feeding opening 1g and the sheet discharge opening 1e into the printer main body 1 is prevented.
In the printer of the first type, preparation for printing is completed only by opening the sheet feeding cover when using the printer. However, when carrying or stocking the printer, since the sheet discharge opening is not covered, penetration of dust or foreign matter into the printer main body cannot be prevented, whereby the operation of the printer may become abnormal.
In the printer of the second type, since both of the sheet feeding opening and the sheet discharge opening are covered by the respective covers, penetration of dust or foreign matter into the printer main body can be substantially prevented. However, when using the printer, both of the sheet feeding cover and the sheet discharge-port-cover must be opened. If printing is performed without opening the sheet-discharge-port cover, there is the possibility that a printed sheet is jammed within the printer.
When intending to place the printer in a state in which the side, the back or the like is made a surface of installation in order to secure a space on a desk when the printer is not used, or to store the printer in a same state, the cables and the like are present in space to become an obstacle and provide an awkward appearance. If the cables and the like are detached, it is necessary to again insert them when using the printer, thereby causing a troublesome operation. Furthermore, since the DC jack and the I/F connector are disposed separately at the back and at the side, respectively, it is necessary to detach the cable from the DC jack when placing the printer in a state in which the back of the printer is placed downward. |
I got an email from Midway at 10:29 am that win primers were in stock. I was on the product page in 30 seconds and they are gone. Are they sending these notices out when product has already sold out???
It sux. I've had that happen more often than not with Midway. It's like playing the lottery.
__________________
-DHR
"They who can give up essential liberty to obtain a little temporary safety, deserve neither liberty nor safety." -Benjamin Franklin
"One loves to possess arms, though they hope never to have occasion for them." -Thomas Jefferson
“Knowledge will forever govern ignorance; and a people who mean to be their own governors must arm themselves with the power which knowledge gives.” -James Madison
The same thing happened to me on back-ordered items. I even pulled over immediately and used my cel-phone but they were out. I called customer service and was told that if they get a shipment and there are only 100 in stock, they will go to the first 100 people. He told me he can't even get supplies and is in the same boat I (we) am.
Same thing with e-notice. I think there are people lurking at their keyboards waiting for in-stock.
__________________
"Lord, make my hand fast and accurate.
Let my aim be true and my hand faster
than those who would seek to destroy me.
Grant me victory over my foes and those who wish to do harm to me and mine.
Let not my last thought be 'If I only had my gun."
And Lord, if today is truly the day you call me home
Let me die in a pile of empty brass."
Amen |
DmG
When there is trap set up for you,
CF
In every corner of this town,
FGCAm
And so you learn the only way to go is underground.
DmG
When there is a trap set up for you,
CF
In every corner of your room,
FGCAm
And so you learn the only way to go is through the roof.
FGCAm
Ooohooooh, through the roof, underground.
FGCAm
Ooohooooh, through the roof, underground.
DmG
And we crossing border after border,
CF
We realise the difference is none.
FG
It's underdogs who, and if you want it,
CAm
You always have to make your own fun.
DmG
And as the upperdog leisurely sighing,
CF
The local cultures are dying and dying.
FG
The programmed robots are buying and buying,
CAm
Secluded freaks they are still trying trying.
FGCAm
Ooohooooh, through the roof, underground.
FGCAm
Ooohooooh, through the roof, underground.
DmG
And as the boy scouts learn to read between the lines,
CF
The silver rabbits hop between their fathers' lies.
FG
And boy scouts ask, "Where? Where do they go?"
CAm
They go to the country that they only know.
DmG
Just like their meanings they lay between the lines,
CF
Between the borders their real countries hide,
FG
Their strategies, they advertise,
CAm
Their strategy of being is one of in-your-face disguise.
FGCAm
Ooohooooh, through the roof, underground.
FGCAm
Ooohooooh, through the roof, underground.
DmG
And when their own walls they will a-crumble,
CF
And all the systems will be discumbumbled,
FG
Around the stump of bigotry, our own,
CAm [CAm ]
Silver rabbits dance the round dance. [Серебряные зайцы водят хоровод]
FGCAm
Ooohooooh, through the roof, underground.
FGCAm
Ooohooooh, through the roof, and underground.
FGCAm
Ooohooooh, through the roof, underground.
FGCAm
Ooohooooh, through the roof, underground!
CAm [CAm ]
Silver rabbits dance the round dance. [Серебряные зайцы водят хоровод]
Through the roof! And underground!
Through the roof! Underground! |
Urgent Care Medicine
Dr. Goy's Overview
Dr. Peter W. Goy graduated from the Medical College of Wisconsin School of Medicine in 1986. He works in Menomonee Falls, WI and specializes in Urgent Care Medicine. Dr. Goy is affiliated with Aurora Medical Center Kenosha and Froedtert & The Medical College Of Wisconsin Community Memorial Hospital Campus. |
IRC-083927 is a new tubulin binder that inhibits growth of human tumor cells resistant to standard tubulin-binding agents.
Tubulin is a validated target for antitumor drugs. However, the effectiveness of these microtubule-interacting agents is limited by the fact that they are substrates for drug efflux pumps (P-glycoprotein) and/or by the acquisition of point mutations in tubulin residues important for drug-tubulin binding. To bypass these resistance systems, we have identified and characterized a novel synthetic imidazole derivative IRC-083927, which inhibits the tubulin polymerization by a binding to the colchicine site. IRC-083927 inhibits in vitro cell growth of human cancer cell lines in the low nanomolar range. More interesting, it remains highly active against cell lines resistant to microtubule-interacting agents (taxanes, Vinca alkaloids, or epothilones). Such resistances are due to the presence of efflux pumps (NCI-H69/LX4 resistant to navelbine and paclitaxel) and/or the presence of mutations on beta-tubulin and on alpha-tubulin and beta-tubulin (A549.EpoB40/A549.EpoB480 resistant to epothilone B or paclitaxel). IRC-083927 displayed cell cycle arrest in G(2)-M phase in tumor cells, including in the drug-resistant cells. In addition, IRC-083927 inhibited endothelial cell proliferation in vitro and vessel formation in the low nanomolar range supporting an antiangiogenic behavior. Finally, chronic oral treatment with IRC-083927 (5 mg/kg) inhibits the growth of two human tumor xenografts in nude mice (C33-A, human cervical cancer and MDA-MB-231, human hormone-independent breast cancer). Together, the antitumor effects induced by IRC-083927 on tumor models resistant to tubulin agents support further investigations to fully evaluate its potential for the treatment of advanced cancers, particularly those resistant to current clinically available drugs. |
Well, I knew this was coming. Forget that Parker is obviously hurting, KY and Danny Green couldn't hit the broadside of a barn and Manu is just simply pathetic on defense and on most shots he takes during a game. It's all Bonner's fault. |
Q:
how to convert this excel formula in english words?
can someone please translate this excel spreadsheet cell formula in english words ?
=ROUND(IF(F28 < 1568,2.5,IF(F28 < 2491,0.004873 * F28-5.142,0.02269*F28^0.7329)),2)
am creating a program based from that formula, but i don't understand which one will go first. atleast i understand this part IF(F28 is less than 1568) ...then what?
A:
Start from the outer if statement and move inwards. The comma in the IF function separates the statements like :
boolean expression, true part, and false part
The following is the psuedo code of the above. All the Round are to two decimal places.
IF (F28 < 1568) THEN
ROUND (2.5)
ELSE IF (F28 < 2491) THEN
ROUND (0.004873 * F28 - 5.142)
ELSE
ROUND (0.02269 * F28^0.7329)
|
Cosmology Machine
The world's first 'cosmology machine' has recently been installed at Durham University, UK. Capable of handling over 456 billion calculations per second, the computer will be able to simulate the creation of the universe and the interactions of hundreds of millions of galaxies thought to be present in the universe. |
Recent advances in bioinformatics in the medical research environment and applications to the study of skin diseases.
The computer has become increasingly intertwined in society for the past 30 years. Within the academic health science centre, there is an increasing need for researchers to become skilled at using the Internet as a mechanism for the retrieval of scientific results and the underlying data. The discipline of bioinformatics, which uses computer technology to provide answers to biological questions, has been expanding in scope and utility for the past decade. Increasing numbers of research groups have been investing in bioinformatics infrastructure to aid in the research process. These continuing investments have led to the establishment for the first time of a supercomputing facility within a hospital. Such computational power is being used for the mapping of genes and the study of human disease. A discussion of the increasing role of computational biology in the research environment of the clinician scientist is presented here. Though the investment in a supercomputer may not be possible in most research settings, several less expensive alternatives relying on existing desktop computers can provide supercomputer-like performance within nearly any environment. |
Not So Off The Shoulder
14 September, 2016
As I've mentioned it before, bare shoulders have been one of my favorite trends this year. Being the potato that I am when it comes to fashion, I keep my eye on what seems easy to style and doesn't need much added help, such as this top.
Last Saturday I had a friend's birthday celebration that went from a day to night event. To be honest, I didn't consider that, specially because I mostly go for black and white. However, I realized that this outfit worked perfectly for both occasions, the only difference was taking off the hat and putting on a bold lipstick at night. I got this top from SheIn and I wanted to wear it immediately. The sleeves are a lot looser than a normal off the shoulder top and that makes it very flowy. I love the lace detail that it has on the neck, I feel like it adds more decor to it without disturbing its simplicity. I paired up the top with these shorts that I got on a Zara summer sale a few months ago, they're so comfy and slightly high waisted, which came in quite handy because the top is a little short.
The day event was at a garden so I was sure I didn't want to wear heels but neither flat shoes. I opted for these sandal platforms that I got about two years ago at Bershka, they used to be my ride or die and totally forgot about them after a couple of months. I was doing a wardrobe clean up when I found them again, but unfortunately we only have a few more weeks of hot weather so I better wear them every single day until then. |
The underwhelming form of Manchester United in the Premier League this season may have been predictable as the squad David Moyes inherited, regardless of their status as reigning champions, is 'a mess'.
United take on title-challenging Chelsea at Stamford Bridge this afternoon knowing that any result other than victory will see greater distance created between themselves and a lucrative top four finish that would secure Champions League football next season.
@Sultan_Alfuhaid I want David to succeed - badly. He's a good guy. But stop pretending he was left with a top squad. He wasn't. It's a mess
Former Sky Sports presenter and current talkSPORT personality Richard Keys believes the squad is 'a mess' and, perhaps due to the enhancing ages of established stars such as Ryan Giggs, Rio Ferdinand and Nemanja Vidic, is not the 'top squad' that they once were.
Thus far, due to only being in his current role for a half-season, manager David Moyes has not created his own team with his only notable signing being his former pupil at Everton; Marouane Fellaini.
According to the latest tabloid speculation, Moyes will be entrusted with a transfer fund of £150m to even £200m, with players such as Chelsea playmaker Juan Mata, Toffees wonderkid Ross Barkley and Paris Saint Germain striker Edinson Cavani all linked with a switch to Old Trafford
For Keys, he may be hoping that at least one of these high-profile signings can be made as he wants 'a strong United'. |
Q:
asp.net EF core MVC cant access data from third foreign key table
Hello playing around with asp.net core MVC but got stuck and need some help.
I'm trying to get my index for courses to show the last name of the instructor but can't figure out how to access the information in the third table. I manage to get the InstructerID from the Departmenttable
public class CourseIndexData
{
public IEnumerable<Course> Courses { get; set; }
public IEnumerable<Department> Departments { get; set; }
public IEnumerable<Instructor> Instructors { get; set; }
}
public async Task<IActionResult> Index()
{
var viewModel = new CourseIndexData();
viewModel.Courses = await _context.Courses
.Include(i => i.Department)
.ThenInclude(i => i.Instructor)
.ToListAsync();
return View(viewModel);
}
@model ContosoUniversity.Models.SchoolViewModels.CourseIndexData
@{
ViewData["Title"] = "Index";
}
<h1>Index</h1>
<p>
<a asp-action="Create">Create New</a>
</p>
<table class="table">
<thead>
<tr>
<th>
Instructor
</th>
<th>
Department
</th>
<th>
Title
</th>
<th>
Credits
</th>
<th></th>
</tr>
</thead>
<tbody>
@foreach (var item in Model.Courses) {
<tr>
<td>
????????
</td>
<td>
@Html.DisplayFor(modelItem => item.Department.Name)
</td>
<td>
@Html.DisplayFor(modelItem => item.Title)
</td>
<td>
@Html.DisplayFor(modelItem => item.Credits)
</td>
A:
It's a graph of data, so it would be:
@Html.DisplayFor(modelItem => item.Department.Instructor.LastName)
You do not really need to have Departments and Instructors properties on your ViewModel (CourseIndexData) because all of that data is effectively inside of Courses and linked via the navigation properties.
|
Von Jürgen Fritz
Jürgen Fritz berichtet von einem persönlichen, sehr seltsamen und tief verstörenden Erlebnis, welches ihn noch immer beschäftigt und ratlos zurück lässt.
Irgendwie sind die Leute manchmal komisch
Kennen Sie dieses Gefühl, man ist gut gelaunt, freundlich zu seinen Mitmenschen, grüßt sie, lächelt ihnen zu, aber irgendwie kommt gar nichts Positives zurück, ja mehr noch als das, sie begegnen einem regelrecht mit Ablehnung und kaum verborgener Antipathie? Mir macht das immer zu schaffen und ich frage mich: Warum nur? Irgendwie sind die Leute manchmal komisch. Ich gestehe, ich verstehe sie oft nicht.
Mein süßer kleiner Stinker
Heute erst wieder. Ich gehe mit meinem süßen kleinen Hündchen im hiesigen Park spazieren. Der kleine ist ja sooo süß. Wie kann man denn ein solches Hündchen nicht lieb haben, knuddeln und drücken wollen? Ich gehe also mit ihm spazieren, der kleine freut sich wie Bolle, rennt hin und her, tobt sich so richtig aus, der kleine Stinker, wälzt sich auf dem Rasen hin und her, kommt wieder zu mir zurückgelaufen, springt an mir hoch, ja ist ganz außer sich vor Freude, die regelrecht ansteckend auf mich wirkt, auf mich überspringt und auch mich erfasst und durchdringt. Ach, das Leben kann so schön sein! Und sind es nicht die kleinen Dinge, die uns zufriedener und glücklicher machen als vieles andere?
Eine unerhörte Begebenheit
Dann aber geschieht das Seltsame, für mich nicht Nachzuvollziehende, das ich mir noch immer nicht erklären kann. Wir gehen also zusammen durch den Park, na ja ich gehe, der kleine rennt und springt, und ich rufe ihn laut, wenn er zu weit von mir weg rennt. Was dann aber geschieht, das werden Sie mir nicht glauben! Die Leute werfen sich voller Angst auf den Boden, manche rennen vor Entsetzen weg, ohne sich überhaupt umzudrehen, Frauen beginnen hysterisch zu schreien, einige beginnen augenblicklich zu weinen, eine Mutter wirft sich auf ihr Kind, erdrückt es fast. Ein junger sportlicher Mann schmeißt sich sogar regelrecht in eine große Abfalltonne, an der er gerade vorbei läuft.
Wie soll ein Mensch das verstehen?
Und dann, keine Minute später haben die Leute sich wieder halbwegs beruhigt, verhalten sich zumindest nicht mehr so völlig überdreht, schauen mich aber ganz böse und vorwurfsvoll an, geben mir mit ihrem Blick ihre Missbilligung und ihre Wut klar zu verstehen, ja lassen mich diese förmlich körperlich spüren, also ob ich Schuld wäre an ihrem vollkommen verrückten Verhalten. Verstehen Sie das? Ich jedenfalls nicht. Und das Schlimmste von allem: das macht dann sogar meinen kleinen Stinker ängstlich und traurig, raubt zunächst mir und dann auch ihm die Unbeschwertheit und Unbefangenheit, ja sogar unsere gute Stimmung. Ach, die Leute sind einfach komisch. Dabei ist er doch sooo lieb, mein kleiner Allahu Akbar.
*
Bild: Pixabay, CC0 Creative Commons
**
Spendenbitte: Wenn Sie diesen Blog (völlig werbefrei) und meine Arbeit wichtig finden und finanziell unterstützen möchten, dann können Sie entweder einmalig oder regelmäßig (Patenschaft) einen Betrag Ihrer Wahl auf das folgende Konto überweisen.
Jürgen Fritz, IBAN: DE44 5001 0060 0170 9226 04, BIC: PBNKDEFF, Verwendungszweck: Spende für Blog. Oder über Paypal: 5 EUR – 10 EUR – 25 EUR – 50 EUR – 100 EUR |
Q:
Django admin listview Customize Column Name
Ok so I have a custom django admin built from a Author Model:
class AuthorAdmin(admin.ModelAdmin):
"""
Author Admin
"""
form = AuthorForm
list_display = ['profile_photo', 'first_name', 'last_name', 'title']
search_fields = ['first_name', 'last_name', 'title', 'credential']
prepopulated_fields = {'slug': ('first_name', 'last_name', 'title')}
def profile_photo(self, obj) :
return '<img src="%s" title="%s" />' % (resize_image(obj.photo, '100x100'), obj.title)
profile_photo.allow_tags = True
But in the django admin listview the column title for the custom column does not have proper capitalization.
Does anyone know how to override the column headers that are built from custom function's names?
I've tried:
def my_function(self, obj) :
"""My Custom Title"""
...
and
def my_function(self, obj) :
class Meta:
verbose_name = _(u"My Custom Title")
A:
Use:
def my_function(self, obj) :
"""My Custom Title"""
...
my_function.short_description = 'This is the Column Name'
It's buried in the admin docs. short_description, specifically, is barely mentioned under the discussion of list_display (more by example than actually called out). The other items like this are similiarly buried in the admin docs, but here's a summary:
short_description: the column title to use (string)
allow_tags: what the name says... let's you use HTML (True or False)
admin_order_field: a field on the model to order this column by (string, field name)
boolean: indicates the return value is boolean and signals the admin to use the nice graphic green check/red X (True or False)
|
Thanksgiving is almost here, and though it's a happy time to be with family and friends, it can be stressful if you're trying to eat healthy. There's a lot of pressure and temptation to indulge. How you handle holiday feasting is an individual decision and can depend on a number of factors, but one thing is certain—the better prepared you are, the better you'll feel when the holidays are over.
The way I see it, there are 4 options for how to eat:
1. No holds barred. Eating unhealthy food in large quantities for a number of meals or days. This is one extreme.
Advertisement
2. Depriving yourself of enjoying some unhealthy food and sticking to a strict eating regime. This is the opposite extreme.
3. Allowing yourself one cheat meal over the holiday time but sticking to your "diet" the rest of the time.
4. Eating a number of unhealthy meals/food but choosing to have smaller portions sizes and saying no to certain things.
For me, I generally choose option 4. I like the idea of eating some special holiday food, but I make sure I take smaller portions of the less clean food and load up on veggies. However, like I mentioned, it depends on certain factors. For instance:
What are your goals?
Are you training for a fitness competition or a race? In that case, you should stick to your diet (maybe have one cheat meal). For example, when I was 3 weeks out from my figure competition I went to New York City with my sisters. I went to a grocery store as soon as I arrived and prepped all my meals for the weekend. I stuck to my diet except for 2 slices of pizza. I love pizza and couldn't pass up NYC pizza! Maybe you're looking to lose weight. If that's the case, think about how you eat during the holidays will affect this goal.
Are you exercising over the holidays?
If you're exercising over the break, it can give you a little more latitude to indulge a bit more than if you aren't exercising at all.
What's your regular workout schedule?
Will you be back at the gym your first day after the holidays or will it take a few days? The answer to this question should play a factor in how you eat.
Ultimately, choose what's right for your situation. And make sure you enjoy the time with family and friends! What about you? How do you handle food during the holidays? Share your strategy in the discussion!
Lydia Di Francesco is a certified Fitness Instructor and Nutrition and Wellness Specialist. She is passionate about helping others get fit with exercise and healthy eating. You can follow her blog or connect with her on Facebook, Twitter or Pinterest. |
[Rapid spontaneous postpartum remission in a case of chronic inflammatory demyelinating polyradiculoneuropathy associated with pregnancy].
A 28-year-old woman developed numbness and weakness of the hands and arms when she was 8 months pregnant, and weakness worsened gradually. However, weakness started to spontaneously subside immediately after delivery and she felt almost recovered several hours later. But weakness and numbness recurred one week after delivery and she was admitted to our hospital. Neurological examination revealed moderate weakness and disturbance of the deep and cutaneous sensations in the upper and lower extremities, and marked decrease of the deep tendon reflexes. The upper extremities were more severely affected. Nerve conduction study showed marked decrease in the motor conduction velocities. Cerebrospinal fluid showed increase of protein without pleocytosis. Teased preparation of the biopsied sural nerve showed occasional internodal segments with thin myelination, indicating demyelination and remyelination. A diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy was made. Treatment with prednisolone markedly improved the weakness and hyporeflexia as well as the cerebrospinal fluid protein. Postpartum rapid remission may have been produced by rapid increase of endogenous steroid hormone in the blood by its massive excretion during delivery. |
Q:
How to change header/page title of change list page in Django Admin
I'm working on Django.And I wanted to change the header in the change list page in the Django admin as marked with red color in the pic
and my admin.py file looks like this:
from django.contrib import admin
from django.contrib.auth.admin import UserAdmin
from .models import CustomUser
class CustomUserAdmin(UserAdmin):
change_form_template = 'change_form.html'
add_form_template='add_form.html'
list_display = ('first_name','last_name','email','is_staff', 'is_active',)
list_filter = ('first_name','email', 'is_staff', 'is_active',)
search_fields = ('email','first_name','last_name','a1','a2','city','state','pincode')
ordering = ('first_name',)
add_fieldsets = (
('Personal Information', {
# To create a section with name 'Personal Information' with mentioned fields
'description': "",
'classes': ('wide',), # To make char fields and text fields of a specific size
'fields': (('first_name','last_name'),'email','a1','a2','city','state','pincode','check',
'password1', 'password2',)}
),
('Permissions',{
'description': "",
'classes': ('wide', 'collapse'),
'fields':( 'is_staff', 'is_active','date_joined')}),
)
So my question is how to change the header in the change list page as shown in the pic??
Thanks in advance!!
A:
To makes changes like that first you have to add a line in the admin file :
change_list_template='change_list_form.html'
So your admin file will look like this:
from django.contrib import admin
from django.contrib.auth.admin import UserAdmin
from .models import CustomUser
class CustomUserAdmin(UserAdmin):
change_list_template='change_list_form.html'
change_form_template = 'change_form.html'
add_form_template='add_form.html'
list_display = ('first_name','last_name','email','is_staff', 'is_active',)
list_filter = ('first_name','email', 'is_staff', 'is_active',)
search_fields = ('email','first_name','last_name','a1','a2','city','state','pincode')
ordering = ('first_name',)
add_fieldsets = (
('Personal Information', {
# To create a section with name 'Personal Information' with mentioned fields
'description': "",
'classes': ('wide',), # To make char fields and text fields of a specific size
'fields': (('first_name','last_name'),'email','a1','a2','city','state','pincode','check',
'password1', 'password2',)}
),
('Permissions',{
'description': "",
'classes': ('wide', 'collapse'),
'fields':( 'is_staff', 'is_active','date_joined')}),
)
After that go to the templates folder and add a html file with name : change_list_form.html
Then in the file change_list_form.html add following code:
{% extends "admin/change_list.html" %}
{% load i18n admin_urls static admin_list %}
{% block content_title %}<h1>Type here your new header</h1>{% endblock %}
That's all you have to do.
|
package com.jennifer.andy.simpleeyes.ui.common
import android.os.Bundle
import android.widget.RelativeLayout
import androidx.recyclerview.widget.LinearLayoutManager
import androidx.recyclerview.widget.RecyclerView
import com.alibaba.android.arouter.facade.annotation.Autowired
import com.alibaba.android.arouter.facade.annotation.Route
import com.alibaba.android.arouter.launcher.ARouter
import com.jennifer.andy.simpleeyes.R
import com.jennifer.andy.simpleeyes.entity.AndyInfo
import com.jennifer.andy.simpleeyes.entity.Content
import com.jennifer.andy.simpleeyes.ui.base.BaseActivity
import com.jennifer.andy.simpleeyes.ui.base.adapter.BaseDataAdapter
import com.jennifer.andy.simpleeyes.utils.bindView
import com.jennifer.andy.simpleeyes.widget.CustomLoadMoreView
/**
* Author: andy.xwt
* Date: 2019-08-26 19:47
* Description:公共RecyclerView界面，根据url加载数据
*/
@Route(path = "/AndyJennifer/common")
class CommonRecyclerActivity : BaseActivity<CommonView, CommonPresenter>(), CommonView {
@Autowired
@JvmField
var title: String? = null
@Autowired
@JvmField
var url: String? = null
private val mToolbar: RelativeLayout by bindView(R.id.tool_bar)
private val mRecycler by bindView<RecyclerView>(R.id.rv_recycler)
private var mCommonAdapter: BaseDataAdapter? = null
override fun initView(savedInstanceState: Bundle?) {
ARouter.getInstance().inject(this)
initToolBar(mToolbar, title)
mPresenter.loadDataInfoFromUrl(url)
}
override fun showLoadSuccess(itemList: MutableList<Content>) {
with(mRecycler) {
mCommonAdapter = BaseDataAdapter(itemList).apply {
setOnLoadMoreListener({ mPresenter.loadMoreInfo() }, mRecycler)
setLoadMoreView(CustomLoadMoreView())
}
layoutManager = LinearLayoutManager(mContext)
adapter = mCommonAdapter
}
}
override fun loadMoreSuccess(data: AndyInfo) {
mCommonAdapter?.addData(data.itemList)
mCommonAdapter?.loadMoreComplete()
}
override fun showNoMore() {
mCommonAdapter?.loadMoreEnd()
}
override fun getContentViewLayoutId() = R.layout.activity_common_recyclerview
} |
8 comments
None of us wants to move but if I couldn't afford my mortgage or to live in my house, then I'd have to - but then again, I work and pay tax. Shall I pay a bit more so that these people who don't want to do this or that, can do as they please? I'm getting sick of the moaning now. It's tough for all of us!
i am sick and tired of listening to all this bedroom tax council tax well tuff get a job or down size stop giving imigrants money send them back to where they came from look after what should be britain and british people if they dont want to work dont pay them cant work find them jobs they can do at home
answer to bobble 1956 is (probably) that the because the wife hasn't had to work before, she probably planned her next years living off benefits and thus never thought to equip herself with skills and knowledge that would allow her to enter the job market. Hence there is probably no job suitable for her. I do have sympathy with the family and put the blame firmly at the governments of the last 20 years who helped make benefit claiming socially acceptable.
I presume that being in the house for thirty years is a mistake as Roy would have been eighteen and Cheryl would have been seven years old when they moved in. How can someone live in council accommodation for thirty years? Obviously when working Roy had a good job, spending £11,000 on his council owned property, why didn't they put that as a deposit and purchase a house at the time, they would be mortgage free by now. If you moved to another house I'm sure that you would be entitled to single payment benefit to cover carpets and curtains to make it habitable.
£764 a month in benefits for doing nothing, only paying twenty percent of the council tax, something others can only dream of.
People who say they smoke 5 a day really smoke at least 10 so by my reckoning if she gives up the fags she's got to be at least £25 a week better off, it's a good start and will more than cover the 'bedroom tax'. Stop complaining about how difficult life is when you still smoke which is both expensive and kills you. |
GENOME ANNOUNCEMENT {#h0.0}
===================
*Rummeliibacillus stabekisii* is constituted of strains of aerobic, Gram-positive, rod-shaped, round-spore-forming bacteria. Strains first described to belong to this species were isolated from different geographical locations (the type strain was isolated from the Payload Hazardous Servicing Facility at the Kennedy Space Center, FL), and their 16S rRNA gene sequence similarities demonstrated that they were most closely affiliated with *Bacillus pycnus* NRRL NRS-1691^T^ (98%), *Kurthia* sp. (96%), and *Viridibacillus* sp. (94 to 96%) ([@B1]). *R. stabekisii* was proposed as the type species of the genus *Rummeliibacillus* ([@B1]).
Strain PP9 was isolated from a soil sample collected from Punta Plaza located in King George Island, which is part of the South Shetlands archipelago in Maritime Antarctica, and identified as *R. stabekisii* ([@B2]). No genome sequences were available for any of the recognized species of the genus *Rummeliibacillus*. To gain a better understanding of this poorly studied species, the whole-genome sequence of *R. stabekisii* PP9 was determined.
The genome of *R. stabekisii* PP9 was sequenced in Seoul, South Korea, by DNA Link, Inc., using the PacBio RSII platform and two single-molecule real-time (SMRT) cells of P6-C4 chemistry with a 20-kb size-selected library; 450,876 raw reads resulted in 237,668 quality-filtered trimmed reads yielding 3,295 Mb, with a mean genome-wide coverage of about 969×. The filtered reads were assembled using HGAP version 2.3 ([@B3]). Annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline version 3.1 to predict protein-coding genes, structural RNAs, small noncoding RNAs, and tRNAs ([@B4]).
The genome of *R. stabekisii* PP9 consists of a circular chromosome of 3,412,092 bp and a circular plasmid of 8,647 bp, with a G+C content of 37.6%. The genome includes 3,244 protein-coding genes, 101 tRNA genes, 6 noncoding RNAs (ncRNAs), and 12 copies of the 16S-23S-5S rRNA operon. From the assigned protein-coding genes, only 1,939 have a putative function, while the remaining 1,305 genes (about 40%) were annotated as hypothetical proteins.
Genes related to sporulation and flagellar assembly were found in the PP9 genome. Round spores and motility observed by light microscopy and in soft agar stabbing, respectively, confirmed that their products are expressed and functional.
The average nucleotide identity (ANI) was calculated based on MUMmer ([@B5]) between the PP9 chromosome and six other complete chromosomes of strains belonging to the *Planococcaceae* family. *Kurthia* (accession no. CP013217.1) was the genus most closely related to *R. stabekisii* PP9 (accession no. CP014806.1), with 85.64% ANI, followed by *Solibacillus* (84.85%; accession no. NC_018065.1), *Jeotgalibacillus* (84.5%; accession no. CP009416.1), *Planococcus* (83.84%; accession no. CP013661.2), *Sporosarcina* (82.64%; accession no. CP014616.1), and *Planomicrobium* (82.45%; accession no. NZ_CCXS01000001.1). The resulting averages reflect a high degree of evolutionary distance between *R. stabekisii* and other members of its family.
Nucleotide sequence accession numbers. {#s1}
--------------------------------------
The genome sequences have been deposited at GenBank under the accession numbers [CP014806](http://www.ncbi.nlm.nih.gov/nuccore/CP014806) (chromosome) and [CP014807](http://www.ncbi.nlm.nih.gov/nuccore/CP014807) (plasmid). Strain PP9 is deposited in The Culture Collection of *Bacillus* and Related Genera (CCGB), under the accession no. CCGB1722.
**Citation** da Mota FF, Vollú RE, Jurelevicius D, Seldin L. 2016. Whole-genome sequence of *Rummeliibacillus stabekisii* strain PP9 isolated from Antarctic soil. Genome Announc 4(3):e00416-16. doi:10.1128/genomeA.00416-16.
|
1. Field of the Invention
This invention relates to a new and useful method for synthesizing a highly siliceous form of crystalline ferrierite-type material identified as having a ZSM-35 structure, the new ZSM-35 synthesized, and use of the crystalline material synthesized in accordance herewith as a catalyst component for organic compound, e.g. hydrocarbon compound, conversion.
More particularly, this invention relates to a method for preparing the crystalline ZSM-35 structure whereby synthesis is facilitated and reproducible and the product exhibits high purity and catalytic utility.
2. Discussion of the Prior Art
Ferrierite is a naturally occurring zeolite with an intersecting 10-ring by 8-ring structure. ZSM-35 is shown to be synthesized from reaction mixtures containing ethylene diamine or pyrrolidine directing agent in U.S. Pat. Nos. 4,016,245 and 4,107,195. Synthetic ferrierite is directed from a certain reaction mixture in U.S. Pat. No. 4,000,248 containing N-methylpyridinium; and another reaction mixture containing choline in U.S. Pat. No. 4,046,859. Piperidine is the directing agent in U.S. Pat. No. 4,251,499 for synthetic ferrierite. U.S. Pat. Nos. 4,323,481 and 4,390,457 teach synthesis of ferrierite from reaction mixtures comprising directing agents of 2,4-pentanedione and 2-aminopyridine, respectively. U.S. Pat. No. 4,377,502 shows morpholine or dioxane used as directing agent for ferrierite synthesis. U.S. Pat. No. 4,584,286 teaches a method for synthesis of ZSM-35 from a reaction mixture comprising bis(N-methylpyridyl) ethylinium as directing agent. Both U.S. Pat. Nos. 4,578,259 and 4,695,440 show use of pyridine plus ethylene glycol as directing agent for synthetic ferrierite; and both U.S. Pat. Nos. 4,721,607 and 4,855,270 show use of pyridine plus ethylene diamine and ethanol as directing agent for synthetic ferrierite. U.S. Pat. No. 4,925,548 teaches synthesis of ZSM-35 with hexamethyleneimine directing agent.
The above disclosures are incorporated herein by reference as to ZSM-35, synthetic ferrierite and their synthesis.
Various organic directing agents are taught for synthesis of various crystalline materials. For example, U.S. Pat. No. 4,139,600 teaches a method for synthesis of zeolite ZSM-5 from a reaction mixture comprising, as a directing agent, an alkyldiamine. U.S. Pat. No. 4,296,083 claims synthesizing zeolites characterized by a Constraint Index of 1 to 12 and an alumina/silica mole ratio of not greater than 0.083 from a specified reaction mixture containing an organic nitrogen-containing cation provided by an amine identified as being selected from the group consisting of triethylamine, trimethylamine, tripropylamine, ethylenediamine, propanediamine, butanediamine, pentanediamine, hexanediamine, methylamine, ethylamine, propylamine, butylamine, dimethylamine, diethylamine, dipropylamine, benzylamine, aniline, pyridine, piperidine and pyrrolidine.
Piperidine is disclosed as an organic directing agent for mordenite synthesis by P. A. Jacobs and J. A. Martens, Studies of Surface Science and Catalysis, 33, 12 (1987); and 2-amino-pyridine is taught for this purpose in U.S. Pat. No. 4,390,457. Tetra-n-propylammonium salts are taught to be mordenite directing agents in U.S. Pat. Nos. 4,707,345 and 4,788,380.
U.S. Pat. No. 4,151,189 claims a method for synthesizing zeolites ZSM-5, ZSM-12, ZSM-35 and ZSM-38 containing an organic nitrogen cation from a specified reaction mixture containing a primary amine having 2 to 9 carbon atoms as a directing agent. U.S. Pat. No. 4,341,748 shows synthesis of ZSM-5 structure from reaction mixtures comprising ethanol, ZSM-5 seeds, ethanol and seeds, ethanol and ammonium hydroxide, and ethanol, ammonium hydroxide and ZSM-5 seeds. U.S. Pat. No. 4,100,262 teaches synthesis of ZSM-5 from a reaction mixture comprising a tetraalkylammonium source and a tetraureacobalt (II) complex.
Lok et al. (3 Zeolites, 282-291 (1983)) teach numerous organic compounds which act as directing agents for synthesis of various crystalline materials, such as, for example, ZSM-5, ZSM-11, ZSM-12, ZSM-20, ZSM-35, ZSM-48, AlPO.sub.4 -5, AlPO.sub.4 -8, AlPO.sub.4 -20 and others. The article does not show the presently required organic for synthesis of ZSM-35. The article does show that cyclohexylamine, N-methylcyclohexylamine, dicyclohexylamine and ethyl-n-butylamine may direct synthesis of AlPO.sub.4 -5 (U.S. Pat. No. 4,310,440).
The zeolitic compositions labeled PSH-3 in U.S. Pat. No. 4,439,409 are synthesized from reaction mixtures containing hexamethyleneimine as directing agent. U.S. Pat. No. 4,954,325 utilizes hexamethyleneimine in another reaction mixture to direct synthesis of MCM-22. That organic is used in U.S. Pat. No. 4,981,663 for synthesis of yet another crystalline structure labelled MCM-35. As mentioned above, ZSM-35 is directed by this same compound in U.S. Pat. No. 4,925,548.
Other publications teaching various organic directing agents for synthesis of various crystalline materials include, for example, U.S. Pat. No. 4,592,902, teaching use of an alkyltropinium directing agent, alkyl being of 2 to 5 carbon atoms, for synthesis of ZSM-5; U.S. Pat. No. 4,640,829, teaching use of dibenzyldimethylammonium directing agent for synthesis of ZSM-50; U.S. Pat. No. 4,637,923, teaching use of (CH.sub.3).sub.2 (C.sub.2 H.sub.5)N.sup.+ (CH.sub.2).sub.4 N.sup.+ (C.sub.2 H.sub.5)(CH.sub.3).sub.2 directing agent for synthesis of another novel zeolite; U.S. Pat. No. 4,585,747, teaching use of bis (N-methylpyridyl) ethylinium directing agent for synthesis of ZSM-48; U.S. Pat. No. 4,585,746, teaching use of bis (N-methylpyridyl) ethylinium directing agent for synthesis of ZSM-12; U.S. Pat. No. 4,584,286, mentioned above, teaching use of bis (N-methylpyridyl) ethylinium directing agent for synthesis of ZSM-35; U.S. Pat. No. 4,568,654, teaching use of cobalticinium, dimethylpiperidinium, trimethylene bis trimethylammonium or tetramethylpiperazinium directing agents for synthesis of ZSM-51; U.S. Pat. No. 4,559,213, teaching use of DABCO-C.sub.4-10 -diquat directing agent for synthesis of ZSM-12; U.S. Pat. No. 4,482,531, teaching synthesis of ZSM-12 with a DABCO-C.sub.n -diquat, n being 4,5,6 or 10, directing agent; and U.S. Pat. No. 4,539,193, teaching use of bis (dimethylpiperidinium) trimethylene directing agent for synthesis of ZSM-12.
Various diquaternary ammonium compounds have been identified as directing agents for a particular assortment of crystalline materials. For instance, U.S. Pat. Nos. 4,490,342 and 4,619,820 show synthesis of ZSM-23 from a reaction mixture containing the organic of U.S. Pat. No. 4,531,012, i.e. (CH.sub.3).sub.3 N.sup.+ (R)N.sup.+ (CH.sub.3).sub.3, where R is a saturated or unsaturated hydrocarbon having 7 carbon atoms. U.S. Pat. No. 4,665,250 teaches the use of linear diquaternary ammonium compounds of the structure (CH.sub.3).sub.3 N.sup.+ (Ch.sub.2).sub.m N.sup.+ (CH.sub.3).sub.3, m being 5, 6, 8, 9 or 10, for synthesis of ZSM-48. U.S. Pat. No. 4,623,527 teaches numerous diquaternary ammonium compounds and shows use of (CH.sub.3).sub.3 N.sup.+ (CH.sub.2).sub.7 N.sup.+ (CH.sub.3).sub.3 directing agent for synthesis of MCM-10.
U.S. Pat. No. 4,632,815 teaches numerous diquaternary ammonium compounds and shows use of (CH.sub.3).sub.3 N.sup.+ (CH.sub.2).sub.4 N.sup.+ (CH.sub.3).sub.3 to direct synthesis of a Silica-X structure type. U.S. Pat. No. 4,585,639 teaches use of the diquaternary (C.sub.2 H.sub.5)(CH.sub.3).sub.2 N.sup.+ (CH.sub.2).sub.4or6 N.sup.+ (CH.sub.3).sub.2 (C.sub.2 H.sub.5) as directing agent for synthesis of ZSM-12. Synthesis of ZSM-5 is directed by the diquaternary (alkyl).sub.3 N.sup.+ (CH.sub.2).sub.6 N.sup.+ (alkyl).sub.3, alkyl being propyl or butyl, in U.S. Pat. No. 4,585,638.
EPA 42,226 and U.S. Pat. No. 4,537,754 teach existence of numerous diquaternary ammonium compounds, but show use of (CH.sub.3).sub.3 N.sup.+ (CH.sub.2).sub.6 N.sup.+ (CH.sub.3).sub.3 as directing agent for synthesis of EU-1. EPA 51,318 teaches use of the same diquaternary for synthesis of TPZ-3. It is noted that EU-1, TPZ-3 and ZSM-50 (synthesized with dibenzyldimethylammonium directing agent) have the same structure.
Applicants know of no prior art method for preparing a highly siliceous crystalline ferrierite-type structure identified as ZSM-35 utilizing the present method. |
Do you want to take the night off? This is what you need to know. Willard Romney will "do what he has to do" to maintain the illusion of his being a carbon-based life form. The president, on the other hand, because he is "more comfortable with the town-hall format," also will do "everything he has to do" to recover from the fiasco he handed the nation in the first of the presidential debates not yet thirteen days ago. The debate likely "will not move the needle" in any direction, although Romney's performance likely "may allay some doubts about his ability to connect with ordinary voters," while the president's performance likely "will reassure nervous Democrats who only last week wondered if he really wanted the job."
There. Done. Now you can all watch Sons of Anarchy, or re-runs of NCIS, and wonder how Illya Kuryakin grew up to reek of formaldehyde on behalf of the U.S. Navy.
I have yet to leave home for the events in Hemptstead on Tuesday evening, and still the horse-race template of this second debate is already set. It is a paint-by-numbers deal for the elite political media, most of which, I guarantee you, already knows what it's going to say at the end of the night. Unless Romney comes out dressed like Anton LaVey, or the president starts reading, unbidden, from Soul On Ice, nothing either of these guys say to Candy Crowley and a room full of New Yorkers will be treated as being of any real consequence. Romney has so battered the political dialogue — and the English language — with his 100-pound bullshit sledge that he has pretty much shaped the narrative of the campaign in such a fashion that his fanatical devotion to barefaced non-facts has become a weird kind of status quo. Far too many people in this business have accepted the Etch-A-Sketch argument to the point at which whether something is true or not is measured by its effectiveness as a tactic. "He had to run to the right in the primaries and then 'pivot' to the center in the general" — that's something that makes the political wiseguys look smart, but, taken literally, it means that the entire election process in the world's oldest self-governing republic is a contest to find out who can most smoothly move from one set of lies to another, and it is also a recipe for depriving the people who ultimately will make that decision of the kind of information they need to do so. How this is in any way good for democracy is not for small minds to ponder, I guess.
Which is why, in my goofy optimistic way, I am clinging to the town-hall format as my last hope that anything resembling something new and interesting will break through the din of a campaign aimed almost completely at the twelve undecided voters left in America. (The "ordinary voters" selected to participate on Tuesday evening are all "undecided voters" chosen by Gallup. All due respect to their honesty, and to Gallup's due diligence, but that data indicate that, given the fact that they need enough people to fill up a ninety-minute debate, some of those people lied their asses off to get the gig.) It is the last stand for spontaneity, the last possibility of a human moment before both candidates climb back into their bubbles and bounce across the landscape the way that white blob on The Prisoner used to do it. It will be the last chance for flesh and blood before the election roars to its inevitable conclusion as a bloodbath of decimal points.
It is possible, though admittedly unlikely, that one of the folks tasked to ask questions on Tuesday evening will ask a question for which one or both of these guys is unprepared. (At this point, I'll settle for someone who asks anything about the global climate crisis, because nobody has so far, and because watching Romney's inevitable "pivot" from climate change to his support for the Keystone XL pipeline would be as entertaining as watching him break-dance.) That kind of human moment wouldn't, please god, be so much a "boxers or briefs" question — although Romney would be the only candidate in history whose answer actually might be interesting — as something drawn from the real lives of real people, from someone who, say, actually has to take her kids to the emergency room for basic medical care, or someone else who actually works in a soup kitchen and is thus qualified and capable to call out the endless fraudulence of how we run the day-to-day operations of this quadrennial design competition.
It's a thin reed, I know. The people asking the questions at Hofstra University have been poked and prodded and vetted and, for all I know, fed tranquilizers, so that nothing will disturb the peaceful and blessed "centrism" and "balance" for which the organizers strive to the exclusion of all that nasty human unpredictability that can get people so damned worked up. The problem, of course, is that we should all want these people to be worked up. The country is still a very tough room for far too many people. Living in America has become hard, grinding work, not altogether because of factors beyond out control, but also because of what we have let our politics become. People should be angry. People should be grim. If, as always happens, somebody comes up with a genuine tale of sorrow and woe, the candidates should be allowed to emphasize for one entire minute and then, dammit, they should be expected either to tell this person specifically how their policies would alleviate the person's situation, or why that person is simply going to have to suck it up while we get things in order for the bond traders.
Spontaneity ends on Tuesday night. The rest is scripted and tailored and approximately as spontaneous as the Orange Bowl halftime show. It should be encouraged and cheered. And, if someone asks the one question that stumps both these guys, the reaction should not be that avoiding the answer was good politics. The reaction should be just answer the damn question.
--
POLL UPDATE: With National Lead, Romney's Vision for Etch-A-Sketch America Takes Hold
DEBATE LIVE: Follow Charles P. Pierce on Twitter from Hofstra and Watch on the Blog >
Charles P. Pierce Charles P Pierce is the author of four books, most recently Idiot America, and has been a working journalist since 1976.
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/*!
* @license
* Copyright 2019 Alfresco Software, Ltd.
*
* Licensed under the Apache License, Version 2.0 (the "License");
* you may not use this file except in compliance with the License.
* You may obtain a copy of the License at
*
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*
* Unless required by applicable law or agreed to in writing, software
* distributed under the License is distributed on an "AS IS" BASIS,
* WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
* See the License for the specific language governing permissions and
* limitations under the License.
*/
import { Person } from '@alfresco/js-api';
import { EcmCompanyModel } from './ecm-company.model';
export class EcmUserModel implements Person {
id: string;
firstName: string;
lastName: string;
description: string;
avatarId: string;
email: string;
skypeId: string;
googleId: string;
instantMessageId: string;
jobTitle: string;
location: string;
company: EcmCompanyModel;
mobile: string;
telephone: string;
statusUpdatedAt: Date;
userStatus: string;
enabled: boolean;
emailNotificationsEnabled: boolean;
constructor(obj?: any) {
this.id = obj && obj.id || null;
this.firstName = obj && obj.firstName;
this.lastName = obj && obj.lastName;
this.description = obj && obj.description || null;
this.avatarId = obj && obj.avatarId || null;
this.email = obj && obj.email || null;
this.skypeId = obj && obj.skypeId;
this.googleId = obj && obj.googleId;
this.instantMessageId = obj && obj.instantMessageId;
this.jobTitle = obj && obj.jobTitle || null;
this.location = obj && obj.location || null;
this.company = obj && obj.company;
this.mobile = obj && obj.mobile;
this.telephone = obj && obj.telephone;
this.statusUpdatedAt = obj && obj.statusUpdatedAt;
this.userStatus = obj && obj.userStatus;
this.enabled = obj && obj.enabled;
this.emailNotificationsEnabled = obj && obj.emailNotificationsEnabled;
}
}
|
Background {#Sec1}
==========
Although remarkable advances have been made in the evolution of therapeutic strategies and molecular elucidation of breast malignancies, recurrence and distant metastasis are still remained hard to cure and poorly understood \[[@CR1], [@CR2]\]. In 2016, it was reported that there will be 249,260 new breast cancer cases, of which 40,890 cases will be lethal to the U.S. female population \[[@CR3]\]. Metastasis accounts for over 90% of the cases of mortality in cancer patients \[[@CR4]\], and women with distant metastatic lesions have a significantly decreased 5-year survival rate compared with women with localized breast tumors (26.3% versus 98.8%) \[[@CR5]\]. Strikingly, only 6% of the patients were initially diagnosed with metastatic breast cancer, and 20% to 50% of primary breast cancer patients would eventually develop invasive phenotypes \[[@CR2]\]. Although overwhelming experimental evidence suggests that metastasis usually occurs at the advanced stages of cancer, some studies implied that tumor cells could be detected in the circulatory system as early as breast cancer initiation \[[@CR6], [@CR7]\]. However, the phenotype of circulating tumor cells is not necessarily uniform, only a minority of cancer cells could migrate to the distant organs and finally form the metastatic lesions \[[@CR8], [@CR9]\]. Hence, the identification of the metastatic cells that are capable of forming metastatic nodules and seeking targeted therapies to eliminate early metastasis are urgently needed in clinical management.
The discovery of CSCs sheds novel light in limiting metastasis. The CSC hypothesis supports the belief that cancer is largely driven by a subpopulation of malignant cells with the distinct ability of self-renewal, heterogeneous population generation, and tumor initiation \[[@CR10]\]. Further, mounting studies suggest that CSCs exhibit high metastatic potential. [Weng D](https://www-ncbi-nlm-nih-gov-u.vtrus.net/pubmed/?term=Weng%20D%5BAuthor%5D&cauthor=true&cauthor_uid=22277639) et al. revealed that early metastatic cells that disseminated in the lungs displayed stem cell markers in the MMTV-PyMT mice model \[[@CR9]\]. Liu et al. compared the gene expression differences between breast CSCs and normal mammary epithelial cells. There were 186 "invasive" genes that were identified, and they revealed a tight correlation to the overall survival and metastasis-free survival in breast cancer and other malignancies, including lung carcinoma, medulloblastoma, and prostate cancer \[[@CR11]\]. [Balic M](https://www-ncbi-nlm-nih-gov-u.vtrus.net/pubmed/?term=Balic%20M%5BAuthor%5D&cauthor=true&cauthor_uid=17020963) et al. identified the existence of the stem-like cells within the bone marrow of breast cancer patients in the early phase \[[@CR12]\]. All of these findings suggest that CSCs might be the precursors guiding cancer metastasis, and identification of key targets mediating CSCs metastasis is of great significance for improving cancer prognosis.
Target identification with natural phytochemicals has become an important strategy leading to the novel discovery of pathological biomarkers nowadays. Although there still is not a specific CSCs-targeting drug in the clinic at present, a lot of naturally occurring compounds have exhibited promising CSCs-limiting effects. Meanwhile, dietary supplements are specifically favored due to their wide safety profile, focus on multi-targets, and economic applications \[[@CR13]\]. EA is one of the representative dietary polyphenols widely found in fruits and vegetables. Our previous study indicated that EA had significant suppressive effects on breast cancer growth and neoangiogenesis \[[@CR14]\]. Meanwhile, extensive studies also proved EA to be an anti-cancer compound with multiple functions such as antioxidant, apoptosis induction, carcinogen-DNA binding blockage, and metastasis inhibition \[[@CR15], [@CR16]\]. However, little investigation has been carried out to explore its CSCs-limiting effects and the precise molecular target involved.
In the current study, we aimed to determine the direct molecular target of EA and its potential regulation effects on CSCs metastasis. DARTS analysis identified ACTN4 as the direct target of EA, and ACTN4 was found to closely correlate with CSCs' self-renewal and metastatic abilities, poor survival period, and basal-like phenotype. Mechanism exploration revealed that interruption of ACTN4/β-catenin interaction will result in the activation of β-catenin proteasome degradation. Our findings would not only facilitate present understanding on the critical role of ACTN4 in breast CSCs metastasis, but also benefit for designing potentially more effective therapeutic strategies against breast cancer.
Methods {#Sec2}
=======
Chemicals and reagents {#Sec3}
----------------------
EA (over 97% purity) was provided by Alpha Aesar Company (Alfa Aesar, WardHill, MA). Dimethylsulfoxide (DMSO) was used to prepare the stock solution of EA and kept at −20 °C. 3-(4, 5-dimethylthiazol −2-yl) -2, 5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), insulin, 4′,6-diamidino-2-phenylindole (DAPI),
lithium chloride (LiCl), Akt inhibitor LY294002, cycloheximide (CHX) and proteasome inhibitor MG132, and were all purchased from Sigma (St. Louis, MO, USA).
Cell culture {#Sec4}
------------
The human breast cancer cell lines MCF-7, BT-549, MDA-MB-231, SKBr3, MDA-MB-453, T47D, BT-474, HCC1937, and MCF-10A were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in medium (L15 for MDA-MB-231; RPMI-1640 for BT-549 and MCF-7; DMEM for SKBr3, MDA-MB-453, T47D, BT474, and HCC1937; and F12 for MCF-10A) at 37 °C in a humidified incubator with or without 5% CO2. 10% FBS and 1% penicillin and streptomycin (Gibco Life Technologies, Lofer, Austria) were added as supplements. The CSCs were sorted from MDA-MB-231 and BT-549 cells and maintained in DMEM/F12 medium. B27 (Invitrogen, Carlsbad, CA, USA), 5 μg/ml of insulin, 20 ng/ml of hEGF (BD Bioscience, Bedford, MA, USA), 1% penicillin/streptomycin and 0.4% BSA were added to DMEM/F12 medium for in vitro propagation.
Breast cancer mice models and EA intervention {#Sec5}
---------------------------------------------
All animal procedures were performed in accordance with the institutional guidelines for the care and use of laboratory animals approved by the Animal Care and Use Committee of Guangdong Provincial Hospital of Chinese Medicine (the Ethics Approval Number 2015008) and the National Institutes of Health guide for the care and use of Laboratory animals. For MMTV-PyMT transgenic mice, 3-week-old female mice were randomly divided into the vehicle and the EA treatment groups, and EA (50 mg/kg/d) was given by intragastric administration from the Weeks 3--18 (*n* = 10 mice, total of 100 glands). For xenograft models, 10^6^ cells of MDA-MB-231 or BT-549 with or without genetic alternations were subcutaneously implanted into the mammary fat pads of female NOD/SCID mice at 4-week-old. After the tumor size reached over \~5 × 5 mm, EA (50 mg/kg/d) was given by intragastric administration for an additional 4 weeks. We monitored tumor formation and tumor size twice a week, and removed the tumors at the end-point of experiments for additional studies.
India ink assay {#Sec6}
---------------
Pulmonary metastatic lesions were imaged and calculated by India ink staining (15% India ink, 85% water, 3 drops of NH~4~OH/100 ml) through intra-tracheal injection. Feket's solution (300 ml of 70% EtOH, 30 ml of 37% formaldehyde, and 5 ml of glacial acetic acid) were then prepared and washed the ink-stained lungs overnight to develop the white tumor nodules against a blue lung background.
Immunohistochemistry and Immunofluorescence analysis {#Sec7}
----------------------------------------------------
Tumor samples were paraffin-embedded and cut into 4 μm sections, and subsequently mounted onto poly-L-lysine-coated slides for immunohistochemistry analysis. The sections were then treated with xylene twice for 10 min and rehydrated with ethanol from 100% to 70% gradually and finally immersed in distilled water. H&E staining was applied to identify the lung metastatic lesions. For immunohistochemistry analysis, the sections were firstly treated with methanol (with 0.3% hydrogen peroxide) for 30 min to inactivate the endogenous peroxide at room temperature. Antigen retrieval was performed by heating the slides in sodium-citrate buffer, followed by permeabilization with 0.2% Triton X-100 for 15 min. After blocking with 10% goat serum, the sections were incubated with indicated primary antibody ACTN4 (Abcam, Cambridge, USA) at 4 °C overnight. DAB detection system (Dako A/S, Glostrup, Denmark) was applied as chromogenic agents according to the manufacturer's instructions. Finally, sections were counterstained using Mayer's hematoxylin, dehydrated, cleared, and mounted before examination. With regard to cellular immunofluorescence detection, 4% paraformaldehyde and 0.2% triton X-100 were firstly administrated to cells for 10 min. Following goat serum blocking for 60 min, the samples were co-incubated with primary antibodies including ACTN4 (Abcam, Cambridge, USA) and β-catenin (Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight, and subsequently labeled with fluorescence-conjugated secondary antibodies for 2 h at room temperature. 4′,6-diamidino-2-phenylindole (DAPI) was finally applied for nuclear staining and the signals were detected with NIKON TS2R fluorescence microscopy.
Flow cytometry analysis {#Sec8}
-----------------------
Single cell suspension were prepared and suspended in wash buffer (PBS containing 1% FBS) at a density of 10^7^/ml. Aldehyde dehydrogenase-based cell detection kit (Stem Cell Technologies, Grenoble, France) was used to detect the subpopulation of CSCs. Briefly, Aldehyde dehydrogenase (ALDH) substrate (Bodipy-aminoacetaldehyde) was co-incubated with 10^6^ cells for 45 min at 37 °C. Diethylaminobenzaldehyde (DEAB), an ALDH1 enzyme inhibitor, was added as a negative control. ALDH fluorescence was detected by a 488-nm blue laser. For CD44^+^/CD24^−^ detection or sorting assay, the cells were incubated with primary antibodies CD44-FITC, CD24-PE, and/or ACTN4-APC (BD Biosciences, San Diego, CA, USA) at 4 °C for 40 min. After washing with PBS, the cells were re-suspended and analyzed with FACSAria SORP (BD Biosciences, San Jose, CA, USA). FlowJo software was applied for data analysis.
Cell proliferation and colony formation assays {#Sec9}
----------------------------------------------
MTT assay was conducted according to the manufacturer's instructions. EA-treated or gene-modified cells were also seeded into 6-well plates at a density of 1000 cells/well to form colonies. After 2 weeks, the colonies were stained with Coomassie Blue and counted.
Wound healing and transwell migration assays {#Sec10}
--------------------------------------------
For wound healing assay, the wound gap was recorded at 0, 24, or 48 h after the scratch made with a 10-μl pipette tip. Transwell assay was examined using 8-mm pore Transwell chambers (Milipore, Billerica, MA, USA).
Mammosphere formation and tumorigenic evaluation assays {#Sec11}
-------------------------------------------------------
The sorted breast CSCs were subjected into mammosphere formation assay at a density of 1000 cells/well in ultra-low attachment plates. For tumorigenic assay, a series dilution of CSCs (10^5^, 10^4^, 10^3^) were injected into the mammary pads of NOD/SCID mice to compare the tumorigenic ratio and tumor initiating cell frequency calculated by Extreme Limiting Dilution Analysis (ELDA) software.
Western blotting and co-immunoprecipitation analysis {#Sec12}
----------------------------------------------------
Total protein extraction and western blot analysis were performed as described previously \[[@CR17]\]. Antibodies for western blotting included ACTN4 (Abcam, Cambridge, USA), vimentin, E-cadherin (Abclonal, Cambridge, MA, USA), β-catenin, *p*-β-catenin (Ser33/37/Thr41), Akt, *p*-Akt (Ser473), GSK3β, *p*-GSK3β (Ser9), lamin B, β-actin (Cell Signaling Technology, MA, USA) as well as secondary anti-rabbit or anti-mouse antibodies. The interaction between ACTN4 and β-catenin were analyzed by co-immunoprecipitation analysis according to the instruction provided by Dynabeads Protein G immunoprecipitation kit (Invitrogen, Carlsbad, CA, USA).
Darts {#Sec13}
-----
DARTS strategy was applied to identify the precise protein target of EA according to the protocol provided by *Lomenick* et al. \[[@CR18]\]. Protein lysates from CSCs were quantified using bicinchoninic acid protein assay. Different concentrations of EA were then co-incubated with the lysates for 60 min at room temperature. The drug-protein interaction system was then treated with pronase (1:50, Roche Applied Science) to undergo proteolysis for 30 min at 4 °C. The lysates were finally denatured and subjected to western blotting analysis. The protective band was visualized by Coomassie bule staining and identified by MALDI-TOF-MS.
In vitro ubiquitination assay {#Sec14}
-----------------------------
Ubiquitination detection assay was carried out according to the instruction provided by Ubiquitination Kit (UW9920, BioMol). The prepared cell lysates were firstly incubated with 100 nM E1, 2.5 mM UbcH5a as E2 \[[@CR19]\], 20 U/ml of inorganic pyrophophatase (Sigma-Aldrich), 5 mM dithiothreitol, 5 mM Mg-ATP and 2.5 mM biotin-labelled ubiquitin in a 50 ml reaction system at 37 °C. After 4 h incubation, 50 ml of 2 × non-reducing gel-loading buffer was added to quench the reaction and subjected to SDS-PAGE analysis. After the proteins smaller than 70 kDa ran out, the gel was transferred onto PVDF membrane and immunoblotted with β-catenin antibody.
Real-time PCR {#Sec15}
-------------
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was applied to extract the total RNA, followed by reverse transcription reaction using the first-strand cDNA synthesis kit (Roche, Mannheim, Germany). SYBR Green kit (Roche, Mannheim, Germany) was utilized to perform real-time PCR analysis on Roche LightCycler 480 detector. PCR reaction condition was set as 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 1 min. The target gene expression was calculated by 2^-△△Ct^ and normalized to the housekeeping gene control. The primers' sequences were listed in Additional file [1](#MOESM1){ref-type="media"}: Table S1.
Plasmids and siRNA construction and transfection {#Sec16}
------------------------------------------------
The pENTER vector plasmid carrying *ACTN4-*cDNA cloning and shACTN4 were purchased from Vigene Biosciences (Jinan, China) and transfected into cells using LipoFiter™ reagent (Hanbio Biotechnology Co, LTD. Shanghai, China). Scrambled plasmids were set as negative control. ACTN4 siRNA and their scrambled ones were bought from Invitrogen (Carlsbad, CA, USA) and transfected by X-tremeGENE siRNA transfection reagent (Roche Diagnostics, IN). The target protein expression was confirmed by western blotting. For the TCF/LEF luciferase assay, the TOPFLASH or FOPFLASH plasmids (Promega, Madison, WI) were transiently transfected into cells with or without EA treatment. pRL-TK plasmid was co-transfected for normalizing the transfection efficiency.
RNA sequencing {#Sec17}
--------------
RNA preparation, library construction and sequencing on BGISEQ-500 platform was performed at Beijing Genomics Institution ([www.genomics.org.cn](http://www.genomics.org.cn), BGI, Shenzhen, China). Genes expression levels were quantified by a software package called RSEM \[[@CR20]\]. NOISeq method was used to screen differentially expressed genes (DEGs) between groups. Gene Ontology (GO) and pathway annotation and enrichment analyses were based on the Gene Ontology Database (<http://www.geneontology.org> [/]{.ul}) and KEGG pathway database ([http://www.genome.jp/kegg/](http://www.genome.jp/kegg)), respectively. Statistical analysis was performed and DEGs were selected with the criteria of fold change ≥ 1.2, *P* ≤ 0.05.
TMA and TCGA analysis {#Sec18}
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The tissue microarray HBre-Duc060CS-03 is commercially bought from Shanghai Outdo Biotech Company (<http://www.superchip.com.cn/technology/Default.aspx>, Shanghai, China) with National Human Genetic Resources Sharing Service Platform (Grant NO.: YCZYPT \[2017\] 02). All of the procedures were followed with the aforementioned immunohistochemistry assay as well as the manufacturer's instructions. TCGA invasive breast carcinoma cancer study based on Agilent microarrays (TCGA, 2015) was applied to analyze *ACTN4* mRNA expression changes by the software cBioPortal ([http://www.cbioportal.org](http://www.cbioportal.org/)). All the data retrieved from TCGA was supported by the guidelines built by TCGA Ethics, Law and Policy group, which are in compliance with the Helsinki Declaration ([http://www.wma.net.u.vtrus.net/en/ 30publications/10policies/b3/index.html](http://www.wma.net.u.vtrus.net/en/%2030publications/10policies/b3/index.html)).
Statistical analysis {#Sec19}
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Data analysis was performed with SPSS 13.0 software. The data were expressed as mean ± SD. Student's *t*-test was used to compare the statistical difference between groups. The significance of multiple groups was compared using the one-way analysis of variance (ANOVA) followed by the Dunnett's post hoc test. A value of *P* \< 0.05 was considered significant.
Results {#Sec20}
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EA retards tumor growth and pulmonary metastasis {#Sec21}
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We initially aimed to characterize the anti-cancer activities of EA on the model of MMTV-PyMT transgenic mice, which produces spontaneous breast cancer. This mouse model is internationally well-accepted for preclinical breast cancer study based on its similar pathogenesis and characteristics with human breast malignancies. Here, descriptors (grainy, granules, pea, or tumor) are used to define the palpable phenotype for all mice glands each week \[[@CR21], [@CR22]\]. In particular, atypical hyperplasia is expected to be present in the 4-week-old mice with fine grains in the mammary glands. Since EA is a dietary compound, we began to feed the female MMTV-PyMT mice with EA (50 mg/kg/d) as early as 3 weeks of age by oral gavage every day. As shown in Fig. [1a](#Fig1){ref-type="fig"}, stage-specific progressions were significantly delayed by EA treatment compared with the vehicle group. Grainy lesions were found in 100% of the glands in Week 8 (vehicle) and week 9 (EA), respectively. The median time to 50% granules was 7 weeks for the vehicle group, whereas the EA group developed 50% granules on Week 8. By the week 14, all mammary glands (*n* = 100) developed pea-sized tumors in the vehicle group, while more than 10 glands in the EA treatment group were identified as free of lesions. EA treatment also obviously delayed the onset of measurable tumors by approximately 2 weeks compared to the vehicle. In addition, mammary tumors in the vehicle group exhibited a significantly increased tumor volume with a more necrotic and hemorrhagic region, suggesting that EA resulted in significant tumor growth retardation (Fig. [1b](#Fig1){ref-type="fig"}). Cancer progression was also slowed down if the EA treatment was started after tumor growth in MMTV-PyMT mice at ages ranging from 6th to 15th weeks (Additional file [2](#MOESM2){ref-type="media"}: Figure. S1A).Fig. 1EA retards tumor growth and pulmonary metastasis. **a** Tumor incidence ratios between the vehicle and EA-treated groups in the MMTV-PyMT mice were compared using a log-rank test, highlighting the median time and the hazard ratio in each curve for grainy, granules, pea, or tumor phenotypes, respectively (*n* = 10 mice, total of 100 glands); **b** Representative images of tumors dissected from the vehicle or the EA-treated mice by 15 weeks (*left*); The curve showed EA resulted in significant tumor growth retardation compared with control at mice ages ranging from 3th to 15th weeks (*Upper right*); Tumor volume in the vehicle group (1383.53 ± 81.10 mm^3^) showed a significant difference compared with the EA-treated group (731.53 ± 59.32 mm^3^) (*Lower right*); **c** Representative images of lungs dissected from the vehicle or the EA-treated mice from the 9th to 18th weeks of mice age. India ink staining was used to identify pulmonary metastatic nodules of each group. H&E staining was utilized to observe tumor micromorphology of each group with 40-fold magnification; **d** The number and volume of metastatic nodules were measured from the 9th to 18th weeks of mice age. The results indicated that EA significantly inhibited the number and volume of metastatic nodules; **e** Mice overall survival (OS, %) in the control and EA treatment groups is shown by the Kaplan-Meier curve; **f** The ALDEFLUOR assay was to identify the population of CSCs in the metastatic nodules of the vehicle or the EA-treated MMTV-PyMT mice. ALDH^hi^ cells were significantly reduced in the EA-treated group (\**P* \< 0.05, \*\**P* \< 0.01, \*\*\**P* \< 0.001, values represented as the Mean ± SD, *n* = 10)
Previous studies have indicated that the MMTV-PyMT mice tend to develop luminal-type mammary adenomas with a tendency of lung metastasis lesions on week 10--15 \[[@CR21], [@CR22]\]. Therefore, we prolonged the administration periods of EA for an additional 3 weeks to examine its inhibition effects on pulmonary metastasis. Visual differences in the lung specimens were observed from Weeks 9--18 between the 2 groups. India ink staining and hematoxylin and eosin (H&E) staining further confirmed the suppressive effects of EA on the pulmonary metastasis, especially at the end-stage progression (Fig. [1c](#Fig1){ref-type="fig"}). In particular, EA administration obviously inhibited the number and volume of metastatic nodules. The mean metastatic nodules in the EA intervention group on Week 15 was significantly decreased to over 50% compared with the vehicle group, while EA elicited a dramatic 90% reduction in the nodule volume compared to the vehicle groups by Week 18 **(**Fig. [1d](#Fig1){ref-type="fig"} **)**. We additionally investigated the overall survival (OS) of mice with or without EA (50 mg/kg/day) at the end of treatment on week 18. The death ratio is 5/10 and 1/10 in control and EA group, respectively. The Kaplan-Meier curve analysis demonstrated that EA significantly prolonged the survival time of MMTV-PyMT mice, possibly due to the decreased tumor burden and the limited lung metastasis (*P* = 0.0265, Fig. [1e](#Fig1){ref-type="fig"}). To thoroughly investigate the potent anti-metastasis effects of EA, we harvested metastatic nodules to detect the CSC population by the ALDH assay. Compared with the DEAB negative control, the CSCs in metastatic nodules of the vehicle group reached approximately 66%, while EA treatment dramatically ALDH^hi^ cells to 38.5% **(**Fig. [1f](#Fig1){ref-type="fig"} **)**. This information hinted that EA might antagonize breast cancer progression and subsequent pulmonary metastasis via suppressing breast CSC activity. Finally, little adverse events, including decreased appetite, weight loss, ruffling fur, or abnormal behavior, was observed in the mice throughout the whole experiment, suggesting that the anti-cancer bioactivities of EA might not be attributed to its toxicity effects (Additional file [2](#MOESM2){ref-type="media"}: Figure. S1B).
EA inhibits breast cancer metastasis associating with CSC limitation {#Sec22}
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After in vivo validation, we continued to validate whether EA could exert anti-proliferative effects on the representative breast cancer cell lines MDA-MB-231 (basal-like), BT-549 (basal-like), and MCF-7 (luminal-like) by MTT assay. As shown in Fig. [2A](#Fig2){ref-type="fig"}, EA could inhibit cell proliferation in a dose-dependent manner, with an IC50 of approximately 25 μM for MDA-MB-231, 20 μM for BT-549, and 30 μM for MCF7 within 24 h. EA administration continued to significantly suppress breast cancer cells over the next 72 h, whereas it did not significantly affect the proliferation of the normal mammary cells MCF-10A from 24 to 72 h. Furthermore, flow cytometry analysis indicated that EA increased the subpopulation of cells undergoing late apoptosis, and arrested the cell cycle in the breast cancer cells mainly at the S&G2/M phases (Additional file [3](#MOESM3){ref-type="media"}: Figure. S2). These data proved that EA elicited a cell growth--inhibitory effect on the breast cancer cell lines, particularly on basal-like phenotypes.Fig. 2EA inhibits breast cancer cell proliferation, migration, and invasion abilities associated with CSC limitation. **(A)** EA suppressed the proliferation of the breast cancer cell lines MDA-MB-231, BT-549, and MCF-7, while posing little inhibitory effects on the human normal mammary epithelial cell MCF-10A (\*\**P* \< 0.01 versus control, values represented as the mean ± SD, *n* = 3) at different dose- and time-intervals indicated; **(B)** The wound healing and chamber invasive assay revealed that breast cancer cell migration and invasion were inhibited by EA in a time- and dose-dependent manner (\*\**P* \< 0.01 versus control at 12 h, ^\#^ *P* \< 0.05, ^\#\#^ *P* \< 0.01 versus control at 24 h, ^&&^ *P* \< 0.01 versus control at 48 h, values represented as the mean ± SD, *n* = 3); **(C)** EA dose-dependently reduced the CSC populations in the breast cancer cells. Representative dot plots of CD44^+^CD24^−/low^ cell surface markers in the breast cancer cells MDA-MB-231 and BT-549 (\*\**P* \< 0.01 versus control, values represented as the mean ± SD, *n* = 3); **(D)** EA limited the primary and secondary CSC mammosphere of the MDA-MB-231 and BT-549 cells in a dose-dependent manner (\**P* \< 0.05, \*\**P* \< 0.01, values represented as the mean SD, *n* = 3); **(E) a.** Western blotting analysis validated that EA elevated p-β-catenin, while inhibiting *p*-GSK-3β and *p*-AKT in the MDA-MB-231 stem-like cell lysates time-dependently; **b.** EA did not suppress the β-catenin mRNA level after 24 h administration; **c.** EA inhibited its transcriptional activity as detected by the TCF/LEF luciferase assay (\*\**P* \< 0.01, values represented as the mean ± SD, *n* = 3); **d.** The relative mRNA expressions of the β-catenin downstream genes were inhibited by EA in the MDA-MB-231 stem-like cell lysates (\**P* \< 0.05, \*\**P* \< 0.01, values represented as the mean ± SD, *n* = 3)
Because EA administration resulted in the decreased metastatic nodules, we next investigated the in vitro effects of EA on breast cancer cell migration and invasion. The influence of EA on the migrate ability of breast cancer cells was assessed by wound healing assay. The gap width in the control group was narrowed more rapidly than that of EA group from 0 to 48 h, indicating that EA could dose- and time-dependently inhibit the migration ability of both MDA-MB-231 and BT-549 cells. For the chamber invasion assay, the results showed that the number of invasive cells was significantly reduced following EA treatment, demonstrating that EA treatment profoundly impaired the invasion capacity of the cancer cells **(**Fig. [2B](#Fig2){ref-type="fig"}; Additional file [4](#MOESM4){ref-type="media"}: Figures. S3 and S4**)**. Since metastasis is closely associated with stemness properties, we then sought to determine if the anti-metastasis ability of EA could be due to its pharmacological targeting of CSCs. As expected, EA dose-dependently decreased the CD44^+^CD24^−/low^ phenotypes in both CSC-enriched MDA-MB-231 and BT549 cells after 24 h **(**Fig. [2C](#Fig2){ref-type="fig"} **)**. To further assess the influences of EA on the self-renewal ability of CSCs, we sorted CD44^+^/CD24^−/low^ subpopulations from MDA-MB-231 cells and conducted mammosphere formation assay. We exposed mammospheres to increasing concentrations of EA and then cultured them for 1 additional passage without EA administration. EA intervention was found to be associated with a decreased number and size of the stem-like cell spheres in MDA-MB-231cells. In particular, the decreased number and size of sphere-forming cells in the subsequent passages further demonstrated that the abrogated CSC self-renewal capacity elicited by EA could possibly be effective and heritable **(**Fig. [2D](#Fig2){ref-type="fig"} **)**.
β-catenin is one of the structural molecules mediating the cell-cell adhesion for cancer cell migration, and is also known for a mediator of cellular signaling pathways for CSC maintenance. Given its distinct role in the epithelial-mesenchymal transition (EMT) process and CSC properties, we tested the influences of EA on β-catenin expression and distribution in CSCs derived from MDA-MB-231 cells. Our results found that the cytosolic and nuclear β-catenin expressions were simultaneously downregulated by EA via the Akt/GSK-3β pathway **(**Fig. [2E](#Fig2){ref-type="fig"}(a)**)**, whereas it posed little effect on the β-catenin mRNA level after 24 h EA treatment **(**Fig. [2E](#Fig2){ref-type="fig"}(b)), indicating that the β-catenin suppression by the EA treatment might have possibly occurred at the posttranslational level rather than transcriptionally. A TCF/LEF luciferase reporter study also revealed that EA potently inhibited β-catenin transcription activity, and possibly resulting in the impaired expressions of the stem-like markers (Nangog, C-myc, Oct-4, and Survivin), as well as the cancer growth and metastasis-associated molecules (Cyclin D1, Snail, ZEB1, and Slug) (Fig. [2E](#Fig2){ref-type="fig"}(c) and 2E(d**))**. Collectively, EA inhibits breast cancer metastasis possibly by limiting CSCs.
Identification of ACTN4 as the direct target of EA in breast CSCs {#Sec23}
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Although it was demonstrated that EA suppressed breast CSCs through inhibiting β-catenin signaling, the direct molecular target of EA was still remained unclear. The DARTS strategy is designed to identify the direct binding target of small molecules based on the lower susceptibility of chemical-protein complex to proteolysis \[[@CR18], [@CR23]\]. Following EA treatment, DARTS identified a protective band near 100 kDa presenting as a dose-dependent manner **(**Fig. [3a](#Fig3){ref-type="fig"} **)**. MALDI-TOF-MS subsequently identified the band as ACTN4 **(**Fig. [3b and c](#Fig3){ref-type="fig"} **)**. Immunoblotting analysis further revealed that EA treatment significantly led to an average suppression of ACTN4 expression in tumors from six pairs of MMTV-PyMT transgenic mice, accompanying with decreased expressions of vimentin and β-catenin **(**Fig. [3d](#Fig3){ref-type="fig"} **)**. The reduced expression of ACTN4 was further confirmed at protein levels in EA--treated breast cancer cell lines **(**Fig. [3e](#Fig3){ref-type="fig"} **)**. Taken together, our results showed that EA could suppress the ACTN4 expression both in vitro and in vivo, suggesting that ACTN4 might be a critical contributor to breast cancer metastasis.Fig. 3Identification of ACTN4 as the direct target of EA in breast CSCs. **a** MDA-MB-231 CSCs were treated with EA at varying concentrations, a 100-kDa protein band was protected from digestion in a dose-dependent manner. The arrowhead indicates the 100-kDa protein; **b** Mass spectrogram identified the 100-kDa-targeted protein of EA as ACTN4; **c** Amino acid sequence of actinin-4. *Bold letters*: peptides that correspond to peaks identified by mass spectrometry; **d** EA administration significantly led to an average suppression in the ACTN4 expression levels, accompanied by inhibited expressions of β-catenin and vimentin in six tumors from EA-treated MMTV-PyMT transgenic mice tumors. A quantitative measurement was conducted to analyze with Image J (\*\**P* \< 0.01, values represented as the mean ± SD, *n* = 6). Cell lysates of Lanes 1, 3, 5, 7, 9 and 11 came from six MMTV-PyMT transgenic mice in vehicle groups, while proteins of Lanes 2, 4, 6, 8, 10 and 12 belonged to six mice in EA treatment groups; **e** EA inhibited ACTN4 expression in the MDA-MB-231, BT-549, and MCF-7 cells by western blot analysis
To directly demonstrate whether ACTN4 is critical for EA action, we transfected ACTN4 recombinant plasmid in MDA-MB-231, BT-549 and MCF-7 cells with or without EA. According to previous studies, some known inhibitor particularly AKT inhibitor LY294002 could potently suppress breast cancer proliferation, migration and invasion \[[@CR17], [@CR24], [@CR25]\], therefore we also included LY294002 (10 μM) as positive control in the following cell proliferation and migration assays. Proliferation analysis revealed that ACTN4 overexpression enhanced breast cancer cell number, and effectively restored the inhibition effects of EA on breast cancer cells **(**Fig. [4a](#Fig4){ref-type="fig"} **)**. In addition, ACTN4 overexpression endowed EA-treated cells with increased migratory and invasive ability, further implying the significant role of ACTN4 in mediating the metastasis inhibition effects of EA **(**Fig. [4b](#Fig4){ref-type="fig"} **)**. The results also gave us a comparison picture about EA and AKT inhibitor LY294002 on their anti-cancer potential. Meanwhile, compared with cells transfected with empty vector, ACTN4 overexpression not only promoted CSC properties but also abrogated the CSC suppressive effects induced by EA, presenting as increased CSC proportion and enhanced sphere formation capability **(**Fig. [4c](#Fig4){ref-type="fig"} **)**.Fig. 4ACTN4 overexpression abrogated the anti-cancer and anti-metastatic effects induced by EA on breast cancer. **a** MDA-MB-231, BT-549 and MCF-7 cells were transfected with ACTN4 recombinant plasmids and treated with or without 25 μM EA for 24 h. LY294002 (10 μM) was used as a positive control for this assay. The findings revealed that ACTN4 overexpression enhanced breast cancer cell number, and effectively restored the inhibition effects of EA on breast cancer cells (\**P* \< 0.05, \*\**P* \< 0.01, values represented as the mean ± SD, *n* = 3); **b** ACTN4 overexpression endowed EA-treated cells with increased migratory and invasive ability (\*\**P* \< 0.01, values represented as the mean ± SD, *n* = 3). LY294002 (10 μM) was used as a positive control for this assay; **c** ACTN4 overexpression abrogated the CSC suppressive effects induced by EA, presenting as increased CSC proportion and enhanced sphere formation capability (\*\**P* \< 0.01 EA + empty vector group versus EA + ACTN4 group in 1st CSC sphere generation, ^\#\#^ *P* \< 0.01 EA + empty vector group versus EA + ACTN4 group in 2nd CSC sphere generation, values represented as the mean ± SD, n = 3)
Together, these data demonstrated the significant role of ACTN4 in mediating the metastasis inhibition effects of EA. The findings were further supported by the fact that siACTN4 did not significantly aggravate the inhibition effects of EA on cell proliferation **(**Fig. [5a and b](#Fig5){ref-type="fig"} **)** and migration **(**Fig. [5c and d](#Fig5){ref-type="fig"} **)** of breast cancer cells. For xenograft models, 10^6^ cells of MDA-MB-231 with or without ACTN4 knockdown were subcutaneously implanted into the mammary fat pads of female NOD/SCID mice at 4-week-old in the presence or absence of EA (50 mg/kg/d). There was no additive inhibitory effects on tumor growth when combining EA with ACTN4 knockdown **(**Fig. [5e](#Fig5){ref-type="fig"} **)**. Given that CSCs represent high metastatic potential \[[@CR9], [@CR10]\], we further detected the CSC population in the tumor essence by ALDH assay. The ALDH^+^ subpopulations in EA plus shACTN4 group showed no significant difference with those in EA group **(**Fig. [5f](#Fig5){ref-type="fig"} **)**. To thoroughly investigate the global effects of EA treatment and ACTN4 knockdown on breast cancer, we additionally perform a global RNA-seq for MDA-MB-231 cells treated with EA or shACTN4 using a BGISEQ-500 by the Beijing Genomic Institution ([www.genomics.org.cn](http://www.genomics.org.cn), BGI, Shenzhen, China). Subsequent analysis identified 510 differentially expressed genes (DEGs) in control *v.s.* EA-treated group as well as 399 DEGs in control *v.s.* ACTN4 knockdown group, respectively (fold change ≥ 1.2, *P* ≤ 0.05). The heat map columns and venn diagrams of altered DEGs were shown in Additional file [5](#MOESM5){ref-type="media"}: Figure. S5A and B. For Gene Ontology (GO) analysis (Additional file [5](#MOESM5){ref-type="media"}: Figure. S5C), the terms existing in both DEG groups encompassed various biological processes during cancer progression, such as localization/locomotion, cell death/proliferation, extracellular region/space, cytoskeleton as well as binding activity, etc. For Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (Additional file [5](#MOESM5){ref-type="media"}: Figure. S5D), the interfered pathways co-existing in both DEGs were mainly responsible for supporting cancer growth and invasion like focal adhesion signaling, signaling pathways regulating pluripotency of stem cells, Wnt signaling, PI3K-Akt signaling, etc. Of note, the results demonstrated that EA treatment and ACTN4 knockdown possibly changed the same group of target genes, suggesting that EA treatment and ACTIN4 knockdown might change the same group of target genes.Fig. 5ACTN4 knockdown exerted no additive anti-cancer and anti-metastatic effects induced by EA on breast cancer. **a** MDA-MB-231 and (**b**) BT-549 cells were transfected with siACTN4 for 48 h and treated with or without 25 μM EA for additional 24 h. LY294002 (10 μM) was used as a positive control substance for this assay. The findings revealed that siACTN4 did not aggravate the inhibition effects of EA on breast cancer cells when used in combination; **c-d** siACTN4 did not significantly aggravate the inhibition effects of EA on cell migration and invasion of MDA-MB-231 cells. LY294002 (10 μM) was used as a positive control substance for this assay; **e** For xenograft models, 10^6^ cells of MDA-MB-231 with or without ACTN4 knockdown were subcutaneously implanted into the mammary fat pads of female NOD/SCID mice at 4-week-old in the presence or absence of EA (50 mg/kg/d). There was no additive inhibitory effects on tumor growth when combining EA with ACTN4 knockdown; **f** The ALDEFLUOR assay showed that the ALDH^+^ subpopulations in the tumor essence between EA alone and EA plus shACTN4 groups
ACTN4 promotes breast cancer growth and metastasis {#Sec24}
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We next explored whether the anti-cancer and anti-metastasis effects of EA were ACTN4-dependant. Immunoblotting and real-time PCR analysis revealed that the enhanced expression of ACTN4 was observed in several basal-like breast cancer cell lines (MDA-MB-231, BT-549, and HCC1937) compared with the normal mammary cell line MCF-10A **(**Fig. [6a](#Fig6){ref-type="fig"} **)**. The stable ACTN4-knockdown cells (MDA-MB-231 shACTN4 and BT-549shACTN4) and ACTN4-overexpressing cells (MCF-7ACTN4) were established by shRNA transfection and plasmids. ACTN4 expression in the gene-modified cells was confirmed by western blot analysis and real-time PCR **(**Fig. [6b](#Fig6){ref-type="fig"} **)**. It was found that the ACTN4-knockdown cells maintained an epithelial cell appearance, whereas their shRNA control exhibited a spindle-like mesenchymal cell morphology. In addition, its absence led to a blockage of malignant cell proliferation, impaired colony formation, and ameliorated metastasis potency in vitro (Fig. [6c and d](#Fig6){ref-type="fig"}). Moreover, tumor formation in the nude mice demonstrated that ACTN4 knockdown could significantly inhibit tumor growth and lung metastasis (Fig. [6e](#Fig6){ref-type="fig"}).Fig. 6ACTN4 promotes breast cancer proliferation and metastasis in vitro and in vivo. **a** Intracellular expression of ACTN4 was determined by Western blot (left) and real-time PCR (right) analysis, respectively; **b** ACTN4 expression was modified by transfecting recombinant plasmid or its shRNA in breast cancer cells and subjected to Western blotting (left) and real-time PCR (right) validation; **c** MTT and colony formation assay showed that ACTN4 silencing abrogated breast cancer cell proliferation while its overexpression promoted cell growth; **d** ACTN4 silencing inhibited the migration and invasion abilities of MDA-MB-231 cells; **e** ACTN4 silencing inhibited breast cancer growth and lung metastasis in vivo (\**P* \< 0.05, \*\**P* \< 0.01 versus control, values represented as the mean ± SD, n = 3)
ACTN4 facilitates breast CSCs self-renewal by stabilizing β-catenin signaling {#Sec25}
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The aforementioned results prompted us to explore the correlation between ACTN4 and CSC phenotypes. Among 9 breast cancer cell lines examined, 3 cell lines (MDA-MB-231, BT-549, and HCC1937) contained a relatively higher percentage (\>30%) of CD44^+^/CD24^−^ cells, indicating that the high percentage of CD44^+^/CD24^−^ cells directly associates with a basal phenotype **(**Figure [7A](#Fig7){ref-type="fig"}). Nevertheless, recent studies suggested that CD44^+^/CD24^−^ cells do not correlate with distant metastasis or with the clinical outcome \[[@CR26]\]. Given that ACTN4 might be a bridge connecting CSCs and metastasis function in breast cancer, particularly for the basal-like populations, we then investigated the expression of ACTN4 in ALDEFLOUR-positive cells or CD44^+^/CD24^−^ populations. ALDEFLOUR-positive cells contained a higher ACTN4 expression compared with the ALDEFLOUR-negative cells. ACTN4 expression was also higher in the CD44^+^/CD24^−^ cells in comparison with the non-CD44^+^/CD24^−^ cells. CSC spheroids are differentiated after reseeding under adherent conditions **(**Fig. [7B](#Fig7){ref-type="fig"}(a)). We then detected the ACTN4 expressions in the non-CSCs cells in adherent culture conditions, undifferentiated CSC spheroids, and CSCs in adherence-promoting plates. As expected, the results revealed ACTN4 differences among the three groups following a pattern of CSC spheres \> CSC differentiating forms \> non-CSCs **(**Fig. [7B](#Fig7){ref-type="fig"}(b)). Taken together, these results showed an enrichment of ACTN4 in breast CSC phenotype.Fig. 7ACTN4 is closely correlated with the CSCs phenotype. **(A)** The expression panel of CD44 and CD24 in breast cancer cell lines; **(B) a.** ACTN4 expression was significantly elevated in the ALDEFLUOR-positive cells and CD44^+^/CD24^−^ phenotypes; **b.** ACTN4 expression was greatly enhanced in the undifferentiated CSC spheroids mammospheres (\**P* \< 0.05, \*\**P* \< 0.01 versus CSC spheres, values represented as the mean ± SD, n = 3); **(C)** EpCAM expression was significantly up-regulated in the ACTN4-positive CSCs by flow cytometry analysis; **(D)** CD44^+^/CD24^−^/ACTN4^+^ cells displayed enhanced proliferation in MDA-MB-231 cells (\*\**P* \< 0.01, values represented as the mean ± SD, *n* = 3); **(E)** CD44^+^/CD24^−^/ACTN4^+^ cells represent a distinct population of CSCs with higher self-renewal **(c)** and adherence-promoting **(b)** capacity (\**P* \< 0.05, \*\**P* \< 0.01 versus CD44^+^/CD24^−^/ACTN4^−^ cells, values represented as the mean ± SD, *n* = 3); **(F)** The relative mRNA expressions of EMT markers in CD44^+^/CD24^−^/ACTN4^+^ cell lysates (\**P* \< 0.05, \*\**P* \< 0.01, values represented as the mean u SD, *n* = 3)
To further extend a link between ACTN4 and CSC phenotype, we sorted ACTN4-positive (CD44^+^/CD24^−^/ACTN4^+^) and -negative cells (CD44^+^/CD24^−^/ACTN4^−^) from MDA-MB-231 CSCs and examined them for their stem-like functions. A previous study demonstrated that CD44^+^/CD24^−^/ESA^+^ was 50-fold enriched in the ability to form breast tumors relative to unfractionated tumor cells \[[@CR27]\]. In Fig. [7C](#Fig7){ref-type="fig"}, FACS analyses demonstrated that the proportion of EpCAM^+^ cells in CD44^+^/CD24^−^/ACTN4^+^ populations tended to be larger than that in CD44^+^/CD24^−^/ACTN4^−^ pools, strongly suggesting that the pattern of ACTN4 expression in the CD44^+^/CD24^−^ phenotype may be similar to that of EpCAM, and that ACTN4 may play a significant role in the self-renewal capacity of CD44^+^/CD24^−^ cells. We next explored the growth potential of CD44^+^/CD24^−^/ACTN4^+^ and CD44^+^/CD24^−^/ACTN4^−^ cells. In cell proliferation assays, it was found that ACTN4^+^ CSC-like cells showed a significantly greater proliferation capability than its ACTN4^−^ counterparts **(**Figure [7D](#Fig7){ref-type="fig"}). The ACTN4^+^ cells formed larger and increased the number of mammospheres compared with the ACTN4^−^ cells (Fig. [7E](#Fig7){ref-type="fig"}(a)). Meanwhile, the ACTN4^+^ cells also had a greater ability to reattach than the ACTN4^−^ cells (Fig. [7E](#Fig7){ref-type="fig"}(b)), and influenced the transcription of EMT markers (Figure [7F](#Fig7){ref-type="fig"}). Thus, CD44^+^/CD24^−^/ACTN4^+^ represent a distinctive invasive phenotype with a higher self-renewal and adherence-promoting capacity of CSCs. Moreover, subcutaneous injection of ACTN4-expressing CD44^+^/CD24^−^ cells resulted in an increased incidence of tumor formation, even when only 1000 cells were implanted, further verifying the tumorigenic significance of ACTN4 on breast cancer **(**Additional file [1](#MOESM1){ref-type="media"}: Table S2**)**.
We continued to thoroughly investigate the underlying mechanisms of ACTN4 mediation of the breast CSC properties. ACTN4 expression was also found to be correlated positively with the Akt/GSK-3β/β-catenin levels (Figure [8A](#Fig8){ref-type="fig"}). Moreover, ACTN4 silencing led to a significant inhibition of β-catenin signaling and downregulation of AKT/GSK-3β phosphorylation in MDA-MB-231^CD44+/CD24-^ populations, which was consistent with the molecular mechanisms of EA (Figure [8B](#Fig8){ref-type="fig"}). Double immunofluorescence analysis revealed that β-catenin and ACTN4 were overlapped mainly in the cytosol of breast CSCs sorted from MDA-MB-231 (Fig. [8C](#Fig8){ref-type="fig"}(a), arrowheads). The immunoprecipitation assay further confirmed the direct interaction between β-catenin and actinin-4 (Fig. [8C](#Fig8){ref-type="fig"}(b)). Once the cytosolic interaction between ACTN4 and β-catenin was interrupted, β-catenin stabilization might be destroyed and subsequently result in the decreased nuclear localization. Since the proteasome degradation pathway is the primary posttranslational regulatory mechanism for decreasing the β-catenin level, we therefore investigated whether ACTN4 inactivation could interrupt β-catenin stabilization by activating its proteasome degradation pathway. We firstly examined the effect of the protein synthesis inhibitor CHX on β-catenin level in ACTN4-silenced cells. After CHX treatment, the half-life of β-catenin degradation in shCtrl cells was estimated to be approximately 12.1 h whereas it was 4.5 h in ACTN4 silencing cells, implying that β-catenin degradation was accelerated following ACTN4 knockdown. By contrast, β-catenin degradation induced by ACTN4 knockdown was blocked following the treatment of proteasome inhibitor MG132, further confirming that the proteasome degradation pathway was activated by ACTN4 downregulation (Figure [8D](#Fig8){ref-type="fig"}). To thoroughly understand how ACTN4 silencing induces β-catenin proteasome degradation, we performed the in vitro ubiquitination assay in the presence of E2 ligase enzyme UbcH5a \[[@CR19]\]. ACTN4 overexpression caused a loss of poly-ubiquitination accumulation of β-catenin, while its knockdown was found to increase β-catenin ubiquitination and its consequent degradation, presenting as accumulation of ubiquitinated β-catenin. The results indicated that ACTN4 silencing might activate the activity of E2 ligase enzyme UbcH5a, and subsequently accelerate β-catenin proteasome degradation (Figure [8E](#Fig8){ref-type="fig"}). Furthermore, the ACTN4-mediated stabilization of β-catenin was tightly correlated with the Akt/GSK-3β signaling. When AKT inhibitor LY294002 was administrated, the decreased β-catenin expression following ACTN4 silencing was aggravated. However, β-catenin reduction induced by ACTN4 silencing was recovered following GSK-3β inhibitor LiCl treatment (Figure [8F](#Fig8){ref-type="fig"}). All of these results indicated that ACTN4 sustained breast CSC properties mainly by increasing β-catenin stabilization.Fig. 8ACTN4 sustains CSCs properties by promoting β-catenin stabilization. **(A)** The expressions of ACTN4 and β-catenin pathway signaling were elevated in the sorted ALDEFLUOR-positive and CD44^+^/CD24^−^ cells; **(B)** ACTN4 knockdown in CSCs resulted in decreased β-catenin and p-AKT/GSK3β expressions; **(C) a.** the immunofluorescence assay showed the co-localization of ACTN4 and β-catenin was mainly located in the cytosol (arrowheads); **b.** the immunoprecipitation assay revealed the direct molecular interaction between β-catenin and ACTN4; **(D)** Breast CSCs were transfected with shACTN4, treated with CHX (10 μg/ml), and MG132 (10 μM) for the indicated time and immunoblotted, and the results showed that ACTN4 silencing could promote β-catenin proteasome degradation. A quantitative measurement was conducted to further analyze with Image J (\*\**P* \< 0.01, values represented as the mean ± SD, n = 3). After CHX treatment, the half-life of β-catenin degradation in shCtrl cells was estimated to be approximately 12.1 h, whereas it was 4.5 h in ACTN4 silencing cells based on Engauge Digitizer software analysis; **(E)** ACTN4 overexpression caused a loss of poly-ubiquitination accumulation of β-catenin, while its knockdown was found to increase β-catenin ubiquitination and its consequent degradation, presenting as accumulation of ubiquitinated β-catenin; **(F)** Breast CSCs transfected with shACTN4 were treated with LY294002 (10 μM) and LiCl (10 μM) and immunoblotted, and the results indicated that ACTN4 silencing activated β-catenin proteasome degradation via modulating GSK3β/Akt phosphorylation
ACTN4 predicts a poor survival period and basal-like phenotype {#Sec26}
--------------------------------------------------------------
To determine the ACTN4 tissue distribution and expression pattern in human breast tumors, we investigated the expression of the ACTN4 protein in a TMA containing 60 primary breast cancers and paired tumor adjacent normal (TAN) mammary tissues. As shown in representative images, ACTN4 was slightly expressed in the normal mammary tissues, whereas its expression was gradually increased in stages I--III breast cancer (Fig. [9a](#Fig9){ref-type="fig"}). These data strongly suggested that ACTN4 expression plays critical roles in breast cancer and might be a potential prognosis biomarker.Fig. 9ACTN4 predicts a poor survival and basal-like breast cancer phenotype. **(A)** ACTN4 is overexpressed in breast tumor tissues (T) compared with the tumor adjacent normal (TAN) breast epithelial tissues from the same patient analyzed by the TMA assay, and ACTN4 was significantly correlated with the breast cancer stage; **(B)** Data analysis from the TCGA database implied that ACTN4 mRNA predicted a poor overall survival (OS) **(a)** and disease free survival (DFS) **(b)** in the 1098 breast cancer patients; **(C)** ACTN4 expression was significantly elevated in the metastatic phenotypes (*P* = 0.0443, \**P* \< 0.05) and **(D)** the TNBC patients (38%)
Furthermore, 1098 patients from The Cancer Genome Atlas (TCGA) invasive breast carcinoma cancer study (TCGA, 2015) were analyzed to evaluate the oncogenic alterations of ACTN4. The mRNA alterations (amplification, mutation, and/or overexpression) of *ACTN4* occurred in 113 (10%) of the 1098 patients (data not shown). Cases with ACTN4 alterations had a significantly decreased median overall survival (123.3 months vs 97.9 months, *p* = 0.0374). The 3-year and 5-year overall survival of cases with alternated ACTN4 expression was 81.8 months and 64.5 months, respectively. Elevated ACTN4 mRNA expression was correlated with the shorter disease-free survival of patients (*p* = 3.392e-5) **(**Fig. [9b](#Fig9){ref-type="fig"}). In addition, comparison between M0 and M1patients demonstrated that cases with metastatic disease had greater ACTN4 mRNA expression (*p* = 0.0443) (Fig. [9c](#Fig9){ref-type="fig"}). TNBC phenotypes, which are usually enriched for CD44^+^/CD24^−^ CSCs, also displayed higher ACTN4 expression than other breast cancer subtypes (Fig. [9d](#Fig9){ref-type="fig"}). Overall, ACTN4 promotes breast cancer progression and metastasis, and is an independent prognostic marker associated with the poor clinical outcome in breast cancer patients.
Discussion {#Sec27}
==========
DARTS strategy is a novel drug target identification system based on the susceptibility difference to proteolysis between single drug and drug-protein complex \[[@CR23]\]. Compared with other affinity-based target identification methods, the key advantage of DARTS is that it does not require ligand modification. Therefore, DARTS is not limited by chemical structure. Here, we applied the DARTS technique to identify ACTN4 as the direct bound protein of EA in breast CD44^+^/CD24^−^ phenotypes. The successful target identification of DARTS strategy is dependent on two factors: the target of the small molecule should be highly abundant in cells, and the identified protein should not be extremely sensitive or resistant to the proteases applied \[[@CR18]\]. This indicates that ACTN4 should be a highly abundant protein in breast CSCs, and would be strongly protected by EA from proteolysis and resulted in detectable differences presented as clear variable bands in Fig. [3A](#Fig3){ref-type="fig"}. In other words, ACTN4 is one of the most abundant and important targets of EA in breast CSCs, and this is not to exclude the existence of any other possible targets of EA in cancer cells. According to literature reports, EA had inhibition effects on multiple targets of cancer cells, such as VEGFR-2 \[[@CR14]\], STAT3 \[[@CR28]\], TGF-β \[[@CR29]\], and NF-κB \[[@CR30]\], etc. However, this is the first study to demonstrate the direct target of EA in cancer cells, and a more comprehensive strategy, such as network pharmacology, might be used to establish the anti-cancer network signaling of EA in the future.
ACTN4, an actin-binding protein, has been described to exist in at least 2 different subcellular locations: the cytosol and nucleus. Shao H et al. proposed ACTN4 was largely responsible for the spreading, motility, and contractility of fibroblasts \[[@CR31]\]. Additionally, [Honda K](https://www.ncbi.nlm.nih.gov/pubmed/?term=Honda%20K%5BAuthor%5D&cauthor=true&cauthor_uid=15633123) et al. demonstrated its potent ability to increase cell motility and promote lymph node metastasis in colorectal cancer \[[@CR32]\]. Consistent with their findings, abnormal ACTN4 expression was also correlated to increased tumor invasiveness and metastasis in breast, esophageal, pancreatic, ovarian, and lung carcinomas, indicating that actinin-4 is a promising biomarker for cancer invasion and predictive indicator for patients with metastatic cancer diseases \[[@CR33]--[@CR36]\]. Several studies also identified the crucial role of ACTN4 in transcriptional regulation. ACTN4 transcriptionally potentiated myocyte enhancer factor-2 by antagonizing histone deacetylase \[[@CR37]\]. ACTN4 could also serve as a NF-κB co-activator by interacting with its RelA/p65 subunit \[[@CR38]\]. More importantly, ACTN4 and E-cadherin might compete for the same binding domain of β-catenin \[[@CR39]\]. ACTN4 was found to promote EMT and tumorigenesis by regulating Snail expression and the Akt pathway in cervical cancer \[[@CR40]\]. However, few studies have revealed the mechanisms of ACTN4-mediated CSC activities and tumor metastasis in breast cancer.
In the present study, we demonstrated at least 4 lines of evidence supporting the crucial roles of ACTN4 in facilitating CSCs and metastasis in breast cancer. On the threshold, we identified ACTN4 as the direct target of EA in breast CSCs using the DARTS strategy. Further validation suggested that EA administration significantly suppressed ACTN4 expression in vitro and in vivo in the breast cancer model, accompanied by CSC suppressive effects. Next, increased expression of ACTN4 predicted poor differentiation, high metastatic potential, and short overall survival and disease-free survival durations. ACTN4 expression was strongly expressed in the TNBC subtype, which is usually enriched for CD44^+^/CD24^−^ CSCs. Thirdly, CD44^+^/CD24^−^/ACTN4^+^ phenotypes showed a greater ESA^+^ proportion, increased tumorsphere-formation ability, and stronger in vivo tumorigenesis potential in mice when compared with the CD44^+^/CD24^−^/ACTN4^−^ populations. Lastly, ACTN4 sustained breast CSC properties mainly by β-catenin stabilization. Furthermore, Charpentier et al. recently identified that breast stem-like cells display higher levels of microtentacles (McTN), tubulin-based protrusions of the plasma cell membrane that aids cell reattachment \[[@CR41]\]. Given that some studies proposed that ACTN4 was preferentially localized in moving structures, such as dorsal ruffles, lamellipodia, and filopodia \[[@CR39]\], it is of great significance to further investigate the expression of ACTN4 expression in McTNs.
In addition, our study clearly validated that ACTN4 establishes a direct association between CSC phenotype and metastatic breast cancer. There mainly exists 2 different strategies for CSC isolation and identification \[[@CR42]--[@CR44]\]. The first one is based on unique surface markers in CSCs. Fluorescence-activated cell sorting analysis (FACS) and/or magnetic-activated cell sorting analysis (MACS) are designed to isolate CSCs expressing specific biomarkers, such as CD44^+^, CD24^−^, EpCAM^+^, and ALDH^hi^, etc. \[[@CR45], [@CR46]\]. The second strategy is to utilize biofunctions of CSCs. CSC-like cells can be selected by their resistance to chemodrugs, EMT induction, or 3-D sphere culture systems \[[@CR47], [@CR48]\]. Nevertheless, functional stem-like cells are not real CSCs and possibly require further purification by additional biomarkers. Our study identified that approximately 100% of the basal-like phenotype MDA-MB-231 cells are CD44^+^/CD24^−^ subpopulations, among which CD44^+^/CD24^−^/ACTN4^+^ cells exhibited better CSC-like properties (high self-renewal properties, adherence-promoting capacity, and tumorigenic potential with very few cells) compared with the unfractioned parts. This finding was consistent with recent studies that showed the presence of CD44^+^/CD24^−^ cells does not correlate with tumorigenicity \[[@CR49]\], distant metastasis or clinical outcome \[[@CR26]\]. Thus, it is reasonable that combined utilization of ACTN4 and CD44^+^/CD24^−^ markers might be more sufficient to isolate metastatic cells with CSC properties in breast cancer.
Conclusion {#Sec28}
==========
In conclusion, our study not only identified ACTN4 as the direct target of EA, but it also highlighted its significant role in mediating breast cancer metastasis and CSCs' properties. Mechanism exploration further revealed that interruption of ACTN4/β-catenin interaction will result in the activation of β-catenin proteasome degradation. It is promising to develop ACTN4 targeting strategies to predict or inhibit breast cancer metastasis in the future, especially for the basal-like phenotypes.
Additional files
================
{#Sec29}
Additional file 1: Table S1.Primers for Real-time PCR analysis. **Table S2.** Tumorigenic ability of CD44^+^/CD24^−^/ACTN4^+^ cells (DOC 51 kb) Additional file 2: Figure S1. (A)Cancer progression was slowed down if the EA treatment was started after tumor growth in MMTV-PyMT mice at ages ranging from 6th to 15th weeks (\**P* \< 0.05, values represented as the Mean ± SD, *n* = 3);**(B)** EA treatment did not cause a significant bodyweight loss compared to the vehicle group. (JPEG 241 kb) Additional file 3: Figure S2.Flow cytometry detection indicated that EA dose-dependently arrested cell cycle mainly at the S&G2/M phases, and induced apoptosis in breast cancer cells MDA-MB-231, BT-549 and MCF-7. (JPEG 703 kb) Additional file 4: Figure S3 and S4.The wound healing and chamber invasive assay revealed that breast cancer cell migration and invasion were inhibited by EA in a time- and dose-dependent manner. (ZIP 2039 kb) Additional file 5: Figure S5.RNA-seq showed that EA treatment and ACTN4 knockdown exhibited similar changes of target genes by BGISEQ-500 analysis. **(A)** Heat map columns describing the hierarchical clustering of EA-treated or ACTN4 knockdown group compared to control, respectively (log 2 fold change ≥ 1.2, *P* ≤ 0.05); **(B)** Venn diagrams of up-regulated and down-regulated DEGs beween EA treatment and ACTN4 knockdown groups; **(C)** GO terms analysis of the indicated DEGs containing 3 aspects including molecular function, cellular component and biological process; **(D)** KEGG pathway enrichment analysis of the indicated DEGs. (JPEG 5607 kb)
ALDH
: Aldehyde dehydrogenase
BC
: breast cancer
BSA
: Bovine serum albumin
CHIP
: Chromatin immunoprecipitation
CHX
: Cycloheximide
CSCs
: Breast cancer stem cells
DAPI
: 4′,6-diamidino-2-phenylindole
DARTS
: Drug affinity responsive target stability
DEAB
: Diethylaminobenzaldehyde
DMSO
: Dimethylsulfoxide
EA
: Ellagic acid
ELDA
: Extreme limiting dilution analysis
EMT
: Epithelial-mesenchymal transition
FACS
: Fluorescence-activated cell sorting analysis
H&E
: Hematoxylin and eosin
LiCl
: Lithium chloride
MACS
: Magnetic-activated cell sorting analysis
MTT
: 3-(4, 5-dimethylthiazol −2-yl) -2, 5-diphenyltetrazolium bromide
TAN
: Tumor adjacent normal
TCGA
: The cancer genome atlas
TMA
: Tissue microarray
**Electronic supplementary material**
The online version of this article (10.1186/s13046-017-0635-9) contains supplementary material, which is available to authorized users.
We thank LetPub ([www.letpub.com](http://www.letpub.com)) for its linguistic assistance during the preparation of this manuscript.
Funding {#FPar1}
=======
This work was supported by the National Natural science Foundation of China (81,402,173, 81,573,651, 81,703,764), Pearl River S&T Nova Program of Guangzhou (201506010098), Guangdong Science Foundation for Distinguished Young Scholars (2016A030306025), Combined Scientific Project Funded by Guangdong Provincial Science and Technology Agency and Guangdong Provincial Academy of Traditional Chinese Medicine (2014A020221047), Guangdong High- level university construction project (A1-AFD018161Z1510), Guangdong High-level Personnel of Special Support Program (A1--3002--16-111-003) and the International Postdoctoral Exchange Fellowship Program & the China Postdoctoral Science Foundation (2016 M592585).
Availability of data and materials {#FPar2}
==================================
All data generated or analyzed during this study are included in this published article \[and its supplementary information files\] uploaded onto the Research Data Deposit (RDD) with an RDD number of RDDB2017000057.
WZY and XXM designed the whole experiments; WN conducted molecular biology experiments and wrote the manuscript; WQ, THL and ZFX carried out pharmacological studies and statistical analysis; ZYF and WSQ assisted cell culture and DARTS experiment; ZJ supported animal feeding and experiments. All authors read and approved the final manuscript.
Ethics approval and cosent to participate {#FPar3}
=========================================
For animal experiments, all animal procedures were performed in accordance with the institutional guidelines for the care and use of laboratory animals approved by the Animal Care and Use Committee of Guangdong Provincial Hospital of Chinese Medicine (the Ethics Approval Number 2015008) and the National Institutes of Health guide for the care and use of Laboratory animals. For TMA and TCGA analysis, the tissue microarray HBre-Duc060CS-03 is commercially bought from Shanghai Outdo Biotech Company (<http://www.superchip.com.cn/technology/Default.aspx>, Shanghai, China) with National Human Genetic Resources Sharing Service Platform (Grant NO.: YCZYPT \[2017\] 02). All of the procedures were followed with the aforementioned immunohistochemistry assay as well as the manufacturer's instructions; All the data retrieved from TCGA was supported by the guidelines built by TCGA Ethics, Law and Policy group, which are in compliance with the Helsinki Declaration ([http://www.wma.net.u.vtrus.net/en/ 30publications/10policies/b3/index.html](http://www.wma.net.u.vtrus.net/en/%2030publications/10policies/b3/index.html)).
Consent for publication {#FPar4}
=======================
Not applicable.
Competing interests {#FPar5}
===================
The authors declare that they have no potential financial competing interests that may in any way gain or lose financially from the publication of this manuscript at present or in the future. Additionally, no non-financial competing interests are involved in the manuscript.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
|
7 Essential Vitamins You Need After Age 40
Vitamins and minerals are very important for our overall health – they help us ward off numerous ailments and are also important for the proper functioning of our organs. According to Kristin Kirkpatrick, MD, these nutrients are important are always important for our health, but especially after we turn 40. As she says, at this time in our life, the rules start to change.
“Your body probably isn’t working the same way at 40-plus as it was at 20,” she says. Muscle mass starts to deteriorate, we’re much more likely to put on weight, menopause may (or may soon) start, and risk of chronic diseases like cancer, heart disease, and diabetes begins to increase—which means your battle plan needs to start looking a little different. One solution is getting enough of the right vitamins and nutrients, which is possible through healthy eating—and food sources are typically (but not always) a better bet than supplements because they’re better absorbed,” she says.
Here are the vitamins and minerals our body requires after the age of 40:
Calcium
For years, we all thought that calcium is only important for the bones. However, several studies have shown that increasing the calcium intake doesn’t reduce the risk of fractures. Even worse, calcium supplements have been found to raise the risk of stroke and cardiac arrest. Although are body can absorb all the calcium it needs under the age of 30, it is also important in the later stages of life. Calcium is required for proper muscle contractions and proper nerve and muscle function as well as other processes in the body. Calcium is still important after the age of 40, but you shouldn’t take too much of it. More calcium is not always better and may even harm your heart health. Women over 40 needs about 1000 mg. a day, while women older than 50 need 1200 mg. You can get the mineral from broccoli, almonds, spinach, dairy products and sardines.
Vitamin B12
The body relies on vitamin B12 for proper blood and brain function. Although younger people can absorb it from food, things change after turning 40 as the levels of stomach acid deplete. This is why it’s best to take multivitamin or vitamin B12 supplements after 40. According to Kirkpatrick, you can’t get too much of it – vitamin B12 is a water soluble vitamin so you’ll just excrete the excess.
Vitamin D
Vitamin D is one of the most important nutrients for our health. Vitamin D deficiency has been linked to serious health problems and the risk only rises as we age. The vitamin is also important for proper calcium absorption. It can be found in cereal, fortified grains and dairy products as well as fish, but the best source of vitamin D is the sun. However, not many people get enough sunlight. Some live in areas with low sunlight, so Kirkpatrick suggests taking vitamin D3 supplements. You should aim for 600 IU (International Units) per day, although you can go as high as 4000 UI. Take vitamin D seriously – it has been associated with heart disease, multiple sclerosis and several types of cancer.
Magnesium
After the age of 40 the risk of high blood pressure rises significantly, and things are even worse if you’re magnesium deficient. This mineral is important for proper muscle, nerve and heart function, but it can also regulate the blood sugar levels. Lack of it has been linked to diabetes, inflammation and heart disease. The levels of magnesium can be increased by eating more beans, soy, nuts, avocado and various seeds. However, make sure to limit the intake to about 320 mg. a day as too much of the mineral may cause cramps, nausea and diarrhea.
Omega-3 fatty acids
Although they’re not vitamins or minerals, omega-3 fatty acids are important for our health. They can reduce the signs of aging and control the blood pressure, while also reducing the level of cholesterol and preventing the risk of numerous cardiovascular diseases. Studies have also discovered that omega-3s can keep the brain properly functioning and the memory sharp. One study showed that people with high levels of omega-3 fatty acids have larger brains and performed better in memory tests, which means that the compounds play a big role in proper brain health.
Omega-3 fatty acids are present in walnuts, flaxseeds, fish and leafy green veggies, but taking supplements is usually better. Kirkpatrick recommends getting 500 mg. if you’re healthy, 800-1000 mg. if you’re suffering from heart disease and 2000-4000 mg. if you have high triglyceride levels. Your doctor can suggest the optimal dosage according to your age and health status.
Potassium
Potassium is important for the cardiovascular system. It keeps the blood pressure under control and lack of it has been related to increased risk of stroke and other cardiovascular diseases. In the past, 3.1 mg. of potassium a day has been considered high, but this is still lower than the actual safe amount of 4.7 mg. a day. Some studies have shown that taking only 2 mg. of potassium a day can be beneficial as well.
Potassium is one of the most important minerals for our health. However, Kirkpatrick suggests avoiding potassium supplements and relying on food sources instead. Potassium supplements have been known to cause arrhythmia and problems in the gastrointestinal tract. The best sources of potassium are bananas, dark leafy greens, beans and lentils. If you choose potassium supplements, your doctor should monitor your intake and how they affect your health.
Probiotics
Just like omega-3s, probiotics aren’t vitamins or minerals, but they’re just as important for our overall health, especially after turning 40. There’s an increasing amount of evidence that relates probiotics with lower risk of heart disease, stroke or diabetes, and getting them after the age of 40 is important due to the decrease in muscle mass and the higher risk of insulin resistance and weight gain.
Probiotics are present in dairy products and fermented soy products, it’s better to take them as a supplement. Foods don’t contain as much strains as supplements and each one comes with its own benefits – some are for weight control, while others prevent diarrhea. As they’re actually live cultures, they can be destroyed by cooking, so it’s best to take them in the form of supplements.
Eddie is the founder and owner of www.WorldTruth.TV. This website is dedicated to educating and informing people with articles on powerful and concealed information from around the globe. I have spent the last 38 years researching Bible, History, Alternative Health, Secret Societies, Symbolism and many other topics that are not reported by mainstream media. |
Fast Food Catering
Doughnuts: What Makes a Great One and How to Eat It?
Doughnuts are dangerous! Of course, we mean it is a good way, particularly in the sense that you will likely not get enough of it so overindulgence is a danger. Even a bad doughnut isn’t too bad that you will immediately throw it away after the first bite.
Great Doughnut Debate
This begs the question: What makes for a great doughnut? With as many personal preferences in doughnuts as there are many types of doughnuts, the debate rages on albeit in a more amicable way. After all, everybody can agree to disagree since there are several types of people in the doughnut world – raised doughnuts people, glazed doughnuts people, and fritter people, for example.
But doughnut people also agree that there are certain characteristics that make for a great doughnut.
Mouthwatering look. You should immediately be tempted to eat the entire doughnut in a few bites as soon as you look at it. You don’t want to eat a sad-looking doughnut because it’s meant to be a “happy food”.
Sweet aroma. Your nose should pick up on the sweet smells coming from it at the same time that your eyes are feasting on its overall look.
Yeasty flavor with a nice mouth-feel. Your mouth experiences an explosion of flavors from the doughnut itself to the frosting and toppings. You shouldn’t feel overwhelmed with any one of its components since each one complements the others.
Most importantly, the best doughnut can be eaten on its own without the benefit of milk, coffee and juice. But obviously you can pair a doughnut with your favorite beverage since it’s about personal preferences.
And don’t forget about the frosting either. It shouldn’t be so sweet it drowns out the yeasty flavors of the base, not to mention that it shouldn’t leave a shiny film in and around your mouth. Otherwise, it means that there’s too much hydrogenated oils in it.
Good Tips to Remember When Eating
Doughnuts are among the ultimate finger food, too. You should ideally eat them with your hands but you can also eat them with utensils, such as when you don’t want to mess up your hands.
A few tips:
Pick up your doughnut with your hand. Use both your hands if it’s too large, or too hard to hold, or too sticky.
Wrap its bottom in a napkin or parchment paper so that you can keep your hands clean, if you want, while you eat.
Bring it to your mouth and take a small bite. Doughnuts are best enjoyed in a slow manner, not to mention that stuffing your mouth doesn’t look pretty.
Watch out that the filling doesn’t drip to your clothes, among others.
Eating doughnuts at chains like Dunkin Donutsisn’t a dainty task. But you can still enjoy them without being a pig.
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CNN's Anderson Cooper takes a look at racially-charged statements made by President Donald Trump following his claim that he is the "least racist" person. |
[The primary disability of children population in Kaluga oblast].
The article considers the primary disability of children population of Kaluga oblast at the age up to 18 years during period of last five years. On the oblast and district levels primary cases, prevalence, dynamics and characteristics of children disability indicators in total and according classes of diseases were detected. The indicators of primary disability in children depending of age and gender were calculated and comparatively analyzed. It is established that primary disability of children population has a distinct trend to increase, especially in adolescents. |
Carotid artery stenting is associated with a higher incidence of major adverse clinical events than carotid endarterectomy in female patients.
The optimal approach to carotid revascularization in female patients with carotid artery stenosis is widely debated. Information available is largely derived from clinical trials that include only highly selected patients. The goal of this study was to compare the early clinical outcomes in women who undergo carotid artery stenting (CAS) vs carotid endarterectomy (CEA). Female patients undergoing CAS or CEA between January 1, 2012 and December 31, 2015, and who were included in the Procedure Targeted American College of Surgeons National Surgical Quality Improvement Program were assessed for their incidence of early postoperative complications. The primary outcome measure was 30-day incidence of a major adverse clinical event (MACE; defined as death, stroke, transient ischemic attack, or myocardial infarction/arrhythmia). Univariable analyses were used to compare results between female patients undergoing CEA and those undergoing CAS. Propensity score matching techniques were used to create a cohort of 125 CAS and CEA patients who were well matched for all known patient-, disease-, and procedure-related factors. Analysis of comparative outcomes between the propensity-matched groups was then performed. The overall study population consisted of 5620 female CEA patients and 131 female CAS patients. Of these patients, 290 (5.2%) from the CEA group and 16 (12.2%) from the CAS group sustained a MACE in the first 30 days after their procedures. Within the propensity-matched cohort, the 30-day incidence of postoperative MACE in the CAS group of this cohort was 11.2% (14 patients) compared with 4.0% (5 patients; odds ratio, 1.01 [95% confidence interval, 1.01-7.77]; P = .04) in the CEA group. Our analysis of a "real-world" clinical registry suggests that CAS may be inferior to CEA in female patients who require carotid artery revascularization. |
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Looking for something new to do with your potatoes? How about 'smashed' potatoes? I have been wanting to try smashed potatoes ever since I saw them on Pioneer Woman and it was past time that I gave them the 'loaded' treatment by topping them with all of the classic loaded baked potato toppings including bacon, cheese, sour cream and green onions. The basic idea behind smashed potatoes is that you take small cooked potatoes and you 'smash' them, by pressing down on them with a glass or something, into thin pancakes and then you bake them until they are golden brown and crispy! There is very little that can compare with potatoes that are nice and crispy on the outside and soft and tender on the inside, think french fries, fritters, etc. and these smashed potatoes are no exception! Of course you can top smash potatoes however you like from simple things like herbs to more elaborate like these but no matter how you top them, they will disappear quickly!
If you use really small potatoes, they work as a really nice 1-2 bit appetizer!
Loaded Smashed Potatoes
New potatoes that are cooked, 'smashed' and then baked until golden brown and crispy and the topped with melted cheddar, sour cream, bacon and green onions.
ingredients
1 pound new potatoes (about 1-1 1/2 inches across), washed
2 tablespoons oil
salt and pepper to taste
2 strips bacon (optional)
1/2 cup cheddar cheese, shredded
1/4 cup sour cream
2 green onions, sliced
directions
Bring the potatoes and enough water to cover them by an inch to a boil, reduce the heat and simmer until a fork can easily be pushed into them, about 15-30 minutes depending on the size of the potatoes, before draining them.
Place the potatoes on a greased baking sheet and 'smash' them by pressing down on them with something like a potato masher, the bottom of a gloss or bowl, etc.
Brush the potatoes with oil, season with salt and pepper and bake them on the top shelf of a preheated 450F/230C oven until golden brown and crispy on top, about 20 minutes.
Meanwhile cook and crumble the bacon.
Sprinkle the cheddar onto the potatoes and optionally broil for a minute to melt the cheese.
Top with the sour cream, bacon and green onions and enjoy as a side or as an appetizer!
Option: You can cook and smash the potatoes a day ahead of time.Option: Add some chopped steamed broccoli.
Anonymous: I find that when you smash them, they do tend to fall apart but after you bake then the second time, especially with a bit of cheese, they firm up again to the point where you can easily pick them up and eat them as two bite appetizers.
I'd definitely recommend smashing them with a glass and not a potato masher. I used a masher and they got stuck between the "rungs" of the masher, and I kept having to try to pry them out of the masher. DEEEEELISH!
I just place a piece of wax paper over each previously simmered potatoe and press down till they are as flat as you want. the flatter the better as they look like fat potatoe chips. Great tasting with olive oil on top. Bake till crispy
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Related literature {#sec1}
====================
For background to the biological activity of aromatic amides, see: Saeed *et al.* (2008[@bb8]); Brunsveld *et al.* (2001[@bb3]); Prins *et al.* (2001[@bb7]). For the anti-emetic activity of *N*-substituted benzamides, see: Vega-Noverola *et al.* (1989[@bb10]). For related structures, see: Escalada *et al.* (2004[@bb5]); Pertlik (1992[@bb6]). For standard bond lengths, see: Allen *et al.* (1987[@bb2]).
Experimental {#sec2}
==============
{#sec2.1}
### Crystal data {#sec2.1.1}
C~8~H~9~NO~2~*M* *~r~* = 151.16Monoclinic,*a* = 13.576 (3) Å*b* = 16.964 (3) Å*c* = 11.025 (2) Åβ = 120.11 (3)°*V* = 2196.5 (10) Å^3^*Z* = 12Mo *K*α radiationμ = 0.10 mm^−1^*T* = 100 K0.42 × 0.28 × 0.22 mm
### Data collection {#sec2.1.2}
Agilent Xcalibur diffractometer with a Ruby (Gemini Cu) detectorAbsorption correction: multi-scan (*CrysAlis PRO* and *CrysAlis RED*; Agilent, 2012[@bb1]) *T* ~min~ = 0.634, *T* ~max~ = 1.0004810 measured reflections2802 independent reflections2545 reflections with *I* \> 2σ(*I*)*R* ~int~ = 0.015
### Refinement {#sec2.1.3}
*R*\[*F* ^2^ \> 2σ(*F* ^2^)\] = 0.059*wR*(*F* ^2^) = 0.192*S* = 1.102802 reflections305 parameters2 restraintsH-atom parameters constrainedΔρ~max~ = 0.58 e Å^−3^Δρ~min~ = −0.56 e Å^−3^
{#d5e582}
Data collection: *CrysAlis PRO* (Agilent, 2012[@bb1]); cell refinement: *CrysAlis PRO*; data reduction: *CrysAlis PRO*; program(s) used to solve structure: *SHELXS97* (Sheldrick, 2008[@bb9]); program(s) used to refine structure: *SHELXL2012* (Sheldrick, 2008[@bb9]); molecular graphics: *OLEX2* (Dolomanov *et al.*, 2009[@bb4]); software used to prepare material for publication: *OLEX2*.
Supplementary Material
======================
######
Click here for additional data file.
Crystal structure: contains datablock(s) global, I. DOI: [10.1107/S1600536813009781/hg5306sup1.cif](http://dx.doi.org/10.1107/S1600536813009781/hg5306sup1.cif)
######
Click here for additional data file.
Structure factors: contains datablock(s) I. DOI: [10.1107/S1600536813009781/hg5306Isup2.hkl](http://dx.doi.org/10.1107/S1600536813009781/hg5306Isup2.hkl)
######
Click here for additional data file.
Supplementary material file. DOI: [10.1107/S1600536813009781/hg5306Isup3.cml](http://dx.doi.org/10.1107/S1600536813009781/hg5306Isup3.cml)
Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?hg5306&file=hg5306sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?hg5306sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?hg5306&checkcif=yes)
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [HG5306](http://scripts.iucr.org/cgi-bin/sendsup?hg5306)).
BN thanks Mangalore University and the UGC SAP for financial assistance for the purchase of chemicals. HSY thanks the UOM for sabbatical leave. RJB acknowledges the NSF MRI program (grant No. CHE-0619278) for funds to purchase the X-ray diffractometer.
Comment
=======
Aromatic amides have found extensive application in synthetic organic chemistry and have a wide range of biological activities (Saeed *et al.*, 2008, Brunsveld *et al.*, 2001; Prins *et al.*, 2001). Various N-substituted benzamides exhibit potent antiemetic activity (Vega-Noverola *et al.*, 1989). The crystal structure of N-methylbenzamide, viz., 2,3-dihydroxy-N-methylbenzamide monohydrate has been reported (Escalada *et al.*, 2004). Also the crystal structures of 2-hydroxy-N-methylbenzamide and 2-hydroxy-N-methylthiobenzamide have been published (Pertlik, 1992). In view of the importance of aromatic amides, we report the crystal structure of the title compound, C~8~H~9~NO~2~, (I).
In (I), three independent molecules (A, B. C) crystallize in the asymmetric unit (Fig. 1). Bond lengths are in normal ranges (Allen *et al.*, 1987). The dihedral angle between the amide group and the benzene ring is 3.0 (2)°, 4.0 (3)° and 3.3 (9)°, respectively. In the crystal, O---H···O hydrogen bonds and weak C---H···N intermolecular interactions are observed (Table 1) forming infinite 1-D chains along (101) and contribute to packing stability (Fig. 2). The closest intercentroid distance between two π-ring systems is 5.214 (6) Å.
Experimental {#experimental}
============
4-Hydroxybenzoyl chloride (1.56 g, 0.01 mole) and methylamine (0.31 g, 0.01 mole) were dissolved in 20 ml methanol and stirred at room temperature for 3 h (Fig. 3). Then the reaction mass was poured into 50 ml ice cold water. The solid obtained was filtered and dried. Single crystals were grown from acetone by the slow evaporation method with a yield of 76%. (m.p. 395 K). Analytical data: Found (Calculated): C % : 63.54 (63.56); H% : 5.98 (6.00) ; N% : 9.21 (9.27).
Refinement {#refinement}
==========
All of the H atoms were placed in their calculated positions and then refined using the riding model with Atom---H lengths of 0.93Å (CH), 0.96Å (CH~3~), 0.86Å (NH) or 0.82Å (OH). Isotropic displacement parameters for these atoms were set to 1.2 (CH, NH) or 1.5 (CH~3~, OH) times *U*~eq~ of the parent atom. Aromatic/amide H refined with riding coordinates: N1A(H1AA), C1A(H1AB), C2A(H2A), C4A(H4A), C5A(H5A), N1B(H1BA), C1B(H1BB),C2B(H2B), C4B(H4B), C5B(H5B), N1C(H1CA), C1C(H1CB), C2C(H2C), C4C(H4C), C5C(H5C). Idealised Me refined as rotating group: C8A(H8AA,H8AB,H8AC), C8B(H8BA,H8BB,H8BC), C8C(H8CA,H8CB,H8CC). Idealised tetrahedral OH refined as rotating group: O1A(H1A), O1B(H1B), O1C(H1C).
Figures
=======
{#Fap1}
{#Fap2}
{#Fap3}
Crystal data {#tablewrapcrystaldatalong}
============
------------------------ ---------------------------------------
C~8~H~9~NO~2~ *F*(000) = 960
*M~r~* = 151.16 *D*~x~ = 1.371 Mg m^−3^
Monoclinic, *Cc* Mo *K*α radiation, λ = 0.71073 Å
*a* = 13.576 (3) Å Cell parameters from 3483 reflections
*b* = 16.964 (3) Å θ = 4.6--77.5°
*c* = 11.025 (2) Å µ = 0.10 mm^−1^
β = 120.11 (3)° *T* = 100 K
*V* = 2196.5 (10) Å^3^ Block, colourless
*Z* = 12 0.42 × 0.28 × 0.22 mm
------------------------ ---------------------------------------
Data collection {#tablewrapdatacollectionlong}
===============
-------------------------------------------------------------------------------------- --------------------------------------
Agilent Xcalibur diffractometer with a Ruby (Gemini Cu) detector 2545 reflections with *I* \> 2σ(*I*)
Detector resolution: 10.5081 pixels mm^-1^ *R*~int~ = 0.015
ω scans θ~max~ = 26.8°, θ~min~ = 2.1°
Absorption correction: multi-scan (*CrysAlis PRO* and *CrysAlis RED*; Agilent, 2012) *h* = −17→12
*T*~min~ = 0.634, *T*~max~ = 1.000 *k* = −21→20
4810 measured reflections *l* = −13→13
2802 independent reflections
-------------------------------------------------------------------------------------- --------------------------------------
Refinement {#tablewraprefinementdatalong}
==========
------------------------------------- --------------------------------------------------------------------------------------------------
Refinement on *F*^2^ Hydrogen site location: inferred from neighbouring sites
Least-squares matrix: full H-atom parameters constrained
*R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.059 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.1461*P*)^2^ + 0.3673*P*\], where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3
*wR*(*F*^2^) = 0.192 (Δ/σ)~max~ \< 0.001
*S* = 1.10 Δρ~max~ = 0.58 e Å^−3^
2802 reflections Δρ~min~ = −0.56 e Å^−3^
305 parameters Extinction correction: *SHELXL*, Fc^\*^=kFc\[1+0.001xFc^2^λ^3^/sin(2θ)\]^-1/4^
2 restraints Extinction coefficient: 0.020 (6)
------------------------------------- --------------------------------------------------------------------------------------------------
Special details {#specialdetails}
===============
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords}
==================================================================================================
------ ------------- -------------- ------------ -------------------- --
*x* *y* *z* *U*~iso~\*/*U*~eq~
O1A −0.1230 (3) 0.69501 (19) 0.1817 (4) 0.0616 (8)
H1A −0.1628 0.7033 0.0974 0.092\*
O2A 0.2475 (3) 0.42236 (17) 0.4097 (3) 0.0612 (8)
N1A 0.1573 (2) 0.39940 (16) 0.1817 (3) 0.0404 (6)
H1AA 0.1024 0.4109 0.0992 0.048\*
C1A 0.0968 (3) 0.5501 (2) 0.3666 (4) 0.0471 (8)
H1AB 0.1499 0.5376 0.4589 0.056\*
C2A 0.0254 (4) 0.6130 (3) 0.3400 (4) 0.0504 (9)
H2A 0.0306 0.6425 0.4141 0.060\*
C3A −0.0548 (3) 0.6331 (2) 0.2026 (4) 0.0452 (8)
C4A −0.0623 (3) 0.5876 (2) 0.0918 (4) 0.0468 (8)
H4A −0.1158 0.6002 −0.0004 0.056\*
C5A 0.0102 (3) 0.5240 (2) 0.1201 (4) 0.0439 (8)
H5A 0.0048 0.4938 0.0467 0.053\*
C6A 0.0917 (3) 0.5048 (2) 0.2590 (3) 0.0402 (7)
C7A 0.1730 (3) 0.4393 (2) 0.2940 (4) 0.0431 (8)
C8A 0.2365 (5) 0.3355 (3) 0.2037 (6) 0.0549 (9)
H8AA 0.2520 0.3063 0.2861 0.082\*
H8AB 0.3062 0.3570 0.2156 0.082\*
H8AC 0.2034 0.3010 0.1239 0.082\*
O1B 1.1116 (3) 0.8638 (2) 0.7338 (3) 0.0617 (9)
H1B 1.1397 0.8800 0.8145 0.093\*
O2B 0.7422 (3) 0.58944 (17) 0.5125 (3) 0.0576 (8)
N1B 0.8321 (3) 0.56916 (17) 0.7436 (3) 0.0406 (7)
H1BA 0.8845 0.5830 0.8261 0.049\*
C1B 0.9770 (3) 0.6941 (2) 0.7987 (4) 0.0455 (8)
H1BB 0.9815 0.6651 0.8729 0.055\*
C2B 1.0491 (3) 0.7578 (2) 0.8255 (4) 0.0464 (9)
H2B 1.1014 0.7717 0.9175 0.056\*
C3B 1.0433 (3) 0.8006 (2) 0.7158 (4) 0.0466 (9)
C4B 0.9661 (4) 0.7791 (3) 0.5779 (5) 0.0540 (10)
H4B 0.9628 0.8074 0.5038 0.065\*
C5B 0.8946 (3) 0.7156 (2) 0.5518 (4) 0.0486 (9)
H5B 0.8434 0.7010 0.4598 0.058\*
C6B 0.8987 (3) 0.6735 (2) 0.6626 (4) 0.0422 (8)
C7B 0.8164 (3) 0.6074 (2) 0.6300 (4) 0.0437 (9)
C8B 0.7582 (4) 0.5039 (2) 0.7230 (5) 0.0562 (11)
H8BA 0.7827 0.4587 0.6927 0.084\*
H8BB 0.6817 0.5173 0.6530 0.084\*
H8BC 0.7606 0.4919 0.8096 0.084\*
O1C 0.3742 (3) 0.4691 (2) 0.6850 (4) 0.0665 (9)
H1C 0.3295 0.4643 0.6007 0.100\*
O2C 0.7454 (3) 0.74129 (17) 0.9069 (3) 0.0630 (8)
N1C 0.6574 (3) 0.76016 (17) 0.6775 (3) 0.0433 (7)
H1CA 0.6057 0.7459 0.5949 0.052\*
C1C 0.5934 (4) 0.6151 (3) 0.8683 (4) 0.0532 (9)
H1CB 0.6448 0.6298 0.9601 0.064\*
C2C 0.5220 (4) 0.5522 (3) 0.8445 (4) 0.0558 (10)
H2C 0.5255 0.5244 0.9193 0.067\*
C3C 0.4443 (3) 0.5305 (2) 0.7068 (4) 0.0484 (9)
C4C 0.4387 (4) 0.5731 (2) 0.5946 (4) 0.0522 (9)
H4C 0.3865 0.5590 0.5027 0.063\*
C5C 0.5114 (4) 0.6359 (2) 0.6212 (4) 0.0491 (9)
H5C 0.5075 0.6644 0.5469 0.059\*
C6C 0.5906 (3) 0.6570 (2) 0.7589 (4) 0.0434 (8)
C7C 0.6724 (3) 0.7228 (2) 0.7924 (4) 0.0463 (8)
C8C 0.7327 (5) 0.8257 (2) 0.6993 (6) 0.0567 (10)
H8CA 0.7403 0.8575 0.7756 0.085\*
H8CB 0.7016 0.8571 0.6156 0.085\*
H8CC 0.8061 0.8061 0.7211 0.085\*
------ ------------- -------------- ------------ -------------------- --
Atomic displacement parameters (Å^2^) {#tablewrapadps}
=====================================
----- ------------- ------------- ------------- -------------- ------------- --------------
*U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^
O1A 0.068 (2) 0.0641 (17) 0.0477 (17) 0.0157 (15) 0.0253 (16) −0.0002 (14)
O2A 0.0643 (19) 0.0652 (17) 0.0364 (15) 0.0055 (14) 0.0122 (13) −0.0009 (13)
N1A 0.0413 (15) 0.0462 (13) 0.0254 (13) 0.0085 (12) 0.0107 (11) 0.0014 (11)
C1A 0.050 (2) 0.057 (2) 0.0305 (16) −0.0027 (16) 0.0168 (16) 0.0005 (14)
C2A 0.051 (2) 0.062 (2) 0.0323 (19) −0.0003 (18) 0.0161 (17) −0.0064 (17)
C3A 0.047 (2) 0.0462 (18) 0.039 (2) −0.0024 (14) 0.0196 (17) 0.0009 (14)
C4A 0.048 (2) 0.055 (2) 0.0309 (17) 0.0003 (17) 0.0146 (15) 0.0058 (15)
C5A 0.050 (2) 0.0504 (18) 0.0297 (18) −0.0048 (15) 0.0187 (16) −0.0044 (14)
C6A 0.0413 (17) 0.0466 (16) 0.0304 (17) −0.0068 (14) 0.0161 (14) −0.0005 (13)
C7A 0.0399 (17) 0.0477 (18) 0.0364 (18) −0.0064 (13) 0.0152 (15) 0.0020 (14)
C8A 0.055 (2) 0.0538 (18) 0.050 (2) 0.0078 (16) 0.0221 (17) 0.0012 (16)
O1B 0.061 (2) 0.0612 (18) 0.053 (2) −0.0118 (15) 0.0207 (17) 0.0036 (14)
O2B 0.0545 (17) 0.0564 (16) 0.0410 (17) −0.0079 (13) 0.0084 (13) −0.0068 (13)
N1B 0.0472 (17) 0.0416 (14) 0.0294 (14) −0.0108 (13) 0.0165 (12) −0.0029 (12)
C1B 0.046 (2) 0.0509 (19) 0.038 (2) 0.0015 (16) 0.0199 (17) 0.0043 (15)
C2B 0.0418 (19) 0.055 (2) 0.037 (2) −0.0008 (16) 0.0159 (17) −0.0020 (16)
C3B 0.044 (2) 0.0481 (19) 0.046 (2) 0.0016 (16) 0.0217 (18) 0.0039 (16)
C4B 0.060 (3) 0.058 (2) 0.039 (2) 0.0001 (19) 0.021 (2) 0.0109 (17)
C5B 0.048 (2) 0.055 (2) 0.0306 (18) 0.0001 (17) 0.0108 (17) 0.0027 (15)
C6B 0.0440 (19) 0.0419 (16) 0.038 (2) 0.0047 (14) 0.0188 (17) 0.0015 (14)
C7B 0.048 (2) 0.0435 (18) 0.036 (2) 0.0085 (15) 0.0189 (17) 0.0025 (14)
C8B 0.057 (2) 0.049 (2) 0.065 (3) −0.0082 (19) 0.032 (2) −0.009 (2)
O1C 0.069 (2) 0.0711 (19) 0.0519 (19) −0.0204 (17) 0.0249 (16) 0.0013 (15)
O2C 0.070 (2) 0.0558 (15) 0.0412 (17) −0.0042 (14) 0.0114 (14) 0.0022 (13)
N1C 0.0464 (16) 0.0442 (13) 0.0334 (14) −0.0093 (12) 0.0156 (12) −0.0013 (12)
C1C 0.053 (2) 0.059 (2) 0.0360 (19) 0.0029 (17) 0.0141 (17) 0.0029 (16)
C2C 0.055 (3) 0.066 (2) 0.037 (2) −0.0010 (19) 0.0153 (19) 0.0130 (18)
C3C 0.044 (2) 0.0509 (19) 0.045 (2) 0.0008 (15) 0.0184 (17) 0.0005 (16)
C4C 0.053 (2) 0.059 (2) 0.037 (2) 0.0008 (18) 0.0176 (18) −0.0027 (16)
C5C 0.055 (2) 0.053 (2) 0.034 (2) 0.0017 (17) 0.0187 (18) 0.0030 (15)
C6C 0.0439 (18) 0.0445 (16) 0.039 (2) 0.0077 (15) 0.0185 (16) 0.0020 (14)
C7C 0.046 (2) 0.0417 (17) 0.047 (2) 0.0048 (14) 0.0198 (17) −0.0018 (14)
C8C 0.063 (3) 0.0435 (18) 0.066 (3) −0.0039 (17) 0.034 (2) 0.0017 (18)
----- ------------- ------------- ------------- -------------- ------------- --------------
Geometric parameters (Å, º) {#tablewrapgeomlong}
===========================
----------------------- ------------ ----------------------- ------------
O1A---H1A 0.8200 C2B---C3B 1.379 (5)
O1A---C3A 1.341 (5) C3B---C4B 1.395 (6)
O2A---C7A 1.199 (5) C4B---H4B 0.9300
N1A---H1AA 0.8600 C4B---C5B 1.379 (6)
N1A---C7A 1.331 (5) C5B---H5B 0.9300
N1A---C8A 1.460 (5) C5B---C6B 1.393 (5)
C1A---H1AB 0.9300 C6B---C7B 1.493 (5)
C1A---C2A 1.371 (6) C8B---H8BA 0.9600
C1A---C6A 1.386 (5) C8B---H8BB 0.9600
C2A---H2A 0.9300 C8B---H8BC 0.9600
C2A---C3A 1.394 (6) O1C---H1C 0.8200
C3A---C4A 1.405 (5) O1C---C3C 1.349 (5)
C4A---H4A 0.9300 O2C---C7C 1.191 (5)
C4A---C5A 1.386 (5) N1C---H1CA 0.8600
C5A---H5A 0.9300 N1C---C7C 1.338 (5)
C5A---C6A 1.405 (5) N1C---C8C 1.446 (5)
C6A---C7A 1.474 (5) C1C---H1CB 0.9300
C8A---H8AA 0.9600 C1C---C2C 1.376 (6)
C8A---H8AB 0.9600 C1C---C6C 1.385 (5)
C8A---H8AC 0.9600 C2C---H2C 0.9300
O1B---H1B 0.8200 C2C---C3C 1.395 (6)
O1B---C3B 1.364 (5) C3C---C4C 1.402 (6)
O2B---C7B 1.215 (5) C4C---H4C 0.9300
N1B---H1BA 0.8600 C4C---C5C 1.380 (6)
N1B---C7B 1.330 (5) C5C---H5C 0.9300
N1B---C8B 1.434 (5) C5C---C6C 1.397 (5)
C1B---H1BB 0.9300 C6C---C7C 1.484 (5)
C1B---C2B 1.387 (6) C8C---H8CA 0.9600
C1B---C6B 1.380 (6) C8C---H8CB 0.9600
C2B---H2B 0.9300 C8C---H8CC 0.9600
C3A---O1A---H1A 109.5 C4B---C5B---H5B 119.9
C7A---N1A---H1AA 121.2 C4B---C5B---C6B 120.2 (4)
C7A---N1A---C8A 117.5 (4) C6B---C5B---H5B 119.9
C8A---N1A---H1AA 121.2 C1B---C6B---C5B 119.6 (4)
C2A---C1A---H1AB 119.2 C1B---C6B---C7B 121.8 (3)
C2A---C1A---C6A 121.5 (4) C5B---C6B---C7B 118.6 (4)
C6A---C1A---H1AB 119.2 O2B---C7B---N1B 122.5 (4)
C1A---C2A---H2A 119.8 O2B---C7B---C6B 124.4 (4)
C1A---C2A---C3A 120.4 (3) N1B---C7B---C6B 113.1 (3)
C3A---C2A---H2A 119.8 N1B---C8B---H8BA 109.5
O1A---C3A---C2A 118.2 (3) N1B---C8B---H8BB 109.5
O1A---C3A---C4A 122.6 (4) N1B---C8B---H8BC 109.5
C2A---C3A---C4A 119.2 (3) H8BA---C8B---H8BB 109.5
C3A---C4A---H4A 120.1 H8BA---C8B---H8BC 109.5
C5A---C4A---C3A 119.8 (4) H8BB---C8B---H8BC 109.5
C5A---C4A---H4A 120.1 C3C---O1C---H1C 109.5
C4A---C5A---H5A 119.7 C7C---N1C---H1CA 121.7
C4A---C5A---C6A 120.6 (3) C7C---N1C---C8C 116.6 (4)
C6A---C5A---H5A 119.7 C8C---N1C---H1CA 121.7
C1A---C6A---C5A 118.5 (3) C2C---C1C---H1CB 119.2
C1A---C6A---C7A 119.0 (3) C2C---C1C---C6C 121.6 (4)
C5A---C6A---C7A 122.5 (3) C6C---C1C---H1CB 119.2
O2A---C7A---N1A 121.6 (4) C1C---C2C---H2C 120.4
O2A---C7A---C6A 125.4 (4) C1C---C2C---C3C 119.2 (4)
N1A---C7A---C6A 113.0 (3) C3C---C2C---H2C 120.4
N1A---C8A---H8AA 109.5 O1C---C3C---C2C 118.6 (4)
N1A---C8A---H8AB 109.5 O1C---C3C---C4C 121.3 (4)
N1A---C8A---H8AC 109.5 C2C---C3C---C4C 120.1 (4)
H8AA---C8A---H8AB 109.5 C3C---C4C---H4C 120.2
H8AA---C8A---H8AC 109.5 C5C---C4C---C3C 119.6 (4)
H8AB---C8A---H8AC 109.5 C5C---C4C---H4C 120.2
C3B---O1B---H1B 109.5 C4C---C5C---H5C 119.7
C7B---N1B---H1BA 121.3 C4C---C5C---C6C 120.6 (3)
C7B---N1B---C8B 117.3 (3) C6C---C5C---H5C 119.7
C8B---N1B---H1BA 121.3 C1C---C6C---C5C 119.0 (4)
C2B---C1B---H1BB 119.8 C1C---C6C---C7C 118.6 (4)
C6B---C1B---H1BB 119.8 C5C---C6C---C7C 122.4 (3)
C6B---C1B---C2B 120.4 (4) O2C---C7C---N1C 122.0 (4)
C1B---C2B---H2B 120.0 O2C---C7C---C6C 125.6 (4)
C3B---C2B---C1B 120.0 (4) N1C---C7C---C6C 112.4 (3)
C3B---C2B---H2B 120.0 N1C---C8C---H8CA 109.5
O1B---C3B---C2B 123.4 (4) N1C---C8C---H8CB 109.5
O1B---C3B---C4B 116.7 (4) N1C---C8C---H8CC 109.5
C2B---C3B---C4B 119.9 (4) H8CA---C8C---H8CB 109.5
C3B---C4B---H4B 120.1 H8CA---C8C---H8CC 109.5
C5B---C4B---C3B 119.9 (4) H8CB---C8C---H8CC 109.5
C5B---C4B---H4B 120.1
O1A---C3A---C4A---C5A −179.7 (3) C3B---C4B---C5B---C6B 0.5 (6)
C1A---C2A---C3A---O1A 179.9 (4) C4B---C5B---C6B---C1B −1.8 (6)
C1A---C2A---C3A---C4A 0.6 (6) C4B---C5B---C6B---C7B 177.3 (3)
C1A---C6A---C7A---O2A −2.4 (5) C5B---C6B---C7B---O2B −3.0 (6)
C1A---C6A---C7A---N1A 178.5 (3) C5B---C6B---C7B---N1B 176.9 (4)
C2A---C1A---C6A---C5A −0.7 (5) C6B---C1B---C2B---C3B −0.5 (6)
C2A---C1A---C6A---C7A 178.3 (3) C8B---N1B---C7B---O2B 0.8 (6)
C2A---C3A---C4A---C5A −0.3 (5) C8B---N1B---C7B---C6B −179.1 (3)
C3A---C4A---C5A---C6A −0.4 (5) O1C---C3C---C4C---C5C 179.6 (4)
C4A---C5A---C6A---C1A 0.9 (5) C1C---C2C---C3C---O1C −179.6 (4)
C4A---C5A---C6A---C7A −178.0 (3) C1C---C2C---C3C---C4C −0.6 (6)
C5A---C6A---C7A---O2A 176.5 (4) C1C---C6C---C7C---O2C 3.7 (6)
C5A---C6A---C7A---N1A −2.6 (4) C1C---C6C---C7C---N1C −177.5 (3)
C6A---C1A---C2A---C3A 0.0 (6) C2C---C1C---C6C---C5C 1.6 (6)
C8A---N1A---C7A---O2A −1.9 (6) C2C---C1C---C6C---C7C −178.3 (4)
C8A---N1A---C7A---C6A 177.2 (3) C2C---C3C---C4C---C5C 0.6 (6)
O1B---C3B---C4B---C5B 179.8 (4) C3C---C4C---C5C---C6C 0.4 (6)
C1B---C2B---C3B---O1B −179.7 (4) C4C---C5C---C6C---C1C −1.5 (6)
C1B---C2B---C3B---C4B −0.9 (6) C4C---C5C---C6C---C7C 178.4 (3)
C1B---C6B---C7B---O2B 176.1 (4) C5C---C6C---C7C---O2C −176.2 (4)
C1B---C6B---C7B---N1B −3.9 (5) C5C---C6C---C7C---N1C 2.7 (5)
C2B---C1B---C6B---C5B 1.8 (6) C6C---C1C---C2C---C3C −0.5 (7)
C2B---C1B---C6B---C7B −177.3 (3) C8C---N1C---C7C---O2C −1.5 (6)
C2B---C3B---C4B---C5B 0.9 (6) C8C---N1C---C7C---C6C 179.6 (3)
----------------------- ------------ ----------------------- ------------
Hydrogen-bond geometry (Å, º) {#tablewraphbondslong}
=============================
---------------------------- --------- --------- ----------- ---------------
*D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A*
O1*A*---H1*A*···O2*C*^i^ 0.82 1.94 2.749 (5) 170
C2*A*---H2*A*···N1*A*^ii^ 0.93 2.66 3.267 (5) 124
C4*A*---H4*A*···N1*B*^i^ 0.93 2.60 3.371 (5) 141
O1*B*---H1*B*···O2*B*^iii^ 0.82 1.98 2.784 (5) 166
C2*B*---H2*B*···N1*C*^iii^ 0.93 2.63 3.404 (5) 142
O1*C*---H1*C*···O2*A* 0.82 1.96 2.750 (5) 163
---------------------------- --------- --------- ----------- ---------------
Symmetry codes: (i) *x*−1, *y*, *z*−1; (ii) *x*, −*y*+1, *z*+1/2; (iii) *x*+1/2, −*y*+3/2, *z*+1/2.
###### Hydrogen-bond geometry (Å, °)
*D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A*
--------------------------- --------- ------- ----------- -------------
O1*A*---H1*A*⋯O2*C* ^i^ 0.82 1.94 2.749 (5) 170
C2*A*---H2*A*⋯N1*A* ^ii^ 0.93 2.66 3.267 (5) 124
C4*A*---H4*A*⋯N1*B* ^i^ 0.93 2.60 3.371 (5) 141
O1*B*---H1*B*⋯O2*B* ^iii^ 0.82 1.98 2.784 (5) 166
C2*B*---H2*B*⋯N1*C* ^iii^ 0.93 2.63 3.404 (5) 142
O1*C*---H1*C*⋯O2*A* 0.82 1.96 2.750 (5) 163
Symmetry codes: (i) ; (ii) ; (iii) .
|
Cryptocurrency exchange Binance has resumed trading a day after suspending activities due to system upgrade, followed by a warning from their risk management system, a statement confirms June 27.
Trading at Binance, which usually ranks within the top three exchanges in the world by daily trading volume, ground to a halt Tuesday following a planned system upgrade.
Developers had forecast trades and withdrawals stopping for a four-hour period, but when automated system signalled an undisclosed vulnerability, the services remained unavailable longer.
The cause of the problem remains unknown, Binance stating that it would resume full functionality at 2:45 PM UTC Wednesday, June 27.
On social media, users continued to report discrepancies while attempting to use the platform, officials however not signalling any major issues as of press time, and stating laconically “Binance System Upgrade Complete”.
Cryptocurrency exchanges often encounter unexpected technical challenges, this month also seeing Bitfinex enter a prolonged period of downtime in order to counter malicious attempts to hack its systems.
Binance is currently the second largest cryptocurrency exchange by daily trade volumes, seeing $844 mln in trades over the past 24 hours to press time. |
The Krassenstein brothers, famous for trolling President Trump on Twitter, were banned from the platform Thursday.
“The Twitter Rules to apply to everyone. Operating multiple fake accounts and purchasing account interactions are strictly prohibited. Engaging in these behaviors will result in permanent suspension from the service,” a Twitter spokesperson told the Washington Examiner.
Ed and Brian Krassenstein, who had 926,000 and 698,000 followers respectively, were known for their quick replies to Trump’s tweets, attacking him and promoting themselves.
The brothers responded to Twitter’s claim that they broke the rules to magnify their profiles on the platform and boost content.
“Twitter claims that we manipulated our interactions through the purchase of fake accounts and fake interactions. We have never once acquired anything for the purpose of increasing our Twitter presence,” the brothers said in a statement Thursday. “In fact, we avoided using any platforms residing outside of Twitter’s own technology to manage our accounts for fear we would be accused of using automated tools, which we have avoided since launching our accounts.”
The pair admitted to operating secondary accounts, but said it was for business purposes and to monitor threats against themselves. They said they were confident Twitter would conclude the suspension was given wrongfully.
In 2016, federal agents seized almost a half-million dollars of assets from the brothers, saying there was “reasonable cause” that the two had committed online financial scams. The brothers were never charged after the government decided forfeiting alleged criminal assets related to the scam was enough. |
WASPnest: a worldwide assessment of social Polistine nesting behavior.
Cooperative breeding decreases the direct reproductive output of subordinate individuals, but cooperation can be evolutionarily favored when there are challenges or constraints to breeding independently. Environmental factors, including temperature, precipitation, latitude, high seasonality, and environmental harshness have been hypothesized to correlate with the presence of cooperative breeding. However, to test the relationship between cooperation and ecological constraints requires comparative data on the frequency and variation of cooperative breeding across differing environments, ideally replicated across multiple species. Paper wasps are primitively social species, forming colonies composed of reproductively active dominants and foraging subordinates. Adult female wasps, referred to as foundresses, initiate new colonies. Nests can be formed by a single solitary foundress (noncooperative) or by multiple foundress associations (cooperative). Cooperative behavior varies within and among species, making paper wasps species well suited to disentangling ecological correlates of variation in cooperative behavior. This data set reports the frequency and extent of cooperative nest founding for 87 paper wasp species. Data were assembled from more than 170 published sources, previously unpublished field observations, and photographs contributed by citizen scientists to online natural history repositories. The data set includes 25,872 nest observations and reports the cooperative behavioral decisions for 45,297 foundresses. Species names were updated to reflect modern taxonomic revisions. The type of substrate on which the nest was built is also included, when available. A smaller population-level version of this data set found that the presence or absence of cooperative nesting in paper wasps was correlated with temperature stability and environmental harshness, but these variables did not predict the extent of cooperation within species. This expanded data set contains details about individual nests and further increases the power to address the relationship between the environment and the presence and extent of cooperative breeding. Beyond the ecological drivers of cooperation, these high-resolution data will be useful for future studies examining the evolutionary consequences of variation in social behavior. This data set may be used for research or educational purposes provided that this data paper is cited. |
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I Can’ts
16052014
Have you ever been hemmed in by your I Can’ts? Sometimes my I Can’ts loom large. One I Can’t tumbles into another until they create an obstacle that seems too big to overcome. My I Can’ts press in on my heart, making me feel like I have weights in my soul. My I Can’ts create shadows that dim my vision and weaken my courage. One I Can’t seems to open the door to another one and before long, I am boxed in by the impossible.
I Can’ts are real, but grow when I feed them. The more I look at them, define them, study their boundaries, and trace their ripples, the more discouraged I become. They can make me feel stuck, incapable of moving forward or discovering any open doors for my heart.
When I come face-to-face with my I Can’ts, I have two choices. I can slump under the weight of all that I can’t do or be, or I can look for the I Can’s that God empowers me to experience. God is not limited by my I Can’ts. He is a God of possibilities, opportunities, and promises. He invites me to look past the I Can’ts that seem so overwhelming, and rest in the truth that He is bigger, stronger, and more powerful than any I Can’t on my horizon.
My Jesus Resolutiontoday is to focus on my I Can’s. Because of Jesus, I can choose worship over worry, prayer over a pity party, and counting my blessings over complaining about my circumstances. I can choose to see what God is doing instead of focusing on things I can’t do. It may even be in my I Can’ts that God’s glory might be magnified, His grace revealed, and His love most deeply experienced. |
Solvingwise this is equivalent to the King Ring. This one has additional which could be identified by touching although they are quite small. Imprints on the packaging prooves that the symbols have this intention.There are at least three differently colored bodies known.
The back side of the packaging contains instructions in:hungarian, english, german, french, italian and spanishThe english instruction read:
1. The colours of the spheres that are identical in origin are mixed along the two halves of the disc turning the segments.2. The work of the player is to set the colour of the spheres again identical within the shortest time. This can be done by the rotation of the two dis halves and the segments rapidly and routine-like.3. The game is over when all the spheres have again uniform colour. The player that reaches this condition in a sorter time, is the better.
Price Tracking Data
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On Media Blog Archives Select Date… December, 2015 November, 2015 October, 2015 September, 2015 August, 2015 July, 2015 June, 2015 May, 2015 April, 2015 March, 2015 February, 2015 January, 2015
32.2 million watched Trump accept GOP nomination
An average of 32.2 million people watched Donald Trump accept the Republican nomination on Thursday night across ABC, CBS, NBC, Fox News, CNN, MSNBC, Univision, Fox Business Network, CNBC and NBC Universo, according to ratings data from Nielsen. Millions more likely tuned in on other networks that are not rated by Nielsen (such as C-SPAN), and on streaming video services, which are not included in the average.
That average is up slightly from the final night of the 2012 RNC, when Mitt Romney accepted the GOP nomination. That night 30.2 million people watched across 11 networks. The 2016 ratings are down substantially from 2008's final night, when John McCain's address averaged just under 39 million viewers.
During the 10 p.m. hour up through 11:45 p.m. last night, which made up Trump's speech and his introduction from daughter Ivanka, Fox News averaged 9.35 million viewers, NBC 4.59 million viewers, CNN 5.48 million viewers, ABC 3.87 million viewers, CBS 3.81 million viewers, and MSNBC 2.95 million viewers.
In the adults 25-54 demographic that TV news channels sell against, Fox News also led the pack, with CNN placing second. |
Q:
Order of growth of the code below
What is the order of growth of the worst case running time of the following code fragment as a function of N? 
int sum = 0;
for (int i = 0; i*i < N; i++)
for (int j = 0; j*j < 4*N; j++)
for (int k = 0; k < N*N; k++)
sum++;
A:
outer loop runs until i*i >= N, that means it runs total of
sqrt(N) times.
For each iteration of outer loop, inner loop runs until j*j >= 4*N, similarly that means it runs sqrt(4N) = 2sqrt(N) times.
For each iteration of middle loop, inner loop runs until k>=N*N, this means N^2 iterations.
Increasing sum is done in constant time.
Multiply the above because you do (3) for each iteration of (2), and (2) for each iteration of (1), and you get sqrt(N)*2sqrt(N)*N^2 = 2N^3, which is in O(N^3)
|
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LAND O LAKES, FL
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This LUXURY Home was just Totally Renovated and looks like a BRAND NEW home with Features that include all the latest design trends "Better than HGTV CUSTOM KITCHEN" with Sparkling NEW GRANITE,and STAINLESS APPS, *SPARKLING Renovated POOL with NEW...
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This attractive Agusta model with 2 bedrooms and den is available for immediate occupancy. The home features a living room big enough for you and your friends, neat, bright, airy family room, and a cozy dining room close to the kitchen. The 10 X 8...
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Welcome home to this amazing BRAND NEW, never lived in, Tampa Town home in the gorgeous Club of Hidden River! It has 3 bedrooms, 2.5 bath, 1 car garage. The kitchen and designing came straight from the model home!! This town home certainly has it all!! It's in one of Tampa's "Best kept Secret" areas surrounded around beautiful lakes and landscaping with a large community pool, spa, fitness center, and game room. You couldn't ask for a better area to commute from!! It's in North East Tampa area close by USF, I-75, I-4 and the Hillsborough River.
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Listing data comes from My Florida Regional MLS. |
Estimating the Lumbar Puncture Needle Depth in Children.
Background: Lumbar puncture's (LP) success is dependent on the skill of the physician, anatomy, size, and posture of the patient. Aims: The purpose of this study was to describe a method that could be used to help estimate the correct depth of needle (Y) insertion in children based on age, weight (W), and height (H). Methods: The study consisted of 200 children American Society of Anesthesiologist class I-II aged 0-12 years who underwent spinal block for orthopedic, pediatric, and genitourinary surgery. The distance from the skin entry point to the tip of the spinal needle was measured after the LP was performed. The relationship between the Y and W, H and body mass index (BMI) was calculated. Predictive statistical models were used to determine the LP needle depth. A paired sample t-test was conducted to compare the findings of the developed model with those of earlier models. Results: The patients were aged 2-144 months, with H and W of 43-154 cm and 2.5-48 kg, respectively. The BMI was 10.75-37.72 kg/m2. Before the Y was estimated, the relationship between the independent variables and the depth variable, which was the dependent variable, was examined. According to the obtained results, the model consists of strong relationships with H, W, and H + W. The formula for predicting Y based on W plus H was as follows: for all patients: Y (cm) = 0.861 + 0.012 × H (cm) + 0.035 × W (kg). Based on H, the formula for predicting the required Y was as follows: For all patients: Y (cm) = 0.393 + 0.023 × H (cm). Based on W, the formula for predicting the required Y was as follows: For all patients: Y (cm) = 1.460 + [0.067 × W (kg)]. Conclusion: The formula may provide a more reliable estimate of the required LP depth in children than that obtained using current models. However, larger studies are needed to standardize the formula. |
Imagine, just for a moment, buying a new 70″ 1080P LED HDTV with a premium JBL sound system, hanging it in your living room, and while installing the cables seeing a sticker on the back that says
“Made in the USA”
A television. Made in the USA.
It sounds like a pipe dream, since major consumer electronics just simply aren’t made here anymore. Other than Olevia’s brief stint in California, no TV has been manufactured in the US for decades. There’s, of course, the idea that labor costs are too high to bother, that unions will somehow ruin everything, and that there’s no point in building electronics anywhere other than Asia, but at least one company we met with at CES 2012 plans to change those perceptions.
HDTVs are large, fragile items. At 46″ and bigger, the cost of insuring and transporting a large, sensitive electronic device across the ocean becomes prohibitive. Thus opens a serious case for manufacturing them a bit closer to home.
That’s what Element Electronics CEO Mike O’Shaughnessy figured, and that’s where the journey to bring television manufacturing back to the United States begins.
Blue Collar Beginnings
Mike was born in the blue-collar town of Warren, Ohio. Warren shares northeastern Ohio with Akron, Cleveland, and Youngstown. It’s as rust belt as you can get. You can therefore begin to see how someone who was born and raised in this kind of area would think of trying to revive high tech manufacturing in America.
Element Electronics is based in Minnesota, and Mike told me candidly that the decision to find manufacturing in America was partially economic and partially patriotic. Of course, any CEO would be foolish to make a move like this if it wasn’t a smart financial decision, but Mike believes that all the pieces are in place for this to make sense.
The electronic components inside the televisions are still being manufactured offshore, but the final assembly will be done in Canton, Michigan (a suburb of Detroit). This is very similar to how the automotive process works.
“Our hope is that by ramping up production on a small scale, we’ll start to see it make sense for suppliers to think about opening up their doors in Michigan to accommodate our production. We have to start small, and this is our first step.”
Mike talks about big plans; the Element plant is starting with a single manufacturing line that will be able to crank out 500,000 units. The factory has room for up to five lines. “Think about it, if we start rolling out five full production lines, suppliers are going to take notice and we can start seeing these other factors come into play. There’s a huge labor and talent pool in the midwest. You’ve got workers with high-tech experience here. Plastic injection molding, assembly, fabrication, you name it. It can be done here, it’s just a matter of economy of scale.”
Since I knew Detroit-area residents would want to know, I asked about using union labor. Mike was again perfectly candid with me. “Not initially, but I want you to know that I’m not against union labor.” and then he told me about his blue collar beginnings, which was a roundabout way of saying that he would support union labor if the economics made sense.
Element televisions are being manufactured in a partnership with Lotus International Company. The Lotus plant is where the TVs will be built.
But are they good?
This is all well and good, but we all know that Element is not exactly a premium brand—and features, quality, and support will win out over fleeting feelings of patriotism. I was brought into a room to demo their product line, where I was surprised to find out that a JBL representative was on hand to reveal that Element has partnered with them to provide premium audio solutions for the sets.
An Element LED HDTV was next to a Samsung unit. The picture quality on both sets was essentially the same; the colors were of course different, but these things are user adjustable. The Element set looked slightly better, but I attributed that to careful setup from their technicians. I’m reasonably sure they didn’t tweak the competitor’s image settings. The sound demo began. The difference between the two was remarkable. The JBL system on the Element TV was far more possessed of depth, bass, and “room-fillingness” than the Samsung set. Mike explained in his up-front manner:
“We have tremendous respect for the big guys,” presumably referring to Samsung, Sony, LG, and other mainstream TV manufacturers. “These guys have refined their product down to shaving millimeters off the thickness. We’re not there, and we can’t compete on that. We felt that we could compete on a feature basis, and partnering with a strong brand like JBL enables us to do that.” He feels that many consumers will be reassured by a recognizable brand name like JBL, and that the superior audio experience might tip the scales given that both displays are very similar on an image quality basis.
And he may be right. Given a handful of televisions side-by-side, with tweaked image settings, your average consumer will be hard pressed to explain exactly why this or that set “looks” better. It’s all going to be personal preference. However, most will agree on what sounds better, especially when it’s a high-end premium audio solution rather than two cheap speakers in the bottom bezel.
Element televisions that will be manufactured in Michigan are 46″ and up. In their suite at CES I saw 70″ sets and a gorgeous 80″ display as well. The Element product line contains both LCD and LED 1080p displays,. Production of the 46″ and up sets will begin in Detroit in March. They will be available at major retailers such as Wal-mart, Sears, and Costco.
If you’ll be shopping for an HDTV in 2012, it meets all your feature requirements, and it would make you feel good to buy a TV made in the USA, go check out an Element display in a few months. You may be able to proudly say that you bought an American-made television.
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[Changes in platelet production of malondialdehyde in diabetics treated with dilazep].
We have investigated the effect of dilazep on the prostaglandin synthesis determined as indicator malondialdehyde (MDA) formation. Twenty patients with type 2 diabetes, aged 25-65 years were orally given dilazep at 100 mg X 3/day for 56 daily. The platelet activity has valued before and after the treatment with the production of the MDA. We conclude that the dilazep inhibited statistically significant biosynthesis of prostaglandin endoperoxides from arachidonic acid. |
Q:
Starting a Python script from bash with a string placed into stdin
How do I start a Python script with some input already waiting in stdin. For example, say I have this simple script located in main.py, using Python3:
mystr = input()
print(mystr)
If you just called python3 main.py from the terminal, the script would pause and wait for you to type something. After hitting enter it would continue and print out what you typed. But is there a way to place something into stdin before the script executes? That way the script wouldn't pause, it would accept what's already there. I was envisioning something like:
python3 main.py | my string but that doesn't work.
Note that I am not asking about command line arguments: I am writing an application which will have to take input from stdin, so I am looking for a convenient solution to test out that environment.
A:
This doesn't depend on the fact that a Python script is involved, and the same methods work for feeding bytes into the stdin of any process started under bash (you might want to add a bash tag to your question to reflect this):
To feed a short string into it
echo 'my string' | python3 main.py
To feed a file of sample input into it
cat my_input | python3 main.py
You can also feed a file like this
python3 mail.py < my_input
[Note: Some people like to spend a lot of time arguing that method 3 is better than method 2, (search for 'useless use of cat' if you're interested), but I've come to prefer method 2 (for reasons that are beyond the scope of your question). Use whichever makes the most sense to you.]
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The long-term outlook for hydrocephalus in childhood. A ten-year cohort study of 155 patients.
Despite the fact that ventriculoperitoneal shunt insertion is the most commonly performed surgical operation in the pediatric neurosurgeon's repertoire, there is a surprising paucity of long-term outcome studies for these patients detailing either the complication rate over a predetermined time period or more importantly their intellectual outcome. The aims of this study, therefore, were to determine the 10-year outcome in a cohort of 155 children with shunted hydrocephalus, both in terms of the number and time sequence of shunt complications and also the long-term academic (schooling) outcome of these individuals. This was a cohort study of 155 hydrocephalic children who underwent first-time ventriculoperitoneal shunt insertion between the years 1978 and 1983, who were then followed up on an annual outpatient basis for a period of 10 years or until death. Their academic records and the surgical morbidity and mortality encountered over the 10-year study period were used as the main outcome measures. For those children surviving until schoolage, 59% were able to attend a normal school. The academic outlook for those children with hydrocephalus secondary to infection (postmeningitic) or intraventricular hemorrhage was less favorable with 52 and 60% requiring special schooling compared to those children with congenital hydrocephalus (29%; p = 0.036). 44% (68/155) of patients in this cohort did not require a shunt revision. The commonest reasons for shunt revision were blockage (49%) and infection (19%) which predominantly occurred within the first year of their original shunt procedure. Overall the infection rate was 12% (44/380 procedures). Furthermore an increased incidence of shunt infection was noted in those under 6 months old (p = 0.040). There was an 11 % mortality during the 10-year follow-up period for those with nontumor-related hydrocephalus. |
1984 United States presidential election in New York
The 1984 United States presidential election in New York took place on November 6, 1984, as part of the 1984 United States presidential election. All 50 States and the District of Columbia participated in this election. Voters in New York chose 36 representatives, or electors to the Electoral College, who selected president and vice president.
New York was won by Ronald Reagan with 53.84% of the popular vote over Walter Mondale with 45.83%, a victory margin of 8.01%.
In the 1984 election, New York turned out more Democratic than the nation at-large by about 10%. The county results indicate a typical (for the time) split between the less-populated counties upstate, and the urban centers of New York City, Buffalo and Albany. While Mondale carried the heavily populated boroughs of New York City overall with 61% of the vote, the strong Republican performance in most upstate counties as well as in the populated suburban areas around NYC was able to pull the state's electoral votes in favor of Reagan.
A portent of the future was seen in Mondale carrying Tompkins County, home of the college town of Ithaca. He was only the third Democrat to do so since the Civil War, after Woodrow Wilson in 1912 and Lyndon Johnson in 1964, and Mondale managing this in a near 50-state landslide loss illustrates how much this county was trending. It is also noteworthy that this was the last time that New York voted more Republican than neighboring Pennsylvania. , it is the last time New York has voted for a Republican in a presidential election, as well as the last time Schenectady County did so.
Results
Results by county
References
New York
1984
Category:1984 New York (state) elections |
, Vol. 29 (2015) — Calling Science Pseudoscience: Fleck’s Archaeologies of Fact and Latour’s ‘Biography of an Investigation’ in AIDS Denialism and Homeopathy , by Babette Babich’s crime may have been to continue when other scholars simply gave up disputing what had become standard or received conventionality ...has rather a lot of evidence on his side, epidemiological (including public health analyses) and clinical but also theoretical. Even more credibly (so one might think), he also has the support ofwho along withwon the Nobel Prize for discovering the HIV virus (note that, himself investigated for failing to credit others, and who was also in the same department asat Berkeley, ought to be named, despite his own dicinclination to name others, among those who ‘discovered’ HIV).supports’s claims and in fact’s claims too accord with’s ..."
This morning May 31, 2013, I listened with great attention to the interesting segment on HIV detection and analysis using electron microscopy in your Science in Action programme. I was surprised however to realise that your interviewee failed to reveal the actual scientific state of opinion about the application of electron microscopy to HIV analysis ...
September 2013 — Joan Shenton has announced the launch of a new version of the Immunity Resource Foundation website, which now includes a blog and a news page and welcomes contributions. Mo Aziz — of vitamin D "Prescribing Sunshine" fame — will be co-administrator of the new site. The site includes many videos and full downloadable PDF archives of Continuum magazine.
— The highest U.S. military court's reversal of a Kansas airman's aggravated assault conviction for exposing multiple sex partners to HIV at swinger parties in Wichita will effectively end such prosecutions in the armed forces, his attorney said. The U.S. Court of Appeals for the Armed Forces unanimously ruled Monday that prosecutors failed to prove that any of David Gutierrez's acts were likely to transmit HIV to his partners. That decision overturns a 25-year precedent that had allowed military personnel to be convicted of aggravated assault solely on the basis of a positive HIV test, attorney Kevin McDermott said Tuesday. Gutierrez was not accused of infecting anyone with HIV.
November 2013 — " The Gutierrez case has the potential to remap the landscape of HIV testing and prosecution in the United States military. With any luck, we will soon see the end of HIV test results being used as a basis to convict a serviceman for aggravated assault."
"I have no idea how anyone could possibly still hang onto the belief that HIV=AIDS after reading even the first 20 pages of this well done book ... This horrible mistake in blaming HIV, has lead the world down the wrong path, with the wrong medication. It's caused inordinate human suffering ... If you can only read a single book, this could be the one. Read it. You won't believe it at first. But go ahead and read some of the actual lab tests. You will realize that no reasonable person could possibly buy into the false story that HIV=AIDS when you see the actual source data, tests, experiments, and the real life statistics ..."
Helen thanks Rethinking AIDS board members Christian Fiala and David Rasnick for key sources, and also the podcast work of Elizabeth Ely and David Crowe.
— In a rarified and politically correct discussion of African health policy, which always ends with Western NGOs demanding more money to save Africa (which somehow never happens), Rethinking AIDS board member Helen Lauer addresses the reality that Health SWAT teams and vaccines aren’t going to solve the development problems of Africa. If Africans don’t get access to simple things like clean water, no high tech health interventions can bring real change. While the discussion is mostly about Ebola, it equally applies to AIDS and other epidemics blamed on infectious agents ignoring all signs of social, environmental and economic factors.
January 2020
The AZT Wikipedia Page – Unsafe Despite the Lies
RA President David Crowe writes: AZT is an incredibly dangerous drug because it directly interferes with DNA synthesis...Hence the name, “DNA chain terminator” for this class of drugs...This is not a complete analysis of all the errors in the Wikipedia AZT page, there are probably many more. Given the high quality of the English, and the accuracy of claims that do not put AZT in a negative light, it is hard to believe that the authors were ignorant of the extensive literature showing that AZT is not effective, and is highly unsafe. David Crowe continues...
June 2019
Rethinking AIDS Addresses the UK Haemophilia Inquiry
David Crowe, President of Rethinking AIDS, informs the UK Haemophilia Inquiry, that it wasn’t HIV infected blood that killed haemophiliacs in the 1980s and 1990s, but the impurities in the Factor VIII that haemophiliacs needed to avoid bleeding to death and, most importantly, the widespread prescribing of AZT, starting in 1987. This was the year when the death rate in HIV-positive haemophiliacs soared. This is not new information, AIDS critics were noting the scientific evidence in the 1990s, but the message has still not got through, probably because the people responsible for panicking when the HIV=AIDS theory was first postulated, would have to admit that they were the guilty parties, not the heroes. RA President David Crowe speaks about the inquiry
From the Archives
Increases in reported death in South Africa from 1997 to 2002
Rodney Richards In conclusion, everything put forth in this analysis supports the contention that the majority of the observed increases in reported death in SA over the past several years—even among the sexually active—are the result of improvements in death reporting...Advocacy campaigns based on the unfounded proposition that these increases are primarily the result of HIV/AIDS may serve a valuable purpose in drawing attention to a potentially alarming problem, but to sacrifice an accurate understanding of South Africa’s mortality data to this end can ultimately be of no value to public health.
October 2018
Valencia, Spain
Meeting of AIDS Dissidents It was a small gathering of 16 people. Nearly all of the attendees had been diagnosed “HIV” antibody positive and many had been heroin addicts. Some were taking antivirals, some were not, and some had given them up altogether. Everybody wanted to learn about the history of the challenge to the virus/AIDS hypothesis. Read the English report
Read the Spanish report
November 2018 Case (17-1537) Tommy Morrison versus Quest Et Al has reached the U.S. Supreme Court: "The case will argue Quest labs never tested for the HIV virus, and its lab reports falsifying HIV infected status were misleading and dangerous, to the detriment and ruination of former world heavyweight champion Tommy Morrison’s boxing career." Trisha Morrison, widow of former WBO heavyweight champion Tommy Morrison describes the truth about Tommy's incorrect diagnosis: "The final report on Tommy, when they did the test, showed no HIV virus in him, no AIDS disease, but yet ESPN still runs with the story that Tommy Morrison had AIDS."
June 2018
Challenging Viral Paradigms
CONFERENCE
Legendary cartoonist R. Crumb has illustrated a theme of the VERS PONT DU GARD France conference — Medicine meets the business concept: unmasking the flaws behind profit-driven research and practice. HIV/AIDS is just one example of many viral paradigms which coincide with $millions in research money and pharma income. But is each virus proven to be pathogenic? What are the implications for safer treatments other than dangerous antiviral medicines and vaccines? CONFERENCE PRESS RELEASE
2012 Conference Report
November 2017 Two Rethinking AIDS board members, Helen Lauer and Joan Shenton, have published a paper in the Madridge Journal of Immunology criticizing the management of disease in Africa by foreigners. Some of the problems identified are poorly defined and overly flexible disease definitions (most notably ‘AIDS’), diagnostic tests that result in many false positives, poor statistics that skew understanding of the severity of health crises, and the marginalization of local health authorities: "The considerations collated here suggest that gross errors persist in the One-Virus-Fits-All model of addressing poverty-related illness in Africa, re-categorized under umbrella labels such as 'HIV/AIDS' and 'West African Ebola'. The study of infectious disease at the genomic level is gradually turning away from surveillance of HIV variation and prevalence; and the discovery of new Ebola strains is not a high priority."
IN MEMORIAM D.C. Journalist Terry Michael — "...Michael, who was gay, startled some mainline gay and AIDS activists when he began writing commentaries aligning himself with a small faction of AIDS researchers who disputed the effectiveness and widespread use of anti-retroviral drugs that most experts say ended AIDS as a terminal illness.... 'Having spent the past three years studying mysteries of the HIV=AIDS paradigm, I am ready to admit I am a full-fledged, out-of-the-closet, HIV-AIDS denialist,' Michael wrote in a Jan. 3, 2010 column in the Washington Times..."
August 2017 Initiatives to Improve the Quality of Life of HIV Positive Diagnosed Subjects: "We have found much scientific information that differs substantially from what is commonly disseminated about the risks of HIV transmission. Diagnostic methods are far from achieving the reliability required to establish lifelong high-risky therapies for patients. The pathogenicity of the virus is not well determined. Nor have we been able to establish how the antigens and antibodies used to make prospective or confirmatory diagnoses are obtained. Eight years after Luc Montagnier has informed us that 'a normal body gets rid of HIV naturally', we could not find any scientific publication that has elaborated this idea. Currently, a person who is diagnosed positive for HIV is never fully re-tested, as it is assumed that this condition can never be reversed. As a result of our research and the issues we mentioned, we strongly recommend putting this assumption under scrutiny."
Gordon Stewart 1918-2016 Rethinking AIDS wishes to honor the memory of Dr. Gordon Stewart, one of our board members until his death in October, 2016. He was a highly respected, well credentialed member of the medical establishment who was never afraid to criticize when he thought some mainstream dogma was wrong. He did this with the Whooping Cough vaccine, but most importantly and most notably, with HIV/AIDS, starting in 1984.
May 2017 Index on Censorship has released a case study detailing the repeated censorship of the film Positive Hell. A reader's comment: "If this documentary was made by flat-earthers, no one would imagine that NASA would pressure film festivals to pan the documentary. What makes this documentary dangerous is that it deals with facts that — when widely understood — erodes confidence in a pathological industry and threatens the credibility of thousands of incompetent doctors, universities, dangerous bureaucracies, and the United States as well. The CDC wouldn’t care if Joan Shenton was wrong — what makes them nervous is that she's right."
IN MEMORIAM April 2017 — Liam Scheff was one of the most penetrating and fearless investigative reporters of our time and one of the funniest YouTube satirists of fake science. He broke a story on HIV/AIDS that was bigger, darker, and more devastating than anything before or since. It came to be known as "Guinea Pig Kids."
April 2017
MAHA STAR
Rethinking AIDS
in Indonesia
30,000 Member
Facebook Group
The movement is supported by the work of Luc Montagnier, winner of the Nobel Prize in Medicine and Physiology, and Kary Mullis, Nobel laureate in chemistry.
March 25, 2017
Controversial HIV film
takes prestigious award
at New York film festival
"The HIV/AIDS documentary film Positive Hell added a new trophy to its collection of film festival awards last weekend receiving the Special Jury Prize for World Social Impact at the Queens World Film Festival"
March 2017
CENSORED A video describing the censoring of "Positive Hell", an award-winning 30 minute documentary. PRESS RELEASE
December 2016
HIV+: Alive and Well Without Drugs French journalist Pryska Ducoeurjoly has posted the article she published in the June-July issue of NEXUS magaxine: People with HIV and no symptoms have studied the flaws in the HIV=AIDS theory and stopped taking their medications — without any harmful effects.
Andrew Herxheimer 1926-2016 April 2016 — Dr Herxheimer was always alert to claims made by the pharmaceutical industry about safety and efficacy of their products: "I think zidovudine [AZT] was a drug that was never really evaluated properly and that its efficacy has never been proved but its toxicity certainly is important. And I think it has killed a lot of people — especially at the higher doses. I personally think that it is not worth using alone or combination at all."
IN MEMORIAM January 2016 — Robert Laarhoven, a long-time AIDS rethinker, and founder of the VirusMyth website, died of lung disease in November, 2014. He will be missed by the entire community. Rethinking AIDS will ensure that the VirusMyth website lives on.
September 2015 — Joan Shenton's film Positive Hell has been officially selected for inclusion in Los Angeles CineFest, a monthly international online event. Positive Hell is the story of five individuals who have defied their doctors and lived on for nearly thirty years with a diagnosis of death. The film highlights a network of people diagnosed HIV Positive in the province of Galicia in Northern Spain.
August 2015 — Alumnae News features a full-page review of Joan Shenton 's film Positively False : "The US government was unable to prove a likelihood that an HIV person is at risk, even during unprotected sex, because there is no proof. And if the transmission of HIV is now in such doubt, the entire edifice of the infectious hypothesis for AIDS will surely come tumbling down."
March 2015 — Commentary on the Penrose Inquiry Report, by M. Aziz: "The dissident view is that the more severe the haemophilia the more likely chance there is of testing HIV+, partly because of the 'antigen overload' of clotting treatment."
September 23, 2014 — Patricia Goodson: “The HIV/AIDS hypothesis is one hell of a mistake”, wrote Kary Mullis in 1996. Mullis – Nobel Laureate in Chemistry, 1993 – and other distinguished scientists have claimed the HIV-causes-AIDS hypothesis is false, unproductive, and unethical. Questioning the HIV-AIDS hypothesis: 30 years of dissent , by |
Archive - Aug 8, 2012
August 8, 2012
By
Ted Lutz
MT. JEWETT â âSwedish by Heritage, Swedish by Heart.â
Thatâs the theme for this yearâs three-day Swedish Festival in Mt. Jewett.
The festival begins Friday with the opening ceremony at 6 p.m. at St. Matthews Lutheran Church on East Main Street. The closing ceremony is at 6 p.m. Sunday at the Center Street Stage.
There is no admission charge to attend the festival.
The annual festival, which brings thousands of visitors to Mt. Jewett, features no less than 30 events.
August 8, 2012
By
Ted Lutz
LUDLOW â Unpatriotic thieves have stolen two aluminum flagpoles at the Gibbs Hill Cemetery near Ludlow.
The families of Tom VanGiesen and Frank Sirianni of Kane donated the flagpoles in honor of Staff Sgt. Kenneth R. VanGiesen. The Kane native was killed last year in Afghanistan while serving with the Pennsylvania Army National Guard.
The soldier was the son of Tom and Sue VanGiesen of Lincoln Street, Kane. The soldier was the longtime girlfriend of Erin Sirianni, daughter of Frank and Sherry Sirianni of Highland Road, Kane.
August 8, 2012
By
Ted Lutz
Citing a "lack of support and cooperation" from the other two board members, Supervisor Steve Dyne resigned Tuesday as the volunteer roadmaster for Wetmore Township.
"At this time, I will not be able to fulfill the daily duties of roadmaster," Dyne said in his letter of resignation. "Therefore, I resign from the volunteer position."
The letter was submitted near the end of the monthly meeting of the three-member Board of Supervisors.
August 8, 2012
By
Ted Lutz
The brush drop-off site at Glenwood Park in Kane has been closed, Kane Borough Streets Department Foreman Mike Beane said Tuesday.
A sign posted at the closed gate at the entrance to the brush drop-off site says the closing is due to âabuseâ in not following the rules.
The brush drop-off site had been open Monday through Friday from 6 a.m. to 1:30 p.m., Beane said.
Using their own vehicles, borough residents hauled leaves, tree branches and brush to the appropriate piles at the drop-off site at the park on Hacker Street (Route 321). |
Management of acute asthma in the emergency department.
Asthma is primarily a clinical diagnosis that is made from a combination of historical features and clinical examination findings. The mainstay of asthma treatment includes short-acting beta agonist therapy (albuterol) and steroids. Handheld inhalers are sufficient for most inhaled therapy; all patients on inhalers should be provided with a spacer. The severity of asthma exacerbations is determined by 3 features: (1) clinical presentation, (2) peak expiratory flow rates, and (3) vital signs. Additional testing, such as chest x-ray and blood gas measurements, is reserved for select patients. Spirometry aids in the diagnosis of asthma and measurement of severity, but it is not always required, nor should it be solely relied upon to make disposition decisions. Inhaled ipratropium decreases hospitalization rates, and it should be routinely used. Levalbuterol provides little to no advantage over less-expensive racemic albuterol. Noninvasive positive pressure ventilation may be utilized in patients with moderate to severe exacerbations. Ketamine may be considered in severe exacerbations, but it should not be used routinely. Magnesium sulfate may be beneficial in severe asthma exacerbations, but routine use for mild to moderate exacerbations is not indicated. |
Kaeo Pongprayoon
Kaeo Pongprayoon (, , ; born 28 March 1980 in Kamphaeng Phet) is a Thai amateur boxer who won a silver medal at the 2012 Summer Olympics.
Pongprayoon won the 2009 Asian Amateur Boxing Championships and the 2009 Southeast Asian Games and 2011 at light flyweight.
At the 2009 World Amateur Boxing Championships he lost his third fight to José Kelvin de la Nieve.
At the 2011 World Amateur Boxing Championships he beat two opponents, then lost 8:14 to Zou Shiming.
At the 2012 Summer Olympics (results) he won his first fight against Algerian Mohamed Flissi 19:11, then defeated Ecuador's Carlos Quipo and Bulgarian Aleksandar Aleksandrov. He reached the final by edging out Russian David Ayrapetyan 13:12. He controversially lost the final to Zou Shiming 10:13.
External links
AIBA Bio
References
Category:1980 births
Category:Living people
Category:Boxers at the 2012 Summer Olympics
Category:Olympic boxers of Thailand
Category:Olympic silver medalists for Thailand
Category:Olympic medalists in boxing
Category:Members of the Order of the Direkgunabhorn
Category:Medalists at the 2012 Summer Olympics
Category:Boxing trainers
Category:People from Kamphaeng Phet Province
Category:Thai male boxers
Category:Southeast Asian Games medalists in boxing
Category:Southeast Asian Games gold medalists for Thailand
Category:Southeast Asian Games bronze medalists for Thailand
Category:Competitors at the 2001 Southeast Asian Games
Category:Competitors at the 2003 Southeast Asian Games
Category:Competitors at the 2005 Southeast Asian Games
Category:Competitors at the 2007 Southeast Asian Games
Category:Competitors at the 2009 Southeast Asian Games
Category:Competitors at the 2011 Southeast Asian Games |
Spring Fundraising Campaign
Did you know, 75% of mental health issues begin before the age of 25, and mental health disorders affect 1 in 4 young people?
That is why for the past 5 years, gpac:ed has been working with headspace Geelong to help young people find their voice.
By empowering young people to express themselves and feel comfortable in their world we build resilience and improve the health of our young people. This means demand on headspace Geelong services is reduced.
Over the five years we have already empowered 1500 students, across 5 unique arts-based programs that aren’t run anywhere else in Australia. At the heart of these programs is the opportunity for young people to express themselves in a safe and supportive environment. Students also get to use their imaginations and creativity to solve social problems. They contribute and feel valued. It’s pretty powerful stuff.
Our goal is to do more. We want to connect with more young people in 2018 and that’s where you can help to raise $10,000 to support our work with headspace.
With your help, our partnership can grow its support for the health and wellbeing of the next generation of leaders. |
Morphological-cytochemical and molecular genetic analyses of mitochondria in isolated human oocytes in the reproductive age.
Molecular genetic, cytochemical and morphometric analyses have been performed on isolated oocytes from 41 women (27-39 years of age) in order to detect mutations of mitochondrial DNA (mtDNA), defects of the respiratory chain (ubiquinone-cytochrome-c-oxidoreductase = complex III; cytochrome-c-oxidase = complex IV) and alterations of mitochondrial volume during cellular ageing. Morphometric analyses showed an increase in mitochondrial numerical density with age from the mean values of 7.36 per micron2 and 6.97 per micron3 up to 30 years to 10.74 per micron2 and 11.66 per micron3 in the age group 31-40 years (P < 0.001). Similarly, an increase in the mitochondrial profile area from 0.074 per micron2 in the age group < 30 years to 0.101 per micron2 was noted in the fourth decade. The mitochondrial volume fraction was also significantly increased in the elder age group. Neither point mutations of mtDNA (nucleotide pairs 3243, 8344) nor the common deletion (4977 bp, nucleotide pairs 8482-13460) could be detected. In parallel, ultra- and immunocytochemical studies of the complexes III-IV failed to reveal functional defects. In conclusion there is an age-related increase in the volume fraction of the mitochondria which might reflect subtle changes in the oxidative phosphorylation capacity, but is not linked to mutations of mtDNA or functional defects of the respiratory chain enzymes in mature human oocytes from women of reproductive age. |
---
abstract: |
This paper shows a new phenomenon in higher cluster tilting theory. For each positive integer $d$, we exhibit a triangulated category $\sC$ with the following properties.
On one hand, the $d$-cluster tilting subcategories of $\sC$ have very simple mutation behaviour: Each indecomposable object has exactly $d$ mutations. On the other hand, the weakly $d$-cluster tilting subcategories of $\sC$ which lack functorial finiteness can have much more complicated mutation behaviour: For each $0 \leq \ell
\leq d-1$, we show a weakly $d$-cluster tilting subcategory $\sT_{\ell}$ which has an indecomposable object with precisely $\ell$ mutations.
The category $\sC$ is the algebraic triangulated category generated by a $( d+1 )$-spherical object and can be thought of as a higher cluster category of Dynkin type $A_{\infty}$.
address:
- 'Institut für Algebra, Zahlentheorie und Diskrete Mathematik, Fakultät für Mathematik und Physik, Leibniz Universität Hannover, Welfengarten 1, 30167 Hannover, Germany'
- 'School of Mathematics and Statistics, Newcastle University, Newcastle upon Tyne NE1 7RU, United Kingdom'
author:
- Thorsten Holm
- Peter Jørgensen
title: 'Cluster tilting vs. weak cluster tilting in Dynkin type A infinity'
---
Introduction {#sec:introduction}
============
This paper shows a new phenomenon in higher cluster tilting theory. For each integer $d \geq 1$, we exhibit a triangulated category $\sC$ whose $d$-cluster tilting subcategories have [*very simple*]{} mutation behaviour, but whose weakly $d$-cluster tilting subcategories can have [*much more complicated*]{} mutation behaviour which we can control precisely.
To make sense of this, recall that if $\sT$ is a full subcategory of a triangulated category, then $\sT$ is called [*weakly $d$-cluster tilting*]{} if it satisfies the following conditions where $\Sigma$ is the suspension functor. $$\begin{aligned}
t \in \sT & \; \Leftrightarrow \;
\Hom( \sT , \Sigma t ) = \cdots = \Hom( \sT , \Sigma^d t ) = 0, \\
t \in \sT & \; \Leftrightarrow \;
\Hom( t , \Sigma \sT ) = \cdots = \Hom( t , \Sigma^d \sT ) = 0.\end{aligned}$$ If $\sT$ is also left- and right-approximating in the ambient category in the sense of Remark \[rmk:approximating\], then it is called [ *$d$-cluster tilting*]{}. These definitions are due to Iyama [@I] and have given rise to an extensive homological theory, see for instance [@BMR] and [@IY]. Note that if $\sT = \add t$ for an object $t$, then $\sT$ is automatically left- and right-approximating, but we will study subcategories which are not of this form since they have infinitely many isomorphism classes of indecomposable objects.
One remarkable property of $d$-cluster tilting theory is [ *mutation*]{}. If $t \in \sT$ is an indecomposable object, then it is sometimes possible to remove $t$ from $\sT$ and insert an indecomposable object $t^* \not\cong t$ in such a way that the subcategory remains (weakly) $d$-cluster tilting. This is called [ *mutation of $\sT$ at $t$*]{}, see [@IY sec. 5].
In good cases, there are exactly $d$ different choices of $t^*$ up to isomorphism. That is, there are $d$ ways of mutating $\sT$ at $t$, see [@IY sec. 5].
To be more precise, one hopes(!) that this happens for $d$-cluster tilting subcategories. Indeed, it does happen for $d = 1$ by [@IY thm. 5.3], but can fail for $d \geq 2$, see [@IY thms.9.3 and 10.2]. The situation for weakly $d$-cluster tilting subcategories is less clear.
We can now explain the opening paragraph of the paper. Let us first define $\sC$ which, as we will explain below, can be thought of as a $d$-cluster category of type $A_{ \infty }$.
\[def:blanket\] For the rest of the paper, $k$ is an algebraically closed field, $d \geq 1$ is an integer, and $\sC$ is a $k$-linear algebraic triangulated category which is idempotent complete and classically generated by a $(d+1)$-spherical object $s$; that is, $$\dim_k \sC( s , \Sigma^{\ell} s )
= \left\{
\begin{array}{cl}
1 & \mbox{ for $\ell = 0, d+1$, } \\[1mm]
0 & \mbox{ otherwise. }
\end{array}
\right.$$ Note that $\sC( - , - )$ is short for the $\Hom$ functor in $\sC$.
We prove the following three theorems about $\sC$, where Theorems A and B show [*very simple*]{}, respectively [*much more complicated*]{} mutation behaviour.
[**Theorem A.** ]{} [ *Let $\sT$ be a $d$-cluster tilting subcategory of $\sC$ and let $t
\in \sT$ be indecomposable. Then $\sT$ can be mutated at $t$ in precisely $d$ ways.* ]{}
[**Theorem B.** ]{} [*Let $0 \leq \ell \leq d-1$ be given. Then there exists a weakly $d$-cluster tilting subcategory $\sT_{\ell}$ of $\sC$ with an indecomposable object $t$ such that $\sT_{\ell}$ can be mutated at $t$ in precisely $\ell$ ways.* ]{}
[**Theorem C.** ]{} [ *Let $\sT$ be a weakly $d$-cluster tilting subcategory of $\sC$ and let $t \in \sT$ be indecomposable. Then $\sT$ can be mutated at $t$ in at most $d$ ways.* ]{}
The interest of Theorems A and C depends on a rich supply of (weakly) $d$-cluster tilting subcategories in $\sC$. Indeed, such a supply exists by the following two theorems. As a prelude, note that there is a bijection between subcategories $\sT \subseteq \sC$ closed under direct sums and summands, and sets of $d$-admissible arcs $\fT$; see Section \[sec:arcs\], in particular Proposition \[pro:subcategory\_bijection\]. A $d$-admissible arc is an arc connecting two integers $t$, $u$ with $u - t \geq 2$ and $u - t \equiv
1 \pmod d$.
[**Theorem D.** ]{} [ *The subcategory $\sT$ is weakly $d$-cluster tilting if and only if the corresponding set of $d$-admissible arcs $\fT$ is a $( d+2
)$-angulation of the $\infty$-gon.* ]{}
[**Theorem E.** ]{} [*The subcategory $\sT$ is $d$-cluster tilting if and only if the corresponding set of $d$-admissible arcs $\fT$ is a $( d+2 )$-angulation of the $\infty$-gon which is either locally finite or has a fountain.* ]{}
We defer the definition of “$( d+2 )$-angulation of the $\infty$-gon” and other unexplained notions to Definition \[def:arcs\] and merely bring Figure \[fig:4-angulation\] which shows part of a $4$-angulation of the $\infty$-gon with a fountain at $0$.
$$\xymatrix @-5.6pc @R=-9ex @! {
\rule{0ex}{10.0ex} \ar@{--}[r]
& *{}\ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^-1.5pc/@{-}[lll]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^-1.5pc/@{-}[lll] \ar@/^-2.5pc/@{-}[lllll]\ar@/^-4.5pc/@{-}[lllllllll]\ar@/^1.5pc/@{-}[rrr]\ar@/^3.5pc/@{-}[rrrrrrr]\ar@/^4.5pc/@{-}[rrrrrrrrr]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^-1.5pc/@{-}[lll]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{}\ar@{--}[r]
& *{} \\
& & & & & & & & & & & 0 \\
}$$
Note how the arcs divide the upper half plane into a collection of ‘quadrangular’ regions, each with four integers as ‘vertices’. Some of the vertices sit at cusps.
We end the introduction with a few remarks about the category $\sC$ which has been studied intensively in a number of recent papers [@FY], [@HJ], [@HJY], [@J], [@KYZ], [@Ng], [@ZZ]. It is determined up to triangulated equivalence by [@KYZ thm. 2.1]. It is a Krull-Schmidt and $( d+1 )$-Calabi-Yau category by [@HJY rmk. 1 and prop. 1.8], and a number of other properties can be found in [@HJY secs. 1 and 2]. Theorems A and E are two reasons for viewing $\sC$ as a cluster category of type $A_{\infty}$, since they are infinite versions of the corresponding theorems in type $A_n$; see [@T thm. 3] for Theorem A and [@M prop. 2.13] and [@T thm. 1] for Theorem E. See also [@HJ] for the case $d = 1$.
The paper is organised as follows: Section \[sec:arcs\] introduces $d$-admissible arcs into the study of the triangulated category $\sC$ and proves Theorem D. Section \[sec:approximations\] proves Theorem E. Section \[sec:combinatorics\] shows some technical results on $(
d+2 )$-angulations of the $\infty$-gon. Section \[sec:proofs\] proves Theorems A, B, and C.
\[not:lax\] We write $\ind( \sC )$ for the set of isomorphism classes of indecomposable objects in $\sC$. We will follow the custom of being lax about the distinction between [*indecomposable objects*]{} and [*isomorphism classes of indecomposable objects*]{}. This makes the language a bit less precise, but avoids excessive elaborations.
The word [*subcategory*]{} will always mean [*full subcategory closed under isomorphisms, direct sums, and direct summands*]{}. In particular, a subcategory is determined by the indecomposable objects it contains.
The arc picture of $\sC$ {#sec:arcs}
========================
By [@HJY prop. 1.10], the Auslander-Reiten (AR) quiver of $\sC$ consists of $d$ components, each of which is a copy of $\BZ
A_{\infty}$, and $\Sigma$ acts cyclically on the set of components.
\[con:coordinates\] We pick a component of the AR quiver of $\sC$ and impose the coordinate system in Figure \[fig:coordinate\_system\].
$$\xymatrix @-4.0pc @! {
& \vdots \ar[dr] & & \vdots \ar[dr] & & \vdots \ar[dr] & & \vdots \ar[dr] & & \vdots \ar[dr] & \\
\cdots \ar[dr]& & {\scriptstyle (-4d-1,0)} \ar[ur] \ar[dr] & & {\scriptstyle (-3d-1,d)} \ar[ur] \ar[dr] & & {\scriptstyle (-2d-1,2d)} \ar[ur] \ar[dr] & & {\scriptstyle (-d-1,3d)} \ar[ur] \ar[dr] & & \cdots \\
& {\scriptstyle (-4d-1,-d)} \ar[ur] \ar[dr] & & {\scriptstyle (-3d-1,0)} \ar[ur] \ar[dr] & & {\scriptstyle (-2d-1,d)} \ar[ur] \ar[dr] & & {\scriptstyle (-d-1,2d)} \ar[ur] \ar[dr] & & {\scriptstyle (-1,3d)} \ar[ur] \ar[dr] & \\
\cdots \ar[ur]\ar[dr]& & {\scriptstyle (-3d-1,-d)} \ar[ur] \ar[dr] & & {\scriptstyle (-2d-1,0)} \ar[ur] \ar[dr] & & {\scriptstyle (-d-1,d)} \ar[ur] \ar[dr] & & {\scriptstyle (-1,2d)} \ar[ur] \ar[dr] & & \cdots\\
& {\scriptstyle (-3d-1,-2d)} \ar[ur] & & {\scriptstyle (-2d-1,-d)} \ar[ur] & & {\scriptstyle (-d-1,0)} \ar[ur] & & {\scriptstyle (-1,d)} \ar[ur] & & {\scriptstyle (d-1,2d)} \ar[ur] & \\
}$$
We think of coordinate pairs as indecomposable objects of $\sC$, and extend the coordinate system to the other components of the quiver by setting $$\label{equ:Sigma}
\Sigma( t , u ) = ( t - 1 , u - 1 ).$$ By [@HJY prop. 1.8], the Serre functor of $\sC$ is $S =
\Sigma^{d+1}$. The actions of $S$ and the AR translation $\tau = S\Sigma^{-1}$ are given on objects by $$\label{equ:S_tau}
S( t , u ) = ( t - d - 1 , u - d - 1 ),
\;\;\;
\tau( t , u ) = ( t - d , u - d ).$$ Like $\Sigma$, the Serre functor $S$ acts cyclically on the set of components of the AR quiver. Indeed, since there are $d$ components, the two functors have the same action on the set of components. The AR translation $\tau$ is given on each component of the AR quiver by moving one vertex to the left.
We also think of the coordinate pair $( t,u )$ as an arc connecting the integers $t$ and $u$. The ensuing geometrical picture is illustrated by Figure \[fig:4-angulation\]. However, not all values of $( t , u )$ are possible. Indeed, it is easy to check that the coordinate pairs which occur in Construction \[con:coordinates\] are precisely the $d$-admissible arcs in the following definition.
\[def:arcs\] A pair of integers $( t , u )$ with $u - t \geq 2$ and $u - t \equiv 1
\pmod d$ is called a [*$d$-admissible arc*]{}.
The [*length*]{} of the arc $( t , u )$ is $u - t$.
The arcs $( r , s )$ and $( t , u )$ [*cross*]{} if $r < t < s < u$ or $t < r < u < s$. Moreover, $( r,s )$ is an [*overarc*]{} of $( t,u )$ if $( r,s ) \neq ( t,u )$ and $r \leq t < u \leq s$.
Let $\fT$ be a set of $d$-admissible arcs.
We say that $\fT$ is a [*$( d+2 )$-angulation of the $\infty$-gon*]{} if it is a maximal set of pairwise non-crossing $d$-admissible arcs.
We say that $\fT$ is [*locally finite*]{} if, for each integer $t$, there are only finitely many arcs of the form $( s , t )$ and $( t , u
)$ in $\fT$.
An integer $t$ is a [*left-fountain of $\fT$*]{} if $\fT$ contains infinitely many arcs of the form $( s , t )$, and $t$ is a [*right-fountain of $\fT$*]{} if $\fT$ contains infinitely many arcs of the form $( t , u )$. We say that $t$ is a [*fountain*]{} of $\fT$ if it is both a left- and a right-fountain of $\fT$.
The first part of the following proposition is a consequence of what we did above. The second part follows from the first because our subcategories are determined by the indecomposable objects they contain, see Notation \[not:lax\].
\[pro:subcategory\_bijection\] Construction \[con:coordinates\] gives a bijective correspondence between $\ind( \sC )$ and the set of $d$-admissible arcs.
This extends to a bijective correspondence between [(i)]{} subcategories of $\sC$ and [(ii)]{} subsets of the set of $d$-admissible arcs.
Let $x \in \ind( \sC )$ be given. Figure \[fig:5\] defines two infinite sets $F^{\pm}( x )$ consisting of vertices in the same component of the AR quiver as $x$. Each set contains $x$ and all other vertices inside the indicated boundaries; the boundaries are included in the sets.
$$\vcenter{
\xymatrix @-3.0pc @! {
&&&*{} &&&&&&&& \\
&&&& *{} \ar@{--}[ul] & & & & *{} \ar@{--}[ur] \\
&*{}&& F^-(x) & & & & & & F^+(x) && *{}\\
&&*{}\ar@{--}[ul]& & & & {x} \ar@{-}[ddll] \ar@{-}[uull] \ar@{-}[ddrr] \ar@{-}[uurr]& & &&*{}\ar@{--}[ur]&\\
&& \\
*{}\ar@{--}[r]&*{} \ar@{-}[rrr] && & *{}\ar@{-}[uull]\ar@{-}[rrrrrr]& & & & \ar@{-}[uurr]\ar@{-}[rrr]&&&*{}\ar@{--}[r]&*{}\\
}
}$$
Recall that $S = \Sigma^{d+1}$ is the Serre functor of $\sC$.
\[pro:Hom\] Let $x, y \in \ind( \sC )$. Then $$\dim_k \sC( x , y ) =
\left\{
\begin{array}{cl}
1 & \mbox{for $y \in F^+( x ) \cup F^-( Sx )$}, \\[2pt]
0 & \mbox{otherwise}.
\end{array}
\right.$$
See [@HJY prop. 2.2].
In other words, $x$ has non-zero maps to a region $F^+( x )$ in the same component of the AR quiver as itself, and to a region $F^-( Sx )$ in the “next” component of the AR quiver. Note that if $d = 1$ then the quiver has only one component so $F^-( Sx )$ is in the same component as $x$.
\[rmk:Hom\] It is not hard to check that $y \in F^+( x ) \Leftrightarrow x \in
F^-( y )$. So the proposition is equivalent to $$\dim_k \sC( x , y ) =
\left\{
\begin{array}{cl}
1 & \mbox{for $x \in F^+( S^{ -1 } y ) \cup F^-( y )$}, \\[2pt]
0 & \mbox{otherwise}.
\end{array}
\right.$$
The following proposition is simple but crucial since it leads straight to Theorem D.
\[pro:crossing\] Let $\fx, \fy$ be $d$-admissible arcs corresponding to $x, y \in
\ind( \sC )$. Then $\fx$ and $\fy$ cross if and only if at least one of the $\Hom$-spaces $$\sC( x , \Sigma^1 y )
\;\; , \;\; \ldots \;\; , \;\;
\sC( x , \Sigma^d y )$$ is non-zero.
For $1 \leq \ell \leq d$, the condition that $\sC( x , \Sigma^{ \ell }y ) \neq 0$ is equivalent to $\Sigma^{ \ell
}y \in F^+( x )$ or $\Sigma^{ \ell }y \in F^-( Sx )$ by Proposition \[pro:Hom\]. If we write $\fx = ( r,s )$, $\fy = ( t,u )$, then, using equations and and the coordinate system on the AR quiver of $\sC$, it is elementary to check that $$\begin{aligned}
\label{equ:b}
\Sigma^{ \ell }y \in F^+( x )
& \Leftrightarrow
\left\{
\begin{array}{l}
u \equiv s + \ell \!\!\!\!\! \pmod d, \\[1mm]
r + \ell \leq t \leq s + \ell - d - 1, \\[1mm]
s + \ell \leq u,
\end{array}
\right. \\
\label{equ:c}
\Sigma^{ \ell }y \in F^- ( Sx )
& \Leftrightarrow
\left\{
\begin{array}{l}
u \equiv s + \ell - 1 \!\!\!\!\! \pmod d, \\[1mm]
t \leq r + \ell - d - 1, \\[1mm]
r + \ell \leq u \leq s + \ell - d - 1.
\end{array}
\right.\end{aligned}$$ The condition that at least one of the $\Hom$ spaces $\sC( x ,
\Sigma^1 y ), \ldots, \sC( x , \Sigma^d y )$ is non-zero is hence equivalent to the existence of at least one $\ell$ with $1 \leq \ell
\leq d$ such that the right hand side of or is true. It is again elementary to check that this is equivalent to the condition that $\fx = ( r,s )$ and $\fy = ( t,u )$ cross.
[**Proof of Theorem D.** ]{} Combine the definition of weakly $d$-cluster tilting subcategories with Propositions \[pro:subcategory\_bijection\] and \[pro:crossing\]. $\Box$
Left- and right-approximating subcategories {#sec:approximations}
===========================================
\[pro:forward\_factorization\] Let $x, y \in \ind( \sC )$ be such that $y \in F^+( x )$.
1. Each morphism $x \rightarrow y$ is a scalar multiple of a composition of irreducible morphisms.
2. A morphism $x \rightarrow y$ which is a composition of irreducible morphisms is non-zero.
Keeping $x$, $y$ as above, let $z \in \ind( \sC )$ be such that $z
\in F^+( x ) \cap F^+( y )$.
3. Non-zero morphisms $x \rightarrow y$, $y \rightarrow z$ compose to a non-zero morphism $x \rightarrow z$.
4. If $y \stackrel{\psi}{\rightarrow} z$ is a non-zero morphism, then each morphism $x \rightarrow z$ factors as $x \rightarrow y
\stackrel{\psi}{\rightarrow} z$.
\(i) Let $x \stackrel{\varphi}{\rightarrow} y$ be a morphism. If $y
\cong x$ then it follows from Proposition \[pro:Hom\] that $\varphi$ is a scalar multiple of the identity, and then we can take the claimed composition of irreducible morphisms to be empty.
If $y \not\cong x$, then let $\tau y \rightarrow y_1
\stackrel{\theta}{\rightarrow} y$ be the AR triangle ending in $y$. Since $x$, $y$ are indecomposable, the morphism $\varphi$ is not a split epimorphism so it factors as $x \rightarrow y_1
\stackrel{\theta}{\rightarrow} y$.
We can repeat this factorization process for the direct summands of $y_1$ to which $x$ has non-zero morphisms, that is, the direct summands of $y_1$ which are in the rectangle $R$ shown in Figure \[fig:rectangle\]; cf. Proposition \[pro:Hom\].
$$\xymatrix @-3.2pc @! {
& & & & & & & *{} & \\
& & & & & & *{} \ar@{--}[ur] & *{} & *{} &&\\
& & & & & & & F^+(x) & & & \\
& & & & & & y \ar@{-}[dddlll] \ar@{-}[ul] & & *{}\ar@{--}[ur] & *{} & *{}\\
& & & & R & & & & & \\
*{} & & x \ar@{-}[ddrr] \ar@{-}[uuuurrrr]& & &&&&&\\
& & & *{} & & & & *{} \\
*{} \ar@{--}[r] & *{} \ar@{-}[rrr] & & & *{} \ar@{-}[uuuurrrr]\ar@{-}[rrrrr] &*{}&&&&*{}\ar@{--}[r]&*{}\\
}$$
Successive repetitions show that the morphism $\varphi$ is a linear combination of compositions of irreducible morphisms within $R$. However, the mesh relations imply that any two such compositions are scalar multiples of each other, so $\varphi$ is a composition of irreducible morphisms.
\(ii) By Proposition \[pro:Hom\] there is a non-zero morphism $x
\rightarrow y$. By part (i), it is a scalar multiple of a composition of irreducible morphisms. But as remarked in the proof of part (i), two morphisms $x \rightarrow y$ which are both compositions of irreducible morphisms are scalar multiples of each other, so it follows that any such composition is non-zero.
\(iii) By part (i), each of the morphisms $x \rightarrow y$ and $y
\rightarrow z$ is a scalar multiple of a composition of irreducible morphisms, so the same is true for the composition $x \rightarrow z$. But $z$ is in $F^+(x)$, so $x \rightarrow z$ is non-zero by part (ii).
\(iv) By Proposition \[pro:Hom\] there is a non-zero morphism $x
\stackrel{\varphi}{\rightarrow} y$. The composition $x
\stackrel{\psi\varphi}{\rightarrow} z$ is non-zero by part (iii). But the space $\sC( x , z )$ is $1$-dimensional by Proposition \[pro:Hom\], so any morphism $x \rightarrow z$ can be factored as $\psi \circ \alpha \varphi$ with $\alpha$ a scalar.
\[pro:backward\_factorization\] Let $x, y, z \in \ind( \sC )$ be such that $x \in F^+( S^{ -1 }y ) \cap F^+( S^{ -1 }z )$ and $z \in F^+(y)$.
If $y \stackrel{\psi}{\rightarrow} z$ is a non-zero morphism, then each morphism $x \rightarrow z$ factors as $x \rightarrow y
\stackrel{\psi}{\rightarrow} z$.
We must show that $\sC( x,\psi ) : \sC( x,y ) \rightarrow \sC( x,z )$ is surjective. By Serre duality, it is equivalent to show that $\sC(
\psi,Sx ) : \sC( z,Sx ) \rightarrow \sC( y,Sx )$ is injective. For this it is enough to show that $\sC( \psi,Sx )$ is non-zero, since the $\Hom$ spaces $\sC( z,Sx )$ and $\sC( y,Sx )$ have dimension $0$ or $1$ over the ground field $k$ by Proposition \[pro:Hom\].
We must hence show that if $z \rightarrow Sx$ is non-zero, then so is the composition $y \stackrel{\psi}{\rightarrow} z \rightarrow Sx$. And this holds by Proposition \[pro:forward\_factorization\](iii) since we have $z \in F^+(y)$ and $Sx \in F^+( y ) \cap F^+( z )$; the latter condition holds because it is equivalent to the assumption $x
\in F^+( S^{ -1 }y ) \cap F^+( S^{ -1 }z )$.
\[rmk:approximating\] Recall that if $\sS$ is a subcategory of $\sC$ and $x \in \sC$ is an object, then a right-$\sS$-approximation of $x$ is a morphism $s
\stackrel{\sigma}{\rightarrow} x$ with $s \in \sS$ such that each morphism $s' \rightarrow x$ with $s' \in \sS$ factors through $\sigma$.
If each $x \in \sC$ has a right-$\sS$-approximation, then $\sS$ is called right-approximating. There are dual notions with “left” instead of “right”.
The following is a generalization of [@HJ thm. 4.4] and [@Ng thm. 2.2], and we follow the proofs of those results.
\[pro:basic\_d\_neq\_1\] Let $\sS$ be a subcategory of $\sC$ and let $\fS$ be the corresponding set of $d$-admissible arcs. The following conditions are equivalent.
1. The subcategory $\sS$ is right-approximating.
2. Each right-fountain of $\fS$ is a left-fountain of $\fS$.
For $d = 1$ this is [@Ng thm. 2.2] so assume $d \geq 2$.
Recall the notion of a slice: If $( t , u )$ is a vertex on the base line of the AR quiver of $\sC$, then the slice starting at $( t , u )$ is $( t , * )$; that is, it consists of the vertices with coordinates of the form $( t , u' )$. The slice ending at $( t , u )$ is $( * , u
)$.
This means that $t \in \BZ$ is a right-fountain of $\fS$ if and only if $\sS$ has infinitely many indecomposable objects on the slice $( t
, * )$ starting at $( t , t + d + 1 )$. Likewise, $t$ is a left-fountain of $\fS$ if and only if $\sS$ has infinitely many indecomposable objects on the slice $( * , t )$ ending at $( t - d - 1 , t ) = S( t , t + d + 1 )$. Hence (ii) is equivalent to the following condition on $\sS$.
- Let $v \in \ind( \sC )$ be on the base line of the AR quiver of $\sC$. If $\sS$ has infinitely many indecomposable objects on the slice starting at $v$, then it has infinitely many indecomposable objects on the slice ending at $Sv$.
Strictly speaking, we should say “infinitely many isomorphism classes of indecomposable objects” but as mentioned in Notation \[not:lax\] we are lax about this.
\(i) $\Rightarrow$ (ii’). Let $v \in \ind( \sC )$ be on the base line of the AR quiver. Note that $v$ and $Sv$ are in different components of the AR quiver since there are $d \geq 2$ components and $S$ moves vertices to the “next” component; cf. Construction \[con:coordinates\].
$$\begin{aligned}
&
\vcenter{
\xymatrix @-3.6pc @! {
&&&{S^{-1}a}&&&&&&&*{}&&&&&&&&&{b} \\
&&&&&&&&*{}&&&&&&&&*{}&&*{}&&&&*{}\\
&&&&&&&*{}&&&&&&&&&&*{}&&&&&*{}&*{}\\
&&&&&&&&*{} &&&&&*{}& *{} & *{} & *{s_3} \ar@{-}[uuurrr]\ar@{.}[dddddrrrrr] &&&&*{}&&*{} \\
&&&&&&&& & *{} & & & & & & *{} &&&&*{}&*{}&*{}&&&&*{}\\
&&&&&&&&*{S^{-1}z} \ar@{-}[uuuuulllll]\ar@{.}[uuuuurrrrr]& & & & *{}& & *{s_2} \ar@{-}[uurr] \ar@{.}[dddrrr]& & && *{}&&&&&&\\
&&&&&&&& & & & *{} & & *{s_1} \ar@{.}[ddrr] \ar@{-}[ur]& & & &*{}&&&&&&*{}\\
&&&&&&& & & & & & & &&&& \\
*{}\ar@{--}[r]&*{} \ar@{-}[rrrrrrrr] &&&&&&& & *{}\ar@{-}[rr]& *{} & *{v} \ar@{-}[uuulll]\ar@{-}[uurr]\ar@{-}[rrrr]& & & & *{} \ar@{.}[uuuuuurrrrrr]\ar@{-}[rrrrrrrrr]&&\ar@{.}[uuuuurrrrr]&*{}&*{}&&*{}\ar@{.}[uuurrr]&&&*{}\ar@{--}[r]&*{}\\
}
}
& \\
& & \\
& & \\
&
\vcenter{
\xymatrix @-2.6pc @! {
&&&{a}&&&&&&&*{}&&&&&&&&&{Sb} \\
&&&&&&&&*{}&&&&&&&&*{}&&*{}&&&&*{}\\
&&&&&&&*{}&&&&&&&&&&*{}&&&&&*{}&*{}\\
&&&&&&&&*{} &&&&&*{}& *{} & *{} & *{Ss_3} \ar@{-}[uuurrr] &&&&*{}&&*{} \\
&&&&&&&& & *{} & & & & & & *{} &&&&*{}&*{}&*{}&&&*{}\\
&&&&&&&&*{z} \ar@{.}[dddlll]\ar@{-}[uuuuulllll]& & & & *{}& & *{Ss_2} \ar@{-}[uurr] & & && *{}\\
&&&&&&&& & & & *{} & & *{Ss_1} \ar@{.}[uuuuuullllll] \ar@{-}[ur]& & & &*{}&&&&&&*{}\\
&&&&&&& & & & & & & &&&& \\
*{}\ar@{--}[r]&*{} \ar@{-}[rrrrrrrr] &&&&\ar@{.}[uuulll]&&& & *{}\ar@{-}[rr]& *{} & *{Sv} \ar@{-}[uuulll]\ar@{-}[uurr]\ar@{-}[rrrr]& & & & *{} \ar@{-}[rrrrrrrr]&&&&*{}&*{}&&*{}&*{}\ar@{--}[r]&*{}\\
}
}
&\end{aligned}$$
Figure \[fig:1\] shows the components of the quiver containing $v$ and $Sv$. As indicated, $b$ is the slice starting at $v$ and $a$ the slice ending at $Sv$. Assume that (i) holds and that $\ind( \sS )
\cap b$ is infinite. To show (ii’), we must show that $\ind( \sS )
\cap a$ is infinite.
Let $z$ be an indecomposable object on $a$ with right-$\sS$-approximation $s \stackrel{\sigma}{\rightarrow} z$. If $s_1$ is an object on $b$ then as shown by outlines in the figure we have $z \in F^-(Ss_1)$. Hence there is a non-zero morphism $s_1
\rightarrow z$ by Proposition \[pro:Hom\]. So each of the infinitely many objects in $\ind( \sS ) \cap b$ has a non-zero morphism to $z$, and each such morphism factors through $\sigma$ because $\sigma$ is a right-$\sS$-approximation. Since $\sC$ is a Krull-Schmidt category, this implies that there is an indecomposable direct summand $s'$ of $s$ such that the component $s'
\stackrel{\sigma'}{\rightarrow} z$ of $\sigma$ is non-zero and such that there are infinitely many objects $s_1$, $s_2$, $s_3$, $\ldots$ in $\ind( \sS ) \cap b$ which have non-zero morphisms to $s'$. Note that $s' \in \sS$ since $\sS$ is closed under direct summands by assumption.
We claim that this forces $s'$ to be on $a$, higher up than $z$. Hence, by moving $z$ upwards we obtain infinitely many objects in $\ind( \sS ) \cap a$.
To prove the claim, note that since $s'
\stackrel{\sigma'}{\rightarrow} z$ is non-zero, Remark \[rmk:Hom\] gives $s' \in F^+( S^{ -1 }z ) \cup F^-( z )$. The sets $F^+( S^{
-1 }z )$ and $F^-( z )$ are outlined in Figure \[fig:1\]. And $s'
\in F^+(S^{-1}z)$ is impossible because there would not be infinitely many objects in $\ind( \sS ) \cap b$ with a non-zero morphism to $s'$, as one sees by considering the sets $F^+(s_i)$ which are also outlined in the figure.
So we have $s' \in F^-(z)$. We already know $s' \in F^-(Ss_i)$ for each $i$. Hence, as one sees in Figure \[fig:1\], we have $s'$ on $a$. Finally, since there is a non-zero morphism $s'
\stackrel{\sigma'}{\rightarrow} z$, it follows that $s'$ is higher up on $a$ than $z$.
(ii’) $\Rightarrow$ (i). Assume that (ii’) holds and that $z \in \ind( \sC )$ is given. We will show (i) by constructing a right-$\sS$-approximation $s
\stackrel{\sigma}{\rightarrow} z$. We must ensure that each morphism $s' \rightarrow z$ with $s' \in \sS$ factors through $\sigma$, and we will do so by considering the possibilities for $s'$ and building up $\sigma$ accordingly.
We only need to consider those $s' \in \ind( \sS )$ which have non-zero morphisms to $z$. By Remark \[rmk:Hom\] there are the cases $s' \in F^-( z )$ and $s' \in F^+( S^{ -1 }z )$, see Figure \[fig:10\]. Note that $z$ and $S^{ -1 }z$ are in different components of the AR quiver; cf. the previous part of the proof.
$$\begin{aligned}
&
\vcenter{
\xymatrix @-4.6pc @! {
&&&&&&&&& \\
&&&&&&&& *{} \ar@{--}[ur] && b \\
&& \\
&&&&&&&&&&&& \\
&&&&&&&&&&& *{} \ar@{--}[ur] \\
&&&& *{S^{-1}z} \ar@{-}[uuuurrrr] \\
&&&&&&&&&&& *{F^+(S^{-1}z)} \\
&& \\
*{}\ar@{--}[r] & *{} \ar@{-}[rr] & & *{v} \ar@{.}[uuuuuuurrrrrrr] \ar@{-}[rr] && *{}\ar@{-}[rr]& *{} & *{} \ar@{-}[uuulll]\ar@{-}[uuuurrrr]\ar@{-}[rrrr]& & & & *{} \ar@{-}[rr]&&*{}\ar@{--}[r]&*{}\\
}
}
& \\
& & \\
& & \\
&
\vcenter{
\xymatrix @-3.1pc @! {
&&&&&&&&&&& \\
&&&&&& *{} \ar@{--}[ul] &&\\
&&& a &&&& \\
&&&&&&& \\
&&& \ar@{--}[ul] &&&& \\
&&&&&&&&&&*{z} \ar@{-}[dddlll]\ar@{-}[uuuullll]\\
&&&& *{F^-(z) \;} &&&&&&& \\
&&&&&&&&& \\
*{}\ar@{--}[r]&*{} \ar@{-}[rrrrrrrr] &&&&&& \ar@{-}[uuuullll] && *{Sv} \ar@{.}[uuuuuullllll]\ar@{-}[rr] && *{}\ar@{--}[r]&*{}\\
}
}
&\end{aligned}$$
First, assume $s' \in \ind( \sS ) \cap F^-( z )$. The slice $a$ in Figure \[fig:10\] determines a half line $F^-( z ) \cap a$. If there are objects of $\sS$ on this half line, then let $s_a$ be the one which is closest to the base line of the quiver and let $s_a
\stackrel{ \sigma_a }{ \rightarrow } z$ be a non-zero morphism. If $s'$ is on $a$ then it is above $s_a$ and Proposition \[pro:forward\_factorization\](iv) implies that each morphism $s'
\rightarrow z$ factors through $\sigma_a$. There are only finitely many slices $a$ intersecting $F^-( z )$. Including the corresponding morphisms $\sigma_a$ as components of $\sigma$ ensures that each morphism $s' \rightarrow z$ with $s' \in \ind( \sS ) \cap F^-( z )$ factors through $\sigma$.
Secondly, assume $s' \in \ind( \sS ) \cap F^+( S^{ -1 } z )$. The slice $b$ in Figure \[fig:10\] determines a half line $F^+( S^{ -1
}z ) \cap b$, and we split into two cases.
The case where $\sS$ has finitely many objects on $F^+( S^{ -1 }z )
\cap b \;$: Let $s_b$ be the direct sum of these objects and let each component of the morphism $s_b \stackrel{ \sigma_b }{ \rightarrow } z$ be non-zero. If $s'$ is on $b$, then $s'$ is one of the direct summands of $s_b$ and each morphism $s' \rightarrow z$ factors through $\sigma_b$ since each non-zero $\Hom$ space in $\sC$ is $1$-dimensional.
The case where $\sS$ has infinitely many objects on $F^+( S^{ -1 }z )
\cap b \;$: Then $\ind( \sS ) \cap b$ is infinite. If $b$ is the slice starting at $v$ and $a$ the slice ending at $Sv$, then condition (ii’) says that $\ind( \sS ) \cap a$ is also infinite. In particular it is non-empty so we have already included the non-zero morphism $s_a
\stackrel{ \sigma_a }{ \rightarrow } z$ as a component of $\sigma$ in the previous part of the proof. If $s'$ is on $b$ then it is straightforward to use Proposition \[pro:backward\_factorization\] to check that each morphism $s' \rightarrow z$ factors through $\sigma_a$.
As above, there are only finitely many slices $b$ intersecting $F^+(
S^{ -1 }z )$. Including the relevant morphisms $\sigma_b$ as components of $\sigma$ ensures that each morphism $s' \rightarrow z$ with $s' \in \ind( \sS ) \cap F^+( S^{ -1 }z )$ factors through $\sigma$.
A similar proof establishes the following dual result.
\[pro:basic\_d\_neq\_1\_dual\] Let $\sS$ be a subcategory of $\sC$ and let $\fS$ be the corresponding set of $d$-admissible arcs. The following conditions are equivalent.
1. The subcategory $\sS$ is left-approximating.
2. Each left-fountain of $\fS$ is a right-fountain of $\fS$.
[**Proof of Theorem E.** ]{} Given a subcategory $\sT$ of $\sC$ and the corresponding set of $d$-admissible arcs $\fT$, Theorem D says that $\sT$ is weakly $d$-cluster tilting if and only if $\fT$ is a $( d+2 )$-angulation of the $\infty$-gon. It is not hard to see that since $\fT$ is a set of pairwise non-crossing $d$-admissible arcs, it is locally finite or has a fountain if and only if it satisfies conditions (ii) in Propositions \[pro:basic\_d\_neq\_1\] and \[pro:basic\_d\_neq\_1\_dual\]. By the propositions, this happens if and only if $\sT$ is left- and right-approximating. $\Box$
Arc combinatorics {#sec:combinatorics}
=================
\[con:i\] Let $\fT$ be a $( d+2 )$-angulation of the $\infty$-gon and let $p_0
\in \BZ$ be given. We define integers $p_1, p_2, \ldots$ inductively as follows: If $p_{ \ell }$ has already been defined, then
- if $\fT$ contains no arcs of the form $( p_{ \ell } , q )$, then let $p_{ \ell + 1 } = p_{ \ell } + 1$;
- if $\fT$ contains a non-zero, finite number of arcs of the form $( p_{ \ell } , q )$, then let $( p_{\ell} , p_{\ell+1} )$ be the one with maximal length;
- if $\fT$ contains infinitely many arcs of the form $( p_{
\ell } , q )$, that is, if $p_{ \ell }$ is a right-fountain of $\fT$, then stop the algorithm and do not define $p_{ \ell + 1 }$.
If the algorithm stops, then it defines a sequence with finitely many elements, $$p_0 < \cdots < p_m.$$ If it does not stop, then it defines a sequence with infinitely many elements, $$p_0 < p_1 < \cdots,$$ and we set $m = \infty$. Let us sum up the properties of the sequence.
1. If $m < \infty$, then $p_m$ is a right-fountain of $\fT$.
2. $( p_{\ell} , p_{ \ell+1 } )$ is either a pair of consecutive integers or an arc in $\fT$.
3. $p_{\ell} - p_0 \equiv \ell \pmod d$.
To see (iii), note that the length of a $d$-admissible arc is $\equiv
1 \pmod d$.
Collin Bleak proved that a triangulation of the $\infty$-gon has a left-fountain if and only if it has a right-fountain, and his method also works for $( d+2 )$-angulations. We thank him for permitting us to bring a proof of the following lemma which establishes the “only if” direction. “If” follows by symmetry. See also [@G lem.4.11].
\[lem:fountains2\] Let $\fT$ be a $( d+2 )$-angulation of the $\infty$-gon. Suppose that $p_0$ is a left-fountain of $\fT$ and perform Construction \[con:i\].
1. The construction gives a finite sequence $p_0 < \cdots < p_m$ with $0 \leq m \leq d$.
2. $p_m$ is a right-fountain of $\fT$.
3. Let $\ft \in \fT$ and assume $\ft \neq ( p_{\ell} , p_{\ell +
1 })$ for $\ell \in \{ 0 , \ldots, m-1 \}$. Then $\ft$ has an overarc $\fr \in \fT$.
\(i) Assume to the contrary that $m \geq d + 1$; this includes the possibility $m = \infty$. Construction \[con:i\](iii) shows that $(
p_0 , p_{ d+1 } )$ is a $d$-admissible arc. If $( p_0 , p_1 )$ is a $d$-admissible arc, then $( p_0 , p_{ d+1 } )$ has strictly greater length than $( p_0 , p_1)$ and by Construction \[con:i\] we have $(
p_0 , p_{ d+1 } ) \not\in \fT$. If $( p_0 , p_1 )$ is not a $d$-admissible arc, then by Construction \[con:i\] there are no arcs in $\fT$ of the form $( p_0 , q )$ so we have $( p_0 , p_{
d+1 } ) \not\in \fT$ again. In either case there must be an arc $(
r,s ) \in \fT$ which crosses $( p_0 , p_{ d+1 } )$, that is, $r < p_0
< s < p_{ d+1 }$ or $p_0 < r < p_{ d+1 } < s$. But $p_0$ is a left-fountain of $\fT$ so $r < p_0 < s < p_{ d+1 }$ is impossible since it would imply that $( r,s )$ crossed an arc in $\fT$.
We must therefore have $p_0 < r < p_{ d+1 } < s$. However, this also leads to a contradiction: We cannot have $p_{\ell} < r < p_{\ell+1}$ for any $\ell \in \{ 0, \ldots, d \}$, for if we did then $( p_{\ell}
, p_{ \ell+1 })$ would not be consecutive integers whence $( p_{\ell}
, p_{\ell+1} ) \in \fT$ by Construction \[con:i\](ii), but this arc would cross $( r,s ) \in \fT$. So we must have $r = p_{\ell}$ for an $\ell \in \{ 1 , \ldots , d \}$. Hence $\fT$ contains arcs of the form $( p_{\ell},q )$, and by Construction \[con:i\] the one with maximal length is $( p_{\ell} , p_{ \ell+1 })$. But this contradicts $( r,s ) \in \fT$ because we know $r = p_{\ell}$ and $p_{ \ell+1 }
\leq p_{ d+1 } < s$.
\(ii) See Construction \[con:i\](i).
\(iii) Let us write $\ft = ( t,u )$ and search for $\fr$.
Since $p_0$ and $p_m$ are a left-fountain and a right-fountain of $\fT$, we must have $t < u \leq p_0$ or $p_0 \leq t < u \leq p_m$ or $p_m \leq t < u$.
If $t < u \leq p_0$, then we can choose $\fr = ( r,p_0 ) \in \fT$ with $r < t$. If $p_m \leq t < u$, then we can choose $\fr = ( p_m,s
) \in \fT$ with $u < s$.
Now assume $p_0 \leq t < u \leq p_m$.
If $t = p_{\ell}$ for an $\ell \in \{ 0 , \ldots , m-1 \}$, then $\ft
\in \fT$ is an arc of the form $( p_{ \ell } , q )$. Among the arcs in $\fT$ of this form, by Construction \[con:i\] the one with maximal length is $( p_{\ell} , p_{\ell+1} )$. Since $\ft \neq (
p_{\ell} , p_{\ell+1} )$ by assumption, we get that $\fr = ( p_{\ell}
, p_{\ell+1} ) \in \fT$ is an overarc of $\ft$.
If $p_{\ell} < t < p_{\ell+1}$ for an $\ell \in \{ 0 , \ldots , m-1
\}$, then we must have $u \leq p_{\ell+1}$, since otherwise $( t,u )
\in \fT$ and $( p_{\ell} , p_{\ell+1} ) \in \fT$ would cross. But then $\fr = ( p_{\ell} , p_{\ell+1} ) \in \fT$ is again an overarc of $\ft = ( t,u )$.
\[lem:fountains\] Let $\fT$ be a $( d+2 )$-angulation of the $\infty$-gon. Then $\fT$ is either locally finite or has precisely one left-fountain and one right-fountain.
If $\fT$ is not locally finite, then it has a left- or a right-fountain. By Lemma \[lem:fountains2\](ii) and its mirror image, it has both a left- and a right-fountain. And it is easy to see that in any event, it has at most one left- and at most one right-fountain.
\[lem:overarc\] Let $\fT$ be a $( d+2 )$-angulation of the $\infty$-gon which is locally finite or has a fountain. Then each arc $\ft \in \fT$ has an overarc $\fr \in \fT$.
The case where $\fT$ has a fountain at $p_0 \;$: Then we must have $m =
0$ in Lemma \[lem:fountains2\], and Lemma \[lem:fountains2\](iii) implies the present result.
The case where $\fT$ is locally finite: Let us write $\ft = ( t,u )$ and search for $\fr$. We can assume that, among the arcs in $\fT$ of the form $( t,v )$, the one of maximal length is $( t,u )$, since otherwise there is obviously an overarc. Let $p_0 = t$ and perform Construction \[con:i\]; then $ ( p_0 , p_1 ) = ( t,u )$. Since $\fT$ is locally finite, it has no right-fountain, so Construction \[con:i\](i) implies $m = \infty$. Construction \[con:i\](iii) implies that $( p_0 , p_{ d+1 } )$ is a $d$-admissible arc. It has strictly greater length than $( p_0 , p_1)$, so $( p_0 , p_{ d+1 } )
\not\in \fT$ follows.
There must hence be an arc $( r,s ) \in \fT$ which crosses $( p_0 ,
p_{ d+1 } )$, that is, $r < p_0 < s < p_{ d+1 }$ or $p_0 < r < p_{ d+1
} < s$.
First, assume $r < p_0 < s < p_{ d+1 }$. Note that we cannot have $p_0 < s < p_1$ since then $( r,s ) \in \fT$ and $( p_0 , p_1 ) \in
\fT$ would cross. So $p_1 \leq s$ whence $\fr = ( r,s ) \in \fT$ is an overarc of $( p_0 , p_1 ) = ( t,u )$.
Secondly, assume $p_0 < r < p_{ d+1 } < s$. This leads to a contradiction in the same way as in the second paragraph of the proof of Lemma \[lem:fountains2\].
\[con:polygon\] Let $\fT$ be a $( d+2 )$-angulation of the $\infty$-gon and let $\fr =
( r,s ) \in \fT$. We can view $\{ r , \ldots , s \}$ as the vertices of an $( s-r+1 )$-gon $R$. Each pair $( r , r+1 ), ( r+1,r+2 ),
\ldots, ( s-1,s )$ is viewed as an edge of $R$, and so is the arc $(
r,s )$; that is, $r$ and $s$ are viewed as consecutive vertices of $R$. Each $d$-admissible arc $\ft$ of which $\fr = ( r,s )$ is an overarc is viewed as a $d$-admissible diagonal of $R$. See Figure \[fig:polygon\].
$$\begin{aligned}
\parbox[t]{3cm}{
\vspace{-17ex}
$
\xymatrix @-3.0pc @R=-4ex @! {
& *{\rule{0ex}{5.5ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^1.5pc/@{-}[rrrrr]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^1.0pc/@{-}[rrr]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^1.0pc/@{-}^{\textstyle\ft}[rrr]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^-3.0pc/@{-}_{\textstyle \fr}[lllllllll]
& *{} \\
& & r & & & & & & & & & s \\
}
$
}
& \;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\;\; &
\begin{tikzpicture}[auto]
\node[name=s, shape=regular polygon, regular polygon sides=10, minimum size=4cm, draw] {}; \draw[shift=(s.corner 2)] node[above] {$r$};
\draw[shift=(s.corner 1)] node[above] {$s$};
\draw[shift=(s.side 1)] node[above] {$\fr$};
\draw[thick] (s.corner 2) to (s.corner 7);
\draw[thick] (s.corner 4) to (s.corner 7);
\draw[thick] (s.corner 7) to node[left=0pt] {$\ft$}(s.corner 10);
\end{tikzpicture} \end{aligned}$$
In particular, the set $$\fR = \{\, \ft \in \fT \,|\, \mbox{ $\fr$ is an overarc of $\ft$ } \,\}$$ is a $( d+2 )$-angulation of $R$.
Observe that $\fR$ divides $R$ into $( d+2 )$-gons, and that one of these $( d+2 )$-gons, say $T$, has $r$ and $s$ among its vertices. We can write the whole set of vertices of $T$ as $$r < t_1 < \cdots < t_d < s,$$ and hence each of $$( r,t_1 ) \;,\; ( t_1,t_2 )
\;,\; \ldots \;,\;
( t_{ d-1 },t_d ) \;,\; ( t_d,s )$$ is either a pair of consecutive integers or a diagonal in $\fR$, that is, an arc in $\fT$.
\[lem:mutation\] Let $\fT$ be a $( d+2 )$-angulation of the $\infty$-gon and let $\ft
\in \fT$.
1. If $\fU$ is a set of $d$-admissible arcs not in $\fT \setminus
\ft$ such that $( \fT \setminus \ft ) \cup \fU$ is a $( d+2
)$-angulation of the $\infty$-gon, then $\fU = \{ \ft^* \}$ for a single $d$-admissible arc $\ft^*$.
2. If $\ft$ has an overarc in $\fT$ then there are $d + 1$ choices of $\ft^*$.
3. If $\ft$ has no overarc in $\fT$ then there are $\leq d$ choices of $\ft^*$.
Suppose that $\ft$ has the overarc $\fr \in \fT$. We will establish (i) and (ii) for $\ft$.
The set of all arcs in $\fT$ of which $\fr \in \fT$ is an overarc can be viewed as a $( d+2 )$-angulation $\fR$ of a polygon $R$ by Construction \[con:polygon\]. When $( \fT \setminus \ft ) \cup \fU$ is a $( d+2 )$-angulation of the $\infty$-gon, $\fr$ is an overarc of each arc in $\fU$ since removing $\ft$ does not create any room above its overarc $\fr$. It follows that $\fU$ can be viewed as a set of $d$-admissible diagonals of $R$ such that $( \fR \setminus \ft)
\cup \fU$ is a $( d+2 )$-angulation of $R$. Then it is well known that, as desired, $\fU$ has one element which can be chosen in $d+1$ different ways.
Now suppose that $\ft$ has no overarc in $\fT$. We will establish (i) and (iii) for $\ft$.
Lemma \[lem:overarc\] shows that $\fT$ is not locally finite and does not have a fountain. Lemma \[lem:fountains\] shows that $\fT$ has a left-fountain $p_0$ which is not a right-fountain. We can perform Construction \[con:i\]. By Lemma \[lem:fountains2\](i+ii) this gives a sequence $p_0 < \cdots < p_m$ with $m \leq d$ where $p_m$ is a right-fountain of $\fT$. Note that $1 \leq m$ since $p_0$ is not a right-fountain. By Construction \[con:i\](ii), each $( p_{ \ell }
, p_{ \ell+1 } )$ is either a pair of consecutive integers or an arc in $\fT$, and it follows from Lemma \[lem:fountains2\](iii) that $\ft = ( p_{j} , p_{j+1} )$ for a $j \in \{ 0 , \ldots , m-1 \}$.
By Construction \[con:polygon\] applied to $\ft = ( p_{ j } , p_{
j+1 } )$, there is a sequence of integers $p_{j} < q_1 < \cdots
< q_d < p_{ j+1 }$ such that each of $( p_{j} , q_1 )$, $( q_1 ,
q_2 )$, $\ldots$, $( q_{ d-1 } , q_d )$, $( q_d , p_{ j+1 } )$ is either a pair of consecutive integers or an arc in $\fT$. We hence have a sequence of integers $$\label{equ:pq}
p_0 < p_1 < \cdots < p_{j}
< q_1 < \cdots < q_d
< p_{ j+1 } < \cdots < p_m$$ where each pair of neighbouring elements is either a pair of consecutive integers or an arc in $\fT \setminus \ft$. In particular, each pair of neighbouring elements has a difference which is $\equiv 1
\pmod d$.
Now consider a $d$-admissible arc $\ft^* = ( v,w ) \not\in \fT
\setminus \ft$ which crosses no arc in $\fT \setminus \ft$.
We cannot have $w \leq p_0$. For if we did, then $\ft^* = ( v,w )$ would not cross $\ft = ( p_{j} , p_{ j+1 } )$, and hence $\ft^*$ would cross no arc in $\fT$ whence $\ft^* \in \fT$. Since $\ft^* \not\in
\fT \setminus \ft$, this would force $\ft^* = \ft$, but this contradicts $v < w \leq p_0 \leq p_{ j }$. We also cannot have $v <
p_0 < w$ because $p_0$ is a left-fountain of $\fT$. Similarly, we cannot have $p_m \leq v$ or $v < p_m < w$.
We conclude that $p_0 \leq v < w \leq p_m$. We claim that, in fact, $v$ and $w$ must be among the elements of the sequence .
Namely, assume that at least one of $v$ and $w$ is not an element of the sequence. Then it is strictly between two such elements. For the sake of argument, say $q_{ \ell } < v < q_{ \ell+1 }$. Then we cannot have $q_{ \ell+1 } < w$, for then $\ft^* = ( v,w )$ and $( q_{ \ell }
, q_{ \ell+1 } ) \in \fT \setminus \ft$ would cross. So we must have $v < w \leq q_{ \ell+1 }$. Hence $( q_{ \ell } , q_{ \ell+1 } )$ is an overarc of $\ft^* = ( v,w )$.
However, the set of all arcs in $\fT$ of which $( q_{ \ell } , q_{
\ell+1 } ) \in \fT \setminus \ft$ is an overarc can be viewed as a $( d+2 )$-angulation $\fR'$ of a polygon $R'$ by Construction \[con:polygon\], and $\ft^*$ can be viewed as a $d$-admissible diagonal of this polygon. Note that $( q_{ \ell } , q_{ \ell+1 } )$ is not an overarc of $\ft = ( p_j , p_{ j+1 } )$ and so $\fR'
\subseteq \fT \setminus \ft$. Hence the assumption that $\ft^*$ crosses none of the arcs in $\fT \setminus \ft$ means that it crosses none of the diagonals in $\fR'$. But then $\ft^* \in \fR'$ whence $\ft^* \in \fT \setminus \ft$ which is a contradiction.
So we have $\ft^* = ( v,w )$ with $v$, $w$ elements in the sequence . However, we saw that each pair of neighbouring elements in this sequence has a difference which is $\equiv 1 \pmod
d$. Hence, for $\ft^*$ to be a $d$-admissible arc, $v$ and $w$ must either be neighbours in the sequence, or $nd+1$ steps apart for an integer $n \geq 1$. But they cannot be neighbours for then we would have $\ft^* \in \fT \setminus \ft$, so $v$ and $w$ must be $nd+1$ steps apart in the sequence.
Since $m \leq d$ by Lemma \[lem:fountains2\](i), the sequence has $m + 1 + d \leq 2d + 1$ elements. It follows that $n = 1$ and hence two different choices of $\ft^*$ must cross each other; this shows part (i) of the present lemma. It also follows that there are at most $(2d + 1) - (d+1) = d$ different choices for $\ft^*$, showing part (iii) of the present lemma.
Proofs of Theorems A, B, and C {#sec:proofs}
==============================
Let $\sT$ be a weakly $d$-cluster tilting subcategory of the triangulated category $\sC$ and let $\fT$ be the corresponding $( d+2
)$-angulation of the $\infty$-gon.
Lemma \[lem:mutation\](i) says that if we drop one arc from $\fT$, then we must add precisely one other $d$-admissible arc to get a new $( d+2 )$-angulation.
So if we drop one indecomposable object from $\sT$, then we must add precisely one other indecomposable object to get a new weakly $d$-cluster tilting subcategory of $\sC$.
That is, “mutating $\sT$ at $t$” has the expected effect of replacing $t$ by a single other indecomposable object.
[**Proof of Theorem A.** ]{} By Theorem E which was already proved in Section \[sec:approximations\], a $d$-cluster tilting subcategory $\sT$ of $\sC$ corresponds to a $( d+2 )$-angulation of the $\infty$-gon $\fT$ which is locally finite or has a fountain.
By Lemma \[lem:overarc\], each $\ft \in \fT$ has an overarc $\fr
\in \fT$.
By Lemma \[lem:mutation\](ii), this means that there are $d + 1$ different choices of a $d$-admissible arc $\ft^*$ such that $( \fT
\setminus \ft ) \cup \ft^*$ is a $( d+2 )$-angulation of the $\infty$-gon.
Excluding the trivial choice $\ft^* = \ft$ leaves $d$ choices for $\ft^*$ and translating back to $\sT$ shows Theorem A. $\Box$
[**Proof of Theorem B.** ]{} Let $\ell \in \{ 0, \ldots, d-1 \}$ be given. Figure \[fig:bad\] shows part of a $( d+2 )$-angulation $\fT_{ \ell }$ of the $\infty$-gon.
$$\vcenter{
\xymatrix @-3.5pc @R=-6ex @! {
\rule{0ex}{5.5ex} \ar@{--}[r]
& *{\rule{0ex}{4ex}^{\lefteqn{\textstyle \cdots}}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^2.0pc/@{-}[rrrr]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^1.5pc/@{-}[rrr]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^1.0pc/@{-}[rr]
& *{} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^1.0pc/@{-}[rr]
& *{} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r]
& *{} \ar@{-}[r]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^-1.0pc/@{-}[ll]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^-1.5pc/@{-}[lll]
& *{\rule{0.1ex}{0.8ex}} \ar@{-}[r] \ar@/^-2.0pc/@{-}[llll]
& *{\rule{0ex}{4ex}^{\lefteqn{\textstyle \!\!\!\!\!\!\! \cdots}}} \ar@{--}[r]
& *{} \\
& & & & {\scriptscriptstyle -d-1} & & {\scriptscriptstyle 0} & & {\scriptscriptstyle d+1} & {\scriptscriptstyle d+1+\ell} & & {\scriptscriptstyle 2d+2+\ell}
}
}$$
It contains the arc $\ft = ( 0 , d+1 )$ and has a left-fountain at $0$ and a right-fountain at $d + 1 + \ell$.
There are $\ell + 1$ different choices of a $d$-admissible arc $\ft^*$ such that $( \fT \setminus \ft ) \cup \ft^*$ is a $( d+2 )$-angulation of the $\infty$-gon; namely, $\ft^* = ( p , p + d + 1 )$ for $p \in \{
0, \ldots, \ell \}$.
Excluding the trivial choice $\ft^* = \ft$ leaves $\ell$ choices. By Theorem D which was already proved in Section \[sec:approximations\], the $( d+2 )$-angulation $\fT_{ \ell }$ therefore corresponds to a weakly $d$-cluster tilting subcategory $\sT_{ \ell }$ with the property claimed in Theorem B. $\Box$
[**Proof of Theorem C.** ]{} Similar to the proof of Theorem A, but $\ft \in \fT$ may or may not have an overarc, so both parts (ii) and (iii) of Lemma \[lem:mutation\] are needed. $\Box$
[**Acknowledgement.**]{} We are grateful to Collin Bleak for permitting us to bring a proof that a $( d+2
)$-angulation of the $\infty$-gon has a left-fountain if and only if it has a right-fountain, see Lemmas \[lem:fountains2\] and \[lem:fountains\]. Collin Bleak originally proved this for $d =
1$.
This work was supported by grant number HO 1880/4-1 under the research priority programme SPP 1388 [ *Darstellungstheorie*]{} of the Deutsche Forschungsgemeinschaft (DFG).
[19]{}
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T. Holm, P. Jørgensen, and D. Yang, Sparseness of t-structures and negative Calabi–Yau dimension in triangulated categories generated by a spherical object, preprint (2011). [ math.RT/1108.2195v1.]{}
O. Iyama, [*Maximal orthogonal subcategories of triangulated categories satisfying Serre duality*]{}, Oberwolfach Rep. (2005), 353–355.
O. Iyama and Y. Yoshino, [*Mutation in triangulated categories and rigid Cohen-Macaulay modules*]{}, Invent. Math. [**172**]{} (2008), 117–168.
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|
WASH GARMENTS BEFORE WEARING !!
Please make it a habit to wash your just purchased undergarments before wearing them.
This is sensitive..
Our undergarments are made in different parts of the world, sit in boxes and go through many hands and exchanges before we purchase them for ourselves.
ALL PLEASE WASH ALL BRAS, UNDERWEAR AND OTHER CLOTHS WHEN YOU BUY BEFORE WEARING THEM. WE DO NOT KNOW WHAT PARASITE IS IN OUR CLOTHES WHEN WE BUY THEM.
Read the article first before looking at the picture. This looks horrible.
After anthropologist Susan McKinley came back home from an expedition in South America , she noticed a very strange rash on her left breast. Nobody knew what it was and she quickly dismissed it believing that the holes would leave in time.
Upon her return she decided to see a doctor after she started developing intense pains. The doctor, not knowing the exact severity of the disease, gave her antibiotics and special creams. As time lapsed the pain did not subside and her left breast became more inflamed and started to bleed.
She decided to bandage her sores however as Susan’s pain grew more intense she decided to seek help from a more certified doctor. Dr. Lynch could not diagnose the infection and told Susan to seek the aid of one of his colleagues who specialized in dermatology whom was sadly on vacation. She waited for two weeks and finally was able to react the dermatologist.
Sadly, a life-changing event was about to unfold during her appointment.
To Miss McKinley’s surprise, after she removed the bandages, they found larva growing and squirming within the pores and sores of her breast.
Sometimes these wicked creatures would all together simultaneously move around into different crevices. What she didn’t know was that the holes were in fact, deeper than she had originally thought for these larvae were feeding off the fat, tissue, and even milk canals of her bosom. |
Effects of acrylonitrile on rat liver cytochrome P-450, benzo(a)pyrene metabolism and serum hormone levels.
Intraperitoneal injections of acrylonitrile (AN), 33 mg/kg, into male rats daily for 3 consecutive days resulted in a 20% decrease in the liver microsomal content of cytochrome P-450. The in vitro formation of 9-hydroxy and 9,10-dihydrodiol metabolites of benzo(a)pyrene in liver microsomes was inhibited. Serum corticosterone levels were markedly decreased (30% of th values for control animals) demonstrating a disturbed pituitary-adrenal axis. A central effect of AN is supported by the finding that prolactin concentrations fell by approximately 60% in exposed animals. AN also induced a doubling FSH levels, whereas LH concentrations were the same as in the control animals. These results suggest an impaired spermatogenesis in the exposed animals. |
116 N.W.2d 275 (1962)
174 Neb. 204
Kenneth NORDAHL, Appellant-Cross-Appellee,
v.
Gordon A. ERICKSON, Wayne Skinner, and Mid-States Equipment Company, a Partnership, Appellees-Cross-Appellants.
No. 35264.
Supreme Court of Nebraska.
July 13, 1962.
*276 Kanouff & Brown, Wahoo, for appellant.
Cline, Williams, Wright, Johnson, Old-father & Thompson, Lincoln, for appellees.
Heard before SIMMONS, C. J., and CARTER, MESSMORE, YEAGER, SPENCER, BOSLAUGH and BROWER, JJ.
SIMMONS, Chief Justice.
This is a workmen's compensation case. The plaintiff sought a permanent total disability award. The one-man compensation court awarded a 45 percent permanent partial disability to the right foot. Plaintiff refused to accept the award and had a hearing before the Workmen's Compensation Court, en banc. There he received an award of 45 percent permanent partial disability of his right foot, an award for the loss of a small toe on the left foot, and a 12 percent permanent partial disability to his body as a whole.
Plaintiff took the matter to the district court where, under our procedure, it became an error proceeding. Defendants cross-appealed, praying that the order of the compensation court be set aside. The district court found no error in the decision of the compensation court and dismissed both the appeals of the plaintiff and defendants.
Plaintiff appeals here where, under our procedure, the matter is for trial de novo on the record made before the compensation court. Defendants cross-appealed.
In effect, the plaintiff contends here, as he has throughout, that he is entitled to permanent total disability under the provisions of section 48-121(1), R.R.S.1943. Defendants, in effect contend for an award under the provisions of section 48-121(3), R.R.S.1943, and for the right foot only.
We find that the plaintiff is entitled to an award of permanent total disability. We reverse the judgment of the trial court and remand the cause with directions to enter an award in accord with this opinion.
Plaintiff, at the time of the accident, was about 36 years of age. He was married and the father of two children whose ages are not shown. He weighed about 200 pounds. He quit school in the 10th or 11th grade. He was born and raised on a farm. All his life he had engaged in hard physical labor for a livelihood.
There is no suggestion in this case that plaintiff is malingering.
The accident here involved occurred September 3, 1958. Up until about 2 years prior thereto, he had lived and worked on a farm, first as a laborer, then in some sort of partnership with his mother. After marriage, he rented a farm and farmed for himself.
Twenty years or more, before this accident, he had broken a bone in the small toe of his left foot. It resulted in a "hammertoe." It, in no sense, was disabling. He also had had some lower back trouble for *277 which he was examined at Mayo's some years before this accident. It was not disabling. It is not involved here.
Somewhere in this period, he and his family inherited a farm of 200 acres of which about 100 acres was pasture land and about 100 acres tillable land. The plaintiff's interest in the farm or whether it was owned free from encumbrance is not shown.
About 2 years before this accident, plaintiff quit farming and engaged in common labor in the construction industry. At the time of the accident, he was working as a carpenter, cement finisher, and anything they wanted him to do. He was working at the top of a grain elevator and above a shaft which was rectangular in shape and about 20×27 inches in dimension. The shaft was 180 feet high. Beneath it was an open space of some 18 feet to a concrete floor which at the time had debris of construction material on it.
A ladder on which plaintiff was standing worked loose and plaintiff fell, feet downward, the entire length of the shaft and open area beneath. His size was such that he about fitted the shaft. He was able to slacken his fall by body and limb pressures on the sides. He was at all times fully conscious. He first struck the concrete floor on his feet, then his buttocks, and then his back. His body had multiple abrasions of the skin, particularly on his arms and legs from contact with the walls of the shaft. With the exception of his right hand, they do not enter into the problem here.
In view of the fact that the question here is whether he had a single permanent injury to the right foot or multiple injuries causing permanent injury to his body as a whole, we discuss each injury separately.
The physician and surgeon who cared for plaintiff throughout became defendants' expert witness.
Plaintiff's right ankle was obviously smashed as a result of the impact. Efforts were made to restore it and finally, after two or more operations, it was fixed permanently immobile. There is no dispute about that, neither is there dispute that his right leg is three-quarters of an inch shorter than the left. It is the above injury for which defendants admit liability under the provisions of section 48-121(3), R.R.S.1943, as a scheduled injury.
In addition to the above, plaintiff's expert witness testified that there was a "marked atrophy of the muscles of his right leg." This was not limited to a below the knee situation and we do not find it denied by defendants' expert witness.
The other of plaintiff's expert witnesses testified that plaintiff, in such movements as getting down on his legs, was awkward and there was some weakness or loss of co-ordination of the right side. He illustrated this by reference to his ability in getting down to replace "a nut in a machine or anything like that."
Defendants' witness makes no reference to this testimony and was not asked.
When doing work, plaintiff wears a steel brace from his shoe to the right knee, as prescribed by defendants' expert witness.
We go now to the left foot. At the time of his first examination, the surgeon found a moderate swelling of the left foot with complaint of tenderness over the dorsum of the foot and some tenderness of the hammertoe. Shortly after the accident, the surgeon operated on and removed a bone from the hammertoe and found "probability" of reinjury to it in the accident. He testified, however, that he performed the operation as a favor to the plaintiff and undertook as a witness for defendants to remove injury to the left foot from plaintiff's disabilities caused by the accident. However, the surgeon prescribed, and plaintiff wears, an arch support on his shoe under the instep of his left foot.
Plaintiff testified that he could not walk barefoot in the house without pain in both feet. When he walks at all with a shoe on, *278 he does so with the weight put on the big toe and heel of the left foot, to avoid pain.
As to the use of his feet, the undisputed evidence is that plaintiff could not stand on his feet for more than 10 minutes without pain often followed by edema. Walking, particularly on rough surfaces, about the farmyard is a constant source of pain and danger.
Plaintiff's right hand, and particularly the index finger, received the worst of the abrasions in the fall. No bones were broken. The testimony is that plaintiff's right hand has lost some of its strength, there is a numb area in the heel of the hand, and misplacement of bones in the wrist. Plaintiff has complained to the doctors of pain in that wrist every time he uses it, and concededly, it has a restricted use. There is also some evidence of pain in the right arm area. Plaintiff is a right-handed man.
At the time of his first examination, plaintiff complained of tenderness throughout his back. He has continuously complained of tenderness in his upper back between his shoulder blades and in his neck. We will make further reference to this in connection with his use of a farm tractor.
He can neither lift nor carry articles of much weight.
Such then is the present condition of a man who, prior to this accident, could do the hardest of hard common labor, and, in his own language, there were few who could follow him. Defendants would have us find that he has only a permanent injury to his right ankle.
We have no difficulty in finding that he has a disability to his body as a whole. Plaintiff's expert witness so testified. In a report to the insurance carrier, defendants' expert witness so stated. The divergence of views here seems to be that plaintiff's expert witnesses find subjective symptoms of pain and disability, whereas defendants' witness undertook to limit his ratings to objective symptoms only.
Plaintiff's expert witness fixed the disability to the body as a whole as a 25 percent permanent partial disability. The question then arises as to how much compensation plaintiff is entitled.
We have held: "Disability under section 48-121, R.S.Supp., 1953, subdivisions (1) and (2), is defined by our statute in terms of employability and earning capacity rather than in terms of loss of bodily function. * * * In defining total disability, losses in bodily function are not important in themselves but are only important insofar as they relate to earning capacity and the loss thereof." Miller v. Peterson, 165 Neb. 344, 85 N.W.2d 700.
As we stated the question in Pavel v. Hughes Brothers, Inc., 167 Neb. 727, 94 N.W.2d 492: "What, then, is the evidence as to plaintiff's ability to perform labor and to earn an income in his particular line of work or in any other for which he is fitted?"
There is no contention here that plaintiff is employable as a common laborer, carpenter, or cement finisher, being the work in which he was engaged when injured.
The defendants contend here that plaintiff is a trained, experienced farmer and can do that work. This is not based upon any evidence of employability as a farm hand. He operates a farm, owned by himself and family and, apparently, had done so in the years 1960-1961. The evidence is that it is not a profitable operation because of the necessity of employing labor. His herd of cattle has increased from 25 to 70, with no evidence as to whether they are encumbered or free from debt. The sole evidence as to plaintiff's activity in this regard is that he, apparently, accompanies some shipments of cattle to the market at Omaha and sells them.
Plaintiff's wife and children do the chores, attend the cattle, and work in the fields. At the time of this hearing in July 1961, plaintiff was employing four hired men.
*279 Plaintiff's tillable ground is devoted to row crops, partly irrigated. Some of the work in connection with it is done by tractor. Defendants stress the testimony of plaintiff that he did about 75 percent of the tractor work in 1961 and less in 1960. But that is not the whole story. The question is as to how and to what extent, he was able to do it. The undisputed testimony is that he could drive the tractor for 2 or 3 hours and then was required to rest. He was unable to use his right foot in manipulating the tractor brake, etc. He sat sideways when that was necessary and did the foot work with his left foot. If he was pulling a following implement requiring that he look back and turn to do so, he immediately had pain in his neck and upper back, and sometimes a locking of the neck. The amount of work which plaintiff was able to perform in hours, days, or seasons is not shown. There is no showing of employability as a farm laborer except insofar as he works for himself, when able, and to the extent above-shown. On occasion, where necessity required, he would sometimes work as many as 8 hours a day with the tractor, followed by spending the next day in the house.
We think the evidence shows that plaintiff is not employable.
Our comment in Elliott v. Gooch Feed Mill Co., 147 Neb. 309, 23 N.W.2d 262, is applicable: "He is, therefore, what is called an `odd lot' man, the nondescript in the labor market, with which industry has little patience, and rarely hires. Work, if he finds any that he can do, is casual and intermittent. He is utterly unable to do the work he did before this accident. Under these facts, we think we are justified in finding that in his present handicapped condition he is entitled to an award for total disability under the Nebraska Compensation Act."
We quoted from the above case in Rapp v. Hale, 170 Neb. 620, 103 N.W.2d 851, as follows: "`For workmen's compensation purposes, "total disability" does not mean a state of absolute helplessness, but means disablement of an employee to earn wages in the same kind of work, or work of a similar nature, that he was trained for, or accustomed to perform, or any other kind of work which a person of his mentality and attainments could do. * * * A workman who, solely because of his injury, is unable to perform or to obtain any substantial amount of labor, either in his particular line of work, or in any other for which he would be fitted except for the injury, is totally disabled within the meaning of the workmen's compensation law.'"
We find that plaintiff is permanently and totally disabled and entitled to compensation as such under the provisions of section 48-121(1), R.R.S.1943.
We do not undertake to tabulate the period of the beginning of compensation payments. We anticipate that the parties will be able to do that without difficulty.
The judgment of the trial court is accordingly reversed and the cause remanded with directions to enter judgment in accord with this opinion.
Reversed and remanded with directions.
CARTER, Justice (dissenting).
I do not agree that the record in this case sustains a finding that plaintiff was totally and permanently disabled. The case involves schedule injuries and other claimed injuries to other members, and a further contention of permanent injury to the body as a whole.
We have few cases in this state in which the relation of a schedule injury to injury to the body as a whole has been considered. The opinion of the majority appears deficient in the consideration of this problem.
The plaintiff sustained an injury to his right foot which, standing alone, would be compensated for as a schedule injury as defined in subdivision (3) of section 48-121, R.R.S.1943. There is no medical evidence *280 in the record that the injury to the right foot constituted more than 40 to 50 percent permanent disability of the right foot.
The evidence shows that plaintiff's physician removed a hammertoe from his left foot which was a condition that did not result from the accident. There was some evidence of injury to the top of the left foot. Examination by X-ray showed no fracture, but revealed an arthritic condition which had developed over a period of time before the accident. No objective symptoms of injury to the left foot were found. No doctor testified as to any amount of permanent disability to the left foot.
There was evidence of some injury to the right wrist. An orthopedic physician testified that the distal end of the ulna lies dorsal-ward from the usual relationship with the radius. There was no evidence of fracture. There is medical evidence that this condition is occasionally seen as a natural development in a normal wrist. There is no medical evidence that the wrist condition was the result of the accident and plaintiff's doctor made no separate permanent disability rating as to the right hand and wrist. In fact, the medical experts considered the condition of the right wrist of no importance.
Plaintiff asserts that he suffered injury to his back. He was treated at the Mayo Clinic for lower back discomfort in February 1958, prior to the accident. No objective evidence of traumatic injury to the back was found and no doctor attempted to fix any percentage of permanent injury to the back or to the body as a whole resulting from the claimed back injury. There was evidence of tenderness in the shoulder blade and dorsal spine area. The medical evidence is that this was caused by an osteoarthritic condition and the use of crutches during the period of convalescence, without any indication of permanency. There is some medical evidence that plaintiff would suffer pain in his back in the future. The disability from such pain is not rated nor is there medical evidence that it was the result of the accident.
I think the provision of subdivision (3) of section 48-121, R.R.S.1943, providing total and permanent disability for the loss of two feet means a complete loss of use of such two members. What then if one or both members under subdivision (3) have not been injured to the extent of permanent total loss of use. Under such circumstances I think the rule stated in Radford v. Smith Bros., 123 Neb. 13, 241 N.W. 753, applies. The announced rule in that case is that a claimant for compensation who has sustained injury to both legs and both hands is entitled to recover such proportion of compensation allowed for total disability as the extent of loss of the several members bears to the total loss of two such members. See, also, Frost v. United States Fidelity & Guaranty Co., 109 Neb. 161, 190 N.W. 208; Johnson v. David Cole Creamery Co., 109 Neb. 707, 192 N.W. 127; Schlesselman v. Travelers Ins. Co., 112 Neb. 332, 199 N.W. 498; Ashton v. Blue River Power Co., 117 Neb. 661, 222 N.W. 42. Under the foregoing rule the plaintiff is entitled to recover permanent partial disability benefits under subdivision (1) in the proportion that the extent of his loss to such members would bear to the total loss of two such members.
A traumatic injury to the back resulting from the accident would raise a question that we do not have before us, as I view the evidence. There is no evidence that the back injury was caused by the accident, although it is one that could well have been lighted up by the accident in view of its nature. The evidence of back injury was subjective only. No objective symptoms were found, other than the preexisting osteoarthritic condition previously mentioned. An injury to a back based only on the complaints of pain by the claimant should not be accepted as proven in the absence of medical support. Magnolia Pipe Line Co. v. Smith, 167 Okl. 316, 29 P.2d 569.
There is no medical proof that the claimed injury to the right wrist or to the back *281 was the result of the accident and, what is more important still, there is no medical proof that such injuries result in any disability chargeable to the accident. The burden of proof is on plaintiff to prove these facts. The plaintiff testifies that he was able to do 20 percent of his tractor work in his farming operations during the first year following the accident and that he was able to do 75 percent of it the second year. This is a clear indication of physical improvement and a confirmation of the medical evidence finding partial permanent disability only. As I view the record, the case should be determined as a two-member injury requiring an award of partial permanent disability under the rule announced in Radford v. Smith Bros., supra, and other cases announcing the rule of that case.
The Workmen's Compensation Act provides that the loss of use of two feet shall constitute total and permanent disability and shall be compensated for according to the provisions of subdivision (1) of section 48-121, R.R.S.1943. It does not authorize the granting of total permanent disability where the use of one or both injured members are not wholly disabled. This court has held on many occasions that it is immaterial whether or not industrial disability is present in a schedule injury. Bronson v. City of Fremont, 143 Neb. 281, 9 N.W.2d 218. Since the competent evidence before the court in the present case relates to the injury of two schedule members, the matter of industrial loss and employability is not material. Paulsen v. Glenn L. Martin-Nebraska Co., 147 Neb. 1012, 26 N.W.2d 11. The plaintiff is therefore entitled to an award of permanent partial disability calculated under the rule of Radford v. Smith Bros., supra. See, also, Paulsen v. City of Lincoln, 156 Neb. 872, 58 N.W.2d 336. The failure of the majority to apply the rule of that case affords the basis of this dissent.
Assuming a compensable injury to the back, a basis for an additional award exists. A 40 to 50 percent disability of the right foot, a lesser unrated disability to the left foot, and an unrated disability to the back based on subjective symptoms not supported by medical evidence do not in my opinion support a finding of permanent total disability.
|
Ultraviolet B radiation induces activation of neutral and acidic sphingomyelinases and ceramide generation in cultured normal human keratinocytes.
The sphingomyelin pathway is an ubiquitous, evolutionary conserved signaling system which transduces an extracellular signal into the cell. During the past few years increasing evidence has shown that the sphingolipid ceramide may play a role as a second messenger in intracellular signal transduction. The ceramide generation via sphingomyelinase (SMase) is followed by three major cellular responses: cell growth arrest, induction of cell differentiation and/or induction of programmed cell death or apoptosis. The aim of this study is to investigate whether activation of SMases and generation of ceramide can be induced by UVB radiation in normal human keratinocytes. The present data show that exposure to UVB radiation results in rapid generation of ceramide. The ceramide accumulation starts 15 min after UV exposure and progressively increases up to 24 h. In vitro measurement of SMase activity following exposure to UVB evidences an activation of both neutral and acidic SMases. Moreover, UVB induces apoptosis in normal human keratinocytes as shown by TUNEL technique and FACS analysis. These data indicate that UVB induced ceramide generation and activation of both neutral and acidic SMases, suggesting that sphingolipids metabolism may be involved in the UVB signaling pathway. |
Australian architects Workroom Design collaborated with Agushi Builders to create Oban House, an urban house in South Yarra near Melbourne, Australia. The contemporary style home takes shape as a simple... |
Friday, November 22, 2013
The Soviet Union was the first government to place a satellite into orbit in 1957 which put enormous pressure on President Eisenhower and the United States to create a civilian space program to match Soviet efforts. The US military would have preferred to continue American's manned and unmanned space program themselves. And even America's famous rocket scientist, Wernher von Braun, who helped place America's first satellite into orbit, was reluctant to abandon the Army's space program for the new civilian space program (NASA).
But President Eisenhower was afraid that a US military industrial complex that was already spending astronomical amounts of money on Earth-- would do the same in space. So creating a peaceful civilian space program was Eisenhower's attempt to curtail massive expenditures on space travel by the US military.
But people forget that during John Kennedy's administration, the US was still well behind the Soviet Union in space achievements and in space technology. The Soviets placed the first human being in orbit soon after Kennedy came into office, further demonstrating their growing technological and political superiority over the United States and the non-communist world. Afterwards, the Soviet Union paraded Yuri Gagarin around the world as an international super star, a political ambassador for the wonders of communism!
Kennedy's reason for entertaining the idea of cooperating with the Soviet Union in space was because no one really knew what the manned Soviet goals in space really were. Would the Soviets claim the Moon and eventually the rest of the Solar System as part of the growing Soviet communist empire? And with the US so far behind in space technology, could the US even stop them from doing so!
Kennedy was told that America was so far behind the Soviet Union in space technology that they would have to spend massive amounts of money on space if the US was to avoid Soviet domination of the new frontiers of space. So that's why he did it! That's why Kennedy used the Moon as a way of leaping ahead of Soviet efforts in space-- just a week after the Soviets humiliated the US again by placing the first human into orbit around the Earth.
International communism and Soviet influence at that time was threatening the United States in practically every region of the planet. We were still in the middle of the Cold War-- and it wasn't a joke! Just a year after Kennedy came into office, the Cuban missile crisis between the US and the Soviet Union almost started World War III and a possible thermonuclear war.
However, the race between the US and Soviet Union in space ended during the Johnson administration when both the US and the Soviet Union signed the Outer Space Treaty in 1967. The law forbid any country from placing weapons of mass destruction on the lunar surface or on any other celestial body and also made it illegal for any single country to own the Moon or any other celestial body in space. So the US fear of a militarized communist Moon was finally over and so were the reasons for a space race between the US and the Soviet Union.
But America's commitment and momentum to reach the Moon was still there even though America was consumed in its tragic military commitment in Vietnam. But US astronauts Neil Armstrong and Buzz Aldrin landed on the lunar surface in July of 1969. The two lunar astronauts plus astronaut Michael Collins, who remained in lunar orbit during the lunar excursions, returned safely to the Earth a few days later-- fulfilling Kennedy's goal and cementing his legacy as one of the most insightful and inspirational presidents in the history of our nation.
Monday, November 11, 2013
Tuscany is renowned for its beautiful cities of Florence and Siena, and is historically famous as the birthplace of the Italian Renaissance. But amongst the paleontological community, Tuscany is also known as the birthplace of an extinct Late Miocene ape-- that some paleontologist controversially believe may have been the earliest primate to walk predominantly on just two legs-- and possibly the earliest bipedal ancestor of humankind.
Oreopithecus bambolii first emerged on an ancient island bioprovince known as Tuscany-Sardinia (Tusco-Sardinia) sometime after 9 million years ago where it existed as the sole primate on the island until approximately 7 million years ago. Tuscany-Sardinia was isolated from the rest of the Italian peninsula and from the rest of continental Europe by the marine waters of the Late Miocene Mediterranean.
Skull of Oreopithecus bambolii
Between 11 to 9 million years ago, sea levels fell during a glacial period caused by the deposition of ice and snow on polar land masses. The lower sea levels allowed various mammals from North Africa and Southern Europe to enter the Tuscany region. Small mammals from Europe apparently entered the isolated island region through rafting or swimming. But fauna from North Africa appears to have entered the region via ephemeral land bridges.
The mostly likely ancestor of Oreopithecus was a small African ape know as Mabokopithecus which appears in the fossil record in Africa about 15 million years ago. Oreopithecus and Mabokopithecus were both folivores (planet eaters) that shared a unique dental attribute in their upper molars: a well-defined hypocone-metaconule crest on the upper molars-- a unique characteristic that has never been found in any other monkey or ape living or extinct.
Because its fossil remains are strongly associated with the wetland freshwater environments that existed on Tuscany-Sardinia at that time, Oreopithecus is frequently referred to as the 'swamp ape'. Dryer areas did exist on the island, but Oreopithecus was rare in those deposits while being abundantly represented in the swampy wetland coastal environments on the island.
It is interesting that the African oreopithecine ancestor, Mabokopithecus, existed in an ecological niche largely restricted to riparian woodland areas. This is a niche similar to that of the extant folivorous primate Colobus guereza (the Colobus monkey). Although the Colobus monkey is highly arboreal, it is known to come down from the trees in order to feed on aquatic plants in nearby swamps.
Oreopithecus bambolii has long been at the center of controversy principally because of its remarkable cranio-dental and post cranial similarity to African hominins (human ancestors) which was first fully noted by Johannes Hurzeler as far back as the 1950s. Further adding to the controversy is the argument that Oreopithecus may have also been the earliest obligatory bipedal primate and the first ape to walk exclusively on just two legs.
Oreopithecine remains display a significant number of post cranial characteristics that could be associated with either arboreal or bipedal locomotion. However, there is one post cranial characteristic that is clearly related to bipedality and that's lumbar lordosis, a curvature of the spine that is unique to the hominins (humans and their bipedal ancestors). However, a recent paper by Russo and Shapiro has question the existence of lumbar lordosis in Oreopithecus, arguing that the supposed features may be simply be an artifact of the skeleton's distortion caused by its compression during fossilization. Russo and Shapiro also argue that the arboreal three toed sloth (Bradypus) would be a better convergent model for the skeletal anatomy and locomotive behavior of the extinct oreopithecines rather than the bipedal hominins.
Three-toed sloth (Bradypus)
While slow climbing suspensory behavior has long been argued as an explanation for some of the anatomical attributes of Oreopithecus, such locomotive behavior appears to be contradicted by the exceptionally robust metatarsals of the oreopithecine foot along with the proportions of the foot's entocuneiform which was proximally-distally short and dorso-ventrally high. The length-height index of the oreopithecine entocuneiform related to the mass placed on the hind limbs and is most similar to that of the gorilla. The gorilla's low entocuneiform length-height index is related to the large amount of mass placed on the hind limbs due to its large body size. Oreopithecus, however, was a very small ape, with males typically weighing about 32 kilograms and females approximately half that size. The average Gorilla male weighs 175 kg with average female gorillas weighing about 85 kilograms. So in order to explain, the gorilla-like entocuneiform index, in Oreopithecus, the swamp ape must have been carrying its entire body weight on just its hind limbs. Kohler and Moya- Sola also noted that the power armload arm ratio strongly suggest that Oreopithecus carried its entire body weight on its hind limbs.
There's also strong evidence in the oreopithecine feet of a significantly reduced arboreal ability. The Oreopithecus foot had robust metatarsals plus a non helical ankle joint, characteristics typically associated with terrestrial catarrhines. Kohler and Moya-Sola have also noted that the mobility and grasping ability of the oreopithecine foot had been appreciably reduced relative to arboreal primates and even more so than in terrestrial baboons.
It would also be difficult to understand why Oreopithecus would expend the energy and the risk of being a highly arboreal tree living primate on an island where there were no terrestrial predators. But even on the ground, why would Oreopithecus abandon terrestrial quadrupedalism for bipedalism? The answer may come from its food source.
Chimpanzee wading bipedally with a stick
Orangutan wading bipedally
Gorilla wading bipedally
The coastal wetlands that Oreopithecus preferred were rich in aquatic plants. Harrison and Rook have suggested that Oreopithecus may have specialized in feeding on aquatic plants which were abundant in the wetland coastal areas of the island such as: sedges, water lilies, reeds, cattail, pond- weeds, horestails, and stoneworts which were abundantly represented in the fossil pollen spectrum.
Extant primates that feed on aquatic plants, usually wade bipedally in the shallow waters to access the food resource. Bipedal wading in primates has been observed in: baboons, macaques, the gorilla, orangutan, and in the chimpanzee. Such aquatic bipedalism in order to access aquatic plants comprises as much as 27% of the feeding behavior of the Western Gorilla.
Additionally, freshwater mollusk and turtles were also abundant in the wetland areas of Tuscany-Sardinia island-- along with predatory crocodiles. Turtle and crocodile eggs may have served as a good source of protein for Oreopithecus.
Marine biologist, Alister Hardy, hypothesized that the power precision grip in humans originally evolved as an adaptation for picking up benthic invertebrates. Wading bipedally in shallow water for food resources such as shellfish
and aquatic plants is, of course, common behavior in many
hunter-gatherer human populations. But such behavior has also been observed in non-human primates such as baboons, macaques, guenons, and capuchin monkeys.
Curiously, Oreopithecus also had a hominin-like power precision grip. Such manual dexterity could have evolved in Oreopithecus as a feeding adaptation for picking up the shelled invertebrates that existed in the wetland areas of Tuscany-Sardinia while it was also feeding on aquatic plants.
As the sole primate on the ancient island of Tuscany-Sardinia for nearly two million years, it seems unlikely that Oreopithecus would have avoided the exploitation of freshwater aquatic food resources that were so rich in carbohydrates and proteins.
Sea levels in the Mediterranean began to fall sometime after 7.4 million years ago and the island's isolation from Europe and Africa appears to have ended sometime between 6.9 to 7.2 million years ago. By 6.1 million years ago, global sea levels fell to such an extent that the Mediterranean Sea became completely isolated from in the inflow of marine waters from the Atlantic Ocean. During this period of lowering sea levels, land bridges were created between Europe and North Africa.
The earliest African hominin, Sahelanthropus, appeared in the fossil record in North Africa sometime between 6.8 and 7.2 million years ago. While not much is known about the postcranial remains of Sahelanthropus, its craniodental morphology isremarkably similarto that of Oreopithecus bambolii.
"The knowledge that we have now is but a fraction of the knowledge we must get, whether for peaceful use or for national defense. We must depend on intensive research to acquire the further knowledge we need ... These are truths that every scientist knows. They are truths that the American people need to understand." (Harry S. Truman 1948). |
INTRODUCTION {#s1}
============
Prostate cancer is the most common cancer in men and one of the leading causes of cancer-related death in the United States \[[@R1]\]. Although localized prostate cancer is often cured, patients with distant metastases have a 5-year survival rate of 28.4%. New approaches for the treatment of advanced and metastatic prostate cancer are needed. In addition, although serum prostate-specific antigen (PSA) has been used as a biomarker to screen for prostate cancer, measurement issues lead to over-diagnosis, resulting in overtreatment of prostate lesions that do not necessarily require therapy \[[@R2]\]. Therefore, alternative or additional methods are needed for accurate diagnosis and prognosis of prostate cancer to determine whether medical treatment is needed.
Antibody-based therapeutics have evolved for cancer therapy \[[@R3]\]. In particular, tumor cell-specific antibodies such as anti-CD20 (rituximab) and anti-erbB2/neu (trastuzumab) antibodies have proven to be effective in clinical cancer treatment \[[@R4], [@R5]\]. These targeted therapeutic antibodies not only have inhibitory effects on tumor cells by binding to targeted molecules, but also lead to heightened host immunity against tumors \[[@R6]\]. Recently, we demonstrated that anti-erbB2/neu targeted antibody can promote the development of tumor specific CD8+ T cells in a syngeneic MMTV-neu mouse model \[[@R7]\].
There are several prostate cancer-specific antigens that can serve as targets for antibody therapy \[[@R8]\]. Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer cells and increased expression correlates with advanced disease and metastasis \[[@R9]\]. Accordingly, PSMA is being actively exploited as a possible target for treatment of prostate cancer \[[@R10]\]. Prostate stem-cell antigen (PSCA), a glycosylphosphatidylinositol (GPI)-anchored protein overexpressed in prostate cancer cells \[[@R11]\], has also been exploited as a possible target for immunotherapy. Monoclonal antibodies against PSCA showed significant inhibition of growth of androgen-dependent tumor xenografts \[[@R12]\]. However, targeting PSCA was less effective in clinical trials \[[@R8]\]. In addition, anti-PSCA antibodies are ineffective against androgen-independent tumors, since PSCA is minimally expressed in such cells \[[@R12]\].
We have developed prostate cancer-specific monoclonal antibody (mAb) named F77 by immunizing mice with the androgen-independent prostate cancer cell line PC3 \[[@R13]\]. Immunohistological analysis showed that this antibody stained 112 of 116 primary and 29 of 34 metastatic human prostate cancer specimens with marked specificity for prostate tumor cells \[[@R14]\]. Some staining of breast tumors \[[@R13]\] and cold agglutinin type binding to erythrocyte antigens has been observed \[[@R15]\].
F77 can inhibit the *in vivo* growth of PC3 tumors and other androgen-independent prostate tumors such as DU145 \[[@R14]\]. F77 recognizes protein O-glycan modifications catalyzed by glycosyltransferases such as fucosyltransferase 1 (FUT1) and glutaminyl (*N*-acetyl) transferase (GCNT)1--3 \[[@R15], [@R16]\]. In this study, we sought to identify glycosylated proteins recognized by F77 as well as the possible glycosylation sites on the protein, and assessed the potential use of the F77 glycoprotein antigen as a novel diagnostic biomarker for prostate cancer.
RESULTS {#s2}
=======
CD44 expresses the antigen for mAb F77 {#s2_1}
--------------------------------------
By immunoprecipitation and mass spectrometry, previous studies identified CD13, CD44, Na^+^/K^+^ ATPase, and cytoskeleton proteins as proteins that expressed the antigen for the prostate cancer-specific mAb F77 \[[@R14]\]. The present studies have focused on CD44, a transmembrane glycoprotein that is important for cell signaling and tumor stemness and metastasis \[[@R17], [@R18]\].
In the prostate cancer cell line PC3, an isoform of CD44 with the molecular weight of 85--90 kDa was predominantly precipitated by mAb F77 (Figure [1A](#F1){ref-type="fig"} and [Supplementary Figure 1](#SD1){ref-type="supplementary-material"}). This CD44 species exists in both cytoplasmic and membrane fractions. We employed CRISPR technology to specifically knock-out (KO) CD44. As shown in Figure [1B](#F1){ref-type="fig"} and [1C](#F1){ref-type="fig"}, CD44 KO only modestly reduced F77 staining in PC3 cells (a reduction of 15%) (Figure [1B](#F1){ref-type="fig"}), indicating that CD44 is one of many proteins or glycolipids that are targets of F77-specific glycosylation.
{#F1}
FUT1 and one of GCNT1, GCNT2 or GCNT 3 are essential enzymes for F77-specific O-linked glycosylation in prostate cancer cells \[[@R16]\]. As expected, knockout of FUT1 dramatically decreased F77 binding activity (Figure [1B](#F1){ref-type="fig"}). Interestingly, FUT1 KO also moderately reduced CD44 expression in PC3 cells (35--50%), suggesting that FUT1 modifications are necessary for CD44 expression (Figure [1C](#F1){ref-type="fig"}).
We investigated whether the F77 antigen and CD44 were co-localized in PC3 cells. As shown in a confocal imaging study, F77 staining co-localized with anti-CD44 signals ([Supplementary Figure 2](#SD1){ref-type="supplementary-material"}), and pre-incubation of F77 with PC3 cells induced the formation of clusters in PC3 membranes ([Supplementary Figure 2](#SD1){ref-type="supplementary-material"}, white arrows).
Identification of the F77-epitope region in CD44 {#s2_2}
------------------------------------------------
One of CD44's functions is to bind to hyaluronic acid. Previous studies have shown that blocking hyaluronic acid binding to CD44 induces apoptosis \[[@R19]--[@R21]\]. To investigate if F77 prevents CD44 binding to hyaluronic acid, we performed competitive binding assays using FITC-labeled hyaluronic acid and F77 mAb. In the presence of F77, binding of FITC-labeled hyaluronic acid to CD44 was not significantly diminished ([Supplementary Figure 3A](#SD1){ref-type="supplementary-material"}). In addition, high concentrations of hyaluronic acid did not inhibit PE-labeled F77 binding ([Supplementary Figure 3B, 3C](#SD1){ref-type="supplementary-material"}), indicating that F77 does not block CD44 from binding to hyaluronic acid. We speculated that F77 binding to CD44 occurred at a distinct region from the hyaluronic acid binding site.
CD44 protein has different isoforms ranging from 85 to 230 kDa. We evaluated which isoform and region of glycosylated CD44 mediates F77 binding. Among the 10 CD44 variant exons (v1-v10), exon v1 protein only exists in mouse but not in human, due to a stop codon in the human gene \[[@R17]\]. We annotated the human CD44 exons according to the various isoforms \[[@R22]\], and constructed pIPHA2 vectors carrying different species of CD44 to co-express with FUT1 in 293T cells (Figure [2A](#F2){ref-type="fig"}): full length, fragments with deletion of exons 6--13 or 6--14 (Figure [2A, 2B](#F2){ref-type="fig"}). In the absence of ectopically expressed FUT1, full length CD44 was expressed as a 130-kDa band and could only be weakly precipitated by F77. This can be explained by the fact that 293T cells express minimal amounts of endogenous FUT1 \[[@R16]\]. In contrast, in the presence of FUT1, although 130-kDa CD44 remained the dominant band in the lysate, F77 preferentially immunoprecipitated a high molecular weight form of CD44 (∼250-kDa). For the CD44 construct with exons 6--13 deleted, F77 was able to precipitate the dominant 75-kDa band from the lysate, as well as the highly glycosylated form of ∼130 kDa. The sizes of both bands were smaller than the corresponding bands from the full length construct. The CD44 construct with the deletion of exons 6--14 expressed a dominant 65-kDa band in the lysate, but F77 only very weakly immunoprecipitated this band and failed to precipitate a highly glycosylated band (Figure [2B](#F2){ref-type="fig"}). These data indicate that exon 14 of CD44 is important for F77-specific glycosylation.
{#F2}
We further studied potential glycosylation sites in exon 14. Using the pIPHA2-CD44exon6-13del construct, we mutated every single potential glycosylation site in exon 14: Serine (S) and Threonine (T) were mutated to Alanine (A), and Tyrosine (Y) was mutated to Phenylalanine (F) (Figure [2C](#F2){ref-type="fig"}). Mutation of most of these sites, except those on constructs M2, M4, M9, and M11, resulted in decreased F77 immunoprecipitation of CD44 (Figure [2D](#F2){ref-type="fig"}), indicating that the F77-specific glycosyltransferase modification occurs on various sites in exon 14. Our studies revealed that a short sequence of about 21 amino acids from T561 toT581 was critical for glycosylation. When amino acid residues T561, S562, T577, S578 and T581 were simultaneously mutated to alanine, the density of the F77-precipitated CD44 band was diminished by ∼ 80% ([Supplementary Figure 4](#SD1){ref-type="supplementary-material"}).
It was interesting to notice that several mutations, especially the M2 variant of CD44, appeared to lead to increased F77 signals (Figure [2D](#F2){ref-type="fig"}). In the study of a herpes simplex virus type 1 (HSV-1) glycoprotein, replacing basic residues around the O-linked glycosylation site also increased glycosylation \[[@R23]\]. *In vitro* assay confirmed that these mutations increased glycosylation efficiency. In our study, the replaced amino acid residues were Ser, not basic residues, but an analysis of the O-glycosylation sites revealed that the Ala residue is preferred around O-glycosylated Ser/Thr, especially in the -10, -4, -2, -1, and +2 positions \[[@R24]\]. Clearly, Ser at these positions were unlikely glycosylation sites, but they might be close to the real ones.
Knockout of CD44 or FUT1 in PC3 limits F77-induced apoptosis {#s2_3}
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Previously, we found that F77 could induce modest apoptosis in PC3 cells \[[@R14]\]. In a study using glycolipid microarrays, F77 appeared to have higher affinity for its antigen at low temperatures (4°C), which is in accord with the cold agglutinin behavior of this antibody \[[@R15]\]. We evaluated the ability of F77 to induce apoptosis at different temperatures. As expected, we noticed that F77 induced much more dramatic apoptosis at 4°C than at 37°C, with greater than 80% of cell death in PC3 cells (Q2 and Q3, Figure [3](#F3){ref-type="fig"}). This elevated apoptosis at the low temperature was also observed in the tumorigenic and F77-positive prostate epithelial cell line RWPE-2, which was derived from RWPE-1 human epithelial cells transformed by the *K-ras* oncogene. We did not detect apoptosis in the parental F77-negative RWPE-1 cell line, indicating the effect was F77-binding dependent ([Supplementary Figure 5](#SD1){ref-type="supplementary-material"}).
{#F3}
We generated CD44 or FUT1 CRISPR knockout PC3 cells (Figure [1B](#F1){ref-type="fig"}), and then tested F77- induced apoptosis in these lines. As shown in Figure [3](#F3){ref-type="fig"}, elimination of CD44 or FUT1 greatly limited F77 mAb-induced apoptosis at low temperature. While 45% apoptosis was observed in the control cell line PC3_CR, only about 15% cell death was detected in FUT1 or CD44 KO cell lines. CD44 has been shown to promote resistance to apoptosis in some cancer cells \[[@R25]\]. These data further confirm that F77-induced apoptosis is highly dependent on glycosylation mediated by FUT1 and that CD44 is a main carrier protein for F77-specific glycosylation.
We further examined whether the monovalent F77 Fab fragment could also induce cell death *in vitro*. The Fab species was prepared by pepsin digestion followed by sieve preparative chromatography. It was observed that the monovalent Fab was only able to induce very limited cell death. However, in the presence of a goat anti-mouse Fab antibody, which crosslinks Fab into a bivalent binding unit, significant cell death was observed again (Figure [4](#F4){ref-type="fig"}). These data suggest that bivalency of F77 mAb is required for induction of apoptosis in the tumor cells.
{#F4}
A novel ELISA to detect F77-glycosylated CD44 in prostate cancer cell culture media and sera from prostate cancer patients {#s2_4}
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Since CD44 is a target of F77-related glycosylation, we next investigated whether this glycosylated form of CD44 could be detected as a secreted protein in the culture media of prostate cancer cells, or in sera from men with prostate cancer. A sandwich ELISA was designed in which F77 was used as the capture antibody and an anti-CD44 antibody (clone IM-7) was used as the detection antibody. Using this assay, F77- glycosylated CD44 was detected in the media from 3 prostate cell lines (PC3, PC3-MM2 and DU145) (Figure [5A](#F5){ref-type="fig"}). Culture medium from the normal prostate cell line RWPE-1 was negative, but culture medium from the *K-ras* transformed clone RWPE-2 was positive. The result is consistent with the mAb F77 staining on these cells by FACS \[[@R14]\]. Of note, medium in which the breast cancer cell line TB129 was cultured was also positive in the ELISA. This is also consistent with the observation of some levels of F77 staining in certain breast cancer cell lines and tissues \[[@R26]\]. As expected, the ELISA signal was reduced in the FUT1 KO PC3 line and not detected in the CD44 KO line and 293T cells.
{#F5}
F77-glycosylated CD44 was also detected in pre-prostatectomy sera from 22 men with Gleason score 7 prostate cancer (Figure [5B](#F5){ref-type="fig"}). Of the 22 sera, 18 had higher levels of F77-glycosylated CD44 compared to normal healthy controls. Ten of 22 men had biochemical (PSA) recurrence following radical prostatectomy. Levels of F77-glycosylated CD44 were not significantly different in the sera of the 10 men who failed to be cured by radical prostatectomy compared to the 12 men who were cured by surgery (Figure [5C](#F5){ref-type="fig"}). However, a correlation of F77-glycosylated CD44 values and time to biochemical recurrence in the sera of the 10 men who failed to be cured was noted (Figure [5D](#F5){ref-type="fig"}).
DISCUSSION {#s3}
==========
Previous studies from our lab have shown that the prostate cancer-specific mAb F77 is potentially useful for therapy of prostate cancer \[[@R13], [@R14]\]. Based on our previous reports, F77 recognizes glycolipids as well as fucosylated O-glycan proteins \[[@R14]--[@R16]\]. In the present study, we found that FUT1 expression is essential for the presentation of F77 antigens. We also confirmed CD44 as a target glycosylated protein for F77 in prostate cancer cells (Figure [1](#F1){ref-type="fig"}), and we further mapped the critical F77 binding region to exon 14 (Figure [2](#F2){ref-type="fig"}). Through a point mutation strategy, we are able to narrow glycosylation patterns relevant to F77 binding to a 21- amino acid region within exon 14, which contains potential glycosylation sites for FUT1- mediated modification (Figures [2](#F2){ref-type="fig"} and [Supplementary Figure 3](#SD1){ref-type="supplementary-material"}).
CD44 is a single pass trans-membrane glycoprotein involved in cell-cell and cell-matrix communication. It is also known that CD44 is highly expressed in cancer stem cells, which are highly resistant to apoptosis and are thought to be essential for metastasis \[[@R18]\]. CD44 consists of twenty exons and has different isoforms with molecular weights ranging from 85 to 230 kDa due to alternative splicing, post-translational modification and partial cleavage \[[@R27]\]. CD44 standard (CD44s) is the shortest form of CD44 and is present in most vertebrate cells. In contrast, the splice variant isoform of CD44 (CD44v) is only expressed in some epithelial cells during embryonic development, in lymphocytes during maturation and activation, and in several types of carcinoma \[[@R28], [@R29]\]. Some CD44v species in tumors are associated with *K-*ras-mediated transformation \[[@R29]\] and represent attractive targets for immunotherapy. Several therapeutic strategies targeting CD44 have led to clinical trials \[[@R18]\], but none seems to lead to successful therapies at this time point \[[@R30], [@R31]\]. As shown in our previous study \[[@R14]\], F77 is highly specific to prostate cancer, and the current study indicates that F77 recognizes a special glycosylated form of CD44, CD44v10 at the exon 14 region (Figure [2](#F2){ref-type="fig"}).
Our study also showed that bivalent F77 can induce apoptosis of prostate cancer cells, especially at 4°C (Figure [3](#F3){ref-type="fig"}). The increased activity at low temperature correlates with the higher binding affinity of F77 to its antigen at 4°C \[[@R15]\]. Meanwhile, CD44 deletion almost completely abrogated F77 --induced apoptosis (Figure [3](#F3){ref-type="fig"}). An anti-CD44 mAb A3D8, which binds to CD44 at the non- hyaluronic acid binding region, has also been observed to induce apoptosis in acute myeloid leukemia cells by inducing lipid raft clustering \[[@R32]\].
We detected F77-glycosylated CD44 in the sera of men diagnosed with primary prostate cancer. All of these men had Gleason score 7 cancer and were treated by radical prostatectomy. While levels of F77-glycosylated CD44 were higher in the majority of men with cancer compared to the value of serum from healthy controls, our study only involved a small number of clinical samples. Similarly, levels of F77-glycosylated CD44 did not predict progression (biochemical recurrence) following surgery in this small study, although there was a correlation between higher levels and longer time to failure. Since our data suggest that CD44v10 may be most relevant to F77 specific glycosylation in prostate cancer, future studies using an antibody specific to this form of CD44 may yield more relevant data than our current ELISA.
In conclusion, we have discovered CD44 as a major protein carrying the F77 epitope at the exon 14 region. F77 can induce apoptosis on prostate tumor cell lines in a bivalent- and CD44-dependent manner. Our study also suggests that serum levels of F77-glycosylated CD44 may have potential utility to facilitate the diagnosis or prognosis of prostate cancer. Since we have identified the specific region that harbors the glycosylation sites, a much more specific diagnostic assay can be developed to monitor the special glycosylated form of CD44 during prostate cancer progression.
MATERIALS AND METHODS {#s4}
=====================
Cells and antibodies {#s4_1}
--------------------
Prostate tumor cell lines were cultured as described previously \[[@R14]\]. The F77 antibody was purified in our laboratory \[[@R13]\]. All cell lines were routinely checked for mycoplasma. PC3 cell lines were confirmed virus free by the IMPACT II test (IDEXX Bioresearch). Anti-CD44 (IM-7), biotinylated anti-CD44 (IM-7) and anti-mouse IgG were purchased from Biolegend. Anti-mouse IgG FITC was purchased from Jackson ImmunoResearch. Anti-rat AF594 was purchased from Invitrogen.
Flow cytometry assays {#s4_2}
---------------------
Cells were incubated with the anti-CD44 PE or the F77 antibody for 20 min on ice. Cells were then washed with FACS buffer (0.5% BSA/PBS), then incubated with anti-mouse IgG AF488 for 20 min at 4°C to detect F77. After washing and collection, the cells were examined on an Accuri C6 flow cytometry (BD Bioiscience) and analyzed using Flowjo software.
Plasmids and CRISPR-Cas9 gene knock out {#s4_3}
---------------------------------------
All CRISPR-CAS9 constructs were purchased from Genecopoeia. CD44 expression vector was obtained from GE Life Sciences, then subcloned into pIPHA2 vector as described previously \[[@R33]\]. FUT1 expression vector was provided by Dr. Minoru Fukuda \[[@R16]\]. Knockout of targeted genes in PC3 cells was performed according to manufacture's instructions. In brief, PC3 cells were seeded in 6-well plates at 3 × 10^5^ cells/well the night before transfection. Cells were then transfected with 2 μg/well of the all-in-one CRISPR-CAS9 vectors using Fugene6 (Roche) according to manufacturer's instructions. After 24 hr, media were changed and cells were incubated with 600 μg/ml of G418 for 24 hr, then media were changed to normal growth media and cells were cultured until stable growth was observed. The cells were then sorted and collected as CD44-negative or F77-low (for FUT1) fractions and termed as knocked down cells using FACS Aria (BD Bioscience). For the FUT1 deletion, genomic PCR was used as a further validation of FUT1 gene deletion and confirmed as the genomic deletions of targeted sequences. For the exon deletion and point mutations of CD44, the vectors were constructed using the QuikChange Site- Directed Mutagenesis Kit (Stratagene) and verified by sequencing. The primers used in this work are listed in [Supplementary Table 1](#SD1){ref-type="supplementary-material"}.
Immunoprecipitation and western blotting {#s4_4}
----------------------------------------
PC3 cells were lysed with low salt lysis buffer \[20 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% TritonX-100, and complete mini protease inhibitor cocktail (Roche)\] and subjected to immunoprecipitation. Five micro gram of F77 antibody were bound to Dynabead protein G magnetic beads (Invitrogen) according to manufacturer's instructions, then incubated with the lysate for 2 hr. For membrane proteins, we first incubated mAb F77 (5 μg) with PC3 cells, then lysed cells with low salt lysis buffer and precipitated with protein G magnetic beads. The precipitates were then washed three times with the buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.01% Tween-20) and boiled for 5 minutes in SDS loading buffer. Samples were analyzed by SDS-PAGE, transferred to Immobilon-P (Millipore) PVDF membranes, and probed using anti-CD44 antibody, and detected with anti-rat IgG HRP (Santa Cruz Biotechnology) and Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Millipore). For glycosylation site analysis, 293T cells were transfected with FUT1 and CD44 mutant plasmids using Fugene6. The cells were then lysed with low salt lysis buffer and subjected to SDS PAGE and western blotting as described above. HA-tagged proteins were detected with anti-HA Peroxidase (3F10; Roche) using ECL.
Apoptosis assay {#s4_5}
---------------
Cells were detached and incubated with either 10 μg/mL mAb F77 or the control antibody mouse IgG3 for 30 min at 4°C or 37°C. After washing with FACS buffer, cells were stained with 5 μL FITC-Annexin V or 7-AAD (Biolegend) according to manufacturer's instructions. Positive controls were established by heat-shocking PC3 cells at 56°C for 20 min before staining with FITC-Annexin V or 7-AAD. Samples were analyzed by BD Accuri C6 flow cytometry (BD Biosciences) and Flowjo software.
ELISA assay {#s4_6}
-----------
High binding 96-well ELISA plates (ISC T3021--4) were coated with 100 mL mAb F77 for a final concentration of 5 ug/mL, and incubated overnight at 4°C. The next day, the plate was washed three times with 200 mL PBS-T (PBS containing 0.05% Tween-20), and incubated with 300 mL blocking buffer (1x Casein in PBS, SurModics, BioFX Phosphate Buffered Saline/Casein Block and Diluent) for 1 hr at room temperature. The plate was washed again, and 50 mL of cell culture medium or serum were added for 1 hr incubation at RT. After washing, biotin anti-CD44 was diluted 1:1000 (stock is 0.5 mg/mL) in PBS, and 100 mL were added to the wells and incubated 1 hr at RT. After washing, SA-HRP (R&D) was diluted 1:200, and 100 mL were added to the wells for incubation at RT for 1 hr. The substrate solution was prepared by dissolving 1 capsule of Phosphate-Citrate Buffer with Sodium Perborate Capsules (Sigma), in 50 ml of water to yield 2X solution. Five mL of the 2X solution was mixed with 5 mL of water to make phosphate-citrate buffer (0.05M, pH 5.0). One tablet of 3,3, 5,5′-Tetramethylbenzidine dihydrochloride (TMB) (Sigma) was dissolved in the dark in the phosphate-citrate solution. The plate was washed six times, and 100 mL developer substrate solution was added to each well and the plate was incubated at RT in the dark for 30 min. Finally, 50 mL of 2 M H~2~SO~4~ was added to stop the reaction. The plate was read at 450 nm with the reference wave length at 620 nm. A batch of PC3 cell culture medium, from confluent cells, was collected and used to define the standard F77-CD44 unit for future study. The reading of undiluted medium for this batch of PC3 cells was defined as 100 units. This standard medium batch was aliquoted and stored at --80°C.
Confocal microscopy {#s4_7}
-------------------
PC3 cells (50,000/well) were seeded into 8 well Lab-Tek chamber slides (Corning) and cultured for 2 days. The cells were washed once with FACS buffer, and then treated with a gradient of 0, 1, and 5 μg/ml of F77 for 30 min at 4°C. After washing 3 times with the FACS buffer, cells were fixed with 4% paraformaldehyde/PBS for 10 min at 4°C. After washing 3 times, cells were incubated with the blocking buffer (3% normal goat serum/PBS) for 1 hr at RT, then incubated with anti-CD44 antibody (1:250) for 2 hr at RT. Cells were then washed three times with the FACS buffer and then incubated with anti-mouse-FITC IgG (1:200 dilution) and Alexa Fluor 594 anti-rat IgG (1:400 dilution). After washing, the chamber was removed and coverslips were loaded using VECTASHIELD mounting medium (Vector Laboratory). The stained cells were examined with a Leica STED 3X Super-Resolution Microscope.
Serum samples {#s4_8}
-------------
Normal human sera (NHS) was a pooled sample from healthy donors \[[@R34]\]. Sera from 22 men who had undergone radical prostatectomy at Stanford University between 1993 and 2003 to treat prostate cancer were used to measure F77-glycosylated CD44. All patients were consented with Institutional Review Board-approved protocols. Pre-prostatectomy blood was drawn in tubes with silica clot activator. Following centrifugation, the sera were collected, aliquoted, and stored at --80°C until analyzed. Patient characteristics and histopathological/morphological variables were obtained from an existing database.
Biochemical failure, also called PSA failure, is used here as a longitudinal outcome measure. Biochemical failure is defined as two consecutive PSA values above a cutoff point of 0.07 ng/ml (for PSA measured by the Tosoh method) or 0.2 ng/ml (for measurements by less sensitive methods). When a subject experienced PSA failure, time to failure was calculated as the number of days between the date of surgery and the first of the two consecutive PSA values that exceeded the cutoff point.
SUPPLEMENTARY MATERIALS FIGURES AND TABLE {#s5}
=========================================
**Author contributions**
X.C. performed the majority of experiments, interpreted the results and wrote the manuscript; Y.N. interpreted the results, wrote the manuscript and performed *in vivo* and *in vitro* experiments; Z.Z. and R.H. participated in some experiments. D.M.P. provided the serum samples and participated in data analysis and manuscript preparation. M.I.G designed experiments, interpreted the results and wrote the manuscript; H.Z. designed experiments, interpreted the results and wrote the manuscript.
Flow cytometry was performed at the Abramson Cancer Center Flow Cytometry and Cell Sorting Shared Resource, a component of Path BioResource, in the Perelman School of Medicine of the University of Pennsylvania, which was established in part by equipment grants from the NIH Shared Instrument Program, and receives support from NIH P30 CA016520 from the National Cancer Institute. Confocal microscopy was performed at the CDB microscopy core at the University of Pennsylvania.
**CONFLICTS OF INTEREST**
The authors declare that they have no competing interests.
**FUNDING**
Work in this study was supported by the NIH grant (U01CA168925) and the Abramson Sigel fund.
[^1]: These authors contributed equally to this work
|
UnBooks:Ill Met By Moonlight
From Uncyclopedia, the content-free encyclopedia
Ill Met By Moonlight (not to be confused with "I'll Met By Moonlight", the story of a tense-challenged, dyslexic, nocturnal gambler) tells the story of two lovers, Bernie Shovel and Doris Felch, who meet under cover of darkness in war torn Crete, braving minefields, searchlights, gullies and German food so that they might snatch a few stolen moments together. Why they do this remains unclear, since they actually live just around the corner from each other in Manchester, England. The book was written by Major Col. Brigadier Stungunner as a reminder, he claims, for young people to cut their hair and get proper jobs.
Contents
Bernie Shovel is exhausted after four months of clandestine hiking. He has climbed up the Cretan mountains and was nearly shot by a passing German patrol. He has dodged minefields unsuccessfully and lost a foot. Hopping over a ridge, he spots the ocean beneath and realises he has made it. And there she is, Doris Felch, with the moon glinting off her false teeth like the rear lights on a Morris Minor. In an urgent whisper, he says:
"Doris, it is me, Bernie"
"Bernie, oh my Bernie, you made it! Yet, we have only a minute for I must go!"
"Only a minute. A minute of stolen pleasure, and then we must part. It's this war. This damned awful war. This damned useless bastard war. This damned stupid bloody bloody damned..."
"Oh, no Bernie, it's not the war, it's just that I think I left the gas on. I was pretty sure I'd turned it off when I left, but now I can't be so sure. I've got to go and check. How about we meet here again later, say 1943?"
"Oh... ok, then, See you later."
Chapter 2: 1943
Bernie Shovel is exhausted after four months of clandestine hiking. He has climbed up the Cretan mountains and had his ears shot off by a passing German patrol. He has dodged minefields unsuccessfully and lost another foot. Crawling over a ridge, he spots the ocean beneath and realises he has made it. And there she is, Doris Felch, her hunchback silhouetted against the sky like an odd shaped mountain. In an urgent whisper, he says:
"Doris, it is me, Bernie"
"Bernie, oh my Bernie, you made it! Yet, we have only a minute for I must go!"
"Eh?"
"Bernie, oh my Bernie, you made it! Yet, we have only a minute for I must go!"
"Sorry Doris, you'll have to speak up a bit! I've had both my ears shot off by a German patrol!"
"But Bernie, how will you wear your glasses?
"My glasses. I know. Never again will they fit me. It's this war. This damned awful war. This damned useless bastard war. This damned stupid bloody bloody damned..."
"Don't worry, you can always tape them to your head. Anyway, time's up. How about we meet here again later, say 1944?"
"Oh... ok, then, See you later."
Chapter 3: 1944
Bernie Shovel is exhausted after four months of clandestine hiking. He has climbed up the Cretan mountains and had his glasses shot off by a passing German patrol. He has dodged minefields unsuccessfully and lost a leg. Dragging himself over a ridge, he spots the ocean beneath and realises he has made it. And there she is, Doris Felch, with her hearing aid gently humming like a large injured mammal. In an urgent whisper, he says:
"Doris, it is me, Bernie"
"Bernie! ... Where are you?"
"Down here."
"Oh, there you are. Bernie, oh my Bernie, you made it! Yet, we have only a minute for I must go!"
"Oh for Christ's sake! Why can't we just meet in the chippy like everyone else?"
"Oh, no Bernie, it's not the war, it's just that I think I left the front door unlocked. I was pretty sure I'd locked it when I left, but now I can't be so sure. There's been a spate of burglaries in my area recently, and I just bought a new toaster. How about we meet here again later, say 1945?"
"Oh... ok, then, See you later."
Chapter 4: 1945
Bernie Shovel is exhausted after four months of clandestine hiking. He has climbed up the Cretan mountains and had his head shot off by a German patrol. He has dodged minefields unsuccessfully and lost his last leg. Dragging himself blindly over a ridge, he realises he has made it. And there she is, Doris Felch, gently cradling her colostomy bag like a newborn infant. In an urgent whisper, he says:
"Doris it's me, Ber"
before he is obliterated by the last Allied bomb to fall on Crete.
Such is the tragedy of war. This damned awful war. This damned useless bastard war. This damned stupid bloody bloody damned... |
ITURI PROVINCE, Democratic Republic of the Congo – Several weeks ago, a woman brought her 11-year-old daughter, Anne*, to a UNFPA-supported health facility. Their family had been uprooted by the conflict in Ituri Province and, in the chaos, Anne was sexually assaulted.
A nurse welcomed the girl, who was too terrified to speak. With a lot of time and patience, clinic staff helped Anne feel comfortable enough to tell her story and consent to an examination. A social worker began to help her through the healing process.
Tragically, Anne’s experience is far from unique. Thousands of women and girls in Ituri Province have been subjected to gender-based violence, as have women and girls in conflict-affected North Kivu and South Kivu.
“Since the beginning of the year we have seen an escalation of various forms of violence against women and girls in conflict-affected areas of Ituri, North Kivu and South Kivu,” said Sennen Hounton, UNFPA’s representative in the Democratic Republic of the Congo.
Extreme deprivation has increased the risk of exploitation, as well. “Women and girls are not only exposed to sexual violence during conflict or displacement,” he noted. “Given a lack of opportunities, displaced women and girls are turning to survival sex to meet basic needs such as health care and food.”
A case worker takes information from young survivors of violence. © UNFPA DRC
In the first half of 2019, some 17,000 survivors of gender-based violence received support from humanitarian actors in the country, including UNFPA, with major gaps in service observed in key hotspots.
Preventing sexual exploitation and abuse
The United Nations Office of the Coordination for Humanitarian Affairs (OCHA) estimates that hundreds of thousands of people have been displaced by the crises in Ituri, North Kivu and South Kivu. The ongoing Ebola outbreak has only exacerbated the vulnerabilities of women and girls.
UNFPA is working with partners to extend access to vital reproductive health services, including care for survivors of gender-based violence, and to protect women and girls from sexual exploitation and abuse.
Anne was able to receive medical and psychosocial care through services funded by the UN Central Emergency Response Fund (CERF) and implemented by UNFPA. Between the end of April and the end of July, UNFPA was able to provide reproductive health care to 70,000 people, and provide critical information and outreach to 600,000 more. These funds will also be able to support an estimated 19,000 safe deliveries and extend access to modern contraceptives to 17,000 women in six crisis-affected provinces of the country.
But the needs continue to outstrip available resources. In North Kivu, for instance, fewer than 30 per cent of health zones affected by Ebola are covered by services to prevent and respond to gender-based violence, even though such violence tends to increase in humanitarian crises.
“We estimate that the humanitarian community needs $91.7 million US dollars, including $44 million for North Kivu, South Kivu and Ituri, to protect women and girls from gender-based violence risks and care for at least 37,500 survivors of violence in 2019,” said Noemi Dalmonte, an expert with UNFPA. “But we are far from reaching this target.”
Urging action
Today, Anne and her mother are living in a camp for displaced persons in Bunia. It is not safe for them to return home, but they have found safety in a temporary shelter. There, Anne has access to a playground and friends.
UNFPA is urging more support for the women and girls of the country.
“I hereby request the donor community to activate commitments that were made at the recent conference in Oslo on Ending Sexual and Gender Based Violence in Emergencies, in the DRC,” Mr. Hounton said. “Responders to gender-based violence need renewed support to ensure that life-saving and survivor-centred care is available for women and girls at the very start of a crisis.”
He added, “We need to prioritize the current hotspots to save lives of Congolese women now.”
The overall humanitarian action plan in the Democratic Republic of the Congo is funded at only 30 per cent.
*Name changed for protection and privacy
|
{% extends base_template %}
{% load i18n xadmin_tags %}
{% load crispy_forms_tags %}
{% block body %}
<div class="container">
<div class="panel panel-default panel-single" style="max-width: 500px;">
{% if validlink %}
<div class="panel-heading">
<h3 class="form-signin-heading">{% trans 'Enter new password' %}</h3>
</div>
<form action="" method="post" id="password-reset-confirm" class="form-horizontal">
<div class="panel-body">
{% csrf_token %}
<p class="text-info">{% trans "Please enter your new password twice so we can verify you typed it in correctly." %}</p>
{% crispy form %}
<button class="btn btn-lg btn-primary btn-block" type="submit">{% trans 'Change my password' %}</button>
</div>
</form>
{% else %}
<div class="panel-heading">
<h2 class="form-signin-heading">{% trans 'Password reset unsuccessful' %}</h2>
</div>
<div class="panel-body">
<p class="text-danger">{% trans "The password reset link was invalid, possibly because it has already been used. Please request a new password reset." %}</p>
</div>
{% endif %}
</div>
</div> <!-- /container -->
{% endblock %} |
Related Search
Schroders Quickview: US economy motors into 20151
Another month and another solid employment report, with the US economy clocking up an increase of 252k in non-farm payrolls in December.
09 Jan 2015
Keith Wade
Chief Economist & Strategist
Contributes toUnstructured Learning Time
Figures for October and November were revised up and for the year as a whole the US added 2.95 million jobs.
Given the added stimulus which will come from the latest fall in oil prices and mortgage rates, we are sticking with our view that they start to move in June and begin the process of normalising interest rates.
The gains in payrolls were slightly ahead of expectations as was the drop in the unemployment rate to 5.6%. Some will dismiss the latter as it partly reflects a fall in the participation rate to 62.7%, levels not seen since the late 1970s. However, as regular readers of our reports will know, we see this as a structural trend, which has been in place since 2008 and reflects the ageing of the US workforce, rather than a sign of weakness as workers become discouraged and give up searching. We are unlikely to see a change in this trend in the near future, with the result that the unemployment rate is likely to fall further in the coming months and end the year below 5%.
The crumb of comfort for the doves in this month’s report was the weakness of wage growth, with average hourly earnings actually falling by 0.2% and decelerating to 1.7% year-on-year. Tepid wage growth remains a puzzle as a tighter labour market should be causing pay to accelerate. Recent research from the San Francisco Federal Reserve suggests that there has been a delayed reaction in pay growth as firms were unable to adjust wages as much as they would have wished to during the downturn due to nominal wage rigidity. Nonetheless, one would expect this period to eventually come to an end: if growth remains solid and the unemployment rate continues to fall, wages will pick up. And growth is solid: total hours worked in the economy, often seen as an indicator of real GDP, rose at an annualised 3.5% in the fourth quarter.
The Federal Reserve will continue to balance low inflation and a weak external environment against the strength of the domestic economy. However, given the added stimulus which will come from the latest fall in oil prices and mortgage rates (now back to May 2013 levels, pre-taper tantrum) we are sticking with our view that they start to move in June and begin the process of normalising interest rates.
Important Information
This website is for UK professional financial advisers only. Retail clients should go to the Investor Centre You should not rely on the views and information on the site when making investment decisions.
Views and Opinions are Schroders' only and may change.
Schroders uses all reasonable skill and care to ensure information is accurate. However, errors or omissions may occur that are outside of our control, such as unauthorised access to this internet service, or the effects of machine, software or operator error or malfunction in connection with data transmission. Information is accurate only on the date shown on the page it appears and we advise that you contact us before you rely on any information to confirm its accuracy.
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Chapter 127: The Girl Worth An S-Class License The reason he chose Devil Desert was that there were not many creatures that could fly, hence it was relatively safe for Han Sen who could. Few people would go there, so there was no shortage of mutant creatures, if not sacred-blood creatures.
Han Sen studied the conditions of the desert and was ready to ask Qin Xuan for a leave. Qin Xuan smiled and said, "Perfect. Our squad needs to protect someone in hunting and I was just thinking where we should take her. You could lead the team to Devil Desert then. It is your first task in the squad, make sure you do a good job."
"What is the reward for this task?" Han Sen blinked and asked, he did not want to do a job that would not pay well.
"If you can make the girl satisfied and designate you as her protector in Steel Armor Shelter, you could get an S-Class license of Saint Hall." Qin Xuan looked at Han Sen and laughed. "Any interest?"
"Yes, I am very interested," Han Sen quickly said. Babysitting alone would pay an S-Class license. Such a good thing could only happen in a dream.
"Then do well. You will get nothing if she is not satisfied with you." Qin Xuan handed him the portfolio.
After reading the materials, Han Sen felt he did not learn anything, as these people's personal information was all confidential. All he knew was the name, age and gender.
"Wang Mengmeng, female, 16 years and 47 days old. Stationmaster, you are not asking me to take a girl who has just entered God’s Sanctuary to a place like Devil Desert. I can’t guarantee her safety." Han Sen looked sullen.
"Of course not. This is your first task, so I will ask Gambler to follow you. You could also pick some people from Steel Armor Shelter." Qin Xuan smiled. "About Wang Mengmeng, I can tell you one more thing. She is your schoolmate."
"My schoolmate? Blackhawk? You must be kidding me. She just entered God’s Sanctuary and with that kind of fitness, she could not even have reached the standard for any special enrolment program." Han Sen did not believe her.
"Some people do not need to pass the exam. We all have different starting points, like you are to protect her, and she is to be protected." Qin Xuan smiled. "Remember to keep a good relationship with her."
"I'm not interested in little girls," Han Sen shrugged and said.
Qin Xuan rolled her eyes at him and said, "Go pick some team members in Steel Armor Gang. You can choose anyone but the team leaders. There should be less than ten members and the fees will be covered by the squad."
Han Sen asked Su Xiaoqiao and some others who had a mutant mount. He did not want to waste his time on walking and would not consider those without mounts.
As for Gambler, Han Sen was confident in him. He was an old member in the squad and had amazing skills, like Sleeveblade.
When Han Sen saw Wang Mengmeng, he finally knew what privileges meant. A girl just over 16 who had just entered God’s Sanctuary for one month had mutant shapeshifting beast soul, mutant armor, mutant weapon and one sacred-blood beast soul mount, making Han Sen and the rest rather jealous.
Although Lin Beifeng was rich, he was only an upstart when compared with Wang Mengmeng. The beast souls she had probably could not be bought with money.
Fortunately, Wang Mengmeng was not an annoying princess. She was delightful and lovely.
She also fought well. Although she had only been in God’s Sanctuary for a month, it looked like that she had gained quite a few geno points already. She must have practiced multiple advanced hyper geno arts since childhood.
Using her advanced beast souls, she could probably beat Su Xiaoqiao in a combat.
The team had been traveling all the time and Wang Mengmeng never complained.
"Brother Han, I heard from Sist
er Qin that you are also in Blackhawk. I’m in Department of Warframe, which department are you in?" Wang Mengmeng called Han Sen brother in a natural and sweet tone.
In fact, kids from prominent families like Wang Mengmeng, Qing and Yuan were all quite well mannered and easy going.
"Ahem, you can call me Sen. I was enrolled this year as well. We are both freshmen and I am an archery major." Han Sen enjoyed talking to an adorable girl like her, which make the journey less boring.
"Archery Department is the department our school is trying to enhance this year and I’m sure the change will happen fast. You must be specially enrolled?" Wang Mengmeng started to chat with him.
"Oh right, Brother Han, you came to Steel Armor Shelter early, so you must have seen Dollar himself?" Wang Mengmeng asked hopefully.
"Yes, we have all seen him at the contest. Why do you ask about him?" Han Sen looked at Wang Mengmeng, surprised.
"I am a fan of Dollar’s, but unfortunately he never showed up since I came here, so I never got to see him," Wang Mengmeng said with some disappointment. |
Q:
How to gzip static content in ASP.NET Core in a self host environment
Is there are way to serve gzip static cotent when using self host environment to publish an ASP.NET Core website?
A:
[Edit 2016-11-13]
There is another way to serve gzipped files that replaces steps 2 and 3. It's basically quite the same idea, but there is a nuget package that does it all for you readily available. It basically checks if the is .gz or .br file that matches the requested one. If it exists it returns it with the appropriate headers. It does verify that the request has a header for the corresponding algorithm. Github code if you want to compile it yourself is here.
There is also an issue to support that in the official repository, so I really hope Microsoft will have the standard plugin to do that, since it's rather common and logical to use that nowadays.
I think I have found the most optimized way of serving the compressed content. The main idea is to pre-compress the files and since the default ASP.NET 5 way is to use gulp to build js, it is as easy to do as this:
1. Add a gulp step to gzip the bundled libraries:
gulp.task("buildApplication:js", function () {
return gulp.src(...)
...
.pipe(gzip())
...
});
This will produce something like libraries.js.gz in your bundles folder
2. Refernce the libraries.js.gz instead of libraries.js in the cshtml file
3. Amend the static file handler to fix the returned headers
We need to add Content-Encoding and change the Content-Type from default application/x-gzip to application/javascript because not all browsers are smart enough to read js properly from x-gzip
app.UseStaticFiles(new StaticFileOptions
{
OnPrepareResponse = context =>
{
if (headers.ContentType.MediaType == "application/x-gzip")
{
if (context.File.Name.EndsWith("js.gz"))
{
headers.ContentType = new MediaTypeHeaderValue("application/javascript");
}
else if (context.File.Name.EndsWith("css.gz"))
{
headers.ContentType = new MediaTypeHeaderValue("text/css");
}
context.Context.Response.Headers.Add("Content-Encoding", "gzip");
}
}
});
Now all there is no CPU cycles to waste to gzip the same content all the time and it's the best possible performance in serving the files. To improve it even further all of js has to be bunlded and minified before gzipping. Another upgrade is to set CacheControl max age in the same OnPrepareResponse to cache for one year and add asp-append-version="true" in the cshtml.
P.S. If you will host behind IIS you might need to turn off the static compression of js and css not to double compress, I am not sure how it will behave in this situation.
A:
This is a fixed version of method 3 from Ilyas answer that works with ASP.NET Core 1 RTM, and it serves pre-zipped javascript files:
app.UseStaticFiles(new StaticFileOptions
{
OnPrepareResponse = context =>
{
IHeaderDictionary headers = context.Context.Response.Headers;
string contentType = headers["Content-Type"];
if (contentType == "application/x-gzip")
{
if (context.File.Name.EndsWith("js.gz"))
{
contentType = "application/javascript";
}
else if (context.File.Name.EndsWith("css.gz"))
{
contentType = "text/css";
}
headers.Add("Content-Encoding", "gzip");
headers["Content-Type"] = contentType;
}
}
});
A:
@Ilya's Answer is very good but here are two alternatives if you are not using Gulp.
ASP.NET Core Response Compression Middleware
In the ASP.NET Core BasicMiddlware repository, you can find (at time of writing) a pull request (PR) for Response Compression Middleware. You can download the code and add it to you IApplicationBuilder like so (at time of writing):
public void Configure(IApplicationBuilder app)
{
app.UseResponseCompression(
new ResponseCompressionOptions()
{
MimeTypes = new string[] { "text/plain" }
});
// ...Omitted
}
IIS (Internet Information Server)
IIS (Internet Information Server) has a native static file module that is independent of the ASP.NET static file middleware components that you’ve learned about in this article. As the ASP.NET modules are run before the IIS native module, they take precedence over the IIS native module. As of ASP.NET Beta 7, the IIS host has changed so that requests that are not handled by ASP.NET will return empty 404 responses instead of allowing the IIS native modules to run. To opt into running the IIS native modules, add the following call to the end of Startup.Configure.
public void Configure(IApplicationBuilder app)
{
// ...Omitted
// Enable the IIS native module to run after the ASP.NET middleware components.
// This call should be placed at the end of your Startup.Configure method so that
// it doesn't interfere with other middleware functionality.
app.RunIISPipeline();
}
Then in your Web.config use the following settings to turn on GZIP compression (Note that I included some extra lines to compress things like .json files which are otherwise left uncompressed by IIS):
<?xml version="1.0" encoding="utf-8"?>
<configuration>
<system.webServer>
<!-- httpCompression - GZip compress static file content. Overrides the server default which only compresses static
files over 2700 bytes. See http://zoompf.com/blog/2012/02/lose-the-wait-http-compression and
http://www.iis.net/configreference/system.webserver/httpcompression -->
<!-- minFileSizeForComp - The minimum file size to compress. -->
<httpCompression directory="%SystemDrive%\inetpub\temp\IIS Temporary Compressed Files" minFileSizeForComp="1024">
<scheme name="gzip" dll="%Windir%\system32\inetsrv\gzip.dll" />
<dynamicTypes>
<add mimeType="text/*" enabled="true" />
<add mimeType="message/*" enabled="true" />
<add mimeType="application/x-javascript" enabled="true" />
<!-- Compress XML files -->
<add mimeType="application/xml" enabled="true" />
<!-- Compress JavaScript files -->
<add mimeType="application/javascript" enabled="true" />
<!-- Compress JSON files -->
<add mimeType="application/json" enabled="true" />
<!-- Compress SVG files -->
<add mimeType="image/svg+xml" enabled="true" />
<!-- Compress RSS feeds -->
<add mimeType="application/rss+xml" enabled="true" />
<!-- Compress Atom feeds -->
<add mimeType="application/atom+xml" enabled="true" />
<add mimeType="*/*" enabled="false" />
</dynamicTypes>
<staticTypes>
<add mimeType="text/*" enabled="true" />
<add mimeType="message/*" enabled="true" />
<add mimeType="application/x-javascript" enabled="true" />
<add mimeType="application/atom+xml" enabled="true" />
<add mimeType="application/xaml+xml" enabled="true" />
<!-- Compress ICO icon files (Note that most .ico files are uncompressed but there are some that can contain
PNG compressed images. If you are doing this, remove this line). -->
<add mimeType="image/x-icon" enabled="true" />
<!-- Compress XML files -->
<add mimeType="application/xml" enabled="true" />
<add mimeType="application/xml; charset=UTF-8" enabled="true" />
<!-- Compress JavaScript files -->
<add mimeType="application/javascript" enabled="true" />
<!-- Compress JSON files -->
<add mimeType="application/json" enabled="true" />
<!-- Compress SVG files -->
<add mimeType="image/svg+xml" enabled="true" />
<!-- Compress EOT font files -->
<add mimeType="application/vnd.ms-fontobject" enabled="true" />
<!-- Compress TTF font files - application/font-ttf will probably be the new correct MIME type. IIS still uses application/x-font-ttf. -->
<!--<add mimeType="application/font-ttf" enabled="true" />-->
<add mimeType="application/x-font-ttf" enabled="true" />
<!-- Compress OTF font files - application/font-opentype will probably be the new correct MIME type. IIS still uses font/otf. -->
<!--<add mimeType="application/font-opentype" enabled="true" />-->
<add mimeType="font/otf" enabled="true" />
<!-- Compress RSS feeds -->
<add mimeType="application/rss+xml" enabled="true" />
<add mimeType="application/rss+xml; charset=UTF-8" enabled="true" />
<add mimeType="*/*" enabled="false" />
</staticTypes>
</httpCompression>
<!-- Enable gzip and deflate HTTP compression. See http://www.iis.net/configreference/system.webserver/urlcompression
doDynamicCompression - enables or disables dynamic content compression at the site, application, or folder level.
doStaticCompression - enables or disables static content compression at the site, application, or folder level.
dynamicCompressionBeforeCache - specifies whether IIS will dynamically compress content that has not been cached.
When the dynamicCompressionBeforeCache attribute is true, IIS dynamically compresses
the response the first time a request is made and queues the content for compression.
Subsequent requests are served dynamically until the compressed response has been
added to the cache directory. Once the compressed response is added to the cache
directory, the cached response is sent to clients for subsequent requests. When
dynamicCompressionBeforeCache is false, IIS returns the uncompressed response until
the compressed response has been added to the cache directory.
Note: This is set to false in Debug mode to enable Browser Link to work when debugging.
The value is set to true in Release mode (See web.Release.config).-->
<urlCompression doDynamicCompression="true" doStaticCompression="true" dynamicCompressionBeforeCache="false" />
</system.webServer>
</configuration>
|
A day after the telecom regulator proposed high spectrum prices, the shares of leading telecom operators tumbled in early trade. The losses were later cut on expectations the proposals might not be accepted. Shares of the largest telecom operator, Bharti Airtel, in morning trade fell as much as 7.6 per cent to Rs 289, its lowest level in nearly two years. The fourth-largest player, Idea Cellular, plunged as much as 14.5 per cent, while Reliance Communications, the second largest, declined as much as 3.4 per cent.
The Telecom Regulatory Authority of India (Trai) had proposed a near-tenfold increase in the reserve price for spectrum over what operators had paid in 2008.
Analysts said the recommendation would push up costs and hurt companies’ profitability. The telecom companies and their industry body slammed the move, saying it would kill the industry.
“These recommendations will pose challenges and hurt earnings but the final impact will depend on a number of variables,” said Anand Shanbhag, head of research, Avendus Securities. Telecom stocks, however, managed to cut losses after the sentiment improved slightly on hope the recommendations would not be implemented.
Trai’s proposals are not binding and the government will decide on the final spectrum rules.
“The recommendations are adverse for the entire telecom industry as well as for consumers. This is the third sensational set of recommendations by the Trai — the earlier ones being in May 2010 and February 2011,” said Naveen Kulkarni and Vivekanand Subbaram, telecom analysts at MF Global, in a note. “The previous recommendations are yet to be implemented and in our view, the current set of recommendations seem too far-fetched to be implemented,” the duo added.
“We find little logic in spectrum being priced so high. We don’t expect broad-based participation in the upcoming auctions if this pricing happens to be final. We believe the government will have to significantly revisit the pricing recommendations,” said HSBC analyst Rajiv Sharma in a report. Bharti Airtel shares finally ended two per cent lower at Rs 306. Idea Cellular closed 3.72 per cent down at Rs 80, while RComm ended at Rs 80, down 1.35 per cent. Telecom analysts expect the stocks to remain range-bound for some time. |
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Modeling studies of ammonia dispersion and dry deposition at some hog farms in North Carolina.
A modeling study was conducted on dispersion and dry deposition of ammonia taking one hog farm as a unit. The ammonia emissions used in this study were measured under our OPEN (Odor, Pathogens, and Emissions of Nitrogen) project over a waste lagoon and from hog barns. Meteorological data were also collected at the farm site. The actual layout of barns and lagoons on the farms was used to simulate dry deposition downwind of the farm. Dry deposition velocity, dispersion, and dry deposition of ammonia were studied over different seasons and under different stability conditions using the short-range dispersion/air quality model, AERMOD. Dry deposition velocities were highest under near-neutral conditions and lowest under stable conditions. The highest deposition at short range occurred under nighttime stable conditions and the lowest occurred during daytime unstable conditions. Significant differences in deposition over crop and grass surfaces were observed under stable conditions. |
Q:
Will gfortran or ifort compilers wisely use SIMD instructions when summing the product of two arrays?
I've got some code written with numpy, and I'm considering porting it to Fortran for better performance.
One operation I do several times is summing the element-wise product of two arrays:
sum(A*B)
It looks like fused multiply-add instructions would help with this. My current processor doesn't support these instructions, so I can't test things yet. However, I may upgrade to a new processor that does support FMA3 (an Intel Haswell processor).
Does anyone know if compiling the program with "-march=native" (or the ifort equivalent) will be enough to get the compiler (either gfortran or ifort) to wisely use SIMD instructions to optimize that code, or do you think I'll have to baby the compilers or code?
A:
If you use -march=native on a machine with SIMD, the compiler should generate SIMD instructions, although I've always used -xHost flag instead with ifort.
But I am not so sure how to make them do it "wisely". My feeling is that at -O3 level ifort and gfortran both tend to be overly aggressive on vectorization (that is, they use the SIMD functionality more often than they should). Very often I have to turn off vectorization to get the most efficient code. This, of course, may or may not be true for you.
It will usually be better to use vector libraries that are optimized for this task. You can use vdmul in MKL or gsl_vector_mul in GSL to do this.
Using -march=NEWARCH will result in a code tuned for the architecture NEWARCH but cannot run on an earlier architecture. You can use the -mtune=NEWARCH flag where NEWARCH is the architecture of your new processor. This will generate code tuned for the new architecture but still executable on the old one. Since you do not yet have the new machine, -mtune is probably what you need at the moment.
With ifort you can use vectorization report flags to show which part of the program has been vectorized. For example, ifort flag -vec-report=1 will give you such information during compilation. I am sure there will be an equivalent flag in gfortran.
A:
Thanks to Xiaolei Zhu's tip, I now know that gfortran will use fused multiply-add to optimize sum(A*B). For example, with this code:
program test
implicit none
real, dimension(7) :: a, b
a = (/ 2.0, 3.0, 5.0, 7.0, 11.0, 13.0, 17.0 /)
b = (/ 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0 /)
print *, sum(a*b)
endprogram
I can compile it with f95 sum.f95 -o sum -O3 -march=core-avx2, and objdump -d sum | grep vfmadd displays
40088b: c4 e2 71 99 44 24 30 vfmadd132ss 0x30(%rsp),%xmm1,%xmm0
400892: c4 e2 69 b9 44 24 34 vfmadd231ss 0x34(%rsp),%xmm2,%xmm0
400899: c4 e2 61 b9 44 24 38 vfmadd231ss 0x38(%rsp),%xmm3,%xmm0
4008a0: c4 e2 59 b9 44 24 3c vfmadd231ss 0x3c(%rsp),%xmm4,%xmm0
4008a7: c4 e2 51 b9 44 24 40 vfmadd231ss 0x40(%rsp),%xmm5,%xmm0
4008ae: c4 e2 49 b9 44 24 44 vfmadd231ss 0x44(%rsp),%xmm6,%xmm0
4008b5: c4 e2 41 b9 44 24 48 vfmadd231ss 0x48(%rsp),%xmm7,%xmm0
So gfortran unrolled the loop and put in 7 fused multiply-add instructions. If I create larger, random, multi-dimensional arrays, I still see vfmadd231ss pop up once (so it doesn't unroll the loop).
|
glabel func_808980C0
/* 00000 808980C0 27BDFFD8 */ addiu $sp, $sp, 0xFFD8 ## $sp = FFFFFFD8
/* 00004 808980C4 00803025 */ or $a2, $a0, $zero ## $a2 = 00000000
/* 00008 808980C8 AFA5002C */ sw $a1, 0x002C($sp)
/* 0000C 808980CC 00A02025 */ or $a0, $a1, $zero ## $a0 = 00000000
/* 00010 808980D0 AFBF001C */ sw $ra, 0x001C($sp)
/* 00014 808980D4 24C50150 */ addiu $a1, $a2, 0x0150 ## $a1 = 00000150
/* 00018 808980D8 AFA50020 */ sw $a1, 0x0020($sp)
/* 0001C 808980DC 0C016EFE */ jal func_8005BBF8
/* 00020 808980E0 AFA60028 */ sw $a2, 0x0028($sp)
/* 00024 808980E4 8FA60028 */ lw $a2, 0x0028($sp)
/* 00028 808980E8 3C07808A */ lui $a3, %hi(D_80898764) ## $a3 = 808A0000
/* 0002C 808980EC 8FA50020 */ lw $a1, 0x0020($sp)
/* 00030 808980F0 24CE0170 */ addiu $t6, $a2, 0x0170 ## $t6 = 00000170
/* 00034 808980F4 AFAE0010 */ sw $t6, 0x0010($sp)
/* 00038 808980F8 24E78764 */ addiu $a3, $a3, %lo(D_80898764) ## $a3 = 80898764
/* 0003C 808980FC 0C017014 */ jal func_8005C050
/* 00040 80898100 8FA4002C */ lw $a0, 0x002C($sp)
/* 00044 80898104 8FBF001C */ lw $ra, 0x001C($sp)
/* 00048 80898108 27BD0028 */ addiu $sp, $sp, 0x0028 ## $sp = 00000000
/* 0004C 8089810C 03E00008 */ jr $ra
/* 00050 80898110 00000000 */ nop
|
INTRODUCTION {#SEC1}
============
*Streptococcus pneumoniae* is a gram-positive, facultative anaerobic bacterium and a significant human pathogen that causes otitis media, sinusitis, pneumonia and meningitis. *Streptococcus pneumoniae* is usually inhaled into the respiratory system and remains in the pharynx and nasal cavity, where it may cause diseases ([@B1],[@B2]). Glycopeptide antibiotics such as vancomycin and as penicillin are frequently used to eradicate the bacteria and relieve symptoms ([@B3],[@B4]). However, substantial evidence of antibiotic resistance in *S. pneumoniae* has accumulated worldwide. Unfortunately, extensively drug-resistant *S. pneumoniae* with resistance to penicillin, cephalosporin, macrolides, β-lactam antibiotics, fluoroquinolone and even vancomycin and linezolid have appeared ([@B5]). Currently, antibiotic combination therapy is used to obtain synergistic treatment effects, but it cannot effectively suppress the growth of drug-resistant bacteria ([@B8]). Therefore, the development of new antibiotic candidates to eradicate *S. pneumoniae* by exploiting new therapeutic strategies is urgently needed.
In bacteria, toxin--antitoxin (TA) systems are strongly correlated with physiological processes such as gene regulation, growth arrest, survival and apoptosis ([@B11]). TA loci were first discovered in 1983 on the mini-F plasmid of *Escherichia coli*, a plasmid addiction module that is responsible for the maintenance of extrachromosomal genetic elements ([@B16]). TA systems consist of toxin genes and antitoxin genes. TA systems are typically located on bacterial plasmids and transferred to daughter cells to yield plasmid stabilization and cell viability ([@B17],[@B18]). In contrast, TA systems located on chromosomes are commonly related to growth arrest, biofilm formation and multidrug tolerance and facilitate the development of persister or dormant cells ([@B19]). A toxin damages the host cell by inhibiting DNA replication, protein synthesis and cell wall synthesis by degrading host RNA or by binding to topoisomerases or ribosomes ([@B23]). In contrast, an antitoxin is an important cellular component because it neutralizes the detrimental activity of its cognate toxin ([@B26]).
TA systems are classified into five groups that are referred to as types I--V. In type I TA systems, the antitoxin is an antisense RNA that binds to the toxin mRNA. The antitoxin inhibits the translation of the toxin and causes it to be degraded. In type II TA systems, both the toxin and the antitoxin are proteins that interact with each other to form a non-toxic protein complex. In type III TA systems, the antitoxin is an RNA that binds directly to the toxin protein to form a non-toxic RNA--protein complex. In type IV TA systems, the toxin and antitoxin do not bind directly to each other but instead compete for the same cellular target. In type V TA systems, the antitoxin is a protein that degrades the toxin mRNA to prevent its expression ([@B27]). HicBA is a type II TA system. In type II TA systems, the toxin is thermodynamically stable, whereas the antitoxin is unstable and is cleaved by cellular proteases because its locally flexible conformation makes it susceptible to proteolysis. Upon proteolysis of the antitoxin, the free toxin is released from the TA complex and binds to host cell components, causing growth arrest or even death of the host cell ([@B32]).
The *S. pneumoniae* strain TIGR4 contains only six TA loci (three RelBE family, one phd-doc family, one HigBA family and one HicBA family) ([@B35]). Overall, *S. pneumoniae* contains a small number of TA loci relative to other bacteria. For example, *Mycobacterium tuberculosis* possesses more than 88 known TA operons. Although the mechanisms of TA activity have been studied for a long time, the physiological roles of TA have not yet been clearly elucidated ([@B36]). Furthermore, the only HicBA TA complex crystal structure determined to date is that of *Yersinia pestis*, Protein Data Bank (PDB) code 4P78 (HicBA3), which lacks the C-terminal region of the antitoxin ([@B39]).
HicB antitoxins consist of two functional motifs: an N-terminal region that binds to the toxin and thereby abolishes its toxicity and a C-terminal region that regulates its TA operon by binding to tis promoter DNA ([@B40],[@B41]). The DNA-binding domain of HicB ([@B42]) forms a ribbon-helix-helix ([@B43]) or helix-turn-helix motif ([@B44]). HicA toxins contain a double-stranded RNA binding domain ([@B45]) and exhibit ribonuclease activity ([@B46]). The active sites of HicA toxins contain a conserved histidine residue ([@B39],[@B47]).
Here, we present the crystal structure of the HicBA complex of *S. pneumoniae* TIGR4 at a resolution of 2.30 Å. The C-terminal DNA-binding domain of the HicB antitoxin shows considerable structural variation, and the HicA toxin forms contacts with the curved back of the HicB antitoxin. This structure reveals the residues that are important in the formation of the HicBA complex. The core residues in HicB that bind to DNA and the properties of the DNA-binding domain were elucidated by nuclear magnetic resonance (NMR). As antibiotic candidates, binding interface-mimicking peptides were designed to act as antimicrobial peptides that suppress the TA interaction. This approach may contribute to the development of novel potent antibiotics as effective treatments for antibiotic-resistant *S. pneumoniae*.
MATERIALS AND METHODS {#SEC2}
=====================
Cloning and transformation {#SEC2-1}
--------------------------
The genes encoding HicB (*SP1786*) and HicA (*SP1787*) were amplified by the polymerase chain reaction (PCR). The primers used in PCR were HicB-F/HicB-R (for HicB) and HicA-F/HicA-R (for HicA) ([Supplementary Table S1A](#sup1){ref-type="supplementary-material"}). The restriction enzymes used for cloning were Nde1 and Xho1. The PCR products of both HicB and HicA were double-cut by Nde1 and Xho1 and ligated into vectors that had been cut by the same enzymes. For structure determination and biological assays, HicB and HicA were ligated into pET21a with no tag and into pET28b, respectively. The pET28b N-terminal tag (MGSSHHHHHHSSGLVPRGSH) was added to HicA as an additional residual tag. For the NMR experiments, the residues of HicB including the DNA-binding domain (HicB^109--150^) were cloned. The primers used in PCR were HicB^109--150^-F and HicB-R ([Supplementary Table S1A](#sup1){ref-type="supplementary-material"}). Restriction digestion was conducted in the same way, and pET28b was used for ligation with the same tag. To confirm the identity of the residue essential to the ribonuclease activity of HicA, His36 of HicA was mutated to Ala36 (HicA-H36A). The primers used in PCR were H36A-F/H36A-R ([Supplementary Table S1A](#sup1){ref-type="supplementary-material"}). Mutation was conducted using the EZchange™ Site-Directed Mutagenesis Kit (Enzynomics, Korea) according to the manufacturer\'s protocol.
Protein expression and purification {#SEC2-2}
-----------------------------------
The cloned plasmids containing HicB and HicA were co-transformed into *E. coli* Rosetta2 (DE3) pLysS competent cells. The cells were grown at 37°C in Luria broth (LB) until the OD~600~ of the cell suspension reached 0.8. Protein overexpression was induced by the addition of 0.5 mM isopropyl 1-thio-β-[d]{.smallcaps}-galactopyranoside (IPTG), and incubation at 37°C was continued for 4 h. The cultured cells were harvested by centrifugation at 11 355 × g at 4°C and stored at --80°C. The harvested cells were suspended in buffer A (20 mM Tris--HCl, pH 7.9, and 500 mM NaCl) containing 5% glycerol by volume and lysed by ultrasonication. After centrifugation for 1 h at 28 306 × g, the supernatant containing soluble proteins was loaded onto a Ni^2+^ affinity open column (Bio-Rad) that had been equilibrated with buffer A, and the column was washed with buffer A containing 50 mM imidazole. The protein bound to the Ni^2+^ column was eluted using an imidazole gradient (100−700 mM), and the TA complex in each fraction was identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the buffer containing the TA complex was exchanged for buffer consisting of 20 mM Tris, pH 7.5 and 150 mM NaCl by size-exclusion chromatography on a HiLoad 16/600 Superdex 200 prep-grade column (GE Healthcare) and concentrated to 10 mg/ml using an Amicon Ultra centrifugal filter unit (Millipore). The purity of the TA protein complex was verified by SDS-PAGE. For the ribonuclease assay, HicA and HicA-H36A were expressed and purified by the same procedure except that HicA-expressing cells were incubated for only 2 h after IPTG induction because of the toxicity of the protein to *E. coli*. The HicBA complex labeled with selenomethionine (SeMet) was obtained by the same procedure, except that cells containing the SeMet-labeled HicBA complex were grown in M9 medium containing extra essential amino acids. For the NMR experiments, the N-terminal (His)~6~-tagged HicB^109--150^ protein was expressed in *E. coli* strain BL21(DE3) using M9 medium mixed with 1.0 g/l \[U-^13^C\] glucose and 1.0 g/l \[^15^N\] NH~4~Cl (Cambridge Isotopes Laboratory) as the sole carbon and nitrogen sources, respectively. The purification of HicB^109--150^ was the same as for the TA complex. To cleave the (His)~6~ tag prior to size-exclusion chromatography, 100 units of thrombin from human plasma (Sigma-Aldrich) were added to 10 mg of purified protein and incubated overnight at 20°C in a buffer consisting of 20 mM sodium phosphate (Na Pi), pH 7.0, and 100 mM NaCl. VapC26 and VapC30 from *M. tuberculosis* were expressed and purified as reported previously ([@B48],[@B49]).
Crystallization, data collection and processing {#SEC2-3}
-----------------------------------------------
Initial crystal screening of the purified HicBA complex was conducted using the Crystal Screening 1, 2 and Index (Hampton Research) kits by mixing 1 μl of protein solution at 10 mg/ml in 20 mM Tris, pH 7.5 and 150 mM NaCl with 1 μl of reservoir solution. Crystals of the HicBA complex were grown using the sitting-drop vapor diffusion method at 4°C. The crystallization solution for HicBA was 0.1 M Tris, pH 8.5 and 2.0 M ammonium sulfate. Cryoprotection of HicBA was achieved by adding 20% glycerol to the solution. The crystals were flash-cooled in liquid nitrogen prior to data collection. The data were collected using an ADSC Quantum Q270r CCD detector at beamlines 5C and 7A of the Pohang Light Source, Republic of Korea. The crystals of the HicBA complex belong to the *orthorhombic* space group *P*2~1~2~1~2 and display unit cell parameters of *a* = 106.263 Å, *b* = 116.541 Å, *c* = 42.490 Å, and α = β = γ = 90.00° for the native HicBA crystal and *a* = 106.926 Å, *b* = 116.602 Å, *c* = 42.680 Å, and α = β = γ = 90.00° for the SeMet-labeled crystals. The calculated total mass of the protein complex, including the N-terminal histidine tag was 26349.7 Da. All raw data were scaled and processed by the HKL2000 ([@B50]). The structure of the HicBA complex from *S. pneumoniae* was determined at 2.80 Å resolution by single-wavelength anomalous dispersion using SeMet-labeled crystals. The final structure of HicBA was determined by molecular replacement. The 2.30 Å data on the native crystal were used for structural analysis, and the SeMet structure was used as a model to determine the native structure. Detailed statistical information on the structures is provided in [Supplementary Table S2](#sup1){ref-type="supplementary-material"}. In the SeMet structure, we determined the positions of 14 selenium sites in the asymmetric unit. *PHENIX* ([@B51]) was first used to automatically build the model, and *COOT* ([@B52]) was utilized to provide the starting model for refinement. *R*~work~/*R*~free~ values ([@B53]) of the SeMet and the native final models using *REFMAC* and *PHENIX* ([@B51],[@B54]) were 20.2/24.0% and 20.5/23.6%, respectively. The overall geometry was validated using *MolProbity* ([@B55]), the results showed that 98% of the residues were in the favored region of the Ramachandran plot and an additional 2% were in the allowed region in the native structure. PyMOL was used to generate all figures ([@B56]). The electrostatic potential surfaces were calculated using the Adaptive Poisson-Boltzmann solver (APBS) method ([@B57]).
Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) {#SEC2-4}
----------------------------------------------------------------------------------
MALS was performed to determine the oligomeric states of HicBA. Size-exclusion chromatography was conducted on a BioSep SEC-s3000 column (Phenomenex) on a 1260 Infinity HPLC system (Agilent Technologies). The scattering data were obtained in a miniDAWN-TREOS line for emission at 657.4 nm (Wyatt Technology) and analyzed using ASTRA 6.0.1.10 software (Wyatt Technology). HicBA hetero-dimer at a concentration of 100 μM was used in the experiment, which was performed in 20 mM Tris, pH 7.5 and 150 mM NaCl, corresponding to the crystallization conditions. The experiments were performed at room temperature.
Electrophoretic mobility shift assay (EMSA) {#SEC2-5}
-------------------------------------------
EMSA was conducted to determine the binding affinity of HicB and HicBA to promoter DNA. A 28-base-pair DNA fragment from the upstream region (promoter DNA) of HicBA was added to the proteins as a palindromic form. The palindromic sequence was as follows: forward---TAATAGAATAATAAGTATCACTCCTTTA; reverse---TAAAGGAGTGATACTTATTATTCTATTA. Two other palindromic sequences (Pal-I and Pal-II) and a control DNA ('X') were also prepared ([Supplementary Table S1B](#sup1){ref-type="supplementary-material"}). The annealed dsDNAs were purchased from Bioneer Innovation (<http://www.bioneer.co.kr/>). The dsDNAs and proteins were added to a binding buffer consisting of 20 mM Tris pH 7.5 and 150 mM NaCl, varying amounts of HicB and HicBA protein were then mixed with the DNA to yield a final volume of 10 μl and the mixtures were incubated for 20 min at 4°C. The total binding solutions were loaded onto 0.8% agarose gels in 0.5× TBE (45 mM Tris-borate, 1 mM EDTA) buffer, and the results were visualized using a Gel Doc (Bio-Rad).
Isothermal titration calorimetry (ITC) measurements {#SEC2-6}
---------------------------------------------------
ITC experiments were performed at 25°C using a MicroCal 200 (GE Healthcare). HicB, HicBA and promoter dsDNA were prepared in a buffer consisting of 20 mM Tris, pH 7.5 and 150 mM NaCl. A dsDNA fragment from the upstream region (promoter DNA) of HicBA was added to the proteins. Affinity measurement was conducted at a protein concentration of 0.1 mM HicB dimer and 0.1 mM HicBA hetero-tetramer in a total volume of 320 μl using the promoter dsDNA solution (2 mM) as the injected titrant. Data collection was performed at 180-s intervals for a total of 19 injections. Curve fitting to calculate the binding affinity (*K*~d~), the enthalpy of binding (△*H*), the entropy of binding (△*S*) and the molar ratio was performed using the MicroCal Origin software. The raw data were fitted to a one-site binding model. The Gibbs free energies (△*G*) were calculated using the standard equation △*G* = △*H* − T△*S*.
NMR study of HicB^109--150^ and DNA titration {#SEC2-7}
---------------------------------------------
NMR spectra of the HicB antitoxin were measured using a Bruker AVANCE DRX 800 spectrometer. All experiments were performed at 298 K. The samples were prepared in a buffer consisting of 20 mM NaPi, pH 7 and 100 mM NaCl containing 10% D~2~O by total volume. The data were processed using NMRPipe/nmrDraw ([@B58]) and further analyzed using NMRviewJ ([@B59]). Data on the carbonyl carbon were obtained using the HNCO and HNCACO spectra, and data on the α/β carbon were acquired using the HNCACB and CBCACONH spectra. Additionally, to explore the structural transition in the ribbon-helix-helix domain that occurs during the binding of HicB to DNA, promoter DNA was added to HicB^109--150^. DNA titration was conducted four times in the measurement of the ^1^H,^15^N-HSQC spectra. The concentration of HicB^109--150^ monomer was maintained at 1 mM, and the DNA concentration was varied from 0 to 0.3 mM (a maximum of 30% of the protein concentration). The chemical shift perturbation (CSP) was calculated by nmrViewJ. The average CSP values of ^15^N and ^1^H were calculated from Equation ([1](#M1){ref-type="disp-formula"}), in which Δδ~N~ and Δδ~H~ represent the CSP values of the amide nitrogen and proton, respectively To determine *K*~d~ by NMR titration, the concentration of the HicB^109--150^ monomer was maintained at 0.5 mM, and the DNA concentration was varied from 0 to 0.6 mM (a maximum of 120% of the protein concentration). $$\documentclass[12pt]{minimal}
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\begin{document}
}{}\begin{equation*}\Delta {\delta _{avg}} = {\rm{ }}{[(0.2{\rm{ }} \times \Delta {\delta ^2}_N + \Delta {\delta ^2}_H)/2]^{1/2}}\end{equation*}\end{document}$$
*In silico* HicBA-DNA docking {#SEC2-8}
-----------------------------
Due to the difficulty of obtaining a DNA-bound crystal of HicBA, *in silico* molecular docking using the High Ambiguity Driven protein--protein DOCKing algorithm (HADDOCK) ([@B60]) was performed to examine the interaction between the promoter DNA and HicBA. The coordinates for a 28-base pair promoter DNA were modeled using the 3D-DART server ([@B61]), and the coordinates for HicBA were obtained from our crystal structure. The hetero-octamer model of the HicBA and the tetramer model of HicB were obtained by symmetry using the HicBA hetero-tetramer structure (PDB code 5YRZ). The residues of the DNA-binding domain inferred from the CSP values (Ile114--Thr117 in β1 for DNA recognition and Asn134--Gln137 in the N-terminal region of α2 for DNA anchoring and facing) and two symmetrical grooves of the DNA were defined as 'active residues' for the generation of the interface contact. Passive residues were defined automatically as the residues around the active residues.
*In vitro* ribonuclease assay after the addition of a peptide mimicking the binding region {#SEC2-9}
------------------------------------------------------------------------------------------
The ribonuclease activity of HicA was confirmed using an RNase Alert Kit (IDT). A fluorescence quenching assay was performed according to the manufacturer\'s protocol. In this system, a fluorophore is covalently attached to one end of a synthetic RNA strand and is quenched by a quencher group at the other end. If synthetic RNA containing a fluorophore--quencher pair interacts with a ribonuclease, the synthetic RNA is digested, and the quencher is released. The released fluorophore emits fluorescence at 520 nm upon excitation at 490 nm. The resulting fluorescence (RFU) was observed on a SPECTRAmax GEMINI XS spectrofluorometer. The concentration range of HicA was varied to determine the ribonuclease activity of HicA and its reaction limits. HicA-H36A was examined by the same method as HicA to show the indispensability of His36 for the ribonuclease activity. Several short peptides that mimic the binding region of HicA were designed and purchased from ANYGEN (<http://www.anygen.com>). Peptides were added to the HicBA complex to release free HicA ([Supplementary Table S3](#sup1){ref-type="supplementary-material"}). The sequence of the peptide used in the regular experiment was 'ELNKYTERGIRKQAG'. Theoretically, mimicking peptides compete with the original protein for binding, if the peptide occupies the specific binding site, the toxin is released, producing fluorescence in the fluorescence quenching assay ([@B62]). The proteins and peptides used in ribonuclease assay were prepared in 20 mM Tris, pH 7.5 and 150 mM NaCl. The HicBA complex was incubated with the peptide for 30 min at 37°C before measurement of fluorescence.
*In vivo* cell growth assay {#SEC2-10}
---------------------------
For the cell growth assay, the plasmids expressing HicBA, HicA and HicA-H36A were transformed into *E. coli* strain BL21(DE3). Transformed cells from single colonies grown on 0.1% glucose-containing M9 medium plates were grown overnight, and the overnight cultures were diluted to an OD~600~ of 0.1. The diluted cells were further grown until the OD~600~ of the cell suspension reached 0.4, at which time 0.5 mM IPTG was added to induce protein expression. The cells were incubated at 37°C for 8 h after induction by IPTG and monitored at 1-h intervals.
*In vivo* HicB neutralization assay {#SEC2-11}
-----------------------------------
To assay neutralization by HicB, a mutational analysis of the binding interface of HicBA was conducted. Six residues of HicB (Phe22, Thr33, Gln34, Glu47, Phe80 and Thr89) were individually mutated to alanine ([Supplementary Table S1A](#sup1){ref-type="supplementary-material"}). The resulting mutant proteins were designated F22A, T33A, Q34A, E47A, F80A and T89A, respectively. The mutations were obtained by the same method used to prepare HicA-H36A, and the mutated HicBA complexes were expressed using the same procedures as those used for the native HicBA complex. The primers used to create these mutations are listed in [Supplementary Table S1A](#sup1){ref-type="supplementary-material"}. pET28b-HicA and pET21a-HicB, and pET28b-HicA and pET21a-HicB mutants were co-transformed into *E. coli* strain BL21 (DE3), respectively. The transformed cells were grown on LB plates containing 0.5 mM IPTG. The plates were incubated at 37°C for 18 h.
Circular dichroism (CD) spectroscopy {#SEC2-12}
------------------------------------
CD measurement of peptides dissolved in 20 mM Tris, pH 7.5 and 150 mM NaCl at a concentration of 25 μM was performed in a Chirascan Plus spectropolarimeter (Applied Photophysics, Ltd.) at 20°C using a cell with a 1 mm light path. CD scans were taken from 260 to 190 nm at bandwidth of 1 nm and a scan speed of 100 nm/min. The results of three scans were averaged, and the solvent signal was subtracted.
Antimicrobial activity test {#SEC2-13}
---------------------------
The antimicrobial activity of the mimicking peptides was evaluated by measuring their minimum inhibitory concentration (MIC) values using the serial dilution method. The activity of the peptides against three strains of gram-positive (*Bacillus subtilis* ATCC 6633, *Staphylococcus aureus* ATCC 6538p, and *Staphylococcus epidermis* ATCC 12228) and five strains of gram-negative (*E. coli* ATCC 25922, *Shigella dysenteriae* ATCC 9752, *Salmonella typhimurium* ATCC 14028, *Klebsiella pneumoniae* ATCC 10031, and *Pseudomonas aeruginosa* ATCC 27853) bacteria was tested. Each bacterial strain was grown overnight in the presence of various concentrations of peptide (0.4--100 μM). The MIC was defined as the lowest peptide concentration that completely inhibited bacterial growth. Each test was conducted in duplicate.
RESULTS {#SEC3}
=======
Overall structure of the HicBA complex {#SEC3-1}
--------------------------------------
The asymmetric unit of the HicBA complex crystal structure contains two hetero-dimeric HicBA complexes. Two HicB antitoxins and two HicA toxins form a hetero-tetrameric assembly (Figure [1A](#F1){ref-type="fig"}). However, the molecular weight of HicBA calculated from size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) was 105 ± 0.1 kDa, which corresponds to the theoretical molecular weight of the hetero-octameric model of HicBA (105.4 kDa) ([Supplementary Figure S1A](#sup1){ref-type="supplementary-material"}). The model of the hetero-octameric assembly of HicBA contains dimeric interfaces between the HicB chains (Figure [1A](#F1){ref-type="fig"} and [Supplementary Figure S2A](#sup1){ref-type="supplementary-material"}). Secondary structure analysis was performed using the 2Struc server ([@B63]).
{#F1}
The *S. pneumoniae* HicB antitoxin contains three α-helices, three 3~10~-helices (η) and four β-strands and the *S. pneumoniae* HicA toxin contains two α-helices and three β-strands with an α-β-β-β-α double-stranded RNA binding domain fold topology (Figure [1B](#F1){ref-type="fig"}). Compared to the structure of the *Y. pestis* HicBA complex ([@B39]), the structure of the *S. pneumoniae* HicBA complex contains an additional long, flexible loop between η1 and η2 of HicB ([Supplementary Figure S2D](#sup1){ref-type="supplementary-material"}). This loop is made up of more than 20 residues of HicB (Figure [1B](#F1){ref-type="fig"}). In particular, the C-terminal region of HicB, which is expected to be a DNA-binding domain, is not present in the structure of the *Y. pestis* HicBA complex ([@B42]) (Figure [1C](#F1){ref-type="fig"}). The structural topologies of the two HicBA complexes are described in [Supplementary Figure S3A and B](#sup1){ref-type="supplementary-material"}. The DNA-binding domain of the type II antitoxin is important in the auto-regulation of the TA operon ([@B41]). Furthermore, the DNA-binding domain of the HicBA system can form either a ribbon--helix--helix ([@B43]) or a helix-turn-helix ([@B44]). In summary, the structure of the *S. pneumoniae* HicBA complex presented here is the first to show the complete conformation of the HicBA TA system and can illuminate the biological function of the HicBA system.
The structural characteristics of HicB antitoxin {#SEC3-2}
------------------------------------------------
Two HicB antitoxins form a homodimer through their N-terminal domains, mainly β1, α1 and β4 of each HicB monomer. However, the molecular weight of HicB calculated from SEC-MALS was 71.8 ± 0.8 kDa, which corresponds to the theoretical molecular weight of a HicB tetramer (71.3 kDa) ([Supplementary Figure S1B](#sup1){ref-type="supplementary-material"}). The model of the HicB tetramer contains an additional C-terminal dimeric interface and an N-terminal interface. ([Supplementary Figure S2B and C](#sup1){ref-type="supplementary-material"}). The average area of the N-terminal dimeric interfaces is 602 Å^2^. The interface area was calculated using the PISA server ([@B64]). The binding is achieved through both hydrophobic interactions and hydrogen bonding networks (Figure [2A](#F2){ref-type="fig"}).
{#F2}
Interestingly, considerable structural deviation is observed in the C-terminal domains of chains A and C of the two HicB antitoxins. The HicB antitoxin consists of an N-domain (residues 3--106), a hinge (residues 107--112), and a C-domain (residues 113--145). The rotation angle of the two C-domains of chains A and C is 151.3°. The root mean square deviation (r.m.s. deviation) value for the two N-domains is 0.81 Å, however, due to the flexibility of the C-domains, the r.m.s. deviation for the whole proteins is 10.63 Å. The N-domain, hinge and C-domains, the rotation angle, and the r.m.s deviation values were obtained using the *DynDom* database ([@B65]) (Figure [2B](#F2){ref-type="fig"}). Notably, the regions of the expected DNA-binding domain and the flexible C-domain are matched, indicating that the flexible nature of these domains is crucial to their role in DNA binding.
Characterization of the interaction of HicB and HicBA with promoter DNA {#SEC3-3}
-----------------------------------------------------------------------
To confirm the DNA-binding properties of HicB and HicBA, an electrophoretic mobility shift assay (EMSA) experiment was conducted. Although we were not able to obtain the dissociation constant from the EMSA experiment, the binding properties of HicB and HicBA were determined based on the results. The concentration of DNA was fixed at 0.01 mM, and the concentration of the proteins was varied from 0 to 0.8 mM (HicB monomer and HicBA hetero-dimer). As the DNA bound to increasing amounts of protein, the bands corresponding to the DNA-protein complex shifted upward. (Figure [3A](#F3){ref-type="fig"}). EMSA showed that DNA binds to both the HicB antitoxin and the HicBA complex, exhibiting slightly higher affinity for HicBA than for HicB. However, no band shifts were observed in the case of the other palindromic sequences or the control 'X' DNA ([Supplementary Figure S3C](#sup1){ref-type="supplementary-material"}).
{#F3}
Isothermal titration calorimetry (ITC) experiments were used to estimate the affinity of binding of HicB and HicBA to the promoter DNA (Figure [3B](#F3){ref-type="fig"}). The promoter DNA-binding reaction of HicB is endothermic and entropically driven, with thermodynamic parameters of 4.3 ± 0.4 kcal mol^−1^ (△*H*) and 33.3 cal mol^−1^ deg^−1^ (△*S*). The measured equilibrium dissociation constant (*K~d~*) for the binding of HicB to DNA was 8.8 ± 1.1 μM. The binding stoichiometry (*n*) is 0.51 ± 0.02, indicating that one HicB dimer binds to each DNA duplex. The experimental values showed that the HicB tetramer interacts with dsDNA. The promoter DNA-binding reaction of HicBA is endothermic and entropically driven, with thermodynamic parameters of 6.5 ± 0.5 kcal mol^−1^ (△*H*) and 44.6 cal mol^−1^ deg^−1^ (△*S*), and the equilibrium dissociation constant (*K~d~*) for the binding of HicBA to DNA was measured as 4.2 ± 0.4 μM. The binding stoichiometry (*n*) is 0.47 ± 0.03, indicating binding of one HicBA hetero-tetramer per DNA duplex. The experimental results showed that the HicBA hetero-octamer interacts with dsDNA and that, the HicBA complex has a higher affinity for DNA than HicB based on the *K*~d~ values. Calorimetric trials were also performed in the absence of proteins under the same experimental conditions. No changes in heat were observed in the control experiments.
DNA-binding domain of HicB {#SEC3-4}
--------------------------
In type II TA systems, the antitoxin alone or in complex with the toxin typically interacts with its corresponding promoter DNA, repressing transcription from the TA operon ([@B41]). Therefore, the DNA-binding properties of the antitoxin are important for the regulation of TA systems. We characterized the interactions between specific residues in HicB and the promoter DNA using NMR titration experiments. In these experiments, HicB^109--150^ was used because of the poor spectral quality of full-length HicB ([Supplementary Figure S4A](#sup1){ref-type="supplementary-material"}).
We predicted the possible DNA-binding domain of HicB and performed backbone assignment of HicB^109--150^ with the exception of the residues Pro113 and Pro121. The secondary structure of HicB^109--150^ in solution was derived from the program TALOS+ ([@B66]) using the assigned chemical shift values ([Supplementary Table S4](#sup1){ref-type="supplementary-material"}). The predicted conformation of HicB^109--150^ includes one β-strand and two α-helices in the following order: β1 (residues 114--119), α1 (residues 122--130), and α2 (residues 135--145). The assigned β1--α1--α2 folding of the C-terminus of HicB corresponds to a ribbon-helix-helix motif ([@B44]). This conformation is highly consistent with the structure of HicB in the HicBA complex. Additionally, the DNA-binding domain of HicBA was superposed with the structures of other ribbon-helix-helix proteins that display DNA-binding affinity ([Supplementary Figure S5A](#sup1){ref-type="supplementary-material"}). The superposition is consistent with the idea that the DNA-binding domain consists of ribbon-helix-helix motif.
We compared the HSQC spectra of HicB^109--150^ (1 mM, monomer concentration) in the absence and presence of increasing concentrations of promoter DNA (0--0.3 mM). These spectra are shown as an overlay in Figure [4A](#F4){ref-type="fig"}. Upon titration with DNA, the spectrum of HicB^109--150^ showed a peak shift indicating that HicB^109--150^--DNA interaction is achieved with fast exchange on the NMR time scale; such rapid exchange is typically observed when molecules bind with low to moderate affinity. The results of the titration experiment were consistent with the ITC data. According to the *K*~d~ determined in the ITC experiment, the binding of HicB to DNA is not strong; thus, binding between HicB^109--150^ and DNA is expected to show rapid exchange. This result is highly consistent with the observed peak movements in the spectra of HicB^109--150^ titrated with DNA. Additionally, as a result of additional titration, *K*~d~ for the binding of HicBA^109--150^ to DNA was measured as 4.5 ± 1.1 μM; this is comparable to the *K*~d~ value calculated from ITC ([Supplementary Figure S4B](#sup1){ref-type="supplementary-material"}).
{#F4}
Based on the assigned backbone ^1^H and ^15^N resonances, we monitored the residues that showed changes in their chemical shifts upon binding of the protein to DNA. We calculated the combined ^1^H and ^15^N CSP values of HicB^109--150^ in the presence of 0.3 mM DNA using Equation ([1](#M1){ref-type="disp-formula"}), (shown in the Materials and Methods section) to quantify the peak shifts. These values are plotted as a function of the HicB^109--150^ residues (Figure [4B](#F4){ref-type="fig"}). The residues on the α2 helix and the C-terminus showed high CSP values. In particular, Phe135 and Gln137 on the N-terminus of the α2 helix and Val148 and Gln149 on the C-terminus exhibited significant changes in their chemical shifts. The residues in the β1 strand showed smaller CSP values than other secondary structure components because the hydrogen bonds between the β strands of the HicB dimer hinder large conformational changes ([@B67]). Based on these data, we characterized the DNA-binding region of HicB^109--150^. The regions showing relatively large CSP values are mapped onto the surface representation of HicB^109--150^ in Figure [4C](#F4){ref-type="fig"}. The darker-colored region in the generated surface is the region that is most affected during interaction with DNA.
The active site of HicA and its ribonuclease activity are dependent on His36 {#SEC3-5}
----------------------------------------------------------------------------
Type II toxins usually show ribonuclease activity, and certain essential residues are required for the organization of the active sites in the VapBC, RelBE, MazEF and PezAT systems ([@B68]). To identify the active site of the HicBA system, sequence alignment was performed using Clustal Omega 1.2.1 ([@B69]) and visualized using ESPript 3.0 ([@B70]) (Figure [5A](#F5){ref-type="fig"}). The sequence of *S. pneumoniae* HicA was aligned with the hypothetical protein from *Thermus thermophilus*, the HicA toxin from *Y. pestis*, the HicA toxin from *Burkholderia pseudomallei*, and the HicA toxin from *E. coli*. The results show a highly conserved histidine residue in the β2 strand and a conserved glycine residue in the loop between β1 and β2, corresponding to a hydrogen-bonded turn. For a detailed comparison, the structural similarity of these proteins was analyzed using DALI ([@B71]), with the exception of *E. coli* HicA, for which no structure has yet been reported.
{#F5}
The structural homologs include (i) the hypothetical protein from *T. thermophilus* \[PDB code 1WHZ (chain A) with an r.m.s. deviation of 1.5 Å, a *Z*-score of 9.0 and a sequence identity of 29%\], (ii) the HicA toxin from *Y. pestis* ([@B39]) \[PDB code 4P78 (chains C and D) with r.m.s. deviations of 1.6--1.7 Å, *Z*-scores of 7.7--8.6 and a sequence identity of 34%\]; and (iii) the HicA toxin from *B. pseudomallei* ([@B47]) \[PDB code 4C26 (chain A) with an r.m.s. deviation of 2.3 Å, a *Z*-score of 5.7 and a sequence identity of 28%\] ([Supplementary Figure S5B](#sup1){ref-type="supplementary-material"}).
In the active site of *S. pneumoniae* HicA, the key residue His36 interacts closely with Thr33 and Glu47 of the HicB antitoxin. The Oη~1~ atom of Thr33 and the Oϵ~1~ atom of Glu47 form hydrogen bonds with the Nϵ~2~ atom and the N atom of this key residue. In addition, the Oϵ~1~ atom of Glu47 greatly stabilizes the active site by forming a salt bridge with the Nδ~1~ atom of the key residue. An intra-protein hydrogen bond between the N atom of the key residue His36 and the O atom of Lys33 in HicA is also observed (Figure [5B](#F5){ref-type="fig"}).
*S. pneumoniae* HicA forms a double-stranded RNA-binding domain ([@B45]). In the structure of the RNA-bound double-stranded RNA-binding domain (PDB code 1DI2) ([@B72]), α1 interacts with the minor groove of the RNA, and α2 interacts with the major groove of the RNA ([Supplementary Figure S5C](#sup1){ref-type="supplementary-material"}). RNA binding is mainly facilitated by these two α-helices; the β-strands make an insignificant contribution to RNA binding ([@B73]). HicA not only binds to RNA substrates but also cleaves them, thereby acting as a ribonuclease toxin ([@B46]). We assayed the RNase activity of HicA using fluorescent RNA substrates. When these substrates are cleaved, they emit fluorescence in proportion to the amount of the substrate cleaved. The ribonuclease activity of *S. pneumoniae* HicA was confirmed by the increase in the resulting fluorescence (RFU) as a function of the increase in the concentration of the HicA monomer protein from 1 μM to 8 μM (Figure [5C](#F5){ref-type="fig"}). At concentrations greater than 8 μM, the reaction was saturated. Due to loss of the key residue, HicA-H36A shows no ribonuclease activity. Compared to other toxin molecules with ribonuclease activity (VapC26 and VapC30 from *M. tuberculosis*), HicA showed the highest activity (Figure [5D](#F5){ref-type="fig"}).
Intermolecular interactions between HicB and HicA related to toxicity {#SEC3-6}
---------------------------------------------------------------------
The HicB antitoxin blocks the enzymatic function of the HicA toxin by forming the TA protein complex. In the toxin-neutralizing complex, HicB covers 1,183 Å^2^ of HicA. Approximately 43% of the residues of HicA toxin chain D participate in this interaction, and the interface area occupies approximately 28% of the total surface area of HicA.
In the area of the interface, hydrophobic interactions are contributed by Phe22, Met44, Phe80, Phe86 and Tyr90 of HicB and by Leu50 and Tyr57 of HicA. The aromatic interactions of Tyr57 of HicA are important in these hydrophobic interaction (Figure [6A](#F6){ref-type="fig"}). The HicB residues involved in hydrophilic interactions are Asp12, Ala19, Tyr30, Thr33, Gln34, Glu47, Thr89, Glu106 and Gln111; these residues interact with Arg30, Lys33, Ser35, His36, Lys38, Glu53, Asn55, Lys56, Tyr57, Thr58 and Gln65 of HicB. Additional salt bridges are formed by Asp12, Glu47, Asp55, and Asp83 of HicB and His36, Lys38, Lys56 and Lys64 of HicA (Figure [6B](#F6){ref-type="fig"}). The overall hydrophilic networks are presented in a schematic diagram.
{ref-type="fig"} are presented. (**C**) Effect of the mutation of residues involved in the interaction between HicB and HicA on the growth of *E. coli* cells. Mutated complexes were induced by IPTG to determine the toxicity to *E. coli* cells. The upper two quadrants indicate the empty vectors and the native HicBA complex. The decreased number of colonies in the lower quadrants indicates that the activity of the mutated HicBA complex was regained.](gky469fig6){#F6}
To verify the key residues that are essential for the interaction between HicB and HicA, six residues of HicB were mutated to alanine based on the interaction analysis shown in Figure [6A](#F6){ref-type="fig"} and [B](#F6){ref-type="fig"}. Four residues involved in hydrophilic interactions with multiple counterparts in HicA (Thr33, Gln34, Glu47 and Thr89) and two other residues involved in hydrophobic interactions (Phe22 and Phe80) were selected. Complexes of HicA with HicB-F22A and HicB-E47A resulted in decreased growth of *E. coli*, suggesting that mutation of Phe22 or Glu47 resulted in loss of the interaction between HicB and HicA. *E. coli* cells containing HicA-HicB-F22A and HicA-HicB-E47A produced far fewer colonies than *E. coli* cells containing the native HicBA complex (Figure [6C](#F6){ref-type="fig"}).
Artificial toxin activation and inhibition of cell growth by HicA {#SEC3-7}
-----------------------------------------------------------------
We designed peptides that mimic the binding interface of HicA to inhibit the interaction between HicB and HicA. Theoretically, these mimicking peptides should compete with HicA and hinder the formation of the TA complex. If a peptide displays high affinity for HicB, more free HicA will be present, resulting in increased RNase activity ([@B62]). The data presented in the main figure were obtained from experiments using the peptide '[ELNKYT]{.ul}E[RGI]{.ul}R[KQ]{.ul}AG', the activity of which was compared with that of other candidate peptides ([Supplementary Figure S6A and B](#sup1){ref-type="supplementary-material"}). The chosen peptide contains 11 residues corresponding to the α2 helix region of HicA (KYTERGIRKQA) and 11 residues corresponding to the interface between HicA and HicB; these residues are underlined. Peptides mimicking the helix are preferred because of their resistance to proteolytic degradation ([@B74]). Due to its membrane affinity and selectivity, the modeled helix should not be divided. Additionally, the optimal length of the helix should be modeled to prevent loss of selectivity and self-aggregation ([@B75]). The circular dichroism spectra of the peptides were determined; the peptide 'ELNKYTERGIRKQAG' showed the highest degree of helicity (39.8%). The helical content of each peptide was quantitatively measured based on the mean residue ellipticity, \[*θ*\]~222~ ([@B76]) ([Supplementary Figure S6C](#sup1){ref-type="supplementary-material"}).
The designed mimicking peptide includes the entire α2 helix of HicA and contains seven residues (Glu53, Asn55, Lys56, Tyr57, Thr58, Lys64 and Gln65) that participate in interactions with HicB. The peptide disrupts the interaction of the HicBA complex by up to 80% (Figure [7A](#F7){ref-type="fig"} and [B](#F7){ref-type="fig"}). The RFU at peptide concentrations greater than 16 μM did not show a marked difference from that observed at 16 μM.
{#F7}
Cell growth assays demonstrated the ability of HicA to arrest bacterial growth and the loss of toxicity of HicA-H36A (Figure [7C](#F7){ref-type="fig"}). *E. coli* cells expressing HicA showed decreased OD~600~ beginning at 2 h after induction. Cells expressing HicA-H36A grew at the same rate as the control cells.
The antimicrobial activity of the mimicking peptide is summarized in Figure [7D](#F7){ref-type="fig"}. The peptide exhibited detectable activities against certain bacterial species. The peptide with the strongest activity against both gram-positive and gram-negative bacteria had the smallest MIC value of 6.3 μM.
DISCUSSION {#SEC4}
==========
Structural uniqueness of the HicBA complex {#SEC4-1}
------------------------------------------
The structure of HicBA of *S. pneumoniae* presented here is the first structure to show the complete conformation of the HicBA TA system. The mechanism of assembly of the HicBA complex involves two dimerization regions of HicB that are located in its N-terminus and its C-terminus. The interface of *Y. pestis* HicBA was studied earlier ([@B39]), but the structural and functional characteristics of the flexible DNA-binding domain of HicB are revealed by our study. NMR assignments confirmed that the C-terminal DNA-binding motif of HicB is a ribbon-helix-helix ([@B43]). The DNA-binding domain corresponds to the flexible C-domain; demonstration of the flexible nature of HicBA provides a supplementary explanation for many flexible-domain-lacking TA complexes ([@B68],[@B77]). In addition, because an intrinsically disordered toxin-neutralizing domain modulates the affinity between the antitoxin and DNA, further study of the relationship between protein flexibility and disorder should yield crucial insights into the reversibility of TA action, including folding upon binding ([@B78]).
According to our SEC-MALS data, *S. pneumoniae* HicBA actually forms a hetero-octamer in solution. The radius of gyration (Rg) calculated from the hetero-octamer model by CRYSOL ([@B79]) (33.6 Å) was consistent with that obtained by analysis using SEC-MALS (35.7 Å). The hetero-tetramer model of HicBA in the crystal structure forms a very flexible and highly mobile structure in which there are no contacts between the C-terminal region of HicB and the remainder of the protein complex (Figure [1A](#F1){ref-type="fig"}). In addition, the functional unit of the ribbon-helix-helix motif is a dimer that contains a tight, stable antiparallel β-sheet ([@B43]). The functional unit of RHH is observed in the hetero-octamer model but not in the hetero-tetramer model (Figure [1A](#F1){ref-type="fig"}). Therefore, the observed conformation of the hetero-tetramer is probably biased by lattice contacts and may not correspond to its true solution structure. Furthermore, HicBs from both *S. pneumoniae* and *Y. pestis* form tetramers as shown by SEC-MALS data ([@B39]), which means that HicB requires homo-dimeric interfaces at both its N- and C-termini to form the tetramer ([Supplementary Figure S2B](#sup1){ref-type="supplementary-material"}). This experimental evidence is consistent with a hetero-octamer model of HicBA.
Insights into the DNA-binding mechanism of HicB {#SEC4-2}
-----------------------------------------------
According to the structural analysis, HicB from *S. pneumoniae* is composed of three domains: an N-domain, a hinge and a C-domain. The hinge is flexible and is formed by residues 107−112. The flexibility of the substrate-binding region of the protein is significantly associated with the specificity of its substrate. Thus, the conformational flexibility of the binding site greatly influences the protein\'s DNA-binding specificity ([@B80]). Based on our study, the flexible nature of HicB plays a crucial role in the protein\'s regulatory dynamics with respect to its target DNA ([@B81],[@B82]). As a transcriptional regulator, the flexible form of HicB is essential for bacterial cell life and death. The flexible domains of the protein undergo a folding transition upon binding to toxins or DNA and thereby exert important regulatory functions. In other words, the intrinsic flexibility of HicB confers functional advantages and enables HicB to bind to its target easily ([@B83],[@B84]).
Stabilization of the conformation of protein upon binding to its substrate might also explain the greater binding affinity of HicBA-DNA than that of HicB-DNA. HicA contributes thermodynamically to the binding affinity and the structural stability of HicB and DNA ([@B85],[@B86]). The mechanical process of binding of HicBA to DNA can be understood by analyzing their interaction at the atomic level ([Supplementary Figure S7A and B](#sup1){ref-type="supplementary-material"}). In our model, two antiparallel β-sheets bind to the major groove of DNA, and the electrostatically positive portions of the two α-helices anchor the DNA facing the phosphates of the DNA backbone. These binding characteristics of HicBA-DNA interactions are mediated by a stabilization-upon-binding mechanism that increases substrate specificity through conformational changes that occur upon the binding of the three binding partners HicB, HicA and DNA ([@B87],[@B88]).
New interactions involved in the active site of HicA {#SEC4-3}
----------------------------------------------------
The active sites of toxins belonging to the VapBC and RelBE families consist of three to four conserved residues and possible additional residues ([@B89],[@B90]). The residues comprising the active site are counteracted by binding of the cognate antitoxins. In proteins of the HicBA family, however, only one conserved histidine is present in the reported HicA toxins, and this histidine residue is likely to contribute to the toxicity of the protein (Figure [5B](#F5){ref-type="fig"}). Our mutation experiments confirmed that His36 is a critical source of toxin activity (Figure [5C](#F5){ref-type="fig"}). Furthermore, HicA toxins are smaller than MazF, RelE and VapC, and their active sites do not include any charged pockets, clusters or cavities ([@B48],[@B91],[@B92]) ([Supplementary Figure S8](#sup1){ref-type="supplementary-material"}). The presence of a conserved glycine and serine near His36 might support the structural integrity of the active site.
In contrast, interesting structural discoveries illuminate the mechanism of blocking of the active site in *Y. pestis* ([@B39]) and *S. pneumoniae* HicBA. In *S. pneumoniae* HicBA, His36 of HicA protrudes but is blocked by Thr33 and Glu47 of HicB. As a result of this interaction, these three residues form a catalytic triad ([@B93]) ([Supplementary Figure S9](#sup1){ref-type="supplementary-material"}). The catalytic triad consists of an acid, a base and a nucleophile. A similar triad is found in *Y. pestis* HicBA. The amino acids acting as the acid and base are the same in the two structures, a glutamate and a histidine, but the residue acting as a nucleophile is a threonine in *S. pneumoniae* and an asparagine in *Y. pestis* ([@B94]). A catalytic triad is usually located within the active site of a protease ([@B95]), but the type II toxin HicA is a ribonuclease and requires His36 for its ribonuclease activity. Therefore, the ribonuclease activity of HicA can be rationally suggested to be inhibited by a folding triad that cannot cut RNA and that is formed via binding to HicB. This explanation is consistent with the results of the mutational study obtained in the HicB neutralization assay, which showed that HicA-HicB-E47A (the HicBA complex with the E47A mutation in HicB) resulted in regeneration of the toxicity of HicA to cells (Figure [6C](#F6){ref-type="fig"}). The role of the acid is estimated to be more important than that of the variable nucleophiles because HicA-HicB-T33A did not show toxicity to cells.
Artificial activation of HicA by a mimicking peptide as an antibacterial strategy {#SEC4-4}
---------------------------------------------------------------------------------
Based on the toxicity of HicA shown in our study (Figure [7C](#F7){ref-type="fig"}), our peptide inhibitors can act as a novel antimicrobial agent by liberating HicA from the HicBA complex. Peptides that mimic the HicBA complex have greater ability to inhibit the interaction between toxin and antitoxin independent of the concentration of peptide than other TA-complex-mimicking peptides ([@B48],[@B49],[@B96]). Furthermore, the MIC of the tested peptides could be obtained via an antimicrobial activity test, despite its relatively high value compared with that of other highly engineered antimicrobial peptides ([@B74],[@B97]). The fact that the antimicrobial activity of peptides with higher α-helical content and better folding was greater ([Supplementary Figure S6C](#sup1){ref-type="supplementary-material"}) suggests that we might be able to obtain a more optimized inhibitor by modifying the peptide using conjugation strategies and surface modulation such as stapling the peptides via hydrocarbon cross-linking to obtain better permeability and structural folding ([@B98],[@B99]).
The expression of HicA protein in *E. coli* has been shown to severely reduce the translation rate ([@B100]), and the expression of HicA in *B. pseudomallei* was associated with modulation of the formation of persister cells ([@B47]). The inhibitory peptides designed for use in this study are based on the structure of the TA interface. Therefore, it is possible that these inhibitory peptides could bind to and inhibit the TA complexes of other bacteria that show structural similarity to HicBA. In conclusion, based on the experimental data obtained in this study, a peptide inducing adequate activation of HicA may serve as a new structure-based antimicrobial agent.
DATA AVAILABILITY {#SEC5}
=================
The structure described in this work has been deposited in the Protein Data Bank (PDB) under the accession code 5YRZ.
Supplementary Material
======================
######
Click here for additional data file.
We thank the beamline staff at Pohang Light Source, Korea (BL-5C and 7A) and SPring-8, Japan (BL44XU).
SUPPLEMENTARY DATA {#SEC6}
==================
[Supplementary Data](https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gky469#supplementary-data) are available at NAR Online.
FUNDING {#SEC7}
=======
National Research Foundation of Korea (NRF) through a grant funded by the Korean government (MEST) \[2018R1A2A1A19018526, 2018R1A5A2024425\]; 2017 BK21 Plus Project for Medicine, Dentistry and Pharmacy. Funding for open access charge: National Research Foundation of Korea.
*Conflict of interest statement*. None declared.
[^1]: The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
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The Ebola virus disease (EVD) outbreak in West Africa claimed \>11,000 lives with nearly 30,000 cases ([@R1]). During the outbreak in Sierra Leone, patients arriving at healthcare facilities were screened for EVD using World Health Organization (WHO) case definitions. Those fulfilling the case definition for suspected EVD were admitted to Ebola holding units (EHUs) to have blood taken for EVD testing and receive medical care until test results were available ([Technical Appendix](#SD1){ref-type="local-data"} Figure 1). Testing was usually performed offsite, with a turnaround time for results of ≈48 hours ([@R2]). During admission, however, EVD-negative patients risked exposure to EVD, raising concerns that EHUs could act as amplification sites for infection ([@R3]--[@R7]). Children, many of whom were unaccompanied, were particularly vulnerable, and, because EHUs were overstretched, supervision to minimize the risk of cross-infection was challenging ([@R4],[@R8]).
An accurate case definition for suspected EVD is critical for future outbreaks. Insufficient sensitivity of case definitions results in EVD-positive patients not being isolated, risking onward transmission in the community. There is an inherent tension between the public health priority to maximize the sensitivity of the case definition (minimizing onward transmission risk) and the individual patient's perspective. The trade-off made by lower specificity means that many EVD-negative patients are kept waiting in EHUs for test results, risking nosocomial infection and delaying treatment for their true underlying condition. Case definitions should be flexible because priorities may change as outbreaks progress. In the 2014--2015 epidemic, the proportion of patients testing positive decreased over time: in October 2014, 77% of those admitted to a Freetown EHU tested positive, versus 1% in April 2015 ([@R5]).
In Sierra Leone, 2 case definitions were used for suspected EVD ([@R9]). Until November 2014, most EHUs used a WHO case definition that was the same for both adults and children, defining anyone who had [\>]{.ul}3 symptoms consistent with EVD and fever, or who had fever and had contact with a person with EVD, as having a suspected case (early-2014 case definition). Beginning in December 2014, the WHO case definition was modified to be age dependent (late-2014 case definition) ([Figure 1](#F1){ref-type="fig"}; [Technical Appendix](#SD1){ref-type="local-data"} Table 1). Under this definition, children only required fever and either 1 symptom (in children \<5 years of age), 2 symptoms (in children 5--12 years of age), or [\>]{.ul}3 symptoms (in children \>12 years of age) ([@R4]). This definition increased the likelihood of admitting EVD-negative children. Furthermore, in overstretched EHUs, children may have been admitted without meeting the criteria for suspected EVD, regardless of definition. In a mixed-age West African cohort, 9% of those admitted did not fulfill the early-2014 case definition ([@R3]).
![World Health Organization screening flowchart for Ebola virus disease used during outbreak in Sierra Leone (late-2014 case definition). Adapted from ([@R9]).](17-1018-F1){#F1}
We aimed to develop a predictive score that could be used to tailor the pediatric case definition for suspected EVD according to the clinical and epidemiologic setting. The goal was to potentially limit unnecessary admissions to EHUs for EVD-negative children without reducing sensitivity.
Methods
=======
Data Sources
------------
We collected data on all children \<13 years of age admitted to 11 EHUs in Sierra Leone (August 2014--March 2015) and built training and validation datasets. We performed multivariable logistic regression on the training dataset to generate a pediatric Ebola predictive (PEP) score, which we tested on the validation dataset. The age cutoff matched the WHO case definition distinguishing between children and adolescents, anticipating that adolescents would have an adult disease phenotype. Settings and data collection methods have been described previously ([@R4],[@R10]). We visited each EHU to extract data from paper clinical records, case investigation forms, and site admission books and to interview staff. We cross-referenced data with the Western Area Ebola Response Centre (WAERC) database and 4 further sources, and single-entered data into a password-protected database (Epi Info version 7.1.4; US Centers for Disease Control and Prevention, Atlanta, GA, USA) ([Technical Appendix](#SD1){ref-type="local-data"}). We removed personal identifiers before analysis and developed a schema for record matching across databases ([Technical Appendix](#SD1){ref-type="local-data"}). We obtained ethics approval for this study from the Sierra Leone Ethics and Scientific Review Committee and the London School of Hygiene and Tropical Medicine Ethics committee (reference 8924).
Statistical Analysis
--------------------
We used Stata version 14.0 (StataCorp LLC, College Station, TX, USA) to perform analyses and limited analysis to children with EVD laboratory test result data. Variables were sex, age, contact history (yes/no), presence of 16 symptoms at EHU admission (yes/no), and days from symptom onset to EHU visit ([@R4]). We included age as a binary variable (\<2 years and [\>]{.ul}2 years), given the higher burden of febrile illnesses that appear similar to EVD (e.g., malaria) in younger children. We considered data to be missing from the analysis if no value had been entered in the source documents (i.e., neither yes nor no).
Descriptive analysis of the cohort comprised the number of children with data available for each variable and the prevalence of signs and symptoms by laboratory-confirmed EVD status. We estimated the proportion of children (for whom we had sufficient data) who met the late-2014 WHO case definition.
Predictive Model Building and Validation and Development of Risk Score
----------------------------------------------------------------------
We split the data randomly into 2 datasets with equivalent proportions of laboratory-confirmed EVD-positive children: a training dataset for predictive score building, and a validation dataset to assess score performance ([@R11]). Using the training dataset, we calculated crude odds ratios (ORs) of association between potential predictive variables and outcome (laboratory-confirmed EVD status) and created an initial multivariable model including all potential predictive variables. A final training model was obtained by removing variables with p\>0.3 from the fully adjusted model in a backward-stepwise fashion. The variables retained for constructing candidate PEP scores were age, gender, contact history, days from first symptoms to admission, and whether all symptoms were systematically documented ([Technical Appendix](#SD1){ref-type="local-data"}).
We created the PEP score by assigning integer scores to variables in the validation dataset on the basis of their regression coefficients in the training dataset model (score = 1 for coefficients \<1, score = 2 for coefficients [\>]{.ul}1) ([@R12]). We calculated each child's overall PEP score by adding together the integer scores for the variables present, which resulted in possible PEP scores of 0--10. To identify the most clinically useful PEP score, we computed the sensitivity, specificity, positive predictive value, negative predictive value, and percentage of children correctly classified (compared with the standard of laboratory confirmation of EVD) of each candidate PEP score. Fully calculating the validity of the WHO case definition would require data on false negatives (those turned away at screening who had EVD), but these data were not available. We compared the PEP score with the WHO case definition as accurately as the available data permitted for completeness ([Technical Appendix](#SD1){ref-type="local-data"}).
To explore the potential effects of PEP scores on the number of correct and incorrect admissions at different times in the epidemic, we applied 2 PEP scores with different levels of sensitivity and specificity to 2 hypothetical populations of children: early in the epidemic when the proportion of suspected cases testing positive in Western EHUs was 77% (high background prevalence, October 2014); and later in the epidemic when the proportion was 4% (low background prevalence, March 2015). We used these hypothetical background prevalences with the sensitivity and specificity for each score to calculate number of true positives and negatives and false positives and negatives obtained by applying each score ([Technical Appendix](#SD1){ref-type="local-data"} Tables 2--5) ([@R5]). We used multiple imputation by chained equations to account for missing data in the analysis of training and validation datasets ([Technical Appendix](#SD1){ref-type="local-data"}) ([@R13]).
Results
=======
Of 1,054 children admitted with suspected cases to 11 EHUs during August 14, 2014--March 31, 2015, no result was available for 48 (5%) ([Technical Appendix](#SD1){ref-type="local-data"}). Of the remaining 1,006 children, 309 (31%) were EVD positive and 697 (69%) EVD negative. Admissions rose from a median 8 (interquartile range \[IQR\] 5--11) per week in August--October 2014 to 50 (40--58) per week in February--March 2015, but the proportion of children that were EVD positive decreased from 57% (95% CI 43%--72%) in October 2014 to 6% (95% CI 2%--9%) in February 2015. At Ola During Children's Hospital (ODCH), the main children's hospital in Freetown, the onsite EHU received 59% of all EHU admissions, increasing from 12% in August--October 2014 to 82% in February--March 2015.
We documented admission of 211 (21%) unaccompanied children. Data were missing for 297 (30%) of the children. EVD-positive children were more likely to be unaccompanied than those who were EVD negative (p\<0.001).
Median patient age was 4 years (IQR 1.3--8.0 years), and 51% of the children were female ([Table 1](#T1){ref-type="table"}). Contact with EVD was reported for 275 (36%) of 754 children who had data available (75% of 1,006 total). Median time from symptom onset to hospital visit was 2 days (IQR 1--4). Fever data were available for 787 (78%) of children ([Table 1](#T1){ref-type="table"}), 775 of whom also had data available on the presence of [\>]{.ul}3 other symptoms. For those with data, fatigue/weakness was most frequently reported (97%), followed by fever (94%), anorexia (80%), vomiting (61%), headache (62%), and diarrhea (46%) ([Table 1](#T1){ref-type="table"}). Bleeding was rare, reported by 3%. Of the 809/1,006 (80%) of children who had sufficient symptom and contact history data recorded to ascertain if they fulfilled the late-2014 WHO suspected case definition, 31 (4%) were admitted despite not meeting the definition ([Technical Appendix](#SD1){ref-type="local-data"}).
###### Overview of 1,006 children who attended an Ebola holding unit and had EVD test results recorded, by final EVD test result status, Sierra Leone, August 14, 2014--March 31, 2015\*
Characteristic All children,
no. (%) or median (IQR) EVD negative EVD positive p value
---------------------------------------------- ---------------------------------------- ----------------------------------- ------------- -------------- ----------- ------------- ---------
No./no. available or median (IQR) \% (95% CI) No./no. available or median (IQR) \% (95% CI)
Total† 1,006 (100) 697 69 309 31 --
Sex
F 512 (51) 348/697 50 (46--54) 164/309 47 (41--53) 0.357
M 494 (49) 349/697 50 (46-54) 145/309 53 (47--59) 0.380
Median age, y (IQR) 4 (1.3--8) 3 (1--7) -- 6 (3--10) -- \<0.001
Age 0--2 y 392 (39) 336/697 48 (44--52) 56/309 18 (14--23) \<0.001
Positive contact, n = 754‡ 275 (36) 108/541 20 (17--24) 167/213 78 (72--84) \<0.001
Days from symptoms to EHU admission, n = 772 2 (1--4) 2 (1--3) -- 3 (2--4) -- 0.001
Admitted with caregiver, n = 822 822 (82) 516/621 83 (80--86) 127/201 63 (56--70) \<0.001
Signs/symptoms§
Fever, n = 787 740 (94) 528/566 93 (91--95) 212/221 96 (92--98) 0.160
Fatigue/weakness, n = 587 568 (97) 393/407 97 (94--98) 175/180 97 (94--99) 0.676
Vomiting/nausea, n = 777 472 (61) 345/556 62 (58--66) 127/221 57 (51--64) 0.238
Diarrhea, n = 763 351 (46) 252/548 46 (42--50) 99/215 46 (39--53) 0.988
Conjunctivitis, n = 669 152 (23) 73/463 16 (13--19) 79/206 38 (32--45) \<0.001
Anorexia, n = 779 621 (80) 452/560 81 (77--84) 169/219 77 (71--83) 0.269
Abdominal pain, n = 594 269 (45) 155/392 40 (35--45) 114/202 56 (49--63) \<0.001
Muscle pain, n = 577 212 (21) 127/377 34 (29--39) 85/200 43 (36--50) 0.037
Joint pain, n = 569 192 (34) 102/368 28 (23--33) 90/201 45 (38--52) \<0.001
Headache, n = 598 370 (62) 256/397 65 (60--69) 114/201 57 (50--64) 0.065
Difficulty breathing, n = 738 199 (27) 169/533 32 (28--36) 30/205 15 (10--20) \<0.001
Difficulty swallowing, n = 687 177 (26) 130/481 27 (23--31) 47/206 23 (17--29) 0.247
Rash, n = 728 98 (13) 88/522 17 (14--20) 10/206 5 (2--9) \<0.001
Cough, n = 587 70 (12) 57/407 14 (11--18) 13/180 7 (4--12) 0.019
Hiccups, n = 723 62 (9) 52/519 10 (8--13) 10/204 5 (2--9) 0.027
Unexplained bleeding, n = 726 22 (3) 19/518 4 (2--6) 3/208 1 (0--4) 0.114
Treatment¶
Antimicrobial drug, n = 657 556 (85) 407/494 82 (79--86) 149/163 91 (86--95) 0.006
Antimalarial drug, n = 657 567 (86) 416/494 84 (81--87) 151/163 93 (87--96) 0.007
IV treatment 115 (11) 101/697 14 (12--17) 14/309 5 (2--7) \<0.001
Malaria RDT+, n = 74 33 (45) 31/57 54 (41--68) 2/17 12 (15--36) 0.002
Median days of EHU stay\# 2 (1--3) 2 (1--2) -- 2 (1--3) -- \<0.001
\*n values and denominators indicate no. children with recorded data available for variable (i.e., for binary variables children with neither "yes" nor "no" populated in their source notes were not included in the denominator, and for the median days symptoms to EHU admission variable those without date of start of symptoms were not included). EHU, Ebola holding unit; EVD, Ebola virus disease; RDT, rapid diagnostic test.
†z-test of proportions, comparing whether the proportion of children with the variable was the same for EVD-negative and EVD-positive children (apart from numerical variables, for which a Wilcoxon rank-sum test was performed to test the hypothesis that the distribution of the variable was the same for EVD-negative and EVD-positive children).
‡Total no. children admitted to holding units with test results available.
§Recorded on presentation at EHU.
¶At EHU.
\#Time from EHU admission until death, discharge, or transfer.
Children who were EVD negative were younger (median age 3 years \[IQR 1--7 years\] vs. 6 years \[IQR 3--10 years\]; p\<0.001) ([Table 1](#T1){ref-type="table"}) and less likely to have conjunctivitis (p\<0.001) than those who were EVD positive. Rash was more common in EVD-negative children (p\<0.001) ([Table 1](#T1){ref-type="table"}; [Figure 2](#F2){ref-type="fig"}). Similar proportions of both groups received antimicrobial and antimalarial drugs, and whereas both spent a median of 2 days in an EHU (admission to death or transfer/discharge), those with EVD tended to stay longer (p\<0.001) ([Table 1](#T1){ref-type="table"}).
{#F2}
Randomly splitting the cohort of 1,006 children generated training and validation datasets of 504 and 502 (descriptive, crude, and adjusted analysis in [Technical Appendix](#SD1){ref-type="local-data"} Table 6). In the training cohort, positive contact (multivariable OR 9.1, 95% CI 4.9--17); age [\>]{.ul}2 years (multivariable OR 2.9, 95% CI 1.4--5.8); and conjunctivitis (multivariable OR 3.8, 95% CI 1.9--7.8) were the strongest positive predictors of EVD. Headache, difficulty breathing, difficulty swallowing, and rash were negative predictors. The final multivariable predictive model included 12 variables: gender; age; positive contact; and presence or absence at hospital visit of fever, diarrhea, conjunctivitis, anorexia, abdominal pain, headache, difficulty breathing, difficulty swallowing, and rash. We present only analysis of the complete records, based on the similarity of receiver operating characteristics (ROC) curves for imputed and complete records analyses ([Technical Appendix](#SD1){ref-type="local-data"} Table 7; [Figure 3](#F3){ref-type="fig"}).
{#F3}
Assigning predictive model values derived from the training dataset to the validation dataset gave a range of PEP scores of 0--10. Plotting the ROC curve as sensitivity (x) against 1 − specificity (y) for all individual child PEP scores (with sensitivity and specificity calculated using the laboratory test as standard) demonstrated that the model had excellent discriminative ability (area under ROC curve = 0.80; [Figure 3](#F3){ref-type="fig"}) ([@R14]). The model coefficients, p values, and assigned integer PEP scores are shown in [Table 2](#T2){ref-type="table"} and the sensitivity, specificity, positive and negative predictive values, and percentage correctly classified for all possible PEP scores within the validation dataset in [Table 3](#T3){ref-type="table"}. A PEP score of 1 was 97% sensitive (95% CI 89%--100%) and 4% specific (95% CI 1%--8%), whereas the maximum PEP of 10 was 5% sensitive (95% CI 1%--13%) and 99% specific (95% CI 96%--100%) ([Table 3](#T3){ref-type="table"}).
###### Scores for each of the variables included in Ebola pediatric predictive model
Variable Coefficient (95% CI) from multivariable model p value Integer score value
----------------------- ----------------------------------------------- --------- ---------------------
Positive contact 2.21 (1.58--2.83) \<0.001 +2
Conjunctivitis 1.34 (0.62--2.05) \<0.001 +2
Age [\>]{.ul}2 y 1.06 (0.37--1.75) 0.003 +2
Fever 0.99 (--0.66 to 2.63) 0.241 +1
Anorexia 0.59 (--0.18 to 1.35) 0.133 +1
Male gender 0.49 (--0.11 to 1.08) 0.111 +1
Abdominal pain 0.42 (--0.23 to 1.08) 0.205 +1
Diarrhea 0.40 (--0.21 to 1.01) 0.197 +1
Difficulty breathing --0.57 (--1.39 to 0.24) 0.168 −1
Difficulty swallowing --0.59 (--1.39 to 0.19) 0.138 −1
Headache --0.63 (--1.29 to 0.35) 0.063 −1
Rash **−**1.00 (--2.13 to 0.14) 0.085 −2
###### Validation of PEP score against a standard of laboratory-confirmed Ebola virus disease status, compared with WHO case definition, based on a cohort of children who attended an Ebola holding unit and had EVD test results recorded, Sierra Leone, August 14, 2014--March 31, 2015\*
Score Sensitivity (95% CI) Specificity (95% CI) PPV (95% CI) NPV (95% CI) \% Correctly classified (95% CI)
---------------------- ---------------------- ---------------------- -------------- -------------- ----------------------------------
0 100 1 (0--4) 31 (25--38) 100 31 (25--38)
1 97 (89--100) 4 (1--8) 31 (25--38) 71 (29--96) 32 (26--39)
2 97 (89--100) 13 (8--20) 33 (27--40) 91 (70--99) 39 (32--46)
3 94 (85--98) 30 (22--37) 37 (30--45) 91 (79--98) 49 (42--56)
4 86 (75--93) 49 (40--57) 43 (34--52) 89 (80--95) 60 (53--67)
5 77 (64--86) 67 (58--74) 51 (40--61) 87 (79--92) 70 (63--76)
6 58 (45--70) 82 (75--88) 59 (46--71) 81 (74--87) 75 (68--80)
7 44 (31--57) 92 (86--96) 70 (54--83) 79 (72--85) 77 (71--82)
8 23 (14--35) 95 (90--98) 68 (45--86) 74 (67--80) 73 (67--79)
9 11 (5--21) 98 (94--100) 70 (35--93) 71 (64--77) 71 (64--77)
10 5 (1--13) 99 (96--100) 75 (19--99) 70 (63--76) 70 (63--76)
WHO case definition† 98 (95--99) 5 (3--7) 30 (27--34) 84 (66--95) 33 (29--36)
\*EVD, Ebola virus disease; NPV, negative predictive value; PEP, pediatric Ebola predictor; PPV, positive predictive value; WHO, World Health Organization.
†Late-2014 WHO case definition with pediatric differentiations.
We considered the effect of using different PEP scores at different times during the outbreak. PEP score 3 (sensitivity of 94% and specificity of 30%) at the high background prevalence time point would have correctly classified 79 patients, with 16 EVD-negative patients admitted unnecessarily and 5 EVD-positive patients being incorrectly not admitted ([Table 4](#T4){ref-type="table"}; [Technical Appendix](#SD1){ref-type="local-data"} Tables 2,3). Using a PEP score of 7 (sensitivity 44% and specificity 92%) at the low background prevalence time point would have correctly classified 90/100 patients, with 8 unnecessary admissions and 2 true EVD-positive patients incorrectly not admitted ([Table 4](#T4){ref-type="table"}; [Technical Appendix](#SD1){ref-type="local-data"} Tables 4,5). Because we only have the true EVD status of patients who were admitted despite screening negative by WHO case definition (not the much larger number who were WHO case definition negatives and not admitted), the sensitivity and specificity calculated may be unreliable ([Technical Appendix](#SD1){ref-type="local-data"}). However, on the basis of the data available, the WHO case definition was estimated to be 98% sensitive and 5% specific ([Table 3](#T3){ref-type="table"}; [Technical Appendix](#SD1){ref-type="local-data"} Tables 8,9).
###### Comparison of 2 different PEP scores on a hypothetical population of 100 suspected EVD patients at different points in EVD outbreak with differing prevalence of EVD\*
PEP score October 2014, 77% of suspected EVD+ cases† March 2015, 4% of suspected EVD+ cases†
------------------------------------- -------------------------------------------- ------------------------------------- ----------------------------------------- ------------------------------- ------------------------------------ ------------------------------------- --------------------------------------- ---- ---
True EVD+,
correctly admitted True EVD--, correctly
not admitted False
EVD+, unnecessarily
admitted False
EVD--, incorrectly not admitted True EVD+, correctly admitted True EVD--, correctly
not admitted False
EVD+, unnecessarily
admitted False EVD--, incorrectly not admitted
3: 94% sensitivity, 30% specificity 72 7 16 5 4 28 68 0
7: 44% sensitivity, 92% specificity 34 21 2 43 2 88 8 2
\*Laboratory-confirmed EVD status figures from Connaught Hospital (Freetown, Sierra Leone) during the 2014--2015 outbreak. EVD, Ebola virus disease; PEP, pediatric Ebola predictive; +, positive; --, negative.
†True or false EVD+ or EVD-- determined by case ascertainment by PEP score. Admission result represents modeled outcome for patients in terms of Ebola holding unit.
Discussion
==========
This large, multicenter study compared symptoms at hospital visit in children \<13 years old who were determined to be positive or negative for EVD during the outbreak in West Africa. As with many childhood diseases, EVD symptoms are nonspecific. The WHO indicators, including fever, breathing difficulties, and gastrointestinal symptoms, are common features in many pediatric pathologies. In this outbreak, gastrointestinal symptoms dominated, whereas bleeding, characteristic of previous outbreaks, was rare ([@R3],[@R15]--[@R19]). This difference meant clinical diagnosis of EVD in the West African outbreak was difficult, which motivated this study. The lack of specificity of both early- and late-2014 WHO case definitions is highlighted by the fact that 69% of the children admitted as suspected EVD cases in this cohort were uninfected; that number increased to 94% in low-prevalence weeks ([@R10]).
Although elegant clinical predictive models have been developed for mixed-age cohorts, the focus of our model is children ([@R3],[@R17],[@R18],[@R20]--[@R22]). The features at presentation that had the strongest association with a positive laboratory test result in this study were positive contact, conjunctivitis (similar to mixed-age cohorts \[[@R17],[@R22]\]), and age [\>]{.ul}2 years. Fever, anorexia, abdominal pain, and diarrhea were weaker predictors of EVD. Certain features in the late-2014 WHO case definition were either not predictive or negative predictors, including bleeding, vomiting/nausea, difficulty breathing or swallowing, muscle or joint pain, headache, or rash ([Table 1](#T1){ref-type="table"}) ([@R9]). These findings emphasize the challenge of diagnosing EVD against high background rates of malaria and respiratory and gastrointestinal infections in children. The early-2014 WHO case definition demonstrated similar lack of specificity (32%) in 1 retrospective mixed-age cohort (sensitivity 80%) ([@R3]), although slightly better figures were documented in 2 smaller mixed-age cohorts ([@R20],[@R23]).
The PEP score model described here could provide the basis for modifying pediatric case definitions as an outbreak evolves, or for different pediatric populations (e.g., at triage in an EHU vs. potentially lower-risk routine outpatient consultations). Similar to the mixed-age, malaria-sensitive score proposed by Hartley et al. ([@R17]), a patient with a high score would be strongly suspected and a low score weakly suspected of having EVD. In times of high community prevalence, children with a PEP score [\>]{.ul}7 ([\>]{.ul}92% specificity, 44% sensitivity) could rapidly be transferred to an ETC while awaiting laboratory confirmation, whereas those with a PEP score of 3 (sensitivity 94%, specificity 30%) could await test results in the EHU. This change could hasten access to specialist care for children with EVD and reduce exposure risk for those who are negative.
Assessing the applicability of our PEP score to future Ebola virus epidemics is important. Ideally, the model should be tested against other datasets from West Africa and prospectively in future outbreaks, because different EVD strains are likely to result in different disease manifestations. Indeed, in another pediatric cohort from Kailahun and Bo, Sierra Leone, containing 91 children \<5 years of age, fever was absent in 25% (compared with 4% in our study) whereas bleeding was seen in 15% ([@R15]). In a large international cohort of 1,371 children \<16 years of age with EVD, fever prevalence was 90% and bleeding 10% ([@R24]). However, it is possible that future pediatric case numbers may be smaller than those seen in this outbreak, which limits opportunities for prospective validation. We suggest governmental and nongovernmental organizations use this non--outbreak period to discuss with local stakeholders the acceptability of the trade-offs inherent within the PEP score, such as public health versus individual risk. One option would be the rapid setup of a triage facility admitting children with a PEP score [\>]{.ul}3 to await test results and fast-tracking those scoring [\>]{.ul}7 to specialized Ebola treatment. However, this decision is highly context-specific, and there are dangers in being too prescriptive without taking into account factors such as local healthcare-seeking behavior.
A key limitation to our study is that PEP scores are derived from a population of children admitted to EHUs, all of whom should have fulfilled either the early- or late-2014 WHO suspected case definition. We do not have information on those not admitted (who were either truly EVD negative or missed EVD-positive cases). Therefore, we could only use data on the small number of children admitted who did not meet the WHO case definition to calculate its sensitivity and specificity, and these children may not have been representative of children who were negative by the WHO case definition but not admitted. Our calculations of WHO case definition validity are therefore only included for completeness and must be treated with caution. A further limitation is reducing EVD contact to a binary variable; more in-depth information (such as whether the child has had contact with a dead body, or whether the child is breastfeeding) could give greater discrimination. However, because 37% EVD-positive children were unaccompanied at hospital admission, an in-depth contact history was unlikely to be reliable.
Missing and unreliable data are another limitation, illustrating the challenge of epidemiologic studies that analyze data from emergency settings. This study was retrospective, using data collected as part of outbreak data gathering rather than as part of a formal prospective study. We accounted for missing data using multiple imputation; reassuringly, imputed analysis gave similar results to a complete records analysis. We are also limited to data from those who sought medical care; thus, the description of EVD/non-EVD cases may be incomplete. External and prospective validation will be key but may be limited by small numbers. Finally, Hartley et al. have demonstrated the crucial importance of malaria testing in diagnostic screening for EVD ([@R17]). We did not have sufficient numbers of children with malaria test results in this cohort to incorporate malaria test results into our predictive score.
We have demonstrated that using a PEP score may help to streamline and improve management for children with suspected EVD, but the score still does not approach the accuracy of laboratory testing. Even by using a sensitive PEP score of 3, at high background prevalence, it is possible that 6% (5/77) of children with EVD could be turned away from an EHU in error ([Table 4](#T4){ref-type="table"}), which would have serious public health implications. Several highly sensitive rapid diagnostic tests (RDT) for EVD underwent preliminary testing toward the end of the West Africa outbreak, although the numbers of children included in these studies were limited ([@R25],[@R26]). Judicious use of EVD RDTs coupled with PCR tests to confirm results could have reduced the scale of the Sierra Leone outbreak ([@R27]). Further development of RDTs, and guidance on selecting the children on whom to use them, is essential for preparing for and responding to future outbreaks. Incorporating screening criteria from an evidence-based clinical prediction model, such as this PEP score model, should contribute to this process.
In conclusion, this study compares features at hospital arrival in EVD-negative and EVD-positive children during the West African epidemic. We describe a predictive PEP score model that would allow for the selection of appropriate case definitions (prioritizing sensitivity or specificity) depending on the clinical and epidemiologic setting. The selected PEP scores had higher positive and negative predictive values than the current WHO case definition. Applying the score in combination with RDTs could be a successful strategy in future outbreaks. External validation of the PEP score will be key to establishing its utility, but because data are scarce, we suggest local stakeholders use this postoutbreak period to reflect how the PEP score might best be used in their context.
######
**Technical Appendix.** Additional information about Ebola infection in children and the development of the Pediatric Ebola Predictive Score (PEP score).
*Suggested citation for this article*: Fitzgerald F, Wing K, Naveed A, Gbessay M, Ross JCG, Checchi F, et al. Developing a pediatric Ebola predictive score, Sierra Leone. Emerg Infect Dis. 2018 Feb \[*date cited*\]. <https://doi.org/10.3201/eid2402.171018>
Preliminary results from this study were presented at the 26^th^ European Congress of Clinical Microbiology and Infectious Diseases, Amsterdam, Netherlands, April 9--12, 2016; and the 34th Annual Meeting of the European Society for Pediatric Infectious Diseases, Brighton, UK, May 10--14, 2016.
These authors contributed equally to this article.
We thank T.B. Kamara, B.E. Parker, V. George, Dr. King, F. Koroma, Z.M. Cooper, I. Sesay, Marion Dumbuya, and the Live Case Management Team of Western Area Emergency Response Centre, Sierra Leone, for their assistance and sharing data. We also thank Brima Kargbo, Marta Lado, Quaanan Kessete, Iza Ciglenecki, Monia Sayah, Rupert Gould, Brian Raleigh, Trina Helderman, David Sinclair, Hana Rohan, Tim Brooks, Charity Garnett, Rachael Cummings, Kate Jarman, and the nurses, cleaners, and community health officers at each medical facility.
This work was supported by Save the Children. F.F. is supported by a grant from the Medical Research Council (MR/K023535/1) and the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. S.Y. was supported by a Wellcome Trust Institutional Strategic Support Fund awarded to the London School of Hygiene and Tropical Medicine.
Dr. Fitzgerald is an NIHR academic clinical lecturer at the UCL Great Ormond Street Institute for Child Health in pediatric infectious diseases. Her research interests include Ebola virus disease, HIV, molecular microbiology, and antimicrobial resistance.
|
Clinical implications of clopidogrel resistance.
The benefits of clopidogrel in the treatment and prevention of coronary artery disease are well established, however, not all individuals respond in the same way to clopidogrel; there are patients who suffer adverse events despite clopidogrel treatment. This review focuses on the definition, potential mechanisms for and clinical implications of clopidogrel resistance, as well as the strategies to improve the response to this antiplatelet drug. There is an inter-individual variability in response to clopidogrel therapy, and a sub-optimal response (clopidogrel resistance) has been associated with adverse cardiovascular events. Nevertheless, there is no clear and consensual definition of clopidogrel resistance. Response to clopidogrel therapy follows a normal, bell-shaped distribution, so a more appropriate description would be variable response rather than clopidogrel resistance. Independent of the term used, lower response to clopidogrel therapy seems to be associated with a higher probability of suffering thrombotic events. Due to the misleading definition of resistance and non-standardized method for assessing platelet inhibition, current guidelines do not recommend the use of platelet function assays to monitor the inhibitory effect of antiplatelet drugs. Current guidelines also do not recommend clopidogrel loading doses higher than 300 mg and daily maintenance doses higher than 75 mg, even though a regimen of 600 mg clopidogrel loading dose seems to be preferred for patients undergoing percutaneous coronary interventions. |
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package oimo.collision.geometry;
import oimo.collision.raycast.*;
import oimo.common.MathUtil;
import oimo.common.Transform;
import oimo.common.Vec3;
import oimo.m.IVec3;
import oimo.m.M;
/**
* A cylinder collision geometry aligned with the y-axis.
*/
@:build(oimo.m.B.bu())
class CylinderGeometry extends ConvexGeometry {
public var _radius:Float;
public var _halfHeight:Float;
/**
* Creates a cylinder collision geometry of radius `radius` and half-height `halfHeight`.
*/
public function new(radius:Float, halfHeight:Float) {
super(GeometryType._CYLINDER);
_radius = radius;
_halfHeight = halfHeight;
_updateMass();
}
/**
* Returns the radius of the cylinder.
*/
public inline function getRadius():Float {
return _radius;
}
/**
* Returns the half-height of the cylinder.
*/
public inline function getHalfHeight():Float {
return _halfHeight;
}
override public function _updateMass():Void {
var r2:Float = _radius * _radius;
var h2:Float = _halfHeight * _halfHeight * 4;
_volume = MathUtil.PI * r2 * _halfHeight * 2;
M.mat3_diagonal(_inertiaCoeff,
1 / 12 * (3 * r2 + h2),
1 / 2 * r2,
1 / 12 * (3 * r2 + h2)
);
}
override public function _computeAabb(aabb:Aabb, tf:Transform):Void {
var axis:IVec3;
var axis2:IVec3;
var eh:IVec3;
var er:IVec3;
M.mat3_getCol(axis, tf._rotation, 1);
M.vec3_abs(axis, axis);
M.vec3_compWiseMul(axis2, axis, axis);
var axis2x:Float = M.vec3_get(axis2, 0);
var axis2y:Float = M.vec3_get(axis2, 1);
var axis2z:Float = M.vec3_get(axis2, 2);
M.vec3_set(er, MathUtil.sqrt(1 - axis2x), MathUtil.sqrt(1 - axis2y), MathUtil.sqrt(1 - axis2z));
M.vec3_scale(er, er, _radius);
M.vec3_scale(eh, axis, _halfHeight);
var max:IVec3;
M.vec3_add(max, er, eh);
M.vec3_sub(aabb._min, tf._position, max);
M.vec3_add(aabb._max, tf._position, max);
}
override public function computeLocalSupportingVertex(dir:Vec3, out:Vec3):Void {
var rx:Float = dir.x;
var rz:Float = dir.z;
var len:Float = rx * rx + rz * rz;
var coreRadius:Float = _radius - _gjkMargin;
if (coreRadius < 0) coreRadius = 0;
var invLen = len > 0 ? coreRadius / MathUtil.sqrt(len) : 0;
var coreHeight:Float = _halfHeight - _gjkMargin;
if (coreHeight < 0) coreHeight = 0;
out.x = rx * invLen;
out.y = dir.y > 0 ? coreHeight : -coreHeight;
out.z = rz * invLen;
}
override public function _rayCastLocal(begin:IVec3, end:IVec3, hit:RayCastHit):Bool {
var p1x:Float = M.vec3_get(begin, 0);
var p1y:Float = M.vec3_get(begin, 1);
var p1z:Float = M.vec3_get(begin, 2);
var p2x:Float = M.vec3_get(end, 0);
var p2y:Float = M.vec3_get(end, 1);
var p2z:Float = M.vec3_get(end, 2);
var halfH:Float = _halfHeight;
var dx:Float = p2x - p1x;
var dy:Float = p2y - p1y;
var dz:Float = p2z - p1z;
// Y
var tminy:Float = 0;
var tmaxy:Float = 1;
if (dy > -1e-6 && dy < 1e-6) {
if (p1y <= -halfH || p1y >= halfH) {
return false;
}
} else {
var invDy:Float = 1 / dy;
var t1:Float = (-halfH - p1y) * invDy;
var t2:Float = (halfH - p1y) * invDy;
if (t1 > t2) {
var tmp:Float = t1;
t1 = t2;
t2 = tmp;
}
if (t1 > 0) tminy = t1;
if (t2 < 1) tmaxy = t2;
}
if (tminy >= 1 || tmaxy <= 0) return false;
// XZ
var tminxz:Float = 0;
var tmaxxz:Float = 1;
var a:Float = dx * dx + dz * dz;
var b:Float = p1x * dx + p1z * dz;
var c:Float = (p1x * p1x + p1z * p1z) - _radius * _radius;
var D:Float = b * b - a * c;
if (D < 0) return false;
var t:Float;
if (a > 0) {
var sqrtD:Float = MathUtil.sqrt(D);
tminxz = (-b - sqrtD) / a;
tmaxxz = (-b + sqrtD) / a;
if (tminxz >= 1 || tmaxxz <= 0) return false;
} else {
if (c >= 0) return false;
tminxz = 0;
tmaxxz = 1;
}
var min:Float;
if (tmaxxz <= tminy || tmaxy <= tminxz) return false;
if (tminxz < tminy) {
min = tminy;
if (min == 0) return false; // the ray starts from inside
hit.normal.init(0, dy > 0 ? -1 : 1, 0);
} else {
min = tminxz;
if (min == 0) return false; // the ray starts from inside
hit.normal.init(p1x + dx * min, 0, p1z + dz * min).normalize();
}
hit.position.init(p1x + min * dx, p1y + min * dy, p1z + min * dz);
hit.fraction = min;
return true;
}
}
|
Viktor Sokol (footballer, born 1954)
Viktor Petrovich Sokol (, ; born 5 December 1954) is a retired Soviet and Belarusian football player and a currently a manager. His son, who is also named Viktor Sokol is also a professional footballer.
Honours
Soviet Top League winner: 1982.
European Cup top scorer: 1983–84.
External links
Profile
Category:1954 births
Category:Living people
Category:Sportspeople from Minsk
Category:Soviet footballers
Category:Belarusian footballers
Category:Belarusian expatriate footballers
Category:Expatriate footballers in Poland
Category:Soviet Top League players
Category:FC Dynamo Brest players
Category:FC Dinamo Minsk players
Category:Jagiellonia Białystok players
Category:FC Dinamo-93 Minsk players
Category:Belarusian football managers
Category:FC Dinamo-93 Minsk managers
Category:FC Dynamo Brest managers
Category:Association football forwards |
Oxidants and antioxidants in disease: oxidative stress in farm animals.
Important infectious diseases in farm animals, such as pneumonia and enteritis, are thought to be associated with the so-called oxidative stress, i.e. a chemical phenomenon involving an imbalance in the redox status of the individual animal. The specifics of oxidative stress and how it may result in disease or be prevented are complex questions with no simple answers. However, the considerable literature on the subject suggests that many researchers consider oxidative stress-related mechanisms to be important early events in disease development. A particularly intriguing aspect is that, at least theoretically, oxidative stress should be easily prevented with antioxidants yet the use of antioxidants as therapy remains controversial. The present knowledge on oxidative stress in farm animals is the topic of this review. |
[Twelve-month-old infants show social preferences for native-dialect speakers].
Recent research demonstrates that social preferences for native language speakers emerge early in development, indicating that infants prefer speakers from their own society. Dialect may also be a reliable cue to group membership because it provides information about an individual's social and ethnic identity. We investigated whether infants showed social preferences toward native-dialect speakers over those with unfamiliar dialects. Infants at 9 and 12 months of age were shown videos in which two adults (a native-dialect speaker and an unfamiliar-dialect speaker) each spoke to and then offered an identical toy to the participating infants. Next, two real versions of the toys were presented to the infants in person. The 12-month-old infants preferentially reached for the toy offered by the native-dialect speaker. The 9-month-old infants also showed a preference for native-dialect speakers but this finding was not statistically significant. Our results suggest that dialects may be a reliable cue to group membership, and that infants' orientation toward members of their native community may guide their social and cultural learning. |
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|
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